WO2024258052A1 - Simultaneous quantification of bk virus and cytomegalovirus in sample - Google Patents

Abstract

The present invention relates to a multiplexing assay method for the quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample. According to the present invention, BKV and CMV in a sample can be simultaneously quantified by one reaction, thereby reducing inspection costs such as a reduction in the amount of the sample, a reduction in inspection time, and a reduction in inspection equipment and administration manpower. In addition, the oligonucleotide set used in the method of the present invention enables more accurate diagnosis of BKV and CMV with high sensitivity and specificity. Furthermore, the method of the present invention can increase the diagnosis rate of opportunistic infection in kidney transplanted patients and contribute to the improvement of the survival rate of transplanted organs and patients in the long term.

Description

Method for simultaneous quantification of BK virus and cytomegalovirus in samples

The present invention relates to a method for simultaneous quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample.

Human polyomavirus type BK virus is a non-enveloped virus with a circular, double-stranded DNA genome of approximately 5,300 bp. The genome encodes three capsid structural proteins, namely viral capsid protein 1 (VP1), VP2, and VP3, as well as the large T and small T antigens.

BK virus was first isolated in 1971 from urine from a patient with ureteric stenosis who had undergone renal transplantation, with the initials "B.K." Although BK virus infection is widespread, infected individuals are usually asymptomatic or present only with mild symptoms (e.g., respiratory infection or fever). After initial infection, BK virus typically remains latent in the uroepithelium and renal tubular epithelial cells. It is thought that more than 80% of the population contains this latent form of the virus.

However, the manifestations of BK virus infection in immunosuppressed and/or immunocompromised individuals are quite severe, for example in the setting of organ transplantation. In organ transplantation, clinical manifestations may include renal dysfunction and the presence of renal tubular cells and inflammatory cells in the urine. In particular, in the setting of immunosuppression and/or immunocompromise, BK virus reactivates and replicates, triggering a series of events beginning with tubular cell lysis and viruria. BK virus then amplifies in the interstitium and crosses the peritubular capillaries, causing viremia and eventually invading the allograft, resulting in a variety of tubulointerstitial lesions and BK virus nephrophathy (BKVN). BK virus significantly increases the likelihood of graft loss. BK virus is an opportunistic viral infection posttransplantation, affecting approximately 15% of renal transplant recipients in the first year after transplantation. If left untreated, BKVN will progress to allograft failure.

Although immunosuppression and/or immunocompromise are the major risk factors for BK virus infection, other risk factors include male sex, older age, previous rejection episodes, degree of human leukocyte antigen mismatch, prolonged cold ischemia time, BK serostatus, and ureteral stent placement. Treatment options for patients with symptomatic BK virus infection are limited, and effective prophylaxis is not available. The cornerstone of treatment is to reduce immunosuppression, which increases the risk of allograft rejection. Antiviral drugs are used, but the results are inconsistent. BK virus is now recognized as a major cause of interstitial nephritis and allograft failure in renal transplant recipients.

BK virus infection is diagnosed by a BK virus blood test or a urine test for decoy cells. The presence of decoy cells is a sensitive measure, but has a low positive predictive value for the diagnosis of BKVN (see Mbianda, et al. Journal of Clinical Virology 71:59-62 (2015)). Quantification of viral load in plasma and urine using DNA or mRNA has been used to diagnose BKVN. However, transplant renal biopsy remains the gold standard for diagnosing BKVN. However, transplant renal biopsy is a time-consuming, invasive, and cumbersome procedure.

Cytomegalovirus (CMV) is a ubiquitous herpes-like virus with a linear, double-stranded DNA genome of approximately 236,000 kb. CMV infects 40-80% of humans before puberty. CMV is latent after primary infection and is often asymptomatic. Even reinfection is often asymptomatic or causes only mild disease in immunocompetent hosts. However, in immunocompromised patients, such as congenitally infected infants and allogeneic transplant recipients or patients with autoimmune deficiency syndrome (AIDS), CMV can cause serious and sometimes life-threatening diseases, such as retinitis, gastrointestinal disorders, and encephalitis. Half of allogeneic stem cell recipients develop CMV infection within 100 days of transplantation. CMV end-organ disease is a serious and frequent complication of allogeneic stem cell transplantation.

Early and sensitive diagnosis is of great importance, as early administration of antiviral drugs (e.g., ganciclovir and foscarnet) can have a significant beneficial effect on the patient's prognosis.

A variety of methods have been used to analyze CMV. Anti-CMV antibodies, especially IgM antibodies, can be used as markers for CMV infection. However, detection of anti-CMV antibodies is limited in distinguishing between latent and active infections.

Most recently used methods of virus detection do not clearly predict whether an infection will be symptomatic. Serological methods are indirect and often lack sensitivity. Viral culture, such as virus culture from blood cells, is a more direct diagnostic parameter for CMV viremia, but this method is technically difficult and time-consuming. Furthermore, virus culture does not necessarily correspond to CMV disease. Isolation of virus from peripheral leukocytes may not predict clinical symptoms in some immunosuppressed patients.

As previously mentioned, while most healthy adults have low levels of BK virus and CMV viruses, these viruses can be activated or opportunistically transmitted in renal transplant recipients when immunosuppressed, resulting in high levels. Therefore, there is a need in the art for a rapid, reliable, and sensitive method for simultaneously quantifying BK virus and CMV viruses in biological samples.

Throughout the specification, numerous references and patent documents are referenced and cited. The disclosures of the cited documents and patents are incorporated herein by reference in their entirety so as to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have endeavored to develop a novel method capable of simultaneously quantifying BKV and CMV in a sample with improved sensitivity and specificity. As a result, the present inventors have confirmed that BKV and CMV in a sample can be effectively and simultaneously quantified in a single reaction by a method using a first oligonucleotide set capable of hybridizing to the VP2 gene of BKV and a second oligonucleotide set capable of hybridizing to the UL55 gene of CMV.

Accordingly, an object of the present invention is to provide a method for simultaneous quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample.

Another object of the present invention is to provide a composition for a method for simultaneous quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample.

Other objects and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the appended claims.

In one aspect of the present invention, a method for simultaneous quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample is provided, comprising the following steps:

(a) performing a nucleic acid amplification reaction comprising contacting a sample in a single reaction vessel with a first set of oligonucleotides hybridizable to the VP2 gene of BKV and a second set of oligonucleotides hybridizable to the UL55 gene of CMV, wherein the nucleic acid amplification reaction generates a first amplification curve representing amplification of the VP2 gene of BKV in the sample and a second amplification curve representing amplification of the UL55 gene of CMV in the sample; and

(b) comparing the generated first amplification curve with a standard curve generated from a reference sample containing a known amount of the VP2 gene to determine the amount of BKV in the sample, and comparing the generated second amplification curve with a standard curve generated from another reference sample containing a known amount of the UL55 gene to determine the amount of CMV in the sample.

In certain embodiments, the first oligonucleotide set comprises a plurality of oligonucleotides hybridizable to the nucleotide sequence of SEQ ID NO: 1 or a complement thereof.

In certain embodiments, at least one of the plurality of oligonucleotides has 1-3 deoxyinosines; one or two of the deoxyinosines are located in a core region ranging from the 3rd nucleotide at the 3'-terminus to the 6th nucleotide at the 3'-terminus of the oligonucleotide, and the remainder are located in a region ranging from the 4th nucleotide at the 5'-terminus to the 7th nucleotide at the 3'-terminus of the oligonucleotide.

In certain embodiments, the first oligonucleotide set does not comprise an oligonucleotide hybridizable to the genomic sequence of JC virus or simian virus 40.

In certain embodiments, the first oligonucleotide set comprises a primer having the nucleotide sequence of SEQ ID NO: 2 and a primer having the nucleotide sequence of SEQ ID NO: 3.

In certain embodiments, the first oligonucleotide set comprises a probe having the nucleotide sequence of SEQ ID NO: 4.

In certain embodiments, the probe comprises a detectable fluorescent label and a quenching moiety capable of quenching a signal from the fluorescent label.

In certain embodiments, the second oligonucleotide set comprises a plurality of oligonucleotides hybridizable to the nucleotide sequence of SEQ ID NO: 5 or the complement thereof.

In certain embodiments, at least one of the plurality of oligonucleotides has 1-3 deoxyinosines; one or two of the deoxyinosines are located in a core region ranging from the 3rd nucleotide at the 3'-terminus to the 6th nucleotide at the 3'-terminus of the oligonucleotide, and the remainder are located in a region ranging from the 4th nucleotide at the 5'-terminus to the 7th nucleotide at the 3'-terminus of the oligonucleotide.

In certain embodiments, the second oligonucleotide set does not comprise an oligonucleotide hybridizable to the genomic sequence of herpes simplex virus 1, human herpesvirus 2, human herpesvirus 4, human herpesvirus 6B, human herpesvirus 6, or human herpesvirus 7 virus.

In certain embodiments, the second oligonucleotide set comprises a primer having the nucleotide sequence of SEQ ID NO: 6 and a primer having the nucleotide sequence of SEQ ID NO: 7.

In certain embodiments, the second oligonucleotide set comprises a probe having the nucleotide sequence of SEQ ID NO: 8.

In certain embodiments, the probe comprises a detectable fluorescent label and a quenching moiety capable of quenching a signal from the fluorescent label.

In certain embodiments, the nucleic acid amplification reaction is a real-time PCR or an isothermal amplification reaction.

In certain embodiments, the reference samples containing the known amount of VP2 gene are three or more, each comprising an amount selected from 10 ¹ copies/uL to 10 ¹⁰ copies/uL.

In a specific embodiment, the other reference samples containing the known amount of the UL55 gene are three or more, each comprising an amount selected from 10 ¹ copies/uL to 10 ¹⁰ copies/uL.

In certain embodiments, the sample is whole blood, plasma or serum collected from a subject who has received a kidney transplant.

In certain embodiments, the subject is less than 1 year post-renal transplant.

In certain embodiments, the sample has a volume of 5 to 15 ul and the total volume of the nucleic acid amplification reaction is 20 to 30 ul.

In another aspect, the present invention provides a composition for simultaneous quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample, comprising:

(a) a first oligonucleotide set capable of hybridizing to the VP2 gene of BKV;

(b) a second oligonucleotide set capable of hybridizing to the UL55 gene of CMV; and

(c) A reagent for nucleic acid amplification comprising a polymerase, dNTPs, and a buffer.

In certain embodiments, the composition further comprises a reference sample containing a known amount of the VP2 gene and another reference sample containing a known amount of the UL55 gene.

In certain embodiments, the first oligonucleotide set comprises a primer having the nucleotide sequence of SEQ ID NO: 2 and a primer having the nucleotide sequence of SEQ ID NO: 3.

In certain embodiments, the first oligonucleotide set comprises a probe having the nucleotide sequence of SEQ ID NO: 4.

In certain embodiments, the second oligonucleotide set comprises a primer having the nucleotide sequence of SEQ ID NO: 6 and a primer having the nucleotide sequence of SEQ ID NO: 7.

In certain embodiments, the second oligonucleotide set comprises a probe having the nucleotide sequence of SEQ ID NO: 8.

The method of the present invention can simultaneously quantify BKV and CMV in a sample with one reaction, and thus exhibits the effects of reducing the cost of testing, such as reducing the amount of sample, shortening the testing time, and shortening the testing equipment and administration personnel. In addition, the oligonucleotide set used in the method of the present invention enables more accurate diagnosis of BKV and CMV with high sensitivity and specificity. Furthermore, the method of the present invention can increase the diagnosis rate of opportunistic infections in renal transplant patients and contribute to improving the survival rate of transplanted organs and patients in the long term.

I. Simultaneous quantitative method

According to one aspect, the present invention provides a method for simultaneous quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample, comprising the following steps:

(a) performing a nucleic acid amplification reaction comprising contacting a sample in a single reaction vessel with a first set of oligonucleotides hybridizable to the VP2 gene of BKV and a second set of oligonucleotides hybridizable to the UL55 gene of CMV, wherein the nucleic acid amplification reaction generates a first amplification curve representing amplification of the VP2 gene of BKV in the sample and a second amplification curve representing amplification of the UL55 gene of CMV in the sample; and

(b) comparing the generated first amplification curve with a standard curve generated from a reference sample containing a known amount of the VP2 gene to determine the amount of BKV in the sample, and comparing the generated second amplification curve with a standard curve generated from another reference sample containing a known amount of the UL55 gene to determine the amount of CMV in the sample.

Below, the contents of the present disclosure are described in more detail for each step:

Step (a): Performing the nucleic acid amplification reaction

In step (a) of the present disclosure, a nucleic acid amplification reaction for target nucleic acids of BKV and CMV is performed.

Specifically, a nucleic acid amplification reaction is performed comprising contacting a sample in a single reaction vessel with a first set of oligonucleotides hybridizable to the VP2 gene of BKV and a second set of oligonucleotides hybridizable to the UL55 gene of CMV.

According to the method of the present invention, the process for amplifying the target nucleic acid of BKV and the process for amplifying the target nucleic acid of CMV occur simultaneously in one reaction vessel through one reaction, and the results thereof are also provided together. Therefore, the method of the present invention is also referred to by terms such as "multiplex analysis", "multiplex reaction", and "multiplex amplification".

The term "vessel, container, carrier" or "reaction vessel" as used herein refers to a physical compartment or space that houses the components used in the nucleic acid amplification reaction for BKV and CMV.

Non-limiting examples of the above containers include tubes, plates, etc. The containers can be sealed, for example, by a cap or by a film via a suitable sealing machine.

In certain embodiments, a “single reaction vessel” is a single well in a well plate, such as a 48-well plate, a 96-well plate, a 192-well plate, or a 384-well plate.

The term "sample" as used herein can mean any analyte that contains or is suspected of containing a nucleic acid to be detected. For example, the sample includes biological samples (e.g., cells, tissues, and body fluids) and non-biological samples (e.g., food, water, and soil), and the biological sample can be, but is not limited to, viruses, bacteria, tissues, cells, blood (including whole blood, plasma, and serum), lymph, bone marrow fluid, saliva, sputum, swabs, aspirations, milk, urine, feces, eye fluid, semen, brain extracts, spinal fluid, joint fluid, thymus fluid, bronchial washings, ascites, or amniotic fluid.

According to one embodiment, the sample can be obtained from a subject, particularly a mammal, more particularly a human, and can be, for example, but not limited to, a swab, saliva, sputum, aspiration, bronchoalveolar lavage (BAL), gargle or blood.

In certain embodiments, the sample is whole blood, plasma or serum.

The above sample can be derived from a subject. The term "subject" as used herein means a subject suspected of being infected with BKV and/or CMV, or a subject requiring testing or diagnosis of the pathogen, from which the target nucleic acid to be analyzed using the methods of the present disclosure is derived. Examples of the subject include, but are not limited to, mammals such as dogs, cats, rodents, primates, and humans, and particularly humans.

In certain embodiments, the subject is a subject who has received a kidney transplant. In more certain embodiments, the subject is a subject who has received a kidney transplant less than 1 year ago.

In the context of the above subject, the sample may be whole blood, plasma or serum collected from a subject who has received a kidney transplant, or whole blood, plasma or serum collected from a subject who has received a kidney transplant less than 1 year ago.

According to one embodiment, the sample may be subjected to a nucleic acid extraction and/or purification process known in the art for efficient amplification reaction (see, e.g., Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)). The nucleic acid extraction and/or purification process may vary depending on the type of sample.

The terms “nucleic acid”, “nucleic acid sequence” or “nucleic acid molecule” as used herein mean a deoxyribonucleotide or ribonucleotide polymer in single-stranded or double-stranded form, which nucleotides may include derivatives of natural nucleotides, non-natural nucleotides or modified nucleotides that can function in the same manner as naturally occurring nucleotides.

The terms "target nucleic acid," "target nucleic acid sequence," or "target sequence," as used herein, refer to a nucleic acid sequence to be detected. The target nucleic acid includes not only one newly generated in the reaction, but also one initially present in the sample.

The target nucleic acid herein includes a target nucleic acid from BKV and a target nucleic acid from CMV, both of which are double-stranded DNA. Such double-stranded target nucleic acids can be separated into single-stranded or partially single-stranded forms for application to the methods of the present invention. Known methods for separating the strands include, but are not limited to, heating, alkali, formamide, urea and glycoxal treatment, enzymatic methods (e.g., helicase action), and binding proteins. For example, strand separation can be achieved by heating at a temperature in the range of 80°C-105°C. A general method for achieving such treatment is provided by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

In one embodiment, there are two target nucleic acids, one of which is a nucleic acid from BK virus (BKV) and the other is a nucleic acid from cytomegalovirus (CMV).

In certain embodiments, the target nucleic acids are two, one of which is the VP2 gene of BKV or a portion thereof, and the other is the UL55 gene of CMV or a portion thereof.

In certain embodiments, the target nucleic acids are two, one of which has a sequence from nucleotide 624 to nucleotide 1679 of the BKV whole genome under Genbank Accession No. NC_001538, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and the other of which has a sequence from nucleotide 624 to nucleotide 1679 of the BKV whole genome under Genbank Accession No. NC_001538, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. A sequence having at least 90% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, to the sequence from nucleotide 82066 to nucleotide 84789 of the CMV whole genome under NC_006273.

In certain embodiments, the target nucleic acids are two, one of which has a sequence from nucleotide 948 to nucleotide 1108 of the BKV whole genome under Genbank Accession No. NC_001538, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and the other of which has a sequence from nucleotide 948 to nucleotide 1108 of the BKV whole genome under Genbank Accession No. NC_001538, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. A sequence having at least 90% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, to the sequence from nucleotide 82871 to nucleotide 83019 of the CMV whole genome under NC_006273.

In other words, there are two target nucleic acids, one of which has a sequence having at least 90% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, to the sequence from the 325th nucleotide to the 485th nucleotide in the VP2 gene of the BKV whole genome under Genbank Accession No. NC_001538, and the other one has a sequence having at least 90% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, to the sequence. A sequence having at least 90% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, to the sequence from the 806th nucleotide to the 954th nucleotide in the UL55 gene of the CMV whole genome under NC_006273.

In certain embodiments, the target nucleic acids are two, one of which has a sequence from nucleotide 948 to nucleotide 1108 of the BKV whole genome under Genbank Accession No. NC_001538, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and the other of which has a sequence from nucleotide 948 to nucleotide 1108 of the BKV whole genome under Genbank Accession No. NC_001538, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. A sequence having at least 90% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, to the sequence from nucleotide 82871 to nucleotide 83019 of the CMV whole genome under NC_006273.

In certain embodiments, the target nucleic acids are two, one of which has a sequence comprising any one nucleotide selected from the group consisting of the 748th nucleotide to the 948th nucleotide to the group consisting of the 1108th nucleotide to the 1308th nucleotide in the BKV whole genome under Genbank Accession No. NC_001538, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and the other of which has a sequence comprising any one nucleotide selected from the group consisting of the 748th nucleotide to the 948th nucleotide to the 1108th nucleotide to the 1308th nucleotide in the BKV whole genome under Genbank Accession No. NC_001538, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. A sequence comprising any one nucleotide selected from the group consisting of the 82671st nucleotide to the 82871st nucleotide in the CMV whole genome under NC_006273 to any one nucleotide selected from the group consisting of the 83019th nucleotide to the 83219th nucleotide, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

In certain embodiments, the target nucleic acids are two, one of which has a sequence comprising any one nucleotide selected from the group consisting of the 848th nucleotide to the 948th nucleotide to the group consisting of the 1108th nucleotide to the 1208th nucleotide in the BKV whole genome under Genbank Accession No. NC_001538, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and the other of which has a sequence comprising any one nucleotide selected from the group consisting of the 848th nucleotide to the 948th nucleotide to the group consisting of the 1108th nucleotide to the 1208th nucleotide in the BKV whole genome under Genbank Accession No. NC_001538, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. A sequence comprising any one nucleotide selected from the group consisting of the 82771st nucleotide to the 82871st nucleotide in the CMV whole genome under NC_006273 to any one nucleotide selected from the group consisting of the 83019th nucleotide to the 83119th nucleotide, or a sequence having at least 90% identity thereto, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

In certain embodiments, the target nucleic acids are two, one of which has a sequence comprising any one nucleotide selected from the group consisting of the 898th to the 948th nucleotide in the BKV whole genome under Genbank Accession No. NC_001538 to any one nucleotide selected from the group consisting of the 1108th to the 1158th nucleotide, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and the other of which has a sequence comprising any one nucleotide selected from the group consisting of the 898th to the 948th nucleotide in the BKV whole genome under Genbank Accession No. NC_001538 to the 1108th to the 1158th nucleotide, or a sequence having at least 90% identity thereto, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. A sequence comprising any one nucleotide selected from the group consisting of the 82821st nucleotide to the 82871st nucleotide in the CMV whole genome under NC_006273 to any one nucleotide selected from the group consisting of the 83019th nucleotide to the 83069th nucleotide, or a sequence having at least 90% identity thereto, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

In certain embodiments, the target nucleic acids are two, one of which has the nucleotide sequence of SEQ ID NO: 1, or a sequence having at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity thereto, and the other of which has the nucleotide sequence of SEQ ID NO: 5, or a sequence having at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity thereto.

In certain embodiments, there are two target nucleic acids, one of which has the nucleotide sequence of SEQ ID NO: 1 and the other of which has the nucleotide sequence of SEQ ID NO: 5.

The present inventors confirmed that among various genes of BKV, targeting the VP2 gene, particularly the nucleotide sequence of SEQ ID NO: 1, can quantify BKV with the highest specificity and sensitivity. In addition, the present inventors confirmed that among various genes of CMV, targeting the UL55 gene, particularly the nucleotide sequence of SEQ ID NO: 5, can quantify CMV with the highest specificity and sensitivity (data not shown).

Although specific sequences of the VP2 gene of BKV and specific sequences of the UL55 gene of CMV are mentioned herein, it should be understood that these sequences are only exemplifying sequences analyzed by the oligonucleotide set according to the present invention, and that various sequences of the VP2 and UL55 genes of subtypes or variants of BKV and CMV are included in the target nucleic acids mentioned herein.

In the method of the present invention, a composition for detecting a target nucleic acid of BKV and a composition for detecting a target nucleic acid of CMV are used for simultaneous quantification of target nucleic acids from BKV and CMV.

The composition for detecting the target nucleic acid of the above BKV is specific for the target nucleic acid of BKV, i.e., the VP2 gene, and the composition for detecting the target nucleic acid of the above CMV is specific for the target nucleic acid of CMV, i.e., UL55.

The phrase "the composition for detecting a target nucleic acid is specific for a target nucleic acid" herein means that the composition for detecting a target nucleic acid is involved in detecting the target nucleic acid, but is not involved in detecting other nucleic acids. In other words, the phrase means that the composition for detecting a target nucleic acid interacts with the target nucleic acid, but does not interact with other nucleic acids.

In the present invention, the composition for detecting the target nucleic acid of BKV is specific for the target nucleic acid of BKV but non-specific for the target nucleic acid of CMV, and the composition for detecting the target nucleic acid of CMV is specific for the target nucleic acid of CMV but non-specific for the target nucleic acid of BKV.

The composition for detecting target nucleic acids of BKV and the composition for detecting target nucleic acids of CMV used in the present invention are used together in one reaction, and therefore exist together in one reaction solution or reaction vessel.

The term "composition for detecting target nucleic acid" as used herein means a composition containing components used for detecting a target nucleic acid.

Examples of components included in the composition for detecting the target nucleic acid include, but are not limited to, an oligonucleotide set used to amplify or detect the target nucleic acid, a label, a nucleic acid polymerase, a buffer, a polymerase cofactor, and dNTPs. Optionally, the composition for detecting the target nucleic acid may include various additional components, such as a substance for promoting a reaction, a molecule for inhibiting nucleic acid polymerase activity, a molecule for preventing contamination, and the like. In addition, the composition for detecting the target nucleic acid may include oligonucleotides or reagents necessary for performing a positive control, a negative control, or an internal control reaction. In addition, the composition for detecting the target nucleic acid may include a reference sample containing a known amount of a standard target nucleic acid for absolute quantification.

The optimal amount of each component used in the method of the present invention can be readily determined by one skilled in the art having the benefit of the present disclosure.

The components of the composition for detecting the target nucleic acid may be stored together in one container or divided into multiple containers prior to the reaction.

According to the method of the present invention, a composition for detecting a target nucleic acid of BKV comprises a first oligonucleotide set capable of hybridizing to the VP2 gene of BKV.

In certain embodiments, the first oligonucleotide set comprises a plurality of oligonucleotides hybridizable to a nucleotide sequence of SEQ ID NO: 1 or a complement thereof: 5'-GTTTCCACTGTAGGCCTCTATCAGCAATCAGGCATGGCTTTGGAATTGTTTAACCCAGATGAGTACTATGATATTCTGTTTCCTGGTGTAAATACTTTTGTTAATAATATTCAATACCTTGATCCTAGGCATTGGGGTCCTTCTTTGTTTGCTACTATTTC-3' (SEQ ID NO: 1). The nucleotide sequence of SEQ ID NO: 1 is a representative example of a conserved region in the VP2 gene of BKV.

In certain embodiments, at least one of the plurality of oligonucleotides included in the first oligonucleotide set has 1-3 deoxyinosines; one or two of the deoxyinosines are located in a core region ranging from the 3rd nucleotide at the 3'-end to the 6th nucleotide at the 3'-end of the oligonucleotide, and the remainder are located in a region ranging from the 4th nucleotide at the 5'-end to the 7th nucleotide at the 3'-end of the oligonucleotide.

It is known that designing some of the multiple oligonucleotides used in a multiplex amplification reaction to have a specific number of deoxyinosines at specific positions as described above suppresses the formation of non-specific amplification products, particularly primer dimers, in the reaction, reduces nucleic acid amplification efficiency, and facilitates the design of oligonucleotides (see International Patent Publication No. WO2018-124665).

The method of the present invention enables more effective detection of target nucleic acids by incorporating deoxyinosine into the oligonucleotides so that some or all of the oligonucleotides in the first oligonucleotide set satisfy the above-described requirements.

According to the method of the present invention, the composition for detecting a target nucleic acid of CMV comprises a second oligonucleotide set capable of hybridizing to the UL55 gene of CMV.

In certain embodiments, the second oligonucleotide set comprises a plurality of oligonucleotides hybridizable to a nucleotide sequence of SEQ ID NO: 5 or a complement thereof: 5'-CAGTACGGTCAACTGGGCGAGGACAACGAAATCCTGTTGGGCAACCACCGCACTGAGGAATGTCAGCTTCCCAGCCTCAAGATCTTCATCGCCGGGAACTCGGCCTACGAGTACGTGGACTACCTCTTCAAACGCATGATTGACCTCAG-3' (SEQ ID NO: 5). The nucleotide sequence of SEQ ID NO: 5 is a representative example of a conserved region in the UL55 gene of CMV.

In certain embodiments, at least one of the plurality of oligonucleotides included in the second oligonucleotide set has 1-3 deoxyinosines; one or two of the deoxyinosines are located in a core region ranging from the 3rd nucleotide at the 3'-terminus to the 6th nucleotide at the 3'-terminus of the oligonucleotide, and the remainder are located in a region ranging from the 4th nucleotide at the 5'-terminus to the 7th nucleotide at the 3'-terminus of the oligonucleotide.

For details regarding the inclusion of the above deoxyinosine, refer to the aforementioned International Patent Publication No. WO2018-124665.

In one embodiment, each of the first oligonucleotide set and the second oligonucleotide set comprises a primer pair and a probe.

The term "primer" as used herein refers to an oligonucleotide that can act as an initiation point of synthesis under conditions that induce synthesis of a primer extension product complementary to a target nucleic acid (template), i.e., the presence of nucleotides and a polymerization agent such as DNA polymerase, and conditions of suitable temperature and pH. The primer must be sufficiently long to prime the synthesis of an extension product in the presence of the polymerization agent. The appropriate length of the primer is determined by a number of factors, such as temperature, application, and the source of the primer.

The term "probe" as used herein refers to a single-stranded nucleic acid molecule comprising a portion or portions that are substantially complementary to a target nucleic acid. According to one embodiment of the present invention, the 3'-end of the probe is "blocked" to prevent its extension. Blocking can be accomplished by any conventional method. For example, blocking can be accomplished by adding a chemical moiety, such as biotin, a label, a phosphate group, an alkyl group, a non-nucleotide linker, a phosphorothioate, or an alkane-diol moiety, to the 3'-hydroxyl group of the last nucleotide. Alternatively, blocking can be accomplished by removing the 3'-hydroxyl group of the last nucleotide or by using a nucleotide that lacks a 3'-hydroxyl group, such as a dideoxynucleotide.

“Complementary” means sufficiently complementary to allow a primer or probe to selectively hybridize to a target nucleic acid sequence under given annealing conditions or hybridization conditions, and includes both “substantially complementary” and “perfectly complementary,” preferably perfectly complementary.

The term "substantially complementary" means that the oligonucleotide is sufficiently complementary so that it can selectively hybridize to a template nucleic acid sequence under designated annealing or hybridization conditions such that the annealed oligonucleotide can be extended by a polymerase to form a complementary copy of the template. Thus, this term has a different meaning than "fully complementary" or related terms.

The term "non-complementary" as used herein means sufficiently non-complementary such that a primer or probe does not selectively hybridize to a target nucleic acid sequence under specified annealing conditions or hybridization conditions, and is intended to encompass both the terms "substantially non-complementary" and "perfectly noncomplementary", preferably completely non-complementary.

The primer or probe may be single-stranded. The primer or probe comprises a deoxyribonucleotide, a ribonucleotide or a combination thereof. The primer or probe used in the present invention may comprise a naturally occurring dNMP (i.e., dAMP, dGMP, dCMP and dTMP), a modified nucleotide or a non-natural nucleotide.

The term "annealing" or "priming" refers to the apposition of an oligonucleotide or nucleic acid to a template nucleic acid, which causes a polymerase to polymerize the nucleotides to form a nucleic acid molecule complementary to the template nucleic acid or a portion thereof.

The term "hybridization" as used herein refers to the formation of a double-stranded structure by two single-stranded polynucleotides through non-covalent bonding between complementary nucleotide sequences under certain hybridization conditions.

The terms “annealing” and “hybridization” are not different and will be used interchangeably throughout this specification.

In certain embodiments, the first oligonucleotide set according to the present invention does not comprise an oligonucleotide hybridizable to the genomic sequence of JC virus or simian virus 40.

The above JC virus and simian virus 40 are known to have high genetic homology with BKV. Therefore, an oligonucleotide designed only considering the target sequence of BKV without considering the sequence having high genetic homology with BKV is likely to hybridize to the genome sequence from JC virus or simian virus 40, and may cause false positive results, for example, for a sample in which BKV is not present but JC virus or simian virus 40 is present.

To prevent this, the first oligonucleotide set according to the present invention is designed taking into account the genome sequences of both JC virus and simian virus 40 so that it does not hybridize to the genome sequences of JC virus or simian virus 40.

In certain embodiments, the first oligonucleotide set comprises a primer having a nucleotide sequence of SEQ ID NO: 2 or a sequence at least 90% homologous thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous thereto, and a primer having a nucleotide sequence of SEQ ID NO: 3 or a sequence at least 90% homologous thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous thereto."

5'-GTTTCCACTGTAGGCCTITATCAGIAATC-3' (SEQ ID NO: 2)

5'-GAAATAGTAGCAAACAAIGAAGGACCICAA-3' (SEQ ID NO: 3).

As can be seen from the above sequence, the two primers can have deoxyinosine (indicated by 'I') incorporated at specific positions.

In calculating the homology above, the deoxyinosine is considered to match the naturally occurring base opposite it in the alignment.

In certain embodiments, the first oligonucleotide set comprises a primer having the nucleotide sequence of SEQ ID NO: 2 and a primer having the nucleotide sequence of SEQ ID NO: 3.

In certain embodiments, the first oligonucleotide set comprises a probe having a nucleotide sequence of SEQ ID NO: 4 or a sequence having at least 90% homology thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology thereto:

5'-TGGAATTGTTTAACCCAGATGAGTACTATGATAT-3' (SEQ ID NO: 4).

As can be seen from the above sequence, the probe does not contain deoxyinosine.

In certain embodiments, the first oligonucleotide set comprises a probe having the nucleotide sequence of SEQ ID NO: 4.

The first oligonucleotide set according to the present invention exhibits 100% target coverage for the VP2 gene sequences of 541 different subtypes and variants of BKV. In addition, the first oligonucleotide set according to the present invention does not detect the genome sequences of 47 strains listed in Table 3 herein. Thus, the first oligonucleotide set according to the present invention detects the target nucleic acid of BKV with high specificity.

In certain embodiments, the second oligonucleotide set according to the present invention does not comprise an oligonucleotide hybridizable to the genomic sequence of herpes simplex virus 1, human herpesvirus 2, human herpesvirus 4, human herpesvirus 6B, human herpesvirus 6, or human herpesvirus 7 virus.

The above herpes simplex virus 1, human herpesvirus 2, human herpesvirus 4, human herpesvirus 6B, human herpesvirus 6, and human herpesvirus 7 viruses are known to have high genetic homology with CMV. Therefore, an oligonucleotide designed only considering the target sequence of CMV without considering the sequence having high genetic homology with CMV is likely to hybridize to the genome sequence from the above herpesviruses, and may cause a false positive result, for example, for a sample in which CMV is not present but the above herpesviruses are present.

To prevent this, the first oligonucleotide set according to the present invention is designed in consideration of the genome sequences of all of the aforementioned herpesviruses so that it does not hybridize to the genome sequences of herpes simplex virus 1, human herpesvirus 2, human herpesvirus 4, human herpesvirus 6B, human herpesvirus 6, or human herpesvirus 7 viruses.

In certain embodiments, the second oligonucleotide set comprises a primer having a nucleotide sequence of SEQ ID NO: 6 or a sequence at least 90% homologous thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous thereto and a primer having a nucleotide sequence of SEQ ID NO: 7 or a sequence at least 90% homologous thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous thereto:

5'-CAGTACIGTCAACTGGGIGAGG-3' (SEQ ID NO: 6)

5'-CTGAGGTIAATCATGCGTTTGAAIAGG-3' (SEQ ID NO: 7).

As can be seen in the above sequence, the two primers have deoxyinosine (indicated by 'I') incorporated at specific positions.

In calculating the homology above, the deoxyinosine is considered to match the naturally occurring base opposite it in the alignment.

In certain embodiments, the second oligonucleotide set comprises a primer having the nucleotide sequence of SEQ ID NO: 6 and a primer having the nucleotide sequence of SEQ ID NO: 7.

In certain embodiments, the second oligonucleotide set comprises a probe having a nucleotide sequence of SEQ ID NO: 8 or a sequence having at least 90% homology thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology thereto:

5'-CCACCGCACTGAGGAATGTCAG-3' (SEQ ID NO: 8).

As can be seen from the above sequence, the probe does not contain deoxyinosine.

In certain embodiments, the second oligonucleotide set comprises a probe having the nucleotide sequence of SEQ ID NO: 8.

The second oligonucleotide set according to the present invention exhibits substantially 100% target coverage for the UL55 gene sequences of 523 different subtypes and variants of CMV. Furthermore, the second oligonucleotide set according to the present invention does not detect the genomic sequences of the 47 strains listed in Table 4 herein. Thus, the second oligonucleotide set according to the present invention detects the target nucleic acid of CMV with high specificity.

In certain embodiments, the composition for detecting a target nucleic acid of BKV according to the present invention, particularly the first oligonucleotide set, generates a signal from the fluorescent label in a manner dependent on the cleavage of an oligonucleotide that specifically hybridizes to the VP2 gene of BKV.

In certain embodiments, the composition for detecting a target nucleic acid of CMV according to the present invention, particularly the second oligonucleotide set, generates a signal from the fluorescent label in a manner dependent on the cleavage of an oligonucleotide that specifically hybridizes to the UL55 gene of CMV.

In particular, generation of a signal by the first oligonucleotide set and the second oligonucleotide set can be achieved by hybridization of an oligonucleotide, e.g., a probe, to a target nucleic acid, followed by cleavage thereof.

Examples of such signal generation include, but are not limited to, TaqMan probe methods (U.S. Patent No. 5,210,015 and U.S. Patent No. 5,538,848).

For signal generation by the TaqMan probe method, a composition for detecting a target nucleic acid comprises a set of oligonucleotides including a primer pair and a probe, as well as a nucleic acid polymerase having 5'-nuclease activity. The probe hybridized to the target nucleic acid is cleaved during amplification of the target nucleic acid to generate a signal indicating the presence of the target nucleic acid.

A specific example of generating a signal by the TaqMan probe method comprises the steps of: (a) hybridizing the target nucleic acid with a probe having a primer pair and an appropriate label (e.g., an interactive dual label); (b) amplifying the target nucleic acid using the product of step (a) and a nucleic acid polymerase having 5' nuclease activity; wherein the probe is cleaved to release the label; and (c) detecting signal generation from the released label.

Signal generation by the first oligonucleotide set and the second oligonucleotide set can be achieved by various methods known to those skilled in the art in addition to the above-described methods.

In one embodiment, each of the probes included in the first oligonucleotide set and the probes included in the second oligonucleotide set comprises a detectable fluorescent label and a quenching moiety capable of quenching a signal from the fluorescent label.

The fluorescent label and the quenching moiety herein may be referred to as an interactive dual label, the fluorescent label may be referred to as a fluorescent reporter molecule, and the quenching moiety may be referred to as a quencher molecule.

A representative example of an interaction label system including the above-mentioned interactive dual labels includes a fluorescence resonance energy transfer (FRET) label system including a fluorescent reporter molecule (donor molecule) and a quencher molecule (acceptor molecule). In FRET, the energy donor is fluorescent, but the energy acceptor can be fluorescent or non-fluorescent. The interaction label system can include a dual label based on "contact-mediated quenching" (Salvatore et al., Nucleic Acids Research, 2002 (30) no.21 e122 and Johansson et al., J. AM. CHEM. SOC 2002 (124) pp 6950-6956). The above-mentioned interactive label system can include any label system that induces a signal change by the interaction between at least two molecules (e.g., dyes).

Reporter molecules and quencher molecules useful in the present invention may include any molecules known in the art. Examples include: Cy2™ (506), YO-PRO™-1 (509), YOYO™-1 (509), Calcein (517), FITC (518), FluorX™ (519), Alexa™ (520), Rhodamine 110 (520), Oregon Green™ 500 (522), Oregon Green™ 488 (524), RiboGreen™ (525), Rhodamine Green™ (527), Rhodamine 123 (529), Magnesium Green™ (531), Calcium Green™ (533), TO-PRO™-1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY TMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3™ (570), Alexa™ 546 (570), TRITC (572), Magnesium Orange™ (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange™ (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red™ (590), Cy3.5™ (596), ROX (608), Calcium Crimson™ (615), Alexa™ 594 (615), Texas Red (615), Nile Red (628), YO-PRO™-3 (631), YOYO™-3 (631), Rphycocyanin (642), C-Phycocyanin (648), TO-PRO™-3 (660), TOTO3 (660), DiD DilC(5) (665), Cy5™ (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705), and Quasar 705 (610). Numbers in parentheses are maximum emission wavelengths in nanometers. Preferably, the reporter molecule and quencher molecule may comprise JOE, FAM, TAMRA, ROX and fluorescein-based labels.

Suitable fluorescent molecules and suitable reporter-quencher pairs are described in various references, including: Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color AND Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, editor, Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); Haugland, R. P., Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996); U.S. Patent Nos. 3,996,345 and 4,351,760.

It is noteworthy that in the present invention, non-fluorescent quencher molecules (e.g., black quenchers or dark quenchers) capable of quenching fluorescence of a broad wavelength or a specific wavelength can be utilized.

In a signaling system comprising a reporter and a quencher molecule, the reporter comprises the donor of FRET and the quencher comprises the remaining partner (acceptor) of FRET. For example, a fluorescein dye can be used as a reporter and a rhodamine dye can be used as a quencher.

In one embodiment, the interactive dual label is linked to a probe within the first oligonucleotide set.

In one embodiment, the interactive dual label is linked to a probe within a second oligonucleotide set.

When the target nucleic acid is not present, each probe in the first oligonucleotide set and the second oligonucleotide set exists in a single-stranded state of a hairpin or random coil structure, and in this case, the quencher molecule comes into proximity to the reporter molecule and quenches the signal from the reporter molecule, so that no signal is generated.

On the other hand, when a target nucleic acid is present, each of the probes hybridizes to the target nucleic acid under appropriate hybridization conditions to form a duplex, thereby dissociating the quencher molecule from the reporter molecule, thereby generating a signal by unquenching a signal from the reporter molecule, and further, as the probe is cleaved by the action of a nuclease, the separation between the quencher molecule and the reporter molecule is enhanced.

According to the present invention, the fluorescent label linked to the probe included in the first oligonucleotide set is different from the fluorescent label linked to the probe included in the second oligonucleotide set. That the two fluorescent labels are different means that signals generated from the fluorescent labels can be easily distinguished using two detection channels due to substantially different signal characteristics (e.g., optical characteristics, emission wavelengths, etc.).

In one embodiment, the fluorescent label linked to the probe included in the first oligonucleotide set is FAM or an equivalent thereof.

In one embodiment, the fluorescent label linked to the probe included in the second oligonucleotide set is Cal Red 610 or an equivalent thereof.

According to the present invention, a nucleic acid amplification reaction is performed for the VP2 gene of BKV and the UL55 gene of CMV.

The above nucleic acid amplification reaction is a multiplex reaction that simultaneously amplifies two target nucleic acids in one reaction vessel.

In one embodiment, amplification of the target nucleic acid can be performed via polymerase chain reaction (PCR).

Polymerase chain reaction is widely used in the art to amplify target nucleic acids, and involves repeated cycles of denaturation of the target nucleic acid sequence, annealing (hybridization) between the target nucleic acid sequence and a primer, and primer extension (U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354).

When the target nucleic acid is double-stranded, it is desirable to make the double-strand into a single-stranded or partially single-stranded form. Methods for separating the double-strands include, but are not limited to, heating, alkali, formamide, urea and glycoxal treatment, enzymatic methods (e.g., helicase action), and binding proteins. For example, separation of the strands can be accomplished by heating at a temperature in the range of 80° C. to 105° C. A general method for accomplishing such treatments is provided by Joseph Sambrook, et. al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

Annealing of the primer and target nucleic acid can be carried out under suitable hybridization conditions, which are generally determined by optimization procedures. Conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength can vary depending on various factors including the length and GC content of the primer and target nucleic acid sequences. Detailed conditions for hybridization can be found in Joseph Sambrook et. al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999).

The primer annealed to the target nucleic acid is extended by a template-dependent polymerase, which comprises the "Klenow" fragment of E. coli DNA polymerase I, a thermostable DNA polymerase and a bacteriophage T7 DNA polymerase. In one embodiment of the present disclosure, the template-dependent polymerase is a thermostable DNA polymerase obtained from various bacterial species.

According to one embodiment of the present invention, a nucleic acid polymerase having nuclease activity (e.g., 5’nuclease activity or 3’nuclease activity) can be used.

Nucleic acid polymerases useful in the present invention include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcus litoralis, Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Pyrococcus A thermostable DNA polymerase obtained from various bacterial species, including Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifex pyrophilus and Aquifex aeolieus. In particular, the thermostable DNA polymerase is Taq polymerase.

When conducting a polymerization reaction, the components required for the reaction may be provided in excess to the reaction vessel. With respect to the components of the extension reaction, excess means an amount of each component such that the ability to achieve the desired extension is not substantially limited by the concentration of said components. It is desirable to provide the necessary cofactors such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP to the reaction mixture in sufficient amounts to cause the desired reaction to occur.

According to the method of the present invention, an increased volume of sample can be used to increase the sensitivity of quantitation. The volume of the sample used can be, but is not limited to, 5 to 15 ul, in particular 8 ul, 9 ul, 10 ul, 11 ul or 12 ul.

Additionally, to increase the sensitivity of the quantification, the method of the present invention may have an increased total reaction volume. The total reaction volume in the nucleic acid amplification reaction according to the present invention may be, but is not limited to, 20 to 30 ul, particularly 25 ul, 26 ul, 27 ul, 28 ul, 29 ul, or 30 ul.

When mRNA is used as a starting material, a reverse transcription step is essential prior to the annealing step, and the details thereof are disclosed in the literature [Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and Noonan, K. F. et al., Nucleic Acids Res. 16:10366 (1988)]. For the reverse transcription reaction, an oligonucleotide dT primer, a random primer or a target-specific primer capable of hybridizing to the poly A tail of mRNA can be used.

According to another embodiment, as a method for amplifying the target nucleic acid, ligase chain reaction (LCR, see Wiedmann M, et al., "Ligase chain reaction (LCR)- overview and applications." PCR Methods and Applications 1994 Feb;3(4):S51-64), gap filling LCR (GLCR, see WO 90/01069, European Patent No. 439182 and WO 93/00447), Q-beta replicase amplification (Q-beta, see Cahill P, et al., Clin Chem., 37(9):1482-5(1991), U.S. Patent No. 5,556,751), strand displacement amplification (SDA, see G T Walker et al., Nucleic Acids Res. 20(7):16911696(1992), Examples of amplification methods that can be used include, but are not limited to, amplification-mediated amplification (e.g., European Patent No. 497272), nucleic acid sequence-based amplification (NASBA, see Compton, J. Nature 350(6313):912(1991)), transcription-mediated amplification (TMA, see Hofmann WP et al., J Clin Virol. 32(4):289-93(2005); U.S. Patent No. 5,888,779), rolling circle amplification (RCA, see Hutchison C.A. et al., Proc. Natl Acad. Sci. USA. 102:1733217336(2005)), recombinase polymerase amplification (RPA), or loop-mediated isothermal amplification (LAMP).

The amplification method described above can amplify target nucleic acids through repetition of a series of reactions with or without changing the temperature. The unit of amplification including repetition of the series of reactions is expressed as a "cycle." The unit of the cycle can be expressed as the number of repetitions or time depending on the amplification method.

For example, detection of a signal can be performed at each cycle of amplification, at a selected subset of cycles, or at the end-point of the reaction.

According to one embodiment of the present invention, amplification of the target nucleic acid is achieved by asymmetric PCR. The ratio of the primers can be selected taking into account cleavage or hybridization of the downstream oligonucleotide.

In one embodiment, the composition for detecting a target nucleic acid according to the present invention further comprises an oligonucleotide set for an internal control reaction. The internal control reaction can be performed simultaneously with the nucleic acid amplification reaction of the VP2 gene of BKV and the UL55 gene of CMV according to the present invention. The term "internal control" herein refers to a substance for confirming the suitability of a reaction, which confirms the presence of a target nucleic acid, the loss of a target nucleic acid during nucleic acid extraction, the presence of an inhibitory substance in an amplification reaction, etc.

In one embodiment, the internal control is human hemoglobin subunit beta (HBB).

In one embodiment, the set of oligonucleotides required for the internal control reaction comprises a primer pair and a probe that are hybridizable with the internal control.

In one embodiment, the probe used in the internal control reaction comprises a detectable fluorescent label and a quenching moiety capable of quenching a signal from the fluorescent label.

In one embodiment, the fluorescent label linked to the probe used in the internal control reaction is Quasar 670 or an equivalent thereof.

In one embodiment, the composition for detecting a target nucleic acid according to the present invention further comprises a positive control for a positive control reaction. The positive control reaction can be performed simultaneously with, before, or after the nucleic acid amplification reaction of the VP2 gene of BKV and the UL55 gene of CMV according to the present invention. The positive control can be the VP2 gene of BKV described above or the UL55 gene of CMV described above. The positive control can be amplified and/or detected by the first oligonucleotide set or the second oligonucleotide set according to the present invention.

In one embodiment, the composition for detecting a target nucleic acid according to the present invention further comprises a negative control for a negative control reaction. The negative control reaction may be performed simultaneously with, before, or after the nucleic acid amplification reaction of the VP2 gene of BKV and the UL55 gene of CMV according to the present invention. The negative control may be sterile water containing no target nucleic acid, particularly ultrapure PCR grade water.

In one embodiment, the composition for detecting a target nucleic acid according to the present invention further comprises UDG (Uracil DNA Glycosylase). UDG can prevent carry-over contamination in which the result of a previous reaction (e.g., cDNA or an amplification product) contaminates a newly performed reaction through various routes. Specifically, dUTP is used instead of dTTP among dNTPs to perform a reverse transcription reaction or an amplification reaction. The result (i.e., cDNA or an amplification product) thereof contains dUTP, and by treating with UDG before performing a new reaction to hydrolyze the previous result containing dUTP, carry-over contamination can be prevented.

As an example of a suitable UDG, heat labile UDG (heat labile Uracil DNA Glycosylase) can be used. The heat labile UDG can include, but is not limited to, UDG derived from a cold-loving organism, such as UDG derived from psychrophilic bacteria or Atlantic cod. These enzymes have the characteristic of being rapidly and irreversibly inactivated when exposed to a temperature of 50°C or 55°C, respectively. Therefore, the heat labile UDG is inactivated before the reverse transcription reaction starts, and only removes carry-over contaminants without affecting the process of cDNA production by the reverse transcription reaction at all.

The composition for detecting a target nucleic acid according to the present invention generates a signal in the presence of its corresponding target nucleic acid, and enables quantification of the target nucleic acid by detecting the generated signal.

According to one embodiment of the present invention, a nucleic acid amplification reaction is performed under conditions in which target amplification is possible together with signal generation by a composition for detecting target nucleic acids.

In one embodiment, the generation of the signal includes "signal generation or disappearance" and "signal increase or decrease" from the label. The generation of the signal herein means the generation of a significant signal, i.e., a signal indicating the presence of the target nucleic acid. For example, a significant signal, i.e., a signal indicating the presence of the target nucleic acid, means a signal having an intensity that exceeds the intensity of a background signal or the intensity of a signal that can occur in the absence of the target nucleic acid, or a significant signal, i.e., a signal indicating the presence of the target nucleic acid, means a signal having an intensity after subtracting the intensity of the background signal or the intensity of a signal that can occur in the absence of the target nucleic acid from the intensity of the generated signal. If a signal is generated but its intensity is similar to or lower than the intensity of the background signal or the intensity of a signal that can occur in the absence of the target nucleic acid, the generation of the signal is not considered as a signal or the generation of a signal in the methods of the present invention.

According to the method of the present invention, the nucleic acid amplification reaction generates a first amplification curve representing amplification of the VP2 gene of BKV when BKV is present in the sample, and a second amplification curve representing amplification of the UL55 gene of CMV when CMV is present in the sample.

In this disclosure, an amplification curve refers to a curve obtained by plotting a data set for a target nucleic acid obtained from an amplification reaction. In one embodiment, the data set includes a plurality of data points including cycle numbers and signal values.

In the present invention, the term "cycle" refers to a unit of change in a condition in a plurality of measurements involving a change in a certain condition. The change in the certain condition refers to an increase or decrease in, for example, temperature, reaction time, number of reactions, concentration, pH, number of replications of a measurement target (e.g., nucleic acid), etc. Accordingly, a cycle may be a time or process cycle, a unit operation cycle, and a reproductive cycle.

For example, when measuring the substrate decomposition ability of an enzyme according to the substrate concentration, the substrate decomposition degree of the enzyme is measured several times by changing the substrate concentration, and the substrate decomposition ability of the enzyme is analyzed from this. At this time, a change in a certain condition is an increase in the substrate concentration, and the unit of substrate concentration increase used is set as one cycle.

As another example, in the case of isothermal amplification of nucleic acids, one sample can be measured several times with different reaction times. In this case, the reaction time is a change in condition, and the unit of reaction time is set as one cycle.

More specifically, the term "cycle" means one unit of repetition, when a reaction of a certain process is repeated or a reaction is repeated at certain time intervals.

For example, in the case of polymerase chain reaction (PCR), one cycle means a reaction including a denaturation step of nucleic acid, annealing step of primer, and extension step of primer. In this case, a change in a certain condition is an increase in the number of repetitions of the reaction, and the unit of repetition of the reaction including the above series of steps is set as one cycle.

The term “signal value” as used herein means a value that is quantified according to a certain scale of the level of a signal (e.g., signal intensity) measured in a cycle of a nucleic acid amplification reaction, or a modified value thereof. The modified value may include a mathematically processed signal value of the measured signal value. Examples of mathematically processed signal values of actually measured signal values (i.e., signal values of a raw data set) may include logarithmic values or derivatives.

In this specification, the term "data point" means a coordinate value that includes a cycle and a signal value. The term "data" means all information that constitutes a data set. For example, each of a cycle and a signal value of an amplification reaction is data.

Data points obtained by the amplification reaction can be expressed as coordinate values that can be expressed in a two-dimensional rectangular coordinate system. In the coordinate values, the X-axis represents the corresponding cycle number, and the Y-axis represents the signal value measured or processed in the corresponding cycle.

The term “data set” as used herein means a collection of said data points. For example, the data set may be a collection of data points obtained directly through an amplification reaction performed in the presence of a set of oligonucleotides specific for a target nucleic acid, or may be a modified data set thereof. The data set may be a part or all of a plurality of data points obtained by the amplification reaction, or a modified data set thereof.

In one embodiment, the amplification curve according to the present invention is obtained by subtracting the background signal according to a conventionally known method.

Step (b): Determination of the amount of BKV and CMV

In step (b) of the present disclosure, the amount of BKV in the sample is determined using the first amplification curve generated above, and the amount of CMV in the sample is determined using the second amplification curve generated above.

Specifically, the amount of BKV in the sample is determined by comparing the first amplification curve generated with a standard curve generated from a reference sample containing a known amount of the VP2 gene, and the amount of CMV in the sample is determined by comparing the second amplification curve generated with a standard curve generated from another reference sample containing a known amount of the UL55 gene.

The standard curve for the VP2 gene and the standard curve for UL55 are prepared prior to or simultaneously with step (b).

In the present invention, a standard curve generated from a reference sample containing the VP2 gene may be referred to as a “standard curve for VP2 gene” or a “standard curve for BKV,” and a standard curve generated from a reference sample containing the UL55 gene may be referred to as a “standard curve for UL55 gene” or a “standard curve for CMV.”

Below, each standard curve is described.

A standard curve for the VP2 gene is obtained from nucleic acid amplification reactions using reference samples containing various known amounts of the VP2 gene.

Similarly, a standard curve for the UL55 gene is obtained from a nucleic acid amplification reaction using another reference sample containing various known amounts of the UL55 gene.

The nucleic acid amplification reaction for obtaining the above standard curve is performed separately from the nucleic acid amplification reaction in the above-described step (a), and may be called a standard nucleic acid amplification reaction or a reference nucleic acid amplification reaction.

In one embodiment, the standard curve for the VP gene or the standard curve for the UL55 gene can be obtained by an absolute quantification method known in the art.

In certain embodiments, the standard curve for the VP gene or the standard curve for the UL55 gene is obtained from the following steps: (i) obtaining an amplification curve by a nucleic acid amplification reaction using a reference sample containing a known amount of the VP gene or the UL55 gene, (ii) determining a threshold cycle (Ct) from the amplification curve, and (iii) plotting the threshold cycle against the log value of the known amount of the VP gene or the UL gene.

In the above-described embodiments, the known amount of VP2 gene or the known amount of UL55 gene used in step (i) may be a commercially or non-commercially available standard material.

Additionally, the amount of the base of said gene can be a dilution series of the amount of said gene.

The dilution series of the above gene refers to a reference sample containing a series of diluted amounts of the gene, which can be prepared taking into account the amount of target nucleic acid known to be commonly present in a sample, particularly a clinical sample.

In one embodiment, the reference samples containing the VP2 gene of the known amount are three or more, each comprising an amount selected from 10 ¹ copies/uL to 10 ¹⁰ copies/uL.

In certain embodiments, the reference samples containing the known amount of VP2 gene include a reference sample comprising 10 ³ copies/uL of VP2 gene, a reference sample comprising 10 ⁵ copies/uL of VP2 gene, and a reference sample comprising 10 ⁷ copies/uL of VP2 gene.

In one embodiment, the other reference samples containing the known amount of the UL55 gene are three or more, each comprising an amount selected from 10 ¹ copies/uL to 10 ¹⁰ copies/uL.

In certain embodiments, the other reference samples containing the known amount of UL55 gene include a reference sample comprising 10 ³ copies/uL of UL55 gene, a reference sample comprising 10 ⁵ copies/uL of UL55 gene, and a reference sample comprising 10 ⁷ copies/uL of UL55 gene.

In the above-described embodiments, the conditions of the nucleic acid amplification reaction for the reference sample are the same as the conditions of the nucleic acid amplification reaction for the sample according to the present invention. For example, the conditions (e.g., temperature, time, etc.), the container (or the type of container (e.g., plate, etc.)) and the device (or the type of device (e.g., thermocycler, etc.)) of the nucleic acid amplification reaction using the reference sample are the same as the conditions, the container and the device used of the nucleic acid amplification reaction using the sample.

In the above-described implementation, determination of the threshold cycle (Ct) in step (ii) can be performed by various methods known in the art.

The above threshold cycle shows a linear correlation with the amount of target nucleic acid present in the sample. A high threshold cycle indicates a small amount of target nucleic acid present in the sample, while a low threshold cycle indicates a large amount of target nucleic acid present in the sample.

Therefore, when determining the threshold cycle from the nucleic acid amplification reactions for a reference sample containing 10 ³ copies/uL of VP2 gene, a reference sample containing 10 ⁵ copies/uL of VP2 gene and a reference sample containing 10 ⁷ copies/uL of VP2 gene, the reference sample containing 10 ³ copies/uL of VP2 gene will exhibit the highest threshold cycle and the reference sample containing 10 ⁷ copies/uL of VP2 gene will exhibit the lowest threshold cycle. The same will apply to the reference sample of UL55. However, it should be noted that even if the amounts of the VP2 gene and the UL55 gene are the same, the threshold cycles obtained from them may be different from each other.

It will be understood by those skilled in the art that the threshold cycle can be determined as a point where the threshold is crossed by applying a predetermined threshold to the amplification curve, or as FDM (first derivative maximum), SDM (second derivative maximum), etc. disclosed in US 6,503,720, US 6,783,934, US 10,176,293, US 8,285,489, US 7,565,250, etc. In addition, it will be understood by those skilled in the art that other parameters, such as Cp (crossing point), Cq (quantification cycle), △Ct, △Cp or △Cq, can be used instead of the threshold cycle.

In the above-described implementations, each standard curve is obtained by plotting the threshold cycle against the known positive log value of the VP gene or UL gene.

In one implementation, the standard curve has the threshold cycle on the y-axis and the amount of each gene (starting amount, log value) on the x-axis.

As described above, each standard curve generated from the reference sample is compared with a corresponding amplification curve among the first amplification curve and the second amplification curve. That is, the first amplification curve is compared with the standard curve for the VP2 gene, and the second amplification curve is compared with the standard curve for the UL55 gene.

In one embodiment, a threshold cycle is obtained from a first amplification curve for a sample, and an amount of VP2 gene (e.g., a value on the x-axis) corresponding to the threshold cycle (e.g., a value on the y-axis) is obtained from a standard curve for the VP2 gene, and the obtained amount is determined as the amount of BKV in the sample.

Similarly, in one embodiment, a threshold cycle is obtained from the second amplification curve, and an amount of the UL55 gene (e.g., a value on the x-axis) corresponding to the threshold cycle (e.g., a value on the y-axis) from a standard curve for the UL55 gene is obtained, and the obtained amount is determined as the amount of CMV in the sample.

As described above, the method of the present invention enables simultaneous quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample by using a first oligonucleotide set capable of hybridizing to the VP2 gene of BKV and a second oligonucleotide set capable of hybridizing to the UL55 gene of CMV.

The quantitative results according to the method of the present invention can be applied to the qualitative analysis of BKV in a sample.

In one embodiment, if the amount of BKV or CMV determined according to the method of the present invention for a particular sample exceeds a predetermined level, the sample is determined to be positive, whereas if the amount of BKV or CMV is less than the predetermined level, the sample is determined to be negative.

As an example, if the amount of BKV determined according to the method of the present invention for a particular sample exceeds a selected level of 500 copies/ml and 1000 copies/ml, the sample may be determined to be positive, whereas if it is below the level, the sample may be determined to be negative.

As another example, if the amount of BKV determined according to the method of the present invention for a particular sample exceeds a selected level of 34.5 IU/ml and 137 IU/ml, the sample may be determined to be positive, whereas if it is below the level, the sample may be determined to be negative.

As mentioned above, the predetermined level can be easily determined by a person skilled in the art.

II. Composition for simultaneous quantification

According to one aspect, a composition is provided for simultaneous quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample, comprising:

(a) a first oligonucleotide set capable of hybridizing to the VP2 gene of BKV;

(b) a second oligonucleotide set capable of hybridizing to the UL55 gene of CMV; and

(c) A reagent for nucleic acid amplification comprising a polymerase, dNTPs, and a buffer.

Since the components (a), (b) and (c) mentioned above have already been explained in Section I mentioned above, their explanation is omitted in this section.

In certain embodiments, the composition further comprises a reference sample containing a known amount of the VP2 gene and another reference sample containing a known amount of the UL55 gene.

In certain embodiments, the first oligonucleotide set comprises a primer having a nucleotide sequence of SEQ ID NO: 2 or a sequence at least 90% homologous thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous thereto, and a primer having a nucleotide sequence of SEQ ID NO: 3 or a sequence at least 90% homologous thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous thereto.

In certain embodiments, the first oligonucleotide set comprises a probe having the nucleotide sequence of SEQ ID NO: 4 or a sequence having at least 90% homology thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology thereto.

In certain embodiments, the second oligonucleotide set comprises a primer having a nucleotide sequence of SEQ ID NO: 6 or a sequence having at least 90% homology thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology thereto, and a primer having a nucleotide sequence of SEQ ID NO: 7.

In certain embodiments, the second oligonucleotide set comprises a probe having the nucleotide sequence of SEQ ID NO: 8 or a sequence having at least 90% homology thereto, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology thereto.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention set forth in the appended claims is not limited by these examples.

Example

Example 1: Confirming target coverage of oligonucleotide sets

The VP2 gene of BKV was determined as a target nucleic acid for detecting BKV. A conserved region with little sequence variation was selected within the VP2 gene of BKV, and a forward primer having a nucleotide sequence of SEQ ID NO: 2 that can hybridize to the conserved region, a reverse primer having a nucleotide sequence of SEQ ID NO: 3, and a probe having a nucleotide sequence of SEQ ID NO: 4 were designed. In the case of the probe, a FAM fluorescent label is linked to one end and BHQ-2 is linked to the other end. The designed primers and probe are collectively referred to as the first oligonucleotide set.

The target coverage of the first oligonucleotide set was analyzed as follows.

First, the genome sequence of BKV (taxonomy ID: 1891762) was collected from the NCBI database. As a result of the collection, a total of 541 target nucleic acid sequences were collected. Thereafter, the sequence of SEQ ID NO: 2, the sequence of SEQ ID NO: 3, and the sequence of SEQ ID NO: 4 in the first oligonucleotide set were compared with each of the collected target nucleic acid sequences to determine the number of mismatches.

The above results are shown in Table 1 below.

Number of mismatches Sequence number 0 1 2 3 4 5 6 7 >=8 Coverage (%) 2 541/541 0/541 0/541 0/541 0/541 0/541 0/541 0/541 0/541 100 3 541/541 0/541 0/541 0/541 0/541 0/541 0/541 0/541 0/541 100 4 541/541 0/541 0/541 0/541 0/541 0/541 0/541 0/541 0/541 100

The above table shows the number of target nucleic acids having 0, 1, 2, 3, 4, 5, 6, 7 and 8 mismatched nucleotides for the sequences of SEQ ID NOS: 2, 3 and 4, respectively. As shown in Table 1, each of the sequences of SEQ ID NOS: 2, 3 and 4 was found to have 0 mismatches with all 541 target nucleic acids, which proves that the primers and probes of SEQ ID NOS: 2, 3 and 4 according to the present invention have 100% match with the collected 541 genome sequences of BKV, thus exhibiting 100% target coverage.

Meanwhile, the UL55 gene of CMV was determined as a target nucleic acid for detecting CMV. A conserved region with little sequence variation within the UL55 gene of CMV was selected, and a forward primer having a nucleotide sequence of SEQ ID NO: 6 that can hybridize to the conserved region, a reverse primer having a nucleotide sequence of SEQ ID NO: 7, and a probe having a nucleotide sequence of SEQ ID NO: 8 were designed. In the case of the probe, a Cal Red 610 fluorescent label is linked to one end and BHQ-2 is linked to the other end. The designed primers and probe are collectively referred to as the second oligonucleotide set.

The target coverage of the second oligonucleotide set was analyzed as follows.

First, the genome sequence of CMV (taxonomy ID: 10359) was collected from the NCBI database. As a result of the collection, a total of 523 target nucleic acid sequences were collected. Thereafter, the sequence of SEQ ID NO: 6, the sequence of SEQ ID NO: 7, and the sequence of SEQ ID NO: 8 in the second oligonucleotide set were compared with each of the collected target nucleic acid sequences to determine the number of mismatches.

The above results are shown in Table 2 below.

Number of mismatches Sequence number 0 1 2 3 4 5 6 7 >=8 Coverage (%) 6 523/523 0/523 0/523 0/523 0/523 0/523 0/523 0/523 0/523 100 7 521/523 2/523 0/523 0/523 0/523 0/523 0/523 0/523 0/523 99.6 8 521/523 0/523 0/523 0/523 0/523 0/523 0/523 0/523 0/523 99.6

The above table shows the number of target nucleic acids having 0, 1, 2, 3, 4, 5, 6, 7 and 8 mismatched nucleotides for the sequences of SEQ ID NO: 6, 7 and 8, respectively.

As shown in Table 2 above, the sequence of SEQ ID NO: 6 was found to have 0 mismatches for all 523 target nucleic acids, and therefore, the sequence exhibited 100% target coverage. In addition, the sequences of SEQ ID NOs: 7 and 8 were found to have 0 mismatches for 521 of the 523 target nucleic acids, and 1 mismatch for 2 target nucleic acids. Since the sequences of SEQ ID NOs: 7 and 8 are expected to be capable of hybridizing even to a target nucleic acid having 1 mismatch, it was confirmed that the primers and probes of SEQ ID NOs: 6, 7, and 8 according to the present invention exhibited substantially 100% target coverage.

Example 2: Confirming the specificity of the oligonucleotide set

The specificity of the first and second oligonucleotide sets designed in Example 1 was confirmed.

First, for the specificity experiment of BK virus, a total of 47 specificity strains were selected, including JC virus and simian virus 40, which belong to polyomavirus and have high genetic homology with BK virus, and other strains known to exist in humans but which should not be detected by the first oligonucleotide set according to the present invention.

A reaction mixture for real-time PCR was prepared by mixing 10 μl of genomic DNA of each of the 47 strains, 6.25 μl of the first oligonucleotide set designed in Example 1, 6.25 μl of 4X Master mix (final, 200 uM dNTPs, 2 mM MgCl ₂ , 2 U of Taq DNA polymerase, 0.1 U of UDG), and 2.5 μl of distilled water.

Each of the above-mentioned prepared reaction mixtures was placed in different tubes and denatured at 95°C for 15 minutes in a real-time thermocycler (CFX96 Real-time Cycler, Bio-Rad), followed by 45 cycles of 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 10 seconds. The signal was measured at 72°C during each cycle to obtain an amplification curve.

The presence or absence of each target nucleic acid was determined by applying a predetermined threshold to the above amplification curve.

The above results are shown in Table 3 below.

No. Analyte Results 1 Acinetobacter baumannii N.D. 2 Citrobacter freundii N.D. 3 Corynebacterium diphtheriae N.D. 4 Corynebacterium striatum N.D. 5 Corynebacterium urealyticus N.D. 6 Escherichia coli (O138:K81(B):H14) N.D. 7 Enterobacter cloacae subsp. cloacae N.D. 8 Enterococcus faecium N.D. 9 Escherichia coli N.D. 10 Escherichia fergusonii N.D. 11 Escherichia hermannii N.D. 12 Escherichia vulneris N.D. 13 Haemophilus aegyptius N.D. 14 Haemophilus aphrophilus N.D. 15 Listeria seeligeri N.D. 16 Listeria innocua Seeliger N.D. 17 Neisseria lactamica Hollis et al. N.D. 18 Neisseria mucosa N.D. 19 Pantoea agglomerans N.D. 20 Serratia marcescens N.D. 21 Staphylococcus capitis subsp. capitis N.D. 22 Staphylococcus hominis subsp. hominis N.D. 23 Staphylococcus epidermidis N.D. 24 Staphylococcus saprophyticus subsp. saprophyticus N.D. 25 Streptococcus pseudopneumoniae N.D. 26 Streptococcus australis N.D. 27 Streptococcus bovis N.D. 28 Streptococcus intermedius N.D. 29 Bacillus cereus N.D. 30 Bacillus subtilis N.D. 31 Salmonella bongori N.D. 32 Herpes simplex virus 1 (Strain: Maclntyre) N.D. 33 Herpes herpesvirus 2 N.D. 34 Human herpesvirus 4, Epstein Barr virus (EBV) N.D. 35 Human herpesvirus 6B N.D. 36 Human herpesvirus 6 N.D. 37 Human Herpes 7 virus (SB strain) N.D. 38 Dengue virus type 2 N.D. 39 La Crosse Virus N.D. 40 Measles Virus N.D. 41 St. Louis Encephalitis Virus N.D. 42 International Standard for Hepatitis B virus N.D. 43 International Standard for Hepatitis C virus N.D. 44 Coxsackievirus B1_titering N.D. 45 Varicella zoster virus N.D. 46 JC virus N.D. 47 Simian virus 40 N.D.

(N.D.: Not Detected)

As shown in Table 3 above, the first oligonucleotide set according to the present invention was found not to detect other clinically important strains, including JC virus and simian virus 40, which have high genetic homology with BKV, demonstrating the high target specificity of the first oligonucleotide set according to the present invention.

Next, for the specificity experiment of CMV virus, a total of 47 specificity strains were selected, including herpes simplex virus 1 (strain: MacIntyre), which belongs to the herpesvirus family and has high genetic homology with CMV virus, human herpesvirus 2, human herpesvirus 4 Epstein-Barr virus (EBV), human herpesvirus 6B, human herpesvirus 6, and human herpesvirus 7 virus (SB strain), and other strains known to exist in humans but which should not be detected by the second oligonucleotide set according to the present invention.

A reaction mixture for real-time PCR was prepared by mixing 10 μl of genomic DNA of each of the 47 strains, 6.25 μl of the second oligonucleotide set designed in Example 1, 6.25 μl of 4X Master mix (final, 200 uM dNTPs, 2 mM MgCl ₂ , 2 U of Taq DNA polymerase, 0.1 U of UDG), and 2.5 μl of distilled water.

Each of the above-mentioned prepared reaction mixtures was placed in different tubes and denatured at 95°C for 15 minutes in a real-time thermocycler (CFX96 Real-time Cycler, Bio-Rad), followed by 45 cycles of 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 10 seconds. The signal was measured at 72°C during each cycle to obtain an amplification curve.

The presence or absence of each target nucleic acid was determined by applying a predetermined threshold to the above amplification curve.

The above results are shown in Table 4 below.

No. Analyte Results 1 Acinetobacter baumannii N.D. 2 Citrobacter freundii N.D. 3 Corynebacterium diphtheriae N.D. 4 Corynebacterium striatum N.D. 5 Corynebacterium urealyticus N.D. 6 Escherichia coli (O138:K81(B):H14) N.D. 7 Enterobacter cloacae subsp. cloacae N.D. 8 Enterococcus faecium N.D. 9 Escherichia coli N.D. 10 Escherichia fergusonii N.D. 11 Escherichia hermannii N.D. 12 Escherichia vulneris N.D. 13 Haemophilus aegyptius N.D. 14 Haemophilus aphrophilus N.D. 15 Listeria seeligeri N.D. 16 Listeria innocua Seeliger N.D. 17 Neisseria lactamica Hollis et al. N.D. 18 Neisseria mucosa N.D. 19 Pantoea agglomerans N.D. 20 Serratia marcescens N.D. 21 Staphylococcus capitis subsp. capitis N.D. 22 Staphylococcus hominis subsp. hominis N.D. 23 Staphylococcus epidermidis N.D. 24 Staphylococcus saprophyticus subsp. saprophyticus N.D. 25 Streptococcus pseudopneumoniae N.D. 26 Streptococcus australis N.D. 27 Streptococcus bovis N.D. 28 Streptococcus intermedius N.D. 29 Bacillus cereus N.D. 30 Bacillus subtilis N.D. 31 Salmonella bongori N.D. 32 Herpes simplex virus 1 (Strain: Maclntyre) N.D. 33 human herpesvirus 2 N.D. 34 Human herpesvirus 4, Epstein Barr virus (EBV) N.D. 35 Human herpesvirus 6B N.D. 36 Human herpesvirus 6 N.D. 37 Human Herpes 7 virus (SB strain) N.D. 38 Dengue virus type 2 N.D. 39 La Crosse Virus N.D. 40 Measles Virus N.D. 41 St. Louis Encephalitis Virus N.D. 42 International Standard for Hepatitis B virus N.D. 43 International Standard for Hepatitis C virus N.D. 44 Coxsackievirus B1_titering N.D. 45 Varicella zoster virus N.D. 46 JC virus N.D. 47 Simian virus 40 N.D.

(N.D.: Not Detected)

As shown in Table 4 above, the second oligonucleotide set according to the present invention was found not to detect other clinically important strains, including herpes simplex virus 1 (strain: MacIntyre), human herpesvirus 2, human herpesvirus 4 Epstein-Barr virus (EBV), human herpesvirus 6B, human herpesvirus 6 and human herpesvirus 7 virus (SB strain), which have high genetic homology with CMV, demonstrating the high target specificity of the second oligonucleotide set according to the present invention.

Example 3: Quantification of target nucleic acid

It was confirmed that the first and second oligonucleotide sets designed in Example 1 were effective in quantifying target nucleic acids.

First, real-time PCR using the first oligonucleotide set according to the present invention was performed on each of a total of 130 plasma samples, including 32 plasma samples clinically confirmed to be positive for BKV and 98 plasma samples confirmed to be negative for BKV.

Genomic DNA was extracted from the above plasma sample using the QIAsymphony DSP Virus/pathogen Midi Kit (Qiagen, Cat. No. 937055). The extracted genomic DNA was then subjected to real-time PCR as described in Example 1 to obtain a first amplification curve.

Next, the Ct value was calculated by applying RFU 300 as a threshold to the first amplification curve.

Thereafter, the amount of BKV was calculated by substituting the Ct value from the first amplification curve into the standard curve for the VP2 gene of BKV prepared in advance.

If the amount of BKV calculated above exceeded 500 copies/ml, the sample was determined to be positive, and if it was below the level, the sample was determined to be negative.

The above results are shown in Table 5 below.

Positive decision Voice Decision Positive samples = 32 32 0 pieces Voice samples = 98 0 pieces 98

As shown in Table 5 above, real-time PCR using the first oligonucleotide set according to the present invention determined all 32 positive samples for BKV as positive, and all 98 negative samples as negative. Therefore, it was confirmed that the first oligonucleotide set according to the present invention can accurately quantify/qualify positive and negative samples of BKV.

Next, real-time PCR using the second oligonucleotide according to the present invention was performed on a total of 130 plasma samples, including 33 plasma samples clinically confirmed to be positive for CMV and 97 plasma samples confirmed to be negative for CMV.

Genomic DNA was extracted from the plasma sample using the QIAsymphony DSP Virus/pathogen Midi Kit (Qiagen, Cat. No. 937055). The extracted genomic DNA was then subjected to real-time PCR as described in Example 1 to obtain a second amplification curve.

Next, the Ct value was calculated by applying RFU 300 as a threshold to the second amplification curve.

Thereafter, the amount of CMV was calculated by substituting the Ct value from the second amplification curve into the standard curve for the UL55 gene of CMV prepared in advance.

If the amount of CMV calculated above exceeded 34.5 IU/ml, the sample was determined to be positive, and if it was below the level, the sample was determined to be negative.

The above results are shown in Table 6 below.

Positive decision Voice Decision Positive samples = 33 33 0 pieces Voice samples = 98 5 pieces 92

As shown in Table 6 above, real-time PCR using the second oligonucleotide set according to the present invention determined all 33 positive samples for CMV as positive, 92 out of 97 negative samples for CMV were determined as negative, but 5 were determined as positive.

For five negative samples determined to be positive by real-time PCR according to the present invention, it was confirmed by sequencing whether they contained CMV. As a result of sequencing, all five negative samples were found to contain CMV. However, whereas the collected negative samples are determined to be negative samples when the amount of CMV is 137 IU/ml or less, the method of the present invention determines them to be negative if the amount of CMV is more than 34.5 IU/ml. The five samples were found to contain amounts of CMV between 34.5 IU/ml and 137 IU/ml. This difference is merely due to the difference in the criteria for determining negative, and it was confirmed that the second oligonucleotide set according to the present invention can accurately quantify/qualify positive and negative samples of CMV.

As can be seen from the above results, the method according to the present invention enables more accurate simultaneous diagnosis of BKV and CMV with high sensitivity and specificity.

Claims (25)

A method for simultaneous quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample, comprising the following steps: (a) performing a nucleic acid amplification reaction comprising contacting a sample in a single reaction vessel with a first set of oligonucleotides hybridizable to the VP2 gene of BKV and a second set of oligonucleotides hybridizable to the UL55 gene of CMV, wherein the nucleic acid amplification reaction generates a first amplification curve representing amplification of the VP2 gene of BKV in the sample and a second amplification curve representing amplification of the UL55 gene of CMV in the sample; and (b) comparing the generated first amplification curve with a standard curve generated from a reference sample containing a known amount of the VP2 gene to determine the amount of BKV in the sample, and comparing the generated second amplification curve with a standard curve generated from another reference sample containing a known amount of the UL55 gene to determine the amount of CMV in the sample. A method in claim 1, wherein the first oligonucleotide set comprises a plurality of oligonucleotides hybridizable to the nucleotide sequence of SEQ ID NO: 1 or a complement thereof. A method according to claim 2, wherein at least one of the plurality of oligonucleotides has 1 to 3 deoxyinosines; wherein one or two of the deoxyinosines are located in a core region ranging from the 3rd nucleotide at the 3'-terminus to the 6th nucleotide at the 3'-terminus of the oligonucleotide, and the remainder are located in a region ranging from the 4th nucleotide at the 5'-terminus to the 7th nucleotide at the 3'-terminus of the oligonucleotide. A method in claim 1, characterized in that the first oligonucleotide set does not contain an oligonucleotide capable of hybridizing to the genome sequence of JC virus or simian virus 40. A method according to claim 2, characterized in that the first oligonucleotide set comprises a primer having a nucleotide sequence of SEQ ID NO: 2 and a primer having a nucleotide sequence of SEQ ID NO: 3. A method according to claim 2, wherein the first oligonucleotide set comprises a probe having a nucleotide sequence of SEQ ID NO: 4. A method according to claim 6, wherein the probe comprises a detectable fluorescent label and a quenching moiety capable of quenching a signal from the fluorescent label. A method in claim 1, wherein the second oligonucleotide set comprises a plurality of oligonucleotides hybridizable to the nucleotide sequence of SEQ ID NO: 5 or a complement thereof. A method according to claim 8, wherein at least one of the plurality of oligonucleotides has 1-3 deoxyinosines; and wherein one or two of the deoxyinosines are located in a core region ranging from the 3rd nucleotide at the 3'-terminus to the 6th nucleotide at the 3'-terminus of the oligonucleotide, and the remainder are located in a region ranging from the 4th nucleotide at the 5'-terminus to the 7th nucleotide at the 3'-terminus of the oligonucleotide. A method in claim 1, characterized in that the second oligonucleotide set does not include an oligonucleotide hybridizable to the genome sequence of herpes simplex virus 1, human herpesvirus 2, human herpesvirus 4, human herpesvirus 6B, human herpesvirus 6, or human herpesvirus 7 virus. A method according to claim 8, characterized in that the second oligonucleotide set comprises a primer having a nucleotide sequence of SEQ ID NO: 6 and a primer having a nucleotide sequence of SEQ ID NO: 7. A method according to claim 8, wherein the second oligonucleotide set comprises a probe having a nucleotide sequence of SEQ ID NO: 8. A method according to claim 12, wherein the probe comprises a detectable fluorescent label and a quenching moiety capable of quenching a signal from the fluorescent label. A method according to claim 1, characterized in that the nucleic acid amplification reaction is a real-time PCR or isothermal amplification reaction. A method according to claim 1, characterized in that the reference samples containing the VP2 gene of the known amount are three or more, each of which contains an amount selected from 10 ¹ copies/uL to 10 ¹⁰ copies/uL. A method according to claim 1, characterized in that the other reference samples containing the known amount of UL55 gene are three or more, each of which contains an amount selected from 10 ¹ copies/uL to 10 ¹⁰ copies/uL. A method according to claim 1, characterized in that the sample is whole blood, plasma or serum collected from a subject who has received a kidney transplant. A method according to claim 17, characterized in that the subject has received a kidney transplant for less than 1 year. A method according to claim 1, wherein the sample has a volume of 5 to 15 ul, and the total volume of the nucleic acid amplification reaction is 20 to 30 ul. Composition for simultaneous quantification of BK virus (BKV) and cytomegalovirus (CMV) in a sample, comprising: (a) a first oligonucleotide set capable of hybridizing to the VP2 gene of BKV; (b) a second oligonucleotide set capable of hybridizing to the UL55 gene of CMV; and (c) A reagent for nucleic acid amplification comprising a polymerase, dNTPs, and a buffer. A composition according to claim 20, characterized in that the composition further comprises a reference sample containing a known amount of VP2 gene and another reference sample containing a known amount of UL55 gene. A composition according to claim 20, wherein the first oligonucleotide set comprises a primer having a nucleotide sequence of SEQ ID NO: 2 and a primer having a nucleotide sequence of SEQ ID NO: 3. A composition according to claim 20, wherein the first oligonucleotide set comprises a probe having a nucleotide sequence of SEQ ID NO: 4. A composition according to claim 1, characterized in that the second oligonucleotide set comprises a primer having a nucleotide sequence of SEQ ID NO: 6 and a primer having a nucleotide sequence of SEQ ID NO: 7. A composition according to claim 1, wherein the second oligonucleotide set comprises a probe having a nucleotide sequence of SEQ ID NO: 8.