WO2016071925A2 - Intégration d'un gène de ss-actine pour la vérification de la qualité d'un échantillon dans un kit de diagnostic de vhs-1 et vhs-2 - Google Patents

Intégration d'un gène de ss-actine pour la vérification de la qualité d'un échantillon dans un kit de diagnostic de vhs-1 et vhs-2 Download PDF

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WO2016071925A2
WO2016071925A2 PCT/IN2015/000408 IN2015000408W WO2016071925A2 WO 2016071925 A2 WO2016071925 A2 WO 2016071925A2 IN 2015000408 W IN2015000408 W IN 2015000408W WO 2016071925 A2 WO2016071925 A2 WO 2016071925A2
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seq
hsv
nucleotide
probes
nucleotide sequence
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WO2016071925A3 (fr
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Amita Jain
Shantanu PRAKASH
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KING GEORGE'S MEDICAL UNIVERSITY
Indian Council of Medical Research
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KING GEORGE'S MEDICAL UNIVERSITY
Indian Council of Medical Research
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • TITLE INTEGRATION OF ⁇ -ACTIN GENE FOR SAMPLE QUALITY CHECK
  • the present invention relates to in vitro for the detection of Herpes Simplex Virus in human samples.
  • the present invention further provides a testing kit based on multiplex PCR, which enables simultaneous detection of herpes simplex virus- 1 (HSV- 1) and herpes simplex virus-2 (HSV-2), essentially comprising probes & primers for the detection of HSV1&2, with an internal control human gene for checking sample quality.
  • HSV- 1 herpes simplex virus- 1
  • HSV-2 herpes simplex virus-2
  • Herpes simplex virus 1 and 2 also known as Human herpes virus 1 and 2 (HHV- 1 and HHV-2), are DNA viruses of the family Herpesviridae.
  • the viral genome is a linear, double stranded DNA molecule of - 152261 to - 154746 nucleotides that contains a single open reading frame which codes for 77 proteins.
  • HSV infections in humans can cause lesions at a variety of sites, e.g., oral-facial, genital, visceral, ophthalmic, cutaneous and the central and peripheral nervous system.
  • HSV- 1 and HSV-2 are genetically and antigenically distinct forms.
  • HSV-2 is the most common cause of genital infections, due to venereal transmission;
  • HSV-1 is commonly associated with other disease locations although both serotypes have been shown to cause disease in all locations of the body.
  • Most HSV 1 infections are contracted during the childhood years, and many infections go completely unnoticed, as the symptoms can be minor or not apparent.
  • HSV 1 is increasingly the cause of genital herpes infections as oral-genital contact becomes a more routine part of sexual expression.
  • HSV infection can range from inconsequential (cold sores in otherwise healthy patients) to highly morbid and fatal (neonates).
  • HSV- 1&2 The currently used methods for the diagnosis of HSV- 1&2 is based on ELISA (Enzyme Linked immune sorbent assay) for detecting the serum markers such as, IgM and IgG specific to HSV1&2.
  • Antibody takes a week or more to become detectable in body fluids. Old style blood tests (called crude antigen tests) could detect antibody to herpes simplex in general but were exceedingly poor at differentiating accurately between types 1 and 2. This inability to distinguish between the two viruses is called cross reactivity. Viral culture is considered to be gold standard but is expensive, time consuming, technically challenging and less sensitive.
  • HSV PCR with its consistently and substantially higher rate of HSV detection, has replaced other tests for diagnosis.
  • PCR test takes a copy of viral genome and amplifies it many times in a visible product. Samples to be tested by PCR are less likely to be influenced by transport issues. Available HSV- 1&2 PCR assays show cross reactivity among HSV- 1 & HSV-2 due to genome similarity among HSV- 1 & HSV-2. There are few assays for HSV- 1 8s HSV-2 detection in uniplex reaction and some have HSV- 1 , HSV-2 85 VZV multiplex. These assays do not have internal quality control system. Moreover due to genotypic diversity many of the assays have limitations in picking up all the existing genotypes. These assays do not provide any provision which can provide assurance that the performed test has run successfully on a good quality sample.
  • US20060141481 Al (US 1 1 /020,676, Brian Mariani), WO 2011 13381 1 A2 (PCT/US2011 /033488, Getman et al) discloses a detection method and kit for detecting HSV- 1 and HSV-2 viruses in a test sample.
  • the test does not have an internal control which provides validity to the reaction.
  • These tests also have limitation of detecting low copy numbers. Since ELISA based methods, culture and available PCR tests have their inherent limitations there is a need for i) a sensitive, reliable and cost effective diagnosis in the first few weeks of infection,
  • test kit which helps in detecting the samples infected with low copy number of viruses
  • test kit which comprises an internal control which enables the checking of the validity of the reaction so as to be ensured that the test has run successfully on a good quality sample
  • reaction mixture for the multiplex PCR which enables the equal intensity of detection of HSV- 1 and HSV-2 viruses separately i.e. the higher presence of one type of herpes virus does not affect the detection of the other herpes virus in the sample.
  • an object of the present invention is to provide a set of probes and primers for the specific detection of human herpes simplex virus 1 and herpes simplex virus 2 simultaneously.
  • Another object of the present invention is to provide a highly specific detection of human herpes simplex virus 1 and herpes simplex virus 2 with no cross reactivity
  • Another object of the present invention is to provide a testing kit using the aforesaid probes and primers with high sensitivity, specificity and cost effectiveness enabling the simultaneous detection of HSV- 1 and HSV-2 virus.
  • Another object of the present invention is to provide a test kit which comprises an internal control which enables the checking of the validity of the reaction so as to be ensured that the test has run successfully and the sample quality is good.
  • Yet another object of the present invention is to provide a reaction mixture for the multiplex PCR which enables the equal intensity of detection of HSV- 1 and HSV-2 viruses separately i.e. the higher presence of one type of herpes virus does not affect the detection of the present other herpes virus in the sample.
  • Primers and probes for the in- vitro detection of Human HSV- 1 virus in a sample comprising: a) a nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO: l or a part of it or a nucleotide having minimum 90% sequence identity with said SEQ ID NO. l, wherein the nucleotide sequence of SEQ ID NO: l represents forward primer to amplify human HSV- 1 virus;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO:2 or a part of it or a nucleotide having minimum 90% sequence identity with said SEQ ID NO:2, wherein the nucleotide sequence of SEQ ID NO: l represents reverse primer to amplify human HSV- 1 ; and c) a nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO: 3 or a part of it or a nucleotide having minimum 90% sequence identity with said SEQ ID NO: 3, wherein the nucleotide sequence of SEQ ID NO: 3 represents probes to detect human HSV- l ; wherein the said nucleotide sequences enables high detection of all genotypes of Human HSV- 1. Wherein said nucleotide sequences enables detection of virus present in low copy number upto >10 2 IU/ml.
  • a reaction mixture for multiplex real time PCR for the simultaneous detection of human herpes virus 1 and 2 comprising: nuclease free water present in an amount of 4.2 ⁇ ; - buffer present in an amount ranging from 12.5 ⁇ 1;
  • ⁇ -actin Primer Forward 10pm present in an amount ranging from 0.20 ⁇ ;
  • HSV- 1 Probe (10pm) present in an amount ranging from 0.175 ⁇
  • HSV-2 Probe (10pm) present in an amount ranging from 0.175 ⁇
  • ⁇ -actin Probe 10pm present in an amount ranging from 0.15 ⁇ ;
  • reaction mixture enables equal intensity detection of all genotypes of human herpes virus- 1852.
  • Fig 1 shows the alignment of HSV- 1 primers and probe with UL36.
  • FIG 2 shows alignment of HSV-2 primers and probe with UL27 [gfi gene).
  • FIG 3 shows alignment of internal control primers and probe with Human ⁇ - actin gene.
  • FIG 4 shows a Real time plot of HSV- 1 positive samples using SEQ ID No. 1, 2 &3 A showing HSV- 1 amplification and B line showing No Template Control.
  • FIG 5 shows a Real time plot of HSV-2 positive samples using SEQ ID No. 4, 5 &6 A showing HSV-2 amplification and B line showing NTC.
  • FIG 6 shows a Real time plot of ⁇ -actin positive samples using SEQ ID No. 7, 8&9 A showing Human ⁇ -actin amplification and B line showing NTC.
  • FIG 7 shows a Real time plot of HSV- 1 positive and HSV-2 negative sample in a multiplex reaction using SEQ ID No. lto9
  • a HSV- 1 positive with NTC (filter 465-510),
  • b HSV-2 negative with NTC (filter 618-660) and
  • c Human ⁇ -actin with NTC (filter 533-580)
  • FIG 8 shows a Real time plot of HSV- 1 negative and HSV-2 positive sample in a multiplex reaction using SEQ ID No. lto9 (a) HSV-1 negative with NTC (filter 465-510), (b) HSV-2 positive with NTC (filter 618-660) and (c) Human ⁇ -actin with NTC (filter 533-580).
  • FIG 9 shows a Real time plot of HSV- 1 negative and HSV-2 negative sample in a multiplex reaction using SEQ ID No. lto9 (a) HSV- 1 negative with NTC (filter 465-510), (b) HSV-2 negative with NTC (filter 618-660) and (c) Human ⁇ -actin with NTC (filter 533-580).
  • FIG 10 shows a Real time plot of HSV- 1 and HSV-2 mixed sample in a multiplex reaction using SEQ ID No. lto9 (a) HSV-1 positive with NTC (filter 465-510), (b) HSV-2 positive with NTC (filter 618-660) and (c) Human ⁇ -actin with NTC (filter 533-580).
  • an in-vitro detection of Herpes simplex virus in human samples provides a multiplex real-time polymerase chain reaction for simultaneous detection of Human Herpes simplex virus 1 and 2 with Human ⁇ -actin gene as internal control.
  • the invention provides a test kit based on multiplex real time PCR for the simultaneous detection of Human Herpes virus 1 & 2.
  • the kit advantageously helps in detecting all the genotypes of HSV1&2, shows no cross reactivity among HSVl & HSV2 and also provides probes and primers that are efficient enough to detect sample with low copy number of viruses.
  • the invention extends to provide a reaction mixture for the multiplex PCR which enables the equal intensity detection of HSV1&2 viruses individually i.e. the higher presence of one type of herpes virus does not affect the detection of the other herpes virus in the sample.
  • the kit comprises nucleotide sequences selected from the group comprising of or any combinations thereof: a) a nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO: l (Tablel) or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO: l ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO: 2 (Table l) or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO:2;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO:3 (Table l) or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO:3;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO: 4 (Table2) or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO:4;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO: 5 (Table2) or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO:5; (Please check if 90% sequence identity is appropriate)
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO:6 (Table2) or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO:6;
  • nucleotide sequences enable high detection of all strains of HSV- 1 and HSV-2 virus.
  • the nucleotide sequences enables detection of HSV- 1 and HSV-2 virus present in low copy number upto > 10 2 IU/ml and > 10 2 IU/ml respectively.
  • the invention also provides primers and probes for the detection of Human HSV- 1 virus in a sample comprising:
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO: l or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO: l, wherein said nucleotide sequence of SEQ ID NO: l represents forward primer to amplify herpes simplex virus 1 ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO:2 or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO:2, wherein said nucleotide sequence of SEQ ID NO: l represents reverse primer to amplify herpes simplex virus 1 ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO:3 or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO:3, wherein said nucleotide sequence of SEQ ID NO:3 represents probes to detect herpes simplex virus 1;
  • nucleotide sequences enables the high detection of all strains of HSV- 1.
  • nucleotide sequences enable detection of Herpes simplex virus- 1 present in low copy number up to > 10 2 IU/ml and ⁇ 10 2 IU/ml respectively.
  • the invention also provides primers and probes for the detection of Human herpes simplex virus 2 in a sample comprising:
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO: 4 or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO:4, wherein said nucleotide sequence of SEQ ID NO:4 represents forward primer to amplify Human herpes simplex virus 2;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO: 5 or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO:5, wherein said nucleotide sequence of SEQ ID NO: 5 represents reverse primer to amplify Human herpes simplex virus 2;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQ ID NO:6 or a part of it or a nucleotide having at least 90% sequence identity with said SEQ ID NO:6, wherein said nucleotide sequence of SEQ ID NO:6 represents probes for the detection of Human herpes simplex virus 2;
  • nucleotide sequences enables high detection of all genotypes of Human herpes simplex virus 2 and wherein said nucleotide sequences enables detection of virus present in low copy number upto > 10 2 IU/ml.
  • the invention further provides a reaction mixture for multiplex real time PCR for the simultaneous detection of Human Herpes virus 1&2 comprising: a) Nuclease free water present in an amount of 4.2 ⁇
  • reaction mixture advantageously enables equal intensity detection of all genotypes of Human Herpes virus 1&2.
  • HSV- 1 Sequences representing different genotypes of HSV- 1 were downloaded from the GenBank nucleotide database and aligned using the program multalin. A highly conserved region of HSV- 1 (UL36) is selected for the design of real-time PCR primers and probe. Different genotypes were aligned and also checked for cross reactivity with human HSV- 1 virus. No degeneracies are done as the primers and probes were binding completely to amplify all genotypes of HSV- 1. The cross reactivity with the HSV-2 genome was tested. The probe is tagged with FAM as a reporter and IABkFQ as a quencher at 3' end. The primers and probes sequences for HSV- 1 are mentioned in table 1 :
  • Nucleotide sequence of the glycoprotein B gene (UL27) region of the human HSV-2 genome from different genotypes was analyzed from large number of isolates obtained throughout the world. The sequences of all the genotypes were aligned using multalin 13 to find conserved sequence of HSV-2 and primers& probe was designed. An alignment of primers & probe with nucleotide sequence of the glycoprotein B gene (UL27) from different HSV-2 genotypes which we studied is presented in Fig.2. No degeneracies are done yet the primers and probe are suitable for all the genotypes. The probe is tagged with Cy5 as a reporter and IAbRQSp as a quencher at 3' end. The primers and probe sequences for HSV-2 are mentioned in table 2.
  • HSV-2 GCTCACCACCAAGGAACTC 19
  • HSV-2 GCCGACACCAAAGCCATA 18
  • HSV-2 Cy5/CCCGCCCCCCTCCGCGCCT/3IAbRQS 19
  • Housekeeping gene is typically a constitutive gene that is required for the maintenance of basic cellular functions and expressed in all cells of an organism, ⁇ -actin (gene name ACTB) is one of six different actin proteins. Actins are highly conserved proteins that are involved in cell motility, structure, and integrity. Actin is a major constituent of the contractile apparatus and one of the two non-muscle cytoskeletal actins. Human ⁇ -actin gene is expressed at relatively constant levels. An alignment of primers & probe with nucleotide sequence of ⁇ -actin, using multalin is shown in Fig. 3. The probe designed is tagged with HEX as reporter and double quenched with ZEN and IABkFQ. The primers and probe sequences for Human ⁇ -actin are mentioned in table 3.
  • a known Human Herpes virus 1&2 controls were amplified with the aforesaid primers and cloned in pTZ57R/T cloning vector using commercial kit then the cloned plasmid was amplified using M 13/pUC sequencing primers (Fwd & Rvs), then the amplified product was sequenced using capillary sequencer to get exact cloned sequence. Further the sequenced segment was checked for the sequence similarity using NCBI Blast program and it was confirmed that the cloned sequence belonged to Human Herpes virus 1&2.
  • a batch of 48 samples was prepared by mixing different viruses in different combinations including Human Herpes virus 1&2 as well as HBV, HCV, Human Parvovirus B19, Human Parvovirus 4, Adenovirus, Dengue, Japanese encephalitis, Influenza virus, Enteroviruses, Varicella zoster virus and was tested for Human Herpes virus by currently designed primer and probes. Results showed that there is no cross reactivity of the designed primers and probes with the above mentioned viruses.
  • the sensitivity of this Real Time based assay was estimated based on a cloned and standardized HSV1&2 DNA with known copy number. Detection of positive samples containing viral load from 10 7 to 10 2 copies/ml, of the HSV1&2 standard in the assay was 100% (10 of 10 tests) respectively, while samples with ⁇ 10 copies/ ml were tested positive 9 out of 10 times for HSV- 1 and 7 out of 10 times for HSV-2. These results shows that the assay has a very high sensitivity and specificity required for detecting Human Herpes virus 1&2.

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Abstract

L'invention concerne des amorces et des sondes pour la détection in vitro des virus VHS-1 et virus VHS-2 humains dans un échantillon, comprenant des séquences nucléotidiques choisies dans le groupe comprenant les suivantes ou l'une quelconque de leurs combinaisons : une molécule d'acide nucléique qui code une séquence nucléotidique de SEQ ID NO: 1 et SEQ ID No. 4 ou une partie ou un nucléotide ayant une identité de séquence d'au minimum de 90 % avec ladite SEQ ID NO: 1, laquelle séquence nucléotidique de SEQ ID NO: 1 ou SEQ ID No. 4 représente une amorce sens pour amplifier le virus VHS-1 humain; une molécule d'acide nucléique qui code une séquence nucléotidique de SEQ ID NO: 2 et SEQ ID No. 5 ou une partie ou un nucléotide ayant une identité de séquence d'au minimum de 90 % avec lesdites SEQ ID NO: 2 et SEQ ID No. 5, laquelle séquence nucléotidique de SEQ ID NO: 1 représente amorce antisens pour amplifier VHS-1 humain; et une molécule d'acide nucléique qui code une séquence nucléotidique de SEQ ID NO: 3 et SEQ ID NO. 6 ou une partie ou un nucléotide ayant une identité de séquence d'au minimum de 90 % avec lesdites SEQ ID NO: 3 et SEQ ID No. 6, laquelle séquence nucléotidique de SEQ ID NO: 3 et SEQ ID No. 6 représente des sondes pour détecter HSV-1 et HSV-2 humains. Les séquences nucléotidiques permettent une détection élevée de tous les génotypes de VHS-1 et HSV-2 humains, lesdites séquences de nucléotides permettant la détection de virus présent à faible nombre de copies jusqu'à > 102 UI/ml.
PCT/IN2015/000408 2014-11-05 2015-11-04 Intégration d'un gène de ss-actine pour la vérification de la qualité d'un échantillon dans un kit de diagnostic de vhs-1 et vhs-2 Ceased WO2016071925A2 (fr)

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Cited By (2)

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US11118236B2 (en) 2018-02-13 2021-09-14 Delta Electronics Int'l (Singapore) Pte Ltd Kit and method for detecting HSV1 and HSV2
CN115927743A (zh) * 2022-07-27 2023-04-07 中国疾病预防控制中心病毒病预防控制所 一种用于同时检测hsv-1、hsv-2和vzv的组合物、试剂盒和方法

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WO2011133811A2 (fr) 2010-04-21 2011-10-27 Gen-Probe Incorporated Compositions, méthodes et kits permettant de détecter l'acide nucléique du virus de l'herpès simplex

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KR20120140069A (ko) * 2011-06-20 2012-12-28 주식회사 맥스바이오텍 단순포진 바이러스 1형 및 2형의 진단을 위한 프라이머 및 프로브, 이를 이용한 검사 방법 및 이를 포함하는 검사 키트
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US20060141481A1 (en) 2004-12-27 2006-06-29 Mariani Brian D HSV-1 and HSV-2 primers and probes
WO2011133811A2 (fr) 2010-04-21 2011-10-27 Gen-Probe Incorporated Compositions, méthodes et kits permettant de détecter l'acide nucléique du virus de l'herpès simplex

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11118236B2 (en) 2018-02-13 2021-09-14 Delta Electronics Int'l (Singapore) Pte Ltd Kit and method for detecting HSV1 and HSV2
CN115927743A (zh) * 2022-07-27 2023-04-07 中国疾病预防控制中心病毒病预防控制所 一种用于同时检测hsv-1、hsv-2和vzv的组合物、试剂盒和方法
CN115927743B (zh) * 2022-07-27 2023-11-03 中国疾病预防控制中心病毒病预防控制所 一种用于同时检测hsv-1、hsv-2和vzv的组合物、试剂盒和方法

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