WO2019135477A2 - Procédé de diagnostic de maladie de tsutsugamushi au moyen de gène multicopie - Google Patents

Procédé de diagnostic de maladie de tsutsugamushi au moyen de gène multicopie Download PDF

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Publication number
WO2019135477A2
WO2019135477A2 PCT/KR2018/012785 KR2018012785W WO2019135477A2 WO 2019135477 A2 WO2019135477 A2 WO 2019135477A2 KR 2018012785 W KR2018012785 W KR 2018012785W WO 2019135477 A2 WO2019135477 A2 WO 2019135477A2
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pcr
primer
dna
tsutsugamushi
gene
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Korean (ko)
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WO2019135477A3 (fr
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김동민
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Chosun University Industry Academic Cooperation Foundation
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Chosun University Industry Academic Cooperation Foundation
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Priority to CN201880084196.3A priority Critical patent/CN111712583B/zh
Priority to JP2020537467A priority patent/JP7065976B2/ja
Publication of WO2019135477A2 publication Critical patent/WO2019135477A2/fr
Publication of WO2019135477A3 publication Critical patent/WO2019135477A3/fr
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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q2549/00—Reactions characterised by the features used to influence the efficiency or specificity
    • C12Q2549/10—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals
    • C12Q2549/113—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals using nested probes
    • 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
    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/113—Real time assay

Definitions

  • the present invention relates to a method for diagnosing Tsutsugamushi disease using multiple replication genes. More particularly, the present invention relates to a primer capable of amplifying a multiple cloning gene sequence of Orientia tsutsugamushi which is a causative organism of Tsutsugamushi disease, The method comprising:
  • Tsutsugamushi disease is an acute febrile disease, infected by a larva of a mite infected by Oriental tsutsugamushi, and exhibits a characteristic clinical pattern due to systemic vasculitis.
  • the main host is rodent and the tick is the host and mediator. It has been reported in Korea since 1951, and it is common in Korea between September and November. Especially, 30% of the patients in autumn are diagnosed as Tsutsugamushi disease.
  • Symptoms include fever, chills, headache, and muscle aches after initial incubation of 1 to 3 weeks after being bitten by larvae of infected flea ticks. If it appears on the face, palms and soles. In the bite of the biting mite, there is eschar which is covered with a black scab within a few days of the onset. In most cases, there are no symptoms such as pain or itching.
  • Tsutsugamushi disease is made by culturing and isolating Orientia tsutsugamushi from the blood of the patient, and a method of detecting an antibody against Orientia tsutsugamushi in the serum of the patient.
  • the method of culturing the cells to diagnose the disease requires more than several weeks for the culturing, which is not suitable for the actual diagnosis of the patient.
  • IFA Indirect immunofluorescent antibody
  • PHA passive erythrocyte agglutination
  • IMC immunochromatography
  • diagnosis using the indirect immunofluorescent antibody method (IFA) in the diagnosis of tsutsugamushi disease disclosed in the Disease Control Headquarters is based on a diagnosis of tsutsugamushi disease when the IFA IgG antibody titer is 1: 256 or more .
  • IFA indirect immunofluorescent antibody method
  • PCR polymerase chain reaction
  • PCR can be performed by conventional PCR using conventional methods and nested PCR method which is 100 times more sensitive than conventional PCR and real-time monitoring of PCR reaction. And real-time PCR method, which can be confirmed as an attribute.
  • PCR was performed using various target protein genes such as 46 kDa, 57 kDa and groEL in the PCR diagnosis method of Tsutsugamushi disease.
  • Nested PCR and real time PCR were mainly used rather than conventional PCR.
  • the sensitivity of the conventional PCR in the diagnosis of Tsutsugamushi disease is very low, within 10%.
  • it is possible to improve the sensitivity by double PCR The disadvantages are that the false positives are increased due to the contamination of the gene because of the disadvantages and the double PCR.
  • the present invention has been conceived to solve the above problems, and it is an object of the present invention to provide a method for diagnosing Tsutsugamushi disease using a multi-copy gene of Orientia tsutsugamushi.
  • it is intended to provide a method for diagnosing Tsutsugamushi disease using a primer pair capable of amplifying the base sequence of a multiple replication gene.
  • the third object of the present invention is to provide a PCR diagnostic kit comprising a primer pair consisting of a specific base sequence.
  • the present invention aims to provide a PCT diagnostic kit capable of rapidly diagnosing Tsutsugamushi bacteria by providing a primer pair composed of a specific base sequence having excellent sensitivity and specificity.
  • the present invention discloses a primer comprising a specific base sequence capable of amplifying the nucleotide sequence of a multicopy gene of Orientia tsutsugamushi.
  • the primer of the present invention comprises at least one base sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 36.
  • the primer pair of the present invention comprises two or more base sequences selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 36.
  • the primer pair may include the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the method for diagnosing Tsutsugamushi disease of the present invention can be carried out using a primer pair capable of amplifying the nucleotide sequence of a multicopy gene.
  • the tsutsugamushi diagnostic PCR kit may include a pair of primers selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 1 to 36.
  • a primer pair comprising a specific base sequence according to the present invention has a high specificity for a multiclip gene.
  • the method for diagnosing Tsutsugamushi disease can detect a high sensitivity and specificity through a PCR reaction using a primer pair containing a specific nucleotide sequence, so that the clinical diagnosis can be quickly and easily performed with high reliability.
  • 1 is a graph of detection sensitivity according to a diagnostic method using an indirect immunofluorescence antibody method.
  • FIG. 2 is a flowchart showing a method for diagnosing Tsutsugamushi disease using PCR detection method according to the present invention.
  • the method for diagnosing Tsutsugamushi disease of the present invention can be carried out using a pair of primers capable of amplifying the nucleotide sequence of a multicopy gene of Orientia tsutsugamushi.
  • the multicopy gene refers to a gene which is repeatedly present at various positions in the Orientia tsutsugamushi gene.
  • the multicopy gene can include 14 kinds of tra-linked gene sequences, more preferably traB , traE, traC, and TchA genes.
  • the multi-copy gene is a target gene of PCR.
  • the target protein genes such as 47kDa, 56kDa, and groEl-STG used in the conventional diagnostic method are present only in one position in the entire gene, the multiple clone gene is repeatedly present at various positions, so PCR is performed to amplify the base sequence As the PCR efficiency increases, the accuracy of diagnosis of Tsutsugamushi disease can be enhanced.
  • the tsutsugamushi diagnostic primer of the present invention comprises at least one base sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 36.
  • the tsutsugamushi diagnostic primer pair of the present invention comprises two or more base sequences selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 36.
  • the primer pair may include the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the primer pair refers to two primers that do not have mutually complementary base sequences.
  • Primers are short-lived oligonucleotides complementary to specific gene sequences and are used for DNA synthesis, PCR, DNA sequencing, and so on. In addition, primers are the most important component of PCR testing and determine accuracy.
  • the primer was designed based on the nucleotide sequence of a multicopy gene of a bacterium strain amplified in the blood of a patient infected with Orientia tsutsugamushi boryong strain, and has a nucleotide sequence complementary to that of a multicopy gene.
  • the method for diagnosing Tsutsugamushi disease comprises the steps of (a) isolating DNA from a sample, (b) performing PCR using the separated DNA as a template using the primer pairs of SEQ ID NOS: 1 to 36, and and c) isolating the PCR product.
  • the DNA extraction in the step (a) may be performed according to a method commonly used in the art, and may be performed using a commercially available DNA extraction kit.
  • PCR may be performed by using isolated DNA as a template and synthesizing complementary DNA strands using the DNA polymerase of the primer pair of the present invention.
  • a real time PCR reaction can be performed by further including a probe based on the base sequence in the product amplified with the pair of primers.
  • the probe refers to a nucleic acid fragment consisting of several to several hundred bases capable of specifically binding to a nucleotide sequence and is labeled so that the presence or absence of a specific nucleic acid can be confirmed.
  • the probe may preferably be labeled with a fluorescent substance at the 5'-terminal and the 3'-terminal, respectively.
  • the fluorescent substance labeled at the 5'-end is selected from the group consisting of 6-carboxyfluorescein (FAM), hexachloro-6-carboxyfluorescein , HEX), tetrachloro-6-carboxyfluorescein, and Cyanine-5 (Cy5), 6-carboxy-4,5-dichloro-2,7-dimethicyl fluorene 6-Carboxy-4 ', 5'-Dichloro-2', 7'-Dimethoxyfluorescein, 6-JOE, tetrachlorofluorescein (TET), tetramethylrhodamine isothiacyanate Carboxy-X-rhodamine (ROX), 6-carboxytetramethyl-rhodamine (TAMRA), and the 3'- The fluorescent substance labeled on the end was 6-carboxytetramethyl-rhodamine (TAMRA) or black holequencher-1,2,3
  • the PCR is an abbreviation for polymerase chain reaction. It is a technique for mass-amplifying a specific region of DNA or RNA in vitro and uses a PCR reaction mixture containing various components known in the art .
  • the PCR reaction mixture may contain an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and water (dH 2 O).
  • the PCR buffer solution may also contain tris-HCl, MgCl 2 , KCl, and the like.
  • PCR typically consists of three reaction steps. The first is the DNA denaturation step, in which two strands of DNA are separated by a single strand of DNA, and the second is the step of binding the primers. After the denatured DNA and the primer are coexisted, And the third is a step of extending the primer by reacting with DNA polymerase in a state where four kinds of substrates (dNTPs) coexist in the elongation reaction.
  • dNTPs substrates
  • the PCR may utilize a conventional PCR reaction, and preferably includes the most common conventional PCR, duplex PCR and nested PCR that perform two or more PCRs simultaneously or sequentially, (Real Time) PCR, which can confirm the presence of the target gene, and most preferably, conventional PCR or real-time PCR can be used.
  • PCR can monitor the increase of the PCR amplification product in real time, it is not necessary to use an additional electrophoresis analysis method, and the PCR amplification product at the end point can be confirmed. Compared with the PCR method, PCR can be analyzed quickly and easily, and the risk of contamination is low.
  • a real-time PCR amplification product detection method is specific to a target sequence and uses a probe coupled with a fluorescent dye. Since the detection specificity is high, even a similar sequence can be detected and detected.
  • PCR products can be isolated according to methods well known in the art. Preferably by agarose gel or polyacrylamide gel electrophoresis or fluorescence analysis (ABI prism 3100 genetic analyzer-elecropherogram). After electrophoresis, electrophoresis results can be analyzed by ethidium bromide staining.
  • the PCR kit for diagnosing Tsutsugamushi disease of the present invention may comprise a pair of primers selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 36.
  • the PCR kit for detection may include an enzyme involved in the polymerase chain reaction, dNTP, and a buffer solution. And may include materials for PCR such as mineral oil, a standard marker, bromophenol blue, or xylene FF as a dyeing reagent, which are necessary components for performing electrophoresis that can be confirmed.
  • the primer is selected from the group consisting of: AM494475.1: O. tsutsugamushi Boryoung complete genome thcA gene (conserved hypothetical protein) was prepared as a forward primer (position 14-34) and a reverse primer (position 171-151) Respectively.
  • the probe was made with a Taqman probe (position 58-78) and represented by SEQ ID NOS: 37 and 38.
  • the primers and probes prepared according to the embodiment of the present invention are shown in Table 1 below.
  • the primers may have a nucleotide sequence complementary to the multiple replication genes tchA, traB, traE, trbC.
  • the sequence length of the primers was 19 nt to 30 nt, the GC content was 20 to 60%, and the Tm value was 50 to 70 ° C.
  • IFA indirect immunofluorescence
  • PCR was carried out as shown in FIG. 2 by detecting the DNA of the positive control group and the negative control group prepared in order to confirm the effectiveness of the prepared primer and the diagnostic method.
  • the whole blood was collected from the patient, and the leukocyte layer was separated, and DNA was isolated using QIA amp DNA mini kit (Qiagen, Germany). Separation method was performed by the manual attached to the kit, and the separated DNA was used as a template for detection of Orientia tsutsugamushi.
  • primers having the nucleotide sequences of SEQ ID Nos. 1 to 36 of the present invention and probes having the nucleotide sequences of SEQ ID NOS: 37 and 38 were used.
  • Real-time PCR was carried out by adding 1 ⁇ l of 2 pmole / ⁇ l probe to the reaction mixture in the same manner as described above. Then, tubes were well mixed and set in a BIONEER Exicycler 96 Real-Time Quantitative Thermal Block (Applied BIONNER) PCR was carried out and labeled with FAM (6-carboxyFluo-rescein) and BHQ-1 (Black Hole Quencher-1) at the 5 'and 3'-ends of the probe.
  • FAM 6-carboxyFluo-rescein
  • BHQ-1 Black Hole Quencher-1
  • agarose gel (Seakem LE agarose, Cambrex Bio Science) containing 0.5 ng / ml EtBr (ethidium bromide, Bioneer) was loaded onto a bioneer electrophoresis apparatus (Bioneer) The PCR product was electrophoresed for 40 min under the condition of the temperature and the result was observed.
  • the PCR amplification product for the negative control group showed no band at any place when electrophoresed.
  • the sizes of the PCR amplification products by the pair of primers of the present invention are shown in Table 3, and the detection of Orientia tsutsugamushi was confirmed by confirming the bands of the corresponding sizes.
  • PCR amplification products were identified by indirect immunofluorescence It was confirmed that a matching result was obtained.
  • the template DNA was used in the same manner as the sample isolated from the above blood samples.
  • conventional PCR and nested PCR for the target protein genes 56 kDa and 47 kDa which are generally used for the detection of Tsutsugamushi, were carried out. are shown in Table 4.
  • the primer pair used for each PCR was prepared according to the reference document or directly designed and used, and PCR was carried out in the same manner as in Example 3.
  • a pair of primers for amplifying the 47 kDa gene was prepared by the method described in Jiang J, Chan TC, Temenak JJ, Dasch GA, Ching WM, Richards AL. Development of a quantitative realtime polymerase chain reaction assay specific for Orientia tsutsugamushi. Am J Trop Med Hyg. 2004 Apr; 70 (4): 351-6.).
  • primer pairs for amplifying the 56 kDa gene are described in reference (Kim DM, Yun NR, Yang TY, Lee JH, Yang JT, Shim SK, et al. Usefulness of nested PCR for the diagnosis of scrub typhus in clinical practice: A Am J Trop Med Hyg. 2006 Sep; 75 (3): 542-5).
  • Table 5 shows the detection sensitivity, specificity and detection time of oriental tsutsugamushi in the method of detecting the oriental tsutsugamushi of Example 3 and the comparative example using the primer of the present invention.
  • sensitivity was 82.7% and 88.5%, respectively, when conventional (C) -PCR proceeded with the TraB1 and TraB2 genes of Example 3 as targets.
  • the sensitivity was 90.4% when conducting conventional PCR (C-PCR), and when the real time PCR (Q-PCR) Sensitivity was 98.1% even though it was only time.
  • the primer pairs of the present invention achieved the objects and effects to be achieved by the present invention.
  • the PCR detection method using the primer pair according to the present invention can provide enough sensitivity to confirm the infection of Orientia tsutsugamushi.

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Abstract

La présente invention concerne un procédé de diagnostic de maladie de tsutsugamushi au moyen de gènes multicopie. De préférence, l'invention concerne un procédé de diagnostic de maladie de tsutsugamushi en utilisant une paire d'amorces basées sur une séquence nucléotidique d'un gène multicopie de Orientia tsutsugamushi en vue de détecter la bactérie Orientia tsutsugamushi provoquant la maladie de tsutsugamushi, et de détecter Orientia tsutsugamushi au moyen de la dite bactérie. Un procédé de diagnostic utilisant une paire d'amorces selon la présente invention peut réaliser une détection avec une sensibilité et une spécificité supérieures par comparaison avec des procédés de test classiques utilisant un gène cible pour la détection, permettant de réaliser un diagnostic clinique rapidement et facilement avec une précision élevée.
PCT/KR2018/012785 2018-01-08 2018-10-26 Procédé de diagnostic de maladie de tsutsugamushi au moyen de gène multicopie Ceased WO2019135477A2 (fr)

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CN201880084196.3A CN111712583B (zh) 2018-01-08 2018-10-26 使用多拷贝基因诊断恙虫病的方法
JP2020537467A JP7065976B2 (ja) 2018-01-08 2018-10-26 マルチコピー遺伝子を用いたツツガムシ病の診断方法

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KR10-2018-0002282 2018-01-08

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KR102509272B1 (ko) * 2021-02-23 2023-03-14 주식회사 인바이러스테크 프라이머와 프로브를 포함하는 쯔쯔가무시 검출용 조성물 및 이를 포함하는 검출 키트

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US20070184460A1 (en) 2006-02-09 2007-08-09 Wei-Mei Ching Diagnostic assay for Orientia tsutsugamushi by detection of responsive gene expression
KR101111621B1 (ko) * 2009-09-14 2012-02-14 주식회사 인트론바이오테크놀로지 역의 상보서열의 특이 구조를 가진 고감도 프라이머를 이용한 오리엔티아 쯔쯔가무시 균의 검출법
KR101185117B1 (ko) * 2010-06-24 2012-10-22 조선대학교산학협력단 쯔쯔가무시병 진단용 프라이머 및 이를 이용한 쯔쯔가무시병의 진단 방법
CN103866013A (zh) * 2014-03-06 2014-06-18 中国人民解放军军事医学科学院放射与辐射医学研究所 一种恙虫病东方体核酸检测试剂盒
CA2982508A1 (fr) * 2015-04-15 2016-10-20 Institut Pasteur Moyen de diagnostic, de prediction ou de surveillance de la pneumonie a pneumocystis

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WO2019135477A3 (fr) 2019-09-06
JP7065976B2 (ja) 2022-05-12
JP2021516039A (ja) 2021-07-01
KR102026553B1 (ko) 2019-09-27
CN111712583A (zh) 2020-09-25
CN111712583B (zh) 2023-10-31

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