WO2019135477A2 - Method of diagnosing tsutsugamushi disease by using multicopy gene - Google Patents

Method of diagnosing tsutsugamushi disease by using multicopy gene Download PDF

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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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PCT/KR2018/012785
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WO2019135477A3 (en
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김동민
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조선대학교산학협력단
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Priority to CN201880084196.3A priority Critical patent/CN111712583B/en
Priority to JP2020537467A priority patent/JP7065976B2/en
Publication of WO2019135477A2 publication Critical patent/WO2019135477A2/en
Publication of WO2019135477A3 publication Critical patent/WO2019135477A3/en

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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

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  • 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

The present invention relates to a method of diagnosing tsutsugamushi disease by using multicopy genes. Preferably, the present invention relates to a method of diagnosing tsutsugamushi disease by providing a primer pair based on the nucleotide sequence of a multi-copy gene of Orientia tsutsugamushi in order to detect Orientia tsutsugamoshi bacteria causing tsutsugamushi disease, and detecting Orientia tsutsugamoshi by using the same. A diagnostic method using a primer pair of the present invention can perform detection with higher sensitivity and specificity compared to conventional test methods using a target gene for detection, thereby being able to perform clinical diagnosis quickly and easily with high accuracy.

Description

다복제유전자를 이용한 쯔쯔가무시병의 진단방법Diagnosis of Tsutsugamushi disease using multiple cloned genes
본 발명은 다복제유전자를 이용한 쯔쯔가무시병의 진단방법에 관한 것으로서, 바람직하게는 쯔쯔가무시병의 원인균인 오리엔티아 쯔쯔가무시( Orientia tsutsugamushi)균의 다복제유전자 염기서열을 증폭시킬 수 있는 프라이머 및 이를 이용한 쯔쯔가무시병을 진단하는 방법에 관한 것이다.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:
쯔쯔가무시병은 급성 발열성 질환으로서 오리엔티아 쯔쯔가무시에 의해 감염된 털진드기의 유충에 물려 감염되며, 전신적 혈관염으로 인한 특징적인 임상 양상을 나타낸다. 주요 숙주는 설치류이며 진드기는 숙주이자 매개체이다. 주요 가을철 열성 질환으로서 우리나라에서는 1951년 처음 보고된 이래, 우리나라 전역에서 9월과 11월 사이에 발생하고 있으며, 특히, 우리나라 가을에 발생하는 환자의 30 %가 쯔쯔가무시병으로 진단될 만큼 흔한 질병이다.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.
증상은 감염된 털 진드기의 유충에 물린 후 1 ~ 3 주의 잠복기를 거친 후 초기증상으로 발열, 오한, 두통, 근육통을 동반하기도 하며, 홍반성 반점과 구진성 발진이 가슴, 배, 몸통 혹은 상하지 및 드문 경우 얼굴이나 손바닥, 발바닥에 나타난다. 털 진드기에 물린 자리에는 발병 수일 내에 까만 딱지로 덮이는 가피(eschar)가 생기며, 대부분의 경우 통증이나 가려움증 등의 증상은 없으며, 가피의 확인은 신속한 진단에 도움이 된다. 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.
대개 병의 경과가 중하지 않고, 항생제 치료에 잘 반응하나, 진단이 늦어질 경우 폐렴, 급성 신부전, 뇌수막염, 뇌염, 상부 위장관 출혈, 다기관 기능 부전, 심지어 심근경색이나 중풍의 형태로 나타날 수 있으며, 이러한 합병증으로 일부 환자에서는 사망을 초래할 수 있다. It is usually due to the progression of the disease and may respond well to antibiotic therapy. However, if the diagnosis is delayed, it may occur in the form of pneumonia, acute renal failure, meningitis, encephalitis, upper gastrointestinal bleeding, multifunctional dysfunction, or even myocardial infarction or paralysis. This complication can lead to death in some patients.
그러므로, 신속하고 정확한 진단이 환자의 예후 개선을 위해 필수적이다.Therefore, rapid and accurate diagnosis is essential for the improvement of the patient's prognosis.
쯔쯔가무시병에 대한 진단은 환자의 혈액으로부터 오리엔티아 쯔쯔가무시를 배양, 분리하여 확인하는 방법, 환자의 혈청 내에서 오리엔티아 쯔쯔가무시에 대한 항체를 검출하는 방법이 있다. 다만, 균체를 배양하여 질병을 진단하는 방법은 배양에 필요한 시간이 수주 이상 요구되고 있어 실제 환자의 진단 목적으로 합당하지 못하다. The diagnosis of 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. However, 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.
주로 진단에 사용되는 검사 방법들은 간접면역형광항체법(IFA), 피동적혈구응집법(PHA) 및 면역크로마토그래피(Immonochromatography)가 가장 흔히 사용되고 있다. Indirect immunofluorescent antibody (IFA), passive erythrocyte agglutination (PHA) and immunochromatography (IMC) are the most commonly used diagnostic methods for diagnosis.
이러한 항체를 이용한 진단법은 증상 발생 1-2주 이후에 검출되는 경우가 많아 민감도가 매우 낮고, 위양성이 많아 진단에 큰 도움이 되지 못하는 단점이 있다.Diagnostic methods using these antibodies are often detected after 1-2 weeks of symptoms and are very low in sensitivity and have a large false positive result.
예를 들어, 도 1을 참조하면 질병관리본부에 개시되어 있는 쯔쯔가무시병을 진단하는 방법 중 간접면역형광항체법(IFA)을 이용한 진단은 IFA IgG 항체가는 1:256 이상이 되면, 쯔쯔가무시병 확진으로 판정한다.For example, referring to Fig. 1, 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 .
그러나, 항체가 상승하여 진단되는 데에 수주가 걸릴 수 있고 민감도 또한 46.7% 비교적 낮으며 증상 발생 1주에 내원할 경우 전체 환자의 42.1% 만 쯔쯔가무시병으로 진단할 수 있어 보다 신속하고 판정이 용이한 새로운 진단법이 요구되고 있다.However, it may take weeks for the antibody to rise, and the sensitivity is also relatively low (46.7%). In the first week of symptoms, only 42.1% of all patients can be diagnosed with Tsutsugamushi disease. New diagnostic methods are required.
대체 진단법으로 PCR(Polymerase Chain reaction)에 기반한 검출법들이 제안되고 있다. PCR은 DNA의 원하는 부분의 복사본(copy)의 수를 기하급수적으로 증폭시킬 수 있는 분자생물학적 방법이다. DNA의 어느 부분이든지 그 서열만 알면 PCR을 통해서 증폭할 수 있다. Detection methods based on polymerase chain reaction (PCR) have been proposed as alternative diagnostic methods. PCR is a molecular biological method that can exponentially amplify the number of copies of a desired portion of DNA. Any part of the DNA can be amplified by PCR if the sequence is known.
PCR법은 전통적인 방법에 의해 이루어지는 컨벤셔널(conventional) PCR과 이를 변형하여 컨벤셔널 PCR법보다 100배 더 민감한 네스티드(nested) PCR법, PCR 반응의 실시간 모니터링(monitoring)이 가능하여 2시간 이내에 결과를 속성으로 확인할 수 있는 리얼타임(real time) PCR법 등이 있다.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.
종래에는 쯔쯔가무시 병의 PCR 진단방법에 있어서, 46kDa, 57kDa, groEL등 다양한 표적 단백 유전자(target protein gene)를 이용한 PCR이 시도되었으며, 컨벤셔널 PCR보다는 네스티드 PCR 및 리얼타임 PCR이 주로 이용되었다. 쯔쯔가무시병 진단에 있어서 컨벤셔널 PCR을 수행할 경우, 민감도가 10 % 이내로 매우 낮다는 단점이 있고, 네스티드 PCR의 경우 PCR을 두 번 시행하여 민감도를 향상시킬 수 있지만, 다른 PCR에 비해 검사시간이 더 소요되는 단점 및 PCR 을 두 번하기 때문에 유전자의 오염(contamination)으로 위양성률이 높아지는 단점이 있다. Conventionally, 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%. In case of nested PCR, 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.
다만, PCR 반응의 실시간으로 모니터링 하는 리얼타임 PCR을 수행하여 검사를 속성으로 진단하는 방법이 있지만, 검사 장비가 매우 고가이기 때문에 의료 기관에서 보편적으로 사용되기에는 어려운 실정이다. However, there is a method of real-time PCR which real-time monitoring of PCR reaction to diagnose the test as an attribute. However, since the testing equipment is very expensive, it is difficult to be universally used in medical institutions.
따라서, 높은 민감도와 특이도를 보이면서, 대체로 검사방법이 간단하고 비용이 적은 진단 방법의 개발이 절실하다.Therefore, it is urgent to develop a diagnostic method which shows high sensitivity and specificity and is simple in principle and low in cost.
본 발명은 상기와 같은 문제점을 해결하기 위하여 안출된 것으로, 본 발명은 오리엔티아 쯔쯔가무시균의 다복제유전자를 이용하여 쯔쯔가무시병을 진단하는 방법을 제공하는 것을 대목적으로 한다. Disclosure of the Invention 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.
바람직하게는, 다복제유전자의 염기서열을 증폭할 수 있는 프라이머쌍을 이용하여 쯔쯔가무시병을 진단하는 방법을 제공하는 것을 목적으로 한다. Preferably, 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.
또한, 본 발명의 두 번째 목적은, 특정 염기서열을 포함하는 프라이머쌍을 제공하는 것을 목적으로 한다.It is a second object of the present invention to provide a primer pair comprising a specific nucleotide sequence.
또한, 본 발명은 세 번째 목적은, 특정 염기서열로 이루어진 프라이머쌍을 포함하는 PCR 진단 키트를 제공하는 것을 목적으로 한다.The third object of the present invention is to provide a PCR diagnostic kit comprising a primer pair consisting of a specific base sequence.
바람직하게는, 본 발명은 민감도와 특이도가 우수한 특정 염기서열로 이루어진 프라이머쌍을 제공함으로써, 쯔쯔가무시균을 신속하게 진단할 수 있는 PCT 진단 키트를 제공하는 것을 목적으로 한다.Preferably, 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.
상술한 첫 번째 목적을 달성하기 위하여, 본 발명은 오리엔티아 쯔쯔가무시 균의 다복제유전자의 염기서열을 증폭할 수 있는 특정 염기서열을 포함하는 프라이머를 개시한다. 본 발명의 프라이머는 서열번호 1 내지 서열번호 36의 염기서열로 이루어진 군 중에서 선택된 1종 이상의 염기서열을 포함한다.In order to achieve the above-mentioned first object, 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.
바람직하게는, 본 발명의 프라이머쌍은 서열번호 1 내지 서열번호 36의 염기서열로 이루어진 군 중에서 선택된 2종 이상의 염기서열을 포함한다.Preferably, 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.
더욱 바람직하게는 상기 프라이머쌍은 서열번호 1 및 서열번호 2의 염기서열을 포함할 수 있다.More preferably, the primer pair may include the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.
본 발명의 두 번째 목적을 달성하기 위한, 본 발명의 쯔쯔가무시병 진단방법은, 다복제유전자(multicopy gene)의 염기서열을 증폭시킬 수 있는 프라이머쌍을 이용하여 검출할 수 있다.In order to achieve the second object of the present invention, 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.
상기 진단방법은 (a) 시료로부터 DNA를 분리하는 단계, (b) 상기 분리된 DNA를 주형으로 서열번호 1 내지 서열번호 36의 프라이머쌍을 이용하여 PCR을 수행하는 단계, 및 (c) 상기 PCR 산물을 분리하는 단계를 포함할 수 있다.(A) separating DNA from the sample, (b) carrying out PCR using the separated DNA as a template using the primer pairs of SEQ ID NOS: 1 to 36, and (c) And separating the product.
본 발명의 세 번째 목적을 달성하기 위한, 쯔쯔가무시 진단용 PCR 키트는 서열번호 1 내지 서열번호 36의 염기서열로 이루어진 군에서 선택된 프라이머쌍을 포함할 수 있다.In order to achieve the third object of the present invention, 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.
본 발명에 따른 특정염기서열을 포함하는 프라이머쌍은 다복제유전자에 대한 높은 특이성을 가진다.A primer pair comprising a specific base sequence according to the present invention has a high specificity for a multiclip gene.
또한, 본 발명에 따른 쯔쯔가무시병 진단방법은 특정염기서열을 포함하는 프라이머쌍을 이용하여 PCR 반응을 통해 높은 민감도와 특이성으로 검출 할 수 있으므로, 높은 신뢰성으로 신속하고 용이하게 임상진단 할 수 있다.In addition, the method for diagnosing Tsutsugamushi disease according to the present invention 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.
도 1은 간접면역형광항체법을 이용한 진단방법에 따른 검출 민감도 그래프이다.1 is a graph of detection sensitivity according to a diagnostic method using an indirect immunofluorescence antibody method.
도 2는 본 발명에 따른 PCR검출방법을 이용한 쯔쯔가무시병 진단방법을 나타내는 순서도이다.2 is a flowchart showing a method for diagnosing Tsutsugamushi disease using PCR detection method according to the present invention.
이하에서는 본 발명을 상세히 설명하고자 한다.Hereinafter, the present invention will be described in detail.
본 발명의 쯔쯔가무시병 진단방법은 오리엔티아 쯔쯔가무시균의 다복제유전자의 염기서열을 증폭시킬 수 있는 프라이머쌍을 이용하여 검출할 수 있다.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.
상기 다복제유전자(multicopy gene)는 오리엔티아 쯔쯔가무시 유전자 내에서 여러 위치에 반복하여 존재하는 유전자를 말하며, 바람직하게는 14 종의 Tra-linked 유전자의 염기서열을 포함할 수 있으며, 더 바람직하게는 traB, traE, traC 및 TchA 유전자의 염기서열을 포함할 수 있다.The multicopy gene refers to a gene which is repeatedly present at various positions in the Orientia tsutsugamushi gene. Preferably, the multicopy gene can include 14 kinds of tra-linked gene sequences, more preferably traB , traE, traC, and TchA genes.
또한, 상기 다복제유전자는 PCR의 표적 유전자(Target gene)이다.In addition, the multi-copy gene is a target gene of PCR.
종래 진단방법에 사용되었던 47kDa, 56kDa, groEl-STG 등의 표적 단백 유전자들은 전체 유전자 내부에 한 위치에만 존재하는 것에 비해 다복제유전자는 여러 위치에 반복하여 존재하기 때문에 PCR을 수행하여 염기서열을 증폭 시 PCR 효율이 높아지므로 쯔쯔가무시병의 진단의 정확도가 높아질 수 있다.Since 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.
본 발명의 쯔쯔가무시 진단용 프라이머는 서열번호 1 내지 서열번호 36의 염기서열로 이루어진 군 중에서 선택된 1종 이상의 염기서열을 포함한다.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.
또한, 본 발명의 쯔쯔가무시 진단용 프라이머쌍은 서열번호 1 내지 서열번호 36의 염기서열로 이루어진 군 중에서 선택된 2종 이상의 염기서열을 포함한다.In addition, 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.
상기 프라이머쌍은 서열번호 1 및 서열번호 2의 염기서열을 포함할 수 있다.The primer pair may include the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.
통상적으로 상기 프라이머쌍은 서로 상보적인 염기서열을 갖지 않는 2개의 프라이머(primer)를 말한다.Usually, the primer pair refers to two primers that do not have mutually complementary base sequences.
프라이머는 특정 유전자 서열에 대하여 상보적인 짧은 단선의 유전자 서열 (oligonucleotide)로 DNA 합성에 기시점이 되며, PCR 검사, DNA시퀸싱(DNA sequencing) 등에 이용된다. 또한, 프라이머는 PCR 검사의 가장 중요한 요소이며, 정확도를 결정한다. 상기 프라이머는 오리엔티아 쯔쯔가무시( Orientia tsutsugamushi) 보령균주에 감염된 환자의 혈액에서 증폭한 보령균주의 다복제유전자의 염기서열에 근거하여 설계하였으며, 다복제유전자와 상보적인 염기서열을 가진다.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.
또한, 상기 쯔쯔가무시병 진단방법은 (a) 시료로부터 DNA를 분리하는 단계, (b) 상기 분리된 DNA를 주형으로 서열번호 1 내지 서열번호 36의 프라이머쌍을 이용하여 PCR을 수행하는 단계, 및 (c) 상기 PCR 산물을 분리하는 단계를 포함할 수 있다.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.
상기 (a)단계에서의 DNA 추출은 당 업계에서 통상적으로 사용되는 방법에 따라 수행될 수 있으며, 상업적으로 판매되는 DNA 추출 키트를 이용하여 수행할 수 있다.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.
상기 (b)단계에서 PCR은 분리된 DNA를 주형으로 하고, 본 발명의 프라이머쌍이 DNA 중합효소를 이용하여 상보적 DNA가닥을 합성하여 PCR 반응을 수행할 수 있다.In the step (b), 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.
또한, 상기 (b)단계에서 한 쌍의 프라이머로 증폭되는 산물 내 염기서열에 근거하여 프로브(Probe)를 더 포함하여 Real time PCR 반응을 수행할 수 있다. In the step (b), 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.
상기 프로브란 염기서열과 특이적으로 결합할 수 있는 수개 내지 수백 개의 염기로 이루어진 핵산단편을 의미하며 라벨링 되어있어 특정 핵산 존재여부를 확인할 수 있다. 프로브는 바람직하게는 5'-말단 및 3'-말단에 각각 형광물질이 표지될 수 있다. 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.
또한, 더욱 바람직하게는 더욱 바람직하게는 상기 5'-말단에 표지된 형광물질은 6-카르복시플루오레세인(6-carboxyfluorescein, FAM), 헥사클로로-6-카르복시플루오레세인(hexachloro-6-carboxyfluorescein, HEX), 테트라클로로-6-카르복시플루오레세인(tetrachloro-6-carboxyfluorescein), 및 Cyanine-5(Cy5), 6-카르복시-4,5-디클로로-2,7-디메쏙시플루오레세인(6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein,6-JOE), 테트라클로로플루오레세인 (tetrachlorofluorescein, TET), 테트라메틸로다민 이소티오시아네이트(tertramethylrhodamine isothiacyanate,Texas Red), 5-카르복시-x-로다민(5-carboxy-X-rhodamine, ROX), 6-카르복시테트라메틸-로다민(6-carboxytetramethyl-rhodamine, TAMRA)으로 이루어진 군에서 선택되고, 상기 3'-말단에 표지된 형광물질은 6-카르복시테트라메틸-로다민(6-carboxytetramethyl-rhodamine, TAMRA) 또는 black holequencher-1,2,3(BHQ-1,2,3)일 수 있으며, 이에 한정 되는 것은 아니다.More preferably still more preferably, 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 (BHQ-1, 2,3), but is not limited thereto.
상기 PCR은 중합효소 연쇄 중합반응(polymerase chain reaction)의 약어로서, DNA 또는 RNA의 특정영역을 시험관 내에서 대량으로 증폭하는 기술이며, 당 업계에 공지된 여러 성분을 포함하는 PCR 반응 혼합액을 이용하여 수행될 수 있다. 상기 PCR반응 혼합액에는 본 발명에서 제공되는 PCR 프라이머쌍 이외에 적당량의 DNA 중합효소, dNTP, PCR 완충용액 및 물(dH 2O)을 포함할 수 있다.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 . In addition to the PCR primer pair provided in the present invention, the PCR reaction mixture may contain an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and water (dH 2 O).
또한, PCR 완충용액은 트리스-HCl(tris-HCl), MgCl 2, KCl 등을 포함할 수 있다.The PCR buffer solution may also contain tris-HCl, MgCl 2 , KCl, and the like.
PCR은 통상적으로 3가지 반응단계로 이루어져 있다. 첫 번째는 DNA 변성단계로, 두 가닥의 DNA를 온도를 가하여 한 가닥의 DNA로 분리시키는 단계이며, 두 번째는 프라이머의 결합단계로, 한 가닥으로 변성한 DNA와 프라이머를 공존시킨 후 온도를 낮추면 프라이머쌍이 각각 상보적인 한 가닥 주형 DNA에 결합되는 단계이고, 세 번째는 신장반응으로 4 종류의 기질(dNTP)이 공존하는 상태에서 DNA 중합효소를 작용시켜 프라이머를 신장시키는 단계로 이루어져 있다.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.
상기 PCR은 통상적인 PCR 반응을 이용할 수 있으며, 바람직하게는 가장 일반적인 컨벤셔널(Conventional) PCR, 두 번의 PCR을 동시에 또는 연속해서 수행하는 듀플렉스(duplex) PCR 및 네스티드(nested) PCR, 실시간으로 결과를 확인할 수 있는 고가의 리얼타임(Real time) PCR을 모두 이용할 수 있으며, 가장 바람직하게는 컨벤셔널 PCR 또는 리얼타임 PCR을 이용할 수 있다.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.
상기 리얼타임(Real time) PCR은 PCR 증폭산물의 증가를 실시간으로 모니터링하여 확인 할 수 있기 때문에 추가적인 전기영동 분석방법을 이용하지 않아도 되며, 앤드 포인트(End point)에서 PCR 증폭산물을 확인하는 기존의 PCR 법에 비해서 신속하고 간편하게 PCR을 해석할 수 있으며 오염의 위험성이 적다.Since the real time 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.
리얼타임 PCR 증폭산물의 검출방법은 타켓 서열(target sequence)에 특이적이며 형광 염료(dye)가 결합된 프로브(Probe)를 이용하며, 검출 특이성이 높아 비슷한 서열까지도 구별하여 검출할 수 있다. 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.
또한, 상기 (c)단계에서는 당 업계에서 널리 공지된 방법에 따라 PCR 산물을 분리할 수 있다. 바람직하게는 아가로스 겔(agarose gel) 또는 폴리아크릴아미드 겔 (polyacrylamide gel) 전기영동 또는 형광분석 장치(ABI prism 3100 genetic analyzer-elecropherogram)에 의해 확인할 수 있다. 전기영동 후 에디듐 브로마이드(ethidium bromide) 염색으로 전기영동 결과를 분석할 수 있다.In the step (c), 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.
본 발명의 쯔쯔가무시병 진단용 PCR키트는 서열번호 1 내지 서열번호 36 의 염기서열로 이루어진 군에서 선택된 프라이머쌍을 포함할 수 있다.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.
또한, 상기 검출용 PCR 키트에는 상기 다복제유전자의 염기서열을 증폭할 수 있는 프라이머쌍 이외에도, 중합효소 연쇄 중합반응에 관여하는 효소, dNTP, 완충 용액을 포함할 수 있으며, PCR 산물의 증폭 여부를 확인할 수 있는 전기영동에 수행에 필요한 구성성분인 미네랄 오일, 표준 마커, 염색시약인 브로모페놀블루 또는 자일렌 FF 등의 PCR용 재료들을 포함할 수 있다.In addition to the primer pair capable of amplifying the nucleotide sequence of the multiple replication gene, 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.
이하, 본 발명을 다음의 실시예에 의하여 균의 검출, 진단 방법에 대해 보다 구체적으로 설명한다. Hereinafter, the present invention will be described in more detail with reference to the methods for detecting and diagnosing bacteria according to the following examples.
다음의 실시예는 단지 본 발명을 자세히 설명하기 위한 것으로, 이들에 의해 본 발명의 내용 및 범위가 실시예에 의해 한정되는 것은 아니라는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.It will be apparent to those skilled in the art that the following examples are merely illustrative of the present invention and that the content and scope of the present invention are not limited by the examples.
<실시예><Examples>
1. 프라이머 제작 1. Primer production
우리나라에서 흔한 쯔쯔가무시 보령균주에 감염된 환자의 혈액에서 증폭된 다복제유전자(multicopy gene) 유전자에 근거하여 설계되었다. It was designed based on the multicopy gene gene amplified in the blood of patients infected with Tsutsugamushi boryong strain common in Korea.
더 바람직하게는, 상기 프라이머는 Genbank Accession No. AM494475.1: O.tsutsugamushi Boryoung complete genome thcA gene(conserved hypothetical protein)에 대하여 정방향의 프라이머(position 14-34) 및 역방향 프라이머(position 171-151)로 제작하였으며, 각각 서열번호 1 내지 서열번호 36으로 표기하였다. More preferably, 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.
또한, 프로브(probe)는 Taqman probe(position 58-78)로 제작하였으며, 서열번호 37 및 38로 표기하였다.In addition, the probe was made with a Taqman probe (position 58-78) and represented by SEQ ID NOS: 37 and 38.
본 발명의 실시예에 따라 제작된 프라이머 및 프로브를 하기 표 1에 나타내었다.The primers and probes prepared according to the embodiment of the present invention are shown in Table 1 below.
서열번호SEQ ID NO: 서열order
1One tttttgcccaaaagccttgtttttgcccaaaagccttg
22 ggcctagcctttatggttggggcctagcctttatggttgg
33 atcctaatggagctggcaatatcctaatggagctggcaat
44 tggagtagctccaggaattgttggagtagctccaggaattgt
55 tggagctggcaataaacctttggagctggcaataaacctt
66 gcattgacgaatggggagtagcattgacgaatggggagta
77 cctccaaccataaaggctagg cctccaaccataaaggctagg
88 catgctacaattgaggctgaagcatgctacaattgaggctgaag
99 agagcaattcctccaaccat agagcaattcctccaaccat
1010 cattatcgaggccgttgaagcattatcgaggccgttgaag
1111 tttagcatgcctgctgtttagtttagcatgcctgctgtttag
1212 tgctacaattgaggctgaaggtgctacaattgaggctgaagg
1313 tgctaaaggaatatggccagttatgctaaaggaatatggccagtta
1414 tcaattgattatgcgtgggttatcaattgattatgcgtgggtta
1515 tcacaggaacaacatcaattttatcacaggaacaacatcaatttta
1616 ttttcaatgagtgatgaggctttttttcaatgagtgatgaggcttt
1717 cgaagctaataccaagctccacgaagctaataccaagctcca
1818 tcattttcaatgagtgatgaggtcattttcaatgagtgatgagg
1919 tggccagttaatttcttggtt tggccagttaatttcttggtt
2020 ctgaatctcaaaaggctggagctgaatctcaaaaggctggag
2121 tggctgcaattaccaaagaatggctgcaattaccaaagaa
2222 gatgccactttcatgtctgcgatgccactttcatgtctgc
2323 tgggcaatttatgtgacgaatgggcaatttatgtgacgaa
2424 tcacctcaaccttctttggaatcacctcaaccttctttggaa
2525 gcagacatgaaagtggcatcgcagacatgaaagtggcatc
2626 cgctaaaccagtagcgaagccgctaaaccagtagcgaagc
2727 ttccaaagaaggttgaggtgattccaaagaaggttgaggtga
2828 cgccagttaacaacaaaaggcgccagttaacaacaaaagg
2929 gctcctctaacttttgtgattgg gctcctctaacttttgtgattgg
3030 agcccagtcgttcttattgcagcccagtcgttcttattgc
3131 tgtggcttcttcttgtgctctgtggcttcttcttgtgctc
3232 attggagatgcgcttcagtcattggagatgcgcttcagtc
3333 cctgacgcaataagaacgactcctgacgcaataagaacgact
3434 gctggaggtcatcaacaacagctggaggtcatcaacaaca
3535 gcgcatctccaatctgaaacgcgcatctccaatctgaaac
3636 ggtgaagatggacgttctggggtgaagatggacgttctgg
3737 cggcctcgataatgcccaacacggcctcgataatgcccaaca
3838 cttcaacggcctcgataatgccccttcaacggcctcgataatgccc
상기 프라이머는 다복제유전자인 tchA, traB, traE, trbC와 상보적인 염기서열을 가질 수 있다.The primers may have a nucleotide sequence complementary to the multiple replication genes tchA, traB, traE, trbC.
또한, 상기 프라이머의 서열길이는 각각 19nt 내지 30nt, GC함량은 20 내지 60% 이며, Tm값은 50 내지 70 ℃가 되도록 제작하였다.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.
2. 검체 준비2. Sample preparation
2007년부터 2008년 사이에 조선대학교 병원을 내원한 환자를 대상으로 당시 가피 또는 반구진성 발진 등이 있으며, 두통, 전신위약감, 근육통, 기침, 오심, 복통과 같은 2가지 이상의 임상증상을 갖고 있는 환자의 혈액샘플을 채취하고 유전자를 추출하여 검사에 이용하였다.Patients who visited Chosun University Hospital between 2007 and 2008 were diagnosed with acute or hemispheric rash and had two or more clinical symptoms such as headache, general weakness, muscle aches, cough, nausea and abdominal pain A blood sample of the patient was taken and the gene was extracted and used for the examination.
혈액 검체에서 간접면역 형광검사법(IFA)에서 오리엔티아 쯔쯔가무시에 대한 lgG 항체가가 4배 이상 상승된 경우를 쯔쯔가무시병 확진으로 정의하였으며, 쯔쯔가무시병으로 확진된 환자 52명의 혈액을 양성대조군으로하고, 쯔쯔가무시가 아닌 타 질환으로 진단된 환자 20명의 혈액을 음성대조군으로 이용하였다.In blood specimens, the case of IgG antibody to Orientia tsutsugamushi in the case of indirect immunofluorescence (IFA) was defined as a diagnosis of tsutsugamushi disease. The blood of 52 patients with tsutsugamushi disease was defined as a positive control, And blood of 20 patients diagnosed as other diseases were used as a negative control group.
3. 오리엔티아 쯔쯔가무시 검출3. Orientation tsutsugamushi detection
제작한 프라이머 및 진단방법의 유효성을 확인하기 위하여 준비한 양성대조군 및 음성대조군에 대한 검체를 대상으로 DNA를 검출하여 도 2에 나타낸 바와 같이 PCR을 진행 하였다.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.
환자로부터 전혈을 채취하고, 백혈구 연층을 분리한 다음, QIA amp DNA 미니키트(Qiagen, Germany)를 이용하여 DNA를 분리하였다. 분리방법은 키트에 동봉된 매뉴얼(manual)에 의해 실시하였으며, 분리한 DNA는 오리엔티아 쯔쯔가무시 균의 검출을 위한 주형(template)으로 사용하였다.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.
추출된 DNA를 주형으로 하여 컨벤셔널 PCR, 네스티드 PCR 및 리얼타임 PCR을 시행하였다. Conventional PCR, nested PCR and real - time PCR were performed using the extracted DNA as a template.
이때, 실험에 사용한 프라이머로는 본 발명의 서열번호 1 내지 서열번호 36의 염기서열을 가진 프라이머 및 서열번호 37 및 38의 염기서열을 가진 프로브를 이용하였다.As the primers used in the experiments, 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.
컨벤셔널 PCR 및 네스티드 PCR은 AccuPowerTM PCR PreMix (1U Top polymerase, 250 ㎛ dNTP, 10 mM Tris-HCl(pH 9.0; Bioneer)에 2 ㎕ 주형 DNA, 각각 1 ㎕씩 10 pmole/㎕ 정방향 프리이머 및 역방향 프라이머, 16 ㎕의 멸균 3차 증류수를 첨가하여 총 20 ㎕의 반응액을 만든 다음 튜브를 잘 혼합하여 Biosystems Veriti TM 96-well Thermal cycle(Applied Biosystems)에 세팅한 다음 PCR을 수행하였다.Conventional PCR and nested PCR were performed using AccuPower 占 PCR PreMix (1U Top polymerase, 250 占 퐉 dNTP, 10 占 Tris-HCl (pH 9.0; Bioneer) with 2 占 퐇 template DNA, 10 pmole / Primer and 16 μl of sterilized third distilled water to prepare a total reaction solution of 20 μl. The tubes were well mixed and set in a Biosystems Veriti ™ 96-well thermal cycle (Applied Biosystems), followed by PCR.
또한, 리얼타임 PCR은 1㎕의 2 pmole/㎕ probe를 포함하여 상기와 동일한 방법으로 반응액을 만든 다음 튜브를 잘 혼합하여 BIONEER Exicycler쪠 96 Real-Time Quantitative Thermal Block(Applied BIONNER)에 세팅한 다음 PCR을 수행하였으며, 프로브의 5'과 3'-말단에 FAM(6-carboxyFluo-rescein)과 BHQ-1(Black Hole Quencher-1)로 표지하여 사용하였다.Real-time PCR was carried out by adding 1 μl of 2 pmole / μl 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.
PCR 수행조건은 표 2에 나타내었다.The PCR conditions are shown in Table 2.
PCR 종료 후, 0.5 ng/ml EtBr (ethidium bromide, Bioneer)을 넣은 1.5 % agarose gel (Seakem LE agarose, Cambrex Bio Science)을 바이오니아 전기영동 기기(Bioneer)로 로딩하고, 100 V(1X TEA, Bioneer)조건으로 40분간 PCR 증폭산물을 전기영동을 진행하여 그 결과를 관찰 하였다. After completion of the PCR, 1.5% 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.
PCR 검사결과, 음성대조군에 대한 PCR 증폭산물은 전기영동 시켰을 때 어떠한 곳에서도 band가 관찰되지 않았다. 또한, 본 발명의 프라이머에 쌍에 의한 PCR 증폭산물의 크기는 표 3에 표기되어 있으며, 해당 크기의 밴드를 확인 함으로써 오리엔티아 쯔쯔가무시 검출 여부를 확인하는 것으로 하였으며, PCR 증폭산물은 간접면역 형광검사법과 일치하는 결과가 도출됨을 확인 할 수 있었다.As a result of PCR, 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.
PCR 반응 조건PCR reaction conditions
예비변성(predenaturation)Predenaturation 94℃94 ° C 10분10 minutes 35 cycles35 cycles
변성(denaturation)Denaturation 94℃94 ° C 30초30 seconds
결합(annealing)Annealing 63℃63 ° C 30초30 seconds
신장(extension)Extension 72℃72 ℃ 1분1 minute
추가-신장(extra-extension)Extra-extension 72℃72 ℃ 7분7 minutes
유전자gene 프라이머쌍 번호Primer pair number 서열order 프라이머 서열번호Primer sequence number 증폭산물 크기(bp) Amplification product size (bp)
tchAtchA 1One tttttgcccaaaagccttgtttttgcccaaaagccttg 1One 149149
ggcctagcctttatggttggggcctagcctttatggttgg 22
22 atcctaatggagctggcaatatcctaatggagctggcaat 33 176176
tggagtagctccaggaattgttggagtagctccaggaattgt 44
33 tggagctggcaataaacctttggagctggcaataaacctt 55 197197
gcattgacgaatggggagtagcattgacgaatggggagta 66
44 cctccaaccataaaggctaggcctccaaccataaaggctagg 77 186186
catgctacaattgaggctgaag catgctacaattgaggctgaag 88
55 agagcaattcctccaaccatagagcaattcctccaaccat 99 142142
cattatcgaggccgttgaag cattatcgaggccgttgaag 1010
66 tttagcatgcctgctgtttagtttagcatgcctgctgtttag 1111 158158
tgctacaattgaggctgaaggtgctacaattgaggctgaagg 1212
traCtraC 1One tgctaaaggaatatggccagttatgctaaaggaatatggccagtta 1313 193193
tcaattgattatgcgtgggttatcaattgattatgcgtgggtta 1414
22 tcacaggaacaacatcaattttatcacaggaacaacatcaatttta 1515 190190
ttttcaatgagtgatgaggctttttttcaatgagtgatgaggcttt 1616
33 cgaagctaataccaagctccacgaagctaataccaagctcca 1717 142142
tcattttcaatgagtgatgaggtcattttcaatgagtgatgagg 1818
44 tggccagttaatttcttggtttggccagttaatttcttggtt 1919 202202
ctgaatctcaaaaggctggag ctgaatctcaaaaggctggag 2020
traEtraE 1One tggctgcaattaccaaagaatggctgcaattaccaaagaa 2121 190190
gatgccactttcatgtctgcgatgccactttcatgtctgc 2222
22 tgggcaatttatgtgacgaatgggcaatttatgtgacgaa 2323 184184
tcacctcaaccttctttggaatcacctcaaccttctttggaa 2424
33 gcagacatgaaagtggcatcgcagacatgaaagtggcatc 2525 169169
cgctaaaccagtagcgaagccgctaaaccagtagcgaagc 2626
44 ttccaaagaaggttgaggtgattccaaagaaggttgaggtga 2727 150150
cgccagttaacaacaaaaggcgccagttaacaacaaaagg 2828
traBtraB 1One gctcctctaacttttgtgattgggctcctctaacttttgtgattgg 2929 172172
agcccagtcgttcttattgc agcccagtcgttcttattgc 3030
22 tgtggcttcttcttgtgctctgtggcttcttcttgtgctc 3131 182182
attggagatgcgcttcagtcattggagatgcgcttcagtc 3232
33 cctgacgcaataagaacgactcctgacgcaataagaacgact 3333 149149
gctggaggtcatcaacaacagctggaggtcatcaacaaca 3434
44 gcgcatctccaatctgaaacgcgcatctccaatctgaaac 3535 199199
ggtgaagatggacgttctggggtgaagatggacgttctgg 3636
<비교예> <Comparative Example>
1. 종래 방법에 의한 오리엔티아 쯔쯔가무시 검출 1. Detection of Orientia tsutsugamushi by conventional methods
본 발명의 프라이머 및 진단방법의 유효성을 비교하기 위하여 하기와 같이 비교 실험을 실행하였다.In order to compare the effectiveness of the primers and diagnostic methods of the present invention, comparative experiments were conducted as follows.
주형 DNA는 상기 혈액검체에서 분리한 것을 동일하게 사용하였으며, 각각의 실험은 쯔쯔가무시 균의 검출에 일반적으로 사용되는 타켓 단백 유전자인 56kDa, 47kDa에 대한 컨벤셔널 PCR 및 네스티드 PCR을 진행 하였으며, 그 결과를 표 4에 나타내었다.The template DNA was used in the same manner as the sample isolated from the above blood samples. In each experiment, 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.
각각의 PCR에 사용한 프라이머쌍은 참고 문헌에 따라 제작하거나, 직접 디자인 하여 사용하였으며, 실시예 3과 동일한 방법으로 PCR을 실시하였다.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.
47kDa 유전자를 증폭시키기 위한 프라이머쌍은 참고문헌 (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.)을 참고하여 제작하였다.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.).
또한, 56kDa 유전자를 증폭시키기 위한 프라이머쌍은 참고문헌 (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 prospective study. Am J Trop Med Hyg. 2006 Sep;75(3):542-5.)를 참고하여 제작하였다.In addition, the 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).
유전자/PCRGene / PCR 프라이머 명칭Name of the primer 서열order 증폭산물 크기(bp)Amplification product size (bp)
47kDa/C-PCR47 kDa / C-PCR OtsuFP630OtsuFP630 AACTGATTTTATTCAACTAATGCTGCTAACTGATTTTATTCAACTAATGCTGCT 118118
OtsuRP747OtsuRP747 TATGCCTGAGTAAGATACRTGAATRGAATTTATGCCTGAGTAAGATACRTGAATRGAATT
47kDa/N-PCR(External Primer)47 kDa / N-PCR (External Primer) OtsuFP555OtsuFP555 TCCTTTCGGTTTAAGAGGAACATCCTTTCGGTTTAAGAGGAACA 238238
OtsuRP771OtsuRP771 GCATTCAACTGCTTCAAGTACAGCATTCAACTGCTTCAAGTACA
47kDa/N-PCR(Internal Primer)47 kDa / N-PCR (Internal Primer) OtsuFP630OtsuFP630 AACTGATTTTATTCAACTAATGCTGCTAACTGATTTTATTCAACTAATGCTGCT 118118
OtsuRP747OtsuRP747 TATGCCTGAGTAAGATACRTGAATRGAATTTATGCCTGAGTAAGATACRTGAATRGAATT
56kDa/C-PCR56 kDa / C-PCR P10P10 GATCAAGCTTCCTCAGCCTACTATAATGCCGATCAAGCTTCCTCAGCCTACTATAATGCC 438438
P11P11 CTAGGGATCCCGACAGATGCACTATTAGGCCTAGGGATCCCGACAGATGCACTATTAGGC
56kDa/N-PCR(External Primer)56 kDa / N-PCR (External Primer) P34P34 TCAAGCTTATTGCTAGATCTGCTCAAGCTTATTGCTAGATCTGC 10031003
P55P55 AGGGATCCCTGCTGCTGTGCTTGCTGCGAGGGATCCCTGCTGCTGTGCTTGCTGCG
56kDa/N-PCR(Internal Primer)56 kDa / N-PCR (Internal Primer) P10P10 GATCAAGCTTCCTCAGCCTACTATAATGCCGATCAAGCTTCCTCAGCCTACTATAATGCC 438438
P11P11 CTAGGGATCCCGACAGATGCACTATTAGGCCTAGGGATCCCGACAGATGCACTATTAGGC
<결과> 쯔쯔가무시병 진단방법의 유효성 확인<Result> Validation of diagnosis method of Tsutsugamushi disease
본 발명의 프라이머를 이용한 실시예 3 및 비교예의 오리엔티아 쯔쯔가무시 검출 방법에 있어서, 오리엔티아 쯔쯔가무시의 검출 민감도(sensitivity), 특이도(specificity) 및 검출시간을 측정한 결과를 표 5에 나타내었다. 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.
표 5에서 알 수 있듯이 실시예 3의 TraB 유전자를 표적으로하여 컨벤셔널(C)-PCR을 진행한 경우, 비교예 1의 56kDa, 47kDa 유전자를 표적으로 한 네스티드(N)-PCR에 비해 적은 시간이 소요되었다.As can be seen from Table 5, when Conventional (C) -PCR was carried out by targeting the TraB gene of Example 3, it was found that compared with the nested-NCR-PCR in which the 56 kDa and 47 kDa genes of Comparative Example 1 were targeted, It took time.
또한, 실시예 3의 TraB1, TraB2 유전자를 표적으로하여 컨벤셔널(C)-PCR을 진행한 경우, 각각 82.7%와 88.5%의 민감도를 나타내었다. In addition, sensitivity was 82.7% and 88.5%, respectively, when conventional (C) -PCR proceeded with the TraB1 and TraB2 genes of Example 3 as targets.
특히, Tch 유전자를 표적으로 이용할 경우에는 컨벤셔널 PCR(C-PCR)을 진행한 경우, 90.4%의 민감도를 보였으며, 리얼타임 PCR(Q-PCR)을 진행한 경우, 검출 시간이 1-2시간에 불과함에도 민감도가 98.1%를 나타내었다. In particular, when the Tch gene was used as a target, 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.
따라서, 하기 표 5에 나타낸 바와 같이, 본 발명의 프라이머쌍은 본 발명이 달성하고자 한 목적 및 효과를 달성하였다.Thus, as shown in Table 5 below, the primer pairs of the present invention achieved the objects and effects to be achieved by the present invention.
상기 결과에 의하면, 본 발명에 따른 프라이머쌍을 이용한 PCR 검출 방법은 오리엔티아 쯔쯔가무시의 감염여부를 확인하기에 충분한 민감도를 제공해 줄 수 있음을 확인할 수 있다.According to the above results, it can be confirmed that the PCR detection method using the primer pair according to the present invention can provide enough sensitivity to confirm the infection of Orientia tsutsugamushi.
유전자/PCRGene / PCR 민감도responsiveness 특이도Specificity 검출 시간Detection time
실시예 3Example 3 TchA6/C-PCRTchA6 / C-PCR 90.4%90.4% 100%100% 3-4h3-4h
TchA6/Q-PCRTchA6 / Q-PCR 98.1%98.1% 100%100% 1-2h1-2h
TrbC1/C-PCRTrbC1 / C-PCR 63.5%63.5% 100%100% 3-4h3-4h
TrbC4/C-PCRTrbC4 / C-PCR 57.7%57.7% 100%100% 3-4h3-4h
TraE1/C-PCRTraE1 / C-PCR 62.5%62.5% 100%100% 3-4h3-4h
TraE3/C-PCRTraE3 / C-PCR 62.5%62.5% 100%100% 3-4h3-4h
TraB1/C-PCRTraB1 / C-PCR 82.7%82.7% 100%100% 3-4h3-4h
TraB2/C-PCRTraB2 / C-PCR 88.5%88.5% 100%100% 3-4h3-4h
비교예 1Comparative Example 1 56kDa/C-PCR56 kDa / C-PCR 1.9%1.9% 100%100% 3-4h3-4h
56kDa/N-PCR56 kDa / N-PCR 84.6%84.6% 100%100% 6-7h6-7h
47kDa/C-PCR47 kDa / C-PCR 1.9%1.9% 100%100% 3-4h3-4h
47kDa/N-PCR47 kDa / N-PCR 84.6%84.6% 100%100% 6-7h6-7h
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였으며, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 정확한 실시의 예시일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아니며, 본 발명의 실질적인 범위는 첨부된 청구항들에 의하여 정의된다고 할 것이다.It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention as defined in the appended claims. The actual scope of the invention will be defined by the appended claims.
<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, CHOSUN UNIVERSITY<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, CHOSUN UNIVERSITY
<120> Diagnosis method of Tsutsugamushi disease using multicopy genes<120> Diagnosis method of Tsutsugamushi disease using multicopy genes
<130> 2018-I181<130> 2018-I181
<150> KR 10-2018-0002282<150> KR 10-2018-0002282
<151> 2018-01-08<151> 2018-01-08
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<223> Primer for PCR<223> Primer for PCR
<400> 16<400> 16
ttttcaatga gtgatgaggc ttt 23ttttcaatga gtgatgaggc ttt 23
<210> 17<210> 17
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 17<400> 17
cgaagctaat accaagctcc a 21cgaagctaat accaagctcc a 21
<210> 18<210> 18
<211> 22<211> 22
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 18<400> 18
tcattttcaa tgagtgatga gg 22tcattttcaa tgagtgatga gg 22
<210> 19<210> 19
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 19<400> 19
tggccagtta atttcttggt t 21tggccagtta atttcttggt t 21
<210> 20<210> 20
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 20<400> 20
ctgaatctca aaaggctgga g 21ctgaatctca aaaggctgga g 21
<210> 21<210> 21
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 21<400> 21
tggctgcaat taccaaagaa 20 tggctgcaat taccaaagaa 20
<210> 22<210> 22
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 22<400> 22
gatgccactt tcatgtctgc 20 gatgccactt tcatgtctgc 20
<210> 23<210> 23
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 23<400> 23
tgggcaattt atgtgacgaa 20 tgggcaattt atgtgacgaa 20
<210> 24<210> 24
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 24<400> 24
tcacctcaac cttctttgga a 21tcacctcaac cttctttgga a 21
<210> 25<210> 25
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 25<400> 25
gcagacatga aagtggcatc 20 gcagacatga aagtggcatc 20
<210> 26<210> 26
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 26<400> 26
cgctaaacca gtagcgaagc 20 cgctaaacca gtagcgaagc 20
<210> 27<210> 27
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 27<400> 27
ttccaaagaa ggttgaggtg a 21ttccaaagaa ggttgaggtg a 21
<210> 28<210> 28
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 28<400> 28
cgccagttaa caacaaaagg 20 cgccagttaa caacaaaagg 20
<210> 29<210> 29
<211> 23<211> 23
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 29<400> 29
gctcctctaa cttttgtgat tgg 23gctcctctaa cttttgtgat tgg 23
<210> 30<210> 30
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 30<400> 30
agcccagtcg ttcttattgc 20 agcccagtcg ttcttattgc 20
<210> 31<210> 31
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 31<400> 31
tgtggcttct tcttgtgctc 20tgtggcttct tcttgtgctc 20
<210> 32<210> 32
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 32<400> 32
attggagatg cgcttcagtc 20attggagatg cgcttcagtc 20
<210> 33<210> 33
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 33<400> 33
cctgacgcaa taagaacgac t 21cctgacgcaa taagaacgac t 21
<210> 34<210> 34
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 34<400> 34
gctggaggtc atcaacaaca 20gctggaggtc atcaacaaca 20
<210> 35<210> 35
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 35<400> 35
gcgcatctcc aatctgaaac 20 gcgcatctcc aatctgaaac 20
<210> 36<210> 36
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Primer for PCR<223> Primer for PCR
<400> 36<400> 36
ggtgaagatg gacgttctgg 20 ggtgaagatg gacgttctgg 20
<210> 37<210> 37
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Probe for PCR<223> Probe for PCR
<400> 37<400> 37
cggcctcgat aatgcccaac a 21cggcctcgat aatgcccaac a 21
<210> 38<210> 38
<211> 23<211> 23
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Probe for PCR<223> Probe for PCR
<400> 38<400> 38
cttcaacggc ctcgataatg ccc 23cttcaacggc ctcgataatg ccc 23

Claims (7)

  1. 오리엔티아 쯔쯔가무시( Orientia tsutsugamushi)균의 다복제 유전자(multicopy gene)를 이용하는 것을 특징으로 하는 쯔쯔가무시병 진단법.A method for diagnosing Tsutsugamushi disease characterized by using a multicopy gene of Orientia tsutsugamushi .
  2. 제 1항에 있어서,The method according to claim 1,
    상기 다복제유전자(multicopy gene)의 염기서열을 증폭시킬 수 있는 프라이머쌍을 이용하여 검출하는 것을 특징으로 하는 쯔쯔가무시병 진단법.A method for diagnosing Tsutsugamushi disease, which comprises detecting a primer pair capable of amplifying the nucleotide sequence of the multicopy gene.
  3. 서열번호 1 내지 서열번호 36의 염기서열로 이루어진 군 중에서 선택된 1종 이상의 염기서열을 포함하는 것을 특징으로 하는 쯔쯔가무시병 진단용 프라이머.And a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 to 36. The primer for the diagnosis of Tsutsugamushi disease.
  4. 서열번호 1 내지 서열번호 36의 염기서열로 이루어진 군 중에서 선택된 2종 이상의 염기서열을 포함하는 것을 특징으로 하는 쯔쯔가무시병 진단용 프라이머쌍.And a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 to 36. 2. A pair of primers for diagnosis of Tsutsugamushi disease.
  5. 제 4항에 있어서,5. The method of claim 4,
    상기 프라이머쌍은 서열번호 1 및 서열번호 2의 염기서열을 포함하는 것을 특징으로 하는 쯔쯔가무시병 진단용 프라이머쌍.Wherein the pair of primers comprises the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2. 2. A pair of primer pairs for the diagnosis of Tsutsugamushi disease.
  6. (a) 시료로부터 DNA를 분리하는 단계;(a) separating DNA from a sample;
    (b) 상기 분리된 DNA를 주형으로 서열번호 1 내지 서열번호 36의 프라이머쌍을 이용하여 PCR을 수행하는 단계; 및(b) performing PCR using the separated DNA as a template using the primer pairs of SEQ ID NOS: 1 to 36; And
    (c) 상기 PCR 산물을 분리하는 단계를 포함하는 것을 특징으로 하는 쯔쯔가무시병 진단법.(c) isolating the PCR product.
  7. 서열번호 1 내지 서열번호 36의 염기서열로 이루어진 군으로 선택된 프라이머쌍을 포함하는 것을 특징으로 하는 쯔쯔가무시병 진단용 PCR 키트.A primer pair selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 1 to 36;
PCT/KR2018/012785 2018-01-08 2018-10-26 Method of diagnosing tsutsugamushi disease by using multicopy gene WO2019135477A2 (en)

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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
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