WO2018026141A1 - Diagnostic method for detection and identification of tuberculosis and non-tuberculosis mycobacteria and simultaneous confirmation of rifampin resistance of tuberculosis bacillus on basis of quanta matrix assay platform and kit therefor - Google Patents

Diagnostic method for detection and identification of tuberculosis and non-tuberculosis mycobacteria and simultaneous confirmation of rifampin resistance of tuberculosis bacillus on basis of quanta matrix assay platform and kit therefor Download PDF

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WO2018026141A1
WO2018026141A1 PCT/KR2017/008168 KR2017008168W WO2018026141A1 WO 2018026141 A1 WO2018026141 A1 WO 2018026141A1 KR 2017008168 W KR2017008168 W KR 2017008168W WO 2018026141 A1 WO2018026141 A1 WO 2018026141A1
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mycobacterium
tuberculosis
ttt ttt
seq
bacteria
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Korean (ko)
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이혜영
왕혜영
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주식회사 옵티팜
연세대학교 원주산학협력단
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Priority to KR1020197003372A priority Critical patent/KR102184637B1/en
Priority to CN201780061679.7A priority patent/CN110168109B/en
Priority to RU2019105616A priority patent/RU2715591C1/en
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    • 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
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
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Definitions

  • the present invention relates to a diagnostic method and kit for detecting and identifying tuberculosis and non-tuberculosis mycobacterium tuberculosis based on a quanta matrix assay platform and whether rifampin resistance of tuberculosis bacteria can be simultaneously confirmed.
  • Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB), and the prevalence of tuberculosis in Korea continues to decrease, but is higher than in foreign countries.
  • MTB Mycobacterium tuberculosis
  • 8 million people suffer from tuberculosis every year and 2 million people die from tuberculosis. Therefore, the rapid determination of resistance of intractable tuberculosis bacteria, which is difficult to treat, is essential for effective treatment and survival of patients.
  • tuberculosis bacteria As well as nontuberculous mycobacteria (NTM) in foreign countries, where non-TB bacteria are isolated from 30% to 50% of antiseptic smear-positive sputum.
  • NTM nontuberculous mycobacteria
  • Mycobacterial bacteriological identification has become a very important issue as it is reported that the incidence of tuberculosis patients due to non-tuberculosis acid bacillus is increased [Wright PW, Wallace RJ Jr, Wright NW, Brown BA, Griffith DE. J Clin Microbiol 1998; 36: 1046-9; Marras TK and Daley CL. Clin Chest Med 2002; 23: 553-67; Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official statement of the American Thoracic Society. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 2000; 55: 210-8].
  • Tuberculosis has been commonly used for the diagnosis of anti-bacterial smear and culture.
  • the antimicrobial smear test has the advantages of rapid results and simple techniques, but it has the disadvantage that sensitivity and specificity are inferior, and that it is impossible to distinguish between tuberculosis bacteria and non-tuberculosis antibacterial bacteria.
  • it is the most accurate method, it takes a long time due to the slow growth of Mycobacterium tuberculosis, and because it is cultured for a long time, it is often contaminated by other bacteria [Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006; 26: 174-8.
  • tuberculosis diagnostic methods using the same have been developed [Chakravorty S and Tyagi JS. Novel multipurpose methodology for detection of Mycobacteria in memelmonary and extrapulmonary specimens by smear microscopy, culture, and PCR. J Clin Microbiol 2005; 43: 2697-702; Kim YJ, Park MY, Kim SY, Cho SA, Hwang SH, Kim HH, et al. Korean J Lab Med 2008; 28: 34-8.
  • the most reliable diagnosis of tuberculosis is to prove infection by Mycobacterium tuberculosis, and therefore, the most reliable method is to detect MTB directly from clinical specimens.
  • Oligonucleotide arrays [Park H, Jang H, Song E, Chang CL, Lee M, Jeong S, et al. J Clin Microbial 2005; 43: 1782-8] has been increasingly used to detect tuberculosis bacteria in direct samples isolated from patients using rapid, highly sensitive and highly specific molecular biological methods.
  • PCR-RFLP PCR-restriction fragment length polymorphism
  • PRA PCR-restriction fragment length polymorphism
  • the PRA method is a method of amplifying a gene region capable of identifying tuberculosis bacteria and non-tuberculosis antibacterial bacteria by PCR, and processing the appropriate restriction enzymes to identify the fragments and identify the bacteria [Lee HY, Park HJ, Cho SN, Bai GH, Kim SJ. J Clin Microbiol. 2000; 38: 2966-71; Bannalikar AS, Verma R. Indian J Med Res. 2006; 123: 165-72; Aravindhan V, Sulochana S, Narayanan S, Paramasivam CN, Narayanan PR. Indian J Med Res. 2007; 126: 575-9.
  • the PRA method has a disadvantage in that identification of fragments treated with restriction enzymes is impossible when two or more bacteria, such as tuberculosis bacteria and non-tuberculosis antibiotics, are mixed.
  • QMAP QuantaMatrix assay Platform
  • the present invention has been made in view of the above necessity, and an object of the present invention is to provide a diagnostic method capable of detecting and identifying novel tuberculosis and non-tuberculosis antibacterial bacteria and whether rifampin resistance of tuberculosis bacteria is confirmed.
  • Another object of the present invention is to provide a diagnostic kit for detecting and identifying novel tuberculosis and non-tuberculosis antibacterial bacteria and confirming the resistance of tuberculosis to rifampin.
  • the present invention provides a method for amplifying DNA from a sample, comprising: a) separating DNA from a sample; b) PCR amplifying from the DNA using primers of SEQ ID NO: 1 to SEQ ID NO: 12; and c) SEQ ID NO: 13 And hybridizing the disks to which the oligomeric probes of Figure 68 are coupled with the PCR amplification products obtained in step b), and then measuring the images of the disks through the Quanta Matrix Assay Platform software. It provides a method for detecting and identifying bacteria and simultaneously checking whether rifampin, eye or resistance of Mycobacterium tuberculosis.
  • the detection and identification strain Mycobacterium tuberculosis H37Rv, Mycobacterium Avium ( M. avium ), Mycobacterium Intracellular ( M. intracellulare ), Foulard when help to Mycobacterium disk (M. scrofulaceum), Mycobacterium pressure wash's (M. abscessus), Mycobacterium kelronayi (M. chelonae), Mycobacterium M. fortuitum , Mycobacterium Justice ( M. ulcerans ), Mycobacterium marinum ( M. marinum ), Mycobacterium Kansasii ( M.
  • kansasii Mycobacterium jenabens ( M. genavense ), mycobacterium simie ( M. simmiae ), mycobacterium terra ( M. terrae ), Mycobacterium nonchromogenicum ( M. nonchromogenicum ), Mycobacterium keratum ( M. celatum ), Mycobacterium gordonae ( M. gordonae ), Mycobacterium zugale ( M. szulgail ), Mycobacterium mucogenicum ( M. mucogenicum ), Mycobacterium Obacens ( M. aubagnense ), Mycobacterium Malmo Enschede (M. malmoense), Pele Mycobacterium (M.
  • M. flavescens is preferably selected from the group consisting of, but not limited to.
  • the primer is preferably labeled with biotin, but is not limited thereto.
  • the present invention is to detect and identify tuberculosis bacteria and non-tuberculosis anti-bacterial bacterium comprising a disk coupled to the primers of SEQ ID NO: 1 to SEQ ID NO: 12 and oligomer probes of SEQ ID NO: 13 to 68, and whether or not rifampin, eye or resistance Provide a kit to verify.
  • the kit is a reagent for performing a PCR amplification reaction, and preferably further includes DNA polymerase, dNTPs, and a buffer, but is not limited thereto.
  • Myco-ID is a species-specific region rpo B gene (Patent Registration No. 10-1377070 (12.03.2014) a biotin group specific for the amplification primers and the resulting PCR product was attached to the probe species polymorphism is present attached 22 non-tuberculosis mycobacteria ( M. avium , M. intracellulare , M. scrofulaceum , M. abscessus ) of 22 3 groups (26 species) including Mycobacterium tuberculosis complex , M. chelonae , M. fortuitum complex, M. ulcerans / M. marinum , M. kansasii , M.
  • the QMAP-based Myco-ID of the present invention can distinguish between tuberculosis bacteria and non-tuberculosis mycobacterium bacteria, and can identify non-tuberculosis acidophilic bacteria and rifampin, tuberculosis bacteria, and resistance to children or fast.
  • the QMAP-based Myco-ID can be very useful for the diagnosis of Mycobacterium tuberculosis and non-tuberculosis mycobacterial infection.
  • Figure 2 is a picture of an example of a REBA Myco-ID, Lane 1-2, 9, 11, 13 M. intracellulare ; lane 3, M. mucogenicum ; lanes 4-8, 16-19, M. tuberculosis ; lane 10, M. massiliense ; lane 12, M. avium ; lane 14, M. fortuitum ; lane 15, M. abscessus ; lane 20, negative control.
  • the specimens of the present invention were solid cultured bacteria (234 samples) and liquid culture fluids (297 samples), which were simultaneously requested for antimicrobial smear and culture tests from suspected tuberculosis bacteria at Seoul Asan Hospital and St. Vincent Hospital from May to September 2009. It was targeted.
  • PCR was performed using nucleic acid-labeled primers for nucleic acid isolated from the specimen. After 5 minutes of reaction at 94 °C for pre-denaturation, 45 times at 94 °C for 30 seconds and 30 seconds at 65 °C for annealing and elongation as a denaturation process, and amplification of 270 bp PCR product at 72 °C for 7 minutes. It was.
  • REBA Myco-ID using the amplified PCR product was carried out using the experimental conditions suggested by the manufacturer.
  • REBA Myco-ID prepared by mixing the same amount of Denaturation solution (0.2N NaOH, 0.2mM EDTA) in the PCR product and allowed to stand at room temperature for 5 minutes, diluted with 2X SSPE / 0.1% SDS. Alkaline phosphatase-labeled diluted in a strip (M & D, Korea) and then reacted for 30 minutes at 50 ° C, washed twice at 62 ° C for 10 minutes using a washing solution (WS), and then diluted 1: 2000 (v / v). The streptavidin conjugate (Roche, Mannheim, Germany) was treated and reacted at room temperature for 30 minutes.
  • the reaction strip was washed twice with TBS solution (pH7.5) for 1 minute at room temperature, and NBT / BCIP (Nitro blue tetrazolium chloride and 5-Bromo-4-chloro-3), a substrate solution for coloring reaction, reacted with alkaline phosphatase. -indolylphosphate, toluidine salt, Roche, Germany) was diluted 1:50 with TBS solution of pH 9.5 and reacted to the strip for 5 minutes at room temperature to detect the PCR product bound to the probe.
  • PCR was performed using nucleic acid-labeled primers from nucleic acids isolated from samples. After 5 minutes of reaction at 94 °C for pre-denaturation, 45 times at 94 °C for 30 seconds and 30 seconds at 65 °C for annealing and elongation as a denaturation process, and amplification of 250 bp PCR product at 72 °C for 7 minutes. It was.
  • the microdisk was washed three times for 1 minute at room temperature with washing buffer, and automatically measured image of disk through the provided QMAP software to detect MTB or NTM, and to identify NTM, Rifampin of tuberculosis bacillus, eye or resistance Can be.
  • each standard strain Mycobacterium tuberculosis H37Rv, M. avium , M. intracellulare , M. scrofulaceum, M. abscessus , M. chelonae , M. fortuitum , M. marinum , M kansasii, M. genavense , M. terrae, M. nonchromogenicum , M. celatum , M. gordonae , M. szulgail , M. mucogenicum , M. aubagnense , M. malmoense , M. phlei , M.
  • nonchromogenicum 10ng-100fg 2310-861.8 1.5 ( ⁇ 0.5) x1000 34.3 13 M. celatum 10ng-100fg 3852-625 2.27 ( ⁇ 1.23) x1000 54.3 14 M. gordonae 10ng-1pg 2157-706 1.56 ( ⁇ 0.59) x1000 37.6 15 M. szulgai 10ng-1pg 3753.5-763.5 2.31 ( ⁇ 1.22) x1000 52.8 16 M. mucogenicum 10ng-1pg 4166.3-898 2.8 ( ⁇ 1.4) x1000 51.5 17 M. aubagnense 10ng-100fg 3683-964.5 2.61 ( ⁇ 0.98) x1000 37.3 18 M.
  • Table 1 shows the analytical sensitivity of the QMAP system.
  • Table 2 shows the specificity test of the QMAP system using 108 standard strains.
  • MTB and NTM detection and NTM identification were tested using 531 strains (234 solid culture bacteria and 297 liquid culture bacteria).
  • 234 solid cultures were identified with 223 MTB and 11 NTM, and the detected 223 MTB measurement ranged from 2074 to 4765 (mean 4255.6 ⁇ SD 458.7) and 11 NTM measurements from 609 to 3569 (1892). ⁇ 923.2) respectively.
  • 212 MTB and 77 NTM in 297 liquid cultures showed positive signals in QMAP test, respectively.
  • 531 strains 527 (99.2%) except 4 strains were correctly identified as MTB and NTM.
  • Table 3 shows the conventional methods for the detection of MTB and NTM in a total of 531 culture DNAs, a comparative table of QMAP system and REBA Myco-ID, abbreviation: AFB, acid-fast bacilli; MTB, M. tuberculosis ; NTM, nontuberculous mycobacteria; ND, not detected.
  • NTM a Two strains of NTM were not detected in the QMAP system and REBA Myco-ID and identified as Rhodococcus erythropolis and R. jostii by rpoB sequencing.
  • M. intracellulare 36 samples (39.1%) of 94 specimens, the M. avium Twenty-one specimens (22.8%), M. abscessus complex , 14 specimens (15.2%), and M. fortuitum were seven specimens (7.6%).
  • Other M. gordonae is or has a four specimens (4.3%), respectively, M. kansasii 2 samples (2.2%), M. chelonae that has the first sample (1.1%) or, M. avium and M. intracellulare, M. avium and M.
  • MTB / NTM differentiation Identification of mycobacterial species Culture methods REBA Myco-ID with culture samples n (%) QMAP system with culture samples n (%) Ranged fluorensce intensity Mean ( ⁇ SD) value
  • NTM (86) M. intracellulare 36 (39.1) 36 (39.1) 513-2610 932.7 ( ⁇ 381.7) M. avium 21 (22.8) 21 (22.8) 517-3569 2065.7 ( ⁇ 885.9)
  • M. abscessus complex 14 (15.2) 14 (15.2) 1338-3543 2534.5 ( ⁇ 654.9)
  • mucogenicum 1 (1.1) 1 (1.1) 625-1625 1125 ( ⁇ 707.1) M. gordonae and M. mucogenicum 1 (1.1) 1 (1.1) 509-2463 1486 ( ⁇ 1381.7) Total 92 (100) 92 (100)
  • Table 4 compares fractions of NTM separated by REBA Myco-ID and QMAP system
  • Table 5 compares the conventional method for detecting rifampin resistance in total 226 culture DNA, the QMAP system and REBA Myco-ID.
  • Table 6 shows a comparison of conventional methods, QMAP system, and DNA sequencing to detect eye or resistance in cultured DNA.
  • the sensitivity to QMAP-based rifampin and eyena resistance was 96.4% (106/110) and 75% (108/144), respectively.
  • the QMAP-based sensitivity was 100% (124/124; 95% CI: 0.9743-1.0000) and 100% (110/110, 95% CI: 0.9711-1.0000) in both rifampin and inna. ) (Table 7).
  • DST drug susceptibility testing
  • PPV positive predictive value
  • NPV negative predictive value
  • CI confidence interval
  • Table 7 compares the QMAP system with the DST and DNA sequencing results
  • kansasii kanII-L1-2 TTT TTT TTT TTT TTT TTT GGCCTCAACACCAAGGACCCGATCACCACG SEQ ID NO: 30
  • M. kansasii Igen / sim-1 TTT TTT TTT TTT TTT TTT TTT ATCTAAATGAACGCGTCGCCGGCAAC GGTTA SEQ ID NO: 31
  • M. genavense / M. simmiae TE2-3 TTT TTT TTT TTT TTT TTT CCGGCCGCACCCGCCGACGTCGAGACGT (SEQ ID NO: 32) M.
  • Table 8 shows the primer and probe sequences used in the present invention.
  • the QMAP-based diagnostic method of the present invention includes seven more species than REBA Myco-ID, so that more species can be separated. That is, in the case of the QMAP system of the present invention, 26 major non-tuberculosis antibiotics including M. tuberculosis ( M. avium , M. intracellulare , M. scrofulaceum , M. abscessus complex , M. chelonae , M. fortuitum complex, M. ulcerans / M. marinum , M. kansasii , M. genavense / M.
  • M. tuberculosis M. avium , M. intracellulare , M. scrofulaceum , M. abscessus complex , M. chelonae , M. fortuitum complex, M. ulcerans / M. marinum , M. kansasii , M. genavense / M.
  • M. simiae M. terrae, M. nonchromogenicum , M. celatum , M. gordonae , M. szulgai , M. mucogenicum , M. aubagnense ,
  • M. malmoense M. phlei , M. smegmatis, M. xenopi , M . peregrinum, M. septicum, M. flavescens may be added to the separation.
  • REBA Myco-ID is distinguished from tuberculosis and NTM in case of REBA Myco-ID.
  • QMAP-based diagnostic method can quickly determine the resistance of the most important rifampin, which is used as the primary drug for tuberculosis in tuberculosis. It can help you determine whether or not you can choose a treatment.
  • the QMAP-based molecular method is able to confirm where resistance was shown in case of rifampin, child or sensitization-resistant, and resistance to tuberculosis.
  • QMAP-based molecular method the test method for the purpose of this application was confirmed that the results compared to the conventional resistance method also shows excellent results.
  • REBA Myco-ID only tuberculosis / NTM can be identified, so in order to confirm the rifampin resistance in the case of tuberculosis, additional tests must be conducted using other molecular diagnostic methods such as REBA MTB-MDR. No test is required.
  • QMAP based diagnostics of the present invention is the reduction of detection time. Normally it takes more than 5 hours for REBA Myco-ID (REBA progression time) to proceed with 96 tests, but for QMAP, the detection time of 1 hour 40 minutes can be shortened, which reduces the most important time in diagnosis. It can be effective.

Abstract

The present invention relates to a diagnostic method for the detection and identification of tuberculosis and non-tuberculosis mycobacteria and the simultaneous confirmation of rifampin and isoniazid resistances of tuberculosis bacillus on the basis of a quanta matrix assay platform and a kit therefor.

Description

퀀타매트릭스 어세이 플랫폼 기반 결핵 및 비결핵 항산균의 검출 및 동정과 결핵균의 리팜핀 내성여부를 동시 확인할 수 있는 진단법 및 그 키트Diagnostics and kits for detecting and identifying tuberculosis and non-tuberculosis mycobacterium tuberculosis based on quanta matrix assay
본 발명은 퀀타매트릭스 어세이 플랫폼 기반 결핵 및 비결핵 항산균의 검출 및 동정과 결핵균의 리팜핀 내성여부를 동시 확인할 수 있는 진단법 및 그 키트에 관한 것이다.The present invention relates to a diagnostic method and kit for detecting and identifying tuberculosis and non-tuberculosis mycobacterium tuberculosis based on a quanta matrix assay platform and whether rifampin resistance of tuberculosis bacteria can be simultaneously confirmed.
결핵은 결핵균(Mycobacterium tuberculosis, MTB)이 원인이 되어 발생하는 만성 감염성 질환이고 우리나라에서 결핵의 유병률은 지속적으로 감소하는 추세이나 외국에 비해 높은 편이다. 또한, 효과적인 항결핵제가 사용되어 왔음에도 불구하고 다제내성 결핵 및 광범위내성 결핵 등 난치성 결핵이 증가함에 따라 아직도 세계적으로 결핵환자가 매년 800만 명이 발병하고 결핵으로 인해서 200만 명이 사망하고 있다. 따라서 치료가 어려운 난치성 결핵균의 내성 여부를 신속하게 판별하는 것이 환자의 효과적인 치료와 생존에 필수적이다. 결핵의 원인균으로는 결핵균뿐만 아니라 외국의 경우 비결핵 항산균(nontuberculous mycobacteria, NTM)의 빈도가 높은 지역에서는 항산균 도말 양성 객담의 30%에서 50%까지 비결핵 항산균이 분리되고 있으며 선진국화될수록 비결핵 항산균에 의한 결핵환자의 발생이 증가하는 것으로 보고되면서 마이코박테리아 균동정이 매우 중요한 문제가 되고 있다[Wright PW, Wallace RJ Jr, Wright NW, Brown BA, Griffith DE. J Clin Microbiol 1998;36:1046-9;Marras TK and Daley CL. Clin Chest Med 2002;23:553-67;Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official statement of the American Thoracic Society. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 2000;55:210-8]. Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB), and the prevalence of tuberculosis in Korea continues to decrease, but is higher than in foreign countries. In addition, despite the use of effective anti-tuberculosis drugs, with the increasing incidence of tuberculosis, including multidrug-resistant tuberculosis and tuberculosis-resistant tuberculosis, 8 million people suffer from tuberculosis every year and 2 million people die from tuberculosis. Therefore, the rapid determination of resistance of intractable tuberculosis bacteria, which is difficult to treat, is essential for effective treatment and survival of patients. The most common causes of tuberculosis are tuberculosis bacteria, as well as nontuberculous mycobacteria (NTM) in foreign countries, where non-TB bacteria are isolated from 30% to 50% of antiseptic smear-positive sputum. Mycobacterial bacteriological identification has become a very important issue as it is reported that the incidence of tuberculosis patients due to non-tuberculosis acid bacillus is increased [Wright PW, Wallace RJ Jr, Wright NW, Brown BA, Griffith DE. J Clin Microbiol 1998; 36: 1046-9; Marras TK and Daley CL. Clin Chest Med 2002; 23: 553-67; Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official statement of the American Thoracic Society. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 2000; 55: 210-8].
국내의 경우 비결핵 항산균 질환의 빈도가 낮다고 알려져 있었기 때문에 항산균 도말양성일 경우 대부분 결핵으로 간주하고 항결핵제 치료를 시행하는 것이 일반적이었으나, 국내에서도 항산균 도말 양성 객담의 10.3-12.2%까지 비결핵 항산균의 검출이 보고되어 왔으나 최근에는 30% 수준까지 그 비율이 높아지고 있는 추세이다[Koh WJ, Kwon OJ, Yu CM, Jeon K, Suh GY, Chung MP, et al. Tuberc Respir Dis 2003;54:22-32]. 또한 비결핵 항산균은 면역 기능 저하자에서 질병을 일으킬 수 있고 진단이 쉽지 않으며 균종에 따라 자연적으로 가지는 약재내성이 다르므로 비결핵 항산균에 의한 결핵의 경우 그 치료법이 결핵균에 의한 결핵의 치료법과 다를 수 있다[Koh WJ, Kwon OJ, Lee KS. J Korean Med Sci 2005;20:913-25;Wagner D and Young LS. Nontuberculous mycobacterial infections: a clinical review. Infection 2004;32:257-70]. In Korea, it was known that the incidence of non-TB disease was low, so it was generally considered that tuberculosis was considered to be tuberculosis, and anti-TB treatment was generally performed. Bacterial detection has been reported, but the rate has recently increased to 30% [Koh WJ, Kwon OJ, Yu CM, Jeon K, Suh GY, Chung MP, et al. Tuberc Respir Dis 2003; 54: 22-32. In addition, since non-TB tuberculosis can cause disease in people with reduced immune function, it is not easy to diagnose, and the drug resistance naturally differs depending on the type of bacteria. May differ [Koh WJ, Kwon OJ, Lee KS. J Korean Med Sci 2005; 20: 913-25; Wagner D and Young LS. Nontuberculous mycobacterial infections: a clinical review. Infection 2004; 32: 257-70.
그러므로 결핵균과 비결핵 항산균을 신속하게 구별할 수 있는 적절한 검사방법이 요구되고 있다.Therefore, there is a need for an appropriate test method that can quickly distinguish between tuberculosis bacteria and non-TB bacteria.
결핵의 진단에는 보편적으로 항산균 도말검사와 배양검사를 이용해왔다. 항산균 도말검사는 신속하게 결과를 얻을 수 있고 기법이 간편하다는 장점이 있으나 민감도와 특이도가 떨어지며 결핵균과 비결핵 항산균을 구분할 수 없다는 단점이 있으며 배양검사는 결핵진단의 표준법으로 결핵을 진단하는 가장 정확한 방법이지만 천천히 자라는 결핵균의 특성상 시간이 오래 걸린다는 단점이 있으며, 장기간 배양을 하기 때문에 다른 균에 의해 오염이 되는 경우가 많다[Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006;26:174-8]. 최근 결핵균 배양시간을 단축하기 위해서 액체배지를 이용한 자동화 검사법이 등장하였으나 이 방법 역시 결핵균과 비결핵 항산균을 구별할 수 없기 때문에 별도의 동정과정이 필요하다는 단점이 있다[Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006;26:174-8]. 또한 약제 내성 여부를 판별하기 위해 Golden standard로 사용되고 있는 미생물 기반의 진단법은 환자로부터 채취한 객담에서 결핵균을 배양한 후, 다시 약제가 포함된 배지에 배양하여 내성여부를 판단하기에 최소 4 ~ 8주가 경과해야 약제 내성 여부를 판별할 수 있다. Tuberculosis has been commonly used for the diagnosis of anti-bacterial smear and culture. The antimicrobial smear test has the advantages of rapid results and simple techniques, but it has the disadvantage that sensitivity and specificity are inferior, and that it is impossible to distinguish between tuberculosis bacteria and non-tuberculosis antibacterial bacteria. Although it is the most accurate method, it takes a long time due to the slow growth of Mycobacterium tuberculosis, and because it is cultured for a long time, it is often contaminated by other bacteria [Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006; 26: 174-8. Recently, automated test method using liquid medium has been introduced to shorten tuberculosis incubation time, but this method also has a disadvantage of requiring a separate identification process because it cannot distinguish between tuberculosis and non-tuberculosis antibacterial bacteria [Yang HY, Lee HJ, Park. WY, Lee KK, Suh JT. Korean J Lab Med 2006; 26: 174-8. In addition, the microbial based diagnostic method, which is used as a golden standard to determine drug resistance, is cultured in a sputum collected from a patient, and then incubated in a medium containing drugs for at least 4 to 8 weeks. It must be passed to determine drug resistance.
내성결핵균에 의한 치료지연은 내성결핵균의 지역 감염확산으로 이어지므로, 초기 결핵진단시 결핵균의 내성 여부를 판단하는 것은 매우 중요하고 이를 위해서는 신속하게 내성여부를 확인할 수 있는 분자진단검사법이 요구되고 있다.The delay in treatment due to M. tuberculosis leads to the spread of M. tuberculosis. Therefore, it is very important to determine whether M. tuberculosis is resistant during the initial diagnosis of M. tuberculosis.
최근에는 분자진단 기술이 발달되면서 이를 이용한 결핵 진단법들이 많이 개발되었다[Chakravorty S and Tyagi JS. Novel multipurpose methodology for detection of Mycobacteria in puilmonary and extrapulmonary specimens by smear microscopy, culture, and PCR. J Clin Microbiol 2005;43:2697-702;Kim YJ, Park MY, Kim SY, Cho SA, Hwang SH, Kim HH, et al. Korean J Lab Med 2008;28:34-8]. 결핵의 확실한 진단방법은 결핵균에 의한 감염을 증명하는 것이므로 임상 검체에서 직접 MTB를 검출하는 방법이 가장 확실하다. Recently, with the development of molecular diagnostic technology, tuberculosis diagnostic methods using the same have been developed [Chakravorty S and Tyagi JS. Novel multipurpose methodology for detection of Mycobacteria in puilmonary and extrapulmonary specimens by smear microscopy, culture, and PCR. J Clin Microbiol 2005; 43: 2697-702; Kim YJ, Park MY, Kim SY, Cho SA, Hwang SH, Kim HH, et al. Korean J Lab Med 2008; 28: 34-8. The most reliable diagnosis of tuberculosis is to prove infection by Mycobacterium tuberculosis, and therefore, the most reliable method is to detect MTB directly from clinical specimens.
따라서 PCR[Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006;26:174-8;Chakravorty S and Tyagi JS. J Clin Microbiol 2005;43:2697-702], Thus PCR [Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006; 26: 174-8; Chakravorty S and Tyagi JS. J Clin Microbiol 2005; 43: 2697-702;
실시간 PCR[Kim YJ, Park MY, Kim SY, Cho SA, Hwang SH, Kim HH, et al. Korean J Lab Med 2008;28:34-8;Jung CL, Kim MY, Seo DC, Lee MA. Korean J Clin Microbiol 2008;1;29-33], Real-time PCR [Kim YJ, Park MY, Kim SY, Cho SA, Hwang SH, Kim HH, et al. Korean J Lab Med 2008; 28: 34-8; Jung CL, Kim MY, Seo DC, Lee MA. Korean J Clin Microbiol 2008; 1; 29-33;
교잡화[Makinen J, Marjamaki M, Marttila H, Soini H. Clin Microbiol Infect. 2006;12:481-3;Padilla E, Gonzalez V, Manterola JM, Perez A, Quesada MD, Gordillo S, et al. J Clin Microbial. 2004;42:3083-8;Sanguinetti M, B Posteraro, F Ardito, S Zanetti, A Cingolani, L Sechi, et al. J Clin Microbiol. 1998;36:1530-3;Tortuli, E, A Mariottini, and G Mazzarelli. J Clin Microbiol. 2003;41:4418-20], 및 Hybridization [Makinen J, Marjamaki M, Marttila H, Soini H. Clin Microbiol Infect. 2006; 12: 481-3; Padilla E, Gonzalez V, Manterola JM, Perez A, Quesada MD, Gordillo S, et al. J Clin Microbial. 2004; 42: 3083-8; Sanguinetti M, B Posteraro, F Ardito, S Zanetti, A Cingolani, L Sechi, et al. J Clin Microbiol. 1998; 36: 1530-3; Tortuli, E, A Mariottini, and G Mazzarelli. J Clin Microbiol. 2003; 41: 4418-20, and
올리고뉴크레오타이드 어레이[Park H, Jang H, Song E, Chang CL, Lee M, Jeong S, et al. J Clin Microbial 2005;43:1782-8] 등, 신속하면서도 민감도와 특이도가 높은 분자생물학적 방법을 이용하여 환자로부터 분리한 직접 검체에서 결핵균을 검출하는 방법의 이용이 점차 늘어나고 있는 추세이다. 또한 비결핵 항산균의 동정법으로 많이 사용되고 있는 방법 중의 하나가 PCR-restriction fragment length polymorphism (PCR-RFLP) analysis (PRA) 방법이다[18]. PRA방법은 결핵균과 비결핵 항산균의 동정이 가능한 유전자 부위를 PCR로 증폭한 후, 적절한 제한효소를 처리하여 그 절편의 양상을 보고 균을 동정하는 방법이다[Lee HY, Park HJ, Cho SN, Bai GH, Kim SJ. J Clin Microbiol. 2000;38:2966-71; Bannalikar AS, Verma R. Indian J Med Res. 2006;123:165-72;Aravindhan V, Sulochana S, Narayanan S, Paramasivam CN, Narayanan PR. Indian J Med Res. 2007;126:575-9]. Oligonucleotide arrays [Park H, Jang H, Song E, Chang CL, Lee M, Jeong S, et al. J Clin Microbial 2005; 43: 1782-8] has been increasingly used to detect tuberculosis bacteria in direct samples isolated from patients using rapid, highly sensitive and highly specific molecular biological methods. In addition, one of the methods widely used for the identification of non-TB antibacterial bacteria is PCR-restriction fragment length polymorphism (PCR-RFLP) analysis (PRA). The PRA method is a method of amplifying a gene region capable of identifying tuberculosis bacteria and non-tuberculosis antibacterial bacteria by PCR, and processing the appropriate restriction enzymes to identify the fragments and identify the bacteria [Lee HY, Park HJ, Cho SN, Bai GH, Kim SJ. J Clin Microbiol. 2000; 38: 2966-71; Bannalikar AS, Verma R. Indian J Med Res. 2006; 123: 165-72; Aravindhan V, Sulochana S, Narayanan S, Paramasivam CN, Narayanan PR. Indian J Med Res. 2007; 126: 575-9.
그러나 PRA방법은 결핵균과 비결핵 항산균 등 두 가지 이상의 균이 섞여 있는 경우에는 제한효소를 처리한 절편의 양상이 복합적으로 나타나기 때문에 동정이 불가능하다는 단점이 있다. 이러한 불편함을 보완하고자 최근에는 결핵균과 비결핵 항산균을 동시에 진단 및 동정할 수 있으며, 다양한 균이 섞여있는 경우에도 동정이 가능한 QuantaMatrix assay Platform (QMAP) 기반 분자진단검사법을 개발하였다. QMAP은 퀀타매트릭스사의 원천기술 (특허 등록번호 1011013100000 (2011.12.26) / 1015823840000 (2015.12.28) 로 suspension array technology를 기반으로 하고 있으며 50μm 크기의 자성을 띠고 있는 디스크 (Microdisk)에 프로브를 결합시키고 PCR산물과 반응시킨 후, 돌연변이를 여부를 형광으로 확인할 수 있는 검사법으로 QMAP의 가장 큰 특징은 디스크에 고유의 코드를 새겨 이 코드로 간섭현상 없이 디스크를 구별하며 기술적으로 1024개의 코드가 가능하므로 각각 고유의 코드가 새겨진 1024종의 디스크를 이용한 다중검사가 가능하며 모든 과정이 96 well plate에서 진행되기 때문에 high throughput이 가능한 시스템이다 (도 1). However, the PRA method has a disadvantage in that identification of fragments treated with restriction enzymes is impossible when two or more bacteria, such as tuberculosis bacteria and non-tuberculosis antibiotics, are mixed. In order to make up for this inconvenience, we recently developed a QuantaMatrix assay Platform (QMAP) -based molecular diagnostic test that can diagnose and identify tuberculosis bacteria and non-tuberculosis antibacterial bacteria at the same time. QMAP is Quanta Matrix's original technology (Patent Registration No. 1011013100000 (2011.12.26) / 1015823840000 (2015.12.28), based on suspension array technology, and combines the probe with a 50μm magnetic disk (Microdisk) After reacting with the product, it is possible to check whether the mutation is detected by fluorescence. The biggest feature of QMAP is to inscribe a unique code on the disc, which distinguishes the disc without interference and technically allows 1024 codes. It is possible to multi-check using 1024 kinds of disks engraved with the code, and since the whole process is performed on a 96 well plate, a high throughput system is possible (FIG. 1).
본 발명은 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 신규한 결핵 및 비결핵 항산균의 검출 및 동정과 결핵균의 리팜핀 내성여부를 확인할 수 있는 진단법을 제공하는 것이다.The present invention has been made in view of the above necessity, and an object of the present invention is to provide a diagnostic method capable of detecting and identifying novel tuberculosis and non-tuberculosis antibacterial bacteria and whether rifampin resistance of tuberculosis bacteria is confirmed.
본 발명의 다른 목적은 신규한 결핵 및 비결핵 항산균의 검출 및 동정과 결핵균의 리팜핀 내성여부를 확인할 수 있는 진단용 키트를 제공하는 것이다.Another object of the present invention is to provide a diagnostic kit for detecting and identifying novel tuberculosis and non-tuberculosis antibacterial bacteria and confirming the resistance of tuberculosis to rifampin.
상기의 목적을 달성하기 위하여 본 발명은 a)검체 시료로부터 DNA를 분리하는 단계;b)서열번호 1 내지 서열번호 12의 프라이머를 사용하여, 상기 DNA로부터 PCR 증폭하는 단계;및 c) 서열번호 13 내지 68의 올리고머 프로브가 커플링된 디스크와 상기 단계 b)에서 얻어진 PCR 증폭산물을 하이브리드형성시키는 후,퀀타매트릭스 어세이 플랫폼 소프트웨어를 통하여 상기 디스크의 이미지를 측정하는 단계를 포함하는 결핵균 및 비결핵 항산균 검출과 동정 및 결핵균의 리팜핀, 아이나 내성 여부를 동시에 확인하는 방법을 제공한다.In order to achieve the above object, the present invention provides a method for amplifying DNA from a sample, comprising: a) separating DNA from a sample; b) PCR amplifying from the DNA using primers of SEQ ID NO: 1 to SEQ ID NO: 12; and c) SEQ ID NO: 13 And hybridizing the disks to which the oligomeric probes of Figure 68 are coupled with the PCR amplification products obtained in step b), and then measuring the images of the disks through the Quanta Matrix Assay Platform software. It provides a method for detecting and identifying bacteria and simultaneously checking whether rifampin, eye or resistance of Mycobacterium tuberculosis.
본 발명의 일 구현예에 있어서, 상기 검출 및 확인 균주는 마이코박테리움 투버큘로시스(Mycobacterium tuberculosis) H37Rv, 마이코박테리움 아비움(M. avium), 마이코박테리움 인트라셀룰라(M. intracellulare), 마이코박테리움 스크로풀라시움(M. scrofulaceum ), 마이코박테리움 압세수스(M. abscessus), 마이코박테리움 켈로나이(M. chelonae), 마이코박테리움 포르투이툼(M. fortuitum), 마이코박테리움 얼서런스 (M. ulcerans), 마이코박테리움 마리눔(M. marinum ), 마이코박테리움 칸사시(M. kansasii), 마이코박테리움 제나벤스(M. genavense ), 마이코박테리움 시미에 (M. simmiae), 마이코박테리움 테라(M. terrae), 마이코박테리움 넌크로모제니쿰(M. nonchromogenicum), 마이코박테리움 켈라툼(M. celatum), 마이코박테리움 고르도네이(M. gordonae), 마이코박테리움 줄게일(M. szulgail ), 마이코박테리움 무코제니쿰(M. mucogenicum), 마이코박테리움 오박넨스(M. aubagnense), 마이코박테리움 말모엔스(M. malmoense), 마이코박테리움 펠레(M. phlei), 마이코박테리움 스메그마티스 (M. smegmatis ), 마이코박테리움 제노피(M. xenopi), 마이코박테리움 페레그리넘 (M. peregrinum) 및 마이코박테리움 프라제센스(M. flavescens)으로 구성된 군으로부터 선택된 것이 바람직하나 이에 한정되지 아니한다. In one embodiment of the invention, the detection and identification strain Mycobacterium tuberculosis (Hycobacterium tuberculosis ) H37Rv, Mycobacterium Avium ( M. avium ), Mycobacterium Intracellular ( M. intracellulare ), Foulard when help to Mycobacterium disk (M. scrofulaceum), Mycobacterium pressure wash's (M. abscessus), Mycobacterium kelronayi (M. chelonae), Mycobacterium M. fortuitum , Mycobacterium Justice ( M. ulcerans ), Mycobacterium marinum ( M. marinum ), Mycobacterium Kansasii ( M. kansasii ), Mycobacterium jenabens ( M. genavense ), mycobacterium simie ( M. simmiae ), mycobacterium terra ( M. terrae ), Mycobacterium nonchromogenicum ( M. nonchromogenicum ), Mycobacterium keratum ( M. celatum ), Mycobacterium gordonae ( M. gordonae ), Mycobacterium zugale ( M. szulgail ), Mycobacterium mucogenicum ( M. mucogenicum ), Mycobacterium Obacens ( M. aubagnense ), Mycobacterium Malmo Enschede (M. malmoense), Pele Mycobacterium (M. phlei), Mycobacterium Smeg Matisse (M. smegmatis), Mycobacterium Geno blood (M. xenopi), Mycobacterium Tampere draw over (M. peregrinum) and Mycobacterium prazecens ( M. flavescens ) is preferably selected from the group consisting of, but not limited to.
본 발명의 다른 구현예에 있어서, 상기 프라이머는 바이오틴이 표지된 것이 바람직하나 이에 한정되지 아니한다.In another embodiment of the present invention, the primer is preferably labeled with biotin, but is not limited thereto.
또 본 발명은 서열번호 1 내지 서열번호 12의 프라이머 및 서열번호 13 내지 68의 올리고머 프로브가 커플링된 디스크를 포함하는 결핵균 및 비결핵 항산균 검출과 동정 및, 결핵균의 리팜핀, 아이나 내성 여부를 동시에 확인하기 위한 키트를 제공한다.In addition, the present invention is to detect and identify tuberculosis bacteria and non-tuberculosis anti-bacterial bacterium comprising a disk coupled to the primers of SEQ ID NO: 1 to SEQ ID NO: 12 and oligomer probes of SEQ ID NO: 13 to 68, and whether or not rifampin, eye or resistance Provide a kit to verify.
상기 키트는 PCR 증폭 반응을 수행하기 위한 시약으로, DNA 폴리머라제, dNTPs 및 버퍼를 더욱 포함하는 것이 바람직하나 이에 한정되지 아니한다.The kit is a reagent for performing a PCR amplification reaction, and preferably further includes DNA polymerase, dNTPs, and a buffer, but is not limited thereto.
이하 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 발명에서는 국내에서 개발된 QMAP기반 dual-ID를 이용하여 고체 및 액체배양액을 가지고 유용성을 평가해 보았다.In the present invention, using the QMAP-based dual-ID developed in Korea, the usefulness of the solid and liquid culture solution was evaluated.
본 발명의 QMAP 기반 Myco-ID는 종특이 다형성이 존재하는 rpoB 유전자 부위 (특허 등록번호 10-1377070 (2014.03.12)를 biotin group이 부착된 프라이머로 증폭하고 얻어진 PCR산물을 종특이 프로브가 부착되어있는 microdisk과 반응시켜 결핵균을 포함한 22종 3그룹 (총26종)의 주요 비결핵 항산균(M. avium , M. intracellulare , M. scrofulaceum , M. abscessus complex, M. chelonae , M. fortuitum complex, M. ulcerans/M. marinum , M. kansasii , M. genavense /M. simiae , M. terrae, M. nonchromogenicum, M. celatum , M. gordonae , M. szulgai , M. mucogenicum , M. aubagnense, M. malmoense , M. phlei , M. smegmatis , M. xenopi , M. peregrinum /M. septicum, M. flavescens)을 검출 및 동정이 가능하며, 리팜핀 내성여부를 알 수 있는 분자진단 검사법으로 자동화가 가능하여 96테스트를 진행시 DNA 추출 (30분), PCR 진행 (1시간), TB/NTM 검출 및 동정과 리팜핀, 아이나 내성여부 (1시간 30분) 총 3시간이면 확인이 가능한 부분이 큰 장점이다.QMAP base of the present invention Myco-ID is a species-specific region rpo B gene (Patent Registration No. 10-1377070 (12.03.2014) a biotin group specific for the amplification primers and the resulting PCR product was attached to the probe species polymorphism is present attached 22 non-tuberculosis mycobacteria ( M. avium , M. intracellulare , M. scrofulaceum , M. abscessus ) of 22 3 groups (26 species) including Mycobacterium tuberculosis complex , M. chelonae , M. fortuitum complex, M. ulcerans / M. marinum , M. kansasii , M. genavense / M. simiae , M. terrae, M. nonchromogenicum, M. celatum , M. gordonae , M. szulgai , M. mucogenicum , M. aubagnense, M. malmoense , M. phlei , M. smegmatis , M. xenopi , M. peregrinum / M. septicum, M. flavescens ) can be detected and automated by molecular diagnostic test to know whether rifampin is resistant. DNA extraction (30 minutes), PCR (1 hour), TB / NTM detection and identification, rifampin, aina resistance (1 hour 30 minutes) a total of 3 hours can be confirmed the great advantage.
본 발명을 통하여 알 수 있는 바와 같이 본 발명의 QMAP기반 Myco-ID는 결핵균 및 비결핵 항산균의 구별이 가능하고, 비결핵 항산균의 동정 및 결핵균의 리팜핀, 아이나 내성여부까지 가능하기 때문에 빠른 시간 내에 원인균을 정확히 동정함으로써 비결핵 항산균에 작용할 수 있는 올바른 약제를 처방할 수 있도록 유용한 정보를 제공하며, 본 발명에서 보았듯이 액체배양의 사용이 점차 늘어남에 따라 결핵균과 빨리 자라는 비결핵 항산균과 섞여있는 경우 비결핵 항산균으로 오진되는 것을 방지할 수 있을 것으로 판단된다. 따라서 QMAP기반 Myco-ID는 결핵균 및 비결핵 항산균 감염증의 진단에 매우 유용하게 사용될 수 있다.As can be seen through the present invention, the QMAP-based Myco-ID of the present invention can distinguish between tuberculosis bacteria and non-tuberculosis mycobacterium bacteria, and can identify non-tuberculosis acidophilic bacteria and rifampin, tuberculosis bacteria, and resistance to children or fast. By accurately identifying the causative organism within the body, it provides useful information for prescribing the correct drug that can act on non-tuberculosis mycobacterium, and as shown in the present invention, as the use of liquid culture increases, If mixed, it can be prevented from being misdiagnosed as non-tuberculosis antibacterial bacteria. Therefore, QMAP-based Myco-ID can be very useful for the diagnosis of Mycobacterium tuberculosis and non-tuberculosis mycobacterial infection.
도 1은 QMAP 시스템에 대한 그림,1 is a diagram for a QMAP system,
도 2는 REBA Myco-ID의 예에 대한 그림, Lane 1-2, 9, 11, 13 M . intracellulare; lane 3, M. mucogenicum; lane 4-8, 16-19, M. tuberculosis; lane 10, M. massiliense; lane 12, M. avium; lane 14, M. fortuitum; lane 15, M. abscessus; lane 20, negative control.Figure 2 is a picture of an example of a REBA Myco-ID, Lane 1-2, 9, 11, 13 M. intracellulare ; lane 3, M. mucogenicum ; lanes 4-8, 16-19, M. tuberculosis ; lane 10, M. massiliense ; lane 12, M. avium ; lane 14, M. fortuitum ; lane 15, M. abscessus ; lane 20, negative control.
이하, 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재된 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the invention and the scope of the present invention is not to be construed as limited by the following examples.
본 발명의 검체는 2009년 5월부터 9월까지 서울아산병원과 성빈센트 병원에서 결핵균 의심환자로부터 항산균 도말검사와 배양검사가 동시에 의뢰된 고체배양균 (234 검체)와 액체배양액 (297 검체)을 대상으로 하였다. The specimens of the present invention were solid cultured bacteria (234 samples) and liquid culture fluids (297 samples), which were simultaneously requested for antimicrobial smear and culture tests from suspected tuberculosis bacteria at Seoul Asan Hospital and St. Vincent Hospital from May to September 2009. It was targeted.
또한, 결핵균 검출을 위한 항산균 도말 검사와 배양검사는 서울아산병원과 성빈센트 병원에서 수행되었다. 항산균 도말검사는 Carbol-fuchsin을 이용한 Ziehl-Neelsen 염색법을 이용하였고 그 결과는 미국 질병예방통제국의 기준에 따라 판독하였다. 배양검사는 NaOH 처리하여 수거한 균체를 3% Ogawa 배지 (아산제약, 한국)와 BACTEC MGIT 960 (Becton Dickinson Microbiology System, Sparks, Md, USA)에 접종함으로써 검체 내에 포함된 원인균을 배양하였다. In addition, the antibacterial smear and culture test for the detection of Mycobacterium tuberculosis were performed at Asan Medical Center and St. Vincent's Hospital. Antimicrobial smears were tested using Ziehl-Neelsen staining with Carbol-fuchsin and the results were read according to the US Centers for Disease Control and Prevention. Cultures were cultured causative bacteria contained in the specimen by inoculating NaOH-treated cells in 3% Ogawa medium (Asan Pharmaceutical, Korea) and BACTEC MGIT 960 (Becton Dickinson Microbiology System, Sparks, Md, USA).
실시예 1. 항산균의 분리 및 동정Example 1.Isolation and Identification of Acid Bacteria
직접 검체를 3% Ogawa 배지 와 BACTEC MGIT 960 system에 접종하고 37℃ 에서 배양한 후, 배양액의 일부를 취하여 100℃ 에서 20분간 가열하고 13,000rpm에서 5분간 원심분리한 후 상층액을 취하는 방법을 이용하였다. MTB-ID PCR kit를 이용한 분자진단방법으로 결핵균과 비결핵 항산균을 구분하였다. Directly inoculate the sample in 3% Ogawa medium and BACTEC MGIT 960 system, incubate at 37 ° C, take a portion of the culture solution, heat at 100 ° C for 20 minutes, centrifuge at 13,000 rpm for 5 minutes, and take supernatant. It was. Molecular diagnostic method using MTB-ID PCR kit was used to distinguish between tuberculosis and non-tuberculosis antibacterial bacteria.
실시예 2. REBA Myco-ID의 수행Example 2 Performance of REBA Myco-ID
REBA Myco-ID를 수행하기 위해서 검체로부터 분리된 핵산을 biotin이 표지된 primer를 이용하여 다음과 같이 PCR을 수행하였다. Pre-denaturation 과정으로 94 ℃ 에서 5분간 반응 후, denaturation 과정으로 94 ℃ 30초와 annealing과 elongation과정으로 65 ℃에서 30초로 45회 시행하고 최종 72 ℃에서 7분간 실시하여 270 bp의 PCR 산물을 증폭하였다.In order to perform REBA Myco-ID, PCR was performed using nucleic acid-labeled primers for nucleic acid isolated from the specimen. After 5 minutes of reaction at 94 ℃ for pre-denaturation, 45 times at 94 ℃ for 30 seconds and 30 seconds at 65 ℃ for annealing and elongation as a denaturation process, and amplification of 270 bp PCR product at 72 ℃ for 7 minutes. It was.
증폭한 PCR 산물을 이용한 REBA Myco-ID는 제조자가 제시한 실험조건을 이용하여 시행하였으면 실험방법은 다음과 같다. PCR 산물에 동량의 Denaturation solution (0.2N NaOH, 0.2mM EDTA)을 섞어 실온에 5분간 방치한 후 2X SSPE/0.1% SDS으로 희석시켜 준비된 REBA Myco-ID strip (M&D, Korea)에 넣은 후 50℃ 에서 30분간 반응시키고, WS (washing solution)을 이용하여 62℃에서 10분간 두 번 씻어준 후 1:2000 (v/v)으로 희석한 alkaline phosphatase-labeled streptavidin conjugate (Roche, Mannheim, Germany)을 처리하여 실온에서 30분간 반응시켰다. 반응이 끝난 strip을 TBS용액 (pH7.5)으로 실온에 1분간 2회 세척하고, alkaline phosphatase에 반응하는 발색용 기질 용액인 NBT/BCIP (Nitro blue tetrazolium chloride and 5-Bromo-4-chloro-3-indolylphosphate, toluidine salt, Roche, Germany)를 pH 9.5인 TBS 용액으로 1:50으로 희석하여 실온에서 strip에 5분간 반응시키면서 프로브에 결합한 PCR 산물을 검출하였다.REBA Myco-ID using the amplified PCR product was carried out using the experimental conditions suggested by the manufacturer. REBA Myco-ID prepared by mixing the same amount of Denaturation solution (0.2N NaOH, 0.2mM EDTA) in the PCR product and allowed to stand at room temperature for 5 minutes, diluted with 2X SSPE / 0.1% SDS. Alkaline phosphatase-labeled diluted in a strip (M & D, Korea) and then reacted for 30 minutes at 50 ° C, washed twice at 62 ° C for 10 minutes using a washing solution (WS), and then diluted 1: 2000 (v / v). The streptavidin conjugate (Roche, Mannheim, Germany) was treated and reacted at room temperature for 30 minutes. The reaction strip was washed twice with TBS solution (pH7.5) for 1 minute at room temperature, and NBT / BCIP (Nitro blue tetrazolium chloride and 5-Bromo-4-chloro-3), a substrate solution for coloring reaction, reacted with alkaline phosphatase. -indolylphosphate, toluidine salt, Roche, Germany) was diluted 1:50 with TBS solution of pH 9.5 and reacted to the strip for 5 minutes at room temperature to detect the PCR product bound to the probe.
실시에 3. QMAP 기반 Dual-ID 수행3. Perform QMAP based Dual-ID
QMAP 기반 Myco-ID 를 수행하기 위해서 검체로부터 분리된 핵산을 biotin이 표지된 primer를 이용하여 다음과 같이 PCR을 수행하였다. Pre-denaturation 과정으로 94 ℃ 에서 5분간 반응 후, denaturation 과정으로 94 ℃ 30초와 annealing과 elongation과정으로 65 ℃에서 30초로 45회 시행하고 최종 72 ℃에서 7분간 실시하여 250 bp의 PCR 산물을 증폭하였다.In order to perform QMAP-based Myco-ID, PCR was performed using nucleic acid-labeled primers from nucleic acids isolated from samples. After 5 minutes of reaction at 94 ℃ for pre-denaturation, 45 times at 94 ℃ for 30 seconds and 30 seconds at 65 ℃ for annealing and elongation as a denaturation process, and amplification of 250 bp PCR product at 72 ℃ for 7 minutes. It was.
증폭한 PCR 산물에 동량의 Denaturation solution (0.2N NaOH, 0.2mM EDTA)을 섞어 실온에 5분간 방치한 후 hybridization buffer에 희석시켜 준비된 커플링된 disk (Quantamatrix, Seoul, Korea)에 넣은 후 40℃ 에서 30분간 반응시키고, WS (washing solution)을 이용하여 25℃에서 1분간 3회 씻어준 후 1:2000 (v/v)으로 희석한 streptavidin R-phycoerythrin conjugate (Prozyme, San Leandro, CA)을 처리하여 실온에서 10분간 반응시켰다. 반응이 끝난 microdisk를 washing buffer로 실온에 1분간 3회 세척하고, 제공되는 QMAP software를 통해 자동으로 disk의 이미지를 측정하여 MTB 또는 NTM 을 검출하고 NTM의 동정, 결핵균의 리팜핀, 아이나 내성여부를 확인할 수 있다.Mix the same amount of Denaturation solution (0.2N NaOH, 0.2mM EDTA) with the amplified PCR product, and leave it at room temperature for 5 minutes, dilute it in hybridization buffer and place it in a coupled disk (Quantamatrix, Seoul, Korea) prepared at 40 ℃. After reacting for 30 minutes, washing with WS (washing solution) three times at 25 ° C. for 1 minute and treating with streptavidin R-phycoerythrin conjugate (Prozyme, San Leandro, CA) diluted 1: 2000 (v / v) The reaction was carried out at room temperature for 10 minutes. After washing the reaction, the microdisk was washed three times for 1 minute at room temperature with washing buffer, and automatically measured image of disk through the provided QMAP software to detect MTB or NTM, and to identify NTM, Rifampin of tuberculosis bacillus, eye or resistance Can be.
본 발명에서 사용된 프라이머 및 프로브(한국 바이오니아에 의뢰하여 합성) 서열은 표 8에 기재하였다.Primers and probes used in the present invention (synthesized by Bioneer Korea) are shown in Table 8.
상기 실시예의 결과를 하기에서 서술한다.The result of the said Example is described below.
QMAPQMAP 기반의 분석적 민감도와 특이도 다중  Multiple analytical sensitivity and specificity 중합효소연쇄반응(multiplex PCR)인Polymerase chain reaction (multiplex PCR) MTB-ID를 이용한 배양 검체 DNA의 결핵균과 비결핵 항산균 감별 Differentiation of Mycobacterium Tuberculosis and Non-tuberculosis Acid Bacteria in Cultured Sample DNA Using MTB-ID
QMAP 기반의 MTB 및 NTM의 분석적 민감도를 알아보기 위해 각 표준 균주 (Mycobacterium tuberculosis H37Rv, M. avium, M. intracellulare , M. scrofulaceum, M. abscessus , M. chelonae , M. fortuitum , M. marinum , M. kansasii, M. genavense , M. terrae, M. nonchromogenicum , M. celatum , M. gordonae, M. szulgail , M. mucogenicum , M. aubagnense , M. malmoense , M. phlei , M. smgmatis , M. xenopi , M. peregrinum , 와 M. flavescens)를 이용하여 10배 (10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg (1 bacilli) 희석하여 테스트를 진행해봤으며 그 결과 1 pg (100 bacilli)에서 100 fg (10 bacilli)로 검출이 확인 되었다 (표 1). QMAP 기반의 특이도를 확인하기 위하여 총 108균주 (MTB H37Rv, 44 NTM 균주, 및 63 non-mycobacterial 균주)를 가지고 테스트를 진행해봤으며 28 표준 균주는 각 특이 프로브가 커플링된 disk 에 정확하게 검출됨이 확인되었으며 3가지 프로브는 M. fortuitum -M. marinum , M. kansasii -M. gastri , 와 M. genavense-M. avium 함께 검출되었다 (표 2).To determine the analytical sensitivity of QMAP-based MTB and NTM, each standard strain ( Mycobacterium tuberculosis H37Rv, M. avium , M. intracellulare , M. scrofulaceum, M. abscessus , M. chelonae , M. fortuitum , M. marinum , M kansasii, M. genavense , M. terrae, M. nonchromogenicum , M. celatum , M. gordonae , M. szulgail , M. mucogenicum , M. aubagnense , M. malmoense , M. phlei , M. smgmatis , M. xenopi , 10 times (10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg (1 bacilli) was diluted using , M. peregrinum , and M. flavescens . Detection of pg (100 bacilli) with 100 fg (10 bacilli) was confirmed (Table 1) .To determine QMAP-based specificity, a total of 108 strains (MTB H37Rv, 44 NTM strains, and 63 non-mycobacterial strains) were identified. 28 standard strains were confirmed that each specific probe was correctly detected on the coupled disk. Three probes were identified as M. fortuitum -M. marinum , M. kansasii- M. gastri , and M. genavense-M. avium were detected (Table 2).
No.No. SpeciesSpecies Detection limitDetection limit Fluorescence intensity Fluorescence intensity Copies,Mean copies (SD)Copies, Mean copies (SD) CV (%)CV (%)
1One Mycobacterium tuberculosis H37RvMycobacterium tuberculosis H37Rv 10ng-100fg10ng-100fg 5108.7-798.35108.7-798.3 2.96 (±1.85) x10002.96 (± 1.85) x1000 62.562.5
22 M. aviumM. avium 10ng-1pg10ng-1pg 2583.3-9112583.3-911 8.01 (±1.6) x10008.01 (± 1.6) x1000 20.020.0
33 M. intracellulareM. intracellulare 10ng-1pg10ng-1pg 2013-697.52013-697.5 1.31 (±0.48) x10001.31 (± 0.48) x1000 36.336.3
44 M. scrofulaceumM. scrofulaceum 10ng-1pg10ng-1pg 3118.7-696.73118.7-696.7 1.82 (±0.89) x10001.82 (± 0.89) x1000 48.748.7
55 M. abscessus M. abscessus 10ng-100fg10ng-100fg 3056-7743056-774 2.24 (±0.84) x10002.24 (± 0.84) x1000 37.437.4
66 M. chelonaeM. chelonae 10ng-100fg10ng-100fg 4850.5-703.84850.5-703.8 2.96 (±1.51) x10002.96 (± 1.51) x1000 51.151.1
77 M. fortuitum complexM. fortuitum complex 10ng-100fg10ng-100fg 2264-6212264-621 1.4 (±0.59) x10001.4 (± 0.59) x1000 41.341.3
88 M. marinumM. marinum 10ng-100fg10ng-100fg 3602.5-7093602.5-709 2.2 (±1.1) x10002.2 (± 1.1) x1000 50.650.6
99 M. kansasiiM. kansasii 10ng-100fg10ng-100fg 4143.5-5314143.5-531 2.12 (±1.21) x10002.12 (± 1.21) x1000 57.257.2
1010 M. genavenseM. genavense 10ng-100fg10ng-100fg 3982-6543982-654 2.17 (±1.4) x10002.17 (± 1.4) x1000 64.964.9
1111 M. terraeM. terrae 10ng-100fg10ng-100fg 3057-603.33057-603.3 1.85 (±0.92) x10001.85 (± 0.92) x1000 49.949.9
1212 M. nonchromogenicumM. nonchromogenicum 10ng-100fg10ng-100fg 2310-861.82310-861.8 1.5 (±0.5) x10001.5 (± 0.5) x1000 34.334.3
1313 M. celatumM. celatum 10ng-100fg10ng-100fg 3852-6253852-625 2.27 (±1.23) x10002.27 (± 1.23) x1000 54.354.3
1414 M. gordonaeM. gordonae 10ng-1pg10ng-1pg 2157-7062157-706 1.56 (±0.59) x10001.56 (± 0.59) x1000 37.637.6
1515 M. szulgai M. szulgai 10ng-1pg10ng-1pg 3753.5-763.53753.5-763.5 2.31 (±1.22) x10002.31 (± 1.22) x1000 52.852.8
1616 M. mucogenicumM. mucogenicum 10ng-1pg10ng-1pg 4166.3-8984166.3-898 2.8 (±1.4) x10002.8 (± 1.4) x1000 51.551.5
1717 M. aubagnenseM. aubagnense 10ng-100fg10ng-100fg 3683-964.53683-964.5 2.61 (±0.98) x10002.61 (± 0.98) x1000 37.337.3
1818 M. malmoenseM. malmoense 10ng-1pg10ng-1pg 3595.5-828.53595.5-828.5 2.5 (±1.06) x10002.5 (± 1.06) x1000 42.642.6
1919 M. phleiM. phlei 10 ng-100 fg10 ng-100 fg 5,009-9285,009-928 3.1 (±1.7) x 10003.1 (± 1.7) x 1000 55.255.2
2020 M. smegmatisM. smegmatis 10 ng-100 fg10 ng-100 fg 3,897-7733,897-773 2.3 (±1.2) x 10002.3 (± 1.2) x 1000 53.153.1
2121 M. xenopiM. xenopi 10ng-100fg10ng-100fg 3404.5-8143404.5-814 2.15 (±0.96) x10002.15 (± 0.96) x1000 44.644.6
2222 M. peregrinumn M. peregrinumn 10ng-100fg10ng-100fg 3063.7-647.33063.7-647.3 2.07 (±0.86) x10002.07 (± 0.86) x1000 41.641.6
2222 M. flavescensM. flavescens 10ng-100fg10ng-100fg 3139.5-833.53139.5-833.5 2.16 (±0.89) x10002.16 (± 0.89) x1000 4141
표 1은 QMAP system의 분석적 민감도를 나타낸 표로, 약어: CV, Coefficients of Variation (상관계수)Table 1 shows the analytical sensitivity of the QMAP system. Abbreviation: CV, Coefficients of Variation
Figure PCTKR2017008168-appb-T000001
Figure PCTKR2017008168-appb-T000001
Figure PCTKR2017008168-appb-I000001
Figure PCTKR2017008168-appb-I000001
Figure PCTKR2017008168-appb-I000002
Figure PCTKR2017008168-appb-I000002
Figure PCTKR2017008168-appb-I000003
Figure PCTKR2017008168-appb-I000003
Figure PCTKR2017008168-appb-I000004
Figure PCTKR2017008168-appb-I000004
표 2는 108 표준균주를 이용한 QMAP system의 특이도 검사를 나타낸 표,Table 2 shows the specificity test of the QMAP system using 108 standard strains.
임상검체에서 QMAP기반 Myco-ID의 유용성 평가 Evaluation of Usefulness of QMAP-based Myco-ID in Clinical Specimens
QMAP기반 Myco-ID의 유용성 평가를 확인하기 위해서 531 균주 (234 고체배양균과 297 액체배양균) 를 이용하여 MTB와 NTM 검출 및 NTM 동정을 테스트하였다. 그 결과 234 고체배양균은 223 MTB와 11 NTM으로 확인되었으며, 검출된 223 MTB 측정값의 (fluorescence intensity) 범위는 2074에서 4765 (mean 4255.6 ± SD 458.7)와 11 NTM 측정값은 609에서 3569 (1892 ± 923.2)으로 각각 나타났다. 또한 297 액체배양액에서 212 MTB 및 77 NTM 은 QMAP 테스트에서 각각 양성 시그날이 나타났다. 총 531 균주 중 4균주를 제외한 527 (99.2%)가 정확하게 MTB와 NTM으로 확인되었으며, 검출되지 않은 4균주 중 2균주는 배양음성이었으며, 나머지 2 균주는 sequencing에 의해 각각 Rhodococcus erythropolis 와 R. jostii 로 확인되었다. QMAP기반 Myco-ID의 결과를 재확인하기 위하여 또 다른 분자진단방법인 REBA Myco-ID (도 2)를 이용하여 그 결과를 비교한 결과 QMAP 기반의 Myco-ID결과와 모두 일치하는 것을 확인하였다 (표 3). In order to confirm the usefulness evaluation of QMAP-based Myco-ID, MTB and NTM detection and NTM identification were tested using 531 strains (234 solid culture bacteria and 297 liquid culture bacteria). As a result, 234 solid cultures were identified with 223 MTB and 11 NTM, and the detected 223 MTB measurement ranged from 2074 to 4765 (mean 4255.6 ± SD 458.7) and 11 NTM measurements from 609 to 3569 (1892). ± 923.2) respectively. In addition, 212 MTB and 77 NTM in 297 liquid cultures showed positive signals in QMAP test, respectively. Of the 531 strains, 527 (99.2%) except 4 strains were correctly identified as MTB and NTM. Two of the 4 strains which were not detected were culture negative, and the remaining 2 strains were Rhodococcus by sequencing. erythropolis and R. jostii . In order to reconfirm the results of QMAP-based Myco-ID, another molecular diagnostic method, REBA Myco-ID (FIG. 2), was used to compare the results. 3).
AFB smear (n)AFB smear (n) Culture (Ogawa), n (%)Culture (Ogawa), n (%) Culture (MGIT 960), n (%)Culture (MGIT 960), n (%) QMAP system, n (%)QMAP system, n (%) Molecutech REBA Myco-ID, n %)Molecutech REBA Myco-ID, n%)
Positive (n=340)Positive (n = 340) MTBMTB 223 (95.3)223 (95.3) MTBMTB 92 (40)92 (40) MTBMTB 315 (59.3)315 (59.3) MTBMTB 315 (59.3)315 (59.3)
NTMNTM 11 (4.7)11 (4.7) NTMNTM 14 (4.7)14 (4.7) NTMNTM 25 (4.7)25 (4.7) NTMNTM 25 (4.7)25 (4.7)
Negative (n=191)Negative (n = 191) MTBMTB 120 (40.4)120 (40.4) MTBMTB 120 (22.6)120 (22.6) MTBMTB 120 (22.6)120 (22.6)
NTMNTM 63 (21.2)63 (21.2) NTMa NTM a 61 (11.5)61 (11.5) NTMaNTMa 61 (11.5)61 (11.5)
Co-infectionb Co-infection b 6 (2)6 (2) Co-infectionb Co-infection b 6 (1.1)6 (1.1) Co-infectionbCo-infectionb 6 (1.1)6 (1.1)
NDC ND C 2 (0.7)2 (0.7) NDC ND C 2 (0.4)2 (0.4) NDcNDc 2 (0.4)2 (0.4)
Total (n=531)Total (n = 531) 　 234 (100)234 (100) 　 297 (100)297 (100) 　 529 (99.6)529 (99.6) 　 529 (99.6)529 (99.6)
표 3은 총 531 배양균 DNA에서 MTB와 NTM 검출을 위한 통상의 방법들, QMAP system과 REBA Myco-ID의 비교 표, 약어: AFB, acid-fast bacilli; MTB, M. tuberculosis; NTM, nontuberculous mycobacteria; ND, 검출 안됨.Table 3 shows the conventional methods for the detection of MTB and NTM in a total of 531 culture DNAs, a comparative table of QMAP system and REBA Myco-ID, abbreviation: AFB, acid-fast bacilli; MTB, M. tuberculosis ; NTM, nontuberculous mycobacteria; ND, not detected.
aNTM의 두 균주는QMAP system과 REBA Myco-ID에서 검출되지 않았으며, rpoB 서열 분석에 의하여 Rhodococcus erythropolis 및 R. jostii로 확인됨. a Two strains of NTM were not detected in the QMAP system and REBA Myco-ID and identified as Rhodococcus erythropolis and R. jostii by rpoB sequencing.
b NTM 및 NTM로 혼합된 케이스. b Case mixed with NTM and NTM.
C 도말-배양음성을 가지는 두 케이스.Two cases with C smear-cultured voice.
비결핵Non-tuberculosis 항산균의Antibacterial 검출분포 Detection Distribution
531 배양균 전체 검체에서 QMAP기반을 통해 94균주가 NTM으로 검출되었으며, NTM의 동정결과를 살펴보면, 전체 94 검체 중 M. intracellulare가 36검체(39.1%)로 가장 많이 동정이 되었으며, M. avium이 21검체(22.8%), M. abscessus complex가 14검체(15.2%), M. fortuitum이 7검체(7.6%)의 순으로 분포하고 있었다. 그 외에 M. gordonae가 각각 4검체(4.3%)로 나왔으며, M. kansasii가 2검체 (2.2%), M. chelonae가 1검체 (1.1%)가 나왔으며, M. avium와 M. intracellulare , M. avium와 M. abscessus이 각각 섞여있는 경우가 2검체 (2.2%)였으며, M. avium와 M. mucogenicum, M. avium과 M. mucogenicum 이 각각 섞여있는 경우가 1검체(1.1%)였다(표 4). 이 결과는 REBA Myco-ID의 결과와 모두 동일하게 나타났으며, 두 종류의 이상의 비결핵 항산균이 섞여 나온 경우까지도 QMAP system에서 모두 정확하게 분리할 수 있음이 확인할 수 있었다.531 were 94 strains through QMAP based on the whole culture sample is detected by NTM, look at the result of identification of NTM, it was the most frequently identified as M. intracellulare is 36 samples (39.1%) of 94 specimens, the M. avium Twenty-one specimens (22.8%), M. abscessus complex , 14 specimens (15.2%), and M. fortuitum were seven specimens (7.6%). Other M. gordonae is or has a four specimens (4.3%), respectively, M. kansasii 2 samples (2.2%), M. chelonae that has the first sample (1.1%) or, M. avium and M. intracellulare, M. avium and M. abscessus are mixed Two samples (2.2%) and one sample (1.1%) containing M. avium and M. mucogenicum and M. avium and M. mucogenicum (Table 4). These results were the same as the results of REBA Myco-ID, and even if two or more types of non-tuberculosis antibiotics were mixed, it was confirmed that they can be accurately separated from the QMAP system.
MTB/NTM differentiationMTB / NTM differentiation 　 Identification of mycobacterial speciesIdentification of mycobacterial species 　
Culture methodsCulture methods REBA Myco-ID with culture samplesREBA Myco-ID with culture samples n (%)n (%) QMAP system with culture samplesQMAP system with culture samples
n (%)n (%) Ranged fluorensce intensityRanged fluorensce intensity Mean ( ± SD) valueMean (± SD) value
NTM (86)NTM (86) M. intracellulareM. intracellulare 36 (39.1)36 (39.1) 36 (39.1)36 (39.1) 513-2610513-2610 932.7 ( ± 381.7)932.7 (± 381.7)
M. aviumM. avium 21 (22.8)21 (22.8) 21 (22.8)21 (22.8) 517-3569517-3569 2065.7 ( ± 885.9)2065.7 (± 885.9)
M. abscessus complexM. abscessus complex 14 (15.2)14 (15.2) 14 (15.2)14 (15.2) 1338-35431338-3543 2534.5 ( ± 654.9)2534.5 (± 654.9)
M. fortuitumM. fortuitum 7 (7.6)7 (7.6) 7 (7.6)7 (7.6) 699-1883699-1883 1082.1 ( ± 424.5)1082.1 (± 424.5)
M. gordonaeM. gordonae 4 (4.3)4 (4.3) 4 (4.3)4 (4.3) 540-1150540-1150 776 ( ± 267.9)776 (± 267.9)
M. kansasiiM. kansasii 2 (2.2)2 (2.2) 2 (2.2)2 (2.2) 537-949537-949 741.5 ( ± 289.2)741.5 (± 289.2)
M. chelonaeM. chelonae 1 (1.1)1 (1.1) 1 (1.1)1 (1.1) 16401640 --
M. mucogenicumM. mucogenicum 1 (1.1)1 (1.1) 1 (1.1)1 (1.1) 26962696 --
Co-infection (6)Co-infection (6) M. avium and M. intracellulareM. avium and M. intracellulare 2 (2.2)2 (2.2) 2 (2.2)2 (2.2) 691-2407691-2407 1188 ( ± 836.5)1188 (± 836.5)
M. avium and M. abscessusM. avium and M. abscessus 2 (2.2)2 (2.2) 2 (2.2)2 (2.2) 608-3013608-3013 2054.8 ( ± 10003.3)2054.8 (± 10003.3)
M. avium and M. mucogenicumM. avium and M. mucogenicum 1 (1.1)1 (1.1) 1 (1.1)1 (1.1) 625-1625625-1625 1125 ( ± 707.1)1125 (± 707.1)
M. gordonae and M. mucogenicumM. gordonae and M. mucogenicum 1 (1.1)1 (1.1) 1 (1.1)1 (1.1) 509-2463509-2463 1486 ( ± 1381.7)1486 (± 1381.7)
TotalTotal 　 92 (100)92 (100) 92 (100)92 (100) 　 　
표 4는 REBA Myco-ID 및 QMAP system에 의해 분리된 NTM의 분표 비교Table 4 compares fractions of NTM separated by REBA Myco-ID and QMAP system
QMAPQMAP 기반  base 리팜핀Rifampin , 아이나 내성의 유용성 평가Usefulness of child, tolerant
총 531 균주 중 결핵균으로 검출된 435 균주에서 Conventional Drug Susceptibility Test (DST) 결과가 있는 고체배양 223 균주를 대상으로 QMAP 기반 리팜핀 내성의 유용성 평가를 진행하였다. 223 결핵 균주 중 리팜핀 내성으로 확인된 균주는 27 (12.1%)이었으며 전통 DST결과와 비교시 모두 일치함을 확인할 수 있었다. 내성으로 확인된 27 균주의 내성 부위는 531TTG가 13균주 (48.1%), △WT4 에서 5 균주 (18.5%), △WT5와 526TAC가 각각 3 (11.1%), 그리고 516GTC, △WT4, △WT2와 △WT4 에서 각각 1 균주 (3.7%)가 내성으로 나타났다. 결과는 리팜핀 내성 확인이 가능한 REBA MTB-MDR을 이용하여 비교하였으며 리팜핀 내성과 내성부위가 모두 일치하게 나타나는 것을 확인할 수 있었다 (표 5).We evaluated the usefulness of QMAP-based rifampin resistance in 435 solid cultures with Conventional Drug Susceptibility Test (DST) results from 435 strains detected as Mycobacterium tuberculosis. Of the 223 tuberculosis strains, 27 (12.1%) were found to be rifampin resistant, and all of them were consistent with the conventional DST results. The resistance sites of 27 strains identified as resistant were 13 strains (48.1%) in 531TTG, 5 strains (18.5%) in ΔWT4, 3 (11.1%) in ΔWT5 and 526TAC, and 516GTC, ΔWT4, △ WT2 and 1 strain (3.7%) each of ΔWT4 was resistant. The results were compared by using REBA MTB-MDR capable of confirming rifampin resistance, and it was confirmed that both rifampin resistance and resistance sites were identical (Table 5).
Conventional method (n=223)Conventional method (n = 223) Molecular DSTMolecular DST 　
REBA MTB-MDRREBA MTB-MDR QMAP systemQMAP system
Rifampin-susceptible (n=196)Rifampin-susceptible (n = 196) 196 (87.9%)196 (87.9%) 196 (87.9%)196 (87.9%)
Rifampin-resistant (n=27)Rifampin-resistant (n = 27) 27 (12.1%)27 (12.1%) 27 (12.1%)27 (12.1%)
531TTG531TTG 13 (48.1%)13 (48.1%) 13 (48.1%)13 (48.1%)
△S4△ S4 5 (18.5%)5 (18.5%) 5 (18.5%)5 (18.5%)
526TAC526TAC 3 (11.1%)3 (11.1%) 3 (11.1%)3 (11.1%)
△S5△ S5 3 (11.1%)3 (11.1%) 3 (11.1%)3 (11.1%)
516GTC516GTC 1 (3.7%)1 (3.7%) 1 (3.7%)1 (3.7%)
△S3△ S3 1 (3.7%)1 (3.7%) 1 (3.7%)1 (3.7%)
△S2, △S4△ S2, △ S4 1 (3.7%)1 (3.7%) 1 (3.7%)1 (3.7%)
표 5는 총 226 배양균 DNA에서 리팜핀 내성여부 검출을 위한 통상적인 방법, QMAP system과 REBA Myco-ID의 비교 Table 5 compares the conventional method for detecting rifampin resistance in total 226 culture DNA, the QMAP system and REBA Myco-ID.
아이나 내성으로 확인된 균주는 모두 108균주였으며, 이 중 82 (75.9%)가 katG 315부분에서내성을 나타냈다 (표 6). 결과는 아이나 내성 확인이 가능한 DNA 시퀀싱을 이용하여 비교하였으며 아이나 내성과 내성부위가 모두 일치하게 나타나는 것을 확인할 수 있었다 (표 6).A total of 108 strains identified as iina were resistant, and 82 (75.9%) of them were resistant to katG 315 (Table 6). The results were compared by using DNA sequencing capable of confirming the resistance of the eye and the resistance and the resistance sites were confirmed to be consistent (Table 6).
Conventional DST (no.)Conventional DST (no.) QMAP systemQMAP system DNA SequencingDNA Sequencing
TypeType no. (%)no. (%) Sequencing resultSequencing result no. (%)no. (%)
INH-susceptible (56)INH-susceptible (56) Wild typeWild type 54 (96.4)54 (96.4) Wild typeWild type 54 (96.4)54 (96.4)
　 Mutant type Mutant type 2 (3.6)2 (3.6) Mutant typeMutant type 2 (3.6)2 (3.6)
　 △katG WT △ katG WT 1 (50/0.9)a1 (50 / 0.9) a katG315 AGC-AACkatG315 AGC-AAC 1 (50/0.9)1 (50 / 0.9)
　 △oxyR-ahpC WT1△ oxyR-ahpC WT1 1 (50/0.9)1 (50 / 0.9) G-A at position -46G-A at position -46 1 (50/0.9)1 (50 / 0.9)
　 　 　 　 　
INH-resistant (144)INH-resistant (144) Wild typeWild type 36 (25)36 (25) Wild typeWild type 36 (25)36 (25)
　 Mutant typeMutant type 108 (75)108 (75) Mutant typeMutant type 108 (75)108 (75)
　 katG315 MTkatG315 MT 75 (69.4/67.6)75 (69.4 / 67.6) katG315 AGC-ACCkatG315 AGC-ACC 75 (69.4/67.6)75 (69.4 / 67.6)
　 △katGWT△ katGWT 7 (6.5/6.3)7 (6.5 / 6.3) katG315 AGC-AACkatG315 AGC-AAC 3 (2.8/2.7)3 (2.8 / 2.7)
　 　 　 katG315 AGC-ACAkatG315 AGC-ACA 2 (1.9/1.8)2 (1.9 / 1.8)
　 　 　 katG315 AGC-AGAkatG315 AGC-AGA 1 (0.9/0.9)1 (0.9 / 0.9)
　 　 　 katG315 AGC-ATCkatG315 AGC-ATC 1 (0.9/0.9)1 (0.9 / 0.9)
　 inhA 15ups MTinhA 15ups MT 22 (20.4/19.8)22 (20.4 / 19.8) inhA -15C-TinhA -15C-T 22 (20.4/19.8)22 (20.4 / 19.8)
　 inhA 8ups MTinhA 8ups MT 1 (0.9/0.9)1 (0.9 / 0.9) inhA -8T-CinhA -8T-C 1 (0.9/0.9)1 (0.9 / 0.9)
　 △oxyR-ahpC WT3△ oxyR-ahpC WT3 2 (1.9/1.8)2 (1.9 / 1.8) C-A at position -11C-A at position -11 2 (1.9/1.8)2 (1.9 / 1.8)
　 katG315 MTinhA 15ups MTkatG315 MTinhA 15ups MT 1 (0.9/NE)1 (0.9 / NE) katG315 AGC-ACCinhA -15C-TkatG315 AGC-ACCinhA -15C-T 1 (0.9/NE)1 (0.9 / NE)
표 6은 배양균 DNA에서 아이나 내성여부 검출을 위한 통상적인 방법, QMAP system과 DNA 시퀀싱의 비교 Table 6 shows a comparison of conventional methods, QMAP system, and DNA sequencing to detect eye or resistance in cultured DNA.
추가로, Phenotypic DST 결과와 비교하여, QMAP 기반의 리팜핀과 아이나 내성에 대한 민감도는 각각 96.4% (106/110)와 75% (108/144)으로 나타났다. 그러나, DNA 시퀀싱 결과와 비교했을 때, QMAP 기반의 민감도는 리팜핀과 아이나에서 모두 100% (124/124; 95% CI: 0.9743-1.0000)와 100% (110/110, 95% CI: 0.9711-1.0000)를 보였다 (표 7).In addition, compared to Phenotypic DST results, the sensitivity to QMAP-based rifampin and eyena resistance was 96.4% (106/110) and 75% (108/144), respectively. However, compared to DNA sequencing results, the QMAP-based sensitivity was 100% (124/124; 95% CI: 0.9743-1.0000) and 100% (110/110, 95% CI: 0.9711-1.0000) in both rifampin and inna. ) (Table 7).
QMAP systemQMAP system DST results, no.DST results, no. Sensitivity, % (no.)(95% CI)Sensitivity,% (no.) (95% CI) Specificity, % (no.)(95% CI)Specificity,% (no.) (95% CI) PPV, % (no.)(95% CI)PPV,% (no.) (95% CI) NPV, % (no.)(95% CI)NPV,% (no.) (95% CI)
ResistantResistant Susceptible Susceptible
RifampicinRifampicin
MutantMutant 106106 1818 96.4 (106/110)(0.9072-0.9888)96.4 (106/110) (0.9072-0.9888) 80 (72/90)(0.7052-0.8705)80 (72/90) (0.7052-0.8705) 85.5 (106/124)(0.7813-0.9070)85.5 (106/124) (0.7813-0.9070) 94.7 (72/76)(0.8684-0.9833)94.7 (72/76) (0.8684-0.9833)
Wild typeWild type 4 4 7272
IsoniazidIsoniazid
MutantMutant 108108 2 2 75 (108/144)(0.6731-0.8139)75 (108/144) (0.6731-0.8139) 96.4 (54/56)(0.8718-0.9972)96.4 (54/56) (0.8718-0.9972) 98.2 (108/110)(0.9321-0.9991)98.2 (108/110) (0.9321-0.9991) 60 (54/90)(0.4966-0.6952)60 (54/90) (0.4966-0.6952)
Wild typeWild type 36 36 5454
DNA-sequencing results, no.DNA-sequencing results, no.
ResistantResistant Susceptible Susceptible
RifampicinRifampicin
MutantMutant 124124 0 0 100 (124/124)(0.9743-1.0000)100 (124/124) (0.9743-1.0000) 100 (76/76)(0.9587-1.0000)100 (76/76) (0.9587-1.0000) 100 (124/124)(0.97432-1.0000)100 (124/124) (0.97432-1.0000) 100 (76/76)(0.9587-1.0000)100 (76/76) (0.9587-1.0000)
Wild typeWild type 0  0 7676
IsoniazidIsoniazid
MutantMutant 110110 0 0 100 (110/110)(0.9711-1.0000)100 (110/110) (0.9711-1.0000) 100 (90/90)(0.9649-1.0000)100 (90/90) (0.9649-1.0000) 100 (110/110)(0.9711-1.0000)100 (110/110) (0.9711-1.0000) 100 (90/90)(0.9649-1.0000)100 (90/90) (0.9649-1.0000)
Wild typeWild type 0  0 9090
DST, drug susceptibility testing; PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval.DST, drug susceptibility testing; PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval.
표 7은 QMAP system과 DST 결과 및 DNA 시퀀싱 결과의 비교Table 7 compares the QMAP system with the DST and DNA sequencing results
NameName Primer sequence (5'-3')Primer sequence (5'-3 ') 　
360long-F360long-f TCAAGGAGAAGCGCTACGACCTGGC(서열번호 1)TCAAGGAGAAGCGCTACGACCTGGC (SEQ ID NO: 1) Myco-IDMyco-id
250R*250R * ACS CGG ATC TGG TTC TGG ATC ARC(서열번호 2)ACS CGG ATC TGG TTC TGG ATC ARC (SEQ ID NO: 2) 5'-Biotin5'-Biotin
I417-FI417-F CTTGGTGGTGGGGTGTGGTGTTTGA (서열번호 3)CTTGGTGGTGGGGTGTGGTGTTTGA (SEQ ID NO: 3) Myco-IDMyco-id
I638R*I638R * GCCAAGGCATYCACCATGYGCCCTTA(서열번호 4)GCCAAGGCATYCACCATGYGCCCTTA (SEQ ID NO: 4) 5'-Biotin5'-Biotin
TR9 TR9 TCGCCGCGATCAAGGAGTTCT(서열번호 5)TCGCCGCGATCAAGGAGTTCT (SEQ ID NO: 5) RifampinRifampin
TR8*TR8 * TGCACGTCGCGGACCTCCA(서열번호 6)TGCACGTCGCGGACCTCCA (SEQ ID NO: 6) 5'-Biotin5'-Biotin
katG-FkatG-F 　 AGCTCGTATGGCACCGGAAC (서열번호 7)AGCTCGTATGGCACCGGAAC (SEQ ID NO: 7) 　IsoniazidIsoniazid
katG-R*katG-R * CCGTACAGGATCTCGAGGAA (서열번호 8)CCGTACAGGATCTCGAGGAA (SEQ ID NO: 8) 5'-Biotin5'-Biotin
inhA-FinhA-F GCTCGTGGACATACCGATTT (서열번호 9)GCTCGTGGACATACCGATTT (SEQ ID NO: 9) 　IsoniazidIsoniazid
inhA-R*inhA-R * ACTGAACGGGATACGAATGG (서열번호 10)ACTGAACGGGATACGAATGG (SEQ ID NO: 10) 5'-Biotin5'-Biotin
ahpC-FahpC-F CCGGCTAGCACCTCTTGGCG (서열번호 11)CCGGCTAGCACCTCTTGGCG (SEQ ID NO: 11) 　IsoniazidIsoniazid
ahpC-R*ahpC-R * ATTGATCGCCAATGGTTAGC (서열번호 12)ATTGATCGCCAATGGTTAGC (SEQ ID NO: 12) 5'-Biotin5'-Biotin
NameName Myco-Probe sequence (5'-3')Myco-Probe sequence (5'-3 ') 　
Myc-14Myc-14 TTT TTT TTT TTT TTT ACCGARGARGACGTCGTCGCCACCATCGA (서열번호 13)TTT TTT TTT TTT TTT ACCGARGARGACGTCGTCGCCACCATCGA (SEQ ID NO: 13) Mycobacterium spp.Mycobacterium spp.
TB-13TB-13 TTT TTT TTT TTT TTT G CAT GTC GGC GAG CCC atc acgT(서열번호 14)TTT TTT TTT TTT TTT G CAT GTC GGC GAG CCC atc acgT (SEQ ID NO: 14) MTB complexMTB complex
IMTBIMTB TTT TTT TTT TTT TTT GAGCATCAATGGATACGCTGCCGGCTAGC (서열번호 15)TTT TTT TTT TTT TTT GAGCATCAATGGATACGCTGCCGGCTAGC (SEQ ID NO: 15) MTB complexMTB complex
Avi-1Avi-1 TTT TTT TTT TTT TTT CAC GCCGGTGAGCCGATCACCA(서열번호 16)TTT TTT TTT TTT TTT CAC GCCGGTGAGCCGATCACCA (SEQ ID NO: 16) M. aviumM. avium
Iavi-1Iavi-1 TTT TTT TTT TTT TTT GCGAGCATCTAGATGAACGCGTGGTCTTCATG(서열번호 17)TTT TTT TTT TTT TTT GCGAGCATCTAGATGAACGCGTGGTCTTCATG (SEQ ID NO: 17) M. aviumM. avium
Int-1Int-1 TTT TTT TTT TTT TTT CTC CGGCCTGCACGCGGGCGAG(서열번호 18)TTT TTT TTT TTT TTT CTC CGGCCTGCACGCGGGCGAG (SEQ ID NO: 18) M. intracellulareM. intracellulare
Iint-2Iint-2 TTT TTT TTT TTT TTT TAGATGAGCGCATAGTCCTTAGGGCTGATGC (서열번호 19)TTT TTT TTT TTT TTT TAGATGAGCGCATAGTCCTTAGGGCTGATGC (SEQ ID NO: 19) M. intracellulareM. intracellulare
SC-long-3SC-long-3 TTT TTT TTT TTT TTT CAC GCCCGTACGGATGGCCAGC (서열번호 20)TTT TTT TTT TTT TTT CAC GCCCGTACGGATGGCCAGC (SEQ ID NO: 20) M. scrofulaceumM. scrofulaceum
Ab-4Ab-4 TTT TTT TTT TTT TTT GC ACC AAT CCG GCTC AGG TG A CCA CCA CC(서열번호 21)TTT TTT TTT TTT TTT GC ACC AAT CCG GCTC AGG TG A CCA CCA CC (SEQ ID NO: 21) M. abscessusM. abscessus
IabsIabs TTT TTT TTT TTT TTT CA TAGCCTCGCTCGTTTTCGAGTGGGGCTG(서열번호 22)TTT TTT TTT TTT TTT CA TAGCCTCGCTCGTTTTCGAGTGGGGCTG (SEQ ID NO: 22) M. abscessusM. abscessus
3a-CH-13a-CH-1 TTT TTT TTT TTT TTT TGCCAACCCGGCTCTGGTGACTG(서열번호 23)TTT TTT TTT TTT TTT TGCCAACCCGGCTCTGGTGACTG (SEQ ID NO: 23) M. chelonaeM. chelonae
IcheIche TTT TTT TTT TTT TTT TAACAAGCCTCGCTCGTTTACGAGTGAGGTTA(서열번호 24)TTT TTT TTT TTT TTT TAACAAGCCTCGCTCGTTTACGAGTGAGGTTA (SEQ ID NO: 24) M. chelonaeM. chelonae
Forts-1Forts-1 TTT TTT TTT TTT TTT GGG CCTGAACGCCGGCCAG(서열번호 25)TTT TTT TTT TTT TTT GGG CCTGAACGCCGGCCAG (SEQ ID NO: 25) M. fortuitumM. fortuitum
Ifort-1Ifort-1 TTT TTT TTT TTT TTT AGTGTGGCTGGGGGCCTTCGGGTTTC (서열번호 26)TTT TTT TTT TTT TTT AGTGTGGCTGGGGGCCTTCGGGTTTC (SEQ ID NO: 26) M. fortuitumM. fortuitum
4a-U/M-L4a-U / M-L TTT TTT TTT TTT TTT AGGCCAGCCCATCACCAGC(서열번호 27)TTT TTT TTT TTT TTT AGGCCAGCCCATCACCAGC (SEQ ID NO: 27) M. ulcerans/M. marinumM. ulcerans / M. marinum
Imari-6Imari-6 TTT TTT TTT TTT TTT AAA TT GGATGCGCTGCCTTT(서열번호 28)TTT TTT TTT TTT TTT AAA TT GGATGCGCTGCCTTT (SEQ ID NO: 28) M. ulcerans/M. marinumM. ulcerans / M. marinum
Ikan-1Ikan-1 TTT TTT TTT TTT TTT ATCAAATGGATGCGTTGCCCTACGGGTA(서열번호 29)TTT TTT TTT TTT TTT ATCAAATGGATGCGTTGCCCTACGGGTA (SEQ ID NO: 29) M. kansasiiM. kansasii
kanII-L1-2kanII-L1-2 TTT TTT TTT TTT TTT GGCCTCAACACCAAGGACCCGATCACCACG(서열번호 30)TTT TTT TTT TTT TTT GGCCTCAACACCAAGGACCCGATCACCACG (SEQ ID NO: 30) M. kansasiiM. kansasii
Igen/sim-1Igen / sim-1 TTT TTT TTT TTT TTT ATCTAAATGAACGCGTCGCCGGCAAC GGTTA(서열번호 31)TTT TTT TTT TTT TTT ATCTAAATGAACGCGTCGCCGGCAAC GGTTA (SEQ ID NO: 31) M. genavense/M. simmiaeM. genavense / M. simmiae
TE2-3TE2-3 TTT TTT TTT TTT TTT CCGGCCGCACCCGCCGACGTCGAGACGT(서열번호 32)TTT TTT TTT TTT TTT CCGGCCGCACCCGCCGACGTCGAGACGT (SEQ ID NO: 32) M. terraeM. terrae
3a-Non-L-13a-Non-L-1 TTT TTT TTT TTT TTT ACCG CCA ACC CGG G TGAGGCGCA(서열번호 33)TTT TTT TTT TTT TTT ACCG CCA ACC CGG G TGAGGCGCA (SEQ ID NO: 33) M. nonchromogenicumM. nonchromogenicum
Cel-ICel-i TTT TTT TTT TTT TTT CGAGAGCCCAATCACCACC(서열번호 34)TTT TTT TTT TTT TTT CGAGAGCCCAATCACCACC (SEQ ID NO: 34) M. celatumM. celatum
Cel-IICel-ii TTT TTT TTT TTT TTT CGCGTCCCCGATCACGAC(서열번호 35)TTT TTT TTT TTT TTT CGCGTCCCCGATCACGAC (SEQ ID NO: 35) M. celatumM. celatum
Igo-2Igo-2 TTT TTT TTT TTT TTT CGAGCATCAAAATGTATGCGTTGTCGTTCTC(서열번호 36)TTT TTT TTT TTT TTT CGAGCATCAAAATGTATGCGTTGTCGTTCTC (SEQ ID NO: 36) M. gordonaeM. gordonae
Szul-4Szul-4 TTT TTT TTT TTT TTT GAACGTCGGCGAGCCGATCACCAGTT (서열번호 37)TTT TTT TTT TTT TTT GAACGTCGGCGAGCCGATCACCAGTT (SEQ ID NO: 37) M. szulgaiM. szulgai
Iszul-4Iszul-4 TTT TTT TTT TTT TTT AAA GGATGCGCTGCCCTCG(서열번호 38)TTT TTT TTT TTT TTT AAA GGATGCGCTGCCCTCG (SEQ ID NO: 38) M. szulgai M. szulgai
Muco-16Muco-16 TTT TTT TTT TTT TTT CACG ACG GCAACCCGGCTCAGGTGACCGCG(서열번호 39)TTT TTT TTT TTT TTT CACG ACG GCAACCCGGCTCAGGTGACCGCG (SEQ ID NO: 39) M. mucogenicumM. mucogenicum
Auba-3Auba-3 TTT TTT TTT TTT TTT GCGAACTCCGACGGCACGCAGCCGCT(서열번호 40)TTT TTT TTT TTT TTT GCGAACTCCGACGGCACGCAGCCGCT (SEQ ID NO: 40) M. aubagnenseM. aubagnense
malmal TTT TTT TTT TTT TTT CGA GTCGGCCGTACCCGCCTCGACCAC(서열번호 41)TTT TTT TTT TTT TTT CGA GTCGGCCGTACCCGCCTCGACCAC (SEQ ID NO: 41) M. malmoenseM. malmoense
IphleiIphlei TTT TTT TTT TTT TTT CCTTT TTTTGGGGGTTCTTGGGTGTTCG (서열번호 42)TTT TTT TTT TTT TTT CCTTT TTTTGGGGGTTCTTGGGTGTTCG (SEQ ID NO: 42) M. phleiM. phlei
IsmegIsmeg TTT TTT TTT TTT TTT TAAGAGTGTGGCTGCCGGCCTTTGAGGT(서열번호 43)TTT TTT TTT TTT TTT TAAGAGTGTGGCTGCCGGCCTTTGAGGT (SEQ ID NO: 43) M. smegmatisM. smegmatis
IxenoIxeno TTT TTT TTT TTT TTT TGCGAGCATCTGGCAAAGACTGTGGTAAGCGG(서열번호 44)TTT TTT TTT TTT TTT TGCGAGCATCTGGCAAAGACTGTGGTAAGCGG (SEQ ID NO: 44) M. xenopiM. xenopi
IflavIflav TTT TTT TTT TTT TTT GAACAGGTGGCTCCCTTTTGGGGGTTGCTT(서열번호 45)TTT TTT TTT TTT TTT GAACAGGTGGCTCCCTTTTGGGGGTTGCTT (SEQ ID NO: 45) M. flavescensM. flavescens
IpereIpere TTT TTT TTT TTT TTT AAAATGTGTGGTCTCACTCCTTGTGGGTG(서열번호 46)TTT TTT TTT TTT TTT AAAATGTGTGGTCTCACTCCTTGTGGGTG (SEQ ID NO: 46) M. peregrinum / M. septicumM. peregrinum / M. septicum
　NameName Rifampin probe sequence (5'-3')　Rifampin probe sequence (5'-3 ') 　positionposition
S1S1 TTT TTT TTT TTT TTT AGCCAGCTGAGCCAATTC(서열번호 47)TTT TTT TTT TTT TTT AGCCAGCTGAGCCAATTC (SEQ ID NO: 47) 509-514509-514
S2S2 TTT TTT TTT TTT TTT CATGGACCAGAACAACCCGC(서열번호 48)TTT TTT TTT TTT TTT CATGGACCAGAACAACCCGC (SEQ ID NO 48) 514-520514-520
S3S3 TTT TTT TTT TTT TTT CCGCTGTCGGGGTTGACC(서열번호 49)TTT TTT TTT TTT TTT CCGCTGTCGGGGTTGACC (SEQ ID NO: 49) 520-525520-525
S4S4 TTT TTT TTT TTT TTT G TTG ACC CAC AAG CGC CGA(서열번호 50)TTT TTT TTT TTT TTT G TTG ACC CAC AAG CGC CGA (SEQ ID NO: 50) 524-529524-529
S5-1S5-1 TTT TTT TTT TTT TTT AAA CTGTCGGCGCTGGGGC(서열번호 51)TTT TTT TTT TTT TTT AAA CTGTCGGCGCTGGGGC (SEQ ID NO: 51) 530-534530-534
S5-3S5-3 TTT TTT TTT TTT TTT ACTGTCGGCGCTGGGGC(서열번호 52)TTT TTT TTT TTT TTT ACTGTCGGCGCTGGGGC (SEQ ID NO: 52) 530-534　530-534
531TTG-3531TTG-3 TTT TTT TTT TTT TTT AAG CGC C GA CTG TTG GC G CTGGGGCC(서열번호 53)TTT TTT TTT TTT TTT AAG CGC C GA CTG TTG GC G CTGGGGCC (SEQ ID NO: 53) 　
526TAC-3526TAC-3 TTT TTT TTT TTT TTT A G G TTG ACC TAC AAG CGCCGA(서열번호 54)TTT TTT TTT TTT TTT A G G TTG ACC TAC AAG CGCCGA (SEQ ID NO: 54) 　
516GTC-2516GTC-2 TTT TTT TTT TTT TTT TTC ATG GTC CAG AAC AAC CC G (서열번호 55)TTT TTT TTT TTT TTT TTC ATG GTC CAG AAC AAC CC G (SEQ ID NO: 55) 　
516TAC-2516TAC-2 TTT TTT TTT TTT TTT CAA TTC ATG TAC CAG AAC AAC CC (서열번호 56)TTT TTT TTT TTT TTT CAA TTC ATG TAC CAG AAC AAC CC (SEQ ID NO: 56) 　
513CCA513CCA TTT TTT TTT TTT TTT G CTG AGC CCA TTC ATG GAC (서열번호 57)TTT TTT TTT TTT TTT G CTG AGC CCA TTC ATG GAC (SEQ ID NO: 57) 　
　NameName Isoniazid probe sequence (5'-3')　Isoniazid probe sequence (5'-3 ') 　positionposition
katG-WTkatG-WT TTT TTT TTT TTT TTT AAATCACCAGCGGCATCGAG (서열번호 58)TTT TTT TTT TTT TTT AAATCACCAGCGGCATCGAG (SEQ ID NO: 58) katG 315 WT-1katG 315 WT-1
katG-MTkatG-MT TTT TTT TTT TTT TTT AAATCACCACCGGCATCGAAG (서열번호 59)TTT TTT TTT TTT TTT AAATCACCACCGGCATCGAAG (SEQ ID NO: 59) katG-315MT-3katG-315MT-3
inhA-15WTinhA-15WT TTTTTTTTTTTTTTT AAA GCCGCGGCGAGACGATAGGTT (서열번호 60)TTTTTTTTTTTTTTT AAA GCCGCGGCGAGACGATAGGTT (SEQ ID NO: 60) inhA-15WT2-3inhA-15WT2-3
inhA-15MTinhA-15MT TTTTTTTTTTTTTTT AAAGCGGCGAGATGATAGGTTGT (서열번호 61)TTTTTTTTTTTTTTT AAAGCGGCGAGATGATAGGTTGT (SEQ ID NO: 61) inhA-15MT-6inhA-15MT-6
inhA-8WTinhA-8WT TTTTTTTTTTTTTTT GACGATAGGTTGTCGGGGTGA (서열번호 62)TTTTTTTTTTTTTTT GACGATAGGTTGTCGGGGTGA (SEQ ID NO: 62) inhA-8WT-1inhA-8WT-1
inhA-8MTinhA-8MT TTT TTT TTT TTT TTT GACGATAGGCTGTCGGG (서열번호 63)TTT TTT TTT TTT TTT GACGATAGGCTGTCGGG (SEQ ID NO: 63) inhA-8MT-1inhA-8MT-1
ahpC-WT1ahpC-WT1 TTT TTT TTT TTT TTT ATATGGTGTGATATATCACCTTTGC (서열번호 64)TTT TTT TTT TTT TTT ATATGGTGTGATATATCACCTTTGC (SEQ ID NO: 64) ahpC-WT1-4ahpC-WT1-4
ahpC-WT2ahpC-WT2 TTT TTT TTT TTT TTT AAACCTTTGCCTGACAGCGACTT (서열번호 65)TTT TTT TTT TTT TTT AAACCTTTGCCTGACAGCGACTT (SEQ ID NO: 65) ahpC-WT2-2ahpC-WT2-2
ahpC-WT3ahpC-WT3 TTT TTT TTT TTT TTT TTCACGGCACGATGGAATGT (서열번호 66)TTT TTT TTT TTT TTT TTCACGGCACGATGGAATGT (SEQ ID NO: 66) ahpC-WT3-6ahpC-WT3-6
ahpC-WT4ahpC-WT4 TTT TTT TTT TTT TTT CGCAACCAAATGCATTGTC (서열번호 67)TTT TTT TTT TTT TTT CGCAACCAAATGCATTGTC (SEQ ID NO: 67) ahpC-WT4-3ahpC-WT4-3
ahpC-WT5ahpC-WT5 TTT TTT TTT TTT TTT TTTGATGATGAGGAGAGTCATGC (서열번호 68)TTT TTT TTT TTT TTT TTTGATGATGAGGAGAGTCATGC (SEQ ID NO: 68) ahpC-WT5-5ahpC-WT5-5
표 8은 본 발명에 사용된 프라이머 및 프로브 서열Table 8 shows the primer and probe sequences used in the present invention.
본 발명의 방법과 기존 Method and Existence of the Invention REBAREBA 비교 compare
도 2에서 알 수 있는 바와 같이, REBA Myco-ID의 경우 검출가능한 종은 결핵균을 포함하여 모두 19종이다. 이에 반하여 본 발명의 QMAP 기반의 진단법은 REBA Myco-ID보다 7개의 균종이 더 포함되어 있어 더 많은 균종을 분리할 수 있다. 즉, 본 발명의 QMAP system의 경우 결핵균을 포함한 26종의 주요 비결핵 항산균(M. avium , M. intracellulare , M. scrofulaceum , M. abscessus complex, M. chelonae , M. fortuitum complex, M. ulcerans /M. marinum , M. kansasii , M. genavense /M. simiae, M. terrae, M. nonchromogenicum , M. celatum , M. gordonae , M. szulgai , M. mucogenicum , M. aubagnense , 이외에 추가로 M. malmoense , M. phlei , M. smegmatis, M. xenopi , M. peregrinum , M. septicum , M. flavescens이 추가로 분리될 수 있다.As can be seen in Figure 2, for the REBA Myco-ID detectable species are all 19 species, including Mycobacterium tuberculosis. In contrast, the QMAP-based diagnostic method of the present invention includes seven more species than REBA Myco-ID, so that more species can be separated. That is, in the case of the QMAP system of the present invention, 26 major non-tuberculosis antibiotics including M. tuberculosis ( M. avium , M. intracellulare , M. scrofulaceum , M. abscessus complex , M. chelonae , M. fortuitum complex, M. ulcerans / M. marinum , M. kansasii , M. genavense / M. simiae, M. terrae, M. nonchromogenicum , M. celatum , M. gordonae , M. szulgai , M. mucogenicum , M. aubagnense , In addition to M. malmoense , M. phlei , M. smegmatis, M. xenopi , M . peregrinum, M. septicum, M. flavescens may be added to the separation.
또한 REBA Myco-ID보다 우수한 점은 REBA Myco-ID경우 결핵과 NTM만을 구별할 수 있으나 QMAP 기반의 진단법은 결핵일 경우 결핵의 1차 약제로 사용되는 가장 중요한 리팜핀의 내성여부까지 확인이 가능하여 빠르게 내성여부를 판단하고 치료제의 선별에 도움을 줄 수 있다.In addition, the superiority of REBA Myco-ID is distinguished from tuberculosis and NTM in case of REBA Myco-ID. However, QMAP-based diagnostic method can quickly determine the resistance of the most important rifampin, which is used as the primary drug for tuberculosis in tuberculosis. It can help you determine whether or not you can choose a treatment.
상기 표 5-7처럼 QMAP기반의 분자법은 결핵이 나온 경우 리팜핀, 아이나 감성-내성 유무, 내성일 경우 어느위치에서 내성을 보였는지 확인이 가능하다. QMAP기반의 분자법, 이번 출원을 목적으로 하는 검사법의 경우 전통적인 내성방법과 비교하였을때의 결과도 우수한 결과를 나타내는 것을 확인할 수 있었다. REBA Myco-ID의 경우 결핵/NTM 의 동정만 가능하기 때문에 결핵일 경우 리팜핀 내성을 확인하기 위해서는 REBA MTB-MDR과 같은 다른 분자진단법을 이용하여 추가 테스트를 진행해야 하나, 본 발명의 방법은 이러한 추가 테스트가 필요하지 아니하다.As shown in Table 5-7, the QMAP-based molecular method is able to confirm where resistance was shown in case of rifampin, child or sensitization-resistant, and resistance to tuberculosis. QMAP-based molecular method, the test method for the purpose of this application was confirmed that the results compared to the conventional resistance method also shows excellent results. In the case of REBA Myco-ID, only tuberculosis / NTM can be identified, so in order to confirm the rifampin resistance in the case of tuberculosis, additional tests must be conducted using other molecular diagnostic methods such as REBA MTB-MDR. No test is required.
또한, 본 발명의 QMAP 기반의 진단법의 또 한가지의 장점은 검출 시간의 단축이다. 통상 96 테스트를 진행하기 위해서 REBA Myco-ID의 경우 5시간 이상이 소요 (REBA진행시간만)가 되나 QMAP의 경우는 1시간 40분의 검출시간을 단축시킬 수 있어서 진단에서 가장 중요한 시간을 단축시킬 수 있는 효과가 있다.In addition, another advantage of the QMAP based diagnostics of the present invention is the reduction of detection time. Normally it takes more than 5 hours for REBA Myco-ID (REBA progression time) to proceed with 96 tests, but for QMAP, the detection time of 1 hour 40 minutes can be shortened, which reduces the most important time in diagnosis. It can be effective.

Claims (6)

  1. a)검체 시료로부터 DNA를 분리하는 단계;a) separating DNA from the sample sample;
    b)서열번호 1 내지 서열번호 12의 프라이머를 사용하여, 상기 DNA로부터 PCR 증폭하는 단계;및b) PCR amplification from the DNA using primers of SEQ ID NO: 1 to SEQ ID NO: 12; and
    c) 서열번호 13 내지 68의 올리고머 프로브가 커플링된 디스크와 상기 단계 b)에서 얻어진 PCR 증폭산물을 하이브리드형성시키는 후,퀀타매트릭스 어세이 플랫폼 소프트웨어를 통하여 상기 디스크의 이미지를 측정하는 단계를 포함하는 c) hybridizing the disk to which the oligomeric probes of SEQ ID NOs. 13-68 are coupled with the PCR amplification product obtained in step b), and then measuring the image of the disk through Quanta Matrix Assay Platform Software.
    결핵균 및 비결핵 항산균 검출과 동정 및 결핵균의 리팜핀, 및 아이나 내성 여부를 동시에 확인하는 방법. A method for detecting and identifying tuberculosis bacteria and non-tuberculosis anti-bacterial bacteria and identifying the rifampin and the resistance of the tuberculosis bacteria.
  2. 제1항에 있어서, 상기 검출 및 확인 균주는 마이코박테리움 투버큘로시스(Mycobacterium tuberculosis) H37Rv, 마이코박테리움 아비움(M. avium), 마이코박테리움 인트라셀룰라(M. intracellulare), 마이코박테리움 스크로풀라시움(M. scrofulaceum), 마이코박테리움 압세수스(M. abscessus), 마이코박테리움 켈로나이(M. chelonae), 마이코박테리움 포르투이툼(M. fortuitum), 마이코박테리움 얼서런스 (M. ulcerans), 마이코박테리움 마리눔(M. marinum ), 마이코박테리움 칸사시(M. kansasii), 마이코박테리움 제나벤스(M. genavense ), 마이코박테리움 시미에 (M. simmiae), 마이코박테리움 테라(M. terrae), 마이코박테리움 넌크로모제니쿰(M. nonchromogenicum), 마이코박테리움 켈라툼(M. celatum), 마이코박테리움 고르도네이(M. gordonae), 마이코박테리움 줄게일(M. szulgail ), 마이코박테리움 무코제니쿰(M. mucogenicum), 마이코박테리움 오박넨스(M. aubagnense), 마이코박테리움 말모엔스(M. malmoense), 마이코박테리움 펠레(M. phlei), 마이코박테리움 스메그마티스 (M. smegmatis ), 마이코박테리움 제노피(M. xenopi), 마이코박테리움 페레그리넘 (M. peregrinum 및 마이코박테리움 프라제센스(M. flavescens)으로 구성된 군으로부터 선택된 것을 특징으로 하는 결핵균 및 비결핵 항산균 검출과 동정 및, 결핵균의 리팜핀 및 아이나 내성 여부를 동시에 확인하는 방법. The method of claim 1, wherein the detection and confirmation strain is Mycobacterium tuberculosis H37Rv, Mycobacterium Abium ( M. avium ), Mycobacterium Intracellulare ( M. intracellulare ), My Foulard when help to M. Te Solarium disk (M. scrofulaceum), Mycobacterium pressure wash's (M. abscessus), Mycobacterium kelronayi (M. chelonae), Mycobacterium M. fortuitum , Mycobacterium Justice ( M. ulcerans ), Mycobacterium marinum ( M. marinum ), Mycobacterium Kansasii ( M. kansasii ), Mycobacterium jenabens ( M. genavense ), mycobacterium simie ( M. simmiae ), mycobacterium terra ( M. terrae ), Mycobacterium nonchromogenicum ( M. nonchromogenicum ), Mycobacterium keratum ( M. celatum ), Mycobacterium gordonae ( M. gordonae ), Mycobacterium zugale ( M. szulgail ), Mycobacterium mucogenicum ( M. mucogenicum ), Mycobacterium Obacens ( M. aubagnense ), Mycobacterium Malmo Enschede (M. malmoense), Pele Mycobacterium (M. phlei), Mycobacterium Smeg Matisse (M. smegmatis), Mycobacterium Geno blood (M. xenopi), Mycobacterium Tampere draw over (M. peregrinum and Mycobacterium prazecens ( M. flavescens ) characterized in that the detection and identification of tuberculosis bacteria and non-tuberculosis acid bacterium, characterized in that selected from the group consisting of, and to simultaneously determine whether the tuberculosis rifampin and eyena resistance.
  3. 제1항에 있어서, 상기 프라이머는 바이오틴이 표지된 것을 특징으로 하는 결핵균 및 비결핵 항산균 검출과 동정 및, 결핵균의 리팜핀 및 아이나 내성 여부를 동시에 확인하는 방법. The method of claim 1, wherein the primer is biotin-labeled and characterized in that detection and identification of tuberculosis bacteria and non-tuberculosis acid bacterium, and whether the tuberculosis rifampin and eye or resistance at the same time.
  4. 서열번호 1 내지 서열번호 12의 프라이머 및 Primers of SEQ ID NO: 1 to SEQ ID NO: 12 and
    서열번호 13 내지 68의 올리고머 프로브가 커플링된 디스크를 포함하는 결핵균 및 비결핵 항산균 검출과 동정 및, 결핵균의 리팜핀, 및 아이나 내성 여부를 동시에 확인하기 위한 키트.A kit for the detection and identification of Mycobacterium tuberculosis and non-tuberculosis antibacterial bacterium comprising a disk coupled with the oligomer probe of SEQ ID NOs.
  5. 제4항에 있어서, 상기 검출 및 확인 균주는 마이코박테리움 투버큘로시스(Mycobacterium tuberculosis) H37Rv, 마이코박테리움 아비움(M. avium), 마이코박테리움 인트라셀룰라(M. intracellulare), 마이코박테리움 스크로풀라시움(M. scrofulaceum), 마이코박테리움 압세수스(M. abscessus), 마이코박테리움 켈로나이(M. chelonae), 마이코박테리움 포르투이툼(M. fortuitum), 마이코박테리움 얼서런스 (M. ulcerans), 마이코박테리움 마리눔(M. marinum ), 마이코박테리움 칸사시(M. kansasii), 마이코박테리움 제나벤스(M. genavense ), 마이코박테리움 시미에 (M. simmiae), 마이코박테리움 테라(M. terrae), 마이코박테리움 넌크로모제니쿰(M. nonchromogenicum), 마이코박테리움 켈라툼(M. celatum), 마이코박테리움 고르도네이(M. gordonae), 마이코박테리움 줄게일(M. szulgail ), 마이코박테리움 무코제니쿰(M. mucogenicum), 마이코박테리움 오박넨스(M. aubagnense), 마이코박테리움 말모엔스(M. malmoense), 마이코박테리움 펠레(M. phlei), 마이코박테리움 스메그마티스 (M. smegmatis ), 마이코박테리움 제노피(M. xenopi), 마이코박테리움 페레그리넘 (M. peregrinum및 마이코박테리움 프라제센스(M. flavescens)으로 구성된 군으로부터 선택된 것을 특징으로 하는 결핵균 및 비결핵 항산균 검출과 동정 및, 결핵균의 리팜핀, 및 아이나 내성 여부를 동시에 확인하기 위한 키트.The method of claim 4, wherein the detection and confirmation strain is Mycobacterium tuberculosis (Mycobacterium tuberculosis) H37Rv, Mycobacterium Avium (M. avium), Mycobacterium intracellular (M. intracellulare), Mycobacterium scrofulasium (M. scrofulaceum),Mycobacterium Absesus (M. abscessus), Mycobacterium Kelowna (M. chelonae), Mycobacterium PortoitumM. fortuitum), Mycobacterium Preferences (M. ulcerans), Mycobacterium marinum (M. marinum ),Mycobacterium KansashiM. kansasii),  Mycobacterium jennavensM. genavense ),Mycobacterium Shimie (M. simmiae), Mycobacterium TerraM. terrae), Mycobacterium nonchromogenicumM. nonchromogenicum), Mycobacterium KelatumM. celatum), Mycobacterium Gordonnai (M. gordonae), Mycobacterium Zulgay (M. szulgail ),Mycobacterium mucogenicumM. mucogenicum), Mycobacterium ObacensM. aubagnense), Mycobacterium MalmoensM. malmoense), Mycobacterium PeleM. phlei), Mycobacterium smegmatis(M. smegmatis ),Mycobacterium GenophyM. xenopi), Mycobacterium Peregrinum (M. peregrinumAnd Mycobacterium prazesenM. flavescensA kit for detecting and identifying tuberculosis bacteria and non-tuberculosis anti-bacterial bacteria characterized in that it is selected from the group consisting of) and, at the same time to check the rifampin, and eye or tolerance of the tuberculosis bacteria.
  6. 제4항에 있어서, 상기 프라이머는 바이오틴이 표지된 것을 특징으로 결핵균 및 비결핵 항산균 검출과 동정 및, 결핵균의 리팜핀, 및 아이나 내성 여부를 동시에 확인하기 위한 키트.According to claim 4, wherein the primer is biotin-labeled, characterized in that the detection and identification of tuberculosis bacteria and non-tuberculosis anti-bacterial bacterium, and a kit for simultaneously confirming the rifampin, and eye or tolerance of Mycobacterium tuberculosis.
PCT/KR2017/008168 2016-08-04 2017-07-28 Diagnostic method for detection and identification of tuberculosis and non-tuberculosis mycobacteria and simultaneous confirmation of rifampin resistance of tuberculosis bacillus on basis of quanta matrix assay platform and kit therefor WO2018026141A1 (en)

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