Ann Clin Microbiol Vol. 20, No. 1, March, 2017 https://doi.org/10.5145/acm.2017.20.1.1 pissn 2288-0585 eissn 2288-6850 Comparative Evaluation of Multiplex Real-Time PCR Assays for Six Pathogens of Sexually Transmitted Infections Hae-Sun Chung, Miae Lee Department of Laboratory Medicine, Ewha Womans University College of Medicine, Seoul, Korea Background: The multiplex real-time PCR assay is a sensitive test for simultaneous detection of various pathogens of sexually transmitted infections (STIs). We evaluated the performance of two multiplex real-time PCR assays for six STI pathogens. Methods: DNA samples after being used to conduct PCR for STI pathogens were stored below 70 o C. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Trichomonas vaginalis (TV) were detected by multiplex real-time PCR with GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR Kits (Infopia, Korea; GeneFinder assay) and Real-Q CT&NG/ MH&TV/MG&UU Kits (BioSewoom, Korea; Real-Q assay). Discrepant results were resolved by another multiplex real-time assay, Anyplex II STI-7 Detection (Seegene, Korea). Any two positive results for the assays were considered true positive. Results: Among 81 samples, the GeneFinder assay detected 63 pathogens from 45 cases (16 CT, 2 NG, 6 MG, 20 MH, 18 UU, and 1 TV) and Real-Q assay detected 66 pathogens from 47 cases (16 CT, 2 NG, 8 MG, 20 MH, 19 UU, and 1 TV). For the results of positive cases and negative cases, the overall concordance rate between the two multiplex real-time assays was 93.8% (Kappa=0.87). For each pathogen, the agreement rates of the two assays ranged from 97.5 to 100% (Kappa>0.8). Conclusion: There was no significant difference between the results of GeneFinder assay and Real-Q assay. Both multiplex real-time PCR assays can be useful methods for the detection of STI pathogens in clinical laboratories. (Ann Clin Microbiol 2017;20:1-6) Key Words: Multiplex real-time polymerase chain reaction, Sexually transmitted infection INTRODUCTION 성매개감염 (sexually transmitted infection, STI) 은흔한감염성질환으로, 사람과사람사이에성접촉을통해전파되는감염을말한다. 성매개감염은심각한이환율과합병증및후유증을유발할수있으며, 임신및출산에도영향을주어태아와신생아건강에도영향을미치기때문에정확한진단과적절한치료가매우중요하다 [1-5]. 성매개감염은원인병원체가세균, 바이러스, 원충, 진균등으로다양하며, 주요원인세균으로는 Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) 등이있고, 원충인 Trichomonas vaginalis (TV) 도흔한원인병원체이다. 원인병원체또는질환별로다양한진단법이 존재하는데, 통상적인배양이나혈청학적검사등기존방법으로는검사가어려운병원체들이많아, 최근에는분자생물학적진단법으로검출하는방법이추천되고있다. 분자생물학적진단법중주로쓰이는것은핵산증폭검사로, Chlamydia 등의병원체에있어서표준진단법으로이용되고있으며, 검체의운반과배양조건이나쁜상황에서유용하게쓰일수있다. 또한, 성매개감염은증상과임상양상이비슷하고중복감염도가능하므로, 다양한병원체를한번에검사하기위해서한검체에서여러표적 DNA를검출하는다중중합효소연쇄반응 (multiplex PCR) 방법이비용과시간을절약할수있다 [2-6]. 실시간중합효소연쇄반응 (real-time PCR) 은중합효소연쇄반응이후전기영동또는부합반응과같은추가과정없이결과분석이가능하므로, 기존중합효소연쇄반응보다증폭산물에의한오염가능성이개선되고검사소요시간도단축되는장점 Received 16 November, 2016, Revised 27 February, 2017, Accepted 28 February, 2017 Correspondence: Miae Lee, Department of Laboratory Medicine, Ewha Womans University College of Medicine, 1071 Anyangcheon-ro, Yangcheon-gu, Seoul 07985, Korea. (Tel) 82-2-2650-5222, (Fax) 82-2-2650-5091, (E-mail) miae@ewha.ac.kr c The Korean Society of Clinical Microbiology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1
2 Ann Clin Microbiol 2017;20(1):1-6 이있다. 또한한쌍의시발체와소식자가함께사용되어비특이적반응이감소되고특이도를향상시킬수있다. 중합효소연쇄반응은한천에전기영동하여결과를판독하는데, 약양성의경우양성또는음성에대한객관적인판독이어려운점이있으며, 이런문제점은특히증폭산물의크기가작은경우에심각하다. 반면, 실시간중합효소연쇄반응은증폭곡선과 threshold cycle (Ct) 값을객관적인지표로활용하여판독하므로판독과정에있어주관적인판단을배제할수있다 [7]. 따라서다중실시간중합효소연쇄반응 (multiplex real-time PCR) 방법은성매개감염의여러원인병원체들을동시에검출할수있는민감하고비용효과적인검사법이라고할수있다. 성매개감염에대한실시간중합효소연쇄반응과기존중합효소연쇄반응과의비교연구에서실시간중합효소연쇄반응이기존의검사방법들보다동일하거나더나은효능을보여주었다 [8,9]. 하지만다수의원인병원체들에대한다중실시간중합효소연쇄반응검사법간의비교평가자료는많지않다. 본연구에서는다중실시간중합효소연쇄반응을이용하여 6 종의성매개감염원인병원체를한검체에서검출하는 GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR (Infopia, Anyang, Korea; GeneFinder assay) 과 Real-Q CT&NG/ MH&TV/MG&UU (BioSewoom, Seoul, Korea; Real-Q assay) 를비교평가하여임상검사실에서의유용성을살펴보았다. MATERIALS AND METHODS 1. 대상및핵산추출 2015년 2월부터 2016년 2월까지서울의한병원진단검사의학과검사실로성매개감염원인병원체 PCR 검사가의뢰되었던검체를대상으로하였다. DNA 추출은상품화된 QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) 를이용하였다. 추출한 DNA로의뢰된검사를시행후잔여 DNA 검체를평가를위한검사전까지 70 o C에냉동하였다. 2. GeneFinder assay GeneFinder assay는 STD I과 STD II 시약각각을한튜브에서검사하여검체당총 2개의튜브를사용한다. STD I은 CT, NG, UU를, STD II는 MG, MH, TV를검출한다. PCR 반응혼합물을준비한후 Applied Biosystems 7500 Real-time PCR Instrument system (Life Technologies, Carlsbad, CA, USA; AB7500) 을이용하여 50 o C 2분, 95 o C 10분, 95 o C 15초 /60 o C 60 초를 40회반복하는조건으로반응시켰다. 검체결과해석은표적의 Ct값이 40 미만인경우는양성으로, 표적의 Ct값이 40 이상이거나, 표적이증폭되지않고내부대조물질 (internal control, IC) 의 Ct값이 40 이하인경우는음성으로판정하였다. 시약내에포함된음성대조가증폭되지않고양성대조의 Ct값 이모두허용범위이내임을확인하고결과를해석하였다. 3. Real-Q assay Real-Q assay는 CT&NG, MH&TV, MG&UU 시약각각을한튜브에서검사하여검체당총 3개의튜브를사용한다. PCR 반응혼합물을준비한후 AB7500을이용하여 50 o C 2분, 95 o C 10분, 95 o C 15초 /60 o C 45초를 45회반복하는조건으로반응시켰다. 검체결과해석은표적의 Ct값이 38 미만인경우는양성으로, 표적의 Ct값이 38 이상이거나, 표적이증폭되지않고 IC 의 Ct값이 38 이하인경우는음성으로판정하였다. 시약내에포함된음성대조가증폭되지않고양성대조의 Ct값이모두허용범위이내임을확인하고결과를해석하였다. 4. 결과분석및통계처리 두검사법의결과가일치할경우진양성 (true positive) 또는진음성 (true negative) 으로판단하였다. 두검사법의결과가불일치할경우, 다중실시간중합효소연쇄반응을이용하여성매개감염원인병원체를검출하는제3의시약인 Anyplex II STI-7 Detection (Seegene, Seoul, Korea; Anyplex assay) 으로검사하여, 두개이상의검사법에서양성인결과를진양성으로판단하였다. 각병원체별로두검사법의일치율을구하고, Kappa 통계량을구하였다. 양성일치율과음성일치율은아래의식으로계산하였다. 양성일치율 = 음성일치율 = 2 양성일치개수전체개수 + 양성일치개수 음성일치개수 2 음성일치개수전체개수 양성일치개수 + 음성일치개수 두검사법의병원체검출성능비교를위해 McNemar 카이제곱분석을시행하였다. 통계분석은 MedCalc Version 10.0 (MedCalc Software bvba, Ostend, Belgium) 을이용하여시행하였다. P값이 0.05 미만인경우통계학적으로의미가있는것으로해석하였다. RESULTS 총 81검체에대해검사를시행하였다. GeneFinder assay에서는 45개의검체에서총 63개의병원체 (CT 16주, NG 2주, MG 6주, MH 20주, UU 18주, TV 1주 ) 가검출되었고, Real-Q assay 에서는 47개의검체에서총 66개의병원체 (CT 16주, NG 2주, MG 8주, MH 20주, UU 19주, TV 1주 ) 가검출되었다. 수집된검체중중복검출검체는양성검체 48개중 16개였다. 단독양성과 2개이상양성을구분했을때각병원체의검출양상은
Hae-Sun Chung and Miae Lee : Multiplex Real-Time PCR Assays for STI 3 Table 1과같다. 중복검출중 MH와 UU 2중양성이 4 검체, CT와 MH 2중양성이 3 검체, CT와 MG, CT와 UU 2중양성이각각 2 검체가있었다. CT와 NG의 2중양성은 1 검체였다. 3중양성은 3 검체에서있었다 (CT, MG, MH; CT, MH, UU; NG, MH, UU). 양성검체 ( 병원체모두보고 ) 와음성검체결과를정확하게보고하는것에대한두검사의일치율은 93.8% 였다 (Kappa= 0.87). 두검사법의검출성능의차이는유의하지않았다 (P= 0.37) (Table 2). 각병원체별일치율은 97.5-100% 였다 (Kappa>0.8). CT 16 주, NG 2주, TV 1주의결과는모두일치하였다. 불일치한결과가있었던 MG, MH, UU에대한두검사의검출성능의차이는통계적으로유의하지않았다 (P 0.5) (Table 3). 두검사에서불일치했던검체에대해 Anyplex assay로확인한결과, 모두진양성으로판정되었다. 불일치했던병원체의 Ct 값은각병원체의양성검체 Ct값중에서가장높은값들이었고, 대부분 Anyplex assay에서의반응강도 (intensity) 가낮은편이었다 (Table 4). DISCUSSION 성매개감염에대한검사실진단검사는증상이있는환자에서정확한원인병원체를진단하는데필수적이다. 또한, 특히임신부를포함한성적으로활동적인여성에서의 CT와 NG를비롯한성매개감염검진은, 합병증을억제하고성매개감염을예방하는데효과적이므로, 선별검사를시행하는것이권고되고있다 [10]. 따라서, 성매개감염의진단검사는정확하고효율적으로진행되어야한다. 이를위해최근다양한병원체를한번에민감하게검출할수있는다중실시간중합효소연쇄반응검사법이유용하게쓰이고있으며, 최근 GeneFinder assay와 Real-Q assay 등의검사가국내검사실에도입되었다. 본연구결과두검사법은우수한일치율을보여주었으며, 성매개감염병원체검출성능에유의한차이는발견되지않았다. 일부두검사에서불일치했던병원체들의 Ct값이모두양성검체중가장높은값을보였던것으로보아, 검체내낮은농도로존재하였던것으로추정할수있다. 판정기준치 (cutoff) 근처의농도에서는양성 / 음성결과가같은검사법내에서도다 Table 1. Results of detection of single and multiple pathogens by GeneFinder and Real-Q assays Pathogen Single pathogen Multiple pathogens 2 pathogens 3 pathogens True positive GeneFinder Real-Q True positive GeneFinder Real-Q True positive GeneFinder Real-Q Chlamydia trachomatis 6 6 6 8 8 8 2 2 2 Neisseria gonorrhoeae 0 0 0 1 1 1 1 1 1 Mycoplasma genitalium 5 3 5 2 2 2 1 1 1 Mycoplasma hominis 11 10 10 7 7 7 3 3 3 Ureaplasma urealyticum 10 10 10 7 7 7 2 1 2 Trichomonas vaginalis 0 0 0 1 1 1 0 0 0 Sum (No. of cases) 32 (32) 29 (29) 31 (31) Abbreviations: GeneFinder, GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR; Real-Q, Real-Q CT&NG/MH&TV/ MG&UU. 26 (13) 26 (13) 26 (13) 9 (3) 8 (2) 9 (3) Table 2. Overall agreement between GeneFinder and Real-Q assays Assays Real-Q Positive cases Negative cases Sum Kappa Agreement rate P pos* P neg* P value GeneFinder Positive cases 43 1 44 0.87 93.8 94.5 93.0 0.37 Negative cases 4 33 37 Sum 47 34 81 *Proportion of positive agreement (P pos), calculated as twice the number of agreed positive cases/(total number of cases+number of agreed positive cases number of agreed negative cases); and proportion of negative agreement (P neg), calculated as twice the number of agreed negative cases/(total number of cases+number of agreed positive cases number of agreed negative cases). by McNemar test. Number of cases of GeneFinder negative and Real-Q positive included one mixed infection case; one of three pathogens was missed. Abbreviations: GeneFinder, GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR; Real-Q, Real-Q CT&NG/MH&TV/ MG&UU.
4 Ann Clin Microbiol 2017;20(1):1-6 Table 3. Comparison of the results of GeneFinder and Real-Q assays according to the pathogens Pathogen Assays Results Real-Q Positive Negative Sum Kappa Agreement rate P pos* P neg* P value Chlamydia trachomatis GeneFinder Positive 16 0 16 1.00 100 100 100 Negative 0 65 65 Sum 16 65 81 Neisseria gonorrhoeae GeneFinder Positive 2 0 2 1.00 100 100 100 Negative 0 79 79 Sum 2 79 81 Mycoplasma genitalium GeneFinder Positive 6 0 6 0.84 97.5 85.7 98.6 0.50 Negative 2 73 75 Sum 8 73 81 Mycoplasma hominis GeneFinder Positive 20 1 21 0.94 97.5 95.2 98.3 1.00 Negative 1 59 60 Sum 21 60 81 Ureaplasma urealyticum GeneFinder Positive 18 0 18 0.96 98.8 97.3 99.2 1.00 Negative 1 62 63 Sum 19 62 81 Trichomonas vaginalis GeneFinder Positive 1 0 1 1.00 100 100 100 Negative 0 80 80 Sum 1 80 81 *Proportion of positive agreement (P pos), calculated as twice the number of agreed positives/(total number of specimens+number of agreed positives number of agreed negatives); and proportion of negative agreement (P neg), calculated as twice the number of agreed negatives/(total number of specimens+number of agreed positives number of agreed negatives). P values were calculated by McNemar test for pathogens with discrepant results. Abbreviations: GeneFinder, GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR; Real-Q, Real-Q CT&NG/MH&TV/ MG&UU. Table 4. Interpretation of discrepant results Pathogen Anyplex Real-Q GeneFinder Results Intensity Results Ct* Interpretation Results Ct Interpretation Mycoplasma genitalium P ++ P 34.62 True positive N No Ct False negative P + P 35.99 True positive N No Ct False negative Mycoplasma hominis P + P 36.43 True positive N No Ct False negative P + N No Ct False negative P 37.50 True positive Ureaplasma urealyticum P + P 36.94 True positive N No Ct False negative *Ct ranges for positive specimens of M. genitalium, M. hominis, and U. urealyticumwere 23.09-35.99 (median 29.23), 19.70-36.43 (median 26.67), and 23.82-36.94 (median 32.49) by Real-Q assay. Ct range for positive specimens of M. hominis was 20.64-37.50 (median 27.35) by GeneFinder assay. Abbreviations: Anyplex, Anyplex II STI-7 Detection; GeneFinder, GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR; Real-Q, Real-Q CT&NG/MH&TV/MG&UU. 르게나올수있다. 민감도의차이도영향을미치는데, MG의경우 GeneFinder assay는제조사의설명서에분석적민감도를 10 copies/μl로제시한반면, Real-Q assay의 probit analysis 분석결과에의한최소검출한계를 28 copies/μl로제시하고있어, GeneFinder assay보다높다. 하지만각제조사에서분석적민감도와최소검출한계를구한방법에차이가있어두수치를단순하게비교하는것은적절하지않으며, 제조사에서제시하 는분석적민감도또는최소검출한계를검사실에서검증 (verification) 해서확인하는것이필요하다. 본연구의결과에서는 2개의 MG이 Real-Q assay에서는검출된반면, GeneFiner assay로는검출되지않았다. 그중한검체는 Anyplex assay에서반응강도가 + (Ct값범위 40-50) 로매우낮은농도로있었을것으로추정할수있었다. 한편, 검사법간차이가있을때, 검사자의오류나검사법에따라검출이어려운특정유전적변
Hae-Sun Chung and Miae Lee : Multiplex Real-Time PCR Assays for STI 5 이주의가능성도고려해야한다. UU은성매개감염의원인균이지만, 상재균으로분자진단검사에서흔히검출되는것으로알려져있어판단에주의를요한다 [11]. 연구자소속병원검사실에서기존에사용한다중중합효소연쇄반응에서 UU는 PCR 증폭산물의크기가 130 bp였으며, 한천에전기영동한결과가육안으로양성여부를판정하기에어려운약한강도의밴드 (band) 를보이는경우가다수있어판독에어려움이많았다. 그에비해, 본연구에서평가한다중실시간중합효소연쇄반응검사법들은객관적인 Ct값을제시해주어판독이용이하였다. 흥미로운점은, 다중실시간중합효소연쇄반응검사법으로양성이나온 UU의 Ct값들이두검사법에서모두다른병원체들 (CT, MH) 의 Ct값에비해유의하게높았다 (data not shown). 즉, UU이양성인경우, 낮은농도로존재하는경우가많은것을알수있었다. 한편, 의료기관별, 환자군별 UU의양성률을비교분석하는연구가 UU의임상적의의를파악하는데도움이될것이다. Ureaplasma species는 UU (formerly known as U. urealyticum biovar 2) 와 U. parvum (formerly known as U. urealyticum biovar 1) 으로세분할수있다. U. parvum은성매개감염에서어떤역할을하는지아직명확하지않고상재균으로있는경우도많으며, CT와같이검출되는경우가많은것으로보고된바있다 [12]. 최근 UU과 U. parvum을구분하여검출할필요성도대두되고있다 [8]. 본연구에서평가한두검사법은모두 UU을검출하도록설계되었으며, U. parvum을검출할수는없다. MH의경우도 U. parvum과유사하게성매개감염의원인균으로서의증거가부족한상황으로상재균으로있는것으로간주되기도한다 [11]. 본연구에서검사하였던검체중 UU과 MH의중복검출건수가 3중양성 2건을포함하여 6건으로중복검출의양상중가장흔한것으로나타났는데, 본연구는임상정보를배제한잔여 DNA 검체를이용하였으므로, 임상적으로의미있는병원균인지는확인할수없었다. 실제임상상황에서는 UU과 MH가양성인경우, 임상증상과치료의필요성과효과등을고려하여치료여부를결정해야하겠다. 기존의국내보고에따르면, 검체종류와환자군에따라다소차이가있었지만, 중복감염이전체감염의 20-40% 에이르는것으로나타났다 [8,13]. 본연구의연구기간동안연구자소속병원검사실의중복양성은전체양성의약 30% 로알려진비율과크게다르지않았다 (data not shown). 본연구에서중복양성검체는전체양성검체중 1/3 (16/48) 이었다. 다중중합효소연쇄반응 (multiplex PCR) 검사법은중복감염을한번에검출할수있다는장점이있어성매개감염의진단에유용하게이용되고있다. 하지만, 다중중합효소연쇄반응검사법은한개의표적을증폭하는단일중합효소연쇄반응 (singleplex PCR) 에비해, 시발체간간섭현상및증폭저해현상으로증폭효율이저하되거나비특이적증폭산물이형성되는등의문제점이초창 기다중중합효소연쇄반응검사법들에서제기된바있다 [14-16]. 본연구에서평가한두검사법은 2개와 3개의튜브를사용하여 1개의튜브에서각각 3개와 2개의병원체를검출하여총 6종의병원체를검출하는다중중합효소연쇄반응검사법을택하고있다. 따라서, 실제하나의튜브에서중복양성이검출된검체는양성 16 검체중 6 검체였다 (data not shown). 각튜브의반응을종합적으로하나의검사결과로생각했을때, 두검사법모두대부분의 2종이상양성검체에서정확하게각각의병원체가검출되어, 중복양성을검출하는데유용하게사용할수있음을확인하였다. 본평가에서는 NG와 TV의양성균주수가매우적어정확한성능을평가하기에는한계가있었다. 본연구의연구기간동안연구자소속병원검사실의 NG와 TV 양성률은각각 1.5%, 0.9% 로매우드물어, 양성검체를확보하는데어려움이있었기때문이다. 두검사법비교시양성균주수가적게포함되었을때일치율과 Kappa 통계량이높게나오는문제점이있다. 추후두병원체에대해서는좀더많은수의양성검체를포함하여평가를진행할필요가있겠다. 또한, 본연구에서검체의임상정보와배양등다른미생물검사결과를포함하지않은점도중요한제한점이다. 추후미생물검사결과와임상정보를종합적으로검토하는비교평가를하여두검사법의임상적의의를밝히는연구가필요하겠다. 본연구에서는 6종의성매개감염의원인병원체를검출하는다중실시간중합효소연쇄반응검사법인 GeneFinder assay와 Real-Q assay를비교평가하였다. 결론적으로, 두검사법간에유의한차이를발견할수없었으며, 두다중실시간중합효소연쇄반응검사법모두임상검사실에서성매개감염의병원체를검출하는데유용하게쓰일것으로기대한다. REFERENCES 1. WHO. Guidelines for the management of sexually transmitted infections. Geneva; World Health Organization, 2003:1-5. 2. Korea Centers for Disease Control and Prevention and Korean Association of Urogenital Tract Infection and Inflammation, ed. Sexually Transmitted Infections Korean Guidelines. Cheongju and Seoul; Korea Centers for Disease Control and Prevention and Korean Association of Urogenital Tract Infection and Inflammation, 2011:3-41. 3. Workowski KA and Bolan GA; Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep 2015;64:1-137. 4. Hong SG. Genital infections. In: Korean Society of Infectoius Diseases, ed. Infectious Diseases. 2nd ed. Seoul; KoonJa Publishing Inc., 2014:239-53. 5. Lee SJ, Lee HJ, et al. Sexually transmitted infections. In: Korean Association of Urogenital Tract Infection and Inflammation, ed. Urogenital Tract Infection and Inflammation. Seoul; Ilchokak Publishing Co., Ltd., 2013:254-358. 6. Trembizki E, Costa AM, Tabrizi SN, Whiley DM, Twin J.
6 Ann Clin Microbiol 2017;20(1):1-6 Opportunities and pitfalls of molecular testing for detecting sexually transmitted pathogens. Pathology 2015;47:219-26. 7. Koo SH, Park KU, et al. Microbiological test methods. In: Korean Society for Laboratory Medicine, ed. Laboratory Medicine. 5th ed, Seoul; Panmun Education, 2014:517-36. 8. Choe HS, Lee DS, Lee SJ, Hong SH, Park DC, Lee MK, et al. Performance of Anyplex TM II multiplex real-time PCR for the diagnosis of seven sexually transmitted infections: comparison with currently available methods. Int J Infect Dis 2013;17: e1134-40. 9. Kim Y, Kim J, Lee KA. Analytical performance of multiplex real-time PCR for six sexually transmitted pathogens. Clin Lab 2015;61:1749-54. 10. LeFevre ML; U.S. Preventive Services Task Force. Screening for Chlamydia and gonorrhea: U.S. Preventive Services Task Force recommendation statement. Ann Intern Med 2014;161:902-10. 11. Lim DH, Lee SJ, Shim BS, Kim CS, Kim ME, Cho YH. The new Korean guideline for sexually transmitted infections. Korean J Korean J Urogenit Tract Infect Inflamm 2011;6:96-113. 12. Wei HB, Zou SX, Yang XL, Yang DQ, Chen XD. Development of multiplex real-time quantitative PCR for simultaneous detection of Chlamydia trachomatis and Ureaplasma parvum. Clin Biochem 2012;45:663-7. 13. Kim Y, Kim J, Lee KA. Prevalence of sexually transmitted infections among healthy Korean women: implications of multiplex PCR pathogen detection on antibiotic therapy. J Infect Chemother 2014;20:74-6. 14. Markoulatos P, Siafakas N, Moncany M. Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal 2002;16: 47-51. 15. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 2000;13:559-70. 16. Gunson RN, Bennett S, Maclean A, Carman WF. Using multiplex real time PCR in order to streamline a routine diagnostic service. J Clin Virol 2008;43:372-5. = 국문초록 = 6 종의성매개감염병원체검출을위한다중실시간중합효소연쇄반응검사법의비교평가 이화여자대학교의과대학진단검사의학교실정혜선, 이미애 배경 : 다중실시간중합효소연쇄반응검사법은다양한성매개감염병원체를동시에검출할수있는민감한검사법이다. 본연구에서는다중실시간중합효소연쇄반응을이용하여 6종의성매개감염병원체를검출하는두검사법을비교평가하였다. 방법 : 성매개감염원인병원체 PCR 검사를시행한후잔여 DNA 검체를 70 o C에냉동하였다. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), Trichomonas vaginalis (TV) 를 GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR Kits (Infopia, Korea; GeneFinder assay) 와 Real-Q CT&NG/MH&TV/MG&UU Kits (BioSewoom, Korea; Real-Q assay) 로검사하였다. 두검사법의결과가불일치할경우, 다중실시간중합효소연쇄반응을이용하여성매개감염원인병원체를검출하는다른시약인 Anyplex II STI-7 Detection (Seegene, Korea) 으로검사하여, 두개이상의검사법에서양성인결과를진양성으로판단하였다. 결과 : 81개검체중 GeneFinder assay에서는 45개의검체에서총 63개의병원체 (16 CT, 2 NG, 6 MG, 20 MH, 18 UU, 1 TV) 가검출되었고, Real-Q assay 에서는 47개의검체에서총 66개의병원체 (16 CT, 2 NG, 8 MG, 20 MH, 19 UU, 1 TV) 가검출되었다. 양성검체와음성검체결과를정확하게보고하는것에대한두검사의일치율은 93.8% 였다 (Kappa=0.87). 각병원체별일치율은 97.5-100% 였다 (Kappa>0.8). 결론 : GeneFinder assay와 Real-Q assay 간에유의한차이를발견할수없었으며, 두다중실시간중합효소연쇄반응검사법모두임상검사실에서성매개감염의병원체를검출하는데유용하게쓰일것이다. [Ann Clin Microbiol 2017;20:1-6] 교신저자 : 이미애, 07985, 서울시양천구안양천로 1071 이화여자대학교의과대학진단검사의학교실 Tel: 02-2650-5222, Fax: 02-2650-5091 E-mail: miae@ewha.ac.kr