Mahony J B, Luinstra K E, Tyndall M, Sellors J W, Krepel J, Chernesky M
Regional Virology and Chlamydiology Laboratory, McMaster University, Hamilton, Canada.
J Clin Microbiol. 1995 Nov;33(11):3049-53. doi: 10.1128/jcm.33.11.3049-3053.1995.
We developed a multiplex PCR (M-PCR) assay for the simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. M-PCR employed C. trachomatis-specific primers KL1-KL2 and N. gonorrhoeae-specific primers HO1-HO3 and produced products of 241 and 390 bp, respectively. PCR products were easily detected by agarose gel electrophoresis and confirmed by Southern hybridization using labelled oligonucleotide probes. M-PCR had a sensitivity of 10 fg of C. trachomatis and N. gonorrhoeae DNA (equivalent to 1 to 2 genome copies). M-PCR detected the presence of C. trachomatis and N. gonorrhoeae DNA in 15 male urethral and 12 female endocervical specimens, 3 of which were positive for C. trachomatis, 18 of which were positive for N. gonorrhoeae and 6 of which were positive for both organisms. M-PCR was evaluated further by testing 200 male first void urine (FVU) specimens, of which 18 were positive by C. trachomatis PCR and Chlamydiazyme and 4 were positive by C. trachomatis PCR but negative by Chlamydiazyme. All 22 FVU specimens were positive by a confirmatory PCR using a second plasmid target and were positive by M-PCR. Ten of 11 men with cultures that were positive for N. gonorrhoeae had FVU specimens that were positive by both N. gonorrhoeae PCR and M-PCR. Two other men with negative N. gonorrhoeae urethral cultures had FVU specimens that were positive by N. gonorrhoeae PCR, by two confirmatory N. gonorrhoeae PCR assays using 165 rRNA and cytosine methyltransferase primers, and by M-PCR. The sensitivity of M-PCR for detecting C. trachomatis was 100% (22 of 22 specimens), compared with 81.8% (18 of 22 specimens) for enzyme immunoassay. Sensitivity of M-PCR for N. gonorrhoeae was 92.3% (12 of 13 specimens) compared with 84.6% (11 of 13 specimens) for urethral culture. The specificity of M-PCR was 100% for both C. trachomatis (178 of 13 specimens) and N. gonorrhoeae (187 of 187 specimens). M-PCR testing of FVU specimens provided a sensitive and noninvasive method for detecting C. trachomatis and N. gonorrhoeae infection in men.
我们开发了一种多重聚合酶链反应（M-PCR）检测方法，用于同时检测沙眼衣原体和淋病奈瑟菌。M-PCR采用沙眼衣原体特异性引物KL1-KL2和淋病奈瑟菌特异性引物HO1-HO3，分别产生241和390 bp的产物。聚合酶链反应产物可通过琼脂糖凝胶电泳轻松检测，并使用标记的寡核苷酸探针通过Southern杂交进行确认。M-PCR对沙眼衣原体和淋病奈瑟菌DNA的灵敏度为10 fg（相当于1至2个基因组拷贝）。M-PCR在15份男性尿道标本和12份女性宫颈标本中检测到沙眼衣原体和淋病奈瑟菌DNA的存在，其中3份沙眼衣原体阳性，18份淋病奈瑟菌阳性，6份两种病原体均阳性。通过检测200份男性首次晨尿（FVU）标本对M-PCR进行了进一步评估，其中18份通过沙眼衣原体聚合酶链反应和衣原体酶免疫测定呈阳性，4份通过沙眼衣原体聚合酶链反应呈阳性但衣原体酶免疫测定呈阴性。所有22份FVU标本通过使用第二个质粒靶标的验证性聚合酶链反应呈阳性，并且通过M-PCR呈阳性。11份淋病奈瑟菌培养阳性的男性中，有10人的FVU标本通过淋病奈瑟菌聚合酶链反应和M-PCR均呈阳性。另外两名淋病奈瑟菌尿道培养阴性的男性，其FVU标本通过淋病奈瑟菌聚合酶链反应、使用165 rRNA和胞嘧啶甲基转移酶引物的两种验证性淋病奈瑟菌聚合酶链反应测定以及M-PCR呈阳性。M-PCR检测沙眼衣原体的灵敏度为100%（22份标本中的22份），而酶免疫测定为81.8%（22份标本中的18份）。M-PCR检测淋病奈瑟菌的灵敏度为92.3%（13份标本中的12份），而尿道培养为84.6%（13份标本中的11份）。M-PCR对沙眼衣原体（13份标本中的178份）和淋病奈瑟菌（187份标本中的187份）的特异性均为100%。对FVU标本进行M-PCR检测为检测男性沙眼衣原体和淋病奈瑟菌感染提供了一种灵敏且非侵入性的方法。
