Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1995 Nov;33(11):3049–3053. doi: 10.1128/jcm.33.11.3049-3053.1995

Multiplex PCR for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Genitourinary specimens.

J B Mahony 1, K E Luinstra 1, M Tyndall 1, J W Sellors 1, J Krepel 1, M Chernesky 1
PMCID: PMC228636  PMID: 8576375

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (313.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anceschi M. M., Falcinelli C., Pieretti M., Cosmi E. V. Multiple primer pairs polymerase chain reaction for the detection of human papillomavirus types. J Virol Methods. 1990 Apr;28(1):59–65. doi: 10.1016/0166-0934(90)90087-v. [DOI] [PubMed] [Google Scholar]
  2. Bauwens J. E., Clark A. M., Loeffelholz M. J., Herman S. A., Stamm W. E. Diagnosis of Chlamydia trachomatis urethritis in men by polymerase chain reaction assay of first-catch urine. J Clin Microbiol. 1993 Nov;31(11):3013–3016. doi: 10.1128/jcm.31.11.3013-3016.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birkenmeyer L., Armstrong A. S. Preliminary evaluation of the ligase chain reaction for specific detection of Neisseria gonorrhoeae. J Clin Microbiol. 1992 Dec;30(12):3089–3094. doi: 10.1128/jcm.30.12.3089-3094.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bobo L., Munoz B., Viscidi R., Quinn T., Mkocha H., West S. Diagnosis of Chlamydia trachomatis eye infection in Tanzania by polymerase chain reaction/enzyme immunoassay. Lancet. 1991 Oct 5;338(8771):847–850. doi: 10.1016/0140-6736(91)91502-l. [DOI] [PubMed] [Google Scholar]
  5. Cano R. J., Palomares J. C., Torres M. J., Klem R. E. Evaluation of a fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae. Eur J Clin Microbiol Infect Dis. 1992 Jul;11(7):602–609. doi: 10.1007/BF01961666. [DOI] [PubMed] [Google Scholar]
  6. Chernesky M. A., Jang D., Lee H., Burczak J. D., Hu H., Sellors J., Tomazic-Allen S. J., Mahony J. B. Diagnosis of Chlamydia trachomatis infections in men and women by testing first-void urine by ligase chain reaction. J Clin Microbiol. 1994 Nov;32(11):2682–2685. doi: 10.1128/jcm.32.11.2682-2685.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chernesky M., Castriciano S., Sellors J., Stewart I., Cunningham I., Landis S., Seidelman W., Grant L., Devlin C., Mahony J. Detection of Chlamydia trachomatis antigens in urine as an alternative to swabs and cultures. J Infect Dis. 1990 Jan;161(1):124–126. doi: 10.1093/infdis/161.1.124. [DOI] [PubMed] [Google Scholar]
  8. De Schryver A., Meheus A. Epidemiology of sexually transmitted diseases: the global picture. Bull World Health Organ. 1990;68(5):639–654. [PMC free article] [PubMed] [Google Scholar]
  9. Hay P. E., Thomas B. J., Gilchrist C., Palmer H. M., Gilroy C. B., Taylor-Robinson D. The value of urine samples from men with non-gonococcal urethritis for the detection of Chlamydia trachomatis. Genitourin Med. 1991 Apr;67(2):124–128. doi: 10.1136/sti.67.2.124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ho B. S., Feng W. G., Wong B. K., Egglestone S. I. Polymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples. J Clin Pathol. 1992 May;45(5):439–442. doi: 10.1136/jcp.45.5.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jaschek G., Gaydos C. A., Welsh L. E., Quinn T. C. Direct detection of Chlamydia trachomatis in urine specimens from symptomatic and asymptomatic men by using a rapid polymerase chain reaction assay. J Clin Microbiol. 1993 May;31(5):1209–1212. doi: 10.1128/jcm.31.5.1209-1212.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Korch C., Hagblom P., Ohman H., Göransson M., Normark S. Cryptic plasmid of Neisseria gonorrhoeae: complete nucleotide sequence and genetic organization. J Bacteriol. 1985 Aug;163(2):430–438. doi: 10.1128/jb.163.2.430-438.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Loeffelholz M. J., Lewinski C. A., Silver S. R., Purohit A. P., Herman S. A., Buonagurio D. A., Dragon E. A. Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction. J Clin Microbiol. 1992 Nov;30(11):2847–2851. doi: 10.1128/jcm.30.11.2847-2851.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mahony J. B., Luinstra K. E., Jang D., Sellors J., Chernesky M. A. Chlamydia trachomatis confirmatory testing of PCR-positive genitourinary specimens using a second set of plasmid primers. Mol Cell Probes. 1992 Oct;6(5):381–388. doi: 10.1016/0890-8508(92)90031-r. [DOI] [PubMed] [Google Scholar]
  15. Mahony J. B., Luinstra K. E., Sellors J. W., Chernesky M. A. Comparison of plasmid- and chromosome-based polymerase chain reaction assays for detecting Chlamydia trachomatis nucleic acids. J Clin Microbiol. 1993 Jul;31(7):1753–1758. doi: 10.1128/jcm.31.7.1753-1758.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mahony J. B., Luinstra K. E., Sellors J. W., Jang D., Chernesky M. A. Confirmatory polymerase chain reaction testing for Chlamydia trachomatis in first-void urine from asymptomatic and symptomatic men. J Clin Microbiol. 1992 Sep;30(9):2241–2245. doi: 10.1128/jcm.30.9.2241-2245.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mahony J. B., Luinstra K. E., Sellors J. W., Pickard L., Chong S., Jang D., Chernesky M. A. Role of confirmatory PCRs in determining performance of Chlamydia Amplicor PCR with endocervical specimens from women with a low prevalence of infection. J Clin Microbiol. 1994 Oct;32(10):2490–2493. doi: 10.1128/jcm.32.10.2490-2493.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nedjar S., Biswas R. M., Hewlett I. K. Co-amplification of specific sequences of HCV and HIV-1 genomes by using the polymerase chain reaction assay: a potential tool for the simultaneous detection of HCV and HIV-1. J Virol Methods. 1991 Dec;35(3):297–304. doi: 10.1016/0166-0934(91)90071-7. [DOI] [PubMed] [Google Scholar]
  19. Ossewaarde J. M., Rieffe M., Rozenberg-Arska M., Ossenkoppele P. M., Nawrocki R. P., van Loon A. M. Development and clinical evaluation of a polymerase chain reaction test for detection of Chlamydia trachomatis. J Clin Microbiol. 1992 Aug;30(8):2122–2128. doi: 10.1128/jcm.30.8.2122-2128.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Palmer H. M., Gilroy C. B., Thomas B. J., Hay P. E., Gilchrist C., Taylor-Robinson D. Detection of Chlamydia trachomatis by the polymerase chain reaction in swabs and urine from men with non-gonococcal urethritis. J Clin Pathol. 1991 Apr;44(4):321–325. doi: 10.1136/jcp.44.4.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schachter J., Pang F., Parks R. M., Smith R. F., Armstrong A. S. Use of gonozyme on urine sediment for diagnosis of gonorrhea in males. J Clin Microbiol. 1986 Jan;23(1):124–125. doi: 10.1128/jcm.23.1.124-125.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sellors J., Mahony J., Jang D., Pickard L., Castriciano S., Landis S., Stewart I., Seidelman W., Cunningham I., Chernesky M. Rapid, on-site diagnosis of chlamydial urethritis in men by detection of antigens in urethral swabs and urine. J Clin Microbiol. 1991 Feb;29(2):407–409. doi: 10.1128/jcm.29.2.407-409.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Singal S. S., Reichman R. C., Graman P. S., Greisberger C., Trupei M. A., Menegus M. A. Isolation of Chlamydia trachomatis from men with urethritis: relative value of one vs. two swabs and influence of concomitant gonococcal infection. Sex Transm Dis. 1986 Jan-Mar;13(1):50–52. doi: 10.1097/00007435-198601000-00011. [DOI] [PubMed] [Google Scholar]
  24. Snijders P. J., van den Brule A. J., Schrijnemakers H. F., Snow G., Meijer C. J., Walboomers J. M. The use of general primers in the polymerase chain reaction permits the detection of a broad spectrum of human papillomavirus genotypes. J Gen Virol. 1990 Jan;71(Pt 1):173–181. doi: 10.1099/0022-1317-71-1-173. [DOI] [PubMed] [Google Scholar]
  25. Tyndall M. W., Nasio J., Maitha G., Ndinya-Achola J. O., Plummer F. A., Sellors J. W., Luinstra K. E., Jang D., Mahony J. B., Chernesky M. A. Leukocyte esterase urine strips for the screening of men with urethritis--use in developing countries. Genitourin Med. 1994 Feb;70(1):3–6. doi: 10.1136/sti.70.1.3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Vandenvelde C., Verstraete M., Van Beers D. Fast Multiplex polymerase chain reaction on boiled clinical samples for rapid viral diagnosis. J Virol Methods. 1990 Nov;30(2):215–227. doi: 10.1016/0166-0934(90)90022-8. [DOI] [PubMed] [Google Scholar]
  27. Zimmerman H. L., Potterat J. J., Dukes R. L., Muth J. B., Zimmerman H. P., Fogle J. S., Pratts C. I. Epidemiologic differences between chlamydia and gonorrhea. Am J Public Health. 1990 Nov;80(11):1338–1342. doi: 10.2105/ajph.80.11.1338. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES