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Nucleic acid amplification tests have the ability to specifically amplify small quantities of DNA and hence have been used successfully in the diagnosis of STDs1, 2 An in-house polymerase chain reaction (PCR) method was developed and evaluated for the detection of Neisseria gonorrhoeae DNA in the urogenital specimens collected (with consent) from patients visiting an STD clinic in India.
The primers (forward primer 5'-CAACTATTCCCGATTGCGA-3' and reverse primer 5'-GTTATACAGCTTCGCCTGAA-3') amplify the 221–480 bp region of orf1 gene. Clinical isolates (n = 40) of N gonorrhoeae were recovered from urethral or cervical swabs by inoculation onto modified Thayer-Martin medium and identified by Gram stain, colony morphology, positive oxidase, and rapid carbohydrate utilisation test. For PCR the clinical samples (n = 489) were centrifuged (30 minutes, 14 000 g) and the cell pellet was lysed with 50 mM TRIS-HCl (pH 7.5) 1% Triton X-100, 1 mM EDTA, 250 μg of proteinase K per ml at 37° for 1 hour, boiled for 10 minutes, and centrifuged. Eight μl of lysate was used for amplification (40 cycles) under standard conditions. Each cycle consisted of 30 seconds at 94°C, 30 seconds at 52°C, and 1 minute at 72°C. The amplified PCR product (10 μl) was analysed by electrophoresis in a 2% agarose gel and characterised by sequencing.
An amplified product of 260 base pairs (bp) of orf1 gene was observed with all N gonorrhoeae isolates but not when DNA from the other non-gonococcal strains (17 closely related Neisseria species, Corynebacterium, Chlamydia trachomatis, Candida, syphilis, and members of Enterobacteriaceae) was used as template. For the 427 clinical swabs collected from men, 379 were positive and 46 were negative by both culture method and orf1-PCR assay. Urethral specimens from two men were culture negative but PCR positive for orf1 gene. Since these two samples tested PCR positive for cppB gene of N gonorrhoeae3 they were considered true positives. Thus, a total of 381 men (89%) were classified as true positives based on the PCR assay (table 1). Of the 62 women tested, 52 were true positives, and five were true negative as they gave concordant results irrespective of the site of collection and the diagnostic method used (table 1). Four culture negative specimens tested positive by the PCR assays using primers specific to orf1 as well as cppB gene and were, therefore, considered positive. One culture negative specimen was positive by the orf1-PCR assay for its endocervical specimen but negative for urethral specimens. For the cppB gene amplification, the specimen yielded a negative result for both the sites. This was therefore classified as true negative. The sensitivity, specificity, positive predictive value, negative predictive value for the PCR method described here would be 100%, 98%, 99.7%, and 100% respectively. The gold standard has been reported as having a sensitivity of 85–95%.4, 5
The high specificity and sensitivity (25 fg DNA per assay, equivalent to 10 cells) coupled with low cost and rapidity of the in-house PCR assay described here can serve as a promising diagnostic method for the detection of gonococcus directly from clinical swab samples.
We thank Dr Krishna Ray and staff at the STD unit, Safdarjung Hospital, New Delhi, for providing the clinical specimens. We extend our appreciation to Professor J W Tapsall, Prince of Wales Hospital, Australia, for providing us with different Neisseria species. One of us (UC) is grateful to CSIR for the award of a senior research fellowship.