Multiplex polymerase chain reaction assay for the detection of Neisseria gonorrhoeae in urogenital specimens

A set of PCR primers were designed to detect a broad range of Neisseria species, including Neisseria meningitidis and Neisseria gonorrhoeae. All the three sets of primers could detect the Neisseria DNA at concentrations as low as 25 fg of bacterial DNA. The specificity of primers for Neisseria species was checked by using DNA from other bacteria found as commensal organisms. The primers targeting the 23S rRNA and nspA genes could amplify when the DNA from any of the Neisseria species commonly found as contaminants of clinical samples was used as template. A third set of primers targeting orf1 gene was also designed and was found to be highly specific for N. gonorrhoeae. The in-house developed multiplex PCR method was compared with culture method for its ability to detect N. gonorrhoeae infections using 238 clinical swabs, obtained from patients visiting Safdarjung Hospital, New Delhi. The sensitivity of the culture method was 96%, while with multiplex-PCR, a sensitivity of 100% was observed. Sequence analysis of the gene for the outer membrane protein, nspA, from 140 clinical isolates of N. gonorrhoeae revealed no intraspecies divergence. The high specificity and sensitivity coupled with low cost and rapidity of the method described here provide a substantial advantage over the labour-intensive and time-consuming culture method currently used in hospitals in India.