Multiplex polymerase chain reaction-based reverse line blot hybridization assay to detect common genital pathogens

The objective of the present paper is to develop and apply a multiplex polymerase chain reaction (mPCR) based reverse line blot (RLB) hybridization assay to facilitate the diagnosis of genital infections by detection of seven recognized or putative genital pathogens (Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma genitalium, Mycoplasma hominis and Trichomonas vaginalis). Species-specific biotin-labelled primer pairs were used in a single mPCR to amplify target regions in each of seven pathogens. The amplified biotin-labelled PCR products were hybridized with membrane-bound-specific oligonucleotide probes and were detected by chemiluminescence. Two hundred and eleven specimens (104 male urethral and 107 female vaginal swabs), collected from patients with suspected genital infections attending the Wuhan First Hospital Sexually Transmitted Diseases (STD) clinic, were tested by mPCR/RLB and results were confirmed by single PCR using different species-specific targets. The sensitivity of the assay was assessed using dilutions of positive DNA controls with known copy numbers, for each target. The assay correctly identified all reference strains and detected potential pathogens in a high proportion of clinical specimens. There was no cross-reaction between the seven pathogens. The mPCR/RLB can detect <= 10(2) copies of the target gene fragments. Comparison of mPCR/RLB and single PCR assays showed discrepant results in six of 211(2.8%) clinical specimens, which were positive by mPCR/RLB assay, but negative by the corresponding single PCR. Nested PCR on the six discrepant specimens gave results consistent with those of mPCR/RLB. In conclusion, the mPCR/RLB hybridization assay is sensitive and specific, and able to rapidly detect genital pathogens in clinical specimens.