The Study On Multiplex PCR Based Reverse Line Blot Hybridization For Detection Of The Common Sexually Transmitted Diseases Pathogens

Objective: To develop and evaluate a new multiplex PCR based reverse line blot hybridization (RLB) for rapid detection of the most common STD pathogens.Methods: 1) Three sets of special primers and three probes directed to the cryptic plasmid of C. trachomatis, 16S rRNA and Opa gene of N. gonorrhoeae were designed respectively, two sets of universal primers and the nine probes directed to the internal transcribed space sequence (ITS) of three Mycoplasmas (U. urealyticum, U. Parvum and M. hominis ) or six Candida (C. alblcans, C. troptcalis, C. krusei, C. glabrata, C. parapsilosis, and C. dubliniensis) were used by checking their specificity against all sequences in Genbank by using http://www.angis.org. Primers and probe have similar melting temperatures. 2) To develop and evaluate PCR/RLB for rapid detection of the six most common Candida. 3: To study and evaluate the multiplex PCR based reverse line blot hybridization (mPCR/RLB) for detection of genitourinary Chlamydia trachomatis, Neisseria Gonorrhea and three Mycoplasmas.Results: 1) The sensitivity of PCR/RLB for detection of Candida is 10 CFU/ml. This method was used to test 100 isolates and 200 vaginal swabs. The results agreed with those of culture for all 100 isolates but 3 specimens which were confirmed their results of PCR/RLB were consistent with those of DNA sequencing. PCR/RLB positive rate (49%) is higher than smear microscopic screening (27%) and culture (39%)(P<0.05). 2) All of C. trachomatis, N. gonorrhoeae and three Mycoplasmas strains were amplified by mPCR/RLB, and the length of PCR products was 208bp～414bp. 93 of 97 FQ-PCR CT-positive specimens were positive by mPCR/RLB, and 35 of 45 FQ-PCR CT-negative specimens were negitive by mPCR/RLB. 34 of 41 FQ-PCR NG-positive specimens were positive by mPCR/RLB, and 98 of 101 FQ-PCR CT-negative specimens were negitive by mPCR/RLB. 36 of 142 specimens were multiple pathogens-positive by mPCR/RLB. 28 of 32 Mycoplasmas-positive specimens by liquid culture were positive by mPCR/RLB. The results of mPCR/RLB agreed with those of liquid culture for all of 13 Mycoplasmas-negitive specimens.Conclusion: We successfully developed a new mPCR/RLB for rapid simultaneous detection of multiple pathogens.