Novel Real-Time PCR For Neisseria Gonorrhoeae Detection Using Self-Quenched Primer

Neisseria gonorrhoeae is one of the most common bacteria of thesexually transmitted infectious disease (STD) with high morbidity. How todetect them quickly and accurately is significant to the diagnosis, treatmentand prevention of the diseases caused by Neisseria gonorrhoeae. Now thegolden standard for diagnosis is culture method, but it costs too much time,which hinders early diagnosis. The Real-time fluorescence quantitativePCR (FQ-PCR) is a new tool basing on the common PCR, which cansupervise the whole PCR process and quantify the adding templateabsolutely and rapidly. Almost all the FQ-PCR kits on the market for NGdiagnosis use the Taqman probe, which is more expensive to synthesize theprobe and label with two fluorescent dyes. The self-quenched primerinvented in 2002 is a new technique with the comparable sensitivity,specificity and far lower price. Objective:To establish a novelfluorescent quantitative PCR method to detect Neisseria gonorrhoeae withhigh sensitivity, specificity, stability and lower price. Method:CPPBgene of Neisseria gonorrhoeae was cloned into the vector pGEM-11Zf. Therecombinant vector was used as standard template. The self-quenchedprimer was designed according to the CPPB gene sequence. After the PCRreaction system was optimized and method evaluation was accomplished,this new method was applied to detect clinical specimens and comparedwith bacterial culture. Results: 1) Restriction maps showed that thestructure of recombinant plasmid pGEM-11Zf–CPPB was consistent withwhat expected. 2) Methodology analysis showed that the new method usingself-quenched primer was successfully established with high sensitivity,specificity and accuracy. 3)After applied in clinical experiments, thismethod has more advantages than classic cultural method. Conclusion:A new real time fluorescence PCR detective method for Neisseriagonorrhoeae has been successfully developed, which would lay asubstantial foundation for investigation of a kit's competition on the market.It also provided a reference to the detection of other pathogenic microbes.