Development Of A Novel Quantitative Real-Time Assay Using Duplex Scorpion Primer For Detection Of Chlamydia Trachomatis

Objective:At present, most of real-time PCR assays for the detection of Chlamydia trachomatis (CT) are based on a fluorescent dye-labeled TaqMan probe-based system and this system has many advantages such as high sensitivity, specificity and is the most wide used clinical gene diagnosis quantitative assay. However, TaqMan probe has two following problems. Firstly, because the probe has both of the fluorophore and quencher on the two ends of the same strand that it is difficult to synthesize and purify. Secondly, the progress of detection has three steps: denaturation, hybridization, extension and enzymolysis, and so costs more time. Unlike TaqMan detection systems, as the fluorescent dye pair and the quencher pair are in different complementary oligonucleotides, duplex probe are simpler to synthesize and significantly easier to purify than TaqMan probe which has the fluorophore and quencher on the ends of the same strand. Furthermore, duplex scorpion probe format is not dependent on enzymic cleavage and, therefore rapid PCR cycling is possible. In present study, we aim to establish a fast, lower price, and easy-to-handle novel real-time PCR assay using the duplex scorpion primer for detection of Chlamydia trachomatis DNA.Methods:The MOMP gene of Chlamydia trachomatis was cloned into the vector pMD18-T,which was used as the standard DNA template. The duplex scorpion probe was designed according to the cloned gene sequence, and then the PCR reaction system was optimized and evaluated. To determine the intra- and interassay coefficient of variations (CV) of the PCR, four standard series and one C. trachomatis positive sample were tested and then the CV was applied to evaluate the reproducibility. The analytical specificity of the assay was investigated by performing PCR with C. pneumoniae, C. psittaci, 15 strains of C. trachomatis and the pathogenic bacteria or commensal organisms of the genital tracts. There were 148 clinical samples, which were confirmed by two FDA-approved NAATs and our new method. Any two positive results out of the possible three-comparator results would define the infected-patient gold standard, and then evaluate detection performance of the assay.Results: (1) The plasmid containing the sequence of interest was constructed successfully. (2) The assay has a quantitative dynamic range of 25 to 1010 genome copies per reaction mixture. The range of intra- and interassay CV of different concentration samples is 4.92%~21.73% and 6.94%~27.17% respectively. The specificity of the method is 100%. (3) The scorpion system can identify 98.6% samples in the validation panel without retest. A quantitative, fast, and easy-to-handle diagnostic approach such as the MOMP-based real-time PCR described here might improve the detection of C trachomatis infection.Conclusion: A new real-time PCR method for C. trachomatis has been successfully developed, which would lay a substantial foundation for the development of a new kit. Our research has showed that the duplex scorpion primer assays are reliable, fast, high sensitivity, specificity, and low-cost. All above suggest that the real-time PCR based on duplex scorpion primer is a novel and excellent method for the gene diagnosis of CT potentially.