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Abstract: A novel quantitative real-time PCR method using the duplex scorpion primer for detection of Chlamydia trachomatis DNA was developed and validated. The assay employs a duplex primer; its most important feature is the intramolecular probe–target interaction. The assay had many prominent characteristics. (i) The duplex probe is simpler to synthesize and significantly easier to purify than TaqMan probe because that the fluorescent dye pair and the quencher pair are in different oligonucleotides. (ii) The method has high sensitivity, specificity, intra- and interassay reproducibilities. (iii) The assay has a quantitative dynamic range of 25 to109 genome copies per reaction mixture. (iv) The scorpion system can identify 98.6% samples in the validation panel without retest. There were 81 positive samples and 67 negative samples, which were confirmed by two FDA-approved NAATs (the Roche Amplicor PCR assay, Abbott LCR kit) and our new method. Any two positive results out of the possible three-comparator results would define the infected-patient gold standard. Of the positive samples, 79 (97.5%) were found positive (ranging from 31 to 227,648 copies/μl, M =4219 copies/μl), whereas no negative samples were found positive by the assay. 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. [Copyright &y& Elsevier]