A novel fluorescent quantitative polymerase chain reaction method of Mycobacterium tuberculosis DNA using duplex scorpion probe

Abstract

Objective To establish a new fluorescent probe-based polymerase chain reaction (PCR) assay (the duplex scorpion probe) to the detect Mycobacterium tuberculosis(TB) DNA. MethodsThe recombinant vector pMD18-T-senX3-regX3 IR was used as the standard template. The duplex scorpion probe was designed according to the cloned gene sequence,and then the PCR reaction system was optimized and evaluated.Results (1) The plasmid containing the sequence of interest was constructed successfully. (2) The assay for Mycobacterium tuberculosis was developed with large detecting range, high sensitivity, specificity, stability and accuracy. (3) After applied in clinical experiments, this novel fluorescent quantitative method has more advantages than the Taqman detecting kit. Conclusions A new real-time PCR detective method for mycobacterium tuberculosis has been successfully developed, which would lay a substantial foundation for the development of a new kit’s competition on the market. Our research has showed that the duplex scorpion probe assays are more accurate, sensitive, specific, and low-cost than the Taqman assay, which suggested that the real-time PCR for TB based on duplex scorpion probe was a novel and excellent method for the gene diagnosis of tuberculosis potentially.