A Novel Fluorescent Quantitative PCR Method Of Mycobacterium Tuberculosis DNA Using Duplex Scorpion Probe

Objective : At present, the Taqman fluorescent quantitative polymerase chain reaction is the most wide used clinical gene diagnosis quantitative assay for mycobacterium tuberculosis but it is still an important issue that how we could increase positive rate, reduce the cost and make the reaction time short. So, it was important to establish a novel real-time PCR assay based on duplex scorpion probe to detect mycobacterium tuberculosis(TB) DNA, with high sensitivity, specificity, stability and lower price, meanwhile three DNA extraction methods were evaluated respectively for finding out the preferred sample processing procedure.Method:The senX3-regX3 IR gene of Mycobacterium tuberculosis was cloned into the vector pMD18-T,which was named as the recombinant vector pMD18-T-senX3-regX3 IR and 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 .The new real-time PCR method was used to detect clinical examples which were prepared by Boiling, Chelex 100 resin treatment and Bacteria DNA Mini Kit respectively, comparing with the Taqman detecting kit.Results: 1) The plasmid containing the sequence of interest was constructed successfully. 2) The assay for mycobacterium tuberculosis DNA was developed with large detecting range, high sensitivity, specificity, stability and accuracy. 3) After applied in clinical experiments, this novel...