Method And Clinical Application Research Of The Antisense Inhibit PCR Detection Of Mycobacterium Tuberculosis Resistant To Rifampin

Mycobacterium tuberculosis (MTB) was discovered by the German scientist Koch in1882and proved to be the pathogen of Tuberculosis. With the standards of hygieneimproving and anti-TB drugs developing continuously, the morbidity and mortality rate ofTB had dropped significantly. Yet since1980s, TB has become active again due to theAIDS epidemic. In recent years, the TB epidemic tends to revive, becoming the primarycause of death and the top killer among infectious disease.Rifampicin (RFP) exerts the antimicrobial effect by first specifically binding with theRNA polymerase β subunit, thereby inhibiting the activity of RNA polymerase, and theninterfering with the Mycobacterium RNA transcription and synthesis, and ultimatelyhindering the protein synthesis. Studies have indicated that more than90%Mycobacteriumtuberculosis resistant RFP could be attributed to gene mutations in the target molecule RNApolymerase gene encoding β-subunit (rpoB). Currently, detecting Mycobacterium tuberculosis mainly relies on such techniques assputum smear, cells culture, medicine sensitivity, and PCR technology. The detailedoperations of these techniques are either tedious or time-consuming with low sensitivityand specificity, easily resulting in misdiagnosis and increasing economic burden of patients.In this study, we apply antisense inhibition PCR technology to detect mutations inrpoB gene of Mycobacterium tuberculosis,531as well as526points. The results indicatethat when the concentrations of wild antisense upstream primer are greater than10μM, thePCR amplification of the wild-type plasmid DNA would be completely inhibited. Underthis condition, with the observed minimum detectable concentration of1-5×103（IU/ml）,its performance fully meets the needs of clinical specimens detection.Taking this method, we test182cases of clinical TB patients, and compare the resultswith those of the traditional susceptibility and PCR direct sequencing method. Theconclusion is that our method, susceptibility method and direct sequencing measured rpoBmutation were64,63and60cases, respectively. As to Chi-square test, statistics indicate nodifference compared to the direct sequencing, illustrating that our method is an accurate andreliable detection.