Analysis Of Rifampin Resistance In Mycobacterium Tuberculosis Isolates And Rapid Detection Of Drug-resistant Gene

Tuberculosis (TB) is a chronic infectious disease caused by theMycobacterium tuberculosis (MTB). Since the middle of 1980's, theepidemiological situation of tuberculosis has been becoming more and moreserious due to the ignoring of tuberculosis, the prevalence of AIDS, the emergenceof drug-resistant strains of MTB and the increase of floating population as well.WHO estimated that approximately 2 billion people are infected with MTBglobally , active TB cases reach 20 million and 10 million new cases of TB occureeach year. Annual TB mortality is 3 million people, and that is the most highmortality in all of the infectious diseases in the world. China is one of 22 TBhighly burden countries and the number of patients in China are second high in theworld. The characteristics of TB prevalence in China are high prevalence rate, highdrug resistant rate, high mortality rate, high infection rate and low reduction rate,low detection rate. The epidemiological situation of TB in Guangxi is very serioustoo. It is estimated that about 15 million people are infected with MTB and thenumber of TB patients reach about 300 thousand. Within them 60 thousand areinfectious TB patient and about 10 thousand people are died of TB per year inGuangxi. Multidrug resistant tuberculosis (MDR-TB) refers to the mycobacteriumtuberculosis that at least resist to rifampin(RFP) and isoniazid(INH) at the sametime. It is regarded as the third factor that causes the rising of TB epidemicsituation globally. Because 90% RFP-resistant isolate strains of MTB are resistantto isoniazid at the same time, RFP drug-resistance is regarded as an importantmarker of MDR-TB. The recent study demonstrates that RFP-resistance isassociated with mutations in rpoB gene of MTB. In order to investigate a methodof detecting gene mutation in MTB associated with resistance, RFP resistancestrains serve as an example to be researched, so that we can investigate a highlyeffective, rapid and simple method for detecting MTB drug resistance. 5Methods At first, RFP resistance in 77 clinical isolate strains of MTBfrom Guangxi are analysed in terms of the results in traditional drug susceptibilitytest. Then the genome DNA of MTB is prepared and the rpoB gene fragment isamplified by PCR. The mutations in the rpoB gene fragment of 77 strains MTB aredetected by DNA sequencing, and the relationship between mutations in the rpoBgene and RFP resistance are analysed. The mutations in the rpoB gene fragment of 77 isolate strains of MTB and27 morning sputa specimens from TB patients are detected by DNA chip. Thetypes of mutations in rpoB gene and the relationship between mutation and drugresistance are analysed. The mutations in the rpoB gene fragment of 63 isolate strains of MTB and27 morning sputa specimens from TB patients are detected by reverse dot blothybridiztion(RDB) membrane chip. The types of mutations in rpoB gene and therelationship between mutation and drug resistance are analysed . Results Within 62 RFP drug-resistant isolate strains, 61 strains areresistant to INH tested by drug susceptibility test. Above 90% RFP-resistant strainshave mutations in RFP resistant determination region(RRDR) of rpoB gene, 531,526 and 516 are the most common positions to be substituted, 59.6%(34/57)RFP-resistant strains have mutation at 531 position, 19.3%(11/57) strains havemutation locate at 526 position, the mutation rate at 516 position is 10.5%(6/57),combinative mutations rate is 5.3%(3/57). No deletion or insertion mutation isfound among all strains. 77 isolate strains are detected by DNA chip. The results show that 57 areidentified as RFP-resistant strains by DNA chip among 62 RFP-resistant strainsidentified by traditional drug susceptibility testing, 12 out of 15 RFP-susceptivestrains, which are identified by traditional drug susceptibility testing, are identifiedas...