The Application Of Bacteriophage-Based Assay Detecting Rifamycin Resistance In Mycobacterium Tuberculosis And Study On Mutation In RpoB Gene

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis(MTB),which is one of the most epidemic human diseases.It is also one of the diseases caused the largest number of death that caused by s single pathogen. The World Health Organization estimates that one-third of the world's population is infected with Mycobacterium tuberculosis which results in 80～100 million new cases ever year.Between 1953 and 1985,the number of tuberculosis cases reported declined in our country,however,a reverse in this downward trend was not prominent in 1990s and became upwards in same areas.Because the drug-resistance Mycobacterium tuberculosis come forth and spread abroad,AIDS prevail and all so,Tuberculosis epidemics situation become serious again in the world.Rifampin(RFP) is one of the most effective one-line drugs that treat tuberculosis.The quick veracious ways that detect the RFP resistance in Mycobacterium tuberculosis is very important to Tuberculosis treating and control.The precise molecular mechanism of RFP-resistance is unclear;to detect the mutation of rpoB is very significative in theory and application. Since RFP is vital in the treatment of tuberculosis,so studies on the mechanism of resistance to RFP and the rapid detection methods are the focuses in the fields of tuberculosis control.【Objective】To evaluate the method of Phage Amplified Biologically Assay(PhaB) in the rapid detection of Rifampin(RFP) resistance.Study the mutation of rpoB gene. Explore the mechanism of RFP-resistance in Mycobacterium tuberculosis.Then,to get more knowledge about the rpoB gene mutations and provide some scientific information to study the molecule mechanism of resistance to Rifampin in Mycobacterium tuberculosis,the rpoB gene sequence of Mycobacterium tuberculosis clinical isolates was analyzed by direct sequencing of PCR products.【Method】The first part,detecting sputum specimens of Mycobacterium tuberculosis of pulmonary tuberculosis patients by Ziehl-Neelsen staining and acidity Lowenstein-Jensen medium.To identify the clinical isolates,in order to obtain the Mycobacterium tuberculosis.The second part,detect the resistance of Rifampin in Mycobacterium tuberculosis by improvement Lowenstein-Jensen culture.To obtain the Rifampin drug strains of clinical isolates Mycobacterium tuberculosis.The third part,detecting RFP resistance in 108 clinical isolates of Mycobacterium tuberculosis and compare it with the result of Lowenstein-Jensen culture method.The forth part,to amplify the rpoB genes of 42 RFP-resistance strains by PCR and sequence rpoB genes of RFP-resistance strains.【Results】All of 108 strains of Mycobacterium tuberculosis,26strains were RFP-susceptible and 82 strains were RFP-resistible detected by PhaB while 24 strains were RFP-susceptible and 84 strains were RFP-resistible by absolute concentration method.21 of 108 strains were RFP-susceptible and 79 strains were RFP-resistible with two methods.The concordant isolates of determination of RFP resistance were 100 strains in two methods and the concordance rate was 92.59%.There were 8 strains in discordant isolates.If the results of absolute concentration method is the golden standard,the sensitivity,specificity,positive and negative predictive value and accuracy,as well as accuracy of PhaB assay was 96.34%,77.78%,94.05%,87.50% and 92.59%respectively.The result of the sequences of the RFP-resistance strains indicates that the mutations of rpoB are detected in 42 of 43;the ratio of mutations is 97.67%.There is 1 new style of mutation formed is founded.【Conclusions】The result of this study indicates that the PhaB assay can be used for rapid method to detect drug susceptibility of RFP in clinical isolates of MTB because it is simplicity,rapidity,low-cost and only for 3 days.The PhaB assay is especially suitable for being popularized and applied in these developing countries and poor areas that the rate of drug-resistant TB is high.The study also shows that the rpoB mutations are the main reason that causes RFP-resistance in Mycobacterium tuberculosis.Further more 1 new style of mutation is founded.It is very valuable for the further research of RFP-resistance and for development of the drug-resistance detecting gene-chip.