Detection Of Second-line Drugs Resistance Mutations In Mycobacterium Tuberculosis By Multicolor Melting Curve Analysis

Tuberculosis,caused by Mycobacterium tuberculosis,is a chronic infectious disease.In recent years,the cases of multidrug resistant tuberculosis(MDR-TB)patients and extensively drug-resistant tuberculosis(XDR-TB)patients have gradually increased.Among them,the treatment time of XDR-TB patients is longer and the cure is more difficult.In addition,more and more cases of heteroresistance of Mycobacterium tuberculosis have been found in clinic,increasing the difficulty of molecular diagnosis.At present,there is still a lack of rapid and highly sensitive diagnostic methods for drug-resistant tuberculosis.In order to realize the early diagnosis of XDR-TB and to guide the clinicians to use drugs effectively,based on the multicolor melting curve analysis,a detection system for second-line drug resistance mutation of Mycobacterium tuberculosis was established.It includes fluoroquinolone resistance detection system and injectable second-line drug resistance detection system.Chapter One,Introduction.This paper introduces the general situation of tuberculosis,clinical diagnosis of tuberculosis,heteroresistance of Mycobacterium tuberculosis and multicolor melting curve analysis.Chapter Two,Detection system for fluoroquinolone resistance mutations.In order to solve the problem of identifying and differentiating the mutant types of fluoroquinolones resistance of Mycobacterium tuberculosis,and to realize the detection of low level heteroresistance mutations,to assist doctors in early diagnosis,and to use drugs accurately,based on the polychromatic melting curve analysis technique,a single and double color detection system is established in this paper.A probe was designed for the drug-resistant determinant region of fluoroquinolones gyrA gene,and the ability of detecting heteroresistant was 5%-20%mutation.781 clinical isolates were used to evaluate the clinical diagnostic performance of the system,and the results showed that the sensitivity and specificity of the system were 92.3%and 97.6%respectively.Chapter Three,Detection system for injectable second-line drug resistance mutations.Based on the multicolor melting curve technology,we established a single-tube and dual-color system,the rrs gene 1401,1402,1484 loci and the eis promoter regions-10,-13,-14,-37 were including,the system realize detect the heteroresistance for 1%mutation about the highest mutation frequency of rrs1401 A>G.488 clinical isolates were used to evaluate the clinical diagnostic performance of the system.The results showed that the sensitivity and specificity of the system were 90.5%and 97.8%respectively.The advantage of this system lies in the detection of multiple loci in a single tube,which greatly reduces the complexity of clinical detection,and at the same time realizes the detection of low level heteroresistance mutation.It is helpful for the early and rapid diagnosis of injectable second-line drug resistance in mycobacterium tuberculosis.