| In recent years, the problem of drug resistance tuberculosis (TB) has become a focus in the areas of public health around the world, especially multi-drug resistance tuberculosis (MDR-TB) and extensively drug resistance tuberculosis (XDR-TB). The incidence and prevalence of drug-resistant TB has become the biggest challenge and matter of TB’s prevention and control today. MDR-TB and XDR-TB have become one of seriously threating the health of people in China.Isoniazid (INH) which is one of the first-line drugs plays an important role in the treatment of TB, so it is essential to research and diagnose INH resistance rapidly, providing the scientific basis for prevention and treatment of TB. The purpose of this study is to establish a TaqMan-MGB real-time PCR assay rapid detecting the drug resistance of Mycobacterium tuberculosis (MTB) to Isoniazid and to be used in clinical specimen test preliminarily, providing the basis for clinical mass screening.To understand their resistance profile, particularly INH-resistance, the drug susceptibility test (DST) of274clinical isolates of MTB collected from12provinces and cities in China were conducted by means of the proportion method. To find out mutant genes’ distribution in clinical MTB isolates in China and screen out the mutant gene which is the greatest contribution to INH-resistance, INH-resistant related genes such as katG, pre-inhA, inhA, ndh and the oxyR-ahpC were amplified by PCR and DNA sequencing.According to mutant locus selected,we designed real-time PCR probes and then an assay of TaqMan-MGB real-time PCR was established for rapid detection of the resistance of MTB to INH based on katG315mutation in MTB. The specificity, sensitivity, repeatability and the minimum detection limit of the method were evaluated with130MTB strains,16standard strains of non-tuberculosis Mycobacterium (NTM),7standard strains of non-Mycobacterium and the positive plasmid control strains. In addition, this method was preliminarily used to tested166sputa including161confirmed patients and5non-TB respiratory disease patients respectively. The result statistical analysis was done with chi-square test.The DST result of274MTB isolates were tested for the resistance to the four first-line anti-tuberculosis drugs (SM、RFP、INH and EMB).56strains were single-drug resistance,152strains were MDR,55strains were sensitive to all the drugs. There were209INH-resistant strains, followed by159RFP-resistant strains in222drug-resistant strains. With the sequence results of209INH-resistant strains, the main SNP mutations occurred in katG and pre-inhA genes, accounting for64.58%and24.88%respectively. At the same time we found that the highest mutant locus is katG315G'C mutation, the the mutation rate was45.93%.The minimum detection limit of the real-time PCR method in this study was10target genes copies/μl which was100times lower than that of conventional PCR. As the results of NTM and non-Mycobacterium control strains tested with this method were negative, the specificity were100%. The coefficients of variation for both intra-and inter-experimental were less than1%, indicating remarkable repeatability. Compared with DST of130strains, the coincidence rate, sensitivity and specificity were68.46%(89/130),59%(59/100) and100%(30/30) respectively. Compared with the results of sequencing, the sensitivity and specificity of wild-type and mutant-type probes were100%respectively.For detection of166sputa, the sensitivity and specificity of the real-time PCR assay were84.47%and100%respectively. For detecting the sputa of patients confirmed with TB, the positive rate of this assay was84.47%, significantly higher than that of the sputa smear microscopy for acid-fast bacilli40.99%(χ2=46.44, P<0.01), the culture with L-J medium40.37%(x2=47.89, P<0.01), IS6110-PCR50.31%(x2=27.39, P<0.01).The positive rate of this method was94.44%in smear-positive or culture-positive sputa, while75.28%in smear-negative and culture-positive sputa. Compared with conventional DST to detect INH-resistance with61culture-positive sputa, the sensitivity and specificity of this method were33.33%(1/3) and100%(58/58) respectively, And the concordance rate between them was96.72%(59/61).The method of TaqMan-MGB real-time PCR which is established in this study could detect katG315G'C mutation of MTB in strains or clinical sputum specificly, sensitively and rapidly to use the diagnosis of TB and MTB drug resistance to INH simultaneously, by which it could improve the positive rate of suspected patients in the clinical diagnosis.The whole process is simple, easy, short time (in3hours) and its result is reliable. The method will be an important adjunct means for clinical diagnosis and treatment of TB. |
PDF Full Text Request