Rapid Detection Of Drug-resistant Mycobacterium Tuberculosis With High-resolution Melting Curves

Background The tuberculosis is global epidemic caused by Mycobacterium tuberculosis, which is more severe in the poverty stricken area. Because of the chronic course of the disease poor patient compliance and inadequate treatment, MTB has developed a drug resistance, which makes a severe public health issue. The traditional diagnosis methods such as sputum smear, sputum culture have some drawbacks including long culture period, low positive rate. Development of rapid diagnosis test of drug-resistance MTB is of great importance in preventing tuberculosis. It has been reported that the mechanism of drug-resistance is DNA mutation mostly. The rifampicin-resistance mutation located at the 81bp core region of rpoB, and the isoniazid-resistance mutation located at katG315,the promoter region of inhA,the promoter region of ahpC and the inhA94 loci. High-resolution melting(HRM) is a newly developed lab test method used in the single nucleotide polymorphism(SNP). It is a cost effective, fast and powerful,accurate, simple and closed-tube technique. In this research, we used the HRM in the detection of MTB drug-resistance related gene:katG, inhA, ahpC and rpoB.Purpose By using the HRM and DNA sequencing to detect the MTB drug-resistance gene include katG,inhA,the promoter region of ahpC,the 81bp core region of rpoB. Results were compared to explore the feasibility of HRM in detecting MTB isoniazid and rifampicin-resistant gene loci.Methods 1. To subcultivate the MTB by improved L-J culture medium, determine their isoniazid and rifampicin susceptibility and resistance.2. To sequence all the 312 samples'target gene:katG, inhA, ohpC, rpoB. Analysis the results with blastn.3. To develop the HRM assay to detect 140 RFP-resistant strains" 81bp core region of rpoB,150 INH-resistant strains' katG315, the promoter region of inhA, the 94 loci of inhA, the promoter region of ahpC, compare the results to the DNA sequencing results, verify the feasibility of HRM in rapid detection of drug-resistant MTB.Results 1. The positive rate of DNA sequencing of rifampicin,isoniazid-resistance strains are 95.7%(134/140),92%(138/150) respectively. The positive rate of HRM of rifampicin,isoniazid-resistance strains are 95.7%(134/140),88.1%(133/150) respectively. There are no significant different between DNA sequencing and HRM(p>0.05).2. The correspondance rate between 140 RFP-resistant strains'HRM results and DNA sequencing is 97.8%(131/134), and the correspondence rate between 150 INH-resistant strains'HRM results and DNA sequencing is 96.4%(133/138)3. The specificity and sensitivity of HRM in detecting RFP-resistance strains are 98.0%(150/153),93.6%(134/140) respectively. The specificity and sensitivity of HRM in detecting INH-resistance strains are98.7%(151/153),88.1%(133/150) respectively.Conclusion 1. The HRM has a similar detection power in detecting drug-resistant MTB when compared to DNA sequencing, while it is more simplified and fast.2. The HRM has a good correspondence with DNA sequencing in detecting drug-resistant MTB strains loci, but it can only be used with a known mutation loci.3. HRM has a good specificity and sensitivity in detecting RFP, INH-resistant MTB.