Study On Reverse Dot Blot Hybridization For Rapid Testing The Resistance Of Mycobacterium Tuberculosis To Eight Anti-tuberculosis Drugs In China

The prevalence of drug resistant tuberculosis, especially multi-drug resistant tuberculosis(MDR-TB) and extensively drug-resistant tuberculosis(XDR-TB), has become significant public health and social hot spot problem. Generally, culture-based traditional drug sensitivity tests have been using in clinical application widely as the gold standard for detecting drug-resistant M. tuberculosis, but it need to take longer time(at least 2-4 weeks) for getting the results after the isolate has been cultivated. New developed molecular biological technology has achieved good detection effect on detecting M. tuberculosis resistance partially to first-line drug, almost as good as the phenotypic methods of culture-based traditional drug sensitivity tests. However, the expensive equipment and consumables has hindered the widespread use in TB high-burden but low-income countries. In addition, these molecular technologies are very few applications in detecting resistance to second-line drugs. PCR-reverse dot blot hybridization(RDBH) is a rapid, simple, efficient, intuitive hybridization method. The aim of this study is to establish a RDBH assay which can detect the drug sensitivity of M. tuberculosis to four first-line and four second-line anti-tuberculosis drugs at the same time, so as to achieve comprehensive and rapid detection of drug-resistant TB, and provide the new technological method for drug-resistant TB control and treatment effectively.According to the major gene mutation sites of the drug resistance-associated genes of eight anti-tuberculosis drugs including isoniazid(INH), rifampicin(RIF), streptomycin(SM), ethambutol(EMB), Ofloxacin(OFX), Kanamycin(KAN), Amikacin(AMK) and Capreomycin(CPM), 46 amino-labeled specific oligonucleotide probes were designed respectively. The more ideal experimental conditions were optimized through the researches with small samples. A total of 326 M. tuberculosis clinical isolates were detected the drug resistance of INH, RIF, SM and EMB and 170 isolates were detected the drug resistance of OFX, KAN, AMK and CPM by the RDBH assay, respectively. The conventional drug susceptibility test(DST) of proportion and DNA sequencing were used to blind evaluate the performance of the RDBH assay preliminarily.After a series of optimized researches, the ideal experimental conditions were determined at following: the amino-labeled specific oligonucleotide probe to each mutation site, the final concentration of the probes were 0.5 ?M; the PCR product was diluted in 5×SSPE/0.1%SDS and hybridized temperature was 60℃; the membrane was washed with buffer at 50 ℃ after hybridization and was detected by TMB colour-developing agent following the manufacturer’s instructions. The drug susceptibility of 326 clinical M. tuberculosis isolates to the first-line drugs and 170 isolates to the second-line drugs were detected by DST, DNA sequencing and the RDBH assay, respectively. Compared with the results of DST, the sensitivity and specificity of the RDBH assay was 84.5%(185/219) and 95.3%(102/107) for INH, 86.5%(166/192) and 95.5%(128/134) for RIF, 72.3%(120/166) and 91.3%(146/160) for SM, 55.4%(51/92) and 82.1%(192/234) for EMB, 62.8%(59/94) and 100.0%(76/76) for OFX, 56.0%(14/25) and 100.0%(145/145) for KAN, 61.5%(8/13) and 98.7%(155/157) for AMK, 45.0%(9/20) and 99.3%(149/150) for CPM, respectively. The Kappa value was 0.75, 0.80, 0.63, 0.37, 0.60, 0.68, 0.67 and 0.57, respectively. Compared with the results of DNA sequencing, the sensitivity and specificity of the RDBH assay was 97.9.%(189/193) and 99.2%(132/133) for INH, 97.7%(171/175) and 98.0%(148/151) for RIF, 97.8%(134/137) and 100.0%(189/189) for SM, 82.0%(91/111) and 99.1%(213/215) for EMB, 93.7%(59/63) and 100.0%(107/107) for OFX, 92.9%(13/14) and 99.4%(155/156) for KAN, 90.0%(9/10) and 99.4%(159/160) for AMK, 90.0%(9/10) and 99.4%(159/160) for CPM, respectively. The Kappa value was 0.97, 0.96, 0.98, 0.84, 0.95, 0.92, 0.89 and 0.89, respectively.The results showed that there was better the performance of the RDBH assay detecting the drug resistances of M. tuberculosis to the first-line and the second-line anti-tuberculosis drugs, providing a good basis for developing the drug resistance detection kit. It has a good application prospect being used for the diagnosis and surveillance of the drug resistance of M. tuberculosis to the first-line and the second-line anti-tuberculosis drugs.