Usefulness of IS6110-based restriction fragment length polymorphism analysis in fingerprinting of Mycobacterium tuberculosis isolates in North India

Objective/background: World Health Organization estimates that approximately one-third of the global community is infected with Mycobatcerium tuberculosis (MTB). Various molecular epidemiology methods were developed and found very promising for assessing the genetic diversity among MTB complex strain. The two major tools restriction fragment length polymorphism (RFLP) and spoligotyping were commonly used in various studies. Some Indian studies raise the question about the utility of IS6110-RFLP in India due to the presence of zero and low copy number of IS6110 element in MTB isolates. In this short study, we attempt to evaluate the usefulness of IS6110-RFLP in genotyping of MTB isolates in North India. Methods: We conducted a short study involving 26 MTB isolates collected from Sawai Madhopur, Rajasthan, India. IS6110-RFLP analysis was performed as previously described method. In brief, the procedure involve MTB DNA digestion (PvuII restriction enzyme), electrophoresis on 1% agarose gel, transfer of DNA fragments on positively charged nylon membrane, hybridization with digoxigenin-labeled IS6110 probe, and detection by digoxigenin nucleic acid labeling and detection kit. Results: IS6110-RFLP analysis of 26 MTB isolates showed presence of IS6110 element in varying range from 0 to 17 copies. Out of the 26 MTB isolates, two (7.8%), three (11.5%), and 21 (80.8%) showed zero, low, and multiple copy numbers, respectively. The isolates, which had IS6110 element, showed 22 different RFLP patterns. Two clusters of two isolates each were found, and 20 isolates showed unique RFLP pattern. The two clusters of isolates had 11 and 13 copy numbers of IS6110. Conclusion: In this study, we found that the majority of isolates were having multiple copy numbers of IS6110 element and showed a very diverse pattern. These results showed that the IS6110-RFLP analysis is still a promising genotyping method and has good discriminatory power to differentiate strains of MTB isolates in India.