Title Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae
Author Hye-young Wang1, Hyunjung Kim2, Yeun Kim2, Hyeeun Bang2, Jong-Pill Kim3, Joo Hwan Hwang4, Sang-Nae Cho5, Tae Ue Kim2, and Hyeyoung Lee2*
Address 1M&D, Inc., Wonju Eco Environmental Technology Center, Wonju 26493, Republic of Korea, 2Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 26493, Republic of Korea, 3Affiliated Hospital, Korean Leprosy Control Association, Euiwang City, Gyeonggi-do 16048, Republic of Korea, 4Occupational Lung Disease Institute, Korea Workers' Welfare Service, Incheon 21417, Republic of Korea, 5Department of Microbiology, College of Medicine, Yonsei University, Seoul 03722, Republic of Korea
Bibliography Journal of Microbiology, 53(10),686-693, 2015,
DOI 10.1007/s12275-015-5057-9
Key Words Mycobacterium leprae, rifampicin, PCR-REBA (Reverse blot hybridization assay), molecular diagnosis
Abstract Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the β-subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507AGC, 513GTG, 516TAT, 531ATG, and 531TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen’s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.