title: Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.
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 -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) 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 531 ATG 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.
authors.affiliation: Wonju Eco Environmental Technology Center, M&D, Inc., Wonju, 26493, Republic of Korea.
authors.forename: Hye-young
authors.initials: HY
authors.lastname: Wang
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, 26493, Republic of Korea.
authors.forename: Hyunjung
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, 26493, Republic of Korea.
authors.forename: Yeun
authors.initials: Y
authors.lastname: Kim
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, 26493, Republic of Korea.
authors.forename: Hyeeun
authors.initials: H
authors.lastname: Bang
authors.affiliation: Affiliated Hospital, Korean Leprosy Control Association, Euiwang City, Gyeonggi-do, 16048, Republic of Korea.
authors.forename: Jong-Pill
authors.initials: JP
authors.lastname: Kim
authors.affiliation: Occupational Lung Disease Institute, Korea Workers Welfare Service, Incheon, 21417, Republic of Korea.
authors.forename: Joo Hwan
authors.initials: JH
authors.lastname: Hwang
authors.affiliation: Department of Microbiology, College of Medicine, Yonsei University, Seoul, 03722, Republic of Korea.
authors.forename: Sang-Nae
authors.initials: SN
authors.lastname: Cho
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, 26493, Republic of Korea.
authors.forename: Tae Ue
authors.initials: TU
authors.lastname: Kim
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, 26493, Republic of Korea. hyelee@yonsei.ac.kr.
authors.forename: Hyeyoung
authors.initials: H
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009166:Mycobacterium leprae / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity
chemical_list: D000904:Antibiotics, Antitubercular; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin
keywords: Mycobacterium leprae; PCR-REBA (Reverse blot hybridization assay); molecular diagnosis; rifampicin