Wang Hye-young, Kim Hyunjung, Kim Yeun, Bang Hyeeun, Kim Jong-Pill, Hwang Joo Hwan, Cho Sang-Nae, Kim Tae Ue, Lee Hyeyoung
Wonju Eco Environmental Technology Center, M&D, Inc., Wonju, 26493, Republic of Korea.
Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, 26493, Republic of Korea.
J Microbiol. 2015 Oct;53(10):686-93. doi: 10.1007/s12275-015-5057-9. Epub 2015 Oct 2.
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.
在麻风病流行的国家，麻风分枝杆菌的耐药性是一个重大问题。我们设计并评估了一种用于检测对利福平（RIF）基因型耐药性的灵敏、特异且高通量的反向杂交检测法（REBA）。研究表明，麻风分枝杆菌对RIF的耐药性涉及编码RNA聚合酶β亚基的rpoB基因突变。PCR-REBA可同时检测6个野生型区域和5种不同突变（507 AGC、513 GTG、516 TAT、531 ATG和531 TTC），包括507位和531位最常见的突变。对韩国汉森病研究所提供的31株临床分离株进行了rpoB基因RIF耐药性的PCR-REBA分析。结果，在一个样本中发现了密码子507 AGC和531 ATG处有2个核苷酸替换的错义突变，在另一个样本中发现了密码子516 TAT处的错义突变和ΔWT6（530 - 534缺失）。这些病例通过DNA序列分析得到了证实。这种快速、简单且高度灵敏的检测方法为麻风分枝杆菌RIF耐药性的基因型评估提供了一种替代测序的实用方法。
