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Rapid detection of rifampicin-resistant Mycobacterium tuberculosis, based on isothermal DNA amplification and DNA chromatography

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Title: Rapid detection of rifampicin-resistant Mycobacterium tuberculosis, based on isothermal DNA amplification and DNA chromatography
Authors: Takarada, Yutaka Browse this author
Kodera, Takuya Browse this author
Kobayashi, Kumi Browse this author
Nakajima, Chie Browse this author →KAKEN DB
Kawase, Mitsuo Browse this author
Suzuki, Yasuhiko Browse this author →KAKEN DB
Keywords: Mycobacterium tuberculosis
Rapid detection
Loop-mediated isothermal amplification
Mutation detection
Issue Date: Oct-2020
Publisher: Elsevier
Journal Title: Journal of microbiological methods
Volume: 177
Start Page: 106062
Publisher DOI: 10.1016/j.mimet.2020.106062
Abstract: Rapid and easy detection of nucleotide point mutations in bacterial pathogens associated with drug resistance is essential for the proper use of antimicrobials. Here, we developed a rapid and simple method for the detection of mutations using Loop-mediated isothermal amplification (LAMP) combined with the single-tag hybridization (STH) chromatographic printed array strips (PAS) method. This procedure is able to detect four mutations (C1349 T, A1295C, G1303 T, A1304 T) in Rifampicin Resistance Determining Region (RRDR) of rifampicin-resistant Mycobacterium tuberculosis (RR-TB), simultaneously. LAMP reactions contained a LAMP primer and eight allele-specific primers for each mutation. The allele-specific primers products were detected by nucleic acid chromatography using PAS. Four detection lines were detected there, one of which was detected at different positions depend on the wild type and the mutant type. We carried out the four mutations detection using 31 genomic DNA (2 A1295T, 1 G1303 T, 6 A1304 T, 22 C1349 T) from clinical isolate. The mutations have been confirmed by sequence analysis. The detection results were completely consistent with the sequence analysis. In the present study, four mutations could be detected, but only 60% of RR-TB could be detected with these four. It is expected that the detection rate will increase by adding more mutant primers. The combined LAMP and STH chromatographic PAS method is a simple and rapid method for detecting point mutations in clinical isolates as a point-of-care testing (POCT) technique. In addition, it does not require special equipment and can meet the demand in areas where drug-resistant bacteria are endemic, such as developing countries.
Rights: © 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license
Type: article (author version)
Appears in Collections:人獣共通感染症国際共同研究所 (International Institute for Zoonosis Control) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
国際連携研究教育局 : GI-CoRE (Global Institution for Collaborative Research and Education : GI-CoRE) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 鈴木 定彦

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