TY - JOUR
T1 - Rapid detection of rifampicin-resistant Mycobacterium tuberculosis, based on isothermal DNA amplification and DNA chromatography
AU - Takarada, Yutaka
AU - Kodera, Takuya
AU - Kobayashi, Kumi
AU - Nakajima, Chie
AU - Kawase, Mitsuo
AU - Suzuki, Yasuhiko
N1 - Funding Information:
This study is based on previous results obtained from a project subsidized by the New Energy and Industrial Technology Development Organization (NEDO) . The research was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan(MEXT) , the Joint Research Program of the Research Center for Zoonosis Control, Hokkaido University to YS, and in part by Japan Agency for Medical Research and Development (AMED) under Grant Number JP20jk0210005 , JP20jm0110021 and JP20wm0125008 to YS.
Funding Information:
We thank Dr. Aki Tamaru for providing DNA extracted from clinically isolated M. tuberculosis. This study is based on previous results obtained from a project subsidized by the New Energy and Industrial Technology Development Organization (NEDO). The research was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan(MEXT), the Joint Research Program of the Research Center for Zoonosis Control, Hokkaido University to YS, and in part by Japan Agency for Medical Research and Development (AMED) under Grant Number JP20jk0210005, JP20jm0110021 and JP20wm0125008 to YS.
Publisher Copyright:
© 2020 Elsevier B.V.
PY - 2020/10
Y1 - 2020/10
N2 - 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.
AB - 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.
KW - DNA-chromatography
KW - Loop-mediated isothermal amplification
KW - Mutation detection
KW - Mycobacterium tuberculosis
KW - Rapid detection
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U2 - 10.1016/j.mimet.2020.106062
DO - 10.1016/j.mimet.2020.106062
M3 - Article
C2 - 32950563
AN - SCOPUS:85091194771
VL - 177
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
SN - 0167-7012
M1 - 106062
ER -