Takarada Yutaka, Kodera Takuya, Kobayashi Kumi, Nakajima Chie, Kawase Mitsuo, Suzuki Yasuhiko
TBA Co., LTD, T-Biz 307, , 6-6-40 Aramaki-Aza-Aoba, Aoba-ku, Sendai 980-8579, Japan.
Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan; Hokkaido University, GI-CoRE Global Station for Zoonosis Control, Sapporo 001-0020, Japan.
J Microbiol Methods. 2020 Oct;177:106062. doi: 10.1016/j.mimet.2020.106062. Epub 2020 Sep 17.
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.
快速、简便地检测与耐药性相关的细菌病原体中的核苷酸点突变对于抗菌药物的合理使用至关重要。在此，我们开发了一种快速简便的突变检测方法，该方法将环介导等温扩增（LAMP）与单标签杂交（STH）色谱印刷阵列条（PAS）方法相结合。此程序能够同时检测耐利福平结核分枝杆菌（RR-TB）的利福平耐药决定区（RRDR）中的四个突变（C1349T、A1295C、G1303T、A1304T）。LAMP反应包含一个LAMP引物和针对每个突变的八个等位基因特异性引物。等位基因特异性引物产物通过使用PAS的核酸色谱法进行检测。在那里检测到四条检测线，其中一条根据野生型和突变型在不同位置被检测到。我们使用来自临床分离株的31份基因组DNA（2份A1295T、1份G1303T、6份A1304T、22份C1349T）进行了这四个突变的检测。这些突变已通过序列分析得到证实。检测结果与序列分析完全一致。在本研究中，可以检测到四个突变，但仅60%的RR-TB可通过这四个突变被检测到。预计通过添加更多突变引物检测率将会提高。LAMP与STH色谱PAS方法相结合是一种简单快速的检测临床分离株中位点突变的方法，可作为即时检测（POCT）技术。此外，它不需要特殊设备，能够满足耐药菌流行地区（如发展中国家）的需求。
