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有关的细菌病原体中的核苷酸点突变对于正确使用抗微生物剂至关重要。在这里，我们开发了一种快速简便的方法，可使用环介导的等温扩增（LAMP）与单标签杂交（STH）色谱印刷阵列条（PAS）方法相结合来检测突变。此程序能够同时检测耐利福平结核分枝杆菌（RR-TB）的利福平耐药测定区（RRDR）中的四个突变（C1349 T，A1295C，G1303 T，A1304 T）。

LAMP反应包含一个LAMP引物和每个突变的八个等位基因特异性引物。通过使用PAS的核酸色谱法检测等位基因特异性引物产物。在那里检测到四个检测线，其中一个在不同位置检测到，取决于野生型和突变型。我们使用来自临床分离株的31个基因组DNA（2个A1295T，1个G1303 T，6个A1304 T，22个C1349 T）进行了四个突变检测。已经通过序列分析确认了突变。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，但是使用这四个突变只能检测到RR-TB的60％。预期通过添加更多的突变引物可以提高检出率。

LAMP和STH色谱相结合的PAS方法是一种简便快速的方法，可作为即时检验（POCT）技术检测临床分离物中的点突变。另外，它不需要特殊的设备，并且可以满足诸如耐药菌等地方性耐药菌流行地区的需求。