Development and clinical application of a rapid and visual loop-mediated isothermal amplification test for tetM gene in Clostridioides difficile strains cultured from feces.

Minyi Lin, Zitong Li, Qianyun Lin, Pu Wang, Wei Liu, Jing Yuan, Zhongsi Hong, Ye Chen
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引用次数: 1

Abstract

Objectives: In this study, we aimed to develop a rapid and visual loop-mediated isothermal amplification (LAMP) assay targeting the tetM gene in Clostridioides difficile strains cultured from feces.

Methods: Primers were designed to recognize the tetM gene in C. difficile by LAMP, using turbidity and visual detection. The sensitivity and specificity of LAMP primers were determined. In addition, we conducted both LAMP and polymerase chain reaction (PCR) for the tcdA, tcdB, cdtA, cdtB, ermB, and tetM genes in 300 toxigenic C. difficile strains cultured from feces.

Results: The target DNA was amplified and visualized within 60 minutes at a temperature of 62°C. A total of 26 bacterial strains were found negative for tetM, which manifested high specificity of the primers. The detection limit of LAMP was 36.1 pg/µl, which was 100-fold more sensitive than PCR. The positive rate of tetM in toxigenic C. difficile strains cultured from feces was 93.3% by both LAMP and PCR. The proportion of toxin types in those C. difficile strains was 95.7% for A+B+CDT-, 4% for A-B+CDT-, and 0.3% for A+B+CDT+, respectively.

Conclusion: This is the first study examining the tetM gene by LAMP in C. difficile strains cultured from feces. Its high specificity, sensitivity, and visual detection make the new assay a powerful diagnostic tool for rapid testing.

粪便培养艰难梭菌tetM基因快速、视觉环介导等温扩增试验的建立及临床应用
目的:在本研究中,我们旨在建立一种针对粪便培养的艰难梭菌菌株tetM基因的快速、视觉环介导的等温扩增(LAMP)方法。方法:设计引物,利用LAMP检测浊度法和目视法识别艰难梭菌tetM基因。测定LAMP引物的敏感性和特异性。此外,我们对300株粪便培养的产毒艰难梭菌的tcdA、tcdB、cdtA、cdtB、ermB和tetM基因进行了LAMP和聚合酶链反应(PCR)。结果:在62°C的温度下,目标DNA在60分钟内扩增并可见。共有26株菌株tetM阴性,表明引物特异性高。LAMP的检出限为36.1 pg/µl,灵敏度为PCR的100倍。从粪便中培养出的难辨梭菌中,LAMP和PCR检测tetM的阳性率为93.3%。A+B+CDT-占95.7%,A-B+CDT-占4%,A+B+CDT+占0.3%。结论:本研究首次采用LAMP检测粪便培养的艰难梭菌的tetM基因。它的高特异性、灵敏度和目视检测使它成为快速检测的有力诊断工具。
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