Department of Infectious Diseases, The Fifth Affiliated Hospital, Sun Yat-Sen University, Zhuhai, 519000, China.
Department of Gastroenterology, Guangdong Provincial Key Laboratory of Gastroenterology, State Key Laboratory of Organ Failure Research, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Int J Infect Dis. 2022 Sep;122:676-684. doi: 10.1016/j.ijid.2022.07.032. Epub 2022 Jul 16.
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.
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.
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.
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.
本研究旨在开发一种针对粪便中艰难梭菌菌株 tetM 基因的快速、可视化环介导等温扩增（LAMP）检测方法。
通过 LAMP 用浊度和可视化检测来识别艰难梭菌中的 tetM 基因，设计引物。测定 LAMP 引物的灵敏度和特异性。此外，我们对 300 株粪便培养的产毒艰难梭菌菌株的 tcdA、tcdB、cdtA、cdtB、ermB 和 tetM 基因进行了 LAMP 和聚合酶链反应（PCR）检测。
目标 DNA 在 62°C 温度下 60 分钟内扩增并可视化。共有 26 株细菌 tetM 为阴性，表现出引物的高特异性。LAMP 的检测限为 36.1 pg/µl，比 PCR 灵敏 100 倍。LAMP 和 PCR 检测粪便中产毒艰难梭菌菌株 tetM 的阳性率分别为 93.3%和 93.3%。这些艰难梭菌菌株的毒素类型比例分别为 95.7%为 A+B+CDT-、4%为 A-B+CDT-和 0.3%为 A+B+CDT+。
这是首次通过 LAMP 检测粪便中艰难梭菌菌株的 tetM 基因。其高特异性、灵敏度和可视化检测使其成为快速检测的有力诊断工具。
