Minyi Lin, Zitong Li, Qianyun Lin, Pu Wang, Wei Liu, Jing Yuan, Zhongsi Hong, Ye Chen
{"title":"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","authors":"Minyi Lin, Zitong Li, Qianyun Lin, Pu Wang, Wei Liu, Jing Yuan, Zhongsi Hong, Ye Chen","doi":"10.1016/j.ijid.2022.07.032","DOIUrl":null,"url":null,"abstract":"<p><strong>Objectives: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>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.</p>","PeriodicalId":505767,"journal":{"name":"International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases","volume":" ","pages":"676-684"},"PeriodicalIF":0.0000,"publicationDate":"2022-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"Development and clinical application of a rapid and visual loop-mediated isothermal amplification test for tetM gene in Clostridioides difficile strains cultured from feces.\",\"authors\":\"Minyi Lin, Zitong Li, Qianyun Lin, Pu Wang, Wei Liu, Jing Yuan, Zhongsi Hong, Ye Chen\",\"doi\":\"10.1016/j.ijid.2022.07.032\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Objectives: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>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.</p>\",\"PeriodicalId\":505767,\"journal\":{\"name\":\"International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases\",\"volume\":\" \",\"pages\":\"676-684\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2022-09-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1016/j.ijid.2022.07.032\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2022/7/16 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.ijid.2022.07.032","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2022/7/16 0:00:00","PubModel":"Epub","JCR":"","JCRName":"","Score":null,"Total":0}
Development and clinical application of a rapid and visual loop-mediated isothermal amplification test for tetM gene in Clostridioides difficile strains cultured from feces.
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.