Toxicity-Based Typing of Clostridioides Difficile by Loop-Mediated Isothermal Amplification (LAMP) in Critically Ill Patients

摘要

This study establishes the loop-mediated isothermal amplification (LAMP) assay for detection of the toxin genes tcdA and tcdB in Clostridioides difficile (C. difficile). Genomic DNA was extracted based on magnetic nanoparticles and amplified by LAMP using the primers designed by us. The specificity and sensitivity of the primers were also determined by agarose gel electrophoresis and fluorescence intensity detection. A total of 11 pathogenic bacterial strains from different species were found to be negative for tcdA and tcdB, whereas only one positive result was obtained for C. difficile. The limit of detection for tcdA and tcdB was determined to be 1 pg/mu L and 10 pg/mu L, respectively. The primers were found to be highly specific and sensitive. The LAMP assay was then applied for the detection of tcdA and tcdB in the feces of critically ill patients and to perform C. difficile typing. In conclusion, the LAMP assay is a reliable tool for the identification of C. difficile toxin genes tcdA and tcdB as well as for direct detection of the toxin genes in feces of critically ill patients with high sensitivity and specificity. This method established by us shows immense potential for clinical application in the future and can greatly reduce the challenges in the detection of C. difficile toxin genes through an expedited process.