Sensitive and Rapid Detection of ermB Gene in Clostridium difficile by Loop-Mediated Isothermal Amplification

Introduction: Macrolide-lincosamide-streptogramin B resistance in Clostridium difficile is mostly due to the ermB resistance determinant. This paper describes a sensitive and rapid molecular method to detect ermB in clinical isolates to guide therapeutic treatment. Methods: Five sets of loop-mediated isothermal amplification (LAMP) primers were designed to target 6 or 8 distinct sequences on erm B gene by Primer Explorer V4 soft ware. The optimal primers were selected for rapid detection and chosen to determine the specificity and sensitivity of the LAMP reactions for erm B by using DNA extracted from reference strain API10463. Twenty-six enteric pathogenic bacteria strains were selected for specificity assays. Both the real-time monitoring of turbidity and the chromogenic reaction using calcein/Mn2+ complex were included to determine negative and positive results. The results demonstrated that target DNA was amplified and visualized by the 2 detection methods within 60 minutes at an isothermal temperature of 62°C. Besides, erm B gene in Clostridium difficile from 62 clinical isolates were tested by the developed LAMP assay followed by conventional PCR. Simultaneously, the tcdA, tcdB, cdtA, cdtB, and tetM genes were detected by PCR. Results: A total of 26 bacteria strains were negative for erm B, which indicated the high-specificity of the primers. The detection limit of LAMP was 36.1 pg/μl DNA, and its sensitivity was 10-fold greater than that of conventional PCR. The positive rate of erm B obtained from 62 clinical isolates of Clostridium difficile were 98.4% (61/62) by LAMP, which was completely consistent with PCR. Besides, among the 62 isolates analyzed in this study, 60 were toxin A-positive and toxin B-positive (A+B+), 1 was toxin Anegative but toxin B-positive (A−B+), 1 was toxin A, toxin B and binary toxin-positives (A+B+CDT+). Both the A−B+ isolate and 2 A+B+ isolates were negative for tet M, while the rest of isolates were positive (95.2%,59/62). Conclusion: This study is the first report regarding the application of the LAMP assay for detection of erm B gene in Clostridium difficile. The developed LAMP method is exhibited to be a potentially valuable, sensitive, specific, simple, and inexpensive assay for the rapid detection of erm B.