Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae by using loop-mediated isothermal amplification

Objective To establish a loop-mediated isothermal mplification (LAMP) assay for the rapid and specific detection of cholera toxin-producing Vibrio cholerae. Methods A set of 6 primers,including 2 outer primers,2 inner primers and 2 loop primers, were designed specifically to recognize the ctxA gene of V.cholerae.The reaction conditions were optimize.Specificity and sensitivity of LAMP were tested by using cholera toxin-producing V. cholerae, non-cholerae vibrio isolates and non-vibrio bacterial isolates. Results The optimized time and temperature conditions for the LAMP assay were 60 min and at 65 ℃, respectively. The LAMP could accurately identify the V. Cholerae isolates carrying the ctxA gene, but fail to detect other non-cholerae vibrio isolates and non-vibrio bacterial isolates. The detection limits of the method were 68 fg of purified genomic DNA/reaction and 42 cfu/ml. In addition,this method was applied to detect artificially contaminated samples and the detection limit was 72 cfu/g for the artificially contaminated samples without precultivation. Conclusion The results suggested that the LAMP is sensitive, specific, inexpensive and rapid in the detection of V. cholerae carrying ctxA gene, which can used in basic clinical laboratory.