Establishment and evaluation of loop-mediated isothermal amplification assay for detecting marine Vibrio vulnificus / 国际检验医学杂志

ABSTRACT

Objective To develop a rapid ,convenient ,sensitive and specific method for the detection of marine Vbrio vulnificus by using loop‐mediated isothermal amplification(LAMP) technique ,and evaluate the specificity and sensitivity of this method . Methods According to marine Vibrio vulnificus cytolysin(vvhA) gene sequence published by GenBank ,a set of LAMP primers were designed .LAMP and polymerase chain reaction(PCR) amplification were carried out in 47 strains of bacteria(including 20 strains of Vibrio) and specificities of LAMP and PCR were compared .Serial ten‐fold dilutions of an overnight Vibrio vulnificus M06 culture were prepared in sterile saline solution ,and compared the sensitivity of LAMP with that of PCR after extracting DNA tem‐plates .A recombinant plasmid containing fragment of vvhA gene was constructed and set as a standard positive control of LAMP assay .Results False‐positive results occurred in the conventional PCR ,while in the LAMP assay positive amplifications were only seen in strains of Vibrio vulnificus and no false‐positive or false‐negative results were generated among 47 strains of bacteria ,which indicated that primers had high specificity .Additionally ,the results of electrophoresis were consistent with those after adding the calcein .The detection limit of LAMP was 4 × 10 CFU each reaction for detecting vvhA gene in pure culture ,which was 10‐fold more sensitive than that of conventional PCR(4 × 102 CFU each reaction) .It indicated that LAMP assay had good sensitivity .Repeated the test twice ,the detection results of LAMP and PCR were both stable .Conclusion An LAMP detection method for marine Vibrio vulnificus has been developed ,which is highly specific ,sensitive ,convenient and suitable for rapid field detection and point‐of‐care testing .