海洋创伤弧菌LAMP快速诊断方法的建立与评价

目的利用环介导等温扩增(LAMP)技术,建立海洋创伤弧菌快速、简便、特异且敏感的检测方法,并对该方法的特异性和灵敏度进行评价。方法选取海洋创伤弧菌溶细胞素(vvhA)基因作为靶基因,根据GenBank公布的序列设计4条LAMP引物;对47株细菌(包括20株弧菌属细菌)进行LAMP和聚合酶链式反应(PCR)扩增,并做特异性比较;对创伤弧菌M06株增菌液10倍倍比稀释,提取DNA后进行灵敏度的检测,并与常规PCR作比较;构建含vvhA基因片段的重组质粒,作为LAMP反应体系的标准阳性对照。结果常规PCR实验出现假阳性结果,而LAMP实验只有海洋创伤弧菌出现阳性扩增,其他样品均为阴性,无假阳性和假阴性结果,表明引物的特异性较好,加入钙黄绿素和电泳结果相一致;针对vvhA基因建立的LAMP技术其最低检测下限为每个反应4×10CFU,是常规PCR(每个反应4×10~2 CFU)的10倍,呈现较好的灵敏度。重复实验过程两遍,PCR和LAMP技术检测结果稳定。结论建立了一种用于检测海洋创伤弧菌的LAMP检测方法,该方法特异性强,灵敏度高,方便快捷,特别适合用于现场和床旁的快速检测。

Establishment and evaluation of loop-mediated isothermal amplification assay for detecting marine Vibrio vulnificus

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 .