海洋创伤弧菌LAMP快速诊断方法的建立与评价

发布时间：2018-03-26 20:34

本文选题：海洋　切入点：创伤弧菌　出处：《国际检验医学杂志》2016年05期

【摘要】：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。
[Abstract]:Objective to establish a rapid, simple, specific and sensitive method for the detection of marine vibrio vulnificus by using loop mediated isothermal amplification (Lampp) technique. The specificity and sensitivity of the method were evaluated. Methods four LAMP primers were designed according to the sequence published by GenBank. 47 strains of bacteria (including 20 strains of Vibrio) were amplified by LAMP and polymerase chain reaction (PCR) and their specificity was compared. The recombinant plasmid containing vvhA gene fragment was constructed as the standard positive control of LAMP reaction system. Results there were false positive results in routine PCR test and positive amplification in LAMP assay only in Vibrio vulnificus. All the other samples were negative, no false positive and false negative results, which showed that the specificity of primers was better, and the results of adding calcium yellowish were consistent with those of electrophoretic electrophoresis. The minimum detection limit of LAMP technique for vvhA gene was 4 脳 10 CFU per reaction, which was 10 times as high as that of conventional PCR (4 脳 10 ~ 2 CFU per reaction). The results of repeated experiments were stable. Conclusion A method for detection of marine Vibrio vulnificus by LAMP was established, which was specific, sensitive, convenient and rapid. It is especially suitable for quick detection on the spot and beside the bed.
【作者单位】：辽宁医学院;威海市立医院;济南博科生物科技有限公司;济南军区总医院;
【基金】：全军“十二五”科研重点项目(BWS12J014)
【分类号】：R440;R378

【相似文献】