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目的建立高危海产品中霍乱弧菌的PCR-激光诱导荧光-毛细管电泳检测方法。方法本研究以高危海产品为研究对象,针对霍乱弧菌肠毒素ctx A基因选择引物,利用PCR反应扩增霍乱弧菌的特征基因片段,用高效毛细管电泳-激光诱导荧光快速检测PCR产物。本研究对PCR条件及毛细管电泳分离条件进行优化,从而实现对高危海产品中霍乱弧菌的快速准确检测。结果在优化的PCR条件下,霍乱弧菌的ctx A基因可得到有效扩增,并且无非特异性条带;PCR扩增产物测序后,与基因库中的序列进行同源性比对,匹配度为99%。所建立的方法用于60份高危海产品中霍乱弧菌的检测,结果与普通凝胶电泳法所得结果一致。结论本方法所需样品量少、快速、灵敏、准确,可以成功用于霍乱弧菌的检测。
Objective To establish a method for the detection of Vibrio cholerae in high-risk seafood by PCR-laser-induced fluorescence-capillary electrophoresis. Methods In this study, high-risk seafood was selected as the research object. Vibrio cholerae enterotoxin ctx A gene-selective primers were used to amplify the characteristic gene fragments of Vibrio cholerae by PCR. The PCR products were rapidly detected by high performance capillary electrophoresis with laser-induced fluorescence. In this study, conditions of PCR and conditions of capillary electrophoresis separation were optimized, in order to achieve rapid and accurate detection of Vibrio cholerae in high-risk seafood. Results Under optimized PCR conditions, the ctx A gene of Vibrio cholerae could be effectively amplified with no specific bands. After sequencing, the PCR products were compared with the sequences in GenBank and the match degree Is 99%. The established method was used for the detection of Vibrio cholerae in 60 high-risk seafoods. The results were consistent with those obtained by ordinary gel electrophoresis. Conclusion The method required less sample volume, fast, sensitive and accurate, can be successfully used for the detection of Vibrio cholerae.