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目的:建立溶藻弧菌TaqMan实时PCR快速检测体系。方法:根据溶藻弧菌胶原酶基因colH的保守序列设计一对引物和一条TaqMan探针,优化反应体系中的引物浓度和探针浓度,评价此反应体系的特异度,并确定其检测下限。结果:优化的反应体系中,引物浓度为200 nmol/L,探针浓度为200 nmol/L,检测下限为1.0×102拷贝/μl,较普通PCR提高了100倍。特异度为100%。结论:本研究所建立的检测体系能够灵敏和特异地检测溶藻弧菌,可以作为溶藻弧菌的快速检测方法。
Objective: To establish a real-time PCR rapid detection system of Vibrio alginolyticus TaqMan. Methods: A pair of primers and a TaqMan probe were designed according to the conserved sequence of colH gene of V. alginolyticus. The primer concentration and probe concentration in the reaction system were optimized. The specificity of this reaction system was evaluated and the detection limit was determined. Results: The optimum reaction conditions were as follows: the concentration of primer was 200 nmol / L, the concentration of probe was 200 nmol / L, the detection limit was 1.0 × 102 copies / μl, which was 100 times higher than that of normal PCR. Specificity is 100%. Conclusion: The detection system established in this study can detect Vibrio alginolyticus sensitively and specifically, which can be used as a rapid detection method for Vibrio alginolyticus.