水中产气荚膜梭菌的荧光定量PCR检测法

发布时间：2018-07-26 06:18

【摘要】：目的研究水中产气荚膜梭菌的荧光定量PCR检测方法,缩短水中产气荚膜梭菌的检测时间。方法根据产气荚膜梭菌的α-toxin基因设计引物并扩增,构建α-toxin-T重组质粒,对重组质粒进行梯度稀释后进行荧光定量PCR检测;用该方法检测水中常见的15种细菌,确定方法的特异性;检测产气荚膜梭菌加标水样,验证方法的可行性。结果成功扩增出产气荚膜梭菌的α-toxin基因并连接T载体(417 bp)。α-toxin-T重组质粒的稀释度在2.17×10~0~2.17×10~9 copies/μl的范围内,PCR反应的特异性良好,且重组质粒各浓度梯度间间隔的Ct值相近;所得回归方程为y=31.69-3.11x,r=0.997 73;该方法的检出限可达到为10 copies/μl。15株受试细菌DNA的检测结果均为阴性,产气荚膜梭菌加标水样检测结果阳性。结论该方法操作快速,具有较好的特异性和灵敏性,适用于水中产气荚膜梭菌的快速检测。
[Abstract]:Objective to study the fluorescence quantitative PCR method for the detection of Clostridium perfringens in water, and to shorten the detection time of Clostridium perfringens in water. Methods according to the 伪 -toxin gene of Clostridium perfringens, primers were designed and amplified to construct 伪 -toxin-T recombinant plasmid. The recombinant plasmid was detected by fluorescence quantitative PCR after gradient dilution, and 15 common bacteria in water were detected by this method, and the specificity of the method was determined. Detection of Clostridium perfringens plus standard water samples to verify the feasibility of the method. Results the 伪 -toxin gene of Clostridium perfringens was successfully amplified and ligated into T vector. The dilution of the recombinant plasmid was 2.17 脳 10 ~ (10) ~ 0 ~ 2.17 脳 10 ~ (9) copies/ 渭 l. The regression equation was obtained as YYI 31.69-3.11xrnr 0.99773.The detection limit of this method was 10 copies/ 渭 l.15 strains of bacteria DNA were negative, and the results of Clostridium perfringens plus standard water samples were positive. Conclusion the method is rapid, specific and sensitive, and can be used for rapid detection of Clostridium perfringens in water.
【作者单位】：中国疾病预防控制中心环境与健康相关产品安全所;
【基金】：国家卫生和计划生育委员会公益性卫生行业科研专项(201302004)
【分类号】：R123

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