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目的 :建立改良分子信标 -实时 PCR检测副溶血弧菌的快速方法 ,应用于副溶血弧菌食物中毒的快速诊断和海产品检验。方法 :根据 Gen Bank公布副溶血弧菌的耐热直接溶血毒素基因 (TDH)的保守序列 ,设计一对引物和改良分子信标探针 ,用 FAM荧光剂标记探针的 5′,并进行特异性和灵敏度分析 ;同时以 11种细菌作对照 ,建立改良分子信标检测副溶血弧菌的实时 PCR反应体系 ,应用于副溶血弧菌食物中毒快速诊断和食品微生物检测。结果 :检测 12种细菌 ,只有副溶血弧菌有荧光信号 ,与其他细菌无交叉反应 ,DNA灵敏度为 16 6 .6 fg/ μl,菌液灵敏度为 6 9cfu/ ml或 6 cfu/ PCR反应体系。改良分子信标 -实时 PCR反应体系检测 4 0株副溶血弧菌均出现特异的荧光信号 ,无干扰。对 3起细菌性食物中毒共 4 8份样品和 10 0份海产品进行检测 ,9份副溶血弧菌实时 PCR阳性 ,其中 7份副溶血弧菌细菌培养阳性 ,其余样品都为阴性。检测时间仅需 2 h。结论 :改良分子信标 -实时 PCR检测体系快速、灵敏度高 ,特异性强 ,可用于副溶血弧菌食物中毒的快速诊断 ,为食源性疾病的分子流行病学调查提供新的检测手段
Objective: To establish a rapid method for the detection of Vibrio parahaemolyticus by modified molecular beacons-real-time PCR, and to apply the rapid diagnosis of Vibrio parahaemolyticus food poisoning and seafood inspection. Methods: According to the conserved sequence of heat-labile direct hemolytic toxin gene (TDH) released by Vibrio parahaemolyticus published by Gen Bank, a pair of primers and modified molecular beacon probes were designed. The probes were labeled with FAM fluorescer 5 ’ Sex and sensitivity analysis. At the same time, 11 kinds of bacteria were used as controls, and a real-time PCR reaction system for detecting molecular Vibrio parahaemolyticus was established. The real-time PCR reaction system was applied to the rapid diagnosis of Vibrio parahaemolyticus food poisoning and food microbe detection. Results: Only 12 Vibrio parahaemolyticus had fluorescence signal and no cross reaction with other bacteria. The sensitivity of DNA was 16 6 .6 fg / μl, and the sensitivity of bacteria was 6 9 cfu / ml or 6 cfu / PCR. The modified molecular beacon - real-time PCR reaction system detected 40 strains of Vibrio parahaemolyticus showed specific fluorescence signal, without interference. A total of 48 samples of 3 bacterial food poisonings and 10 0 samples of seafood were tested. Nine of them were positive by real-time PCR, of which 7 were positive for Vibrio parahaemolyticus, and all the other samples were negative. Detection time only 2 h. Conclusion: The improved molecular beacon-real-time PCR detection system is rapid, sensitive and specific and can be used for rapid diagnosis of food poisoning caused by Vibrio parahaemolyticus, providing a new detection method for molecular epidemiological investigation of foodborne diseases