摘要
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 。
Objective:To develop modified molecular beacon based fluorescence PCR assay for the detection of V. parahaemolyticus.The established method was appliedto the rapid diagnosis of V. parahaemolyticus food poisoning and sea food examination.Methods:Two sets of primers were selected from the core sequence of TDH gene publishedin GenBank,and the modified molecular beacon labeled at 5'end & 3'end with diffferent fluorophors was designed. The molecular beacon and primer set were tested against numerous strains from 12 different bacterial species. Real-time PCR assay for V.parahaemolyticus was established. Then this assay was applied to the food poisoning diagnosis.Results:Only V. parahaemolyticus strains generated fluorescent signals, and no cross-reaction was observed with other 11 bacterias. The sensitivity achieved was 69cfu /ml or 6cfu/PCR reaction. The assay was used to detect 40 V. parahaemolyticus strains, no false signals were observed.Forty-eight food poisoning samples and 100 sea food samples were tested, nine samples were found V. parahaemolyticus positive by real time PCR. Among the tested positive samples, seven samples were detected positive by traditional culture method. The overall test could be finished within one day starting from sample preparation.Conclusion:The modified molecular beacon-based real-time PCR assay was rapid, sensitive, and specific.It required 2 hours for the detection. It could be applied to the rapid diagnosis of V. parahaemolyticu food poisoning & food microbiology examination.
出处
《现代预防医学》
CAS
2004年第3期441-443,共3页
Modern Preventive Medicine
基金
国家自然科学基金资助项目 (3 0 3 0 0 2 81)
广东省卫生厅资助项目 (A2 0 0 3 70 9)
关键词
分子信标
实时PCR
快速检测
副溶血弧菌
食物中毒
V. parahaemolyticus
Modified molecular beacon
TDH
Real-time PCR
Rapid detection
