摘要
目的 建立改良分子信标—实时PCR检测霍乱弧菌的快速方法 ,应用于霍乱监测。 方法 根据GenBank公布的霍乱弧菌肠毒素基因A亚单位 (ctxA)的保守序列 ,设计一对引物和改良分子信标探针 ,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霍乱弧菌的实时PCR反应体系 ,应用于霍乱监测。 结果 霍乱弧菌改良分子信标检测体系检测 12种细菌 ,只有霍乱弧菌有荧光信号 ,与其他细菌无交叉反应 ,DNA灵敏度为10 2 4fg/ul ,菌液灵敏度为 3 2cfu/ml或 3cfu/PCR反应体系。对 10 0份海产品和 3 0份腹泻病人大便标本进行检测 ,结果都为阴性。检测时间仅需 2h。 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 ,可用于霍乱的快速诊断 。
Objective To develop a modified molecular beacons and real-time PCR for rapid detection of Vibrio cholera and surveillance of cholera. Methods One set of primers was selected from the core sequence of ctxA gene in GenBank and the corresponding modified molecular beacon labeled with fluorophors was designed. The molecular beacon and primer set was tested againste numerous strains from 12 different bacterial species. Real-time PCR assay for the detection of V. cholera was established & applied to cholera surveillance. Results Only V. cholera strains possessing ctxA gene generated fluorescent signals, and no cross-reaction was observed with other 11 bacteria. The sensitivity achieved was 32cfu/ml or 3cfu/PCR reaction. 100 sea food samples & 30 stools were tested, all samples were negative by real time PCR & traditional culture method. Conclusion The modified molecular beacons-based real-time PCR assay was rapid, sensitive, and specific. It required 2 hours. It could be applied to the rapid diagnosis of cholera.
出处
《中国热带医学》
CAS
2004年第4期499-501,共3页
China Tropical Medicine
基金
国家自然科学基金资助项目 (30 30 0 2 81 )
广东省卫生厅资助项目 (A2 0 0 370 9)
