改良分子信标实时PCR快速检测大肠杆菌O_(157):H_7

Rapid Detection of E. coli O_(157):H_7 by using modified beacons and real-time PCR

目的建立改良分子信标实时PCR快速检测大肠杆菌O157H7的快速方法。方法根据GenBank公布O157H7的rfbE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’端,建立改良分子信标实时PCR检测O157H7的反应体系,应用于食物中毒快速诊断和食品微生物检测。结果共检测11种细菌,只有大肠杆菌O157H7有荧光信号,其余10种全无荧光信号,且与其他细菌无交叉反应,DNA灵敏度为64fg,菌液灵敏度为59cfu/ml或2cfu/PCR反应体系。应用改良分子信标实时PCR反应体系检测10株O157H7均出现特异的荧光信号,无干扰。对89份食品样品进行检测,5份O157H7实时PCR阳性,其余样品为阴性,检测仅需2h。5份阳性样本,经传统方法培养,有3份检出大肠杆菌O157H7。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,可用于大肠杆菌O157H7食物中毒的快速诊断和食品微生物检测,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 method for rapid detection of E. coli O157 ： H7 from samples of contaminated food by using modified molecular beacons and real - time PCR. Methods The primers and modified molecular beacons were designed based on the core sequence of rfbE gene published on GenBank for detection,of E. coli O157 ： H7. The probe was labeled with FAM at 5＇ end. The molecular beacons and primer set was tested against numerous strains from 11 different bacterial species and used for rapid detection and diagnosis of ffod poisoning, Results For the modified molecular beacons - based real - time PCR assay, the sensitivity was 64fg, 59cfu/ml or 2cfu/PCR reaction. There was no cross - reaction with other bacteria as control. The real - time PCR assay was used to detect 10 E. coli O157 ： H7 and no false signals were observed, 89 food samples were tested and E. coli O157 ： H7 was detected from 5 samples by real time PCR, 3 samples were positive for E. coli O157 ： H7 detected by traditional culture method. The overall test could be finished within 2 hours. Conclusion The modified molecular beacons - based real - time PCR assay is rapid, sensitive and specific for detection of E. coli O157 ： H7 from contaminated food.