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目的:针对活的沙门菌,建立快速检测的方法。方法:以沙门菌OmpC基因RNA为检测对象,针对其设计荧光定量PCR引物和探针,摸索合适的反应体系和反应条件,同时以沙门菌及其亲缘关系较近的对照菌株进行特异性实验,以PCR产物克隆质粒和沙门菌纯培养物梯度稀释进行灵敏度实验,最终建立食源性沙门菌快速检测逆转录实时荧光PCR方法。结果:所设计的S-F/S-R引物和S-Probe探针能有效地将沙门菌与其他亲缘关系较近的肠杆菌科细菌如金黄色葡萄球菌、肺炎克雷伯菌、志贺菌等区分开来。该方法对沙门菌纯培养物梯度稀释后的检测下限为<10 CFU/25 ml。同时,将建立的RNA逆转录荧光定量PCR检测方法与DNA荧光定量检测方法进行比较,DNA荧光定量PCR灵敏度仅为103 CFU/ml,远低于逆转录荧光定量PCR,且逆转录荧光定量PCR针对RNA进行检测,与活的沙门菌相关联,准确度更高。结论:建立沙门菌逆转录荧光定量PCR检测技术,具有特异、灵敏、快捷的特点,适用于食品中沙门菌的快速检测。
Objective: To establish a rapid detection method for live Salmonella. Methods: Salmonella OmpC gene RNA was used as the detection target. Fluorescent quantitative PCR primers and probes were designed, and the suitable reaction system and reaction conditions were explored. At the same time, specific experiments were carried out with Salmonella and its close relatives. Sensitivity experiments were carried out with gradient dilution of PCR product cloning plasmids and Salmonella pure culture, finally, real-time PCR method of rapid detection of reverse transcription by foodborne Salmonella was established. Results: The designed SF / SR primers and S-Probe probes can effectively distinguish Salmonella from other closely related Enterobacteriaceae such as Staphylococcus aureus, Klebsiella pneumoniae and Shigella Come. The method has a lower detection limit of <10 CFU / 25 ml for gradient dilution of Salmonella pure culture. At the same time, the established method of RNA reverse transcription fluorescence quantitative PCR and DNA fluorescence quantitative detection methods compared DNA fluorescence quantitative PCR sensitivity of only 103 CFU / ml, much lower than the reverse transcriptional quantitative PCR, and reverse transcriptional quantitative PCR for RNA is detected, associated with live Salmonella, with greater accuracy. Conclusion: The establishment of Salmonella reverse transcription fluorescence quantitative PCR detection technology has the characteristics of specificity, sensitivity and quickness, and is suitable for the rapid detection of Salmonella in food.