食品中沙门菌PCR检测方法的建立

为建立食品中快速检测沙门菌的PCR方法。选取沙门菌属侵袭性抗原保守基因invA基因上的靶序列设计一对引物,选择最适Mg 浓度和退火温度,建立最适PCR反应体系,用2%琼脂糖,5μl反应产物(包括EB),100V,40min进行电泳,显像。用该引物对已经传统方法鉴定的22种77株沙门菌和24种24株非沙门菌进行特异性检测,并对人工污染的食品进行检测条件的研究。Mg 浓度和退火温度对该反应体系的影响较小,稳定性较好;经传统方法鉴定的22种77株沙门菌和24种24株非沙门菌验证了该检验方法具有很好的特异性;该检测方法可以在19h内检出含有沙门菌102CFUg的食品(火腿肠、鸡蛋、散装肉馅)。与传统方法比较,该方法快速、敏感、特异,能在较短的时间内对大量样品同时进行检测,适用于食品中沙门菌的快速、敏感、特异检测。

Polymerase Chain Reaction (PCR) Method for Detection of Salmonella in Foods

To establish a rapid detection technique for Salmonella in foods by polymerase chain reaction(PCR). Amplification of nucleotide sequences within the invA gene of Salmonella was evaluated as a means of detecting Salmonella. A collection of 77 strains of Salmonella, isolated mostly from humans and animals in China, and a collection of 24 genera of non-Salmonella bacteria were used for the research of detecting Salmonella in foods by the PCR. Results: (1) The PCR method of detecting Salmonella was established, including sample preparation, amplification of Salmonella, DNA extraction, amplification of the DNA and detection of the amplification products. The result showed that the PCR method had good stability because the Mg and the annealing temperature had little impact on the PCR reaction. (2) All the Salmonella strains could be amplified into 284 bp products while none of the non-Salmonella genera yielded specific amplification product. (3) The assay allowed detection of Salmonella from foods containing 102 CFU/g of the bacteria within 19 hours. In contrast to the conventional culture technique, the PCR method is more rapid, sensitive and specific. (4) The assay was able to detect Salmonella from a lot of food at the same time in a shorter period. The method deserves spreading. A rapid, sensitive and specific PCR method is established to detect Salmonella in foods.