PCR法检测肉鸭屠宰加工生产链中沙门氏菌的污染

Detection of Salmonella typhimurium in Duck-slaughtering Chain by PCR

为建立肉鸭加工中快速、准确的沙门氏菌PCR检测方法,并应用于肉鸭屠宰生产加工链沙门氏菌的实时监测。采用Primer Premier 5.0软件针对沙门氏菌特有的fimY基因设计合成一对引物5′-GCATTCCGCTCATTAGAT-3′和5′-TGGAGGCTGATAACAAGG-3′,并对沙门氏菌DNA扩增PCR体系的退火温度、引物浓度、Mg2+浓度和聚合酶浓度进行优化。结果表明:成功扩增出沙门氏菌标准株和分离株的275bp目的片段,灵敏度达到了1.2pg;应用建立的PCR方法对国内某典型肉鸭屠宰厂的肉鸭屠宰链环节进行沙门氏菌污染的检测,发现在屠宰环境、拔毛浸烫水、浸蜡冷却水、清洗池水、宰前毛、食道、粪便和沥水体腔内共有25个样品中扩增出了275bp大小的特异性片段,而空白对照无扩增条带。

In order to establish a rapid and accurate detection method of Salmonella typhimurium contamination in duck-slaughtering chain, PCR was used to monitor duck-slaughtering chain in real time. A pair of primers 5′-GCATTCCGCTCATTAGAT-3′ and 5′-TGGAGGCTGATAACAAGG-3′ were designed to amplifyfimY gene by PCR according to the publishedfimY gene sequence of Salmonella typhimurium. PCR reaction conditions were optimized. Results indicated that a fragment with 275 bp in length was amplified in both standard strain and isolated strain ofSalmoneUa typhimurium. The detection limit of PCR assay was 1.2 pg. This established PCR method was used for the detection of Salmonella typhimurium in a typical duck-slaughtering chain. Totally 25 samples from plucking pool, cooling wax pool, cleaning pool, feathers, esophagus, intestine, colorectal feces, and dripping peritoneal were amplified to produce the 275 bp fragment. In contrast, no amplified fragment was achieved in the negative control. In the present study, a PCR assay was first developed for the detection of Salmonella typhimurium in duckslaughtering chain and typical duck-slaughtering chain with serious contamination of Salmonella typhimurium was observed. These results provided a practical application and useful guidance to the management of duck meat production and food safety.