动物性食品源空肠弯曲杆菌二重PCR检测方法的建立及应用

Development and Application of Multiplex PCR Assay for the Detection of Campylobacter jejuni in Animal Origin Food

根据GenBank空肠弯曲杆菌（Campylobacter jejuni，cj）的16SrDNA及hip O（编码马尿酸酶基因）序列设计两对特异引物，建立检测动物性食品源cj的二重PCR方法，并应用于样品检测。结果显示只对Cj能特异的扩增出699bp和366bp两个基因片段，而大肠杆菌、沙门氏菌等其他11种细菌均未扩增出条带；Cj标准株ATCC33560的16SrDNA及hip O序列与GenBank其他Cj的相应序列具高度相似性（分别为99．7％～99．9％，98．1％～99．7％）；该方法可在27h内完成，其灵敏度为2．4～16CFU／mL；四川省雅安市鸡肉、猪肉、牛肉和牛奶样品中的Ci阳性率分别为38．0％（19／50）、28．3％（15／53）、17．1％（6／35）和8．6％（4／46）。

According to the 16S rDNA and hip 0 gene sequences of C. jejuni in GenBank, two pairs of specific primers were designed and used in multiplex PCR for detection of C. jejuni in animal origin food. Multiplex PCR was developed and applied to the sample testing. The results indicated that two specific fragments （699bp and 366bp） were detected after amplification of the DNA template of C. jejuni, while other bacteria strains （ 11 species tested） were not detected. Meanwhile, the 16S rDNA and hip O gene sequence of C. jejuni ATCC33560 exhibited a high sim- ilarity with those of some strains of C. jejuni presented in GenBank. The total assay could be completed in 27h with a detection limit of 2.4 -16CFU/mL. The chicken, pork, beef and milk from Yahn markets in Sichuan province were detected by the multiplex PCR, and 38.0% , 28.3% , 17.1% and 8.6% of them were found respectively to be positive for C. jejuni. Multiplex PCR assay was specific, sensitive and time -saving, which provided reference for detection of C. jejuni in animal origin food.