空肠弯曲杆菌和结肠弯曲杆菌双重PCR鉴别检测方法的建立

Establishment of double PCR for detection and differentiation of Campylobacter jejuni from Campylobacter coli

为快速检测和鉴定空肠弯曲杆菌和结肠弯曲杆菌,本研究设计并合成了空肠弯曲杆菌马尿酸酶基因hipO和结肠弯曲杆菌cdtC基因的2对特异性引物,建立并优化了快速检测空肠弯曲杆菌和结肠弯曲杆菌的双重PCR方法。结果显示,双重PCR对空肠弯曲杆菌hipO基因和结肠弯曲杆菌cdtC基因的扩增产物的大小分别为653bp和313bp。PCR扩增条件最终确定为退火温度为52℃,dNTP终浓度为0.4mmol/L,hipO和cdtC基因引物的终浓度均为2 5μmol/L。该双重PCR的灵敏度可达到6.6 8×1 0-2μg/mL,对多杀性巴氏杆菌、产单核细胞李氏杆菌、产气荚膜梭菌、伤寒沙门菌和金黄色葡萄球菌扩增均无目的条带,说明该PCR方法具有良好的灵敏性和特异性。符合试验结果与单一PCR方法结果相同。用所建立的方法对本实验室采集的鸡肛拭子划线培养后的可疑菌落进行PCR鉴定,结果显示2株为空肠弯曲杆菌,2株为结肠弯曲杆菌。结果表明,本研究建立的双重PCR可以用于同时检测空肠弯曲杆菌和结肠弯曲杆菌。

To detect and identify Campylobacter jejuni and Campylobacter coli,a double PCR method was developed based on two pairs of primers for hipOof C.jejuni and cdtCof C.coli gene amplification.The results demonstrated that 653 bp and 313 bp DNA fragments of hipOand cdtCgenes were amplified by this protocol,respectively.The PCR amplification conditions ultimately were determined that the annealing temperature was 52 ℃,the dNTP concentration was 0.4mmol/L,the primer concentration of both hipO and cdtC gene were 25μmol/L.The sensitivity of the double PCR can achieve 6.68×10-2μg/mL,no target product was amplified from Pasteurella multocida,Listeria monocytogenes,Clostridium perfringens,Salmonella typhi and Staphylococcus aureus respectively.The single PCR method results were consistent with the kit of the method results.Furthermore,the streaked suspected colonies from chicken anal swabs were tested by the PCR assay and the results showed 2samples were positive for C.jejuni and 2samples was positive for C.coli.In conclusion,the established double PCR method in this study can be used for the rapid detection and identification of C.jejuni and C.coli.