摘要
本研究应用多重PCR反应(mPCR)结合变性高效液相色谱(DHPLC)技术建立食品中空肠弯曲菌和结肠弯曲菌的快速检测方法。以编码嗜热弯曲菌属的16S rRNA基因、编码空肠弯曲菌的gyrA基因和编码结肠弯曲菌的cdt基因为靶基因,选择3对引物,建立并优化了鉴别空肠弯曲菌和结肠弯曲菌的mPCR体系,扩增产物分别为287bp、159bp和173bp。采用22株细菌验证了该mPCR具有特异性。mPCR检测的灵敏度在DNA水平上达到空肠弯曲菌10pg/μL、结肠弯曲菌1pg/μL;在人工模拟污染样品起始污染浓度为1.5个/mL时,42℃微需氧条件下培养24h即可被检出。在随机采集的172份冷冻鸡肉类样品中,检出了18份样品为空肠弯曲菌阳性,7份样品为结肠弯曲菌阳性。本研究建立的mPCR-DHPLC方法可特异、灵敏地实现对空肠弯曲菌和结肠弯曲菌的快速检测。
We report a new molecular technique for the analysis of Campylobacter jejuni and Campylobacter coli based on the separation of target fragments by denaturing high-performance liquid chromatography （DHPLC）. A multiplex PCR （mPCR） assay for detection of Campylobacter jejuni and Campylobacter coli was developed using primers that specifically amplify segments of the 16S rRNA, gyrA and cdt genes. The mPCR had a detection limit of 10 pg/μL genomic DNA for Campylobacterjejuni and 1 pg/μL for Campylobacter coil No cross-reaction was detected with non-thermo-tolerant Campylobacter and other 22 strains tested. The assay could detect 1.5 CFU/mL Campylobacter jejuni and Campylobacter coil in artificial inoculated chicken meat sample after enrichment at 42℃ in microaerophilic condition for 24 h. Test on 172 chicken meat samples detected 18 Carapy- lobacter jejuni positive and 7 Campylobacter coli positive. These results indicated that the multiplex PCR-DHPLC assay could be used for specific detection of Campylobacter jejun and Campylobacter coli.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2009年第3期207-212,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家"十一五"科技支撑计划(2006BAK02A13)
关键词
空肠弯曲菌
结肠弯曲菌
多重PCR
变性高效液相色谱
Campylobacter jejuni
Campylobacter coli
multiplex polymerase chain reaction（mPCR）
denaturing high-performance liquid chromatography （DHPLC）
