变性高效液相色谱检测空肠弯曲菌

目的应用多重PCR反应(multiplex PCR,mPCR)结合变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)技术建立食品中空肠弯曲菌的快速检测方法。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基因为靶基因,选择2对引物,建立并优化了鉴别空肠弯曲菌的多重PCR体系,扩增产物分别为287bp、159 bp。采用22株细菌验证了该多重PCR具有特异性。PCR检测的灵敏度在DNA水平上达到10 pg/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24 h可被检出。结果在随机采集的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。结论本研究建立的多重PCR-DHPLC方法可特异、灵敏地实现对空肠弯曲菌的快速检测。

Detection of Campylobacter jejuni with denaturing high-performance liquid chromatgraphy

A new molecular technique based on the use of multiplex PCR （mPCR） combined with the denaturing highperformance liquid chromatography（DHPLC） was used for the detection of Campylobaeter jejuni, in which the mPCR assay was developed by using 2 sets of primers that could specifically amplify segments of the 16S rRNA and ,gyrA genes. The target genes fragments of the mPCR assay were 287 bp and 159 bps respectively. Analysis of 22 strains of C. jejuni indicated that this PCR system was specific and the detection limit of the mPCR in the DNA level was 10 pg/ L. This mPCR assay did not crossreact with the non-tthermo-tolerant Campylobacter and could detect 1.5 CFU/mL of Campylobacter jejuni in artificially inoculated chicken meat sample after enrichment at 42℃ in microaerophilic condition for 24 hours. By using this method 18 out of 172 chicken meat sarnples showed positive result in the detection of C. jejuni. These results indicated that the multiplex PCRDHPLC assay can be used for the specific and sensitive detection of Campylobacter jejuni.