含内标的多重实时PCR法同时检测空肠弯曲菌和结肠弯曲菌

Simultaneous detection of Campylobacter jejuni and Campylobacter coli using multiplex real-time PCR containing internal amplification control

中文关键词:  弯曲菌  同步快速检测  内部扩增对照

英文关键词:Campylobacter  simultaneous and rapid detection  internal amplification control

中文摘要:

      目的 建立含内标的多重实时荧光PCR法同时检测空肠弯曲菌和结肠弯曲菌。方法 针对空肠弯曲菌特有hipO基因和结肠弯曲菌特有ceuE基因设计引物探针, 设计并优化内标DNA添加量。测试了方法的特异性、灵敏度以及在鸡肉中的检出限。结果 内标的最适添加量为104 copies/PCR。所建立方法对空肠弯曲菌和结肠弯曲菌的灵敏度分别达到4.7 copies/PCR和5.23 copies/PCR; 对115株空肠弯曲菌、49株结肠弯曲菌和42株非目标菌株在3种不同类型的实时荧光PCR仪上的特异性均达到100%; 对鸡肉中空肠弯曲菌和结肠弯曲菌的检出限达到10 CFU/25 g, 与传统检测方法一致。采用所建立的方法对50份市售生鲜鸡肉进行检测发现, 空肠弯曲菌阳性率为12%(6/50), 结肠弯曲菌阳性率为4% (2/50); 传统国标检测方法除了1份空肠弯曲菌阳性样品未得到分离确认, 其余PCR阳性样品均在平板上分离确认。结论 该方法特异性强、灵敏度高、开放性好、含有内标可防止“假阴性”, 可应用于食品中2种重要致病性弯曲菌的快速同步检测。

英文摘要:

      Objective To develop a multiplex real-time PCR method with internal amplification control for the simultaneous detection of Campylobacter jejuni and Campylobacter coli. Methods Primers and probes were designed based on Campylobacter jejuni specific hipO gene and Campylobacter coli specific ceuE gene, and the internal amplification control DNA content was optimized. The specificity, sensitivity, and detection of limit in chicken were determined. Results The optimum addition of internal amplification control was 104 copies/PCR. The sensitivities reached 4.7 copies/PCR and 5.23 copies/PCR for Campylobacter jejuni and Campylobacter coli respectively. It showed 100% specificity for 115 C. jejuni strains, 49 C. coli strains and 42 non-target strains on all 3 kinds of real-time PCR systems. The limit of detection of the real-time PCR method in the artificially contaminated raw chicken meat was 10 CFU/25 g, in accordance with the traditional detection method. The real-time PCR method showed that 12% (6/50) of real chicken meat samples were contaminated with C. jejuni, and 4% (2/50) were C. coli positive, all of the PCR-positive samples were confirmed by traditional method except one C. jejuni positive sample. Conclusion This method has strong specificity, high sensitivity, good openness and contains IAC to prevent “false negatives”, which can be used in the rapid simultaneous detection of two most important Campylobacter species from food.