Hygiene and Technology of Milk, Department of Veterinary Sciences, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Oberschleissheim, Germany.
Mol Cell Probes. 2010 Jun;24(3):124-30. doi: 10.1016/j.mcp.2009.11.004. Epub 2009 Nov 26.
In order to meet the growing demand for fast and reliable detection of potentially toxinogenic Bacillus cereus, we developed a multiplex real-time PCR assay based on SYBR Green I with subsequent melting curve analysis. We designed and selected primers specific for genes of toxins responsible for diarrhoea (nheA, hblD and cytK1) and emesis (ces). A panel of 337 Bacillus strains was applied to the novel method on Light Cycler 2.0 with average melting temperature (T(m)) values of 73.85 degrees C (nheA), 87.01 degrees C (hblD), 78.66 degrees C (ces) and 82.19 degrees C (cytK1). An adapted version of the assay was also successfully run on Light Cycler 480 using one third (113 strains) of the total test panel. Verification of PCR results by conventional PCR as well as immunoassays and cytotoxicity tests gave an overall excellent correlation. Distinct melting peaks were only observed in B. cereus and B. cereus group strains but not in other Bacilli and Gram-positive or Gram-negative bacteria. Artificial contamination of three different food matrices with distinct bacterial counts revealed a detection limit of 10(1) CFU/g B. cereus cells after overnight enrichment. Thus, the novel multiplex real-time PCR turned out to be a reliable method for identification of B. cereus with enteropathogenic potential.
为了满足对快速可靠检测潜在产毒蜡样芽胞杆菌的日益增长的需求，我们开发了一种基于 SYBR Green I 的多重实时 PCR 检测方法，随后进行熔解曲线分析。我们设计并选择了与腹泻（nheA、hblD 和 cytK1）和呕吐（ces）毒素基因负责的毒素基因特异性的引物。一组 337 株蜡样芽胞杆菌应用于新型 Light Cycler 2.0 上，平均熔解温度（T(m)）值分别为 73.85°C（nheA）、87.01°C（hblD）、78.66°C（ces）和 82.19°C（cytK1）。该检测方法的改编版本也成功地在 Light Cycler 480 上运行，使用总测试面板的三分之一（113 株）。通过常规 PCR 以及免疫测定和细胞毒性试验对 PCR 结果进行验证，结果具有极好的相关性。只有在蜡样芽胞杆菌和蜡样芽胞杆菌组菌株中观察到明显的熔解峰，而在其他芽孢杆菌和革兰氏阳性或革兰氏阴性细菌中则没有。对三种不同的细菌计数的人工污染食品基质进行检测，发现经过过夜富集后，检测限为 10(1) CFU/g 蜡样芽胞杆菌细胞。因此，新型多重实时 PCR 是一种可靠的方法，可用于鉴定具有肠道致病性的蜡样芽胞杆菌。
