Poultry Diagnostic and Research Center, University of Georgia, Athens, GA 30602, USA.
J Food Prot. 2012 Apr;75(4):743-7. doi: 10.4315/0362-028X.JFP-11-297.
Conventional culture methods have traditionally been considered the "gold standard" for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis-specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis-specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.
传统的培养方法一直被认为是分离和鉴定食源性病原体的“金标准”。然而，培养方法既费时又费力。最近，国家家禽改进计划（National Poultry Improvement Plan）临时批准了一种用于检测肠炎沙门氏菌（Salmonella enteritidis）的肠炎沙门氏菌特异性实时 PCR 检测方法，该方法与另一种也获得国家家禽改进计划临时批准的培养方法进行了比较，后者用于分析集成禽舍的环境样本。该方法使用通过靴套或拖拭法收集的 422 个现场样本进行了验证。样本通过在四硫磺酸盐肉汤中进行选择性富集培养，然后转移到改良的半固体 Rappaport-Vassiliadis 培养基上，再在含有新生霉素和木糖赖氨酸的亮绿琼脂平板上划线培养。从每个样本的选择性富集肉汤中收集 1 毫升等分试样，通过商业 PrepSEQ 核酸提取试剂盒进行 DNA 提取，并通过肠炎沙门氏菌特异性实时 PCR 检测方法进行分析。实时 PCR 检测方法在靴套和拖拭样本之间未检测到显著差异。相比之下，培养方法从靴套中检测到明显更多的阳性样本。实时 PCR 检测方法对现场样本的诊断灵敏度明显高于培养方法。获得的 Kappa 值为 0.46，表明实时 PCR 检测方法与培养方法之间存在中度一致性。此外，实时 PCR 方法的周转时间为 2 天，而培养方法为 4 至 8 天。这种实时 PCR 方法的高灵敏度以及时间和劳动力的减少，使其成为用于诊断、监测和研究以提高食品安全的传统培养方法的绝佳替代方法。
