开发一种无需 DNA 提取即可定量检测零售鸡肉中弯曲杆菌属的采样和实时 PCR 方法。
Development of a Sampling and Real-time PCR Method for the Quantitative Detection of Campylobacter spp. in Retail Chicken Meat Without DNA Extraction.
机构信息
Faculty of Nutrition, Kobe Gakuin University, Kobe, Hyogo 651-2113, Japan.
Graduate School of Nutritional Sciences, Nagoya University of Arts and Sciences Graduate School, Nagoya, Aichi 470-0196, Japan.
出版信息
J Food Prot. 2023 Jan;86(1):100028. doi: 10.1016/j.jfp.2022.100028. Epub 2022 Dec 14.
Campylobacter food poisoning is caused by consumption of the contaminated foods, especially poultry meat. Continuous quantitative measurement of Campylobacter spp. in contaminated foods is crucial to develop preventive measures. We developed a direct-qPCR method for determining the viable cell counts of Campylobacter spp. using qPCR without DNA extraction from enriched food samples and a sampling method (the wrap procedure) in which the sample is wrapped in a sheet, different from the conventional homogenization procedure. The viable cell counts of Campylobacter spp. before and after enrichment of the samples sampled using the wrap and homogenization procedures from chicken samples inoculated with Campylobacter jejuni were determined using the culture method, and the cycle threshold (C) values after enrichment were determined using the direct-qPCR. An enrichment regression equation was generated from the viable cell counts obtained before and after enrichment, and a direct-qPCR regression equation was generated from the C values and viable cell counts obtained after enrichment, enabling the viable cell counts before enrichment to be estimated from the C values. Estimated viable cell counts were similar for the culture method when sampled by the homogenization procedure, but lower for the wrap procedure. However, the detection rate of direct-qPCR was 37.5% for liver and 89.7% for breast fillet using the homogenization procedure, whereas using the wrap procedure, it was 100% for both samples. The detection rate of direct-qPCR for retail chicken was 30.4-35.7% for the homogenization procedure, and 85.7-100% for the wrap procedure. Colonies were observed using the culture method, but their quantification was difficult due to swarming or their low number. However, estimating viable cell counts using the combination of wrap procedure and direct-qPCR methods is possible. The developed method can provide baseline data for the risk assessment Campylobacter food poisoning.
空肠弯曲菌食物中毒是由食用受污染的食物引起的，尤其是家禽肉类。连续定量检测污染食品中的弯曲菌对制定预防措施至关重要。我们开发了一种直接 qPCR 方法，用于确定使用 qPCR 从富集食品样品中不提取 DNA 的情况下弯曲菌属的活菌数，以及一种不同于传统匀浆程序的采样方法（包裹程序），其中样品用薄片包裹。使用培养方法确定使用包裹和匀浆程序从接种空肠弯曲菌的鸡样品中采样的样品在富集前后的弯曲菌属活菌数，并使用直接 qPCR 确定富集后的循环阈值（C）值。从富集前后获得的活菌数生成富集回归方程，从富集后的 C 值和活菌数生成直接 qPCR 回归方程，从而可以从 C 值估计富集前的活菌数。用匀浆程序采样时，用直接 qPCR 估计的活菌数与培养法相似，但包裹程序的活菌数较低。然而，直接 qPCR 的检测率在使用匀浆程序时肝脏为 37.5%，乳房肉为 89.7%，而使用包裹程序时均为 100%。直接 qPCR 对零售鸡肉的检测率在使用匀浆程序时为 30.4-35.7%，在使用包裹程序时为 85.7-100%。使用培养法观察到了菌落，但由于菌群密集或数量较少，难以对其进行定量。然而，使用包裹程序和直接 qPCR 方法的组合来估计活菌数是可行的。该方法可为弯曲菌食物中毒的风险评估提供基线数据。
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