LI Li-li,SHAN Hong,ZHANG Rui-ying,et al.Establishment and Preliminary Application of Real-time PCR Technology for Rapid Detection of Foodborne Salmonella[J].HEILONGJIANG AGRICULTURAL SCIENCES,2020,(12):93-98.[doi:10.11942/j.issn1002-2767.2020.12.0093]
食源性沙门氏菌Real-time PCR技术快速检测方法的建立及初步应用
- Title:
- Establishment and Preliminary Application of Real-time PCR Technology for Rapid Detection of Foodborne Salmonella
- 关键词:
- Real-time PCR; 沙门氏菌; 快速检测; 初级农产品
- 文献标志码:
- A
- 摘要:
- 近年来分子生物学检测技术发展迅速，本研究通过优化建立了食源性沙门氏菌的Real-time PCR检测方法，并对其结果的特异性、灵敏度、重复性等进行验证，以及初级农产品添加实验的适用性和一致性进行比对。结果表明：优化后的Real-time PCR检测条件为：95 ℃，5 min，95 ℃，30 s；然后65 ℃，30 s，72 ℃，30 s，此步骤为40个循环；最后72 ℃，5 min。总反应体系为20 μL，其中上下游引物的添加量分别为1 μL。建立的Real-time PCR快速检测方法特异性强，重复性好，灵敏度高而且不需要前增菌，较传统检测方法大大缩短了检测时间，整个试验操作过程可在6 h内完成。将此方法应用于人工污染的初级农产品中沙门氏菌的检测，结果表明生鲜猪肉、即食蔬菜和即食水果样品中沙门氏菌的检测灵敏度均较高。通过与微生物传统计数方法的检测结果进行比较，结果显示与传统方法的一致性为100%，可用于食品中沙门氏菌的检测。
- Abstract:
- In recent years,molecular biology detection technology has developed rapidly.In this study,we established a nucleic acid-based fluorescent quantitative PCR detection method,verified the specificity,sensitivity and repeatability of the method,and compared with the results of the experiment of adding the primary agricultural product.The results showed that,the conditions of optimized fluorescence quantitative PCR detection were 95 ℃,5 min; 95 ℃,30 s,65 ℃,30 s,72 ℃,30 s for 40 cycles and the final 72 ℃,5 min.The total reaction system was 20 μL with the addition amount of the upstream and downstream primers 1 μL respectively.The established Real-time PCR rapid detection method had strong specificity,good reproducibility,high sensitivity and does not require pre-enrichment.Compared with traditional detection methods,the detection time was greatly shortened,and the entire experimental operation process could be completed within 6 hours.This method was applied to the detection of Salmonella in artificially contaminated primary agricultural products.The results showed that the detection sensitivity of Salmonella in fresh pork,ready-to-eat vegetables and ready-to-eat fruit samples was high.The consistency with the traditional microbial counting method was 100%,which could be used for the detection of Salmonella in food.
参考文献/References:
［1］方婷子，史贤明，施春雷.沙门氏菌血清型快速 PCR鉴定方法的建立 [J].中国食品学报,2017,17（2）:212-218.
相似文献/References:
[1]甄 珍.微生物能力验证实验结果分析[J].黑龙江农业科学,2011,(06):86.
ZHEN Zhen.Microbe Ability Test Results and Analysis[J].HEILONGJIANG AGRICULTURAL SCIENCES,2011,(12):86.
备注/Memo
收稿日期：2020-07-09