手持式实时荧光定量聚合酶链式反应仪现场快速检测肉类样本猪、鸡、鸭源性成分

Rapid detection of pork, chicken and duck-derived components in meat samples by handheld quantitative real-time polymerase chain reaction

中文关键词:  快速预处理  手持式荧光实时PCR仪  猪、鸡、鸭源性成分  快速检测  食品真实性  现场检测

英文关键词:rapid pretreatment  handheld quantitative real-time polymerase chain reaction  pork, chicken and duck-derived components  rapid detection  food authenticity  on-site detection

中文摘要:

      目的 建立适用于手持式实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)仪肉类成分检测方法, 实现肉源性成分现场快速检测。方法 本研究以猪、鸡、鸭源性成分为研究对象, 探索肉类样本前处理工具、裂解液成分、裂解液浓度、裂解液添加量等对所提DNA的影响, 构建肉类样本快速预处理方法; 筛选特异性引物和探针, 利用手持式实时荧光定量PCR一体机进行特异性、灵敏度、检出限验证, 绘制扩增标准曲线; 对真实单一肉类样品和混合样品进行检测。结果 建立的肉类样本快速预处理方法5 min完成操作, 提取DNA耗时10 min, 提取的DNA满足后续实验; 猪、鸡、鸭检测体系均具有良好的特异性; 对猪、鸡、鸭源性DNA成分的检出限分别为0.010、0.001、0.001 ng/μL; 所建立的标准曲线线性关系良好, 猪肉DNA扩增标准曲线: Y=–3.956X+38.803, r2=0.9956; 鸡肉DNA扩增标准曲线: Y=–3.499X+40.491, r2=0.9909; 鸭肉DNA扩增标准曲线: Y=–3.961X+39.192, r2=0.9962; 对28份肉类样本检测, 成功率为100%。结论 本研究建立的方法可用于在手持式实时荧光定量PCR一体机上对猪、鸡、鸭肉类样本的快速、准确检测, 为肉类食品的市场监管和相关执法提供有力的技术保障。

英文摘要:

      Objective To develop a meat component detection method based on handheld quantitative real-time polymerase chain reaction (PCR) for rapid on-site identification of meat sources. Methods This study focused on the pork, chicken and duck-derived components, explored the influence of the single factors of meat sample pretreatment tools, lysate composition, lysate concentration and lysate addition amount on the extracted DNA, and constructed a rapid pretreatment method for meat samples; screened specific primers and probes, used the handheld quantitative teal-time PCR instrument to verify specificity, sensitivity and limit of detection, and drew amplification standard curves; and detected the real single and mixed meat samples. Results The established rapid pretreatment method for meat samples could be completed within 5 min, the DNA extraction took 10 min, and the extracted DNA could meet the requirements of subsequent experiments. The detection methods exhibited high specificity for pork, chicken and duck samples. The limits of detection of pork, chicken and duck-derived DNA components were 0.010, 0.001 and 0.001 ng/μL. The standard curve showed a sound linear relationship. The amplification standard curve of pig DNA was: Y=–3.956X+38.803, r2=0.9956. The amplification standard curve of chicken DNA was: Y=–3.499X+40.491, r2=0.9909. The amplification standard curve of duck DNA was: Y=–3.961X+39.192, r2=0.9962. The method was successfully applied to detect 28 meat samples with a 100% success rate. Conclusion These results indicate that the rapid meat sample pretreatment method established in this experiment can be used for the rapid and accurate detection of pork, chicken and duck meat on a handheld quantitative real-time PCR all-in-one machine. This approach provides robust technical support for monitoring and controlling meat adulteration at the retail or market level.