食品中单核细胞增生李斯特氏菌ddPCR定量检测方法的建立及应用

Establishment and Application of a ddPCR Method for Quantitative Detection of Listeria Monocytogenes in Foods

本研究针对食品中单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)建立一种快速定量的微滴式数字PCR(droplet digital PCR,ddPCR)检测方法。通过优化反应条件和反应体系,建立食品中LM的ddPCR快速定量检测方法,并对该方法进行特异性、灵敏性以及重复性考察,以即食食品香肠和乳制品奶酪棒为样品进行人工污染,验证该方法的可行性。结果显示:经优化后,确定ddPCR方法的最佳上、下游引物浓度均为900 nmol/L,最佳探针浓度为250 nmol/L,最佳退火温度为58.3℃,延伸时间为60 s,循环数为45个循环。建立的ddPCR方法特异性良好,定量限(LOQ)为294 CFU/mL,重复检测的变异系数均小于12%;ddPCR定值结果与平板计数结果间存在良好的线性相关性(R2=0.998)。在人工添加试验中同时进行ddPCR与平板计数检测,LOQ可达102CFU/g,呈现出良好的重复性、准确性。本研究建立的食品中LM的ddPCR定量检测方法特异性好、灵敏性高、准确性好,能够为LM的快速定量检测提供技术支持。

In this study,a rapid and quantitative droplet digital PCR(ddPCR)method was established for the detection of Listeria monocytogenes(LM)in foods.By optimizing reaction conditions and reaction systems,a fast quantitative ddPCR detection method Biotechnology for LM in foods was established.The specificity,sensitivity,and repeatability of this method were examined,and its feasibility was validated using artificially contaminated ready-to-eat sausage and dairy cheese sticks as samples.The results showed that after op-timization,the optimal upstream and downstream primer concentrations for the ddPCR method were determined to be 900 nmol/L,the optimal probe concentration was 250 nmol/L,the optimal annealing temperature was 58.3°C,the extension time was 60 s,and the cycle number was 45 cycles.The established ddPCR method exhibited good specificity,a limit of quantification(LOQ)of 294 CFU/mL,and a coefficient of variation of less than 12%for repeated testing.There was a strong linear correlation(R²=0.998)between the ddPCR quantification results and the plate count results.The LOQ could achieve 102 CFU/g in the manual spiking test with simultane-ous ddPCR and plate counting assay,which demonstrated good reproducibility and accuracy.The ddPCR method for the quantitative determination of LM in foods established in this study was highly specific,sensitive and accurate,providing technical support for the rapid quantitative determination of LM.