单核细胞增生李斯特氏菌活菌PMAxx-ddPCR检测方法的建立及应用

Establishment and Application of a PMAxx-ddPCR Detection Method for Viable Listeria monocytogenes

摘要:

【目的】将改良版叠氮溴化丙锭（improved propidium monoazide，PMAxx）与微滴式数字PCR（droplet digital PCR，ddPCR）技术相结合，建立一种针对单核细胞增生李斯特氏菌（Listeria monocytogenes，LM）活菌的检测方法。【方法】对PMAxx终浓度、暗处理时间及曝光时间进行优化，建立最佳的PMAxx-ddPCR检测体系，并用不同体积比的LM活菌验证其准确性，考察PMAxx-ddPCR检测方法的特异性、灵敏性和重复性。用不同浓度LM活菌与死菌对奶酪棒样品进行人工污染，比较传统的平板计数法和作者建立的PMAxx-ddPCR检测方法的结果；进一步对市售金针菇和火腿肠进行检测，评估PMAxx-ddPCR检测方法的实际效果。【结果】在PMAxx终浓度为10 μmol/L、暗处理时间和曝光时间均为10 min时，PMAxx可显著抑制死菌DNA扩增，但对活菌DNA扩增没有显著影响。建立的PMAxx-ddPCR检测方法仅对LM产生特异性扩增，定量限为147 CFU/mL，相对标准偏差（relative standard deviation，RSD）均小于20%，重复性较好。人工污染奶酪棒试验结果表明，平板计数法和PMAxx-ddPCR检测方法对LM的定量结果差异不显著（P>0.05）。进一步分析17份金针菇和16份火腿肠样品检测结果，PMAxx-ddPCR检测法和平板计数法均从7份金针菇样品中检出LM，且2种方法定量结果没有显著差异（P>0.05）。【结论】作者建立的LM活菌的PMAxx-ddPCR检测方法可对食品中LM活菌进行特异性定量检测。

Abstract:

[Objective] A detection method for viable Listeria monocytogenes (LM) was established combining improved propidium monoazide (PMAxx) with droplet digital PCR (ddPCR). [Method] After optimization of the final PMAxx concentration, dark treatment time, and exposure time, the optimal PMAxx-ddPCR detection system was established. Different volume ratios of viable LM were employed to verify the accuracy of the PMAxx-ddPCR method, and the specificity, sensitivity, and reproducibility of this method were examined. Cheese stick samples were artificially contaminated with different concentrations of viable and dead LM and then used to compare the detection results of the conventional plate counting method and the PMAxx-ddPCR method established in this study. Additionally, commercially available enoki mushrooms and ham sausages were tested to evaluate the practical detection performance of the PMAxx-ddPCR method. [Result] At a final PMAxx concentration of 10 μmol/L, dark treatment time of 10 min, and exposure time of 10 min, PMAxx significantly inhibited the DNA amplification of dead bacteria, while having no significant effect on that of viable bacteria. The developed PMAxx-ddPCR method only generated specific amplification for LM, with a limit of quantification of 147 CFU/mL. The relative standard deviations (RSD) of the method were consistently less than 20%, indicating good repeatability. In the artificial contamination experiment of cheese sticks, the quantification results of LM by the plate counting method and the PMAxx-ddPCR method had no significant difference (P>0.05). Further detection of 17 enoki mushroom samples and 16 ham sausage samples revealed that LM was detected in 7 enoki mushroom samples by both PMAxx-ddPCR and plate counting methods. Moreover, the quantitative results of the two methods showed no significant difference (P>0.05). [Conclusion] A PMAxx-ddPCR method is established for detecting viable LM, enabling specific quantitative detection of viable LM in food.