DNase处理法结合数字PCR与qPCR对活的非可培养状态副溶血性弧菌检测比较研究
目的 探讨DNase处理法结合微滴化数字PCR(droplet digital PCR, ddPCR)在检测冷冻食品中活的非可培养状态(viable but non-culturable state, VBNC)副溶血性弧菌的适用性, 并与实时荧光定量PCR(qPCR)进行比较。方法 用无菌磷酸盐缓冲液对解冻的大西洋鲑鱼样品进行10倍梯度稀释, 再加入副溶血性弧菌, 终浓度为6.6×105 CFU/mL。?20℃分别诱导10、20和30 d。将DNase试剂与叠氮溴化丙锭(propidium monoazide, PMA)染料分别作用于不同时期的冷冻基质, 结合qPCR和ddPCR进行比较检测, 对比其作用效果。结果 DNase-qPCR和PMA-qPCR检测3个冷冻阶段活性副溶血性弧菌的Cq值分别为31.41±0.06、32.40±0.04、34.59±0.15和31.24±0.06、32.32±0.03、34.25±0.12, 2种方法Cq值均呈现上升的趋势且数值接近。采用DNase-ddPCR和PMA-ddPCR检测各阶段活性副溶血性弧菌, 可直接读出绝对拷贝数, 分别为233±6.43、108±5.57、28±3.21和256±6.56、126±3.06、35±2.52。2种方法检测的拷贝数差异不大, 重复性好。相对标准偏差均在可接受范围内, 符合欧盟定量检测要求。结论 DNase处理试剂与PMA对有活性的副溶血性弧菌检测效果相当, 与PMA相比, DNase处理法无需强光照射, 操作简便快捷, 无试剂毒性。ddPCR不需要标准品即能实现对基质中微量活性副溶血弧菌精准定量检测。
Objective To explore the applicability of DNase treatment combined with droplet digital PCR (ddPCR) in the detection of viable but non-culturable state (VBNC) Vibrio parahaemolyticus, and compare with real-time fluorescence quantitative PCR (qPCR). Methods The thawed Atlantic salmon samples were diluted by 10 times gradient with sterile phosphate buffered saline, and V. parahaemolyticus was added. The final concentration was 6.6×105 CFU/mL induced of 10, 20 and 30 d at ?20℃. The DNase reagent and the propidium monoazide (PMA) dye were applied to the frozen substrates at different stages, and the effects were compared by qPCR and ddPCR, respectively. Results The Cq of V. parahaemolyticus in 3 freezing stages detected by DNase-qPCR and PMA-qPCR was 31.41±0.06, 32.40±0.04, 34.59±0.15 and 31.24±0.06, 32.32±0.03, 34.25±0.12, respectively. The Cq values of both methods showed an upward trend and the values were close to each other. When detected by DNase-ddPCR and PMA-ddPCR for V. parahaemolyticus, the absolute number of copies could be read directly: 233±6.43, 108±5.57, 28±3.21, and 256±6.56, 126±3.06, 35±2.52. There was little difference in the number of copies detected by the two methods, and they had good repeatability. Relative standard deviation was acceptable and met the requirement of quantitative detection in EU. Conclusion The DNase treatment reagent and PMA have the similar effect on the detection of active V. parahaemolyticus. Compared with PMA, the DNase treatment method does not require strong light irradiation, and the operation is simple and rapid, and there is no reagent toxicity. ddPCR can detect trace amounts of active V. parahaemolyticus in the matrix and achieve accurate quantification without the need for standards.
标题：DNase处理法结合数字PCR与qPCR对活的非可培养状态副溶血性弧菌检测比较研究
英文标题：Comparison of DNase treatment combined with digital PCR and qPCR for detection of Vibrio parahaemolyticus in viable but non-culturable state
作者：
杨娟 上海出入境检验检疫局动植物与食品检验检疫技术中心
何宇平 上海出入境检验检疫局动植物与食品检验检疫技术中心
黄新新 上海出入境检验检疫局动植物与食品检验检疫技术中心
彭强辉 浙江清华长三角研究院
曾静 北京出入境检验检疫局检验检疫技术中心
孙晓红 上海海洋大学食品学院
赵勇 上海海洋大学食品学院
李想 上海出入境检验检疫局动植物与食品检验检疫技术中心
郭德华 上海出入境检验检疫局动植物与食品检验检疫技术中心
中文关键词:副溶血性弧菌,活的非可培养状态,DNase处理法,实时荧光定量PCR,微滴化数字化PCR,
英文关键词:Vibrio parahaemolyticus,viable but non-culturable state,DNase treatment,real-time fluorescence quantitative PCR,droplet digital PCR,
发表日期：2019-04-19
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