| 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 |
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| 引用本文： | 杨 娟,何宇平,黄新新,彭强辉,曾 静,孙晓红,赵 勇,李 想,郭德华. 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[J]. 食品安全质量检测学报, 2019, 10(10): 3180-3185 |
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| 作者姓名： | 杨 娟 何宇平 黄新新 彭强辉 曾 静 孙晓红 赵 勇 李 想 郭德华 |
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| 作者单位： | 上海出入境检验检疫局动植物与食品检验检疫技术中心;浙江清华长三角研究院;北京出入境检验检疫局检验检疫技术中心;上海海洋大学食品学院 |
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| 基金项目： | 国家重点研发计划项目(2017YFE0110800)、上海市科技兴农项目(2019-02-08-00-10-F01149)、上海市技术化标准专项(16DZ0500102) |
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| 摘 要： | 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6.6×10~5 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。
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| 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 |
| 收稿时间： | 2019-04-19 |
| 修稿时间： | 2019-04-28 |
Comparison of DNase treatment combined with digital PCR and qPCR for detection of Vibrio parahaemolyticus in viable but non-culturable state |
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| YANG Juan,HE Yu-Ping,HUANG Xin-Xin,PENG Qiang-Hui,Zeng Jing,SUN Xiao-Hong,Zhao Yong,LI Xiang and GUO De-Hua. Comparison of DNase treatment combined with digital PCR and qPCR for detection of Vibrio parahaemolyticus in viable but non-culturable state[J]. Journal of Food Safety & Quality, 2019, 10(10): 3180-3185 |
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| Authors: | YANG Juan HE Yu-Ping HUANG Xin-Xin PENG Qiang-Hui Zeng Jing SUN Xiao-Hong Zhao Yong LI Xiang GUO De-Hua |
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| Affiliation: | Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau,Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau,Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau,Yangtze Delta Region Institute of Tsinghua University,Beijing Inspection Quarantine Testing Center of Beijing Entry-Exit Inspection and Quarantine Bureau,College of Food Science and Technology, Shanghai Ocean University,College of Food Science and Technology, Shanghai Ocean University,Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau and Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau |
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| Abstract: | |
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| Keywords: | Vibrio parahaemolyticus viable but non-culturable state DNase treatment real-time fluorescence quantitative PCR droplet digital PCR |
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