食品科学 ›› 2019, Vol. 40 ›› Issue (4): 332-337.doi: 10.7506/spkx1002-6630-20170915-214

• 安全检测 • 上一篇    下一篇

一步法微滴数字PCR检测生菜中GII型诺如病毒

陈嘉茵1,方?苓2,吴诗微2,唐书泽1,张志强3,李?晖2,*,吴希阳1,*   

  1. (1.暨南大学理工学院食品科学与工程系,广东?广州 510632;2.广东省疾病预防控制中心病原微生物检验所,广东?广州 511430;3.香港中文大学生命科学学院食品及营养学部,香港)
  • 出版日期:2019-02-25 发布日期:2019-03-05
  • 基金资助:
    2015年度广东省公益研究与能力建设专项(2015A030401042);2015年广东省国际合作项目(2015A050502030); 2016年广东省前沿与科技创新专项粤港联合创新项目(2016A050503031)

Detection of Norovirus Genogroup II in Lettuce by Droplet Digital PCR

CHEN Jiayin1, FANG Ling2, WU Shiwei2, TANG Shuze1, Peter C.K. Cheung3, LI Hui2,*, WU Xiyang1,*   

  1. (1. Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China; 2. Institute of Pathogenic Microbiology, Guangdong Provincial Center for Disease Control and Prevention, Guangzhou 511430, China; 3. Food and Nutritional Sciences Program, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong China)
  • Online:2019-02-25 Published:2019-03-05

摘要: 目的:采用逆转录微滴数字聚合酶链式反应(reverse transcription droplet digital polymerase chain reaction,RT-ddPCR)技术和QX200 Droplet Digital PCR System,建立一步法RT-ddPCR高灵敏快速检测生菜中GII型诺如病毒(norovirus genegroup II,NoV GII)的方法。方法:优化RT-ddPCR检测NoV GII的反应体系;通过10?倍梯度稀释的NoV GII线性阳性质粒确定RT-ddPCR的检测范围;利用其他常见的肠道病毒核酸(非GII型诺如病毒)作为反应模板,与NoV GII核酸同时进行RT-ddPCR,判定反应体系的特异性,并通过不同时间多次检测样品的方式判断该检测方法的稳定性。采用ISO/TS 15216-1:2013食品中诺如病毒RNA提取方法,对人工染毒不同水平(高、中、低)的NoV GII生菜样品进行检测,同时研究去除抑制物前后对RT-ddPCR检测的影响,并与逆转录定量聚合酶链式反应(reverse transcription quantitative polymerase chain reaction,RT-qPCR)进行检测回收率的对比,探究RT-ddPCR在食品中NoV?GII快速与定量检测上的发展潜力与应用前景。结果:RT-ddPCR的最高检测限为8.47×104?copies/μL,最低检测限为2.12?copies/μL,RT-ddPCR扩增效率为95.44%,标准曲线相关系数为0.997?3。在不同浓度人工染毒生菜样品中,抑制物的存在对ddPCR回收率检测结果没有显著性差异。在高、中染毒浓度下,抑制物对RT-qPCR与RT-ddPCR回收率检测结果影响不显著。低浓度下,未去除抑制物的RT-qPCR法平均回收率仅1.43%,与去除抑制物后的RT-qPCR检测结果(平均回收率为9.71%)有显著差异,且与RT-ddPCR的检测结果(未去除抑制物的RT-ddPCR平均回收率为11.80%,去除抑制物后平均回收率为12.53%)存在显著性差异。结论:生菜抑制物对RT-ddPCR的检测影响不显著,利用RT-ddPCR可有效测出低浓度的受污染样品,避免用现有的RT-qPCR方法因抑制物带来的“假阴性”检测结果。本实验建立的RT-ddPCR方法能高效、灵敏地检测出受污染食品中NoV?GII的低病毒含量,在食源性病毒检测中具有可观的发展潜力和应用前景。

关键词: 逆转录微滴数字聚合酶链式反应, 一步法, GII型诺如病毒, 生菜, 回收率

Abstract: Objective: To develop a rapid and sensitive one-step reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay for the detection of norovirus genogroup II (NoV GII) in lettuce. Methods: The RT-ddPCR system was optimized; the detection limit was tested using a series of 10-fold diluted enzyme digested plasmids containing the target gene sequence of NoV GII. The specificity was validated by using nucleic acids from Nov GII and other enteric viruses as targets. The repeatability of the method was also examined. Lettuce samples artificially infected with different concentrations (high, moderate, and low levels) of NoV GII were detected by RT-ddPCR following RNA extraction performed as described in ISO/TS 15216-1:2013. The influence of removal of inhibitors on the performance of RT-ddPCR was evaluated in comparison with reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results: The maximum and minimum detection limits of RT-ddPCR were 8.47 × 104 and 2.12 copies/μL, respectively. The amplification efficiency was 95.44% (r = 0.997 3). The existence of inhibitors in lettuce showed no significant difference in the recovery RT-ddPCR at different contamination levels. The recovery rates of RT-qPCR at low contamination level with and without inhibitors were 1.43% and 9.71% respectively, which were significantly different from RT-ddPCR (11.80% and 12.53% respectively). Conclusions: The RT-ddPCR assay developed was stable in detecting food samples regardless of the presence of inhibitors such as lettuce. It could efficiently detect low concentrations of NoV GII virus in food without false-negative results, which may occur in RT-qPCR because of the existence of inhibitors. Therefore, this one-step RT-ddPCR assay was able to detect low levels of NoV GII in contaminated food samples, which indicates a considerable application prospect in the field of foodborne virus detection.

Key words: reverse transcription droplet digital PCR (RT-ddPCR), one-step procedure, norovirus genogroup II (NoV GII), lettuce, recovery rate

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