摘要
目的:应用一步法微滴数字PCR方法(RT-ddPCR),建立一种食品中人星状病毒(HAstV)定量检测方法。方法:筛选引物探针,建立HAstV RT-ddPCR方法;利用其它常见食源性病毒RNA,确定特异性;梯度稀释HAV RNA标准样品确定检测限、准确度和重复性。用已知效价的MS2噬菌体制备人工污染样品,研究RT-ddPCR方法在不同种类食品基质中的检测限和回收率,比较2种RNA提取方法的病毒检出量。结果:HAstV RT-ddPCR方法准确度、灵敏度和重复性良好,检测限105~100拷贝/μL,最低定量限5拷贝/μL;不同种类食品基质的检测限分别为7.2×106~3600拷贝(贝类消化腺),7.2×106~3600拷贝(硬表面食品),7.2×106~7200拷贝(生食蔬菜)、7.2×107~7200拷贝(软质水果);高浓度污染剂量时,Trizol裂解结合磁珠纯化方法病毒检出量显著低于Trizol裂解提取方法(P<0.05);不同种类食品基质中模拟病毒回收率存在显著差异(P<0.05)。结论:本研究建立的食品中HAstV定量检测方法可有效定量检测HAstV,可通过病毒回收率计算样品中HAstV的实际载量。
Objective:To establish a sensitive and rapid one-step reverse transcriptase droplet digital polymerase chain reaction(RT-ddPCR)assay for detection human astrovirus(HAstV)in different food matrices.Methods:The RT-ddPCR quantitative detection assay for HAstV was established and optimized by applying the selected specific primers and probes.The specificity of this quantitative method was validated via using other foodborne viruses RNA as targets.A HAstV RNA reference material was series diluted to evaluate the quantitative detection limit,accuracy and variability of the developed assay.In order to test the detection limit and recovery rate in food,artificial positive samples were prepared by adding MS2 bacteriophage with known titer to different food matrices.Virus elution and purification method in food from CEN ISO/TS 15216-2:2013 was adopted and the virus detection amounts of two RNA extraction methods were compared.Results:The HAstV RT-ddPCR assay had better accuracy,sensitivity and smaller variability with a detection range from 105 to 100(copies/μL)and a lower limit of quantitation of 5 copies/μL.The detection limits in digestive glands of bivalve molluscan shellfish,food with hard surface,raw vegetables and soft fruits were 7.2×106-3600 copies,7.2×106-3600 copies,7.2×106-7200 copies and 7.2×107-7200 copies,respectively.In higher contamination level,detection amount of virus was significantly lower(P<0.05)when RNA was extracted and purified by trizol combined with magnetic beads.A statistically significant difference(P<0.05)in virus recovery rate among different food matrices was indicated by one-way ANOVA and Turkey multiple comparison analysis.Conclusion:The established RT-ddPCR assay in food could effectively detect HAstV in contaminated food.However,the single RT-ddPCR quantitative result could not reflect actual virus load in food which could be calculated by applying recovery rate.
作者
徐蕾蕊
李丹
魏咏新
马丹
魏海燕
汪琦
张西萌
付溥博
刘莉
赵晓娟
曾静
Xu Leirui;Li Dan;Wei Yongxin;Ma Dan;Wei Haiyan;Wang Qi;Zhang Ximeng;Fu Pubo;Liu Li;Zhao Xiaojuan;Zeng Jing(Science and Technology Research Center of China Customs,Beijing 100026)
出处
《中国食品学报》
EI
CAS
CSCD
北大核心
2021年第1期300-308,共9页
Journal of Chinese Institute Of Food Science and Technology
基金
国家重点研发计划项目(2017YFC1601602)。
关键词
人星状病毒
定量检测
一步法反微滴数字PCR
回收率
human astrovirus
quantitative detection
one-step reverse transcriptase droplet digital PCR
recovery rate
