猪肝中戊型肝炎病毒检测方法的比较
Comparison of detection methods for hepatitis E virus in pig liver
王秋媛 1张瑞婷 2尹文娇 2曹经瑗 2李晓梅 3毕胜利2
作者信息
- 1. 中国疾病预防控制中心病毒病预防控制所国家卫生健康委医学病毒和病毒病重点实验室,北京 102206;山东第一医科大学(山东省医学科学院)公共卫生与健康管理学院,济南 250117
- 2. 中国疾病预防控制中心病毒病预防控制所国家卫生健康委医学病毒和病毒病重点实验室,北京 102206
- 3. 山东第一医科大学(山东省医学科学院)公共卫生与健康管理学院,济南 250117
- 折叠
摘要
目的 对猪肝中戊型肝炎病毒(hepatitis E virus,HEV)核酸检测方法进行优化和比较,为动物内脏标本中HEV检测提供技术参考.方法 分别采用三种方法(PBS匀浆处理法、蛋白酶K处理法、氯仿抽提法)对人工污染HEV粪便悬液的猪肝标本进行前处理和病毒核酸提取,通过HEV RT-qPCR检测,比较HEV回收率、抑制率.将筛选出的最优的HEV检测方法应用于市售猪肝检测,对检出的阳性标本进行HEV基因分型.结果 PBS匀浆处理法、蛋白酶K处理法、氯仿抽提法的HEV回收率分别为9.88％、0.19％和17.28％.蛋白酶K处理法的回收率小于1％,舍弃该方法,对其他两种方法进行t检验,结果表明两种方法回收率的差异具有统计学意义(t=26.801,P＜0.001),氯仿抽提法的回收率较高.三种方法的抑制率均小于75％,在ISO/TS 15216-2:2019标准的范围内.在192份市售猪肝标本中检出17份HEV RNA阳性标本,核酸阳性率为8.85％,其中5份标本HEV基因分型成功,均为基因4型.结论 用氯仿抽提法对猪肝进行前处理,病毒回收效果较好,且在市售猪肝标本中检出HEV核酸阳性标本,该方法可用于食品中病毒的检测.
Abstract
Objective To optimize and compare method for hepatitis E virus(HEV)nucleic acid detection from pig liver,and provide technical references for HEV detection in animal viscera specimens.Methods Three methods(PBS homogenization treatment,proteinase K treatment,chloroform extraction method)were used to pretreat and extract viral nucleic acid form pig liver,which was artificially contaminated with HEV fecal suspensions,and HEV RT-qPCR was used to compare the HEV recovery rate and inhibition rate.The optimized HEV method was applied to commercially available pig liver specimens,and HEV genotyping was performed on positive specimens.Results The HEV recovery rate of PBS homogenization treatment,proteinase K treatment and chloroform extraction method was 9.88％,0.19％and 17.28％,respectively.The recovery rate of proteinase K treatment was less than 1％,and it was discarded;t-test was performed to compare recovery rates of the other two methods,which showed statistically significant differences(t=26.801,P＜0.001),the chloroform extraction method had a higher recovery rate.The inhibition rates of the three methods were all less than 75％,within the range of the ISO/TS 15216-2:2019 standard.Among 192 commercially available pig liver specimens,17 specimens were detected positive for HEV RNA,with a nucleic acid positive rate of 8.85％;five specimens were successfully genotyped for HEV,all of which were genotype 4.Conclusions The virus recovery effect was good when chloroform extraction method was used for pig liver pretreatment;moreover,this method could detect HEV RNA from commercially available pig livers,which indicate that it can be used for virus detection in food.
关键词
戊型肝炎病毒/猪肝/病毒回收/核酸检测/基因分型Key words
Hepatitis E virus/Pig liver/Virus recovery/Nucleic acid detection/Genotyping引用本文复制引用
基金项目
国家科技重大专项(2018ZX10201002-009-002)
出版年
2024
NETL
NSTL
万方数据