猪粪便中戊肝病毒实时荧光RT-PCR定性检测方法的建立及其分子流行病学特征
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摘要
目的 建立以TaqMan探针为基础的戊肝病毒(HEV)实时荧光RT-PCR检测方法和构建系统发育树,对戊肝病毒进行基因分型,检测中国部分省市猪场的猪粪便样本HEV污染水平。方法 参照GenBank HEV基因型序列,针对HEV保守区设计引物和探针,优化反应体系,建立实时荧光RT-PCR和巢式RT-PCR检测体系。结果 建立的HEV实时荧光RT-PCR检测方法的灵敏度为19.9拷贝/μL,扩增效率92.9%～109.1%,与札如病毒(Sa)、诺如病毒(Nov)、甲肝病毒(HAV)均不发生交叉反应。荧光RT-PCR检测猪粪便样本342份,其中HEV阳性样本210份,阳性率61.4%,育肥前阳性率56.6%,育肥后阳性率66.9%,育肥前后样本阳性率差异具有统计学意义(χ~2=24.8,P<0.05);经基因分型鉴定体系测定阳性毒株基因型均为HEV-4型,且存在4b、4d、4h三种基因亚型。结论 中国部分省市猪场中HEV感染普遍,基因型均为HEV-4型,各省市猪场感染率和基因亚型存在差异。
OBJECTIVE Real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) assay based on Taqman and phylogenetic tree were developed for detecting hepatitis E virus in swine feces of pig farms from several provinces and city.METHODSDesigning prime and probe refering to HEV genotype sequences of Genbank, we developed a Taqman-based real-time RT-PCR assay and nested RT-PCR according to HEV conserved domain after optimizing reaction system, then detected the prevalence of HEV infection of pig farms. RESULTS The sensitivity of the real-time RT-PCR assay established in this experiment was 19.9 copies/μL, the amplification efficiency was 92.9%-109.1%, there was no cross reaction with Sapovirus, Norovirus and Hepatitis A. A total of 342 samples of swine feces were detected. There were two hundred and ten positive samples, and positive rate was 61.4%. The positive rate of before-fattening was 56.6%, and after-fattening was 66.9%. The positive rate of before and after fattening samples had statistical difference(χ~2=24.8,P<0.05). The genotype identification system determined that the positive strains isolated in this study were HEV-4 type, and three subtypes of 4 b, 4 d and 4 h were detected. CONCLUSION The pig farms of several provinces and city are contaminated by HEV extensively. The genotypes of the isolated strains are all HEV-4 type. The infection rate and infection subtype of pigs in different provinces and cities are different.
OBJECTIVE Real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) assay based on Taqman and phylogenetic tree were developed for detecting hepatitis E virus in swine feces of pig farms from several provinces and city.METHODSDesigning prime and probe refering to HEV genotype sequences of Genbank, we developed a Taqman-based real-time RT-PCR assay and nested RT-PCR according to HEV conserved domain after optimizing reaction system, then detected the prevalence of HEV infection of pig farms. RESULTS The sensitivity of the real-time RT-PCR assay established in this experiment was 19.9 copies/μL, the amplification efficiency was 92.9%-109.1%, there was no cross reaction with Sapovirus, Norovirus and Hepatitis A. A total of 342 samples of swine feces were detected. There were two hundred and ten positive samples, and positive rate was 61.4%. The positive rate of before-fattening was 56.6%, and after-fattening was 66.9%. The positive rate of before and after fattening samples had statistical difference(χ~2=24.8,P<0.05). The genotype identification system determined that the positive strains isolated in this study were HEV-4 type, and three subtypes of 4 b, 4 d and 4 h were detected. CONCLUSION The pig farms of several provinces and city are contaminated by HEV extensively. The genotypes of the isolated strains are all HEV-4 type. The infection rate and infection subtype of pigs in different provinces and cities are different.
引文
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[14] 林虹,管大伟,邓小玲,等.应用TaqMan荧光定量RT-PCR检测猪肝中戊型肝炎病毒的研究 [J].华南预防医学,2015,41(2):171-175.
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[16] 蔡洁新,杨立杰,赵振杰.荧光RT-PCR检测与ELISA检测在戊肝临床诊断效果的比较分析 [J].标记免疫分析与临床,2016,23(9):1080-1082.
[17] 王延臣,赵晨燕.戊型肝炎病毒实验室诊断技术的现状 [J].中华检验医学杂志,2008,31(8):852-854.
[18] KING N J,HEWITT J,PERCHEC-MERIEN A M.Hiding in plain sight?it’s time to investigate other possible transmission routes for hepatitis E virus (HEV) in developed countries [J].Food Environ Virol,2018,10(3):225-252.
[19] 张晓峰,帅江冰,李爱云,等.猪戊型肝炎病毒荧光定量PCR检测方法的建立及其分子流行病学 [J].中国兽医学报,2011,31(6):822-827.
[20] XIA J,ZENG H,LIU L,et al.Swine and rabbits are the main reservoirs of hepatitis E virus in China:detection of HEV RNA in feces of farmed and wild animals [J].Arch Virol,2015,160(11):2791-2798.
[21] 慕春龙,夏平安,张龙现,等.河南省猪场戊型肝炎病毒流行病学调查[J].中国畜牧兽医,2011,38(8):197-201.
[22] LOPEZ P,RISALDE M L A,FRIAS M,et al.Risk factors associated with hepatitis E virus in pigs from different production systems [J].Vet Microbiol,2018,224:88-92.
[23] NING H Q,NIU Z X,YU R S,et al.Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb[J].Vet Microbiol,2006,121:1-2.
[24] 司伏生.猪戊型肝炎病毒分子流行病学分析及基因3型猪戊型肝炎病毒感染性克隆的构建[D].南京:南京农业大学,2011
[2] 朱于敏,董世娟,司伏生,等.我国人和猪戊型肝炎病毒(HEV)的基因型及其流行状况分析[C]//上海市畜牧兽医学会2011年学术年会论文集.上海:上海市畜牧兽医学会,2011:1-4.
[3] VON WULFFEN M,WESTHOLTER D,LUTGEHETMANN M,et al.Hepatitis E:still waters run deep [J].J Clin Translation Hepatol,2018,6(1):40-47.
[4] 胡凤姣,李玥,石蕊寒,等.北京市售猪肝脏戊型肝炎病毒相关抗原检测 [C]//中国畜牧兽医学会兽医食品卫生学分会第十二届学术研讨会论文集.长春:中国畜牧兽医学会兽医食品卫生学分会,2012:278-283.
[5] GUERRA J,KAMPA K C,MORSOLETTO D G B,et al.Hepatitis E:a literature review [J].J Clin Translation Hepatol,2017,5(4):376-383.
[6] 江勇,韩涛.《2016年欧洲儿童胃肠、肝病和营养学会委员会意见书:儿童戊型肝炎》推荐意见[J].临床肝胆病杂志,2016,32(7):1256-1257.
[7] 张小霞,许锋,梁振普,等.河南地区猪戊型肝炎病毒的检测及基因序列分析 [J].河南农业大学学报,2011,45(3):302-306.
[8] HUANG F F,HAQSHENAS G,GUENETTE D K,et al.Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States [J].J Clin Microbiol,2002,40(4):1326-1332.
[9] UKULI A Q,MUGIMBA K K.Seroprevalence of hepatitis E in swine abattoir workers [J].Afr Health Sci,2017,17(4):1022-1028.
[10] 于水生.上海地区猪戊型肝炎流行病学调查和基因型分析 [D].南京:南京农业大学,2008.
[11] 黄芬,禹文海,马天武,等.云南某农村猪戊型肝炎病毒分子流行病学调查 [J].中国人兽共患病学报,2012,28(3):274-277.
[12] SALINES M,ANDRAUD M,ROSE N.From the epidemiology of hepatitis E virus (HEV) within the swine reservoir to public health risk mitigation strategies:a comprehensive review [J].Veterin Res,2017,48(1):31.
[13] 肖爱欢.广西猪戊型肝炎分子流行病学调查及戊型肝炎病毒ORF2的扩增和抗原性分析 [D].南宁:广西大学,2010.
[14] 林虹,管大伟,邓小玲,等.应用TaqMan荧光定量RT-PCR检测猪肝中戊型肝炎病毒的研究 [J].华南预防医学,2015,41(2):171-175.
[15] 陈欣,梁雨然,曹誉龄,等.荧光定量PCR检测戊肝病毒的应用价值探究 [J].现代预防医学,2015,42(22):4162-4164.
[16] 蔡洁新,杨立杰,赵振杰.荧光RT-PCR检测与ELISA检测在戊肝临床诊断效果的比较分析 [J].标记免疫分析与临床,2016,23(9):1080-1082.
[17] 王延臣,赵晨燕.戊型肝炎病毒实验室诊断技术的现状 [J].中华检验医学杂志,2008,31(8):852-854.
[18] KING N J,HEWITT J,PERCHEC-MERIEN A M.Hiding in plain sight?it’s time to investigate other possible transmission routes for hepatitis E virus (HEV) in developed countries [J].Food Environ Virol,2018,10(3):225-252.
[19] 张晓峰,帅江冰,李爱云,等.猪戊型肝炎病毒荧光定量PCR检测方法的建立及其分子流行病学 [J].中国兽医学报,2011,31(6):822-827.
[20] XIA J,ZENG H,LIU L,et al.Swine and rabbits are the main reservoirs of hepatitis E virus in China:detection of HEV RNA in feces of farmed and wild animals [J].Arch Virol,2015,160(11):2791-2798.
[21] 慕春龙,夏平安,张龙现,等.河南省猪场戊型肝炎病毒流行病学调查[J].中国畜牧兽医,2011,38(8):197-201.
[22] LOPEZ P,RISALDE M L A,FRIAS M,et al.Risk factors associated with hepatitis E virus in pigs from different production systems [J].Vet Microbiol,2018,224:88-92.
[23] NING H Q,NIU Z X,YU R S,et al.Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb[J].Vet Microbiol,2006,121:1-2.
[24] 司伏生.猪戊型肝炎病毒分子流行病学分析及基因3型猪戊型肝炎病毒感染性克隆的构建[D].南京:南京农业大学,2011