鉴别猪流行性腹泻病毒基因型的一步法双重qRT-PCR
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摘要
猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)是目前导致猪(Sus scrofa domestica)群严重腹泻的主要病原体之一,临床中危害最严重的是PEDV变异毒株(GⅡ型)。建立快速准确的诊断方法可鉴别临床上PEDV的基因型,有利于开展不同基因型的PEDV防控。本研究针对PEDV的纤突蛋白(spike, S)基因N端的相对保守区设计1对引物,同时针对变异毒株(GⅡ型)和经典毒株(GⅠ型)的差异区域设计2个探针,探针采用5-羧基荧光素(5-carboxyfluorescein, FAM)(GⅡ型)和5-六氯荧光素氨基磷酸酯(5-hexachloro-fluorescein hosphoramidite, HEX)(GⅠ型)荧光信号分别标记,通过扩增条件摸索、特异性、敏感性、重复性等实验,建立一种基于探针的一步法双重q RT-PCR检测技术,用于鉴别猪流行性腹泻病毒的不同基因型。结果显示:本研究建立了该方法的标准曲线,该方法在10~(-1)～10~8拷贝数/μL范围内,检测GⅡ毒株(R2=0.9892)和GⅠ型毒株(R2=0.9914)具有较好的扩增效率,Ct值与浓度之间具有较好线性关系;检测灵敏度分别为GⅡ毒株7.34拷贝数/μL和2.3×10~2半数组织培养感染剂量(tissue culture infective dose,TCID50)/0.1 mL、GⅠ型毒株4.32拷贝数/μL和4.3×103TCID50/0.1 mL;GⅠ型和GⅡ型之间没有交叉反应,且与猪δ冠状病毒(Porcine deltacoronavirus, PDCoV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus, TGEV)、猪瘟病毒(Classic swine fever virus, CSFV)、猪轮状病毒(Porcine rotavirus, RV)、日本脑炎病毒(Japanese encephalitis virus, JEV)及猪细小病毒(Porcine parvovirus, PPV)等病毒核酸之间没有交叉反应,特异性较好;组间和组内变异系数低,重复性好;临床样品检测结果与病毒分离方法符合率为100%。本研究为临床疑似PEDV感染病料的检测提供快速准确的鉴别方法,并可确定其基因型,为防控该病提供技术支持。
Porcine epidemic diarrhea virus(PEDV) is one of the important pathogens that affect severe diarrhea in 3～10 day-old piglets(Sus scrofa domestica). Now the variant strain(GⅡtype) causes serious loss in farms. In order to differentiate the genotypes of PEDV, a rapid and accurate diagnostic method is established and used to detect PEDV from clinical diarrhea samples, those are advantageous to the prevention and control of these diseases. In this study, a pair of primers based the N-terminal domain of the PEDV S gene were designed, and 2 probes were designed in the light of the different regions of PEDV spike gene(S) from the variant strains(GⅡ) and the classical strains(GⅠ). The 5' end of those probes were individually marked by 5-carboxyfluorescein(FAM)(G Ⅱ) and 5-hexachloro-fluorescein hosphoramidite(HEX)(G Ⅰ) fluorescent signals. One single-step duplex qRT-PCR based on specific probes was established to distinguish different genotypes of PEDV by experiments on amplification conditions, specificity, sensitivity, repeatability, and so on. Firstly, the standard curves were established. Within the range of 10-1～108 copies/μL, this method showed good amplification efficiency for G Ⅱ strain(R~2=0.9892) and G Ⅰ strain(R2=0.9914), and there was a good linear relation-ship between Ct value and concentration as well. Secondly, the sensitivity was 7.34 copies/μL or 2.3×102 TCID50(tissue culture infective dose)/0.1 mL for GⅡstrain, 4.32 copies/μL or 4.3×103 TCID50/0.1 mL for GⅠstrain, respectively. Thirdly, there was no cross-reaction between GⅡand GⅠstrains, in addition between PEDV and other pathogens about Porcine deltacoronavirus(PDCoV), Classic swine fever virus(CSFV),Transmissible gastroenteritis virus(TGEV), Porcine rotavirus(RV), Japanese encephalitis virus(JEV), Porcine parvovirus(PPV). Therefore, the specificity of this method was good. In the end, the coefficient of variation between and within groups was low, so the reproducibility was good. In detecting clinical samples by this method, the results were 100% consistent with virus isolation. This study are useful tools for quantifying viral load, detecting PEDV, and differentiating PEDV genotypes, and providing one technique for the prevention and control of PEDV.
Porcine epidemic diarrhea virus(PEDV) is one of the important pathogens that affect severe diarrhea in 3～10 day-old piglets(Sus scrofa domestica). Now the variant strain(GⅡtype) causes serious loss in farms. In order to differentiate the genotypes of PEDV, a rapid and accurate diagnostic method is established and used to detect PEDV from clinical diarrhea samples, those are advantageous to the prevention and control of these diseases. In this study, a pair of primers based the N-terminal domain of the PEDV S gene were designed, and 2 probes were designed in the light of the different regions of PEDV spike gene(S) from the variant strains(GⅡ) and the classical strains(GⅠ). The 5' end of those probes were individually marked by 5-carboxyfluorescein(FAM)(G Ⅱ) and 5-hexachloro-fluorescein hosphoramidite(HEX)(G Ⅰ) fluorescent signals. One single-step duplex qRT-PCR based on specific probes was established to distinguish different genotypes of PEDV by experiments on amplification conditions, specificity, sensitivity, repeatability, and so on. Firstly, the standard curves were established. Within the range of 10-1～108 copies/μL, this method showed good amplification efficiency for G Ⅱ strain(R~2=0.9892) and G Ⅰ strain(R2=0.9914), and there was a good linear relation-ship between Ct value and concentration as well. Secondly, the sensitivity was 7.34 copies/μL or 2.3×102 TCID50(tissue culture infective dose)/0.1 mL for GⅡstrain, 4.32 copies/μL or 4.3×103 TCID50/0.1 mL for GⅠstrain, respectively. Thirdly, there was no cross-reaction between GⅡand GⅠstrains, in addition between PEDV and other pathogens about Porcine deltacoronavirus(PDCoV), Classic swine fever virus(CSFV),Transmissible gastroenteritis virus(TGEV), Porcine rotavirus(RV), Japanese encephalitis virus(JEV), Porcine parvovirus(PPV). Therefore, the specificity of this method was good. In the end, the coefficient of variation between and within groups was low, so the reproducibility was good. In detecting clinical samples by this method, the results were 100% consistent with virus isolation. This study are useful tools for quantifying viral load, detecting PEDV, and differentiating PEDV genotypes, and providing one technique for the prevention and control of PEDV.
引文
Chang S H,Bae J L,Kang T J,et al.2002.Identification of the epitope region capable of inducing neutralizing antibodies against the Porcine epidemic diarrhea virus[J].Molecules and Cells,14(2):295-299.
Chen J,Wang C,Shi H,et al.2011.Complete genome sequence of a Chinese virulent Porcine epidemic diarrhea virus strain[J].Journal of Virology,86(24):11538-11539.
Cho Y Y,Lim S I,Kim Y K,et al.2014.Complete genome sequence of K14JB01,a novel variant strain of Porcine epidemic diarrhea virus in South Korea[J].Genome Announcements,2(3):e00505-14.
Cruz D J,Kim C J,Shin H J.2008.The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize Porcine epidemic diarrhea virus[J].Virus Research 132(1-2):192-196.
Dastjerdi A,Carr J,Ellis R J,et al.2015.Porcine epidemic diarrhea virus among farmed pigs,Ukraine[J].Emerging Infectious Diseases,21(12):2235-2237.
Huang Y W,Dickerman A W,Pineyro P,et al.2013.Origin,evolution,and genotyping of emergent Porcine epidemic diarrhea virus strains in the United States[J].mBio,4(5):e00737-00713.
Jung K,Chae C.2005.RT-PCR-based dot blot hybridization for the detection and differentiation between Porcine epidemic diarrhea virus and transmissible gastroenteritis virus in fecal samples using a non-radioactive digoxigenin cDNA probe[J].Journal of Virological Methods,123(2):141-6.
Kocherhans R,Bridgen A,Ackermann M,et al.2001.Completion of the porcine epidemic diarrhoea coronavirus(PEDV)genome sequence[J].Virus Genes,23(2):137-144.
Li C,Li W,Lucio de Esesarte E,et al.2017.Cell Attachment domains of the Porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies[J].Journal of Virology,91(12):e00273-17.
Li D,Feng H,Liu Y,et al.2018.Molecular evolution of Porcine epidemic diarrhea virus and porcine deltacoronavirus strains in central China[J].Research in Veterinary Science,120:63-69.
Li W,van Kuppeveld F J,He Q,et al.2016.Cellular entry of the Porcine epidemic diarrhea virus[J].Virus Research,226:117-127.
Park S J,Kim H K,Song D S,et al.2011.Molecular characterization and phylogenetic analysis of Porcine epidemic diarrhea virus(PEDV)field isolates in Korea[J].Archives of Virology,156(4):577-585.
Pensaert M B,de Bouck P.1978.A new coronavirus-like particle associated with diarrhea in swine[J].Archives of Virology,58(3):243-247.
Ren X,Li P.2011.Development of reverse transcription loopmediated isothermal amplification for rapid detection of Porcine epidemic diarrhea virus[J].Virus Genes,42(2):229-35.
Sun D,Feng L,Shi H,et al.2008.Identification of two novel B cell epitopes on Porcine epidemic diarrhea virus spike protein[J].Veterinary Microbiology,131(1-2):73-81.
Sun J,Li Q,Shao C,et al.2018.Isolation and characterization of Chinese Porcine epidemic diarrhea virus with novel mutations and deletions in the S gene[J].Veterinary Microbiology,221:81-89.
Temeeyasen G,Srijangwad A,Tripipat T,et al.2014.Genetic diversity of ORF3 and spike genes of Porcine epidemic diarrhea virus in Thailand[J].Infection,Genetics and Evolution,21:205-213.
Van Diep N,Sueyoshi M,Norimine J,et al.2018.Molecular characterization of US-like and Asian non-S INDELstrains of Porcine epidemic diarrhea virus(PEDV)that circulated in Japan during 2013-2016 and PEDVs collected from recurrent outbreaks[J].BMC Veterinary Research,14(1):96.
Wang H,Cong F,Zeng F,et al.2018.Development of a real time reverse transcription loop-mediated isothermal amplification method(RT-LAMP)for detection of a novel Swine acute diarrhea syndrome corona virus(SADS-CoV)[J].Journal of Virological Methods,260:45-48.
Wang L,Zhang Y,Byrum B.2014.Development and evaluation of a duplex real-time RT-PCR for detection and differentiation of virulent and variant strains of Porcine epidemic diarrhea viruses from the United States[J].Journal of Virological Methods,207:154-157.
Wilrich C,Wilrich P T.2009.Estimation of the POD function and the LOD of a qualitative microbiological measurement method[J].Journal of AOAC INTERNATIONAL,92(6):1763-1772.
Xia L,Dai L,Yang Q.2018.Transmissible gastroenteritis virus infection decreases arginine uptake by downregulating CAT-1 expression[J].Veterinary Research,49(1):95.
Yuan W,Li Y,Li P,et al.2016.Development of a nanoparticle-assisted PCR assay for detection of Porcine epidemic diarrhea virus[J].Journal of Virological Methods,220:18-20.
Zhang J,Tsai Y L,Lee P Y,et al.2016.Evaluation of two singleplex reverse transcription-insulated isothermal PCRtests and a duplex real-time RT-PCR test for the detection of Porcine epidemic diarrhea virus and Porcine deltacorona virus[J].Journal of Virological Methods,234:34-42.
Zhao P D,Bai J,Jiang P,et al.2014.Development of a multiplex TaqMan probe-based real-time PCR for discrimination of variant and classical Porcine epidemic diarrhea virus[J].Journal of Virological Methods,206:150-155.
Zuo Q,Zhao R,Liu J,et al.2018.Epidemiology and phylogeny of spike gene of Porcine epidemic diarrhea virus from Yunnan,China[J].Virus Research,249:45-51.
Chen J,Wang C,Shi H,et al.2011.Complete genome sequence of a Chinese virulent Porcine epidemic diarrhea virus strain[J].Journal of Virology,86(24):11538-11539.
Cho Y Y,Lim S I,Kim Y K,et al.2014.Complete genome sequence of K14JB01,a novel variant strain of Porcine epidemic diarrhea virus in South Korea[J].Genome Announcements,2(3):e00505-14.
Cruz D J,Kim C J,Shin H J.2008.The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize Porcine epidemic diarrhea virus[J].Virus Research 132(1-2):192-196.
Dastjerdi A,Carr J,Ellis R J,et al.2015.Porcine epidemic diarrhea virus among farmed pigs,Ukraine[J].Emerging Infectious Diseases,21(12):2235-2237.
Huang Y W,Dickerman A W,Pineyro P,et al.2013.Origin,evolution,and genotyping of emergent Porcine epidemic diarrhea virus strains in the United States[J].mBio,4(5):e00737-00713.
Jung K,Chae C.2005.RT-PCR-based dot blot hybridization for the detection and differentiation between Porcine epidemic diarrhea virus and transmissible gastroenteritis virus in fecal samples using a non-radioactive digoxigenin cDNA probe[J].Journal of Virological Methods,123(2):141-6.
Kocherhans R,Bridgen A,Ackermann M,et al.2001.Completion of the porcine epidemic diarrhoea coronavirus(PEDV)genome sequence[J].Virus Genes,23(2):137-144.
Li C,Li W,Lucio de Esesarte E,et al.2017.Cell Attachment domains of the Porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies[J].Journal of Virology,91(12):e00273-17.
Li D,Feng H,Liu Y,et al.2018.Molecular evolution of Porcine epidemic diarrhea virus and porcine deltacoronavirus strains in central China[J].Research in Veterinary Science,120:63-69.
Li W,van Kuppeveld F J,He Q,et al.2016.Cellular entry of the Porcine epidemic diarrhea virus[J].Virus Research,226:117-127.
Park S J,Kim H K,Song D S,et al.2011.Molecular characterization and phylogenetic analysis of Porcine epidemic diarrhea virus(PEDV)field isolates in Korea[J].Archives of Virology,156(4):577-585.
Pensaert M B,de Bouck P.1978.A new coronavirus-like particle associated with diarrhea in swine[J].Archives of Virology,58(3):243-247.
Ren X,Li P.2011.Development of reverse transcription loopmediated isothermal amplification for rapid detection of Porcine epidemic diarrhea virus[J].Virus Genes,42(2):229-35.
Sun D,Feng L,Shi H,et al.2008.Identification of two novel B cell epitopes on Porcine epidemic diarrhea virus spike protein[J].Veterinary Microbiology,131(1-2):73-81.
Sun J,Li Q,Shao C,et al.2018.Isolation and characterization of Chinese Porcine epidemic diarrhea virus with novel mutations and deletions in the S gene[J].Veterinary Microbiology,221:81-89.
Temeeyasen G,Srijangwad A,Tripipat T,et al.2014.Genetic diversity of ORF3 and spike genes of Porcine epidemic diarrhea virus in Thailand[J].Infection,Genetics and Evolution,21:205-213.
Van Diep N,Sueyoshi M,Norimine J,et al.2018.Molecular characterization of US-like and Asian non-S INDELstrains of Porcine epidemic diarrhea virus(PEDV)that circulated in Japan during 2013-2016 and PEDVs collected from recurrent outbreaks[J].BMC Veterinary Research,14(1):96.
Wang H,Cong F,Zeng F,et al.2018.Development of a real time reverse transcription loop-mediated isothermal amplification method(RT-LAMP)for detection of a novel Swine acute diarrhea syndrome corona virus(SADS-CoV)[J].Journal of Virological Methods,260:45-48.
Wang L,Zhang Y,Byrum B.2014.Development and evaluation of a duplex real-time RT-PCR for detection and differentiation of virulent and variant strains of Porcine epidemic diarrhea viruses from the United States[J].Journal of Virological Methods,207:154-157.
Wilrich C,Wilrich P T.2009.Estimation of the POD function and the LOD of a qualitative microbiological measurement method[J].Journal of AOAC INTERNATIONAL,92(6):1763-1772.
Xia L,Dai L,Yang Q.2018.Transmissible gastroenteritis virus infection decreases arginine uptake by downregulating CAT-1 expression[J].Veterinary Research,49(1):95.
Yuan W,Li Y,Li P,et al.2016.Development of a nanoparticle-assisted PCR assay for detection of Porcine epidemic diarrhea virus[J].Journal of Virological Methods,220:18-20.
Zhang J,Tsai Y L,Lee P Y,et al.2016.Evaluation of two singleplex reverse transcription-insulated isothermal PCRtests and a duplex real-time RT-PCR test for the detection of Porcine epidemic diarrhea virus and Porcine deltacorona virus[J].Journal of Virological Methods,234:34-42.
Zhao P D,Bai J,Jiang P,et al.2014.Development of a multiplex TaqMan probe-based real-time PCR for discrimination of variant and classical Porcine epidemic diarrhea virus[J].Journal of Virological Methods,206:150-155.
Zuo Q,Zhao R,Liu J,et al.2018.Epidemiology and phylogeny of spike gene of Porcine epidemic diarrhea virus from Yunnan,China[J].Virus Research,249:45-51.