猪δ冠状病毒TaqMan荧光定量RT-PCR检测方法的建立与初步应用
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摘要
本研究旨在建立一种灵敏、快速检测猪δ冠状病毒(porcine deltacoronavirus,PDCoV)的TaqMan荧光定量RT-PCR方法。参照GenBank中PDCoV有关基因序列,设计一对特异性引物用于扩增PDCoV M基因。将测序正确的基因片段克隆入pMD18-T载体,构建重组质粒作为建立标准曲线的病毒模板。设计合成一对特异性引物与TaqMan探针,进行反应条件和反应体系的优化,建立快速检测PDCoV的TaqMan实时荧光定量RT-PCR方法,并进行该方法的灵敏性、特异性与重复性验证。结果表明,该方法能有效扩增1.0×10~1～1.0×10~9拷贝·μL~(-1)的PDCoV标准质粒,建立的标准曲线呈现良好的线性关系。该方法的检测灵敏度为1.0×10~1拷贝·μL~(-1);对猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪伪狂犬病病毒等病原不发生交叉反应,具有很好的特异性;重复性试验结果显示变异系数(CV)小于1%,重复性良好。对2017—2018年期间收集的河南省不同猪场的100份腹泻病料进行检测,PDCoV的阳性检出率为23%(23/100),与PDCoV SYBR GreenⅠ荧光定量RT-PCR检测方法的符合率为100%。人工感染PDCoV的仔猪,取攻毒后不同时间的粪便样品,应用本研究建立方法与常规RT-PCR方法对样品进行检测,TaqMan荧光定量RT-PCR检测方法的灵敏度远远优于常规RT-PCR。上述结果表明,本研究所建立的TaqMan荧光定量RT-PCR检测方法能够灵敏、特异地检测PDCoV,可用于临床PDCoV的检测。
This experiment was conducted to develop a TaqMan based real-time fluorescent RT-PCR for the detection of PDCoV. Based on the M gene sequence of PDCoV in GenBank, a pair of primers was designed, and M gene of PDCoV was amplified and cloned into pMD18-T vector to get the recombinant plasmid. Then another pair of primers and a TaqMan probe was designed, and a TaqMan based real-time fluorescent RT-PCR assay was developed by optimizing the conditions and systems of reaction. Sensitivity, specificity and reproducibility assay were determined. The results showed that this assay could detect the PDCoV between 1.0×10~1-1.0×10~9 copies·μL~(-1) with a good linear relation, and the sensitivity limit was 1.0×10~1 copies·μL~(-1). There were no cross-reaction with porcine epidemic diarrhea virus(PEDV), transmissible gastroenteritis virus(TGEV), pseudorabies virus(PRV) and other porcine viruses. The CV(Coefficient of Variation) of intra-assay and inter-assay were no more than 1%. The TaqMan based real-time RT-PCR assay was then used to detect 100 diarrhea clinical samples of porcine that collected from 2017 to 2018, and the PDCoV positive ratio was 23%, and the coincidence rate with SYBR Green Ⅰ real-time PCR assay was 100%. Piglets were inoculated with PDCoV, and fecal samples were collected and detected by TaqMan real-time RT-PCR and common RT-PCR assay, the results showed that the sensitivity of the former was much better than the latter. This assay provides a high sensitivity, specificity and reproducibility method for detection of PDCoV.
This experiment was conducted to develop a TaqMan based real-time fluorescent RT-PCR for the detection of PDCoV. Based on the M gene sequence of PDCoV in GenBank, a pair of primers was designed, and M gene of PDCoV was amplified and cloned into pMD18-T vector to get the recombinant plasmid. Then another pair of primers and a TaqMan probe was designed, and a TaqMan based real-time fluorescent RT-PCR assay was developed by optimizing the conditions and systems of reaction. Sensitivity, specificity and reproducibility assay were determined. The results showed that this assay could detect the PDCoV between 1.0×10~1-1.0×10~9 copies·μL~(-1) with a good linear relation, and the sensitivity limit was 1.0×10~1 copies·μL~(-1). There were no cross-reaction with porcine epidemic diarrhea virus(PEDV), transmissible gastroenteritis virus(TGEV), pseudorabies virus(PRV) and other porcine viruses. The CV(Coefficient of Variation) of intra-assay and inter-assay were no more than 1%. The TaqMan based real-time RT-PCR assay was then used to detect 100 diarrhea clinical samples of porcine that collected from 2017 to 2018, and the PDCoV positive ratio was 23%, and the coincidence rate with SYBR Green Ⅰ real-time PCR assay was 100%. Piglets were inoculated with PDCoV, and fecal samples were collected and detected by TaqMan real-time RT-PCR and common RT-PCR assay, the results showed that the sensitivity of the former was much better than the latter. This assay provides a high sensitivity, specificity and reproducibility method for detection of PDCoV.
引文
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[13] JUNG K,HU H,EYERLY B,et al.Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs[J].Emerg Infect Dis,2015,21(4):650-654.
[14] HU H,JUNG K,WANG Q H,et al.Development of a one-step RT-PCR assay for detection of pancoronaviruses (α-,β-,γ-,and δ-coronaviruses) using newly designed degenerate primers for porcine and avian `fecal samples[J].J Virol Methods,2018,256:116-122.
[15] SONG D,ZHOU X,PENG Q,et al.Newly emerged porcine Deltacoronavirus associated with diarrhoea in swine in China:identification,prevalence and full-length genome sequence analysis[J].Transbound Emerg Dis,2015,62(6):575-580.
[16] 张利卫,曹贝贝,李炳晓,等.新发猪Delta冠状病毒和猪流行性腹泻病毒SYBR Green Ⅰ双重荧光RT-PCR检测方法的建立及应用[J].中国兽医学报,2018,38(4):618-624.ZHANG L W,CAO B B,LI B X,et al.Establishment and application of a duplex SYBR Green Ⅰ real-time RT-PCR assay for simultaneous detection of emerging PDCoV and PEDV[J].Chinese Journal of Veterinary Science,2018,38(4):618-624.(in Chinese)
[17] 韦学雷,梁青青,曹贝贝,等.猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用[J].中国兽医学报,2018,38(1):11-16.WEI X L,LIANG Q Q,CAO B B,et al.Establishment and application of a multiplex RT-PCR assay for simultaneous detection of porcine delta coronavirus,porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J].Chinese Journal of Veterinary Science,2018,38(1):11-16.(in Chinese)
[18] 逄凤娇,俞正玉,何孔旺,等.猪Deltacoronavirus RT-PCR检测方法的建立及其应用[J].中国预防兽医学报,2015,37(9):683-686.PANG F J,YU Z Y,HE K W,et al.Development and application of a RT-PCR assay for detection of porcine deltacoronavirus[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(9):683-686.(in Chinese)
[19] 陈小金,张霞,王玉玲,等.猪德尔塔冠状病毒荧光定量PCR检测方法的建立[J].中国动物检疫,2018,35(9):95-100.CHEN X J,ZHANG X,WANG Y L,et al.Development of fluorescence quantitative PCR assay for detection of porcine deltacoronavirus[J].China Animal Health Inspection,2018,35(9):95-100.(in Chinese)
[20] 韩丽,周雍,李炳晓,等.猪Delta冠状病毒和猪流行性腹泻病毒双重RT-PCR诊断方法的建立及初步应用[J].中国兽医科学,2017,47(5):557-562.HAN L,ZHOU Y,LI B X,et al.Establishment and preliminary application of a duplex reverse transcription-PCR for the detection of porcine deltacoronavirus and porcine epidemic diarrhea virus[J].Chinese Veterinary Science,2017,47(5):557-562.(in Chinese)
[2] SINHA A,GAUGER P,ZHANG J Q,et al.PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine[J].Vet Microbiol,2015,179(3-4):296-298.
[3] WANG L Y,BYRUM B,ZHANG Y.Porcine coronavirus HKU15 detected in 9 US states,2014[J].Emerg Infect Dis,2014,20(9):1594-1595.
[4] HU H,JUNG K,VLASOVA A N,et al.Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States[J].J Clin Microbiol,2015,53(5):1537-1548.
[5] LEE S,LEE C.Complete genome characterization of korean porcine deltacoronavirus strain KOR/KNU14-04/2014[J].Genome Announc,2014,2(6):e01191-14.
[6] SAENG-CHUTO K,LORSIRIGOOL A,TEMEEYASEN G,et al.Different lineage of porcine deltacoronavirus in Thailand,Vietnam and Lao PDR in 2015[J].Transbound Emerg Dis,2017,64(1):3-10.
[7] DONG N,FANG L R,YANG H,et al.Isolation,genomic characterization,and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014[J].Vet Microbiol,2016,196:98-106.
[8] WANG Y W,YUE H,FANG W H,et al.Complete genome sequence of porcine deltacoronavirus strain CH/Sichuan/S27/2012 from mainland China[J].Genome Announc,2015,3(5):e00945-15.
[9] MA Y M,ZHANG Y,LIANG X Y,et al.Origin,evolution,and virulence of porcine deltacoronaviruses in the United States[J].MBio,2015,6(2):e00064-15.
[10] CHEN Q,GAUGER P,STAFNE M,et al.Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets[J].Virology,2015,482:51-59.
[11] LI W T,LI H,LIU Y B,et al.New variants of porcine epidemic diarrhea virus,China,2011[J].Emerg Infect Dis,2012,18(8):1350-1353.
[12] THACHIL A,GERBER P F,XIAO C T,et al.Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies[J].PLoS One,2015,10(4):e0124363.
[13] JUNG K,HU H,EYERLY B,et al.Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs[J].Emerg Infect Dis,2015,21(4):650-654.
[14] HU H,JUNG K,WANG Q H,et al.Development of a one-step RT-PCR assay for detection of pancoronaviruses (α-,β-,γ-,and δ-coronaviruses) using newly designed degenerate primers for porcine and avian `fecal samples[J].J Virol Methods,2018,256:116-122.
[15] SONG D,ZHOU X,PENG Q,et al.Newly emerged porcine Deltacoronavirus associated with diarrhoea in swine in China:identification,prevalence and full-length genome sequence analysis[J].Transbound Emerg Dis,2015,62(6):575-580.
[16] 张利卫,曹贝贝,李炳晓,等.新发猪Delta冠状病毒和猪流行性腹泻病毒SYBR Green Ⅰ双重荧光RT-PCR检测方法的建立及应用[J].中国兽医学报,2018,38(4):618-624.ZHANG L W,CAO B B,LI B X,et al.Establishment and application of a duplex SYBR Green Ⅰ real-time RT-PCR assay for simultaneous detection of emerging PDCoV and PEDV[J].Chinese Journal of Veterinary Science,2018,38(4):618-624.(in Chinese)
[17] 韦学雷,梁青青,曹贝贝,等.猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用[J].中国兽医学报,2018,38(1):11-16.WEI X L,LIANG Q Q,CAO B B,et al.Establishment and application of a multiplex RT-PCR assay for simultaneous detection of porcine delta coronavirus,porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J].Chinese Journal of Veterinary Science,2018,38(1):11-16.(in Chinese)
[18] 逄凤娇,俞正玉,何孔旺,等.猪Deltacoronavirus RT-PCR检测方法的建立及其应用[J].中国预防兽医学报,2015,37(9):683-686.PANG F J,YU Z Y,HE K W,et al.Development and application of a RT-PCR assay for detection of porcine deltacoronavirus[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(9):683-686.(in Chinese)
[19] 陈小金,张霞,王玉玲,等.猪德尔塔冠状病毒荧光定量PCR检测方法的建立[J].中国动物检疫,2018,35(9):95-100.CHEN X J,ZHANG X,WANG Y L,et al.Development of fluorescence quantitative PCR assay for detection of porcine deltacoronavirus[J].China Animal Health Inspection,2018,35(9):95-100.(in Chinese)
[20] 韩丽,周雍,李炳晓,等.猪Delta冠状病毒和猪流行性腹泻病毒双重RT-PCR诊断方法的建立及初步应用[J].中国兽医科学,2017,47(5):557-562.HAN L,ZHOU Y,LI B X,et al.Establishment and preliminary application of a duplex reverse transcription-PCR for the detection of porcine deltacoronavirus and porcine epidemic diarrhea virus[J].Chinese Veterinary Science,2017,47(5):557-562.(in Chinese)