猪德尔塔冠状病毒SYBR GreenⅠ荧光定量PCR检测方法的建立及应用
|
摘要
为建立猪德尔塔病毒(porcine deltacoronavirus,PDCoV)快速灵敏的诊断方法,采用RT-PCR方法扩增猪德尔塔冠状病毒(PDCoV)M基因,将M基因克隆到载体pEASY-Blunt Cloning Kit,以构建出的重组质粒作为标准阳性质粒,并以此为模板建立PDCoV M基因的SYBR GreenⅠ荧光定量PCR检测方法,并对该方法进行敏感性、特异性和重复性等验证。结果,建立的SYBR GreenⅠ荧光定量PCR方法的Ct值与标准品模板在6.57×10~8～6.57×10~1copies/μL范围内呈良好的线性关系,相关系数为0.999,斜率为-4.215;该方法特异性强,对PEDV和TGEV猪常见病原检测均无特异性扩增;可重复性良好,组内和组间变异系数均小于2%;灵敏度可达6.57×10~1copies/μL;对临床样品的检测,本研究建立的SYBR GreenⅠ荧光定量PCR方法的阳性检出率为5.0%。上述结果表明,本研究成功建立了检测PDCoV的SYBR GreenⅠ荧光定量PCR方法,可实现对PDCoV的快速灵敏诊断。
In order to establish a sensitive protocol for the detection of porcine deltacoronavirus,the RT-PCR was used to amplify the porcine deltacoronavirus(PDCo V)M gene,which was cloned into the vector p EASY-Blunt cloning kit,and the recombinant plasmid was constructed as a standard positive plasmid.The recombinant plasmid was used as a template to establish PDCo V SYBR Green I PCR assay and verification tests of sensitivity,specificity and repeatability were carried out.In result,the Ct value of the established SYBR Green I PCR had a good linear relationship with the standard template in the range from 6.57×10~8 to 6.57×10~1 copies/μL with a correlation coefficient of 0.999 and a slope of-4.215.This method is specific and has no specific amplification for common pathogens such as PEDV and TGEV in pigs.The intra-assay and inter-assay coefficients of variation are less than 2% with good repeatability.The sensitivity can reach 6.57×10~1 copies/μL.For the detection of clinical samples,the positive detection rate of the established SYBR Green I PCR method was 5.0%.In conclusion,this experiment successfully established the SYBR Green I PCR for the detection of PDCo V,which can realize the rapid and sensitive diagnosis of PDCo V infection.
In order to establish a sensitive protocol for the detection of porcine deltacoronavirus,the RT-PCR was used to amplify the porcine deltacoronavirus(PDCo V)M gene,which was cloned into the vector p EASY-Blunt cloning kit,and the recombinant plasmid was constructed as a standard positive plasmid.The recombinant plasmid was used as a template to establish PDCo V SYBR Green I PCR assay and verification tests of sensitivity,specificity and repeatability were carried out.In result,the Ct value of the established SYBR Green I PCR had a good linear relationship with the standard template in the range from 6.57×10~8 to 6.57×10~1 copies/μL with a correlation coefficient of 0.999 and a slope of-4.215.This method is specific and has no specific amplification for common pathogens such as PEDV and TGEV in pigs.The intra-assay and inter-assay coefficients of variation are less than 2% with good repeatability.The sensitivity can reach 6.57×10~1 copies/μL.For the detection of clinical samples,the positive detection rate of the established SYBR Green I PCR method was 5.0%.In conclusion,this experiment successfully established the SYBR Green I PCR for the detection of PDCo V,which can realize the rapid and sensitive diagnosis of PDCo V infection.
引文
[1]吴美洲,陈芳洲,朱银杏,等.丁型冠状病毒我国猪源株的遗传变异分析[J].中国兽医科学,2016(6):689-694.WU Mei-zhou,CHEN Fang-zhou,ZHU Yin-xing,et al.Genetic variation analysis of porcine deltacoronavirus in China[J].Chinese Veterinary Science,2016(6):689-694.
[2]ZHANG MJ,LIU DJ,LIU XL,et al.Genomic characterization and pathogenicity of porcine deltacoronavirus strain CHN-HG-2017 from China[J].Archives of Virology.2018.
[3]HU H,JUNG K,VLASOVA A N,et al.Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States[J].Journal of Clinical Microbiology,2015,53(5):1537-1548.
[4]WOO P C Y,LAU S K P,LAM C S F,et al.Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus[J].Journal of Virology,2014,88(2):1318-31.
[5]WANG L,BYRUM B,ZHANG Y.Porcine coronavirus HKU15detected in 9 US States,2014[J].Emerging Infectious Diseases,2014,20(9):1594-1595.
[6]SINHA A,GAUGER P,ZHANG J,et al.PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine[J].Veterinary Microbiology,2015,179(3-4):296-298.
[7]LEE S,LEE C.Complete Genome characterization of korean porcine deltacoronavirus strain KOR/KNU14-04/2014[J].Genome Announc,2014,2(6):e01191-14.
[8]NAN D,FANG L,ZENG S,et al.Porcine deltacoronavirusin mainland China[J].Emerging Infectious Diseases,2015,21(12):2254-2255.
[9]SONG D,ZHOU X,PENG Q,et al.Newly emerged porcine deltacoronavirus associated with diarrhoea in swine in China:Identification,prevalence and fulllength genome sequence analysis[J].Transboundary&Emerging Diseases,2015,62(6):575-580.
[10]WANG Y W,YUE H,FANG W,et al.Complete genome sequence of porcine deltacoronavirus strain CH/Sichuan/S27/2012 from Mainland China[J].Genome Announcements,2015,3(5):e00945-15.
[11]MADAPONG A,SAENGCHUTO K,LORSIRIGOOL A,et al.Complete genome sequence of porcine deltacoronavirus isolated in Thailand in 2015[J].Genome Announc,2016,4(3):e00408-16.
[12]JANETANAKIT T,LUMYAI M,BUNPAPONG N,et al.Porcine deltacoronavirus,Thailand,2015[J].Emerging Infectious Diseases,2016,22(4):757-759.
[13]方谱县,方六荣,董楠,等.猪δ冠状病毒的研究进展[J].病毒学报,2016(2):243-248.FANG Pu-xiang,FANG Liu-rong,DONG Nan,et al.Researchadvances in the porcine deltacoronavirus[J].Chinese Journal of Virology,2016(2):243-248.
[14]易新健,刘准,张翠翠,等.猪Delta冠状病毒的研究进展[J].中国兽医学报,2017(11):2235-2238.YI Jian-xin,LIU Zhun,Zhang Cui-cui,et al.Advances in research on porcine delta coronavirus[J].Chinese Journal of Veterinary Science,2017,37(11)
[15]杨浩,方六荣,董楠,等.基于猪德尔塔冠状病毒重组核衣壳蛋白的ELISA抗体检测方法的建立与评价[J].微生物学通报,2017,44(12):2830-2838.YANG Hao,FAng Liu-rong,DONG Nan,et al.Development and evaluation of an indirect ELISA based on recombinant nucleocapsid protein for detecting antibodies against porcine deltacoronavirus[J].Microbiology China,2017,44(12):2830-2838.
[16]韩丽,周雍,李炳哓,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(05):557-562.HAN Li,ZHONG Yong,LI Bing-Xiao,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(05):557-562.
[17]刘巧玲,黄涛,汤德元,等.猪德尔塔冠状病毒病的研究进展[J].黑龙江畜牧兽医,2018(23):50-53.LIU Qiao-ling,HUANG Tao,TANG De-yuan,et al.Advances in research on porcine delta coronavirus[J].Heilongjiang Animal Science and Veterinary Medicine,2018(23):50-53,267.
[18]肖帅,刘新生,方玉珍,等.猪德尔塔冠状病毒TaqMan荧光定量PCR检测方法的建立与应用[J].中国兽医科学,2017(9):1080-1086.XIAO Shuai,LIU Xin-sheng,FANG Yu-Zhen,et al.Development and application of TaqMan real-time fluorescent PCR for detection of porcine deltacoronavirus[J].Chinese Veterinary Science,2017(9):1080-1086.
[19]POSTIGO A,FERRER P E.Viral inhibitors reveal overlapping themes in regulation of cell death and innate immunity[J].Microbes&Infection,2009,11(13):1071-1078.
[20]ALINIKULA J,LASSILA O,NERA K P.DT40 mutants:a model to study transcriptional regulation of B cell development and function[J].SubcellBiochem,2006,40:189-205.
[2]ZHANG MJ,LIU DJ,LIU XL,et al.Genomic characterization and pathogenicity of porcine deltacoronavirus strain CHN-HG-2017 from China[J].Archives of Virology.2018.
[3]HU H,JUNG K,VLASOVA A N,et al.Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States[J].Journal of Clinical Microbiology,2015,53(5):1537-1548.
[4]WOO P C Y,LAU S K P,LAM C S F,et al.Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus[J].Journal of Virology,2014,88(2):1318-31.
[5]WANG L,BYRUM B,ZHANG Y.Porcine coronavirus HKU15detected in 9 US States,2014[J].Emerging Infectious Diseases,2014,20(9):1594-1595.
[6]SINHA A,GAUGER P,ZHANG J,et al.PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine[J].Veterinary Microbiology,2015,179(3-4):296-298.
[7]LEE S,LEE C.Complete Genome characterization of korean porcine deltacoronavirus strain KOR/KNU14-04/2014[J].Genome Announc,2014,2(6):e01191-14.
[8]NAN D,FANG L,ZENG S,et al.Porcine deltacoronavirusin mainland China[J].Emerging Infectious Diseases,2015,21(12):2254-2255.
[9]SONG D,ZHOU X,PENG Q,et al.Newly emerged porcine deltacoronavirus associated with diarrhoea in swine in China:Identification,prevalence and fulllength genome sequence analysis[J].Transboundary&Emerging Diseases,2015,62(6):575-580.
[10]WANG Y W,YUE H,FANG W,et al.Complete genome sequence of porcine deltacoronavirus strain CH/Sichuan/S27/2012 from Mainland China[J].Genome Announcements,2015,3(5):e00945-15.
[11]MADAPONG A,SAENGCHUTO K,LORSIRIGOOL A,et al.Complete genome sequence of porcine deltacoronavirus isolated in Thailand in 2015[J].Genome Announc,2016,4(3):e00408-16.
[12]JANETANAKIT T,LUMYAI M,BUNPAPONG N,et al.Porcine deltacoronavirus,Thailand,2015[J].Emerging Infectious Diseases,2016,22(4):757-759.
[13]方谱县,方六荣,董楠,等.猪δ冠状病毒的研究进展[J].病毒学报,2016(2):243-248.FANG Pu-xiang,FANG Liu-rong,DONG Nan,et al.Researchadvances in the porcine deltacoronavirus[J].Chinese Journal of Virology,2016(2):243-248.
[14]易新健,刘准,张翠翠,等.猪Delta冠状病毒的研究进展[J].中国兽医学报,2017(11):2235-2238.YI Jian-xin,LIU Zhun,Zhang Cui-cui,et al.Advances in research on porcine delta coronavirus[J].Chinese Journal of Veterinary Science,2017,37(11)
[15]杨浩,方六荣,董楠,等.基于猪德尔塔冠状病毒重组核衣壳蛋白的ELISA抗体检测方法的建立与评价[J].微生物学通报,2017,44(12):2830-2838.YANG Hao,FAng Liu-rong,DONG Nan,et al.Development and evaluation of an indirect ELISA based on recombinant nucleocapsid protein for detecting antibodies against porcine deltacoronavirus[J].Microbiology China,2017,44(12):2830-2838.
[16]韩丽,周雍,李炳哓,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(05):557-562.HAN Li,ZHONG Yong,LI Bing-Xiao,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(05):557-562.
[17]刘巧玲,黄涛,汤德元,等.猪德尔塔冠状病毒病的研究进展[J].黑龙江畜牧兽医,2018(23):50-53.LIU Qiao-ling,HUANG Tao,TANG De-yuan,et al.Advances in research on porcine delta coronavirus[J].Heilongjiang Animal Science and Veterinary Medicine,2018(23):50-53,267.
[18]肖帅,刘新生,方玉珍,等.猪德尔塔冠状病毒TaqMan荧光定量PCR检测方法的建立与应用[J].中国兽医科学,2017(9):1080-1086.XIAO Shuai,LIU Xin-sheng,FANG Yu-Zhen,et al.Development and application of TaqMan real-time fluorescent PCR for detection of porcine deltacoronavirus[J].Chinese Veterinary Science,2017(9):1080-1086.
[19]POSTIGO A,FERRER P E.Viral inhibitors reveal overlapping themes in regulation of cell death and innate immunity[J].Microbes&Infection,2009,11(13):1071-1078.
[20]ALINIKULA J,LASSILA O,NERA K P.DT40 mutants:a model to study transcriptional regulation of B cell development and function[J].SubcellBiochem,2006,40:189-205.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |