小反刍兽疫病毒抗体高敏快速荧光定量检测方法的建立
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摘要
为建立快速定量检测羊血清中小反刍兽疫病毒抗体的方法,本研究通过优化大肠杆菌密码子、优化表达条件等步骤,在大肠杆菌原核表达系统中获得了可溶性的N蛋白与NH融合蛋白,基于纯化的可溶性N蛋白与NH融合蛋白建立了小反刍兽疫病毒抗体高敏荧光免疫定量检测试剂盒。该方法能够快速定量检测绵羊血清中的小反刍兽疫病毒抗体,敏感性高,特异性强,对其他相关的羊类病原无交叉反应,其组内与组间变异系数分别低于10%和15%,具有良好的重复性。对292份临床血清样品进行检测,同时用法国IDVET竞争ELISA试剂盒进行比较,两种方法符合率达到93.84%。与血清中和试验进行比较,高敏荧光快速定量检测方法检测效价值基本与标准阳性血清(OIE参考实验室提供)的中和试验效价值相符合。本试验建立的方法可对小反刍兽疫病毒抗体进行现场快速定量检测,具有较高的实用价值和推广价值。
In an effort to establish a highly sensitive and rapid fluorescence quantitative assay for the detection of peste des petits ruminants virus(PRRSV)antibodies in sheep serum,in the present study we obtained a soluble and purified N protein fusing with NH protein of PRRSV expressed in Escherichia coli.Using this fusion protein,we have successfully developed a highly sensitive and rapid fluorescence quantitative method which was used to detection PPRSV antibodies.We demonstrated clearly that the established assay was quick and quantitative in detecting of PPRSV antibodies in sheep serum.Importantly,our results revealed that this approach had high sensitivity and specificity.The coefficients of variation of batch-to-batch and within-batch was lower than 10% and 15%,respectively.A total of 292 clinical serum samples were detected using our method.The coincidence rate between this method and the competition ELISA kit(offered by IDVET) was 93.84%,indicating high quality of our assay.Finally,we compared our method with the serum neutralization test using positive serum(providedby OIE Reference Laboratory for PPR in France),and the result verified the good quality of our system.In conclusion,the method we established can be used on-site as a point-of-care approach for rapid and quantitative detection of PPRV antibodies.
In an effort to establish a highly sensitive and rapid fluorescence quantitative assay for the detection of peste des petits ruminants virus(PRRSV)antibodies in sheep serum,in the present study we obtained a soluble and purified N protein fusing with NH protein of PRRSV expressed in Escherichia coli.Using this fusion protein,we have successfully developed a highly sensitive and rapid fluorescence quantitative method which was used to detection PPRSV antibodies.We demonstrated clearly that the established assay was quick and quantitative in detecting of PPRSV antibodies in sheep serum.Importantly,our results revealed that this approach had high sensitivity and specificity.The coefficients of variation of batch-to-batch and within-batch was lower than 10% and 15%,respectively.A total of 292 clinical serum samples were detected using our method.The coincidence rate between this method and the competition ELISA kit(offered by IDVET) was 93.84%,indicating high quality of our assay.Finally,we compared our method with the serum neutralization test using positive serum(providedby OIE Reference Laboratory for PPR in France),and the result verified the good quality of our system.In conclusion,the method we established can be used on-site as a point-of-care approach for rapid and quantitative detection of PPRV antibodies.
引文
[1]田克恭.人与动物共患病[M].北京:中国农业出版社,2013.TIAN Ke-gong.Human and Animal Comorbidity[M].Beijing:China Agricultural Press,2013.(in Chinese)
[2]李继东,蒙学莲,朱学亮,等.小反刍兽疫病毒H基因或F基因重组山羊痘病毒活载体疫苗的研究[J].中国兽医科学,2015,45(2):111-117.LI Ji-dong,MENG Xue-lian,ZHU Xue-liang,et al.Study on recombinant goatpox virus vaccines vectored with H or F gene of peste des petits ruminants virus[J].Chinese Veterinary Science,2015,45(2):111-117.(in Chinese)
[3]BOUSSINI H,CHITSUNGO E,BODJO S C,et al.First report and characterization of peste des petits ruminants virus in Liberia,West Africa[J].Tropical Animal Health and Production,2016,48(7):1503-1507.
[4]TAYLOR W.The global eradication of peste des petits ruminants(PPR)within 15 years-is this a pipe dream[J].Tropical Animal Health and Production,2016,48(3):559-567.
[5]BARON M D,DIALLO A,LANCELOT R,et al.Peste des petits ruminants virus[J].Advances in Virus Research,2016,95(3):1-42.
[6]王华,吴晓东,包静月,等.我国小反刍兽疫现状与防控策略[J].动物医学进展,2015,36(6):142-145.WANG Hua,WU Xiao-dong,BAO Jing-yue,et al.Epidemic analysis and control strategies of peste des petits ruminants in China[J].Advances in Virus Research,2015,36(6):142-145.(in Chinese)
[7]AZIZ U R,ABUBAKAR M,RASOOL M H,et al.Evaluation of risk factors for peste des petits ruminants virus in sheep and goats at the wildlife-livestock interface in Punjab Province,Pakistan[J].Biomed Research International,2016,15(5):245.
[8]HOLZER B,HODGSON S,LOGAN N,et al.Protection of cattle against rinderpest by vaccination with wildtype but not attenuated strains of peste des petits ruminants virus[J].Journal of Virology,2016,90(10):5152-5162.
[9]刘玉洪,徐自忠,花群义,等.小反刍兽疫病毒分子生物学研究进展[J].动物医学进展,2006,27(12):1-6.LIU Yu-hong,XU Zi-zhong,HUA Qun-yi,et al.Advance in molecular biology of peste des petits ruminants virus[J].Progress in Veterinary Medicine,2006,27(12):1-6.(in Chinese)
[10]LI L,WU X,LIU F,et al.Rapid detection of lineage IV peste des petits ruminants virus by real-time RT-PCR[J].Journal of Virological Methods,2016,148(1):131-133.
[11]SHARMA K K,KSHIRSAGAR D P,KALYANI I H,et al.Diagnosis of peste des petits ruminants infection in small ruminants through in-house developed indirect ELISA:Practical considerations[J].Veterinary World,2015,8(4):443-448.
[12]WU X,LIU F,LI L,et al.Diagnosis of peste des petits ruminants infection in small ruminants through in-house developed indirect ELISA:Practical considerations[J].Virus Genes,2016,52(3):422-427.
[2]李继东,蒙学莲,朱学亮,等.小反刍兽疫病毒H基因或F基因重组山羊痘病毒活载体疫苗的研究[J].中国兽医科学,2015,45(2):111-117.LI Ji-dong,MENG Xue-lian,ZHU Xue-liang,et al.Study on recombinant goatpox virus vaccines vectored with H or F gene of peste des petits ruminants virus[J].Chinese Veterinary Science,2015,45(2):111-117.(in Chinese)
[3]BOUSSINI H,CHITSUNGO E,BODJO S C,et al.First report and characterization of peste des petits ruminants virus in Liberia,West Africa[J].Tropical Animal Health and Production,2016,48(7):1503-1507.
[4]TAYLOR W.The global eradication of peste des petits ruminants(PPR)within 15 years-is this a pipe dream[J].Tropical Animal Health and Production,2016,48(3):559-567.
[5]BARON M D,DIALLO A,LANCELOT R,et al.Peste des petits ruminants virus[J].Advances in Virus Research,2016,95(3):1-42.
[6]王华,吴晓东,包静月,等.我国小反刍兽疫现状与防控策略[J].动物医学进展,2015,36(6):142-145.WANG Hua,WU Xiao-dong,BAO Jing-yue,et al.Epidemic analysis and control strategies of peste des petits ruminants in China[J].Advances in Virus Research,2015,36(6):142-145.(in Chinese)
[7]AZIZ U R,ABUBAKAR M,RASOOL M H,et al.Evaluation of risk factors for peste des petits ruminants virus in sheep and goats at the wildlife-livestock interface in Punjab Province,Pakistan[J].Biomed Research International,2016,15(5):245.
[8]HOLZER B,HODGSON S,LOGAN N,et al.Protection of cattle against rinderpest by vaccination with wildtype but not attenuated strains of peste des petits ruminants virus[J].Journal of Virology,2016,90(10):5152-5162.
[9]刘玉洪,徐自忠,花群义,等.小反刍兽疫病毒分子生物学研究进展[J].动物医学进展,2006,27(12):1-6.LIU Yu-hong,XU Zi-zhong,HUA Qun-yi,et al.Advance in molecular biology of peste des petits ruminants virus[J].Progress in Veterinary Medicine,2006,27(12):1-6.(in Chinese)
[10]LI L,WU X,LIU F,et al.Rapid detection of lineage IV peste des petits ruminants virus by real-time RT-PCR[J].Journal of Virological Methods,2016,148(1):131-133.
[11]SHARMA K K,KSHIRSAGAR D P,KALYANI I H,et al.Diagnosis of peste des petits ruminants infection in small ruminants through in-house developed indirect ELISA:Practical considerations[J].Veterinary World,2015,8(4):443-448.
[12]WU X,LIU F,LI L,et al.Diagnosis of peste des petits ruminants infection in small ruminants through in-house developed indirect ELISA:Practical considerations[J].Virus Genes,2016,52(3):422-427.