猪圆环病毒2型抗体间接ELISA检测方法的建立
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摘要
为了更加快速灵敏地检测血清中猪圆环病毒2型(Porcine circovirus type 2,PCV2)抗体的水平,我们通过优化反应条件建立了快速检测猪圆环病毒2型抗体的间接ELISA方法。结果显示,本研究研制的试剂盒检测3份PCV2抗体阳性血清的阳性滴度为1∶12 800～1∶25 600,而韩国JBT PCV2试剂盒检测的阳性滴度为1∶3200～1∶6400;对猪繁殖与呼吸综合征病毒、猪瘟病毒、猪伪狂犬病毒、猪口蹄疫病毒等10种其他病原体的20份阳性血清的检测结果均为阴性,表明本研究研制的试剂盒具有良好的特异性;批内重复性试验变异系数(C.V%)为2.26%～7.60%,批间重复试验的变异系数(C.V%)为1.67%～6.41%,变异系数均小于10%,呈现良好的可重复性。因此,我们成功建立了敏感性高、特异性强及重复性好的猪圆环病毒2型抗体间接ELISA检测方法。
In the present study, an indirect ELISA kit was developed and optimized for rapid detection of antibodies against Porcine circovirus type 2(PCV2). The serum samples were collected from PCV2 infected pigs and tested with this indirect ELISA kit and a similar commercial Korean ELISA kit. The results showed that the in-house ELISA kit was more sensitive than the commercial Korean kit. These serum samples were detected positive at dilution of 1∶12 800-1∶25 600 in the in-house ELISA kit and 1∶3200-1∶6400 in the commercial Korean kit. The in-house ELISA kit also showed high specificity as it did not pick up signals from serum samples of Porcine reproductive and respiratory syndrome virus(PRRSV), Classical swine fever virus(CSFV), Porcine Pseudorabies virus(PRV),Foot and mouth disease virus(FMDV) and other 10 pathogens. The coefficients of inter-and intra-assay variations were 2.26%-7.60%and 1.67%-6.41%, suggesting its good repeatability. These results indicated that this indirect ELISA kit developed here was suitable for detection of serum antibodies against PCV2.
In the present study, an indirect ELISA kit was developed and optimized for rapid detection of antibodies against Porcine circovirus type 2(PCV2). The serum samples were collected from PCV2 infected pigs and tested with this indirect ELISA kit and a similar commercial Korean ELISA kit. The results showed that the in-house ELISA kit was more sensitive than the commercial Korean kit. These serum samples were detected positive at dilution of 1∶12 800-1∶25 600 in the in-house ELISA kit and 1∶3200-1∶6400 in the commercial Korean kit. The in-house ELISA kit also showed high specificity as it did not pick up signals from serum samples of Porcine reproductive and respiratory syndrome virus(PRRSV), Classical swine fever virus(CSFV), Porcine Pseudorabies virus(PRV),Foot and mouth disease virus(FMDV) and other 10 pathogens. The coefficients of inter-and intra-assay variations were 2.26%-7.60%and 1.67%-6.41%, suggesting its good repeatability. These results indicated that this indirect ELISA kit developed here was suitable for detection of serum antibodies against PCV2.
引文
[1]Cai J,Xie X,Hu Y,et al.Generation of PCV2 in PK15cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome[J].Acta Vet Hung,2017,65(2):278-290.
[2]Resendes A R,Segalés J.Characterization of vascular lesions in pigs affected by porcine circovirus type2-systemic disease[J].Vet Pathol,2015,52(3):497-504.
[3]Becskei Z,Aleksi?-Kova?evi?S,Rusvai M,et al.Distribution of porcine circovirus 2 Cap antigen in the lymphoid tissue of pigs affected by postweaning multisystemic wasting syndrome[J].Acta Vet Hung,2010,58(4):483-498.
[4]Lekcharoensuk P,Morozov I,Paul P S,et al.Epitope mapping of the major capsid protein of type 2 porcine circovirus(PCV2)by using chimeric PCV1 and PCV2[J].J Virol,2004,78(15):8135-8145.
[5]丛丽媛,蒋智勇,张春红,等.猪圆环病毒2型抗体检测间接ELISA方法的建立[J].中国兽医学报,2008(8):892-896.
[6]高超,高云航,么乃全,等.猪圆环病毒2型ORF2基因原核表达蛋白间接ELISA检测方法的建立及应用[J].中国兽医学报,2008,28(8):888-891.
[7]马超英.4种猪瘟病毒抗体检测方法的比较[J].动物医学进展,2012,33(10):128-131.
[8]黄立,曲哲会,郭晓秋,等.豫南地区规模化猪场猪繁殖与呼吸综合征病毒和猪圆环病毒2型抗体检测与分析[J].中国畜牧兽医,2013,40(12):199-203.
[9]张琪,庞文静,付明哲,等.山羊痘病毒ORF103蛋白的原核表达及间接ELISA抗体检测方法的建立与应用[J].中国兽医科学,2017(5):537-543.
[10]曹增国,王化磊,盖微微,等.埃博拉病毒抗体间接ELISA检测方法的建立及应用[J].畜牧兽医学报,2016,47(3):615-619.
[2]Resendes A R,Segalés J.Characterization of vascular lesions in pigs affected by porcine circovirus type2-systemic disease[J].Vet Pathol,2015,52(3):497-504.
[3]Becskei Z,Aleksi?-Kova?evi?S,Rusvai M,et al.Distribution of porcine circovirus 2 Cap antigen in the lymphoid tissue of pigs affected by postweaning multisystemic wasting syndrome[J].Acta Vet Hung,2010,58(4):483-498.
[4]Lekcharoensuk P,Morozov I,Paul P S,et al.Epitope mapping of the major capsid protein of type 2 porcine circovirus(PCV2)by using chimeric PCV1 and PCV2[J].J Virol,2004,78(15):8135-8145.
[5]丛丽媛,蒋智勇,张春红,等.猪圆环病毒2型抗体检测间接ELISA方法的建立[J].中国兽医学报,2008(8):892-896.
[6]高超,高云航,么乃全,等.猪圆环病毒2型ORF2基因原核表达蛋白间接ELISA检测方法的建立及应用[J].中国兽医学报,2008,28(8):888-891.
[7]马超英.4种猪瘟病毒抗体检测方法的比较[J].动物医学进展,2012,33(10):128-131.
[8]黄立,曲哲会,郭晓秋,等.豫南地区规模化猪场猪繁殖与呼吸综合征病毒和猪圆环病毒2型抗体检测与分析[J].中国畜牧兽医,2013,40(12):199-203.
[9]张琪,庞文静,付明哲,等.山羊痘病毒ORF103蛋白的原核表达及间接ELISA抗体检测方法的建立与应用[J].中国兽医科学,2017(5):537-543.
[10]曹增国,王化磊,盖微微,等.埃博拉病毒抗体间接ELISA检测方法的建立及应用[J].畜牧兽医学报,2016,47(3):615-619.