猪瘟病毒血清抗体间接ELISA检测方法的建立
发布时间：2018-12-15 13:25
【摘要】：为建立快速、准确的猪瘟病毒(CSFV)血清抗体检测方法,根据Gen Bank上猪瘟兔化弱毒全基因序列,设计1对引物,扩增E2蛋白主要抗原区基因片段;然后,将其克隆入载体p ET-28a(+),采用大肠杆菌Rosetta(DE3)表达出含E2蛋白主要抗原区的重组蛋白。以纯化的截短E2蛋白为包被抗原,优化间接ELISA反应条件,建立了检测CSFV血清抗体的间接ELISA。经过优化,确定最佳抗原包被浓度为8 g/m L,待检血清稀释倍数为1∶160,酶标二抗稀释倍数为1∶2000。该方法的阳性临界值为0.37,阴性临界值为0.32。批内变异系数在1.58%~3.33%之间,批间变异系数在2.67%~5.35%之间,说明该方法重复性良好。特异性检验中,该方法与猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪流行性腹泻病毒、猪流感病毒、传染性胃肠炎病毒、乙型脑炎病毒、伪狂犬病病毒、猪细小病毒阳性血清均没有交叉反应。用该方法与IDEXX CSFV抗体检测试剂盒对1 101份临床猪血清样品进行符合性检测,结果,该方法的敏感性、特异性、符合率分别为89.07%(603/677)、77.59%(329/424)和84.65%(932/1101)。上述结果表明,该方法可用于临床CSFV血清抗体的检测,为CSFV血清抗体检测试剂盒的研制奠定了基础。
[Abstract]:In order to establish a rapid and accurate method for detection of (CSFV) serum antibody of CSFV, a pair of primers were designed according to the whole gene sequence of Gen Bank to amplify the main antigen region of E2 protein. Then, the recombinant protein containing the main antigen region of E2 protein was expressed by Escherichia coli Rosetta (DE3). Using the purified truncated E2 protein as the coating antigen and optimizing the reaction conditions of indirect ELISA, an indirect ELISA. for the detection of serum antibodies of CSFV was established. After optimization, the best antigen coating concentration was 8 g / m L, the dilution multiple of serum to be tested was 1: 160, and the dilution multiple of enzyme labeled second antibody was 1: 2000. The positive critical value and negative critical value of this method are 0. 37 and 0. 32 respectively. The coefficient of variation is between 1.58% and 3.33%, and the coefficient of variation between batches is between 2.67% and 5.35%, which indicates that the method has good reproducibility. Specific tests were performed with porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine epidemic diarrhea virus, swine influenza virus, infectious gastroenteritis virus, Japanese encephalitis virus, pseudorabies virus. There was no cross-reaction in serum of porcine parvovirus positive. The method and IDEXX CSFV antibody kit were used to detect the conformance of 1 101 clinical pig serum samples. The results showed that the sensitivity, specificity and coincidence rate of this method were 89.07% (603 / 677), respectively. 77.59% (329 / 424) and 84.65% (932 / 1101). These results indicate that this method can be used for the detection of serum antibodies in clinical CSFV, which lays a foundation for the development of a kit for detection of serum antibodies of CSFV.
【作者单位】： 南京农业大学动物医学院;江苏高校动物重要疫病与人兽共患病防控协同创新中心;
【基金】：江苏省农业科技自主创新资金项目(CX(15)1056;CX(16)1028)
【分类号】：S852.651
[Abstract]:In order to establish a rapid and accurate method for detection of (CSFV) serum antibody of CSFV, a pair of primers were designed according to the whole gene sequence of Gen Bank to amplify the main antigen region of E2 protein. Then, the recombinant protein containing the main antigen region of E2 protein was expressed by Escherichia coli Rosetta (DE3). Using the purified truncated E2 protein as the coating antigen and optimizing the reaction conditions of indirect ELISA, an indirect ELISA. for the detection of serum antibodies of CSFV was established. After optimization, the best antigen coating concentration was 8 g / m L, the dilution multiple of serum to be tested was 1: 160, and the dilution multiple of enzyme labeled second antibody was 1: 2000. The positive critical value and negative critical value of this method are 0. 37 and 0. 32 respectively. The coefficient of variation is between 1.58% and 3.33%, and the coefficient of variation between batches is between 2.67% and 5.35%, which indicates that the method has good reproducibility. Specific tests were performed with porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine epidemic diarrhea virus, swine influenza virus, infectious gastroenteritis virus, Japanese encephalitis virus, pseudorabies virus. There was no cross-reaction in serum of porcine parvovirus positive. The method and IDEXX CSFV antibody kit were used to detect the conformance of 1 101 clinical pig serum samples. The results showed that the sensitivity, specificity and coincidence rate of this method were 89.07% (603 / 677), respectively. 77.59% (329 / 424) and 84.65% (932 / 1101). These results indicate that this method can be used for the detection of serum antibodies in clinical CSFV, which lays a foundation for the development of a kit for detection of serum antibodies of CSFV.
【作者单位】： 南京农业大学动物医学院;江苏高校动物重要疫病与人兽共患病防控协同创新中心;
【基金】：江苏省农业科技自主创新资金项目(CX(15)1056;CX(16)1028)
【分类号】：S852.651
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