彩色免疫金银染色法检测猪繁殖与呼吸综合征病毒的研究

Studies on Porcine Reproductive and Respiratory Syndrome Virus by Colour Immune-gold Silver Staining Assay

试验首次建立了固相载体上的彩色免疫金银染色法(CIGSS)检测猪繁殖与呼吸综合征病毒(PRRSV),并获得成功。用提纯的PRRSV高免兔制备高免血清,按常规方法提取兔抗PRRSVIgG,建立了检测PRRSV抗原的彩色免疫金银染色法。经方阵试验确定最佳兔抗PRRSV IgG工作浓度为1∶400,金标羊抗兔IgG的工作浓度为1∶1 000,银染色的时间为15 min,彩染的时间为2 min。用该法对提纯的PRRSV病毒抗原的最低检出量为1.469μg/ml,其敏感性为DIGFA的5倍。以CIGSS法检测了52份疑似PRRSV感染的猪样本,阳性率为53.84%,而检测猪细小病毒、猪瘟病毒、猪伪狂犬病毒、猪巴氏杆菌以及正常的Marc-145细胞培养物,均为阴性反应。表明该法具有快速、准确、操作简便、敏感性高、易于判断、特异性强、重复性好等优点,可用于大量PRRSV抗原样本的检测。

A color immune-gold silver staining （CIGSS） assay was developed for the detection of porcine reproductive and respiratory syndrome virus （PRRS） antigen the first time. The optimum working concentration of rabbit anti-PRRSV immunoglobulin G （IgG） was determined to be 1 ： 400, that of sheep anti-sheep anti-rabbit IgG labeled with 5 nm colloidal gold to be 1 ： 1 000 and the optimum time of silver staining to be 15 minutes, the optimum of color staining to be 2 minutes. The minimum amount of purified PRRSV antigen detected by the CIGSS was 1. 469 μg/ml,with the sensitivity of 5-fold greater than that of DIGFA. The high specify of the CIGSS was shown by the specific blocking test of rabbit anti-PRRSV serum and cross-reaction test with porcine parvovirus （PPV）, swine fever virus （SFV）, pasteurella multocida, pseudorabies virus （PRV） and normal cell culture. The study indicates that the CIGSS for the detection of PRRSV antigen is economical, facile, rapid, highly sensitive, highly specific and reproductive. It is an efficient mean for rapid diagnosis and epidemiological investigation of PRRSV infection.