摘要
为快速准确地监测1型猪繁殖与呼吸综合征病毒(PRRSV-1)在我国的流行情况,本研究以分子筛纯化灭活的PRRSV-1(ZD-1株)为包被抗原,利用PRRSV-1的特异性单克隆抗体(MAb EU-1)为竞争抗体,经条件优化,建立了检测PRRSV-1的竞争ELISA(EU-C-ELISA)方法。反应条件优化结果显示,最佳抗原包被量为455 ng/孔,PRRSV-1 MAb EU-1的最佳稀释度为1:8。EU-C-ELISA结果判定标准:血清样品抑制率PI≥45%时判定为阳性,PI<45%判定为阴性。特异性试验结果显示,该方法仅能检测PRRSV-1阳性血清,与各谱系PRRSV-2及其他常见猪病毒和细菌阳性血清均无交叉反应;敏感性试验结果显示,该方法能检测出16倍稀释的阳性标准血清,并且最早可在PRRSV ZD-1株感染猪后第7 d检测到血清中PRRSV-1抗体阳转;批内和批间重复性试验的变异系数均小于5%,对比试验结果显示该方法特异性和敏感性均显著优于现有试剂盒对PRRSV-1抗体的检测。利用该方法对2020年采自黑龙江、辽宁、河北、山东省10个猪场的432份临床血清样品进行检测,2个猪场检出PRRSV-1抗体阳性,猪场阳性率达20%,样品总阳性率为15.0%,显示PRRSV-1抗体已存在于我国部分地区猪群中。本研究在我国首次建立了PRRSV-1竞争ELISA抗体检测方法,为监测PRRS-1在我国的流行情况及科学防控该病提供了检测手段。
In order to rapidly and accurately monitor the prevalence of porcine reproductive and respiratory syndrome virus type 1(PRRSV-1)in China,in this study,the purified PRRSV-1(ZD-1 strain)was used as the coating antigen,and the specific monoclonal antibody(EU-1)against PRRSV-1 was used as the competitive antibody.After optimization of the conditions,a specific competitive ELISA(EU-C-ELISA)method was established for the detection of PRRSV-1.The results showed that the optimal antigen coating concentration is 455ng/well,and the optimal dilution of the competitive antibody was 1:8.When percentage inhibition(PI)of the serum sample is more than 45%,it is considered to be positive,otherwise,it is negative.The specificity test showed that this method can only detect antibody of PRRSV-1 strain,and there was no cross reaction with positive sera of PRRSV-2strains,other porcine viruses and bacteria.Besides,the sensitivity test showed that a 16-fold dilution of positive serum can be detected,and PRRSV-1 antibody could be detected as early as 7 days after challenge.The coefficients of variation(CV)of the intra and inter-assay repeated tests were less than 5%.The comparison test showed that the specificity and sensitivity of this method performs better than the existing kits for detection of PRRSV-1 antibodies.Furthermore,432 clinical serum samples that collected from 10 pig farms in Heilongjiang,Liaoning,Hebei and Shandong provinces in 2020 were detected by this method,and PRRSV-1antibody was detected in two of the pig farms.The positive rate of pig farm was 20%,and the positive rate of serum sample was15%,indicating that PRRSV-1 has a certain prevalence in some areas of China.This study established a specific ELISA antibody detection method against PRRSV-1 for the first time in China,which provides a method for monitoring the prevalence of PRRSV-1in China and scientifically preventing and controlling the disease.
作者
许浒
相丽润
张文立
汤艳东
赵静
李超
龚邦俊
李宛生
付军
彭金美
王倩
周国辉
冷超粮
安同庆
蔡雪辉
张洪亮
田志军
XU Hu;XIANG Li-run;ZHANG Wen-li;TANG Yan-dong;ZHAO Jing;LI chao;GONG Bang-jun;LI Wan-sheng;FU jun;PENG Jin-mei;WANG Qian;ZHOU Guo-hui;LENG Chao-liang;AN Tong-qing;CAI Xue-hui;ZHANG Hong-liang;TIAN Zhi-jun(National Key Swine Disease Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Nanyang Normal University,Nanyang 473061,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第6期617-623,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(32002315)
中国博士后科学基金(2020M680788)。
关键词
1型猪繁殖与呼吸综合征病毒
竞争ELISA
流行病学调查
porcine reproductive and respiratory syndrome virus type I
competitive ELISA
epidemiological investigation
