摘要
猪流行性腹泻病毒(PEDV)是引发猪群腹泻的主要病原之一,给我国生猪养殖业造成了重大的经济损失。为建立IgA-ELISA抗体检测方法,本研究采用变异PEDV真核表达的S-N融合蛋白作为包被抗原,通过系列反应条件优化,建立了PEDV的IgA-ELISA抗体检测方法。该方法的最佳反应条件为包被抗原质量浓度2 mg/L,用5%BSA于37℃封闭2 h;将待检血清1∶50稀释于37℃作用1 h,加1∶20000稀释酶标二抗于37℃作用30 min,TMB显色10 min;以D_(450 nm)≥0.326判定为阳性,当D_(450 nm)≤0.277判定为阴性,介于两者判定为可疑。检测猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪传染性胃肠炎病毒(TGEV)、轮状病毒(PoRV)标准阳性血清抗体均为阴性,且重复性变异系数均低于10%。通过对我国福建地区304份样品(血清179份和乳汁125份)进行检测,并与商品试剂盒进行参照对比,总符合率达到93.2%以上,表明该试剂盒检测方法具有较高的特异性、敏感性和重复性,可用于对PEDV的IgA抗体检测。
Porcine epidemic diarrhea virus(PEDV)is one of the main pathogens that cause viral diarrhea in pigs,and has caused significant economic losses to the pig breeding industry in China.In order to establish a PEDV IgA-ELISA antibody detection method,the S-N fusion protein expressed by the variant PEDV was used as the coating antigen,and the reaction conditions were optimized.The optimal reaction conditions of this method were:the concentration of the coating antigen was 2 mg/L,blocking with 5% BSA at 37℃ for 2 h,diluting the serum to be tested 1∶50 at 37℃ for 1 h,incubation with 1∶20000 dilution enzyme labeled secondary antibody at 37℃ for 30 min;the best color development time for TMB was 10 min;D_(450 nm)≥0.326 was judged as positive,when D_(450 nm)≤0.277 was judged as negative,and between the two,it was judged as suspicious.The detection of CSFV,PRRSV,PRV,PoRV,TGEV standard positive serum antibodies were all negative,and the repeatability coefficient of variation was less than 10%.By testing 304 samples(179 serum samples and 125 milk samples)in Fujian,and comparing with the commercial kit,the results showed that the total coincidence rate was more than 93.2%.In conclusion,the PEDV IgA-ELISA antibody detection method established in this study has high specificity,sensitivity and reproducibility,and can be used for clinical IgA antibody detection against PEDV.
作者
王隆柏
陈秋勇
曾宪煜
吴学敏
车勇良
陈如敬
周伦江
WANG Longbai;CHEN Qiuyong;ZENG Xianyu;WU Xuemin;CHE Yongliang;CHEN Rujing;ZHOU Lunjiang(Fujian Research Center of Animal Disease Control Engineering Technology,Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agriculture Sciences,Fuzhou 350013,China;Changting Animal Husbandry Technology Ertension Station,Changting,Fujian 366300,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2023年第2期234-239,共6页
Chinese Journal of Veterinary Science
基金
公益类科研院所专项资助项目(2021R10260016)
福建省重大专项专题资助项目(2021NZ029023)
5511协同创新工程资助项目(XTCXGC2021008)
福建省星火科技计划资助项目(2020S0002)。
