旨在建立检测猪圆环病毒3型(Porcine circovirus type 3,PCV3)血清抗体的间接ELISA方法以及摸清广西地区PCV3的流行情况.试验以PCV3的核衣壳蛋白多肽作为包被抗原,通过对包被抗原的最佳工作浓度、血清稀释度、封闭剂、酶标抗体工作浓度及作用时间、底物反应时间,阴性和阳性判断标准,敏感性、特异性及重复性等进行研究,建立了检测PCV3间接ELISA方法.随后,应用建立的方法检测2021-2023年广西地区采集的912份猪血清中PCV3抗体存在的情况.抗原最佳包被量为1.25 μg/孔、血清稀释度为1:800、封闭剂为5％的脱脂奶粉、酶标抗体工作浓度为1:10 000、二抗作用时间为60 min、底物反应时间为15 min.阴性和阳性判断标准OD450≥0.55,为阳性;OD450 ≤0.48,为阴性;当0.48＜OD450＜0.55时,为疑似阳性,需对疑似样品再次检测.该方法与猪圆环病毒2型(PCV2)、猪瘟(CSFV)、猪繁殖与呼吸综合征(PRRSV)和伪狂犬病(PRV)等病毒抗体无交叉反应.批内与批间重复性试验的变异系数分别低于5％与8％,说明基于PCV3核衣壳蛋白多肽建立的间接ELISA方法具有良好的敏感性、特异性及重复性,检测方法稳定可靠.利用所建立的方法进行血清学调查时发现,广西地区PCV3抗体阳性率达到36.62％,说明广西地区猪群中PCV3的感染率较高,应引起重视.
Establishment and Application of Indirect ELISA Method for the Detection of Porcine Circovirus Type 3 Based on Capsid Protein Polypeptide
The purpose of this trial was to establish an indirect ELISA for the detection of antibodies to Porcine circovirus type 3(PCV3)and to determine the epidemic situation of PCV3 in Guangxi.The experiment studied the method by using the Capsid Protein polypeptide of PCV3 to determine the optimal antigen and serum dilution,sealant,working concentration of ELISA antibody,reaction time of the secondary antibody,reaction time of the substrate,negative and positive criteria,sensitivity,specificity and repeatability of the indirect ELISA.Additionally,912 clinical porcine sera collected from 2021 to 2023 in Guangxi were detected using the established indirect ELISA method.The antigen coating concentration was 1.25 µg/well,the serum dilution was 1:800,the sealant was 5％skim milk powder,the working concentration of the ELISA antibody was 1:10 000,the reaction time of the secondary antibody was 60 min and the reaction time of the substrate was 15 min.The negative and positive criteria were as follows:the value of OD450 ≥ 0.55 indicated a positive result,a value of OD450 ≤ 0.48 indicated a negative result,and a value of 0.48＜OD450＜0.55 indicated a suspected positive result.Suspected samples should be tested again.The ELISA method did not show any cross-reaction with antibodies to PCV2,CSFV,PRRSV,PRV,etc.The coefficient of variation in intra-batch and inter-batch tests was lower than 5％and 8％respectively,which demonstrated that the results in the experiment indicated that the established indirect ELISA method based on the Capsid Protein polypeptide of PCV3 had good sensitivity,specificity,repeatability and reliability.The positive rates of PCV3 were high in pigs from Guangxi.The positive rate of PCV3 antibodies detected in Guangxi was 36.62％using this method,indicating a very high infection rate of PCV3 in pigs in the Guangxi region.Therefore,more attention should be paid to it.
Porcine circovirus virus type 3Capsid ProteinPolypeptidesIndirect ELISA
万方数据
维普
