PCV1-2m嵌合病毒TaqMan荧光定量PCR检测方法的建立及其初步应用

Establishment of PCV1-2m chimeric virus TaqMan FQ-PCR detection method and its preliminary application

为确定感染性克隆质粒免疫小鼠拯救获得的病毒量与诱导抗体之间的关系,以选择最佳免疫剂量,本研究建立了一种快速、灵敏的TaqMan荧光定量PCR检测方法。根据pcDNA3.1(+)-PCV1-2m-V5质粒的ORF2和V5标签序列设计特异性引物和探针,并将该质粒作为阳性标准品,优化荧光定量PCR条件后,按10倍倍比稀释,建立标准曲线,进行灵敏性、特异性和稳定性验证;而后,应用该方法对免疫不同质粒浓度的6组小鼠,分别在免疫后1~9周进行病毒血症检测;同时,应用ELISA方法分别对PCV2抗体滴度进行检测。结果表明,试验建立的荧光定量PCR方法具有良好的灵敏性、特异性和稳定性,检测范围可达1.29×10^1~1.29×10^9拷贝/μL;病毒血症可持续至第5周,抗体可维持至第8周。通过分析抗体滴度变化与病毒载量消长关系,确定使用200μg作为小鼠的最佳免疫剂量。本试验为感染性克隆质粒进一步在本体动物(猪)体内评估免疫效果奠定基础。

In order to determine the relationship between the amount of virus rescued by infectious clone plasmid-immunized mice and the induction antibody,and to select the optimal immunization dose,a fast and sensitive TaqMan fluorescent quantitative PCR(qPCR)detection method was established in this study.Specific primers and probes were designed based on the pcDNA3.1(+)-PCV1-2 m-V5 plasmid ORF2 and V5 tag sequences,and as a positive standard it was used to optimize the qPCR conditions.The sensitivity,specificity and reproducibility of qPCR were determined after the establishment of the standard curve.The method was then used to detect viremia in 6 groups of mice immunized with different plasmid concentrations at 1-9 weeks after immunization.At the same time,the titer of PCV2 antibody was detected by ELISA.The results showed that the qPCR method established in the experiment had good sensitivity,specificity and reproducibility with a detection range of 1.29×10^1-1.29×10^9 copies/μL.The optimal immune dose for mice was 200μg.The rescued virus lasted until the 5 th week in the blood,and the antibody was mantained to a high titration at the 8 th week post inoculation.This experiment can lay the foundation for the evaluation of the immune effect of infectious clone plasmid-immunized in proprioceptive animals(pigs).