猪瘟病毒荧光定量PCR的建立及对病毒体外复制动态的研究

Proliferation dynamic of Classical swine fever virus in vitro using a newly developed fluorescent quantitative PCR assay

为了了解猪瘟病毒(CSFV)在PK-15细胞上的增殖规律。根据GenBank中CSFV Shimen毒株的NS5A基因保守区域序列,设计1对特异性引物,以CSFV总RNA为反转录模板,经优化反应条件,建立了基于实时荧光定量PCR技术的CSFV检测方法,并对其进行了敏感性、特异性、重复性试验,结果显示,该方法重复性好、特异性强,最低检测模板浓度为10拷贝/μL,并且CSFV的最低检测限为1 TCID_(50)/mL。利用所建立的方法研究CSFV在PK-15细胞上清中的增殖动态,并与TCID_(50)方法绘制的复制动态曲线进行比较。结果显示,两种方法测定的CSFV在PK-15细胞上的复制动态具有一定的平行关系,该方法为研究猪瘟病毒的致病机理及利用体外细胞培养毒生产疫苗提供了试验依据。

Classical Swine Fever Virus(CSFV)can grow in the cell line PK-15,but the characterization of proliferation is still unknown.A Real-time quantitative PCR assay for detection of CSFV was developed using the specific primer designed according to the NS5A gene of CSFV.The Real-time quantitative PCR assay was established using the total RNA of CSFV as template.The method was specific and had good reliability for the detection of CSFV.The sensitivity of the assay was 101 copies/μL nucleic acid and 1TCID50 virus.This method was developed for the detection of the replication disciplinarian of CSFV in the PK-15 cell supernatant,which was compared to TCID50 assay.The results showed that the one-step growth curves of CSFV in PK-15 cells were parallel by the developed assay and TCID50 detection.