PCV2和PCV3双重纳米PCR方法的建立及初步应用

摘要

本研究旨在建立一种同时检测猪圆环病毒2型(PCV2)和猪圆环病毒3型(PCV3)的双重纳米PCR(nanodPCR)方法,同时应用该方法对PCV2和PCV3进行检测.参考GenBank中登录的PCV2和PCV3型基因序列分别设计特异性引物,对nano-dPCR的反应条件进行优化;同时考察所建立的nano-dPCR检测方法的特异性和敏感性.结果显示,所建方法的特异性和敏感性良好,对PCV2和PCV3两种病毒的最低核酸检测量分别为93.2和91.6拷贝/μL,其敏感性比普通PCR高100倍.应用该方法对送检的265份临床样本进行检测,结果显示,PCV2和PCV3在猪群中存在普遍感染,PCV3和PCV2的阳性率分别为16.6％和14.7％,混合感染阳性率为6.8％.临床样品的检测结果表明,本试验建立的nano-dPCR方法为PCV2和PCV3早期感染进行快速、敏感鉴别诊断提供了新方法.%This study was aimed to establish a duplex nanoparticle-assisted PCR (nano-dPCR) method for the simultaneous detection of porcine circovirus type 2 (PCV2) and PCV3,and to apply this method in the detection of PCV2 and PCV3.Primers specific to PCV2 and PCV3 were designed by reference to the respective gene sequences in GenBank.The reaction conditions of nano-dPCR were also optimized.An evaluation of the specificity and sensitivity of the established nano-dPCR protocol proved the method to be both specific and sensitive,with the lower detection limit for the amount of nucleic acid in PCV2 and PCV3 to be 93.2 and 91.6 copies/μL,respectively.This sensitivity was 100 times higher than that of conventional PCR.The method was applied to inspect 265 clinical samples sent for testing,and the results showed that PCV2 and PCV3 infection in pig was rather common,with 16.6％ positive for PCV3,14.7％ positive for PCV2,and 6.8％ for mixed infection.The detection results on clinical samples supported the newly established nano-dPCR method as a rapid and sensitive differential diagnosis for the early infection of PCV2 and PCV3.