猪圆环病毒3型TaqMan荧光定量PCR方法的建立和初步应用

摘要

为了灵敏而便捷的检测猪圆环病毒3型(PCV3),根据PCV3 ORF2基因组保守序列设计合成4对引物,先使用荧光染料(SYBR)定量PCR法验证其能否有效扩增目的片段,并根据结果和引物对所扩增序列的交集进一步设计TaqMan探针,通过优化反应条件和反应体系建立快速检测PCV3的实时荧光定量PCR检测方法.结果显示,SYBR染料法和 TaqMan探针法均能有效地扩增 PCV3标准质粒以及其他阳性样品,并且对1.29×102～1.29×109拷贝·μL －1的PCV3标准质粒(Pmd18-t-Cap)呈现良好的线性关系,该方法的检测灵敏度为1.29×102拷贝·μL －1,比常规PCR的灵敏度高100倍,该方法与猪圆环病毒2型、猪轮状病毒、猪繁殖与呼吸综合征病毒等病毒基因以及猪链球菌、猪肺炎支原体等细菌基因均无交叉反应,具有良好的特异性;批次内重复和批次间重复试验结果显示变异系数(CV)均小于1.5％,呈良好的可重复性.用该方法对2016年至2017年间收集的124份来自湖北省等地的猪病料DNA样品进行检测,结果显示本地区PCV3阳性率为8.06％(10/124),PCV2和PCV3共感染率为8.06％(10/124).上述结果表明,本研究建立TaqMan荧光定量PCR能够灵敏特异的检测圆环病毒3型,为圆环病毒3型的临床检测提供有效手段.%To detect the Porcine Circovirus type 3(PCV3)sensitively,rapidly and specifically,four pairs of primers were designed targeting the conserved region of PCV 3 ORF3 gene.SYBR-based quantitative PCR results showed that Real-time PCR with all primer pairs can detect viral genome efficaciously.Then a TaqMan probe were designed according to the PCR products inter-section sequence,and a real-time quantitative PCR method was established by optimizing the reaction conditions and systems.The method presented a good linear relation with the Pmd18-t-Cap vector(Recombinant plasmid harboring PCV3 ORF2 gene)ranging from 1.29×102-1.29× 109copies· μL －1.The assay was highly specific for PCV3,without cross-reaction with other porcine virus and bacteria,such as Porcine circovirus 2(PCV2),Porcine reproductive and respira-tory syndrome virus(PRRSV),Porcine rotavirus(PoRV),Haemophilus parasuis(Hps),Acti-nobacillus pleuropneumoniae(App).And the limit detection of this assay was 1.29×102copies·μL－1,more sensitive than the conventional PCR.The CV(Coefficient of Variation)of intra-assay and inter-assay were less than 2％,showed that the assay was reproducible.The real-time qPCR was further used to detect the DNA sample extracted from clinical sample collected from 2016 to 2017,the PCV3 positive ratio was 8.06％(10/124),and the co-infection rate of PCV3/PCV2 was 8.06％(10/124).The real-time qPCR assay provides a high sensitivity and specificity,repeatability method for detection of PCV3.