【摘要】 试验建立了鉴别猪伪狂犬病病毒(PRV)强弱毒高效纳米PCR检测方法,并对相关条件进行优化,组装了试剂盒。根据PRV保守序列设计3对引物分别扩增PRV基因组的gB(431 bp)、gE(316 bp)和gG(202 bp)3个基因,用于区分PRV强毒与基因缺失毒株,优化反应条件后建立了纳米PCR检测PRV强弱毒的方法并组装试剂盒,对试剂盒进行了特异性、敏感性、批内批间可重复性及保存期评估试验,并对临床样品进行检测。特异性试验表明,此试剂盒对于PRV能够扩增出431(gB)、316(gE)和202 bp(gG)的目的片段,对于PRV-Bartha-K61能够扩增出431和202 bp的目的片段,对于猪捷申病毒、非洲猪瘟病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪细小病毒及大肠杆菌等DNA或cDNA均未扩增出条带。敏感性试验表明,此试剂盒的方法比常规PCR方法敏感100~1 000倍,最低核酸拷贝数检出量可以达到101 copy/μL数量级。试剂盒的批内、批间检测结果无明显差异,稳定性良好。-20℃至少可保存12个月。在对中国黑龙江、吉林等7个省市的临床送检的117份样品进行检测,结果显示,PRV强毒阳性率为51%,阴性率为49%,未发现有弱毒感染。鉴别PRV强弱毒纳米PCR试剂盒的研制,对PRV感染的早期检测、野毒株和疫苗株的鉴别、疾病控制等都有重要意义,而且也可以用于其他动物PRV感染的检测和早期诊断。更多还原

【Abstract】 In this study, a Nanoparticle-assisted Polymerase Chain Reaction(nanoPCR) kit was developed to detect and differentiate wild-type and gene-deleted pseudorabies strains(PRV). Three primers here were designed from conserved regions of PRV with amplicon of gB(431 bp) gE(316 bp) gG(202 bp) and used for differentiate wild-type and gene-deleted PRV, and a nanoPCR kit was then developed with the specificity, sensitivity,repeatability and retention period tested after this method was optimized, then it was tested by clinical samples.DNA or cDNA of PRV, PRV-Bartha-K61, porcine teschovirus(PTV), African swine fever virus(ASFV), porcine circovirus type 2(PCV2), porcine reproductive and respiratory syndrome virus(PRRSV), porcine parvovirus(PPV),and Escherichia Coli(E.coli) were detected by this assay, results showed that the amplicon with size of 431 bp,316 bp and 202 bp were amplified from PRV, 431 bp and 202 bp were amplified in the PRV-Bartha-K61. The sensitivity of the assay for this kit was 100~1 000 fold higher than conventional PCR. The intra- and interrepeatability test showed no significant difference, and no significant change was found after being conserved at-20 ℃ for 12 months. Total of the 117 clinical samples collected from 7 provinces of China showed that 51%(60 of 117) were positive for wild-type PRV, and 49%(57 of 117) were negative. In conclusion, the nanoPCR kit should be a useful method for early and rapid detection of PRV and meanwhile differentiation of swine vaccinated with PRV gE-deleted vaccine from swine infected with wild virus, also of other kind of animals infected with PRV.更多还原