猪德尔塔冠状病毒RT-nPCR检测方法的建立与N基因变异分析  

Development of RT-nPCR Detection Method for Porcine Deltacoronavirus and Analysis of N Gene Variation

作　　者：朱小甫[1] 吴旭锦[1] ZHU Xiaofu;WU Xujin(Key Laboratory of Animal Epidemic Disease Diagnostic Laboratory of Molecular Biology in Xianyang City,Xianyang Vocational Technical College,Xianyang 712000,China)

机构地区：[1]咸阳职业技术学院,咸阳市动物疫病分子生物学诊断技术研究重点实验室,咸阳712000

出　　处：《中国动物传染病学报》2023年第6期72-77,共6页Chinese Journal of Animal Infectious Diseases

基　　金：陕西省科技计划项目(2021NY-037);咸阳职业技术学院重点科研项目(2021KJB01)。

摘　　要：建立一种基于猪德尔塔冠状病毒(PDCo V)N基因的RT-n PCR检测方法,对陕西省猪群PDCo V感染情况进行流行病学调查,分析PDCo V流行毒株N基因变异情况。通过病毒分离、检测引物设计、反应体系与条件优化、灵敏性试验和特异性试验,建立了PDCo V RT-nPCR检测方法,并对N基因片段进行序列分析。结果表明:建立的RT-n PCR方法能够扩增出303 bp的N基因目的片段,检测单链c DNA的极限为2.0194×10^(-3)ng·μL^(-1),该方法检测PEDV、TGEV、PRo V、CSFV与PRRSV均无交叉反应,特异性良好;陕西省猪场184份样品检测结果显示,PDCo V阳性率为6.52%(12/184);N基因序列分析发现,获得的HY2019、QLS2020的N基因片段同源性为99.0%,提示可能为同一来源;进化树分析发现,HY2019、QLS2020与我国四川株SCNC201705、Sichuan2017、Sichuan2019同处于一个小的进化分支上,且均存在168~179位“TGTCTGTCTCTA”共12个核苷酸缺失,推测陕西株与四川株高度同源,可能起源于早期四川毒株。The objectives of the present study were to develop a RT-nPCR detection method based on the N gene of porcine deltacoronavirus(PDCoV)for epidemiological investigation in pigs in Shaanxi province and analyze the Ngene mutation.A PDCoV RT-nPCR detection method was developed through virus isolation,primer design,optimization of reaction conditions.The results showed that the RT-nPCR method developed here amplified 303 bp of N gene target fragment and the detection limit of single-stranded cDNA was 2.0194×10^(-3) ng/µL.This RT-nPCR method had no reaction to PEDV,TGEV,PRoV,CSFV and PRRSV.The test results of 184 samples from pig farms in Shaanxi province showed that the PDCoV positive rate was 6.52%(12/184).Ngene sequence analysis found that the amplified N gene fragments of HY2019 and QLS2020 had a homology of 99.0%,suggesting that they might be from the same source.Phylogenetic tree analysis found that HY2019 and QLS2020 were located in the same small evolutionary branch with the Chinese Sichuan strains SCNC201705,Sichuan2017 and Sichuan2019.In addition,there were 12 nucleotide deletions found in positions 168-179 of"TGTCTGTCTCTA".These results suggested that the Shaanxi strains shared high homology with Sichuan strains and might originate from Sichuan area.

关 键 词：猪德尔塔冠状病毒 仔猪腹泻 检测 反转录-聚合酶链式反应 

分 类 号：S852.65[农业科学—基础兽医学]