猪博卡病毒1型nPCR检测方法的建立与 SXWN20株基因序列分析  

Construction of An nPCR Detection Method for Porcine Boca Virus Type 1 and Gene Sequence Analysis of SXWN20 Strain

作　　者：朱小甫[1] 吴旭锦[1] 郑红青 尹宝英[1] 熊忙利[1] 张文娟[1] 张兆顺[1] ZHU Xiaofu;WU Xujin;ZHENG Hongqing;YIN Baoying;XIONG Mangli;ZHANG Wenjuan;ZHANG Zhaoshun(Xianyang Key Laboratory of Animal Epidemic Disease Diagnostic Laboratory of Molecular Biology,Xianyang Vocational and Technical College,Xianyang,Shaanxi 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,陕西咸阳712000

出　　处：《陕西农业科学》2022年第11期82-86,共5页Shaanxi Journal of Agricultural Sciences

基　　金：陕西省科技计划项目(2021NY-037);咸阳职业技术学院科研基金资助项目(2021KJB01);陕西省自然科学基金项目(2021JQ-900);咸阳市科技计划项目(2020k02-63)。

摘　　要：建立了检测猪博卡病毒1型(PBoV)的nPCR方法,对陕西省部分猪群PBoV1型感染情况进行流行病学调查。通过优化反应体系与条件,测试了方法的灵敏性和特异性,建立了PBoV1型nPCR检测方法,并对阳性样品进行测序分析。结果表明,建立nPCR方法检测PBoV1型单链DNA的极限为4.742×10^(-2) ng/μL,该方法与PCV2、PCV3、PPV、猪链球菌和猪大肠杆菌DNA均无交叉反应,特异性良好。检测了陕西省猪场60份样品,PBoV1型阳性率为3.33%(2/60)。测序获得的SXWN20株VP1/VP2基因序列比对发现,SXWN20株与参比的PBoV1型毒株核苷酸同源性为96.6%~97.5%,与PBoV2/3/4/5型同源性仅为53.8%~59.7%。进化树分析显示,所有参比毒株分为3个大分支,SXWN20株与PBoV1型毒株处在一个进化分支,PBoV2型处在一个进化分支,PBoV3/4/5型处在一个进化分支。A nPCR method for detecting porcine Boca virus type 1 has been constructed,and an epidemiological investigation of PBoV type 1 infection in some pig herds in Shaanxi has been conducted.Through optimization of the nPCR system and conditions,the sensitivity and specificity of the method were tested,the PBoV1 type nPCR detection method was constructed,and the positive samples were sequenced and analyzed.The results showed that the limit of the established nPCR method for detection of PBoV1 single-stranded DNA was 4.742×10^(-2) ng/μL.This method had no cross-reactivity with PCV2,PCV3,PPV,Streptococcus suis and pig Escherichia coli DNA.60 samples from pig farms in Shaanxi Province were tested,and the positive rate of PBoV1 was 3.33%(2/60).The sequence alignment of the VP1/VP2 gene of the SXWN20 strain obtained by sequencing revealed that the nucleotide homology between the SXWN20 strain and the reference PBoV1 strain was 96.6%~97.5%,and the homology was 53.8%~59.7%with PBoV2/3/4/5.Phylogenetic tree analysis showed that all reference strains were divided into three major branches.SXWN20 strain and PBoV1 strain belonged to one evolutionary branch,PBoV2 strain belonged to one evolutionary branch,and PBoV3/4/5 strain belonged to one evolutionary branch.

关 键 词：猪博卡病毒 核酸检测 序列分析 聚合酶链式反应 

分 类 号：S852.65[农业科学—基础兽医学]