摘要
为了能够同时开展对小反刍兽疫病毒(PPRV)、蓝舌病血清8型病毒(BTV8)、鹿流行性出血热血清1型病毒(EHDV1)和非洲马瘟病毒(AHSV)4种外来动物疫病病原体的核酸检测,本试验根据GenBank中相关病毒序列设计引物和探针,建立多重普通逆转录PCR(RT-PCR)和实时荧光定量RT-PCR检测方法,并在采集的临床样本检测中进行初步应用验证。结果显示:建立的PPRV/BTV8/EHDV1三重实时荧光定量RT-PCR检测方法仅对PPRV、BTV8和EHDV1有特异荧光信号,检测敏感度可达10^(1.70)~10^(2.08) copies/μL DNA;建立的PPRV/BTV8/EHDV1/AHSV四重普通RT-PCR方法可特异性同步检测PPRV、BTV8、EHDV1和AHSV,检测敏感度可达10^(3) copies/μL DNA;2种方法对羊痘、羊口疮、口蹄疫、阿卡斑、牛病毒性腹泻等临床相似的病毒均无扩增。在925份临床样本中检测出1例PPRV核酸阳性样本;经核苷酸序列测定及BLAST比对确定为谱系Ⅳ型PPRV毒株序列。本试验所建立的三重实时荧光定量RT-PCR检测方法和四重普通RT-PCR方法可特异性同步检测相应的3种或4种病毒病原,且具有临床样本检测的应用潜力。
For simultaneous detection of exotic peste des petits ruminants virus(PPRV),bluetongue virus serotype 8(BTV8),epizootic haemorrhagic disease virus serotype 1 and African horse sickness virus(AHSV),this study designed primers and probes according to related virus sequences from GenBank,and applied in multiplex routine reverse transcription-polymerase chain reaction(RT-PCR)and real-time fluorescent quantitative RT-PCR for their detection in clinical samples.The results showed that the PPRV/BTV8/EHDV1 triplex real-time fluorescent quantitative RT-PCR specifically recognized PPRV/BTV8/EHDV1,with the limit of detection at 10^(1.70)-10^(2.08) copies/μL DNA;the PPRV/BTV8/EHDV1/AHSV quadruplex routine RT-PCR specifically recognized PPRV/BTV8/EHDV1/AHSV simultaneously and with the limit of detection at 10^(3) copies/μL DNA;neither RT-PCR reacted with viruses causing similar clinical signs including sheep pox virus,orf virus,foot and mouth disease virus(FMDV),Akabane virus and bovine viral diarrhea virus(BVDV).Among 925 clinical samples,one was detected as PPRV by the RT-PCR and confirmed to be PPRV lineage IV by nucleotide sequence and BLAST comparison.Thus,the triplex real-time fluorescent quantitative RT-PCR and quadruplex routine RT-PCR allowed for simultaneous detection of three or four exotic viral pathogens in clinical specimens.
作者
赵文华
李富祥
杨仕标
ZHAO Wen-hua;LI Fu-xiang;YANG Shi-biao(Yunnan Animal Science and Veterinary Institute,Yunnan Tropical and Subtropical Animal Viral Disease Laboratory,Kunming 650224,China)
出处
《中国兽医杂志》
CAS
北大核心
2022年第2期27-33,38,共8页
Chinese Journal of Veterinary Medicine
基金
国家重点研发项目(2017YFD0501805,2017YFD0501801)
国家自然科学基金项目(32060809)。
关键词
小反刍兽疫病毒
蓝舌病病毒8型
流行性出血热病毒1型
非洲马瘟病毒
四重普通RT-PCR
三重实时荧光定量RT-PCR
peste des petits ruminants virus(PPRV)
bluetongue virus serotype 8(BTV8)
epizootic haemorrhagic disease virus serotype 1(EHDV1)
African horse sickness virus(AHSV)
quadruplex routine RT-PCR
triplex fluorescent quantitative real-time RT-PCR
