小反刍兽疫病毒野毒与疫苗毒双重荧光RT-PCR鉴别检测方法的建立  

DEVELOPMENT OF DUAL FLUORESCENCE RT-PCR FOR DIFFERENTIATION OF WILD STRAIN AND VACCINE STRAIN OF PESTE DES PETITS RUMINANTS VIRUS

作　　者：何世成[1] 邹玖零 王卫国[1] 王昌建[1] 唐小明[1] 林源[1] 鲁杏华[1] 邱立新[1] 

机构地区：[1]湖南省动物疫病预防控制中心,长沙410014 [2]永州市动物疫病预防控制中心,永州425000

出　　处：《中国动物传染病学报》2016年第4期31-35,共5页Chinese Journal of Animal Infectious Diseases

摘　　要：对GenBank中的小反刍兽疫野毒(Peste des petits ruminants virus,PPRV)及疫苗毒株的基因序列进行分析,设计两对引物和两条特异探针,建立小反刍兽疫病毒野毒与疫苗毒株的双重荧光RT-PCR鉴别检测方法。特异性检测表明,建立的双重荧光RT-PCR方法,可特异检测野毒(FAM通道)和疫苗毒(VIC通道)。灵敏度实验表明,FAM通道可检测到10^(-5)稀释度的RNA(3.576 ng/mL),VIC通道可检测到10^(-3)稀释度的RNA(27.6 ng/mL)。重复性实验表明,该双重荧光RT-PCR方法重复性较好。用建立的二重荧光RT-PCR方法对20份组织样品进行检测,结果显示,该方法可以有效区分野毒和疫苗毒株,且野毒检测结果与实验室确诊阳性结果一致,可以用于临床检测。Two pairs of primers and two specific probes were designed based on the genome sequences of wild isolates and vaccine strain of Peste des petits ruminants virus（PPRV） published in GenBank.Subsequently,a dual fluorescence RT-PCR assay was developed for differentiation of PPRV wild isolates and vaccine strain.The specificity testing indicated that the dual fluorescence RT-PCR assay revealed difference between wild isolate（FAM） and vaccine strain（VIC）.The sensitivity test showed that its detection limits were 10-（-5） dilution degrees（3.576 ng/mL） RNA in FAM channel and 10-（-3） dilution degrees RNA（27.6 ng/mL） in VIC channel.Additionally,20 tissue samples were examined using the dual fluorescent RT-PCR assay.The positive samples were confirmed through other laboratory tests.The results demonstrated the reliability of the assay to differentiate vaccinated animals from field infected animals.

关 键 词：小反刍兽疫病毒 野毒 疫苗毒 荧光RT-PCR 鉴别 

分 类 号：S852.659.5[农业科学—基础兽医学]