小反刍兽疫疫苗毒与野毒实时荧光RT-PCR鉴别检测方法的建立

Development of a real-time fluorescence RT-PCR assay to differentiate vaccine strain from isolate strain of Peste des petits ruminants virus

为了建立鉴别检测小反刍兽疫疫苗毒与野毒的快速分子生物学检测方法,通过对自行测序的疫苗毒基因组序列及GenBank中登录的野毒基因组序列进行比对分析,设计了2套引物和TaqMan荧光探针,对实时荧光RT-PCR反应条件进行优化,建立了小反刍兽疫疫苗毒与野毒实时荧光RT-PCR鉴别检测方法。特异性试验证实,该检测方法只能检测到目的病毒核酸,表明其具有良好的特异性。灵敏性试验发现,检测疫苗株的最低检测限可达1.38 mg/L的总RNA,检测野毒株的最低检测限为0.16 mg/L的核酸。对同一样品进行重复性检测,检测的荧光扩增曲线阈值完全重合,证明其重复性极好。从180份临床样品中,检测出2份疫苗病毒株阳性样品,其余均为阴性。结果表明,本研究所建立的实时荧光RT-PCR方法能对小反刍兽疫疫苗毒及野毒进行鉴别检测,具有特异性好、灵敏度高、重复性极好的优点,是开展小反刍兽疫疫情监测的有力工具。

In order to developing a differential diagnosis of Peste des petits ruminants （PPR）, two pairs of primers and two TaqMan fluorescence probes were designed basing on analyzing the genome sequence between vaccine strain and isolate strain. After optimizing reaction condition of the real-time RT-PCR,the real-time RT-PCR assay was developed to differentiate vaccine strain of PPR from isolate strain. The specificity test indicated that the assay detected only nucleic acid of the objective viruses. The tests indicated that the detection limits of real-time RT-PCR for PPR vaccine strain and isolate strain were 1.38 mg/L and 0.16 mg/L RNA, respectively. The results illustrated the speci- ficity,sensitivity and reproducibility of the developed methods were high. One hundred and eighty clinical samples were detected by real-time RT-PCR assay for PPR virus,2 samples were positive to vaccine strain, and others were negative. The study demonstrated that the real-time RT-PCR assay for detection of PPR could differentiate immune animal from infected animal,and can be use to clinical test and inspection.