荧光RT-PCR鉴别诊断O型与AsiaⅠ型口蹄疫病毒方法的建立

从GenBank中获取多个口蹄疫病毒（Foot-and-mouth disease virus, FMDV）基因组序列，进行多序列比对之后，设计对应于口蹄疫病毒的3D、VP3和3C区域的引物和探针，分别建立了FMDV群特异性实时荧光RT-PCR、O型特异性实时荧光RT-PCR和AsiaⅠ型特异性实时荧光RT-PCR检测方法。三种FMDV实时荧光RT-PCR在Lightcycler荧光PCR仪上扩增均出现标准的S型曲线，并且具有良好的特异性，可以很好地将FMDV与传染性牛鼻气管炎病毒（Infectious bovine rhinotracheitis virus）、猪传染性胃肠炎病毒（Swine transmissible gastroenteritis virus）、赤羽病病毒（Akabane virus）和猪呼吸系统冠状病毒（Porcine respiratory corona virus）区分。FMDV群特异性实时荧光RT-PCR可以成功扩增O型（O1和O2毒株）和AsiaⅠ型（AsiaⅠ1和AsiaⅠ2毒株）FMDV；O型特异性实时荧光RT-PCR可成功扩增O型（O1和O2毒株）FMDV，AsiaⅠ型（AsiaⅠ1和AsiaⅠ2毒株）FMDV为阴性；AsiaⅠ型特异性实时荧光RT-PCR可成功扩增AsiaⅠ型（AsiaⅠ1和AsiaⅠ2毒株）FMDV，O型（O1和O2毒株）FMDV为阴性。FMDV群特异性实时荧光RT-PCR、O型特异性实时荧光RT-PCR和AsiaⅠ型特异性实时荧光RT-PCR都可以检测到10个拷贝的模板，灵敏度接近检测方法的极限。检测时间短，从加样到反应结束只需要70 min。该方法有潜力用于FMDV的实验室筛查与鉴别诊断。

Establishment of Real-time RT-PCR Assay to Differentiate 0 and Asia I Type Foot-and-mouth disease virus

Several genome sequences of Foot-and-mouth disease virus (FMDV) were obtained from GenBank. After multiplex alignment, three sets of primers and probes were designed according to 3D, VP3 and 3C region of FMDV. Three sets of primers and probes were used to establish FMDV species-specificity, O type-specificity and AsiaⅠtype-specificity Real-time RT-PCR, respectively. Standard S shape curves occurred after Real-time RT-PCR amplification on Lightcycler machine. All three newly established methods had good specification for they could differentiate FMDV from Infectious bovine rhinotracheitis virus, Swine transmissible gastroenteritis virus, Akabane virus and Porcine respiratory corona virus. FMDV species-specificity Real-time RT-PCR could successfully amplify O type(O1 strain and O2 strain) and AsiaⅠtype (AsiaⅠ1 strain and AsiaⅠ2 strain) FMDV. O type-specificity Real-time RT-PCR could successfully amplify O type(O1 strain and O2 strain) FMDV but failed to amplify AsiaⅠ (AsiaⅠ1 strain and AsiaⅠ2 strain) FMDV. AsiaⅠtype-specificity Real-time RT-PCR could successfully amplify AsiaⅠ(AsiaⅠ1 strain and AsiaⅠ2 strain) FMDV but failed to amplify O type (O1 strain and O2 strain) FMDV. All methods could detect 10 copies of DNA template, closely reach the detection limit. The whole Real-time RT-PCR reaction could be finished in 70 min. These methods have potential to apply in FMDV screen and differential detection in laboratory.