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根据基因库中对虾白斑综合症病毒W SSV(AF369029)和传染性皮下及造血器官坏死病毒IHHNV(AF218226)基因序列,设计了W SSV和IHHNV的两对特异性引物和两条用不同荧光基团标记的TaqM an探针。对反应条件和试剂浓度进行优化,建立了能够同时检测W SSV和IHHNV的二重实时荧光PCR方法。该方法特异性好,对W SSV和IHHNV的检测敏感性分别达到2和20个模板拷贝数;此外抗干扰能力强,对W SSV和IHHNV不同模板浓度进行组合,仍可有效地同时检测这二个病毒。对保存的30份经常规PCR检测仅为W SSV或IHHNV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中1份为W SSV和IHHNV混合感染。本研究建立的二重实时荧光PCR方法用于W SSV和IHHNV的检测具有特异、敏感、快速、定量等优点。
Two pairs of specific primers of W SSV and IHHNV were designed according to the sequence of WSSV (AF369029) and IHHNV (AF218226) of infectious subcutaneous and hematopoietic necrosis virus Labeled TaqM an probe. The reaction conditions and reagent concentrations were optimized and a dual real-time PCR method was developed to detect W SSV and IHHNV simultaneously. The specificity of the method is good, and the detection sensitivity to W SSV and IHHNV is respectively up to 2 and 20 copies of the template. In addition, the anti-interference ability is strong. The combination of different template concentrations of W SSV and IHHNV can still effectively detect these two A virus. The results of two real-time fluorescence PCR tests on 30 samples of routine WBC or WNV positive samples that were routinely detected by conventional PCR were all positive. One of them was a mixed infection of W SSV and IHHNV. The double real-time fluorescence PCR method established in this study is specific, sensitive, rapid and quantitative for the detection of W SSV and IHHNV.