PCR结合斑点杂交技术在检测禽网状内皮增生病毒中的应用

Use of PCR and Dot Blot Hybridization to Detect Chicken Reticuloendotheliosis Virus

关键词:  网状内皮增生病毒，狄可辛标记核酸探针  斑点杂交  多聚酶链反应

Key words:  Reticuloendotheliosis virus (REV)  degoxigenin labled probe  PCR  Dot blot hybridization

摘要:

评价PCR结合斑点杂交技术在鸡REV检测中的应用价值。方法：采用PCR法制备禽网状内皮细胞增生症病毒(Reticuloendotheliosis virus，REV)特异性地高辛标记DNA探针，同时用PCR技术、斑点杂交方法和PCR产物斑点杂交方法检测不同地区的病、死鸡的组织样品REV的感染情况。结果：PCR产物斑点杂交法的检出率(45.16%,14/31)高于组织DNA直接斑点杂交法（32.26%,10/31），显著高于单纯PCR扩增法(0%,0/31)。结论：PCR结合斑点杂交检测技术能快速、敏感、准确的进行检测，充分避免PCR中的假阴性和假阳性现象，值得推广应用。

Abstract:

Objective: To evaluate the combination of PCR and Dot blot assays in detection for REV infection in chicken samples. Methods: REV-specific digoxigenin-labled DNA probe was prepared by PCR. DNA samples were extraced and tested by regular PCR and dot blot hibridization, respectively or by PCR plus dot blot. The sensitivity of differenr methods were compared with each other. Results: The positive rate (45.16%,14/31) using PCR-Dot blot was higher than that (32.26%,10/31) by direct Dot blot for tissue DNA, significantly higher than PCR amplification (0%,0/31). Conclusions：the combined PCR-Dot blot assay is a rapid, sensitive and specific diagnosis method. It could overcome false negative and false positive in PCR, and the sensitivity was significantly improved..