检测鹿黏膜病病毒LDR-PCR方法的建立

Development of LDR-PCR assay for deer mucosal disease

为准确特异灵敏地检测鹿黏膜病病毒,本研究建立了一种新的LDR-PCR方法。首先在病毒的保守区内设计1对LDR探针,LDR探针两端各连有1段引物对应序列,以连接产物为模板进行PCR,琼脂糖凝胶电泳检测结果。通过对LDR反应的退火温度、连接酶浓度及探针浓度等反应条件进行优化,确定了LDR最佳的反应体系,并建立了LDR-PCR方法。结果表明,本方法可以特异地检测BVDV,最低检测限为101个拷贝。此方法的建立为BVDV基础研究和临床应用提供了良好的技术平台,为进一步开发高通量的多病原检测技术提供了技术基础。

In order to accurate,specific and sensitive detection of deer mucosal disease virus,this study established a new LDR-PCR method.The conservative region of the virus was used to design a pair LDR probes.At each end the corresponding the primer sequences were attached.Utilized the ligation product as a template PCR to do,agarose gel electrophoresis test.By LDR reaction annealing temperature,the ligase concentration and reaction conditions such as probe concentrations,the optimum LDR reaction system was optimized to determine,and established LDR-PCR method.The results show that this approach can specifically detect BVDV,the lowest detection limit is 101 copies.The established method provides a good technical platform for the research and clinical application of BVDV.and provides the technical foundation for the further development of high-throughput multi-pathogen detection technology.