舞毒蛾核型多角体病毒PCR检测技术的建立
Establishment of PCR Assay for Detecting of Lymantria dispar Multiple-embedded Nucleopolyhedrovirus (LdMNPV)
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摘要: 根据舞毒蛾核型多角体病毒(LdMNPV)的蜕皮甾体尿苷二磷酸葡萄糖基转移酶基因(egt)设计一对特异性引物,成功建立了LdMNPV的PCR检测技术体系,其对LdMNPV基因组的灵敏度可达到1 fg·mL-1。应用该技术体系从舞毒蛾带毒幼虫、卵、蛹的基因组中扩增出目的片段,证明了这一技术的可行性。对不同浓度的多角体悬液的扩增结果显示:其检测最低量为5 OBs·mL-1。形态学研究表明:从内蒙舞毒蛾体内分离到的LdMNPV中,少数表面有凹陷的孔洞,病毒粒子从这些孔洞中游离出来,这也阐明了此技术能够从多角体悬液中扩增出目的片段的原因。在将来LdMNPV的检测中,可用虫体进行研磨过滤离心得到的组织液作为模板进行扩增。
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关键词:
- 舞毒蛾核型多角体病毒
- / PCR
- / 微量检测
- / egt基因
Abstract: A technical architecture used for the microdetermination of Lymantria dispar multiple-embedded nucleopolyhedrovirus was set up. Based on egt gene of LdMNPV, a pair of primers was designed with the sensitivity of 1fg·mL-1 DNA. By using this pair of primers, the partial sequences of egt gene were amplified from the eggs, larvae and pupae of the infected L. dispar, which verified the feasibility of this method. The partial sequences of egt gene were also amplified from the polyhedron suspension and the minimum amount of detection could be as low as 5 OBs·mL-1 of polyhedron. Morphology research indicated that the surface of a few of polyhedrons was rough pitted and released virions. It could be a reason why the target DNA fragment could be amplified from the virus suspension. So in the detection of LdMNPV, suspension after trituration, filtration and centrifugalism can be used as template. -
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