Morphological Characteristics of Erannis ankeraria Nucleopolyhedrovirus and Establishment of Molecular Detection Technique
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摘要:
目的 落叶松尺蛾核型多角体病毒（Erannis ankeraria nucleopolyhedrovirus，EranNPV）是有效调控落叶松尺蛾种群数量和质量的重要生物因子，明确EranNPV超微形态学结构，研究并建立其精准灵敏的分子（PCR）快速检测技术，有助于深入探究该病毒的传播机制和林间流行规律，从而为利用该病毒持续控制落叶松尺蛾提供技术支撑。 方法 利用扫描电镜和透射电镜对EranNPV多角体的外部形态和内部结构特征进行观察；根据EranNPV的ac54基因设计用于PCR检测的特异性引物EaV，通过多种昆虫病毒的PCR扩增结果验证引物特异性，测试该PCR检测技术体系对EranNPV基因组DNA和病毒悬浮液的灵敏性。 结果 电镜观察结果表明，该病毒多角体多为表面光滑的不规则多面体，少数多角体表面散布孔洞凹陷，平均直径为1.43 ± 0.20 μm，多角体内的病毒粒子呈单束分布，平均长度为219.14 ±17.50 nm；以EaV为引物，只有EranNPV能够扩增到目的条带，利用EaV为引物建立的PCR检测技术对EranNPV基因组DNA的检测灵敏度可达10 fg/mL，对EranNPV悬浮液的检测灵敏度可达1×102 OBs/mL。 结论 EranNPV为典型的单粒包埋型核型多角体病毒，利用EaV引物建立的PCR检测技术具有高度的特异性和灵敏度，有望进一步应用于EranNPV的林间传播和流行病学研究当中。 Abstract:Objective Erannis ankeraria nucleopolyhedrovirus (EranNPV) is a significant natural enemy microbial product that effectively regulates the population and quality of E. ankeraria. The elucidation of the ultramicromorphological characteristics of EranNPV and the establishment of a sensitive and convenient PCR-based rapid detection method will help to further investigate the epidemiology and transmission mechanisms of EranNPV in forest ecosystems. This, in turn, provides technical support for the continuous control of E. ankeraria using EranNPV. Method The external morphology and internal structural features of EranNPV polyhedra were observed using scanning electron microscopy and transmission electron microscopy. A pair of PCR detection primers, EaV, was designed based on the ac54 gene of EranNPV. The specificity of EaV was examined by PCR amplification results of various insect viruses. The sensitivity of PCR amplification using EaV primers was determined by testing different concentrations of EranNPV genomic DNA and polyhedrons. Result The electron microscopy results showed that the majority of the virus polyhedra were irregular polyhedra with smooth surfaces, and a few had scattered pore depressions with an average diameter of 1.43 ± 0.20 μm. The virus particles within the polyhedra were distributed in a single bundle with an average length of 219.14 ± 17.50 nm. When EaV is used as the primer, only EranNPV can be amplified to the target band. The PCR assay using EaV primers exhibited a sensitivity of 10 fg/mL for EranNPV genomic DNA and a sensitivity of 1×102 OBs/mL for polyhedrons. Conclusion EranNPV is a typical single nucleocapsid nucleopolyhedrovirus. The PCR assay using EaV primers demonstrates high specificity and sensitivity. It is expected to be further applied to the study of the diffusion and transmission mechanism of EranNPV in forest ecosystems. -
图 1 落叶松尺蛾核型多角体病毒超微结构
Figure 1. Ultrastructure of Erannis ankeraria nucleopolyhedrovirus
图 2 利用EaV引物检测不同核型多角体病毒基因组的扩增结果
M：DL2000 Marker，1～8：EranNPV、HycuNPV、EcobNPV、ApciNPV、LydiNPV、SpliNPV、NezhNPV、阴性对照Negative control （ddH2O） 。
Figure 2. Detection of different nucleopolyhedrovirus genomes using primer pair EaV
图 3 不同浓度EranNPV基因组ac54基因扩增结果
M：DL2000 Marker，1：阴性对照Negative control （ddH2O） ，2～7：1 μg/mL、1 ng/mL、1 pg/mL、100 fg/mL、10 fg/mL、1 fg/mL。
Figure 3. Amplifications of ac54 in different concentrations of EranNPV genomic DNA using primer pair EaV
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