Christian P D, Gibb N, Kasprzak A B, Richards A
CSIRO Division of Entomology, P.O. Box 1700, Canberra ACT 2601, Australia.
J Virol Methods. 2001 Jul;96(1):51-65. doi: 10.1016/s0166-0934(01)00318-4.
A diagnostic method is described for the identification and differentiation of nucleopolyhedrovirus (NPV) pathogens of Helicoverpa species (Lepidoptera: Noctuidae) isolated from the environment. The method is based on the polymerase chain reaction (PCR) used in conjunction with restriction fragment length polymorphism (RFLP) analysis and comprises three parts. The first part describes procedures for obtaining PCR quality viral DNA from individual diseased H. armigera cadavers recovered during bioassay analyses of soil and other types of environmental sample. These procedures were modified from standard techniques used for the routine purification and dissolution of NPV polyhedra and provided an overall PCR success rate of 95% (n=60). The second part describes the design of several sets of PCR primers for generating DNA amplification products from closely and distantly related NPVs. These PCR primers were designed from published DNA sequence data and from randomly cloned genomic DNA fragments isolated from a reference H. armigera SNPV (HaSNPV) isolate. The final part of the method describes how specific PCR products when digested with specific restriction endonuclease enzymes, can be used to generate diagnostic DNA profiles (haplotypes) that can be used both to identify heterologous NPVs e.g. Autographa californica MNPV and related viruses, and to differentiate genotypic variants of Helicoverpa SNPV. In the latter case, only two PCR products and four restriction digests were required to differentiate a reference set of 10 Helicoverpa SNPV isolates known to differ 0.1--3.5% at the nucleotide level. The diagnostic method described below marks the second part of a two-phase quantitative-diagnostic protocol that is now being applied to a variety of ecological investigations. In particular, its application should lead to a significant improvement in our understanding of the distribution and population genetics of Helicoverpa SNPVs in the Australian environment, as well as providing a sound basis for the design of pre- and post-release monitoring systems for genetically enhanced bioinsecticides. It is also likely that this method can be adapted readily to the study of other insect pathogen associations important economically.
本文描述了一种诊断方法，用于鉴定和区分从环境中分离出的棉铃虫属（鳞翅目：夜蛾科）的核型多角体病毒（NPV）病原体。该方法基于聚合酶链反应（PCR）并结合限制性片段长度多态性（RFLP）分析，包括三个部分。第一部分描述了从土壤和其他类型环境样本的生物测定分析过程中回收的单个患病棉铃虫尸体中获取PCR质量病毒DNA的程序。这些程序是从用于NPV多角体常规纯化和溶解的标准技术修改而来的，总体PCR成功率为95%（n = 60）。第二部分描述了几组PCR引物的设计，用于从亲缘关系远近不同的NPV中产生DNA扩增产物。这些PCR引物是根据已发表的DNA序列数据以及从参考棉铃虫单粒包埋核型多角体病毒（HaSNPV）分离株中随机克隆的基因组DNA片段设计的。该方法的最后一部分描述了特定的PCR产物在用特定限制性内切酶消化后，如何用于生成诊断性DNA图谱（单倍型），这些图谱可用于鉴定异源NPV，如苜蓿银纹夜蛾多核衣壳核型多角体病毒（Autographa californica MNPV）及相关病毒，以及区分棉铃虫SNPV的基因型变体。在后一种情况下，仅需两个PCR产物和四次限制性酶切，就能区分一组已知在核苷酸水平上差异为0.1%-3.5%的10个棉铃虫SNPV参考分离株。下文所述的诊断方法是一个两阶段定量诊断方案的第二部分，该方案目前正应用于各种生态调查。特别是，其应用应能显著增进我们对澳大利亚环境中棉铃虫SNPV的分布和群体遗传学的了解，并为设计基因增强型生物杀虫剂释放前和释放后的监测系统提供坚实基础。该方法也很可能可以很容易地适用于对其他具有重要经济意义的昆虫病原体关联的研究。
