A rapid method for the identification and differentiation of Helicoverpa nucleopolyhedroviruses (NPV Baculoviridae) isolated from the environment

A diagnostic method is described for the identification and differentiation of nucleopolyhedrovirus (NPV) pathogens of Helicoverpa species (Lepidopter a: Noctuidae) isolated from the environment. The method is based on the pol ymerase chain reaction (PCR) used in conjunction with restriction fragment length polymorphism (RFLP) analysis and comprises three parts. The first pa rt describes procedures for obtaining PCR quality viral DNA from individual diseased H. armigera cadavers recovered during bioassay analyses of soil a nd other types of environmental sample. These procedures were modified from standard techniques used fur the routine purification and dissolution of N PV 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 genera ting DNA amplification products from closely and distantly related NPVs. Th ese PCR primers were designed from published DNA sequence data and from ran domly cloned genomic DNA fragments isolated from a reference H. armigera SN PV (HaSNPV) isolate. The final part of the method describes how specific PC R products when digested with specific restriction endonuclease enzymes, ca n be used to generate diagnostic DNA profiles (haplotypes) that can be used both to identify heterologous NPVs e.g. Autographa californica MNPV and re lated viruses, and to differentiate genotypic variants of Helicoverpa SNPV. In the latter case, only two PCR products: and four restriction digests we re required to differentiate a reference set of 10 Helicoverpa SNPV isolate s known to differ 0.1-3.5% at the nucleotide level. The diagnostic method d escribed below marks the second part of a two-phase quantitative-diagnostic protocol that is now being applied to a variety of ecological investigatio ns. In particular, its application should lead to a significant improvement in our understanding of the distribution and population genetics of Helico verpa SNPVs in the Australian environment, as well as providing a sound bas is for the design of pre- and post-release monitoring systems for genetical ly enhanced bioinsecticides. It is also likely that this method can be adap ted readily to the study of other insect pathogen associations important ec onomically. (C) 2001 Published by Elsevier Science B.V. All rights reserved .