title: A refined method for the detection of baculovirus occlusion bodies in forest terrestrial and aquatic habitats.
abstract: A sensitive and efficient method was developed for the detection of genetically modified and wild-type baculovirus occlusion bodies (OB) in forest terrestrial and aquatic habitats. The protocol facilitates the analysis of a large number of samples collected and frozen to maintain viral integrity. Lyophilization was used to standardize the size of both field-collected soil samples and test substrates inoculated with OBs for the determination of minimum detection threshold. To simulate natural conditions, terrestrial test substrates were inoculated at a standardized moisture content determined using a soil pressure plate apparatus. OBs, extracted from lyophilized test substrates by washing, sieving and centrifugation, were subjected to alkaline lysis and viral DNA isolated using a purchased DNA purification kit. PCR amplified DNA was visualized using agarose gel electrophoresis. Minimum detection thresholds in terrestrial substrates were 10(3), 10(2), 10(2) and 10(1) OBs from 0.5 g of lyophilized L, F-H and mineral soil horizons, and 1.0 ml of leachate, respectively. Detection thresholds in aquatic substrates were 10(0) and 10(3) OBs from 1.0 ml of pond water and 1.0 g of bottom sediment, respectively.
authors.affiliation: Canadian Forest Service, Natural Resources Canada, 1219 Queen St E, Sault Ste Marie, Ontario, Canada P6A 2E5. pebling@nrcan.gc.ca
authors.forename: Peter M
authors.initials: PM
authors.lastname: Ebling
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Holmes
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004279:DNA, Viral / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D004777:Environment; D007304:Insect Viruses / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D007814:Larva / Q000821:virology; D007915:Lepidoptera / Q000821:virology; D030781:Organisms, Genetically Modified; D016133:Polymerase Chain Reaction; D012988:Soil Microbiology; D014197:Trees / Q000821:virology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D014871:Water Microbiology
chemical_list: D004279:DNA, Viral; D014759:Viral Envelope Proteins