Canadian Forest Service Publications

A refined method for the detection of baculovirus occlusion bodies in forest terrestrial and aquatic habitats. 2002. Ebling, P.M.; Holmes, S.B. Pest Management Science 58: 1266-1222.

Year: 2002

Issued by: Great Lakes Forestry Centre

Catalog ID: 21501

Language: English

Availability: PDF (request by e-mail)

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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 103, 102, 102 and 101 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 100 and 103 OBs from 1.0 ml of pond water and 1.0 g of bottom sediment, respectively.