Canadian Forest Service Publications

DNA hybridization assay for detection of nucleopolyhdrovirus in whitemarked tussock moth (Orgyia leucostigma) larvae. 2001. Ebling, P.M.; Smith, P.A.; van Frankenhuyzen, K. Pest Management Science 57: 66-71.

Year: 2001

Available from: Great Lakes Forestry Centre

Catalog ID: 18630

Language: English

CFS Availability: PDF (request by e-mail)

Abstract

DNA dot-blot hybridization assays utilizing a horseradish peroxide-labelled whole genomic DNA probe and enhanced chemiluminescence were conducted to quantify detection thresholds of nucleopolyhedrovirus (NPV) in whitemarked tussock moth (Orgyia leucostigma) larvae. The minimum detection thresholds for an aqueous suspension of occlusion bodies (OBs), OBs added to macerates of non-infected larvae and OBs in macerates of diseased larvae were 7.8 X 103, 7.8 x 103, and 1.5 x 103 OBs, respectively. Purified viral DNA was detected at a concentration of 1.6 x 10-1 ng in a 20µl volume. The presence of pre-occluded viral nucleocapsids and DNA, inherent to infected larvae, improved the detection threshold five-fold compared with OBs alone. Larval tissues did not block the detection system utilized, nor did they bind non-specifically to the probe. Detection thresholds, upon sequential hybridization of the same membrane, on average deteriorated two-fold between the first and second hybridization and an additional six-fold between the second and third hybridization. NPV infection was detected two days post-inoculation (pi) in about one-third of the larvae examined and in almost all larvae three days pi. Microscopic analysis of stained larval smears missed NPV infection in almost all larvae two days pi and about two-thirds of the larve three days pi. Results from the two methods of analysis were not comparable unitl four days pi. The detection system utilized is a reliable, efficient and simple method for the early detection of NPV infection in large numbers of larvae and may be used for further studies quantifying the role of this baculovirus in the ecology of whitemarked tussock moth populations.

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