Extraction and detection of baculoviral DNA from lake water, detritus and forest litter

Aims: This paper describes a quick, reproducible, sensitive method for baculoviral DNA extraction, purification and detection from freshwater and forest litter environments. Methods and Results: The extraction protocol utilizes enzymatic and chemical lysis and physical disruption. To assess the effi...

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Bibliographic Details
Published in:Journal of applied microbiology 2001-04, Vol.90 (4), p.630-636
Main Authors: England, L.S, Trevors, J.T, Holmes, S.B
Format: Article
Language:English
Subjects:
Baculoviridae
Baculoviridae - genetics
Baculoviridae - isolation & purification
Baculovirus
bioassays
Biological and medical sciences
Biological control
Choristoneura fumiferana
Control
detection limit
DNA
DNA fragmentation
DNA, Viral - analysis
environmental assessment
Environmental Microbiology
forest litter
Freshwater
Fundamental and applied biological sciences. Psychology
genome
insects
lakes
Phytopathology. Animal pests. Plant and forest protection
polymerase chain reaction
Polymerase Chain Reaction - methods
Protozoa. Invertebrates
purification methods
Reproducibility of Results
Water Microbiology
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Summary:Aims: This paper describes a quick, reproducible, sensitive method for baculoviral DNA extraction, purification and detection from freshwater and forest litter environments. Methods and Results: The extraction protocol utilizes enzymatic and chemical lysis and physical disruption. To assess the efficiency of the extraction and purification protocol, PCR was used to detect a 530 bp DNA fragment from the genome of a genetically-modified baculovirus, Choristoneura fumiferana NPVegt(-)/lacZ(+). The detection limit of PCR amplification was routinely about 4.1 x 10(2) occlusion bodies (OBs) 450 microliter(-1) lake water. Template DNA from the detritus and forest litter samples required 100-fold dilutions before use in PCR reactions. The detection limits for detritus and forest litter samples were routinely about 7.41 x 10(3) and 2.08 x 10(4) OBs 0.5 g(-1) dry weight, respectively. Conclusions: The DNA extraction and purification methodology is reproducible, sensitive and can be used in lieu of, or in conjunction with, insect bioassays. Significance and Impact of the Study: The DNA extraction and purification protocol described in this paper will facilitate risk assessment and ecological studies of both wild-type and genetically-modified baculoviruses.
ISSN:1364-5072
1365-2672
DOI:10.1046/j.1365-2672.2001.01289.x