Abstract
A technique using the polymerase chain reaction (PCR) was developed for detection of the nucleopolyhedrovirus (NPV) polyhedrin gene. 152 nucleotide sequences of polyhedrin gene were compared in pairwise and multiple alignment sequences. Eleven highly conserved DNA sequences within the coding region of the polyhedrin gene were identified. Two candidate regions were targeted for amplification and consequently one pair of degenerate PCR primers was designed to produce fragments of about 355 bp. The NPVs tested by this technique were Autographa californica (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea NPV (HcNPV), Lymantria dispar NPV (LdNPV), Spodoptera exigua NPV (SeNPV), S. litura NPV (SlNPV), Spodoptera littoralis NPV (SpliNPV) and nine local NPV isolates. Furthermore, three randomly chosen PCR products were cloned and sequenced. The sequencing data showed that the three PCR products were fragments of polyhedrin gene. Conclusively, this technique would be useful in monitoring the environmental fate, distribution of NPVs, release of the wild type and recombinant NPVs and quality control studies of baculoviral insecticides as well.
Highlights
INTRODUCTIION Baculoviruses have a large circular double-stranded DNA genome ranging from approximately 80 to 180 kb in size (Blissard and Rohrmann, 1990)
Murphy et al (1995) have reported baculovirus infections in over 600 insect species in the order of Lepidoptera, Hymenoptera, Diptera, Coleoptera, Neuroptera, Trichopera and Thysanura, as well as in the Crustaceae order Decapoda, it is recently confirmed that only those derived from orders Lepidoptera, Hymenoptera, and Diptera are members of the family Baculoviridae (ICTV, 2009)
A candidate region was defined as a site within the polyhedrin open reading frame that had 17+ bases from the Universal Primer for Early and Rapid Detection of Nucleopolyhedroviruses of Multiple Species 59
Summary
INTRODUCTIION Baculoviruses have a large circular double-stranded DNA genome ranging from approximately 80 to 180 kb in size (Blissard and Rohrmann, 1990) They are considered to be the largest and most broadly studied insect viruses because they are of great interest and utility to large cross-sections of agricultural and biomedical research community. Several methods have been employed to detect wild type or recombinant nucleopolyhedrovirus (NPV), such as microscopic diagnosis (Traverner MP, Connor, 1992), serological techniques (Brown et al., 1982, Naser and Miltenburger, 1983, Webb and Shelton, 1990), radioimmunoassay techniques (Smith and Summers, 1981, Knell et al, 1983), and DNA dot blot hybridization assays (Ward et al, 1987, Keating et al, 1989) The use of these techniques has been limited because they are either tedious and unreliable, or because they utilize radioactive materials. After the first report about localization of the polyhedrin gene in
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