Quantification of latent Mamestra brassicae nuclear polyhedrosis virus in M. brassicae insects using a PCR-scintillation proximity assay
Identifieur interne : 004326 ( Main/Exploration ); précédent : 004325; suivant : 004327Quantification of latent Mamestra brassicae nuclear polyhedrosis virus in M. brassicae insects using a PCR-scintillation proximity assay
Auteurs : David S. Hughes [Royaume-Uni] ; Robert D. Possee [Royaume-Uni] ; Linda A. King [Royaume-Uni]Source :
- Journal of Virological Methods [ 0166-0934 ] ; 1994.
English descriptors
- Teeft :
- Acceptor molecules, Assay, Baculovirus, Binding radiolabelled ligands, Biotinylated primer, Body cells, Body tissue, Brassicae, Brassicae cells, Brassicae insects, Cell line, Cell lines, Control mbnpv, Copy number, Final concentration, Genome, Genomic, Laboratory culture, Latent baculovirus genomes, Latent baculovirus infection, Latent mbnpv, Latent mbnpv genome, Latent state, Latent virus, Latent virus sequences, Liquid scintillation, Mamestra brassicae, Mamestra brassicae insects, Mblc, Mbnpv, Molecular mass, Panolis frammea, Polyhedrin, Polyhedrin gene coding region, Polyhedrosis virus, Polymerase chain reaction, Primer, Pseudorabies virus, Scintillation, Scintillation counter, Scintillation proximity assay, Serial dilutions, Standard curve, Target copy numbers, Target sequence, Viral genome, Virological, Virological methods, Virus particles.
Abstract
Abstract: A laboratory culture of Mamestra brassicae insects (MbLC) was found to harbour a latent baculovirus infection. The copy number of the occult MbNPV genome in both the MbLC larvae, and in a cell line derived from the fat body of MbLC was determined by the use of a rapid and convenient PCR-scintillation proximity assay (SPA). The SPA system relies on the use of fluomicrospheres (SPA beads) coated with acceptor molecules which are capable of binding radiolabelled ligands in solution. In the assay described, a biotinylated PCR primer is used and [3H]dNTPs are incorporated into the amplified DNA. The SPA beads are coated with streptavidin, and after binding the biotinylated primer, any amplified, radiolabelled DNA will activate the fluor. The amount of amplified DNA from the target sequence can then be directly quantified using a scintillation counter. The number of MbNPV genomes present in a persistently infected M. brassicae cell, as proposed by SPA, suggest between 13 and 20 copies of the viral genome may be present in individual fat body cells.
Url:
DOI: 10.1016/0166-0934(94)90160-0
Affiliations:
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King</name><affiliation wicri:level="1"><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane, Headington, Oxford OX3 OBP</wicri:regionArea><wicri:noRegion>Oxford OX3 OBP</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Virological Methods</title><title level="j" type="abbrev">VIRMET</title><idno type="ISSN">0166-0934</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1994">1994</date><biblScope unit="volume">50</biblScope><biblScope unit="issue">1–3</biblScope><biblScope unit="page" from="21">21</biblScope><biblScope unit="page" to="27">27</biblScope></imprint><idno type="ISSN">0166-0934</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0166-0934</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Acceptor molecules</term><term>Assay</term><term>Baculovirus</term><term>Binding radiolabelled ligands</term><term>Biotinylated primer</term><term>Body cells</term><term>Body tissue</term><term>Brassicae</term><term>Brassicae cells</term><term>Brassicae insects</term><term>Cell line</term><term>Cell lines</term><term>Control mbnpv</term><term>Copy number</term><term>Final concentration</term><term>Genome</term><term>Genomic</term><term>Laboratory culture</term><term>Latent baculovirus genomes</term><term>Latent baculovirus infection</term><term>Latent mbnpv</term><term>Latent mbnpv genome</term><term>Latent state</term><term>Latent virus</term><term>Latent virus sequences</term><term>Liquid scintillation</term><term>Mamestra brassicae</term><term>Mamestra brassicae insects</term><term>Mblc</term><term>Mbnpv</term><term>Molecular mass</term><term>Panolis frammea</term><term>Polyhedrin</term><term>Polyhedrin gene coding region</term><term>Polyhedrosis virus</term><term>Polymerase chain reaction</term><term>Primer</term><term>Pseudorabies virus</term><term>Scintillation</term><term>Scintillation counter</term><term>Scintillation proximity assay</term><term>Serial dilutions</term><term>Standard curve</term><term>Target copy numbers</term><term>Target sequence</term><term>Viral genome</term><term>Virological</term><term>Virological methods</term><term>Virus particles</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: A laboratory culture of Mamestra brassicae insects (MbLC) was found to harbour a latent baculovirus infection. The copy number of the occult MbNPV genome in both the MbLC larvae, and in a cell line derived from the fat body of MbLC was determined by the use of a rapid and convenient PCR-scintillation proximity assay (SPA). The SPA system relies on the use of fluomicrospheres (SPA beads) coated with acceptor molecules which are capable of binding radiolabelled ligands in solution. In the assay described, a biotinylated PCR primer is used and [3H]dNTPs are incorporated into the amplified DNA. The SPA beads are coated with streptavidin, and after binding the biotinylated primer, any amplified, radiolabelled DNA will activate the fluor. The amount of amplified DNA from the target sequence can then be directly quantified using a scintillation counter. The number of MbNPV genomes present in a persistently infected M. brassicae cell, as proposed by SPA, suggest between 13 and 20 copies of the viral genome may be present in individual fat body cells.</div></front></TEI><affiliations><list><country><li>Royaume-Uni</li></country></list><tree><country name="Royaume-Uni"><noRegion><name sortKey="Hughes, David S" sort="Hughes, David S" uniqKey="Hughes D" first="David S." last="Hughes">David S. Hughes</name></noRegion><name sortKey="King, Linda A" sort="King, Linda A" uniqKey="King L" first="Linda A." last="King">Linda A. King</name><name sortKey="Possee, Robert D" sort="Possee, Robert D" uniqKey="Possee R" first="Robert D." last="Possee">Robert D. Possee</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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