Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction

Real-time polymerase chain reaction (RT-PCR) detection of proviral nucleic acid sequences of small ruminant lentiviruses (SRLV) in blood samples was developed and evaluated. Priming oligonucleotides were designed on the highly conserved 5′ untranslated leader-gag region while those on the long termi...

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Bibliographic Details
Published in:Journal of virological methods Vol. 147; no. 2; pp. 338 - 344
Main Authors: Brinkhof, J.M.A., van Maanen, C., Wigger, R., Peterson, K., Houwers, D.J.
Format: Journal Article
Language:English
Published: London Elsevier B.V 01.02.2008
Amsterdam Elsevier
New York, NY
Subjects:
Animals
Biological and medical sciences
Diagnosis
DNA, Viral - blood
Enzyme-Linked Immunosorbent Assay
Fundamental and applied biological sciences. Psychology
Genes, gag
Goat Diseases - diagnosis
Goat Diseases - virology
Goats
Lentivirus
Lentivirus - genetics
Lentivirus - isolation & purification
Lentivirus Infections - diagnosis
Lentivirus Infections - veterinary
Lentivirus Infections - virology
Lentiviruses
Microbiology
Proviruses - genetics
Real-time PCR
Reverse Transcriptase Polymerase Chain Reaction - methods
Ruminantia
Serology
Sheep
Sheep Diseases - diagnosis
Sheep Diseases - virology
Techniques used in virology
Terminal Repeat Sequences
Virology
Sheep
Serology
Diagnosis
Goats
Real-time PCR
Lentiviruses
Microbiology
Method
Real time
Provirus
Virology
Polymerase chain reaction
Vertebrata
Mammalia
Goat
Artiodactyla
LTR sequence
Veterinary
Detection
Ungulata
Nucleic acid
ISSN:0166-0934, 1879-0984
Online Access:Get full text
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Summary:Real-time polymerase chain reaction (RT-PCR) detection of proviral nucleic acid sequences of small ruminant lentiviruses (SRLV) in blood samples was developed and evaluated. Priming oligonucleotides were designed on the highly conserved 5′ untranslated leader-gag region while those on the long terminal repeat (LTR) assay were derived from literature. DNA was extracted from the buffycoat interlayer of centrifuged blood samples. Real-time PCR was performed by means of LightCycler technology (Roche Applied Science) using melting temperature analysis (SYBR Green I) for detection. Results were compared with those of serology using samples from Dutch sheep and goat flocks with known SRLV statuses, with sequential samples from a natural transmission experiment and samples from different regions in Norway, France, Spain and Italy. Real-time PCR testing, especially the application of oligonucleotides for priming the leader-gag region appeared promising in detecting SRLV specific proviral DNA in blood samples from both sheep and goats.
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ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2007.10.013