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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| Vydáno v: | Journal of virological methods Ročník 147; číslo 2; s. 338 - 344 |
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| Hlavní autoři: | , , , , |
| Médium: | Journal Article |
| Jazyk: | angličtina |
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London
Elsevier B.V
01.02.2008
Amsterdam Elsevier New York, NY |
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| ISSN: | 0166-0934, 1879-0984 |
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| Abstract | 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. |
|---|---|
| AbstractList | 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.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. 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. 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. |
| Author | Brinkhof, J.M.A. Wigger, R. Peterson, K. van Maanen, C. Houwers, D.J. |
| Author_xml | – sequence: 1 givenname: J.M.A. surname: Brinkhof fullname: Brinkhof, J.M.A. email: j.brinkhof@gddeventer.com organization: Animal Health Service Ltd., Arnsbergstraat 7, 7418 EZ Deventer, The Netherlands – sequence: 2 givenname: C. surname: van Maanen fullname: van Maanen, C. organization: Animal Health Service Ltd., Arnsbergstraat 7, 7418 EZ Deventer, The Netherlands – sequence: 3 givenname: R. surname: Wigger fullname: Wigger, R. organization: Animal Health Service Ltd., Arnsbergstraat 7, 7418 EZ Deventer, The Netherlands – sequence: 4 givenname: K. surname: Peterson fullname: Peterson, K. organization: Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3508 TD Utrecht, The Netherlands – sequence: 5 givenname: D.J. surname: Houwers fullname: Houwers, D.J. organization: Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3508 TD Utrecht, The Netherlands |
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| Keywords | 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 |
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| SubjectTerms | 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 |
| Title | 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 |
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