Effect of pooling and multiplexing on the detection of bluetongue virus RNA by real-time RT-PCR
| Autor: | Frank Vandenbussche, Nesya Goris, T. Vanbinst, Corinne Sailleau, K. De Clercq, E. Vandemeulebroucke, Stéphan Zientara |
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| Přispěvatelé: | Sciensano [Bruxelles], Réseau International des Instituts Pasteur (RIIP), Virologie, École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence Française de Sécurité Sanitaire des Aliments (AFSSA) |
| Rok vydání: | 2008 |
| Předmět: |
Serotype
040301 veterinary sciences Pooling Cattle Diseases Reoviridae Bluetongue REAL-TIME RT-PCR Virus 0403 veterinary science 03 medical and health sciences Virology Animals Multiplex 030304 developmental biology 0303 health sciences Sheep Orbivirus biology Reverse Transcriptase Polymerase Chain Reaction BLUETONGUE VIRUS 04 agricultural and veterinary sciences RELATION VIRUS-VECTEUR biology.organism_classification POOLING Actins Real-time polymerase chain reaction [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology RNA Viral MULTIPLEX Cattle Viral load |
| Zdroj: | Journal of Virological Methods Journal of Virological Methods, Elsevier, 2008, 152 (1-2), pp.13-17. ⟨10.1016/j.jviromet.2008.06.005⟩ |
| ISSN: | 0166-0934 |
| DOI: | 10.1016/j.jviromet.2008.06.005 |
| Popis: | International audience; Real-time RT-PCR (RT-qPCR) was used routinely for laboratory diagnosis during the 2006/2007 bluetongue virus (BTV) serotype 8 epidemic. In the present study the impact of pooling and multiplexing strategies on RT-qPCR are assessed. To avoid any bias in the pooling experiments, 121 BTV-8 positive blood samples with a low to high viral load were selected and pooled individually with nine negative blood samples. Analyses of the individually and pooled samples indicated an overall mean difference of 4.32 Ct-values. The most pronounced differences were observed in samples with the lowest viral load of which 70% could no longer be detected after pooling. The pooling strategy is therefore not suitable for BTV detection at the individual level since animals infected recently may be missed. An alternative approach to reduce costs and workload is to apply a multiplexing strategy in which the viral RNA and internal P-actin control RNA are detected in a single reaction. Parallel analysis (singleplex versus multiplex) of a 10-fold dilution series and 546 field samples proved that the sensitivity of the BTV RT-qPCR was not affected whereas the beta-actin reaction was reduced only slightly. Without the use of an internal control, 0.6% of 1985 field samples is at risk of being diagnosed incorrectly as negative. |
| Databáze: | OpenAIRE |
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