Construction of an internal standard used in RT nested PCR for Borna Disease Virus RNA detection in biological samples.
The highly neurotropic Borna Disease Virus (BDV), which belongs to the Mononegavirales order--Bornaviridae family--is generally detected using the RT-nested-PCR. If false positive results (often caused by laboratory contaminations) can be avoided, some false negative results which are mostly due to inhibitory effects of some reaction components and/or to sample preparation errors, can occur. Thus, in order to control the RT-PCR sample, an RNA internal standard molecule named "mimic" was constructed with the same primer recognition sites as the viral nucleic acids, flanking a heterologous DNA fragment of distinct molecular weight. Because of their different sizes, the mimic and viral PCR products can be easily discriminated by agarose gel electrophoresis. The co-amplification of both BDV and mimic RNA was performed on infected cells and on biological tissues such as the brain and blood, commonly known to contain PCR inhibitor components. After mimic sensitivity studies were achieved (2.5 fg of "p40 RNA mimic" and 0.25 fg of "p24 RNA mimic"), the competitive amplification reaction between both BDV and mimic RNA was performed on these tissues. The results confirmed that nervous tissue has an inhibitory effect on RT-PCR, which supports the necessity of BDV detection by a higher sensitive method such as RT nested PCR. Moreover, these results confirmed the interest of an internal standard for BDV RNA detection in biological samples.
Legay
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V
Agence Française de Sécurité Sanitaire des Aliments d'Alfort, Maisons-Alfort, France.
Sailleau
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C
Dauphin
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G
Zientara
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S
D001483:Base Sequence; D001769:Blood / Q000821:virology; D001890:Borna Disease / Q000175:diagnosis*; D001891:Borna disease virus / Q000235:genetics / Q000302:isolation & purification*; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D005188:False Negative Reactions; D005784:Gene Amplification; D066298:In Vitro Techniques; D009417:Nerve Tissue / Q000821:virology; D012367:RNA, Viral / Q000032:analysis*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards / Q000662:veterinary*; D012703:Serotyping; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D012367:RNA, Viral; D014764:Viral Proteins; C077347:p40 protein, Borna disease virus