[Hepatitis delta virus RNA detection by one-step RT-PCR].
We have developed a method of detection HDV RNA where the reverse transcription and amplification are carried out in the same tube, thus reducing the handling time and the contamination risk. RNA extracted from serum or plasma on a microcolumn technique (kit QIAamp viral RNA, QIAgen) is submitted to reverse transcription and amplification by using the One-Step RT-PCR kit (QIAgen). Primers and probe were placed in the conserved ribozymic regions of the HDV genome. The sensitivity of the technique was estimated to 420 copies per reaction by using serial dilutions of a tittered sample. Its specificity was shown by the negativity of 24 samples from anti-HDV negative subjects, either healthy or coinfected by HBV, HCV or HGV. A low tittered sample was reproductively detected in different series. This One-step RT-PCR technique is specific, reproducible and allows the fast detection of active HDV RNA replication. Its adaptation to the routine diagnosis in a laboratory of medical virology is possible and will allow its evaluation on a greater number samples.
Ombandza-Moussa
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Laboratoire de virologie, Hôpital Paul Brousse, Université Paris XI, 12, avenue Paul Vaillant Couturier, 94804 Villejuif.
Dussaix
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Roque-Afonso
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AM
D000328:Adult; D005260:Female; D003698:Hepatitis Delta Virus / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors
D012367:RNA, Viral