Amplification of hepatitis delta virus RNA sequences by polymerase chain reaction: a tool for viral detection and cloning.
In this investigation we have evaluated the feasibility of using the polymerase chain reaction (PCR) for hepatitis delta virus (HDV) RNA detection, cloning and sequencing. Total RNA from HDV-infected liver and serum samples was purified and Moloney murine leukaemia virus (M-MLV) reverse transcribed. HDV cDNA was then directly amplified with Taq polymerase using three pairs of specific primers. It was possible to amplify a region of about 1200 bp in three partially overlapping fragments including the whole HDAg-ORF. A DNA fragment of the expected size was repeatedly obtained from an initial sample of less than 0.1 pg of liver RNA and from 10 pl of infected serum. An amplified fragment of 359 bp obtained by PCR from an infected woodchucks' liver was sequenced. The sequence was 91.8% and 98.6% identical to previously published HDV sequences. In addition, amplified and 32P-radiolabelled HDV sequences were shown to hybridize specifically to HDV RNA extracted from HDV-infected liver and serum. In conclusion this technique promises to be of great value in the appraisal of HDV infection, rapid synthesis of HDV probes and analysis of the genetic variability of the virus.
Zignego
A L
AL
Unité de Recombinaison et Expression Génétique (Inserm U 163/CNRS UA 271), Institut Pasteur, Paris, France.
Deny
P
P
Feray
C
C
Ponzetto
A
A
Gentilini
P
P
Tiollais
P
P
Bréchot
C
C
D000818:Animals; D001483:Base Sequence; D003698:Hepatitis Delta Virus / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008099:Liver / Q000382:microbiology; D008392:Marmota; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D002679:Pan troglodytes; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification
D012367:RNA, Viral