A novel, sensitive, and specific RT-PCR technique for quantitation of hepatitis C virus replication.
The detection of negative-strand hepatitis C virus (HCV) RNA is a hallmark of replication. A highly sensitive and specific method is required to quantify the very low level of replication inherent to in vitro infection systems. Based on reverse transcription with a tagged primer in the 5' non-coding region of the HCV genome, followed by a nested PCR with a second round of real-time PCR, a novel method is described with improved sensitivity for negative-strand HCV RNA quantification. The lower detection level was 25 copies per reaction of negative-strand HCV RNA, even in the presence of 1 x 10(5) copies of positive-strand HCV RNA. This protocol was applied to the detection of negative HCV strand RNA in the liver of HCV-infected patients as well as in primary human hepatocytes infected in vitro. In both models, and particularly in each of three, independent in vitro infection experiments, this assay permitted the quantitation of HCV replication.
Carrière
Matthieu
M
UPRESS 1833, Faculté de Médecine Paris 5, France.
Pène
Véronique
V
Breiman
Adrien
A
Conti
Filoména
F
Chouzenoux
Sandrine
S
Meurs
Eliane
E
Andrieu
Muriel
M
Jaffray
Patrick
P
Grira
Lilia
L
Soubrane
Olivier
O
Sogni
Philippe
P
Calmus
Yvon
Y
Chaussade
Stanislas
S
Rosenberg
Arielle R
AR
Podevin
Philippe
P
D002478:Cells, Cultured; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D019698:Hepatitis C, Chronic / Q000821:virology; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D008099:Liver / Q000166:cytology / Q000821:virology; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014779:Virus Replication
D012367:RNA, Viral