Direct detection of hepatitis C virus (HCV) RNA from whole blood, and comparison with HCV RNA in plasma and peripheral blood mononuclear cells.
Hepatitis C virus (HCV) requires reverse transcriptase-polymerase chain reaction (RT-PCR) or branched DNA signal amplification assays to be detected in patient samples. Although conventional methods of RNA isolation are employed for samples of serum, plasma, and peripheral blood mononuclear cells (PBMCs), whole blood is generally considered an unsuitable source of RNA because of abundant RNases and polymerase inhibitors. Using a cationic surfactant, Catrimox-14, we adapted a procedure for RNA isolation from whole blood, plasma, and PBMCs that yields RNA template suitable for HCV RT-PCR. RNA isolation required less than 2 hr, and HCV sequences were easily detected in sample volumes of 50 microliters whole blood or plasma, and in less than 1 x 10(4) PBMC. Following the addition of blood to Catrimox, HCV RNA was stable in the mixture when incubated for at least 7 days at room temperature prior to RNA extraction. Comparison of whole blood HCV RNA and plasma HCV RNA from individuals with chronic hepatitis suggests that HDV RNA can be more reliably detected in whole blood. Three of 15 HCV antibody positive patients (20%) had HCV RNA present in whole blood but simultaneously obtained plasma samples were negative. Two of the HCV antibody negative individuals with chronic hepatitis contained HCV RNA in whole blood, yet one of these patient's plasma was negative for viral RNA. The Catrimox-14 method of RNA purification is useful for detecting HCV RNA in whole blood and blood subfractions, and provides a practical method of measuring plasma and PBMC HCV RNA from clinical specimens.
Schmidt
W N
WN
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, USA.
Klinzman
D
D
LaBrecque
D R
DR
Macfarlane
D E
DE
Stapleton
J T
JT
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D016174:Hepacivirus / Q000302:isolation & purification*; D006526:Hepatitis C / Q000097:blood / Q000821:virology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D008969:Molecular Sequence Data; D010949:Plasma / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood; D012680:Sensitivity and Specificity; D013501:Surface-Active Agents / Q000737:chemistry*; D014158:Transcription, Genetic; D050337:Trimethyl Ammonium Compounds
D017931:DNA Primers; D004279:DNA, Viral; D000644:Quaternary Ammonium Compounds; D012367:RNA, Viral; D013501:Surface-Active Agents; D050337:Trimethyl Ammonium Compounds; C009410:tetradecyltrimethylammonium