Improved methods for quantification of human immunodeficiency virus type 1 RNA and hepatitis C virus RNA in blood using spin column technology and chemiluminescent assays of PCR products.
The quantification of human immunodeficiency virus type 1 (HIV-1) RNA or hepatitis C virus (HCV) RNA has been facilitated by adapting a spin column procedure for sample preparation and the use of chemiluminescent detection of polymerase chain reaction (PCR) products in microtiter plate format. All materials were commercially available and relatively inexpensive. By making a single dilution prior to amplification, concentrations of 500 copies to 2.5 million HIV-1 1 RNA copies per mL and 1,000 copies to 50 million HCV RNA copies per mL could be determined on 140-microL samples. Between-run imprecision employing the improved procedure for HIV-1 RNA was 23%. Correlation of HIV-1 RNA concentrations obtained using chemiluminescent detection with values obtained by colorimetric assay of PCR products was 0.98. Correlation of HCV RNA concentration determined by the spin column-chemiluminescent assay procedure with those obtained by branched DNA methodology was 0.91. Spin columns could be used with serum or plasma containing acid-citrate-dextrose or heparin anticoagulant, but heparinized samples required treatment with heparinase prior to amplification.
Lin
H J
HJ
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030, USA.
Tanwandee
T
T
Hollinger
F B
FB
D000925:Anticoagulants / Q000494:pharmacology; D004247:DNA / Q000032:analysis; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis*; D015497:HIV-1 / Q000302:isolation & purification*; D016174:Hepacivirus / Q000302:isolation & purification*; D006493:Heparin / Q000494:pharmacology; D019763:Heparin Lyase; D006526:Hepatitis C / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011133:Polysaccharide-Lyases / Q000378:metabolism; D012367:RNA, Viral / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*
D000925:Anticoagulants; D012367:RNA, Viral; D006493:Heparin; D004247:DNA; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase