Assays for viral sequences and their value in validation of viral elimination.
Plasma pools and blood products were analysed for hepatitis C virus (HCV) RNA by the polymerase chain reaction (PCR). Initially, samples were amplified by "nested" PCR using two pairs of primers from the highly-conserved 5' non-coding region of the genome. The method was later modified to amplification with only the outer pair of primers and a "hot-start" to eliminate amplification of non-target sequences. The modified method was as sensitive as "nested" PCR. However, with samples containing low levels of RNA, it is necessary to perform repeat amplifications to avoid false-negative results. Ten out of 17 anti-HCV positive pools from paid donors were positive, while none of the 12% pools from unpaid donors was positive for RNA or anti-HCV antibodies. Four i.v. immunoglobulins made from contaminated pools were virus-free, indicating removal of virus during current manufacturing processes. A quantitative PCR method was established using a positive donation. Probit analysis gave an estimated viral titre of 3.6 x 10(6) genomes/ml.
Saldanha
J
J
N.I.B.S.C., Division of Virology, Potters Bar, Herts, UK.
D001483:Base Sequence; D001688:Biological Products / Q000592:standards*; D001769:Blood / Q000382:microbiology; D001782:Blood Donors; D004340:Drug Contamination / Q000517:prevention & control; D005188:False Negative Reactions; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000517:prevention & control / Q000635:transmission; D006801:Humans; D016756:Immunoglobulins, Intravenous / Q000302:isolation & purification; D008403:Mass Screening / Q000379:methods*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012367:RNA, Viral / Q000097:blood*; D012680:Sensitivity and Specificity; D014838:Volunteers
D001688:Biological Products; D016756:Immunoglobulins, Intravenous; D012367:RNA, Viral