CAPTURE AND RT-PCR OF HEPATITIS-C VIRUS-RNA WITH SAFETY PRIMERS

The principle and practice of the polymerase chain reaction (PCR) has had a major impact on medical research. This is a powerful method but it does have its limitations, especially for clinical diagnostic work. We describe some improvements of hepatitis C virus (HCV) amplificatio n such as simplification of specimen preparation, elimination of false negative reactions influenced by point mutations, and fluorimetric de tection. The aim of the method is to make the procedure as easy and as inexpensive as possible for routine laboratories and for blood screen ing. After rapid chemical denaturation of the clinical specimen with g uanidine thiocyanate and simultaneous hybridization of biotinylated pr imers to template HCV RNA, the product was fixed to streptavidin-coate d magnetic beads and potential inhibitors were removed in easy washing steps. To eliminate the influence of point mutations within the prime r binding sites, primer sets with different lengths at their 3'-end we re developed for capture, reverse transcription, and amplification of genomic fragments by PCR. Positive results were identified by fluoresc ence staining. The low cost of the method allows the quantitation of t emplates by testing of dilution series as is common in microbiological laboratories.