NOVEL, LIGATION-DEPENDENT PCR ASSAY FOR DETECTION OF HEPATITIS-C VIRUS IN SERUM

A simple, sensitive, and specific ligation-dependent PCR (LD-PCR) meth od for the detection of hepatitis C virus (HCV) RNA in serum is descri bed. The assay uses two DNA capture probes for RNA isolation and two D NA hemiprobes for subsequent PCR. Each capture probe has a 3' sequence complementary to the conserved 5' untranslated region of HCV RNA and a biotin moiety at the 5' end capable of interacting with streptavidin -coated paramagnetic bends. Each hemiprobe contains a sequence complem entary to the 5' untranslated region in juxtaposition to one another a nd a common sequence for PCR primer binding. In guanidinium thiocyanat e solutions, the capture probes and the hemiprobes form a hybrid with their target, and the hybrid can be isolated from serum by the binding of the capture probes to the paramagnetic beads in the presence of a magnetic field. The hemiprobes can then be linked to each other by inc ubation with T4 DNA ligase to form a full probe that serves as a templ ate for a PCR. When serial 10-fold dilutions of synthetic HCV RNA (10( 7) to 10 molecules) were tested, there was a good correlation between the amount of PCR product and the initial number of RNA molecules, wit h a sensitivity of 100 HCV RNA molecules per reaction. Twenty-four spe cimens that had been tested by either a branched DNA probe (bDNA) assa y (13 specimens) or a reverse transcription PCR (RT-PCR) assay (11 spe cimens) were also analyzed by LD-PCR. The results showed a good correl ation among LD-PCR, RT-PCR, and the bDNA assay, However, both LD-PCR a nd RT-PCR were more sensitive than the bDNA assay when the HCV titer w as low.