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Journal of Clinical Microbiology, Mar 1996, 501-507, Vol 34, No. 3
Copyright © 1996 by the American Society for Microbiology. All rights reserved.

Novel, ligation-dependent PCR assay for detection of hepatitis C in serum

TC Hsuih, YN Park, C Zaretsky, F Wu, S Tyagi, FR Kramer, R Sperling and DY Zhang
Department of Pathology, Mount Sinai Hospital, New York 10029, USA.

A simple, sensitive, and specific ligation-dependent PCR (LD-PCR) method for the detection of hepatitis C virus (HCV) RNA in serum is described. The assay uses two DNA capture probes for RNA isolation and two DNA 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 beads. Each hemiprobe contains a sequence complementary to the 5' untranslated region in juxtaposition to one another and a common sequence for PCR primer binding. In guanidinium thiocyanate 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 incubation with T4 DNA ligase to form a full probe that serves as a template 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, with a sensitivity of 100 HCV RNA molecules per reaction. Twenty-four specimens that had been tested by either a branched DNA probe (bDNA) assay (13 specimens) or a reverse transcription PCR (RT-PCR) assay (11 specimens) were also analyzed by LD-PCR. The results showed a good correlation among LD-PCR, RT-PCR, and the bDNA assay. However, both LD-PCR and RT-PCR were more sensitive than the bDNA assay when the HCV titer was low.

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