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

J Clin Microbiol. 1996 Mar;34(3):501-7. doi: 10.1128/jcm.34.3.501-507.1996.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • DNA Ligases / pharmacology
  • Hepacivirus / genetics
  • Hepacivirus / isolation & purification*
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction*
  • RNA, Viral / analysis
  • Sensitivity and Specificity
  • Viremia / virology*

Substances

  • RNA, Viral
  • DNA Ligases