Novel, ligation-dependent PCR assay for detection of hepatitis C in serum.
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.
Department of Pathology, Mount Sinai Hospital, New York 10029, USA.
T C
TC
Hsuih
Y N
YN
Park
C
C
Zaretsky
F
F
Wu
S
S
Tyagi
F R
FR
Kramer
R
R
Sperling
D Y
DY
Zhang
D001483:Base Sequence; D011088:DNA Ligases / Q000494:pharmacology; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D014766:Viremia / Q000821:virology*
D012367:RNA, Viral; D011088:DNA Ligases