Detection of HCV RNA by the asymmetric gap ligase chain reaction

Abstract

The ligase chain reaction (LCR) and the gap ligase chain reaction (gLCR) are exponential amplification techniques for the detection of DNA sequences in a sample. Both techniques depend on the enzyme, DNA ligase, to join adjacent probes annealed to a DNA molecule. However, DNA ligase joins DNA inefficiency on an RNA target. Consequently, LCR and gLCR cannot amplify RNA efficiency. RNA detection methods using LCR or gLCR require a cDNA synthesis step. The carryover of four dNTPs from the cDNA reaction inhibits gLCR. Although LCR can use cDNA reaction products directly, background generated by blunt-end ligation does not allow the high sensitivity typically needed for HIV or HCV detection. The asymmetric gap ligase chain reaction (AGLCR) is a modification of gLCR that allows for the detection of RNA by using < or = 3 of the 4 nucleotides in the cDNA step and the gLCR step. Fewer than 50 copies of synthetic RNA transcript can be reproducibly detected. HCV, an RNA virus with no DNA intermediate, was chosen as the initial RNA model system. HCV antibody-positive and normal samples were analyzed, and the results were found to correlate with the results obtained using nested RNA-PCR. AGLCR provides a new nucleic acid amplification technique that can aid in the diagnosis of disease when the detection of RNA is critical.