Sensitive and specific HBV genomic DNA detection using RCA-based QCM biosensor

Rolling circle amplification (RCA) is an isothermal amplification technique for small, circular DNA templates with the unique property of the product localization. This research describes the application of RCA-based quartz crystal microbalance (QCM) biosensor for direct detection of hepatitis B virus (HBV) genomic DNA from clinical samples. The covalent bonding between the capture probes and the gold electrode surface guarantees that the linkage between the capture probes and the amplified RCA products is maintained during the assay. Making use of the high amplification efficiency of Phi29 DNA polymerase and the remarkable precision of Escherichia coli DNA ligase in differentiating mismatched bases at the ligation site, as low as 104 copies/mL HBV genomic DNA can be detected. Experimental results show that RCA has significantly enhanced sensitivity for the target strand compared to the single-base mismatch strand. In this paper, with 60 min RCA duration, the proposed sensitive detection shows over 10 times amplification of the original signals. The combination of RCA with QCM biosensor has the potential to become a successful clinical application.