Application of Single‐labelled Probe‐primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

Abstract

AbstractA new method based on the incorporation of a single‐labelled probeprimer into polymerase chain reaction (PCR) for the detection of PCR‐amplified DNA in a closed system is reported. The probeprimer consists of a specific probe sequence on the 5′ ‐end and a primer sequence on the 3′‐end. A fluorophore is located at the 5′‐end. The primer‐quencher is an oligonucleotide, which is complementary to the probe sequence of probe‐primer and labelled with a quencher at the 3′‐end. In the duplex formed by probe‐primer and primer‐quencher, the fluorophore and quencher are kept in close proximity to each other. Therefore the fluorescence is quenched. During PCR amplification, the specific probe sequence of probe‐primer binds to its complement within the same strand of DNA, and is cleaved by Taq DNA polymerase, resulting in the restoration of fluorescence. This system has the same energy transfer mechanism as molecular beacons, and a good quenching efficiency can be ensured. Following optimization of PCR conditions, this method was used to detect hepatitis B virus (HBV) DNA in patient sera. This technology eliminates the risk of carry‐over contamination, simplifies the amplification assay and opens up new possibilities for the real‐time detection of the amplified DNA.