Development And Clinical Applications Of Hepatitis B Virus And Chlamydia Trachomatis DNA Quantitative Detection By Duplex Mutation Primers

Real-time polymerase chain reaction (real-time PCR) assay can be characterized as specific or nonspecific. The nonspecific dsDNA binding dyes methods are prone to"false positives"in that undesired products such as primer dimers and other spurious amplicons can increase fluorescence. The specific probe-based methods confer additional specificity to the detection of the amplification products and are widely used in nucleic acids-based research and clinical molecular diagnostics. Many of the probe-based systems rely on the principle of fluorescence resonance energy transfer (FRET) for signal generation and well-known examples include TaqMan, molecular beacons, scorpion primers, and amplifluor. These multilabeled probes have a fluorophore and quencher placed at their termini and/or located within their nucleotide sequence. The structure of these probes brings fluorophore and quencher moieties into close proximity so that FRET takes place. When separation of the dye pair FRET is destroyed, fluorescence is produced. Although more or less satisfactory for most common uses, these probes are difficult to design, synthesize and purify and are, therefore, expensive. The synthesis and purification of high-quality multilabeled FRET oligonucleotides are often neither trivial nor inexpensive relative to the fluorescent dye pair, and the pair of fluorophore and quencher are in the same oligonucleotide. This prompted scientists to design a probe with a fluorescent dye pair and a quencher pair in different oligonucleotides such as hybridization probes, displacing probes. But both hybridization probes and displacing probes are 5'-terminal labeled with fluorophore or quencher and blocked from extension with a 3'-phosphate group thus they are not a real single-labeled probes, and the costs are increased.Therefore, we have devised a new method based on a primer with a fluorophor attached at its 5'-end, is annealed to a complementary oligonucleotide of a single-base mismatched labeled with a quencher at 3'-end. The duplex mutation primers are more specific than double-strand DNA dyes and, unlike other internal probes, do not require the synthesis of a quencher on the same molecule. It offers several advantages over other multi-labeled probes for real-time detection of amplification products. First, the primers are natural'hot start'primers, since they are duplex form and cannot hybridize with each other at lower temperature than their Tm values. This also minimizes non-specific annealing in course of amplification. Second, the long double-stranded region of the duplex mutation primer is more stable; therefore, the background signal is much lower. Other characteristic of the duplex mutation primers is that after primer-target hybridization, the distance between the fluorophore and the quencher changes from close proximity to totally free separation. This property endows them with much higher sensitivity than those of other internal FRET probes. Third, the fluorophore and quencher are attached to two separate strands. This makes the synthesis of the FRET probe less costly and permits the convenient design.A novel real-time PCR method with the duplex mutation primers was established, and clinical performance of the new real-time PCR assay for hepatitis B virus (HBV) and Chlamydia trachomatis (CT) infection detection was evaluated.The high sensitivity, wide linear range, and good reproducibility, combined with low cost, make this novel real-time PCR technique particularly well suited for application in clinical and epidemiological studies.