Protocol for the use of self-reporting duplex mutation primers to detect PCR products in the diagnosis of HBV.

Quantitative measurements of serum hepatitis B virus (HBV) DNA are useful for tailoring of treatment schedules and the monitoring of HBV replication during therapy. We developed a novel fluorescence-based quantitative real-time PCR for quantitating HBV DNA based on the duplex mutation primers principle, in which signal is generated by melting a duplex mutation primer during renaturation. The duplex mutation primers are much more specific than double-stranded DNA dyes like SYBR Green I and, unlike other probes, do not require the double-labeled synthesis of fluorophore and quencher on the same molecule.

Use of duplex mutation primers for real-time PCR quantification of hepatitis C virus RNA in serum.

Background: The duplex mutation primers offer many advantages over other multi-labeled probes for real-time detection of amplification products.

Objectives: To develop and validate a novel real-time PCR for quantification of HCV RNA based on the duplex mutation primers technology.

Materials And Methods: The duplex mutation primers were selected in the highly conservative 5' non-coding region (5'NCR) of the HCV RNA.

Development of a novel quantitative real-time assay using duplex mutation primers for rapid detection of Candida species.

We developed a novel quantitative real-time PCR for quantitating Candida DNA based on the duplex mutation primer principle, in which a signal is generated by melting a duplex mutation primer during renaturation. The duplex mutation primers are much more specific than double-stranded DNA dyes such as SYBR-Green I and, unlike other probes, do not require the double-labeled synthesis of fluorophore and a quencher on the same molecule. A total of 176 clinical blood specimens were obtained from patients hospitalized in our hospital with clinically proven or suspected systemic Candida infection.

Macrophage-specific overexpression of antimicrobial peptide PR-39 inhibits intracellular growth of Salmonella enterica serovar Typhimurium in macrophage cells.

Although purified and synthesised PR-39 shows potent antibacterial effects in vitro, its ability to kill intracellular bacteria in macrophages, which are a major cause of refractory intracellular infection, has not yet been demonstrated. Both to enhance its antimicrobial potential and to reduce systemic side effects, it would be desirable to deliver PR-39 into macrophage cells and to limit its activation to the site of infection. To address this issue, PR-39 DNA was inserted into the eukaryotic expression plasmid pIRES2-EGFP and the adenoviral vector Ad-MSP, from which PR-39 can be specifically expressed in macrophage cells from the macrophage-specific promoter.

[Screening and identification of proteins interacting with RAR alpha-V via yeast two-hybrid system].

Objective: To screen the protein interacting with retinoic acid receptor variant protein (RAR alpha-V) via the yeast two-hybrid technique (YTHT) and to find out the targets protein and study its biological function.

Methods: The bait vector of pGBKT7-RAR alpha-V was constructed for screening proteins interacting with RAR alpha-V in K562 cell cDNA expression library via YTHT. The protein-protein interaction was confirmed with re-transformation in yeast and GST pull-down in vitro.

Development of a novel quantitative real-time assay using duplex scorpion primer for detection of Chlamydia trachomatis.

A novel quantitative real-time PCR method using the duplex scorpion primer for detection of Chlamydia trachomatis DNA was developed and validated. The assay employs a duplex primer; its most important feature is the intramolecular probe-target interaction. The assay had many prominent characteristics.