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.

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. The presence of DNA from pathogens in the Candida species was detected using real-time PCR targeting of an internal transcribed spacer region of a fungal gene. The assay exhibited a low limit of detection (10 CFU/ml of blood), an excellent reproducibility and specificity. Twenty-eight positive samples exhibited a wide range of Candida species loads, extending from 13 to 90,528 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97.4%, respectively, compared with the results of blood culture. Our data suggest that this assay may be appropriate for use in clinical laboratories as a simple, low-cost and rapid screening test for the most frequently encountered Candida species.

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. The assay was validated with the Viral Quality Control panel, which also includes Chinese HCV RNA standards. RESULTS: The detection limit was 57 IU/mL, and a good linear correlation in the range of 102-108 IU/mL was revealed (r(2) = 0.999) with the novel method. This assay has a dynamic range of at least 8 log10 without the need for specimen dilution, good clinical intra- and inter-run precision, and excellent correlation with a commercially available assay(r(2) = 0.95). CONCLUSIONS: The high sensitivity, wide linear range, and good reproducibility, combined with low cost, make this novel quantitative HCV real-time PCR assay particularly well suited for application to clinical and epidemiological studies.

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. pIRES2-EGFP/PR39 and Ad-MSP/PR-39 were either transduced or infected into macrophage RAW264.7 cells for stable or transient expression of PR-39. PR-39 expression in macrophage cells was subsequently confirmed by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. Furthermore, its antimicrobial activity in macrophage cells was evaluated by colony enumeration assay. Results showed that the macrophage-specific promoter could initiate targeted expression of PR-39 only in macrophage RAW264.7 cells. Moreover, either stable or transient expression of PR-39 in macrophage cells conferred enhanced antimicrobial activity against Salmonella enterica serovar Typhimurium. Our results have demonstrated that macrophage-specific expression of antimicrobial peptide PR-39 in macrophages could inhibit the growth of intracellular S. Typhimurium and indicated it to be a novel and promising approach for the control of refractory intracellular infection.

[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. RESULTS: The bait vector was successfully constructed without toxicity, leakage and self-activation. Sixteen proteins were screened by YTHT and eight positive clones were identified by re-transformation in yeast. The interaction between RAR alpha-V and JTV-1 was confirmed by GST pull-down in vitro. CONCLUSIONS: There are some kinds of proteins interacting with RAR alpha-V in cell. The biological dysfunction caused by certain protein-protein interaction may be involved in the pathogenesis of leukemia.

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. (i) The duplex probe is simpler to synthesize and significantly easier to purify than TaqMan probe because that the fluorescent dye pair and the quencher pair are in different oligonucleotides. (ii) The method has high sensitivity, specificity, intra- and interassay reproducibilities. (iii) The assay has a quantitative dynamic range of 25 to10(9) genome copies per reaction mixture. (iv) The scorpion system can identify 98.6% samples in the validation panel without retest. There were 81 positive samples and 67 negative samples, which were confirmed by two FDA-approved NAATs (the Roche Amplicor PCR assay, Abbott LCR kit) and our new method. Any two positive results out of the possible three-comparator results would define the infected-patient gold standard. Of the positive samples, 79 (97.5%) were found positive (ranging from 31 to 227,648 copies/microl, M=4219 copies/microl), whereas no negative samples were found positive by the assay. A quantitative, fast, and easy-to-handle diagnostic approach such as the MOMP-based real-time PCR described here might improve the detection of C. trachomatis infection.