Correlation of chronic periodontitis in tropical area and IFN-γ, IL-10, IL-17 levels.

OBJECTIVE: To evaluate the correlation of chronic periodontitis in tropical area and IFN-γ, IL-10 and IL-17 levels. METHODS: One hundred and forty-eight patients and one hundred and thirty-two healthy control subjects were included in the study. Clinical parameters (PI, GI and PD) and GCF levels of IFN-γ, IL-10 and IL-17 were measured at baseline, week 8, week 16 and week 24 after mechanical removal of dental plaque. IFN-γ and IL-10 were determined with ELISA methods and IL-17 was determined with the cytometric bead array. RESULTS: Removal of dental plaque resulted in improvement in all clinical parameters. Meanwhile, GCF IL-17 declined to control levels, while GCF IFN-γ and IL-10 levels remained unchanged. CONCLUSIONS: The decline of GCF IL-17 levels in patients with resolution of periodontitis suggests that IL-17 is involved in the periodontal inflammatory process.

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.

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.