sybr-green-i and Acute-Disease

Human astrovirus (HAstV) has been recognized as the second most common cause of diarrhoea among children under 5 years old. To date, the true incidence of HAstV was underestimated when using enzyme immunoabsorbent assays (EIAs) and conventional reverse transcription (RT)-polymerase chain reaction (PCR) methods. The sensitivity of detection of EIA is insufficient and, although RT-PCR is more sensitive than EIA, the time required is a limitation for astrovirus detection. The aim of the study was to develop a real-time RT-PCR method in order to increase the sensitivity, to quantify the viral load and to minimize the time required for HAstV detection. The real-time RT-PCR reported here requires only one rapid step to obtain a high sensitivity (0.0052 infectious units (IU) (0.0026 IU/microl)) in all human astrovirus detected. The real-time RT-PCR detected IUs down to a 10(-6) dilution with an improvement in the detection limit of factor 10(4), whereas the conventional RT-PCR detected down to IUs 10(-2) dilution. This process is able to reduce the time of the assay and avoids the risk of contamination. The method described below has been validated with a panel of 100 clinical samples and the results obtained confirmed the high specificity of the assay; consequently, the application of this assay for molecular diagnosis is feasible as a versatile tool for ascertaining the true implication of HAstV in acute viral gastroenteritis.

Topics: Acute Disease; Astroviridae Infections; Benzothiazoles; Child, Preschool; Diamines; Feasibility Studies; Fluorescent Dyes; Gastroenteritis; Humans; Incidence; Indicator Dilution Techniques; Mamastrovirus; Nucleic Acid Denaturation; Organic Chemicals; Quinolines; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity; Seroepidemiologic Studies; Serotyping; Temperature

To detect the expression of CD95 and CD95L mRNAs after liver transplantation and investigate the role of CD95 and CD95L in acute liver allograft rejection.. The expressions of CD95 and CD95L mRNAs of peripheral blood lymphocyte from 56 liver allograft recipients were examined using SYBR real-time PCR.. CD95 and CD95L mRNA levels in the recipients with acute rejection were significantly higher than those without rejection (P<0.01), and the elevation occurred about 2 days earlier than that of alanine aminotransferase and aspartate aminotransferase.. CD95 and CD95L are related to acute liver allograft rejection and their mRNA expression level may serve as an indicator for prediction and diagnosis of acute rejection episodes.

Topics: Acute Disease; Adult; Aged; Benzothiazoles; Diamines; Fas Ligand Protein; fas Receptor; Female; Gene Expression; Graft Rejection; Humans; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Lymphocytes; Male; Middle Aged; Organic Chemicals; Polymerase Chain Reaction; Quinolines; RNA, Messenger

Interleukin-6 (IL-6) is a pleiotropic acute reactant cytokine involved in inflammatory responses. To explore the role of IL-6 in inflammation, this study examined the efficacy of exogenous IL-6 in preventing intestinal ischemia/reperfusion (I/R) injury associated with small bowel transplantation (SBTx). Syngenic orthotopic SBTx was performed in Lewis rats after 6-h graft preservation in University of Wisconsin (UW) at 4 degrees C. IL-6 mutein (IL-6m, 500 microg/kg), a recombinant molecular variant of human IL-6, was subcutaneously given to donors 2 h before harvesting (IL-6mD) or to excised grafts by luminal infusion (IL-6mG). Animal survival was 100% and 75% in IL-6mD (p<0.05 vs. control) and IL-6mG groups, respectively, compared with 64.3% in untreated controls. The severity of I/R injury (e.g. epithelial denudation, villous congestion) was reduced with IL-6m, in addition to a striking increase in re-epithelization. With IL-6m, neutrophil extravasation was markedly reduced in intestinal grafts and in remote organs (e.g. lung). IL-6m mediated anti-inflammatory effects through the inhibition of I/R-induced up-regulation of intragraft and circulating IL-1-beta, tumor necrosis factor-alpha (TNF-alpha) and IL-6. IL-6m also increased intestinal graft tissue blood flow. These results show that IL-6 is effective in protecting the intestine from cold I/R injury by maintaining graft blood flow and reducing pro-inflammatory cytokine up-regulation and neutrophil infiltration.

Topics: Acute Disease; Animals; Benzothiazoles; Blotting, Western; Cytokines; Diamines; Immunohistochemistry; Inflammation; Interleukin-1; Interleukin-6; Intestinal Mucosa; Intestines; Lung; Male; Mutation; Neutrophils; Organ Transplantation; Organic Chemicals; Peroxidase; Quinolines; Rats; Rats, Inbred Lew; Reperfusion Injury; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Time Factors; Transcription Factors; Tumor Necrosis Factor-alpha; Up-Regulation

A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 10(7) PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.

Topics: Acute Disease; Animals; Antibodies, Viral; Benzothiazoles; Cell Line; Dengue; Dengue Virus; Diamines; DNA Primers; Fluorescent Dyes; Humans; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Serotyping; Species Specificity