ABSTRACT
Objective: To develop a detection method for single nucleotide polymorphisms (SNPs) of bronchial asthma (BA) susceptibility genes (IL-13, IL-33, and GSDMA) based on fluorescence PCR melting curves.Methods: Peripheral blood samples from 33 patients with BA were collected. DNA was extracted, and positive plasmids were constructed. Probes and primers for fluorescence polymerase chain reaction (PCR) were designed according to IL-13, IL-33, and GSDMA sequences, and the SNPs were separately detected by gene sequencing and fluorescence PCR melting curve.Results: The system was successfully divided into 3 SNPs, including IL-13, IL-33, and GSDMA, and a comparison of sequencing methods showed that the results were completely consistent. The lowest detection limit was 1 ng/reaction, the sensitivity and specificity were 100%, and this method had high repeatability (CV = 2.8%).Conclusion: The fluorescence PCR melting curve method is suitable for the rapid and accurate classification of SNPs. The method is economical, simple, and efficient, and is suitable for the screening of the susceptible gene SNPs in a large-scale population of patients with BA.
Subject(s)
Asthma/diagnosis , Genetic Testing/methods , Genotyping Techniques/methods , Mass Screening/methods , Polymerase Chain Reaction/methods , Adolescent , Adult , Asthma/blood , Asthma/genetics , Feasibility Studies , Female , Fluorescence , Genetic Predisposition to Disease , Genotyping Techniques/economics , Humans , Interleukin-13/genetics , Interleukin-33/genetics , Male , Mass Screening/economics , Neoplasm Proteins/genetics , Polymerase Chain Reaction/economics , Polymorphism, Single Nucleotide , Sensitivity and Specificity , Young AdultABSTRACT
<p><b>OBJECTIVE</b>PPAR-gamma is associated with the differentiation, apoptosis, proliferation and cytokine secretion of immunologic cells. This study investigated peripheral blood lymphoblastic PPAR-gamma mRNA expression and its correlation with plasma IL-13 contents in children with acute idiopathic thrombocytopenic purpura (ITP).</p><p><b>METHODS</b>Fifty-three children with acute ITP who were in line with the standard test between September 2007 and July 2008 were enrolled. Fifty healthy children during the same period were used as the control group. PPAR-gamma mRNA expression in peripheral blood lymphocytes were detected by RT-PCR. Plasma IL-13 contents were detected using ELISA.</p><p><b>RESULTS</b>PPAR-gamma mRNA expression in peripheral blood lymphocytes from acute ITP children were significantly higher than that in the control group (0.78 +/- 0.03 vs 0.52 +/- 0.05; P< 0.05). Plasma IL-13 contents in children with acute ITP were also significantly higher than those in the control group (160.21 +/- 34.26 pg/mL vs 121.42 +/- 12.69 pg/mL; P< 0.05). There was a positive correlation between plasma IL-13 level and lymphoblastic PPAR-gamma mRNA expression in children with ITP (r=0.89, P< 0.05).</p><p><b>CONCLUSIONS</b>PPAR-gamma mRNA expression in peripheral blood lymphocytes increased and were positively correlated with plasma IL-13 contents in children with acute ITP, suggesting that PPAR-gamma and IL-13 might participate in the pathogenesis of acute ITP.</p>