Quantitative Analysis of Interleukine-2 Receptor α mRNA Expression in Ankylosing Sondylitis by Fluorescence Quantitative Reverse Transcription Polymerase Chain Reaction and Clinical Significance

Objective:To construct and evaluate real-time fluorescence quantitative poiymerase chain reaction for detecting IL-2Ra mRNA in ankylosing sondylitis(AS) on TaqMan technique. Methods: A pair of primers and a probe with MGB were designed by Primer EXPress V2. O software program . Using the peripheral blood monocytes (PBMC) of homo sapiens as samples , total RNA was isolated from fresh PBMC . RNA was amplified by the real-time FQ-RT-PCR. The specificy of the recombined plasmid was tested by agarose gel eletrophoresis and sequencing analysis . IL-2Rα recombined plasmid was transcribed to cRNA by T7 RNA poiymerase for Serial Standard materials of FQ-RT-PCR. A new method was created to quantify IL-2Rα mRNA in ideal condition sensitivity, reproducibity and efficiency of the FQ-RT-PCR was evaluated and used ,combined with sIL-2R,for clinical application of AS. Results:Minimum limit of FQ-RT-PCR system was 7cells/ml and linear range from (7-107)cells/ml . The intra-and-inter-assay cofficient variation(CV) was 8. 4% and 9. 6% respectively. Recombined plasmid contained the target fragment was specificity and accuracy by BLAST. Standard reference was constructed successfully. RT-PCR product in AS with HLA-B27 positive groups were higher than HLA-B27 negtive groupsand health controls (P 0. 01), HLA-B27 negtive groups and HLA-B27 positive groups were not different (P 0. 05) . The sensitivity of IL-2Rα mRNA was 96. 7%. Conciusions:Real Time FQ-RT-PCR of JL-2Rα mRNA is constructed successfully. This is easy, rapid and more sensitive , accurate and reliable method for quantifing IL-2Rα mRNA. There is highly statistical significance,compared with sIL-2R ,on the expression of IL-2Rα mRNA and inflammatory states between AS and control group and effective information for adminstration of patients.