TaqMan实时荧光定量逆转录聚合酶链反应在强直性脊柱炎白细胞介素2受体mRNA检测中的应用价值  

Quantitative analysis of interleukine-2 receptor α mRNA expression in ankylosing sondylitis by fluorescence quantitative reverse transcription polymerase chain reaction and clinical significance

作　　者：骆方军[1] 吕建新[1] 

机构地区：[1]温州医学院检验医学院浙江省医学遗传学重点实验室,浙江325035

出　　处：《中华检验医学杂志》2006年第4期320-323,共4页Chinese Journal of Laboratory Medicine

基　　金：浙江省自然科学基金资助项目(NO.302769;NO.397550)

摘　　要：目的利用TaqMan实时荧光定量逆转录聚合酶链反应（FQ-RT-PCR）检测强直性脊柱炎（AS）患者外周血单个核细胞白细胞介素2受体α（IL-2Rα）mRNA的表达水平,并进行疾病的活动性相关分析.方法根据GenBank提供的序列设计一对引物和一条TaqMan探针.提取外周血单个核细胞（PBMC）总RNA,加入FQ-RT-PCR反应体系,产物切胶回收与pUCm-T载体连接,用T7RNA聚合酶转成cRNA,制备系列浓度参照物.对其特异性、线性、精密度和探针稳定性进行评价.定量HLA-B27阳性组与阴性组AS患者PBMC的IL-2Rα mRNA基因表达并探讨与血浆可溶性白细胞介素2受体（sIL-2R）的相互关系.结果成功构建了IL-2Rα mRNA的cRNA参照物.线性范围7～107 cells/ml,批内CV 8.4%,批间CV 9.6%.对各30例HLA-B27阳性与阴性AS的IL-2Rα mRNA检测表明,与正常对照组结果比较,阳性组与阴性组IL-2Rα mRNA和sIL-2R有明显差异（P〈0.01）.IL-2Rα mRNA对炎症活动评价的灵敏度为96.7%.结论 FQ-RT-PCR具有灵敏、特异、结果重复性好等优点;IL-2Rα mRNA比sIL-2R更能反映强直性脊柱炎患者的炎症活动程度.Objective To construct and evaluate the real-time fluorescence quantitative polymerase chain reaction for detecting IL-2Rα mRNA in ankylosing spondylitis （AS） based on TaqMan technique. Methods A pair of primers and a TaqMan probe were designed by sequence in GenBank. Total RNA isolated from the fresh peripheral blood monocytes （PBMC） of homo sapiens was amplified by the realtime FQ-RT-PCR. The product was collected by agarose gel electrophoresis and sequencing analysis then ligated with pUCm-T. The recombined plasmid was transcribed to cRNA by T7 RNA polymerase in order to prepare serial standard materials. A new method was created to quantify IL-2Rα mRNA in ideal condition. Sensitivity, reproducibility and efficiency were evaluated and used, combined with sIL-2R, for clinical application of AS. Results The linear range was （ 7 - 10^7 ） cells/ml. The intra-and-inter-assay coefficient variation was 8.4% and 9. 6% respectively. Recombined plasmid contained the target fragment was specific and accurate by BLAST. Standard reference was constructed successfully. The RT-PCR product in AS with HLA-B27 positive groups was higher than that with HLA-B27 negative groups and health controls （ P 〈 0.01 ） , HLA-B27 negative groups and HLA-B27 positive groups were not different （ P 〉 0. 05 ）. The sensitivity of IL-2Rα mRNA was 96.7% . Conclusions Real Time FQ-RT-PCR of IL-2Rα mRNA is constructed successfully. This is an easy, rapid, 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 administration of patients.

关 键 词：逆转录聚合酶链反应 脊柱炎 强直性 受体 白细胞介素2 

分 类 号：R593.23[医药卫生—内科学]