原发性干燥综合征自身抗原SSBmRNA荧光定量检测方法的建立  

Establishment of fluorogenic quantitative method for measurement of SSB mRNA gene expression in Sjgren syndrome patients

作　　者：张慧[1] 孙静慧[2] 耿红莲[3] 周琳[3] 范列英[1] 刘皋林[2] 孔宪涛[3] 谭龙益[3] 

机构地区：[1]上海市东方医院检验科,上海200120 [2]第二军医大学长征医院临床药理研究室 [3]第二军医大学长征医院实验诊断科

出　　处：《临床检验杂志》2006年第6期404-406,共3页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金资助(基金编号:30271180)

摘　　要：目的建立检测原发性干燥综合征(PSS)自身抗原SSBmRNA荧光定量的方法,测定SSB基因表达水平。方法基于TaqMan荧光探针技术,以克隆载体pGEM-T-SSB作为定量模板,在GeneAmp5700型检测仪上通过Ct值定量起始模板,建立实时荧光定量RT-PCR方法;检测20名PSS患者外周血单个核细胞(PBMC)中SSBmRNA的表达。结果本方法的动态检测范围为104～108拷贝/μgRNA(r2≥0.997);低值的批内、批间变异系数(CV)分别为6.8%和16.4%,高值的批内、批间的CV分别为7.6%和18.9%;20名PSS患者外周血中均有SSBmRNA的表达,范围为4.65×105～8.49×106拷贝/μgRNA,均值为4.80×106拷贝/μgRNA,20例健康体检者的SSBmRNA的表达范围为2.71×103～1.30×104拷贝/μgRNA,均值1.92×103拷贝/μgRNA,两组相差显著(P<0.01)。结论成功建立了PSS自身抗原SSB基因表达含量的荧光定量检测方法,检测结果可用拷贝数表示,定量准确可靠。Objective To establish a specific fluorogenic quantitative method for detecting the expression of SSB mRNA in peripheral blood mononuclear cells （PBMCs） of primary Sj ？ gren syndrome （pSS） patients. Methods A real-time fluorescent quantitative reverse transcription PCR（FQ-RT-PCR） was set up based on fluorescent TaqMan methodology. In this method prokaryotic expressing vector pGEM-T was used as standard plasmid and RNA quantification was based on the threshold cycle（Ct） values using GeneAmp 5700 Sequence Detection Systems to examine the specific expression of SSB mRNA in PBMCs. Results The dynamic range of the assay was 10^4 to 10^8 copiesμg RNA,and that of endogenous standards was 10^3 to 10^8 copies/μg RNA. The intra-assay and inter-assay precision for low value was 6. 83% and 16.36% respectively,and was 7. 64% and 18.9% for high value respectively. In 20 patients with primary SjOgren syndrome the SSB mRNA expression range was from 4.65 × 10^5 to 8.49 × 10^6 copies/μg RNA and the mean value was 4.8× 10^6opies/μg RNA. The mean of SSB mRNA copy number in pSS group was significantly higher than that in control. Conclusion The detection for SSB gene expression with FQ-PCR may obtain accurate, speedy results.

关 键 词：原发性干燥综合征 实时荧光定量PCR 自身抗原 

分 类 号：R593.2[医药卫生—内科学]