TaqMan—MGB实时荧光定量PCR检测RIG—G基因方法的建立及其方法学验证  

Establishment and evaluation based of a RIG-G gene detection system by TaqMan-MGB probe real-time PCR

作　　者：庞丽[1] 权文强[1] 吴军录[1] 姚懿雯[1] 李冬[1] 

机构地区：[1]同济大学附属同济医院检验科,上海200065

出　　处：《中华检验医学杂志》2016年第12期936-940,共5页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金（81272603,81472179）;上海申康医院发展中心课题（SHDC22014008）

摘　　要：目的：建立一种以TaqMan－MGB荧光探针为特点，检测人类急性早幼粒细胞白血病（ M3型）中维甲酸诱导基因G（ RIG－G）的实时荧光定量聚合酶链反应（ RT－qPCR）方法，并运用该方法对正常人、M3患者外周血中的RIG－G表达水平进行分析，探讨其在M3诊断中的价值。方法方法学建立。针对人RIG－G基因设计特异性引物和TaqMan－MGB荧光探针，以逆转录的互补DNA为模板，建立TaqMan－MGB实时荧光定量PCR检测方法和标准曲线；在方法特异性、正确度、精密度、分析灵敏度、干扰物质等各方面对检测方法的性能进行评估，并用该法验证20例M3患者外周血与骨髓中RIG－G表达水平相关性，同时检测40份正常人及20份M3患者标本，t检验比较两组外周血RIG－G水平，用ROC曲线分析其对M3的检测效能。结果 RIG－G标准品拷贝数log值与循环阈值数（ Ct ）的标准曲线方程为：Y＝－3．539X＋42．952，R^2＝0．999，呈良好的线性关系。用建立的实时荧光定量PCR方法检测标准品，结果与已知值比较，相关性系数r＝0．999，计算偏倚值均＜5％；分别选取高值（107拷贝／μl）、中值（104拷贝／μl）、低值（10^1拷贝／μl）3个浓度，批内CV分别为1．38％、2．31％、7．56％；批间CV分别为0．71％、1．17％、5．07％，均＜10％；该方法的分析灵敏度为101拷贝／μl。 M3患者外周血与骨髓中RIG－G表达水平相关系数为r＝0．996，斜率b＝0．973，结果相关性良好，t＝0．099，P＞0．05，显示两组标本检测结果差异无统计学意义。40份正常健康体检外周血RIG－G基因的拷贝数[3．62×10^4（1．61×10^4～4．90×10^4）拷贝／μl]与M3患者[7．10×10^2（5．43×10^2～2．21×10^3）拷贝／μl]差异有统计学意义（U＝18，P＜0．001）。结论成功建立了检测人外周血RIG－G基因的TaqMan－MGB荧光实时定量PCR方法。该法具有较好的正确Objective To establish a TaqMan-MGB fluorescent probe characterized real-time polymerase chain reaction （ qPCR） method for detecting retinoic acid induced genes G （ RIG-G） in human acute promyelocytic leukemia （ M3 ） .Analyze RIG-G expression levels in peripheral blood of both normal persons and M3 patients and explore its diagnosis value for M 3.Methods Methodology establishment study.A detection method and standard curve of TaqMan-MGB real-time PCR were established after designing specific primers and TaqMan-MGB fluorescence probe of human RIG-G gene and using reverse transcription complementary DNA （ cDNA） as a template.The performance of this method was evaluated in specificity, accuracy, precision, analytical sensitivity and interference substances . Twenty clinical specimens with M3 were quantified RIG-G expression so as to evaluate the correlation between peripheral blood and bone marrow samples .Meanwhile , the results of RIG-G expression in peripheral blood of 40 normal specimens and 20 patients with M3 were analyzed by t-test.And receiver-operating characteristic curve （ ROC ） was used to analyze the detection efficiency of M 3.Results There was a good linear relationship between log value of RIG-G standard substance and threshold cycle number （ Ct ） （ standard curve equation：Y=-3.539X＋42.952,R2 =0.999）.New method was used to detect standard substance . The deviation between observed and expected values was 〈5% （r=0.999）.Three concentration samples （107 ,104 ,101 copies/μl） were selected for precision test.Intra-assay coefficients of variation were 1.38%, 2.31% and 1.38%, respectively , and intre-assay coefficients of variation were 0.71%, 1.17% and 5.07%, separately.All were less than 10%.The sensitivity of this method was 10^1 copies/μl.There was a good correlation of RIG-G results between peripheral blood and bone marrow in M 3 patients（r=0.996, b=0.973）.But there was no significant difference between this two group results （t=0.099, P〉0.05）. Howe

关 键 词：实时聚合酶链反应 细胞内信号肽和蛋白质类 白血病 早幼粒细胞 急性 

分 类 号：R733.7[医药卫生—肿瘤] R730.43[医药卫生—临床医学]