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度、精密度、灵敏度及特异性，能够为临床M3患者的早期诊断及治疗监测提供必要帮助。
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）. However , there was obvious difference of RIG-G value in peripheral blood between control group and M 3 patient group （U=18,P〈0.001）, 3.62 ×10^4（1.61 ×10^4 -4.90 ×10^4）copies/μl for controls and 7.10 ×10^2 （5.43 ×10^2 -2.21 ×10^3 ） copies/μl for M3 patients, respectively.Conclusions Successfully establishe a TaqMan-MGB real-time PCR method for detecting RIG-G gene in peripheral blood.The accuracy, precision, sensitivity and specificity are good .It could provide necessary help in early diagnosis and monitor treatment of clinical M3 patients.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
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）
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
Real-time polymerase chain reaction
Intracellular signalling peptides and proteins
Leukemia, acute promyeloeytic
