CRLF2实时定量PCR检测方法的建立及初步应用

Establishment and preliminary application of real time PCR assay for quantitative detection of CRLF2

目的建立人细胞因子受体样因子2（CRLF2）实时定量PCR检测方法。方法设计特异性引物扩增目的基因CRLF2及管家基因ABL,将纯化的PCR产物进行TA克隆,经菌落PCR筛选并测序鉴定后,提取重组质粒DNA,紫外分光光度计检测浓度换算成copies/mL浓度,稀释制备成质粒标准品,制作标准曲线观察灵敏度和线性范围,同时对质粒标准品的稳定性进行评估。初步应用该方法检测10例健康儿童和10例急性淋巴细胞白血病（ALL）初诊儿童的骨髓单个核细胞CRLF2水平。结果 CRLF2PCR产物具有单一特异的熔解曲线;标准品的线性检测范围为103~108 copies/mL;质粒标准品反复冻融3次仍旧稳定;临床标本的CRLF2水平均在标准品线性检测范围。结论该实验室建立的CRLF2实时定量PCR检测方法具有良好的特异性、线性范围和稳定性,可应用于临床ALL儿童CRLF2基因的定量检测。

Objective To establish a real-time quantitative PCR method for the detection of cytokine receptor-like factor 2（CRLF2）expression.Methods Specific primers amplification target gene CRLF2 and housekeeping genes ABL were designed,the purified PCR products were performed the TA cloning.After bacterial colony PCR screening and sequencing,then the recombinant plasmids DNA was extracted and measured by using UV spectrophotometer and converted to copies/mL concentration.Finally it was diluted for preparing the plasmid standard substance,then the standard curve was drawn for observing the sensitivity and linear rang,meanwhile the stability of the plasmid DNA was evaluated.This method was initially applied to detect the CRLF2 level of bone marrow mononuclear cells in 10 cases of healthy children and 10 cases of newly diagnosed acute lymphoblastic leukemia（ALL）.Results CRLF2 PCR product had a single specific melting curve;the linear detection range of the standard substance was103- 108copies/ml;the plasmid standard substance by freeze-thawing for 3times remained stable;the CRLF2 level of clinical sample was within the linear detection range of standard substance.Conclusion The real-time quantitative PCR method for CRLF2 established by our laboratory has good specificity,linearity range and stability,which can be applied to the quantitative detection of CRLF2 gene in clinical ALL children.