【摘要】 为了探讨以通用引物应用PCR技术扩增克隆性免疫球蛋白重链基因(IgH)重排的最佳实验条件及SYBRGreenⅠ实时定量PCR检测该基因的可行性,以通用引物对克隆性IgH重排进行扩增,对影响PCR扩增的退火温度、引物浓度、Taq酶用量、dNTP浓度、镁离子浓度、循环次数等实验因素进行了系统研究,找出最佳反应参数,并以通用引物进行了SYBRgreenⅠ实时定量PCR对IgH重排基因的检测,测定了该法检测IgH重排基因的敏感性。结果表明:最佳退火温度为60℃,最佳引物浓度为0.8μmol/L,0.5U的Taq酶量效果满意,最适dNTP浓度为100μmol/L,最适镁离子浓度为3.0mmol/L,最佳循环次数为40次。SYBRGreenⅠ实时定量PCR可以实现对克隆性IgH重排的扩增和荧光信号的检测分析,其对IgH重排基因检测的敏感性为104/ml。结论:确定了应用PCR技术检测克隆性IgH重排的最适反应条件,实现了用通用引物对克隆性IgH重排稳定、特异的扩增,初步实现了以通用引物和应用SYBRGreenⅠ实时定量PCR对IgH重排的检测。更多还原

【Abstract】 To investigate the optimal reaction conditions of clonal rearrangements of immunoglobulin heavy chain （IgH） gene using ploymerase chain reaction （PCR） with consensus primers and the feasibility of detecting this gene using SYBR greenⅠreal-time quantitative PCR with consensus primers, the systemic experiments were performed, which included annealing temperature, concentration of primer, the amount of Taq enzyme, concentration of dNTP and Mg²⁺, number of PCR cycles. Detection of this gene on SYBR greenⅠreal-time quantitative PCR with consensus primers was carried out under the optimal reaction parameters obtained from previous study, and the sensitivity of IgH rearrangements gene was examined by using SYBR greenⅠreal-time quantitative PCR. The results indicated that the optimal annealing temperature was 60℃, the optimal concentration of primers was 0.8 μmol/L, the satisfactory Taq enzyme amount was 0.5 U, the optimal concentration of dNTP was 100 μmol/L, the optimal concentration of Mg²⁺ was 3.0 mmol/L, the suitable number of cycle was 40 cycles. Amplification of IgH rearrangement gene and detection of desired gene fluorescence signal on SYBR greenⅠreal-time PCR were performed. The sensitivity of IgH gene using this quantitative PCR was 10⁴/ml. It is concluded that the optimal reaction parameters for amplification of clonal IgH rearrangements gene by using PCR technique was determined, and stable and specific amplification of desired gene with consensus primers was performed. Basically, IgH rearrangemen gene was successfully detected by SYBR greenⅠreal-time PCR.更多还原