南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (07): 1009-1013.

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实时荧光定量PCR检测外周血TREC的方法研究

阴继霞； 武大林； 许文娟； 孙竞；   

  1. 南方医科大学南方医院医学中心实验科； 南方医科大学南方医院医学中心实验科 广东广州510515； 广东广州510515；
  • 出版日期:2006-07-20 发布日期:2006-07-20

Real-time fluorescent quantitative PCR for detection of peripheral blood T-cell receptor excision circles

YIN Ji-xia, WU Da-lin, XU Wen-juan, SUN Jing Medical Laboratory Center, Nangfang Hospital, Southern Medical University, Guangzhou 510515, China   

  1. 南方医科大学南方医院医学中心实验科； 南方医科大学南方医院医学中心实验科 广东广州510515； 广东广州510515；
  • Online:2006-07-20 Published:2006-07-20

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摘要： 目的建立及优化检测移植后患者外周血TREC拷贝数的实时荧光定量PCR(FQ-PCR)的方法及实验影响条件,为临床研究造血干细胞移植的免疫功能重建机制提供方法学基础。方法在ABI7000PCR仪上实时扩增检测构建的TREC质粒标准品,摸索最佳扩增条件及建立标准曲线,然后用优化的FQ-PCR方法检测临床样本。结果T3、T4引物和R3、R4引物的扩增效率分别优于T1、T2引物和R1、R2引物。TaqMan-MGB探针比普通的TaqMan探针扩增效率高。金牌热启动Taq酶,比较普通Taq酶而言,提高了PCR的特异性和敏感性。FQ-PCR的反应条件设定:95℃10min后95℃5s,53℃30s,40循环结束。结论通过摸索与优化实验条件,建立了实时荧光定量PCR检测外周血单个核细胞中TREC的方法。 

Abstract: Objective To develop and optimize real-time florescence quantitative PCR (FQ-PCR) with the housekeeping gene RAG2 as cell number control to quantify T-cell receptor excision circle (TREC) in the peripheral blood. Methods The real-time PCR system for amplifying TREC and RAG2 genes was established on the basis of ABI 7000 apparatus using Golden Taq system, designed primers, TaqMan-MGB probes and optimized buffer. PCR conditions were optimized with standard samples of TREC plasmid. Results The amplification with the primer pair T3 and T4 was more efficient than that with T1 and T2. More specific and efficient amplification in FQ-PCR was achieved using TaqMan-MGB probes as compared with general Taq-Man probes. Golden Taq was more effective than general Taq in improving the specificity and decreasing the artifact, and 95 ℃ for 10 min, 95 ℃ for 5 s, and 53 ℃ for 30 s for a total of 40 cycles using ABI7000 was found as the optimized thermal parameter setting. Conclusion An optimized real-time PCR protocol for detecting TREC in peripheral blood mononuclear cells is established. 

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