实时荧光定量PCR测定胸腺近期输出功能方法的建立和应用

Estabitishment and application of real-time fluorescence quantitative PCR for detecting recent thymic output function

目的 建立一种准确、快速的检测胸腺输出功能的方法。方法 根据TCRδ基因序列，设计引物和探针，建立实时荧光定量PCR检测T细胞受体重排切除环（TRECs）的方法；并用于检测正常人及慢乙肝PBMCs中TRECs的含量。结果 建立了测定TRECs含量的实时荧光定量PCR方法，产物与预计长度相符，序列正确；最低可扩增出5 copies的模板；重复五次，Ct值的变异系数为1．06％。正常人21-45岁组的TRECs含量为（7767．4±2369．5）copies／10^6 PBMCs，16-20岁组为（28374．4±7820．4）copies／10^6 PBMCs，21-45岁慢乙肝组（6480．9±2031．2）copies／10^6 PBMCs，正常人21-45岁组分别与16-20岁组、同龄慢乙肝组比较差异均有统计学意义，P〈0．05。结论 实时荧光定量PCR检测TRECs的方法特异性强，灵敏度高，重复性好。正常人21-45岁组的胸腺输出功能低于16-21岁组，慢乙肝组低于同龄正常人。

Objective To establish an accurate and efficient method for detecting recent thymic output function and analyze the content of T-cell receptor （TCR） rearrangement excision circles （TRECs） within peripheral blood mononuclear cells（PBMCs）. Method According to the specific sequence of TCRδ, the primers and the fluorescent probe （TaqMan） were designed and synthesized. The standard quantitative template was constructed by T/A cloning. The method for detecting TRECs was established after optimization of reaction condition, then its specificity, sensitivity and stability were tested. Quantitative detection of TRECs in DNA of PBMCs from normal individuals and patients of chronic hepatitis B were preformed by real-time PCR using TaqMan technique. Results Detection of TRECs was quick and accurate by real-time fluorescence quantitative PCR. The CV value of Ct was 1.06%, the product was specific which was confirmed by electrophoresis and sequencing and the method showed high sensitivity. The mean value of TRECs from normal individuals was（7767.4±2369.5） copies/10^6 PBMCs in healthy controls at age 21-45 but （28 374.4±7820.4）copies/10^6 PBMCs in those at age 16- 20（ P 〈 0.05）. The mean value of TRECs from patients with chronic hepatitis B was （6480.9 ±2031.2）copies/ 10^6 PBMCs in those at age 21-45, which was statistically significant as compared with normal individuals at age 21- 45.Conclusion Real-time fluorescence quantitative PCR for detecting the TRECs is an accurate, efficient and stable method and the recent thymic output function might decrease in patients with chronic hepatitis B.