TaqMan-MGB探针实时荧光PCR法检测T细胞受体重排删除环评估慢性乙型肝炎患者胸腺近期输出功能

Detection of T cell receptor rearrangement excision circles(TRECs) by TaqMan-MGB probe real-time fluorescence PCR for thymic recent output function in patients with chronic hepatitis B

目的:建立一种快速、准确检测T细胞受体重排删除环(TRECs)的方法,用来评价慢性乙型肝炎患者胸腺输出功能。方法:根据T细胞受体(TCR)基因序列设计引物与探针,建立TaqMan-MGB双标记探针技术定量测定外周血中TRECs的含量,通过重复性、敏感性、准确性实验评价该方法的可靠性;并将此技术应用于慢性乙型肝炎中型(CHB)患者中评价胸腺输出功能。结果:通过多次试验成功建立了检测TRECs含量的TaqMan-MGB双标记探针实时荧光定量检测方法,并对5×10~5copies的质粒进行10倍法稀释,最低检出可达50 copies;对6. 18 Lg copies/10~6PBMCs和3. 54 Lg copies/10~6PBMCs高低浓度标本同一批次进行25次检测结果平均值为6. 08±0. 14,3. 52±0. 18 Lg copies/10~6PBMCs批内变异系数分别为2. 26%,5. 12%,对这两份标本连续5 d重复20次试验结果平均值为6. 10±0. 23,3. 51±0. 22 Lg copies/10~6PBMCs批间变异系数分别为3. 85%、6. 27%;用双蒸水代替模板进行检测无目的基因扩增。35例健康成年人外周血TRECs平均含量为5. 62±1. 38 Lg copies/10~6PBMCs,45例同年龄段CHB患者外周血TRECs平均含量为4. 09±0. 97 Lg copies/10~6PBMCs,两者差异存在统计学意义(t=11. 25,P <0. 05)。结论:Taq Man-MGB双标记探针时荧光定量PCR技术检测TRECs特异性强,灵敏度高,重复性好;成年CHB中型患者外周血TRECs含量明显低于同龄健康人群,TRECs可用于CHB患者胸腺功能的评估。

Objective: To establish a rapid and accurate quantitative method to detect T-cell receptor excision DNA circles(TRECs) for evaluating the thymic recent output function with CHB patients.Methods: The primers and probes were designed with gene sequences of T-cell receptor(TCR).By establishing Taqman-MGB double labeling probe in the quantitative determination of peripheral blood TRECs content through the repeatability,sensitivity,accuracy experiment evaluation of the reliability,this method was used to evaluate the thymic recent output function among patients with chronic hepatitis.Results: A real-time fluorescence quantitative detection method for Taq Man-MGB double-probe with TRECs content was established.The minimum detection limit was 50 copies by detecting plasmid of 5 × 10^5 copies in 10 times diluted solution.The mean of 6.18 Lg copies/106 PBMCs and 3.54 Lg copies/10^6 PBMCs which had tested 25 times for the same sample was 6.08 ± 0.14,3.52 ± 0.18 Lg copies/10^6 PBMCs,internal coefficient was 2.26%,5.12%.The average of the 20 tested results was 6.10 ± 0.23,3.51 ± 0.22 Lg copies/10^6 PBMCs,external coefficient was 3.85%,6.27%.The detection of distilled water was non-objective gene amplification.There was a statistical difference(t = 11.25,P < 0.05) among patients(n = 45,mean of TRECs 4.09 ± 0.97 Lg copies/10^6 PBMCs) with chronic HBV infections compared with the same age healthy groups(n = 35,mean of TRECs 4.82 ± 1.28 Lg copies/10^6 PBMCs).Conclusion: A real time fluorescence quantitative PCR for Taqman-MGB double labeling probes to detect TRECs had high specificity,sensitivity and repeatability.The content of TRECs in patients with CHB was significantly lower than that of healthy people of the same age,and TRECs can be used to evaluate the thymic function of CHB patients.