Establishment and application of real-time fluorescence quantitative PCR for detecting recent thymic output function / 中华实验和临床病毒学杂志

ABSTRACT

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>According to the specific sequence of TCRdelta, 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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>