[Establishment and application of real-time fluorescence quantitative PCR for detecting recent thymic output function].
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).


METHODS
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.


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.


CONCLUSIONS
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.
Li
Hong-bing
HB
Department of Infection Diseases, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002 China.
Chen
Ming
M
Wang
Li-ping
LP
Zhu
Xue-wen
XW
Ge
Guo-hong
GH
Li
Xiu-hua
XH
D000328:Adult; D017931:DNA Primers; D005260:Female; D015329:Gene Rearrangement, T-Lymphocyte; D019694:Hepatitis B, Chronic / Q000097:blood / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics*; D015203:Reproducibility of Results; D013950:Thymus Gland / Q000276:immunology* / Q000378:metabolism; D055815:Young Adult
D017931:DNA Primers; D016692:Receptors, Antigen, T-Cell, gamma-delta