Gene melting curve spectratyping technique in analyzing polymorphism of t-cell receptor beta chain variable region in cd4+ t cells of tuberculosis patients / 第二军医大学学报

ABSTRACT

Objective To analyze the polymorphism of the T-cell receptor (TCR) beta chain variable region(BV) gene family spectratyping of peripheral blood CD4+T cells in tuberculosis patients by gene melting curve spectratyping (GMCS) technique combined 24 h fluorescence quantitative (FQ)-PCR. Methods The total RNA of peripheral blood mononuclear cells (PBMCs) from 15 healthy blood donors and 30 tuberculosis patients were used to obtain the cDNA. The cDNAs were amplified by FQ-PCRusing 26 different forward primers and the same reverse primer, and the spectratype was analyzed by DNA melting curve. Results The DNA melting curves of each TCR BV family in 15 healthy donors showed the same spectratype. However, the DNA melting curves of 26 TCR BV families showed different spectratypes in 30 tuberculosis patients comparedwith healthy blood donors, and the different rate was from 6. 7% to 33. 3%. The resutt indicated that the difference rate of 26 TCR BV families in tuberculosis patients was significantly different from that of healthy blood donors (P<0. 05, P<0. 01). In addition, the different rates of 13 TCR BV families (totally 26 families) in tuberculosis patients were more than 20%. Conclusion This study suggests that FQ-PCR combinedwithGMCS is a convenient and stable method for detecting the TCR beta gene repertoire in the peripheral blood of tuberculosis patients.