Construction of recombinant plasmid and standard curve for detection of TRECS by real-time quantitative PCR

Objective To construct recombinant plasmid and standard curve for the detection of TCR rearrangement excision circles(TRECs)by real-time quantitative PCR assay.Methods The primer expression software was used to obtain a couple of primer and fluorescent probe after analysis of TCRδ gene sequence.DNA was extracted from PBMCs of normal individuals and then amplified by PCR.The PCR product was connected with pUCM-T vector and then transferred into Ecoil DH5α.The standard recombinant plasmid was gained from the positive plasmid.Results The sequence of target segment in recombinant plasmid was confirmed to be correct by sequence check,which indicated that TRECs segment was cloned successfully.There was a good liner function in statistics between the Ct value and the concentration gradient of standard DNA specimen in 103-107 standard DNA specimen after real-time quantitative PCR amplification(r=-0.998).Conclusion The recombined plasmid and standard curve for real-time quantitative PCR to detect TRECs were well both in linearity and speciality.