Construction of recombinant plasmid and standard curve for detection of TCR VγI-Jγ gene rearrangement by real-time quantitative PCR

Objective To construct recombinant plasmid and standard curve for detection of TCR VγI-Jγgene rearrangement and establish real-time quantitative polymerase chain reaction(PCR) assay for the detection of minimal residual disease(MRD) in lymphoid malignant diseases. Methods Analysis of TCR VγI-Jγ gene rearrangement sequence and material of GENEBANK. The primer expression software was used to obtain a couple of primer and fluorescent probe. DNA was extracted from the bone marrow cells in one patients of acute lymphoblastic leukemia and TCR VγI-Jγgene rearrangement was amplified by PCR . The PCR product was connected with pMD18-T vector and then transferred into Ecoil DH5α. The standard recombinant plasmid was gained from the positive plasmid. Results The fluorescence levels were increased intensitively after real-time quantitative PCR amplification, which indicated that TCR VγI-Jγ rearrangement gene was cloned Successfully. The CT value were 21.08, 24.34, 27.53, 30.58 and 33.25 respectively with 10 0～10 -5 standard DNA specimen after real-time quantitative PCR amplification, which indicated that there is a good linear function in statistics between the Ct value and the concentration gradient of standard DNA specimen. The regression value was 0.998. Conclusion The recombined plasmid and standard curve for real-time quantitative PCR to detect TCR VγI-Jγgene rearrangement were well both in specific and linear function, and it may be united standard in the detection of MRD.