| Objective: Minimal residual disease (MRD) mainly induce relapseof acute lymphoblastic leukemia (ALL) .Therefore it has important significance to monitor MRD of ALL patients for analyzing the development and estimating the effect of treatment, predicting the earlier relapse, and choosing effective treatment strategies. It has been proved that the variable region (V) and joint region (J) of T Cell Receptor (TCR) gene will be rearranged when stem cells differentiate to lymphocytes. This will form the specific V-D-J fragment of lymphocytic clone which can be regarded as the genetic marker for MRD detection of malignant hematopathy such as ALL, etc. At present, the main method to detect rearrangement of TCR gene in China is qualitative polymerase chain reaction (PCR).Although PCR can detect MRD, the clinical sense is limited. Because, some of ALL patients with MRD may relapse and the others are constantly catabatic which show that the quantity of MRD is different in ALL patients. Moreover, PCR has false-positive detection, nonspecific amplification, electrophoresis errors after amplification, harm to human body by EB Staining, etc. In order to overcome above problems and farther improve the clinical value of detection of MRD in ALL patients, we explored to construct a method to detect the arrangement of VyI-Jy gene of TCR quantitatively and more exactly in ALL patients with real time fluorescent quantitative polymerase chain reaction (FQ-PCR).Methods: DNA of mononuclear cell of bone marrow obtained byclassical NP40-protease K assimilation and phenol-chloroform extraction were taken as samples to conduct FQ-PCR from 36 cases in which VyI-Jy gene rearrangement of TCR were positive with5qualitative PCR. The 36 patients come from 3 groups: initial treatment group, complete remission (CR) group and after hematopoietic stem cells transplantation (HSCT) group .10 normal volunteers were treated as TCR VyI-Jy gene rearrangement negative control group. Due to the invariableness of J fragment of TCR VyI-Jy gene in the rearrangement, we mainly aimed at the congenerous conservative rank of it to analyze in 3 randomly selected cases whose TCR VyI-Jy gene rearrangement were positive and products of PCR were purified. Then, combining with the data of GENEBANK, we designed the probe with computer. The PCR product from a ALL case, in which TCR VyI-Jy gene rearrangement was positive and leucocythemia cells of marrow ratio was 98%, was purified and connected to pMD 18-T Vector. After the plasmid was transformed to OH 5a of escherichia coli, we extracted the plasmid DNA and measured its OD value. 0.5ug/2.5ul plasmid DNA corresponded to 10?2.5ul gene level. The plasmid DNA was diluted to 10皛10~5 concentration gradient and conducted FQ-PCR amplification on ABI PRISM 7000 according to the settings and instructions. The results were protracted standard curve, and then the 36 cases above were detected with FQ-PCR and the results were compared with the standard curve to quantitatively analyze the TCR Vyl-Jy gene rearrangement.Results:l.The Ct values of standard samples which dilute concentration gradient ranged from 10?to 10's are 21.08,24.34,27.53,30.58 and 33.25 respectively.Statistical analysis revealed that the Ct value and initial concentration of a standard sample had a linear relationship. The higher the initial concentration was, the lower the Ct value. Regression coefficient was 0.998.2.An exact Ct value of standard sample which was diluted to KT46concentration could still be obtained. The sensitivity for the established real time fluorescent quantitative PCR were 10"4 level.3. The standard samples of 10皛10 concentration gradient were repeatedly measured six times. The stability was good and the CV was 7.44%. 5.43% 6.01% and 7.28% respectively. The standard sample of 10~3 concentration was quantitatively measured with reagent K IK III IV N V VI. The stability of reagent which was confected on different time was good. Its CV was 6.23%.4.The amount of TCR Vyl-Jy gene rearrangement in initial tre...
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