PCR-RNA转录本分子杂交用于急性淋巴细胞白血病微小残留病的检测

目的为了快速、敏感地检测急性淋巴细胞白血病（急淋）微小残留病。方法以Ｔ细胞受体γ链基因重排为标志基因，将确诊时标本扩增后的ＰＣＲ产物转录成放射标记的ＲＮＡ探针，而不同病期标本的ＰＣＲ产物转录成待测ＲＮＡ，待测ＲＮＡ与探针杂交后经ＲＮａｓｅＡ消化，之后再经聚丙烯酰胺凝胶电泳和放射自显影。结果急淋细胞系ＤＮＡ对数稀释试验表明本方法的敏感度在１０－５水平。１例急淋完全缓解期微小残留病也被成功地检测出来。而当探针和待测ＲＮＡ并非来自同一个体时，交叉杂交的结果为阴性。结论为临床检测急淋微小残留病提供了一种有效手段

DETECTION OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOBLASTIC LEUKEMIA USING PCR MOLECULE HYBRIDIZATION OF RNA TRANSCRIPTS

Objective To detect acute lymphoblastic leukemia's (ALL's)minimal residual disease(MRD) rapidly and effectively. Methods In this assay, the gamma T cell receptor gene rearrangements serve as marker genes. The gene rearrangements are amplified from the diagnostic specimens using a consensus V segment primer and a consensus J segment primer to which the promoter T7 RNA polymerase has been appended. The PCR product from this amplification is transcribed into a radiolabeled RNA probe. The opposite DNA strand is transcribed into test RNA from the PCR product of different staged specimens. The test RNA is hybridized with the probe, and later the digestion with RNase A, Polyacrylamide gel electrophoresis and autoradiography are in progress. Results According to the mechanism, the perfectly matched RNA duplex can prevent the digestion of RNase A, and the presence of the leukemia cells in the test specimen can be determined. Logarithmical dilution experiments with DNA of a cell line from ALL have shown that this assay's sensitivity is at the 10 -5 level. Minimal residual disease was successfully detected in a case of ALL during its complete remission stage. But if the probe and test RNA are not from the same individual, the results of this kind of cross hybridization are negative. Conclusion The above results suggest that this assay can become an effective measure in the detection of ALL MRD clinically.