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目的寻找一种敏感、特异的方法检测弥漫性大B细胞淋巴瘤(DLBCL)bcl-2/IgH基因重排,并通过测定产物的序列,以了解所建方法的可靠性。方法运用专业引物设计软件设计bcl-2/IgH基因重排半巢式PCR引物,对52例经临床病理确诊的DLBCL石蜡包埋组织及10例慢性扁桃体炎患者的新鲜扁桃体组织通过半巢式递降温度梯度PCR(touch down PCR)扩增,检测bcl-2/IgH基因重排,并对其产物进行克隆和序列分析。结果通过一步法检测到bcl-2/IgH基因重排8例,其中DLBCL6例,新鲜扁桃体组织2例;进行第2次半巢式PCR时仅在DLBCL中发现5例阳性,新鲜扁桃体组织均阴性。将阳性产物在网上序列分析显示:一步法检测到的8例中有3例为假阳性,而半巢式PCR扩增出的5例均为bcl-2/IgH基因重排片段。3例假阳性的片段分别与人类第19号染色体BAC331191,LLNLR-245D11基因片段及1号染色体RP11-498P10基因片段同源。结论常用的检测bcl-2/IgH基因重排引物扩增结果存在一定假阳性,其机制可能是因为人类基因组中存在与常用引物同源性较高的序列。为研究设计的引物与传统的引物结合,进行半巢式PCR可以排除这种假阳性扩增,提高诊断的准确性。
Objective To search for a sensitive and specific method to detect the gene rearrangement of bcl-2 / IgH in diffuse large B cell lymphoma (DLBCL) and to determine the reliability of the constructed method by measuring the sequence of the product. Methods The bcl-2 / IgH gene rearrangement PCR primers were designed by using the professional primer design software. 52 patients with pathologically confirmed DLBCL paraffin-embedded tissue and 10 patients with chronic tonsillitis were treated by semi-nested descending Temperature-gradient PCR (touch down PCR) amplification, detection of bcl-2 / IgH gene rearrangement, and its products were cloned and sequence analysis. Results Eight cases of bcl-2 / IgH gene rearrangement were detected by one-step method, including 6 cases of DLBCL and 2 cases of fresh tonsil tissue. In the second half-nested PCR, only 5 cases were found positive in DLBCL, and the fresh tonsil tissues were negative . Sequence analysis of the positive products showed that three of the 8 cases detected by one-step method were false-positive, and all 5 cases amplified by semi-nested PCR were bcl-2 / IgH gene rearrangement fragments. Three false positives were homologous to human chromosome 19 BAC331191, LLNLR-245D11 and chromosome 1 RP11-498P10, respectively. Conclusion The commonly used detection of bcl-2 / IgH gene rearrangement primer amplification results there is a certain false positive, the mechanism may be due to the presence of human genome sequences commonly used primers and high homology. For the study of primers designed to combine with traditional primers, semi-nested PCR can exclude this false-positive amplification and improve the diagnostic accuracy.