半巢式聚合酶链反应方法检测弥漫性大B细胞淋巴瘤bcl-2/IgH基因重排

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。

Detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma by hemi-nested PCR

Objectives To explore a sensitive and specific method for detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma(DLBCL), and verify the credibility of the established method. Methods bcl-2/IgH hemi-nested PCR primers were designed using the professional primer design software. Fifty-two samples of pathologically diagnosed DLBCL and 10 fresh tonsil tissues were amplified using hemi-nested touch down-PCR to detect bcl-2/IgH gene rearrangement. The PCR products were cloned and sequenced. Results bcl-2/IgH gene rearrangement was detected in 6 of 52 DLBCL samples and 2 of 10 fresh tonsil tissues using one-way method. By using the hemi-nested PCR for the second round amplification, 5 of DLBCL were positive, but all of the fresh tonsil tissues were negative. The positive PCR products were sequenced and analyzed on the Internet, 3 of 8 cases obtained by one-way method were false positive, 5 positive cases amplified using hemi-nested PCR were all bcl-2/IgH gene rearrangement. PCR products of 3 false positive cases were homologous to BAC331191 and LLNLR-245D11 in human chromosome 19 and RP11-498P10 in chromosome 1. Conclusion There are false positive results using common primers for detecting bcl-2/IgH gene rearrangement. The mechanism may be that highly homological sequences to human genome exist in commonly used primers. The specificity of the diagnosis could be improved by hemi-nested PCR using the combination of primers we designed and the traditional ones.