Kreuzer K A, Lass U, Bohn A, Landt O, Schmidt C A
Abteilung für Innere Medizin und Poliklinik m.S. Hämatologie und Onkologie, Medizinische Fakultät Charité der Humboldt-Universität zu Berlin, Germany.
Cancer Res. 1999 Jul 1;59(13):3171-4.
Quantifying bcr/abl fusion transcripts in chronic myelogenous leukemia is thought to serve as a powerful parameter for monitoring the kinetic nature of this clonal disease in vivo and in vitro. Recently, we demonstrated the technical advantages as well as the clinical relevance of quantitating bcr/abl fusion mRNA using the 5-nuclease assay and a real-time fluorescence reverse transcriptase-PCR (RT-PCR) detection system (ABI PRISM 7700 SDS). Meanwhile, another technique was introduced (LightCycler technology) that may be used for the same purpose. To investigate whether this method may be an appropriate alternative to the described procedure, we have established bcr/abl LightCycler RT-PCR for major and minor bcr/abl fusion transcripts. We found that, with only minor modifications, TaqMan RT-PCR and fluorescent probe design can be used to obtain comparable results in the LightCycler system. The developed method could quantitate as little as 10 bcr/abl copies per 100 ng cDNA and was as safe and reproducible as the previously described technique. Because reaction efficiency was identical within different bcr/abl major fusions, one single RT-PCR could be established that simultaneously detects b2a3, b2a2, b3a2, and b3a3 fusion RNA with equal specificity and sensitivity. Compared to results generated by the ABI PRISM 7700 SDS, absolute amounts of bcr/abl did not differ significantly, and there was a linear correlation between the respective values. We conclude that TaqMan chemistry can be used in the LightCycler and that both real-time fluorescence PCR detection systems equally fulfill the criteria for the safe and reliable quantitation of bcr/abl fusion RNA in clinical samples. This may be of help for further standardization of quantitative bcr/abl RT-PCR, which, again, is necessary for the comparison of results generated by different investigators.
定量慢性粒细胞白血病中的bcr/abl融合转录本被认为是监测这种克隆性疾病在体内和体外动力学特性的一个有力参数。最近，我们展示了使用5-核酸酶分析和实时荧光逆转录聚合酶链反应（RT-PCR）检测系统（ABI PRISM 7700 SDS）定量bcr/abl融合mRNA的技术优势及其临床相关性。同时，另一种技术（LightCycler技术）被引入，可用于相同目的。为了研究该方法是否可能是所述方法的合适替代方法，我们建立了用于主要和次要bcr/abl融合转录本的bcr/abl LightCycler RT-PCR。我们发现，只需进行少量修改，TaqMan RT-PCR和荧光探针设计就可用于在LightCycler系统中获得可比结果。所开发的方法能够定量低至每100 ng cDNA中10个bcr/abl拷贝，并且与先前描述的技术一样安全且可重复。由于不同bcr/abl主要融合体之间的反应效率相同，因此可以建立一个单一的RT-PCR，同时以相同的特异性和灵敏度检测b2a3、b2a2、b3a2和b3a3融合RNA。与ABI PRISM 7700 SDS产生的结果相比，bcr/abl的绝对量没有显著差异，并且各自的值之间存在线性相关性。我们得出结论，TaqMan化学方法可用于LightCycler，并且两种实时荧光PCR检测系统同样满足临床样本中bcr/abl融合RNA安全可靠定量的标准。这可能有助于进一步标准化定量bcr/abl RT-PCR，而这对于比较不同研究者产生的结果而言又是必要的。
