Multiplex fluorescent RT-PCR to quantify leukemic fusion transcripts
Multiplex fluorescent RT-PCR to quantify leukemic fusion transcripts
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DOI：
10.2144/02331md04
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发表时间：
2002-07-01
期刊：
影响因子：
2.7
通讯作者：
Lavabre-Bertrand, T
中科院分区：
文献类型：
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作者：
Dupont, M;Goldsborough, A;Lavabre-Bertrand, T
对来自异常染色体融合事件的嵌合转录物的检测为白血病的诊断提供了非常有价值的工具。我们开发了一种简单、廉价、可重复和自动化的方法来定量RT-PCR产物。我们的方法利用荧光PCR，与内部控制（HPRT）的特定融合转录的共扩增。我们还结合了实时定量PCR的优点，即PCR产物的连续荧光检测与终点测定的低成本，通过以新的方式检查在扩增的指数期期间产生的荧光PCR产物的量。这是通过使用激光诱导荧光毛细管电泳系统ABI PRISM（R）310 A的自动加载和定量能力实现的，因此我们可以在指数期期间以低成本、可重复和灵敏的方式有效地监测扩增。我们已经使用五种白血病细胞系仔细验证了我们的新技术，每种细胞系都表达不同的融合转录本。已对专属性和重现性（CV在10%以内）进行了检查，证明了我们的技术具有出色的精密度。对于五种细胞系的连续稀释获得的至少10（-4）至10（-6）的高灵敏度水平证实了我们的荧光PCR作为与其他更复杂和昂贵的方法相当的方法的选择。我们的研究结果允许定量PCR产物和源自易位断点的嵌合mRNA的量。我们证明，我们的新的荧光方法是有用的检测和定量残留的白血病细胞在接受治疗的患者。
The detection of chimeric transcripts derived from aberrant chromosomal fusion events provides an exceptionally valuable tool for the diagnosis of leukemia. We have developed a simple, inexpensive, reproducible, and automated method to quantify RT-PCR products. Our approach utilizes fluorescent PCR,for the co-amplification of the specific fusion transcript with an internal control (HPRT). We have also combined the advantages of real-time quantitative PCR, namely continuous fluorescent detection of PCR products with the low cost of an end-point assay by examining in a novel manner the amount of fluorescent PCR product generated during the exponential phase of amplification. This has been achieved by, using the automated loading and quantification capacity of a laser-induced fluorescence capillary electrophoresis system, the ABI PRISM(R) 310A, so that we can effectively monitor amplification during the exponential phase cheaply reproducibly, and in a sensitive manner. We have, carefully verified our new technique using five leukemia cell lines, each expressing a different fusion transcript. Specificity and reproducibility (cv within 10%) have been examined and demonstrate the excellent precision of our technology. The high sensitivity levels of at least 10(-4) to 10(-6) obtained for the serial dilutions of the five cell lines validate the choice of our fluorescent PCR as a comparable method to other more complicated and expensive methods. Our results have allowed as to quantify PCR products and the amount of chimeric mRNA originating from the translocation breakpoint. We demonstrate that our novel fluorescent method is useful to detect and quantify residual leukemic cells in patients undergoing therapy.