Development and validation of a 3-Plex RT-qPCR assay for the simultaneous detection and quantitation of the three PML-RARa fusion transcripts in acute promyelocytic leukemia.
Development and validation of a 3-Plex RT-qPCR assay for the simultaneous detection and quantitation of the three PML-RARa fusion transcripts in acute promyelocytic leukemia.
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DOI：
10.1371/journal.pone.0122530
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Xia Z
中科院分区：
文献类型：
--
作者：
Chen Z;Tong Y;Li Y;Gao Q;Wang Q;Fu C;Xia Z
快速诊断急性早幼粒细胞白血病(APL)伴早幼粒细胞白血病-维甲酸受体α(PML-RARA)有助于全反式维甲酸(ATRA)的高效治疗。实时定量逆转录聚合酶链式反应(RT-qPCR)是诊断APL合并PML-RAR A的有价值的工具。然而，单一的RT-qPCR分析既费力又昂贵，必须在三个反应中进行，以确定是否存在三个PML-RARA转录本中的一个，并对涉及的转录本进行量化。本文介绍了一种新的基于TaqMan MGB探针的三重RT-qPCR方法，可以同时检测三种PML-RARA转录本。设计了特异的引物和探针，并将结果进一步归一化为Abelson基因。对串联稀释质粒的检测结果表明，PML-RARA bcr1、bcr2和bcr3的分析灵敏度为每次反应10个拷贝。用该方法分析NB4细胞的bcr1转录本，灵敏度可达10-4。重现性令人满意，因为循环阈值的变异系数在批间和批内均小于3%。对319例初诊白血病患者(包括61例急性早幼粒细胞白血病)进行检测后，三重RT-qPCR检测结果与传统的PML-RAR检测方法完全一致。三重RT-qPCR的定量结果与单一RT-qPCR的结果高度相关，对60例PML-RARA阳性的APL初诊标本和57例患者的199例随访标本具有相似的检测敏感性。有趣的是，1例断裂点在1579处的PML-RARA bcr2病例，在单次RT-Q-PCR中未被检测到，却被3-plex RT-qPCR检测到。3-plex RT-qPCR方法是一种特异、灵敏、稳定、经济的方法，可用于APL合并PML-RAR A的快速诊断和疗效监测。
Rapid diagnosis of acute promyelocytic leukemia (APL) with promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa) contributes to a highly effective therapy with all-trans retinoic acid (ATRA). Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is a valuable tool to diagnose APL with PML-RARa. However, a single RT-qPCR analysis, which is laborious and costly, has to be performed in three reactions to determine whether one of the three PML-RARa transcripts is present and to quantify the involved transcript. This paper describes a novel TaqMan MGB probe-based 3-plex RT-qPCR assay in a single reaction to detect simultaneously the three PML-RARa transcripts. Specific primers and probe were designed, and the results were further normalized to the Abelson gene. The detection results for the serially diluted plasmid indicate that the analytical sensitivity was 10 copies per reaction for PML-RARa bcr1, bcr2, and bcr3. A relatively high sensitivity of 10-4 was achieved with this assay when analyzing the bcr1 transcripts obtained from the NB4 cell line. The reproducibility was satisfactory because the coefficients of variation of cycle threshold values were less than 3% for both inter- and intra-assays. After testing 319 newly diagnosed patients with leukemia (including 61 APL cases), the results of the 3-plex RT-qPCR assay completely agreed with the traditional methods used for the detection of PML-RARa. The quantitative results of the 3-plex RT-qPCR were highly correlated with the single RT-qPCR and showed similar assay sensitivity for 60 PML-RARa positive APL samples at diagnosis and 199 samples from 57 patients during follow-up. Interestingly, one PML-RARa bcr2 case at diagnosis with breakpoint at 1579, which was not detected by the single RT-q-PCR, was detected by the 3-plex RT-qPCR assay. The 3-plex RT-qPCR assay is a specific, sensitive, stable, and cost-effective method that can be used for the rapid diagnosis and treatment monitoring of APL with PML-RARa.
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