Melting curve analysis after T allele enrichment (MelcaTle) as a highly sensitive and reliable method for detecting the JAK2V617F mutation.
Melting curve analysis after T allele enrichment (MelcaTle) as a highly sensitive and reliable method for detecting the JAK2V617F mutation.
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DOI：
10.1371/journal.pone.0122003
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Komatsu N
中科院分区：
文献类型：
--
作者：
Morishita S;Takahashi K;Araki M;Hironaka Y;Sunami Y;Edahiro Y;Tsutsui M;Ohsaka A;Tsuneda S;Komatsu N
JAK2V617F突变的检测对诊断经典型骨髓增生性肿瘤(MPN)至关重要。然而，由于必须区分真阳性和假阳性结果，检测低频JAK2V617F突变是一项具有挑战性的任务。在这里，我们建立了一种高灵敏、准确的JAK2V617F检测方法，并将其命名为T等位基因富集化后熔化曲线分析(MelcaTle)。MelcaTle包括三个步骤：1)两个循环的JAK2V617F等位基因通过PCR扩增和BsaXI消化，2)在桥联核酸(BNA)探针存在下选择性扩增JAK2V617F等位基因，3)使用BODIPY-FL标记的寡核苷酸进行熔融曲线分析。使用这种方法，我们使用10 ng基因组DNA标准成功地检测到了JAK2V617F等位基因的近单一拷贝，没有假阳性信号。此外，MelcaTle在90次筛查健康人JAK2V617F的试验中未显示阳性信号。当将MelcaTle应用于27名根据等位基因特异性PCR分析最初被归类为JAK2V617F阳性并因此被怀疑患有MPN的患者时，我们发现其中两名患者实际上JAK2V617F阴性。更仔细的临床数据分析显示，这两名患者发生了原因不明的一过性红细胞增多症，但没有真性红细胞增多症，这是MPNS的一种亚型。这些发现表明，新开发的MelcaTle检测方法将显著提高JAK2V617F阳性MPNS的诊断水平。
Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.
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影响因子：
11.4
作者：
Quentmeier, H;Macleod, RAF;Drexler, HG
通讯作者：
Drexler, HG
影响因子：
4.1
作者：
Huijsmans, Cornelis J. J.;Poodt, Jeroen;Hermans, Mirjam H. A.
通讯作者：
Hermans, Mirjam H. A.
影响因子：
4.1
作者：
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通讯作者：
Yoo, Nam Jin
影响因子：
2.9
作者：
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通讯作者：
Nielsen, PE
影响因子：
64.8
作者：
James, C;Ugo, V;Vainchenker, W
通讯作者：
Vainchenker, W