Barra Gustavo Barcelos, Santa Rita Ticiane Henriques, Almeida Ana Luisa Santa Cruz, Jácomo Rafael Henriques, Nery Lídia Freire Abdalla
Sabin Medicina Diagnóstica, SAAN, quadra 3, lote 145/185, Brasilia 70632-300, Brazil.
Post-Graduation in Health Science, University of Brasilia, Brasilia 70910-900, Brazil.
Diagnostics (Basel). 2020 Mar 12;10(3):153. doi: 10.3390/diagnostics10030153.
Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2 method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2 method-the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.
检测Janus激酶2（JAK2）V617F突变是骨髓增殖性肿瘤的诊断标准，突变等位基因水平高与预后较差相关。该突变通常通过等位基因特异性定量PCR（AS-qPCR）在血液DNA上进行检测，并使用绝对定量法进行测量。然而，一些自动化DNA提取方法会从血液中共同提取PCR抑制剂，为了维持标准曲线，qPCR绝对定量需要付出更多努力。JAK2 V617F也可以使用液滴数字PCR（ddPCR）在血清中检测，血清这种样本中的抑制剂较少，有利于自动化提取，但ddPCR仪器不像qPCR热循环仪那样广泛可用。在此，我们评估是否可以使用两种方法通过AS-qPCR对血液DNA中的JAK2 V617F进行准确定量，并使用金标准分子诊断方案验证该检测方法。接下来，我们应用经过验证的方法评估该突变是否可以在血清中可靠地检测/定量。JAK2 V617F可以使用两种方法通过AS-qPCR进行定量——与商业试剂盒相比，该检测方法具有高度准确性（偏差为1.91%）、高度精确性（对于突变等位基因比例为93%、54%和2.5%的样本，总变异系数分别为0.40%、1.92%、11.12%）、高度敏感性（检测限为0.15%），并且在1.1%至99.9%之间呈现线性检测响应。与配对的全血相比，血清呈现出更高的突变等位基因负荷（平均为4%），这使得JAK2突变检测率提高，并有利于增加JAK2 V617F的高通量分析。
