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Open AccessArticle

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

1
Sabin Medicina Diagnóstica, SAAN, quadra 3, lote 145/185, Brasilia 70632-300, Brazil
2
Post-Graduation in Health Science, University of Brasilia, Brasilia 70910-900, Brazil
*
Author to whom correspondence should be addressed.
These authors contributed equally to the study.
Diagnostics 2020, 10(3), 153; https://doi.org/10.3390/diagnostics10030153
Received: 30 December 2019 / Revised: 28 February 2020 / Accepted: 9 March 2020 / Published: 12 March 2020
(This article belongs to the Collection Biomarkers in Medicine)
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-∆∆Cq 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-∆∆Cq 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. View Full-Text
Keywords: JAK2V617F; 2-∆∆Cq method; allele-specific qPCR; relative quantification; myeloproliferative neoplasms JAK2V617F; 2-∆∆Cq method; allele-specific qPCR; relative quantification; myeloproliferative neoplasms
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Barra, G.B.; Santa Rita, T.H.; Almeida, A.L.S.C.; Jácomo, R.H.; Nery, L.F.A. Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method. Diagnostics 2020, 10, 153.

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