Research on point mutations in onco-hematology plays a critical role in understanding neoplasias and formulating effective treatment strategies. In this context, investigating the V617F mutation in the JAK-2 gene is of particular significance for diagnosing BCR-ABL negative myeloproliferative syndromes, notably Polycythemia vera. A range of techniques, including sequencing, PCR + RFLP, and real-time PCR, can be employed for this purpose. This study aims to describe the utilization of real-time PCR with a specific MGB probe to detect the V617F mutation in the JAK-2 gene. We also present the retrospective results obtained from peripheral blood samples of 250 patients who were assessed between July 2022 and July 2023 due to suspected myeloproliferative disorders (MPD).

The primary objectives of this study are to validate a rapid, cost-effective, and sensitive MGB TaqMan probe-based real-time PCR method, coupled with automatic magnetic DNA extraction (EXTRACT MPTA, Loccus) compared with column manual extraction (Promega), for detecting the JAK-2 V617F mutation in an onco-hematology remote laboratory (ROLF). Additionally, we aim to evaluate the incidence of the V617F mutation in the 250 cases submitted to ROLF.

For the real-time PCR reaction, we utilized a specific MGB probe (Thermo) targeting the V617F mutation (rs77375493) along with the SNP detection master mix reagent. The PCR was performed using a Rotor-Gene Q instrument (Qiagen), and the raw fluorescence data were analyzed at cycle 45. To assess sensitivity, we conducted tests using a homozygous cell line for the mutation (HEL 92).

Among the 250 cases (126 females and 124 males) originating from 28 different hospitals in Brazil, 63 (25.2%) tested positive for the V617F mutation, with a relatively equal distribution between genders (p = 0.1459). The mean age of V617F positive samples was 66 years compared to 55 years for negative samples (p < 0.0001). Analysis of fluorescence intensity at cycle 45 of real-time PCR in positive and negative samples for the V617F mutation revealed remarkable sensitivity, detecting the presence of the mutated allele down to 1% (limit of detection - LOD). The average fluorescence intensity was 0.52 for mutated samples and 0.04 for non-mutated samples (p < 0.0001, Mann-Whitney test). DNA from 32 samples and diluted controls were extract manually and automatically (magnetic) and 100% agreement was obtained.

Our study demonstrates the clinical relevance and high sensitivity of real-time PCR with the MGB probe for detecting the V617F mutation in the JAK-2 gene. It accurately identifies V617F mutation even at low levels (1% LOD). The automatic magnetic DNA extraction setting offers a rapid and cost-effective alternative to manual column extraction. Notably, we observed a significant correlation between the V617F mutation and older age, with positive samples having a mean age of 66 years compared to 55 years for negative samples (p < 0.0001). These findings have important implications for molecular diagnostics in onco-hematology, enabling early and precise diagnosis of myeloproliferative disorders (MPD).