VALIDATION OF A RAPID AND SENSITIVE MGB PROBE-BASED REAL-TIME PCR FOR DETECTING JAK-2 V617F MUTATION

Introduction: 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). Objectives: 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. Methods: 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). Results: 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. Conclusion: 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).