JAK2 V617F 신속 검출을 위한 대립유전자중합효소연쇄반응법 및 융합곡선분석법의 유용성

Abstract

ABSTRACT

Rapid allele-specific PCR and melting curve analysis for detection of JAK2 V617F

Park, Woo Kwan Advisor: Prof. Geon Park Department of Bio new drug development Graduate School of Chosun University

Objectives: Detection of the JAK2 V617F is an essential diagnostic tool for BCR/ABL-negative myeloproliferative neoplasms (MPNs). Despite many techniques for detection of JAK2 V617F have introduced in clinical laboratory fields, any techniques is required for purified DNA as template. We develop a rapid detection technique using direct real-time allele-specific PCR (ASP) and melting curve analysis without DNA preparation from whole blood.

Methods: Fifty venous samples of Patients with BCR/ABL-negative MPNs were used in the study. Because hemoglobin is a strong quencher, we treated 300 μL of whole blood by 600 μL of rapid RBC lysis solution at room temperature for 3 min. After centrifugation and discard of supernant, cell pellet was diluted with 100 μL of phosphate-buffer saline. A leukocyte suspension was used directly in the direct real-time ASP. For confirmation of results of direct real-time melting curve analysis, we also performed an in-house JAK2 V617F ASP and a BsaXI-treated nested PCR-direct sequencing using purified DNA. For investigating detection limit and specificity of this technique, we used HEL 92.1.7 cell-line and 96 samples of normal persons, respectively.

Results: Melting temperatures of JAK2 V617F mutant melting peak and internal control melting peak were 77.0±0.3℃ and 83.5±0.2℃, respectively. Concordance rate with direct real-time melting curve analysis and in-house conventional ASP was 100% (60% vs 60%, respectively). Total running time and handling time of direct real-time melting curve analysis for 10 samples was 1.5 hr and only 10 min. The detection limit and specificity of real-time melting curve analysis was 2% and 100% according to BsaXI-treated nested PCR-direct sequencing.

Conclusions: Direct real-time ASP and melting curve analysis for detection of JAK2 V617F is a rapid, reliable, and economic technique.