title: Advantages of the quenching probe method over other PCR-based methods for detection of the JAK2 V617F mutation.
abstract: The detection of a V617F mutation (G to T exchange at nucleotide 1,849) in the JAK2 gene is crucial for the diagnosis of myeloproliferative neoplasms (MPN) such as polycythemia vera. Although sequence analysis is the standard method for detection, it is not suitable for clinical examinations due to the requirement of expensive equipment. In this study, we evaluated the efficiencies of four PCR-based methods to detect JAK2 V617F: allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), high-resolution melting analysis (HRM) and the quenching probe method (QP). The HEL cell line, which harbors a homozygous JAK2 V617F mutation, as well as bone marrow samples from 16 MPN patients and normal control samples, were used in this assessment. The sensitivity of the detection limit of all four methods was also examined using samples of HEL cells mixed in a variety of ratios with cells containing wild-type JAK2. The results of all four methods were found to be concordant. AS-PCR was shown to be the most sensitive; however, it produced false positive results. Although PCR-RFLP demonstrated high specificity, it was time consuming. By contrast, results were obtained using HRM and QP in only 2 h. It was easier to recognize the curves derived from the mutant allele obtained using QP. QP is also suitable for the rough estimation of allele burden. JAK2 V617F assays are mainly used for diagnosis at presentation in clinical settings. We therefore conclude that in situations where high sensitivity is not required, QP is the preferable method for the detection of JAK2 V617F. To the best of our knowledge, this is the first report to demonstrate the efficiency of the QP method for the detection of JAK2 V617F using a standard thermal cycler.
authors: Department of Laboratory Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan.
authors: Aya
authors: A
authors: Ono
authors: Yuki
authors: Y
authors: Okuhashi
authors: Yusuke
authors: Y
authors: Takahashi
authors: Mai
authors: M
authors: Itoh
authors: Nobuo
authors: N
authors: Nara
authors: Shuji
authors: S
authors: Tohda