Development and Validation of a Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction Combined
- PDF / 520,612 Bytes
- 7 Pages / 595.276 x 790.866 pts Page_size
- 82 Downloads / 226 Views
ORIGINAL RESEARCH ARTICLE
Development and Validation of a Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction Combined with Melting Analysis-Assay for Clinical JAK2 V617F Mutation Detection Weiwei Liu • Tingting Hu • Yuming Chen Xinju Zhang • Xiaoye Gu • Ming Guan
•
Published online: 12 July 2014 Ó Springer International Publishing Switzerland 2014
Abstract Background and objective JAK2 V617F mutation is a molecular marker for myeloproliferative neoplasms (MPNs). As there are no China Food and Drug Administrationapproved assays for the detection of JAK2 V617F mutation in China, validation of the analytic performance of this assay is important for the clinical laboratory before its clinical implementation. We have established a method for detecting JAK2 V617F using tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR) combined with melting-curve analysis. Methods A total of 202 blood samples and 20 bone marrow aspirates were obtained from MPNs patients at Huashan Hospital, Fudan University. The accuracy, precision, reproducibility, analytical sensitivity, lower limit of
detection, analytical specificity, interfering substances, ruggedness, robustness, reportable range and reporting of this assay were validated. Results There was a close agreement between the reference method (sequencing) and melting-curve analysis (j = 0.89). The precision was 100 % and the results of the assay were unaffected by lipoprotein (\27 mmol/L) or bilirubin (\450 lmol/L). The analytical sensitivity of the JAK2 mutation was 1.25 %. Conclusions Tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR) combined with melting-curve analysis can be used in the clinical laboratory for detection of the JAK2 V617F mutation.
Key Points W. Liu and T. Hu contributed equally to this work. W. Liu (&) Department of Laboratory Medicine, Shanghai Tenth People’s Hospital, Tongji University, 301 Yanchang Road, Shanghai 200072, People’s Republic of China e-mail: [email protected] W. Liu Department of Laboratory Medicine, Shanghai Skin Disease Hospital, 1278 Baode Road, Shanghai 200071, People’s Republic of China T. Hu Y. Chen M. Guan (&) Department of Laboratory Medicine, Huashan Hospital, Fudan University, 12 Central Urumqi Road, Shanghai 200040, People’s Republic of China e-mail: [email protected] X. Zhang X. Gu Central Laboratory, Huashan Hospital, Fudan University, Shanghai, People’s Republic of China
A rapid and sensitivity assay for detecting the JAK2 V617F mutation was developed and validated. A major advantage of this assay is its one-step, highly sensitive and lower-cost compared with other real-time PCR with hybridization probe melting. This assay may prove a useful tool for diagnosis and therapy monitoring of MPNs. 1 Introduction Myeloproliferative neoplasms (MPNs) represent a heterogeneous group of clonal stem cell diseases with abnormal hematopoietic cell proliferation [1]. Somatic mutations in the Janus ki
Data Loading...
