Establishment and analytical validation of JAK2 V617F mutation assay by fluorescence PCR combined with melting curve analysis
10.3760/cma.j.issn.1009-9158.2013.09.006
- VernacularTitle:荧光PCR结合熔解曲线法检测JAK2 V617F突变方法的建立及其方法学验证
- Author:
Tingting HU
;
Yuming CHEN
;
Xinju ZHANG
;
Xiaoye GU
;
Ming GUAN
;
Weiwei LIU
- Publication Type:Journal Article
- Keywords:
Janus kinase 2;
Polymerase chain reaction;
Mutation
- From:
Chinese Journal of Laboratory Medicine
2013;36(9):796-800
- CountryChina
- Language:Chinese
-
Abstract:
Objective According to the requirements of the College of American Pathologists (CAP),to validate the laboratory developed test-JAK2 V617F mutation.Methods This is a clinical laboratory diagnosis study.Fifty-seven peripheral blood and bone marrow samples were collected from the BCR/ABL-negative myeloproliferative neoplasms (MPN) patients in Huashan Hospital from Apr 2010 to Dec 2010.JAK2 V617F mutation was detected by fluorescence PCR combined with melting curve analysis.The accuracy,precision,analytical sensitivity,and anti-interference ability of the assay were validated.The Kappa test was used to evaluate accuracy.Coincidence rate was used to evaluate the repeatability.Paired t test was used to evaluate analytical sensitivity.Results There was a close agreement between the reference method(sequencing) and the test method (fluorescence melting curve analysis) (Kappa =0.89).The coincidence rate was 100％.The results of the assay was not affected by lipoprotein (＜ 24 mmol/L) or bilirubin (＜450 μmol/L).The detection limit of the assay was 5％.Conclusion JAK2 V617F mutation assay by fluorescence PCR combined with melting curve analysis could be applied to the clinical laboratory.
