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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

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Abstract

Background and objective

JAK2 V617F mutation is a molecular marker for myeloproliferative neoplasms (MPNs). As there are no China Food and Drug Administration-approved 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 (κ = 0.89). The precision was 100 % and the results of the assay were unaffected by lipoprotein (<27 mmol/L) or bilirubin (<450 µmol/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.

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Acknowledgments

This work was supported by the grants from the Outstanding Young Talent Plan of Shanghai (XYQ2013095), Pandeng Talent Plan of the Shanghai Tenth People’s Hospital (2013), Fudan University Zhuoxue Talent Plan (2011) and the National Science Foundation (Grant No. 81001059 and 81370067).

Conflict of interest

We declare that none of the authors have any conflict of interests regarding the publication of this study.

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Authors and Affiliations

  1. Department of Laboratory Medicine, Shanghai Tenth People’s Hospital, Tongji University, 301 Yanchang Road, Shanghai, 200072, People’s Republic of China

    Weiwei Liu

  2. Department of Laboratory Medicine, Shanghai Skin Disease Hospital, 1278 Baode Road, Shanghai, 200071, People’s Republic of China

    Weiwei Liu

  3. Department of Laboratory Medicine, Huashan Hospital, Fudan University, 12 Central Urumqi Road, Shanghai, 200040, People’s Republic of China

    Tingting Hu, Yuming Chen & Ming Guan

  4. Central Laboratory, Huashan Hospital, Fudan University, Shanghai, People’s Republic of China

    Xinju Zhang & Xiaoye Gu

Authors
  1. Weiwei Liu
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  2. Tingting Hu
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  3. Yuming Chen
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  4. Xinju Zhang
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  5. Xiaoye Gu
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  6. Ming Guan
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Corresponding authors

Correspondence to Weiwei Liu or Ming Guan.

Additional information

W. Liu and T. Hu contributed equally to this work.

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Liu, W., Hu, T., Chen, Y. et al. 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. Mol Diagn Ther 18, 579–585 (2014). https://doi.org/10.1007/s40291-014-0111-6

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  • DOI: https://doi.org/10.1007/s40291-014-0111-6

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