Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2<sup>V617F</sup> Mutation
Precise quantification of the JAK2V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of...
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| Format: | Article |
| Language: | English |
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KeAi Communications Co., Ltd.
2024-06-01
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| Series: | Global Medical Genetics |
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| Online Access: | http://www.thieme-connect.de/DOI/DOI?10.1055/s-0044-1785537 |
| _version_ | 1826995597799849984 |
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| author | Yupeng Liu Cong Han Jie Li Shicai Xu Zhijian Xiao Zhiyun Guo Shuquan Rao Yao Yao |
| author_facet | Yupeng Liu Cong Han Jie Li Shicai Xu Zhijian Xiao Zhiyun Guo Shuquan Rao Yao Yao |
| author_sort | Yupeng Liu |
| collection | DOAJ |
| description | Precise quantification of the JAK2V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988). |
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| issn | 2699-9404 |
| language | English |
| last_indexed | 2025-02-18T09:34:51Z |
| publishDate | 2024-06-01 |
| publisher | KeAi Communications Co., Ltd. |
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| series | Global Medical Genetics |
| spelling | doaj.art-e185824e9a014d1295d9b1f57b8b07c32024-11-02T18:14:02ZengKeAi Communications Co., Ltd.Global Medical Genetics2699-94042024-06-01110213214110.1055/s-0044-1785537Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2<sup>V617F</sup> MutationYupeng Liu0Cong Han1Jie Li2Shicai Xu3Zhijian Xiao4Zhiyun Guo5Shuquan Rao6Yao Yao7School of Life Sciences and Engineering, Southwest Jiaotong University, Chengdu, ChinaState Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, ChinaDepartment of Laboratory Medicine, Peking University Shenzhen Hospital, Shenzhen, ChinaState Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, ChinaState Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, ChinaSchool of Life Sciences and Engineering, Southwest Jiaotong University, Chengdu, ChinaState Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, ChinaState Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, ChinaPrecise quantification of the JAK2V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988).http://www.thieme-connect.de/DOI/DOI?10.1055/s-0044-1785537myeloproliferative neoplasmsJAK2 V617F mutationddPCRoptimization |
| spellingShingle | Yupeng Liu Cong Han Jie Li Shicai Xu Zhijian Xiao Zhiyun Guo Shuquan Rao Yao Yao Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2<sup>V617F</sup> Mutation Global Medical Genetics myeloproliferative neoplasms JAK2 V617F mutation ddPCR optimization |
| title | Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2<sup>V617F</sup> Mutation |
| title_full | Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2<sup>V617F</sup> Mutation |
| title_fullStr | Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2<sup>V617F</sup> Mutation |
| title_full_unstemmed | Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2<sup>V617F</sup> Mutation |
| title_short | Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2<sup>V617F</sup> Mutation |
| title_sort | laboratory developed droplet digital pcr assay for quantification of the jak2 sup v617f sup mutation |
| topic | myeloproliferative neoplasms JAK2 V617F mutation ddPCR optimization |
| url | http://www.thieme-connect.de/DOI/DOI?10.1055/s-0044-1785537 |
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