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Rapid and Accurate Detection of Molecular Markers for Myelop...

Rapid and Accurate Detection of Molecular Markers for Myeloproliferative Neoplasms Based on Digital LAMP

作     者:Guojun Cao Jinze Li Yajun Qiu Zhiqi Zhang Wei Zhang Lianqun Zhou Ming Guan 

作者单位:Department of Laboratory Medicine Huashan Hospital Shanghai Medical College Fudan University Key Laboratory of biomedical detection technology Suzhou Institute of Biomedical Engineering and Technology Chinese Academy of Sciences 

会议名称:《第二届中国临床分子诊断大会》

会议日期:2019年

学科分类:1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学] 

关 键 词:myeloproliferative neoplasm digital LAMP (dLAMP) single-channel chip multi-channel chip quantitative detection 

摘      要:Objective: According to the diagnostic criteria of the WHO updated in 2016, the gene mutations of JAK2, CALR and MPL had been used as an important reference index for the diagnosis of classical myeloproliferative neoplasm(MPN), which was of great significance to improve the laboratory diagnosis rate of MPN. The aim of this study was to establish a digital LAMP(dLAMP) platform for rapid, high throughput and absolute quantitative detection of MPN molecular markers by integrating the advantages of LAMP(rapid), microfluidics(high throughput) and digital PCR(absolute quantification). Methods: A kind of single-channel and multi-channel(four-channel) silicon-based high-throughput capillary array chips were designed and fabricated simultaneously. In order to improve the sample injection effect, the surface of silicon chip was hydrophobically modified, and the inner wall of micropore was hydrophilically treated. Besides on the basis of traditional LAMP reagent formulation, additives were introduced to improve the performance of existing LAMP reagent so as to make it suitable for nanoscale amplification reaction. Amplification completed, the chip was transferred to the in-house platform for results observation and photography. To determine the accuracy of the in-house dLAMP detection platform, results consistency of the in-house dLAMP platform and commercial digital PCR platform was compared. Results: Duo to the chemical modification of the chip surface and the inner wall of micropores, the filling rate of the chips increased from 85.5% to 95.8%. The injection effect was improved significantly. The single channel chip and multi-channel chip could meet the requirements of single and multi-index detection at one time respectively. Additives such as formamide and nanoparticles were of great significance in improving the detection performance of nanoliter level LAMP reaction system. The lowest detection sensitivity of CALR-1 mutation burden was 0.5%, that of CALR-2 muta

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