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标题：CRISPR/Cas12a-Based Ultrasensitive and Rapid Detection of JAK2 V617F Somatic Mutation in Myeloproliferative Neoplasms
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作者：Miaomiao Chen ; Chunhua Zhang ; Zhiqing Hu 等
期刊名称：Biosensors
电子版ISSN：2079-6374
出版年度：2021
卷号：11
期号：8
DOI：10.3390/bios11080247
语种：English
出版社：MDPI Publishing
摘要：The J AK2 V617F mutation is a major diagnostic, therapeutic, and monitoring molecular target of Philadelphia-negative myeloproliferative neoplasms (MPNs). To date, numerous methods of detecting the JAK2 V617F mutation have been reported, but there is no gold-standard diagnostic method for clinical applications. Here, we developed and validated an efficient Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 12a (Cas12a)-based assay to detect the JAK2 V617F mutation. Our results showed that the sensitivity of the JAK2 V617F/Cas12a fluorescence detection system was as high as 0.01%, and the JAK2 V617F/Cas12a lateral flow strip assay could unambiguously detect as low as 0.5% of the JAK2 V617F mutation, which was much higher than the sensitivity required for clinical application. The minimum detectable concentration of genomic DNA achieved was 0.01 ng/μL (~5 aM, ~3 copies/μL). In addition, the whole process only took about 1.5 h, and the cost of an individual test was much lower than that of the current assays. Thus, our methods can be applied to detect the J AK2 V617F mutation, and they are highly sensitive, rapid, cost-effective, and convenient.
关键词：enJAK2V617F;CRISPR/Cas12a;mutation detection;fluorescence detection system;lateral flow strip;Philadelphia-negative myeloproliferative neoplasms