Rapid detection of CALR type 1 and type 2 mutations using PNA-LNA clamping loop-mediated isothermal amplification on a CD-like microfluidic chip
Rapid detection of CALR type 1 and type 2 mutations using PNA-LNA clamping loop-mediated isothermal amplification on a CD-like microfluidic chip
复制标题
在类 CD 微流控芯片上使用 PNA-LNA 钳环介导的等温扩增快速检测 CALR 1 型和 2 型突变
DOI：
10.1016/j.aca.2018.04.022
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发表时间：
2018-09-18
影响因子：
6.2
通讯作者：
Guan, Ming
中科院分区：
文献类型：
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作者：
Cao, Guojun;Kong, Jilie;Guan, Ming
出血和血栓形成是骨髓增生性肿瘤（MPN）的常见并发症，并显著增加发病率和死亡率。分子标志物，包括CALR突变，不仅被认为是诊断标志物，而且是MPN相关出血和血栓形成的危险因素，特别是对于偏远基层医院的患者。我们试图开发一种易于使用的检测方法，用于快速检测费城染色体阴性MPN患者的CALR 1型（CALR-1）和CALR 2型（CALR-2）突变。建立了肽核酸锁核酸（PNA-LNA）夹持环介导等温扩增（LAMP）检测方法，将其集成到离心光盘（CD）微流控平台中。通过该微流控平台和内部的真实的时间PCR分析，同时测试了总共158个临床血液样品。验证了LAMP阵列的检测性能，并通过桑格测序鉴定了矛盾的结果。结果表明，我们开发的LAMP方法具有良好的灵敏度，特异性和精密度。用真实的时间荧光法检测CALR-1和CALR-2突变的检出限分别可达1%和0.5%，用目测法检测CALR-1和CALR-2突变的检出限分别为10%和5%。不同检测系统间无非特异性背景扩增。对于CALR-1和CALR-2 LAMP检测系统，1%突变质粒的批内CV值分别为10.56%和10.51%，批间CV值分别为19.55%和18.39%。产物均通过解链曲线分析和电泳分析，然后进行桑格测序分析，与数据库序列一致。该微流控平台可在60 min内完成CALR-1/2突变的快速检测。对临床标本的检测结果显示，CALR野生型133例，突变型15例，突变型9例。两种测定法的相关系数值（Kendall's tau_B）为0.99。有趣的是，通过新建立的检测平台，我们惊讶地发现一名中国患者同时携带CALR-1和CALR-2突变。通过桑格测序分析验证了该结果。本文开发的LAMP检测系统显示出良好的灵敏度、特异性和稳定性。此外，检测结果可以直接通过反应体系的颜色变化来判断，无需任何辅助设备。因此，我们开发的平台具有广泛应用于偏远和经济欠发达地区的潜力。(C)2018 Elsevier B. V.版权所有。
Bleeding and thrombosis represent common complications in myeloproliferative neoplasms (MPN) and significantly contribute to morbidity and mortality. Molecular markers, including CALR mutations, were considered not only as diagnostic markers, but also as risk factors for bleeding and thrombosis associated with MPN, especially for patients in remote primary hospitals. We sought to develop an easy-to-use assay for the rapid detection of CALR type 1 (CALR-1) and type 2 (CALR-2) mutations in Philadelphia chromosome-negative MPN patients. Peptide nucleic acid-locked nucleic acid (PNA-LNA) clamping loop-mediated isothermal amplification (LAMP) assays were established, which were integrated into a centrifugal compact disc (CD) microfluidic platform. A total of 158 clinical blood samples were tested simultaneously by this microfluidic platform and an in-house real time PCR assay. The detection performance of the LAMP arrays was validated and conflicting results were identified by Sanger sequencing. The results suggested that the LAMP methods we developed exhibited good sensitivity, specificity, and precision. By real time fluorescence assay the detection limit for CALR-1 and CALR-2 mutations could reach as low as 1% and 0.5% respectively, and 10% and 5% respectively by visual method. There were no nonspecific background amplifications among different detection systems. For the CALR-1 and CALR-2 LAMP detection systems, intra-batch CV values of 1% mutated plasmid were 10.56% and 10.51% respectively, and the inter-batch CV values were 19.55% and 18.39%, respectively. The products were all analyzed by melting curve analysis and electrophoresis followed by Sanger sequencing analysis, which were consistent with the database sequences. The microfluidic platform could complete rapid detection of CALR-1/2 mutations within 60 min. The results of clinical samples detected by our CD-like microfluidic chipLAMP assay and rtPCR assay suggested that 133 samples were CALR wild type, 15 were CALR-1 mutation type, and 9 were CALR-2 mutation type. The correlation coefficient value (Kendall's tau_b) of the two assays was 0.99. Interestingly, by the newly established detection platform, we were surprised to find that one patient of Chinese origin harbored both CALR-1 and CALR-2 mutations. This result was verified by Sanger sequencing analysis. The LAMP detection systems developed herein displayed good sensitivity, specificity, and stability. Additionally, the detection results could be directly judged by color changes of the reaction systems without any auxiliary equipment. Thus, the platform we developed has the potential of being widely used in remote and economically undeveloped areas in the future. (C) 2018 Elsevier B.V. All rights reserved.