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Capillary-based integrated digital PCR in picoliter droplets†
Lab on a Chip ( IF 6.1 ) Pub Date : 2017-12-15 00:00:00 , DOI: 10.1039/c7lc01160a
Jinyu Chen 1, 2, 3, 4 , Zhaofeng Luo 2, 3, 4, 5 , Lin Li 3, 4, 6, 7 , Jinlong He 1, 2, 3, 4 , Luoquan Li 1, 2, 3, 4 , Jianwei Zhu 1, 2, 3, 4 , Ping Wu 4, 8 , Liqun He 1, 2, 3, 4
Affiliation  

The droplet digital polymerase chain reaction (ddPCR) is becoming more and more popular in diagnostic applications in academia and industry. In commercially available ddPCR systems, after they have been made by a generator, the droplets have to be transferred manually to modules for amplification and detection. In practice, some of the droplets (∼10%) are lost during manual transfer, leading to underestimation of the targets. In addition, the droplets are also at risk of cross-contamination during transfer. By contrast, in labs, some chip-based ddPCRs have been demonstrated where droplets always run in channels. However, the droplets easily coalesce to large ones in chips due to wall wetting as well as thermal oscillation. The loss of droplets becomes serious when such ddPCRs are applied to absolutely quantify rare mutations, such as in early diagnostics in clinical research or when measuring biological diversity at the cell level. Here, we propose a capillary-based integrated ddPCR system that is used for the first time to realize absolute quantification in this way. In this system, a HPLC T-junction is used to generate droplets and a long HPLC capillary connects the generator with both a capillary-based thermocycler and a capillary-based cytometer. The performance of the system is validated by absolute quantification of a gene specific to lung cancer (LunX). The results show that this system has very good linearity (0.9988) at concentrations ranging from NTC to 2.4 × 10−4 copies per μL. As compared to qPCR, the all-in-one scheme is superior both in terms of the detection limit and the smaller fold changes measurement. The system of ddPCR might provide a powerful approach for clinical or academic applications where rare events are mostly considered.

中文翻译:

皮升液滴中基于毛细管的集成数字PCR

液滴数字聚合酶链反应(ddPCR)在学术界和工业界的诊断应用中越来越受欢迎。在市售的ddPCR系统中,在用发生器将其滴下后,必须将液滴手动转移到模块中进行扩增和检测。在实践中,一些液滴(约10%)在手动转移过程中会损失掉,从而导致目标的低估。另外,液滴在转移过程中也有交叉污染的风险。相比之下,在实验室中,已经证明了一些基于芯片的ddPCR,其中液滴始终在通道中运行。但是,由于壁润湿和热振荡,液滴很容易在芯片上聚结成较大的液滴。当将此类ddPCR用于绝对定量稀有突变时,液滴的损失会变得很严重,例如在临床研究的早期诊断中或在细胞水平上测量生物多样性时。在这里,我们提出了一种基于毛细管的集成ddPCR系统,该系统首次用于以这种方式实现绝对定量。在该系统中,HPLC T型接头用于产生液滴,长的HPLC毛细管将发生器与基于毛细管的热循环仪和基于毛细管的细胞仪连接起来。该系统的性能通过对肺癌(LunX)特异的基因的绝对定量来验证。结果表明,该系统在NTC至2.4×10的浓度范围内具有很好的线性(0.9988)。我们提出了一种基于毛细管的集成ddPCR系统,该系统首次用于以这种方式实现绝对定量。在该系统中,HPLC T型接头用于产生液滴,长的HPLC毛细管将发生器与基于毛细管的热循环仪和基于毛细管的细胞仪连接起来。该系统的性能通过对肺癌(LunX)特异的基因的绝对定量来验证。结果表明,该系统在NTC至2.4×10的浓度范围内具有很好的线性(0.9988)。我们提出了一种基于毛细管的集成ddPCR系统,该系统首次用于以这种方式实现绝对定量。在该系统中,HPLC T型接头用于产生液滴,长的HPLC毛细管将发生器与基于毛细管的热循环仪和基于毛细管的细胞仪连接起来。该系统的性能通过对肺癌(LunX)特异的基因的绝对定量来验证。结果表明,该系统在NTC至2.4×10的浓度范围内具有很好的线性(0.9988)。该系统的性能通过对肺癌(LunX)特异的基因的绝对定量来验证。结果表明,该系统在NTC至2.4×10的浓度范围内具有很好的线性(0.9988)。该系统的性能通过对肺癌(LunX)特异的基因的绝对定量来验证。结果表明,该系统在NTC至2.4×10的浓度范围内具有很好的线性(0.9988)。每微升−4拷贝。与qPCR相比,多合一方案在检测限和较小的倍数变化测量方面均具有优势。ddPCR系统可能为临床或学术应用程序提供强大的方法,这些应用程序通常会考虑罕见事件。
更新日期:2017-12-15
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