Capillary-based integrated digital PCR in picoliter droplets
Capillary-based integrated digital PCR in picoliter droplets
复制标题
基于毛细管的皮升液滴集成数字 PCR
DOI：
10.1039/c7lc01160a
复制
发表时间：
2018-02-07
期刊：
影响因子：
6.1
通讯作者：
He, Liqun
中科院分区：
文献类型：
--
作者：
Chen, Jinyu;Luo, Zhaofeng;He, Liqun
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，在它们已经由发生器制成之后，必须将液滴手动转移到用于扩增和检测的模块。在实践中，一些液滴（约10%）在手动转移过程中丢失，导致低估目标。此外，液滴在转移过程中也存在交叉污染的风险。相比之下，在实验室中，已经证明了一些基于芯片的ddPCR，其中液滴总是在通道中运行。然而，由于壁润湿以及热振荡，液滴容易在芯片中聚结为大液滴。当这种ddPCR应用于绝对量化罕见突变时，例如在临床研究的早期诊断中或在细胞水平测量生物多样性时，液滴的损失变得严重。在这里，我们提出了一个基于毛细管的集成ddPCR系统，用于第一次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，并且长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。通过对肺癌特异性基因（LunX）的绝对定量来验证系统的性能。结果表明，该系统在NTC至2.4 × 10−4拷贝/μL的浓度范围内具有非常好的线性（0.9988）。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都是上级的。ddPCR系统可能为临床或学术应用提供一种强有力的方法，其中大多数考虑罕见事件。
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.