Capillary-based integrated digital PCR in picoliter droplets

Abstract

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 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.

Comparison of the intensity of biofilm formation by Listeria monocytogenes using classical culture-based method and digital droplet PCR

“…New capillary-based integrated ddPCR system limits crosscontamination. 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 (Chen et al 2018).…”

Off‐Chip Vertical Step Emulsification Droplets Preparation Device Applied for Droplet Digital PCR

“…One is chamber-based, [6][7][8] and the other is droplet-based. [9][10][11] The chamberbased dPCR devices need to process tens of thousands of microreactors with the same volume on substrates of different materials (such as polydimethylsiloxane [PDMS], [12] polymethyl methacrylate [PMMA], [13] fused silica, [10] silicon, [14] etc. ), and then the reagents are separated into each microreactors for PCR reaction.…”

Emulsion PCR (ePCR) as a Tool to Improve the Power of DGGE Analysis for Microbial Population Studies

“…After the ePCR reaction, obtained by microfluidics technology, the droplets stream individually pass through the reader for fluorescence analysis. Although this innovative automation of the system allows efficient process management, which reduces the risk of laboratory errors or cross-contamination, it does not currently allow population studies, for which further processing of the generated amplicons are essential [ 23 , 24 , 25 , 26 , 27 ].…”