Controlled Rehydration of Dried Reagents for Robust Multiplex Digital PCR

Abstract

Digital polymerase chain reaction (dPCR) is emerging as a powerful method for nucleic acid detection due to its unprecedented sensitivity and precision. However, most current dPCR platforms are inherently limited by their low multiplexing ability due to primer-pair cross interactions and spectral overlap of available fluorophores. Here, we present a novel and robust method for multiplexing dPCR that is free from primer dimerization and fluorescence channel number limitation, enabling highly precise and multiplexed detection of nucleic acid targets. By prestoring target-specific primers and probes in different storage chambers, the method physically separates reactions and thus avoids the primer-pair cross interactions and spectral overlap of different fluorescent probes that usually occur within a single-tube reaction. Furthermore, a dissolvable delay valve (DDV) is embedded between each pair of the reagent prestorage chamber and reaction microwell array. Such a DDV configuration allows full reconstitution of the prestored reagents and then generates a uniform concentration distribution of the reconstituted reagents across the entire reaction microwell array, which is favorable for achieving reliable and robust multiplex dPCR assays. We demonstrated the feasibility of this method by performing an eight-plex dPCR assay targeting the seven most common point mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and a reference sequence (wild-type KRAS allele).

An Evaluation of the Sensitivity and Applicability of a Droplet Digital Polymerase Chain Reaction Assay to Simultaneously Detect Pseudomonas aeruginosa and Pseudomonas fragi in Foods

“…Liu et al designed a dual-channel sensor, with represents positive P. fragi and P. aeruginosa reaction and established a multiplex digital PCR method without extracting ctDNA to reduce the reaction steps [37]. Xie Tengbao et al avoided the interference of cross primers and the overlap of fluorescence in a single tube through physical separation [38]. Simpler and more practical multiple detection methods still urgently need to be developed.…”

Advances in droplet digital polymerase chain reaction on microfluidic chips

“…Moreover, the probes are prestored in the store part on the chip and can be rehydrated using a dissolvable delay valve which enhances the spatial uniformity of reaction conditions. 151 In addition, in the detection of circulating tumor DNA, which is present in very small amounts in biological samples, denaturation-enhanced ddPCR is used to obtain results that improve the stability and precision of the detection of mutated genes. With the continuous efforts of researchers, ddPCR will eventually replace qPCR as a widely used nucleic acid detection tool in the future and play an increasingly important role in the field of life analysis.…”

A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets

“…In addition, dominant research methodologies in nucleic acid detection include droplet digital loop-mediated isothermal amplification (ddLAMP) and droplet digital recombinase polymerase amplification (ddRPA). , The reaction can be completed at a constant temperature because ddLAMP does not require costly, complicated thermal cyclers. The complete reaction is rapid, usually in 10 min, and enough detectable amplification products are produced. , However, the Poisson distribution is a fundamental limitation that all reported approaches share, in addition to their strengths.…”