Droplet digital PCR of viral ‎DNA/RNA, current progress, challenges, and future perspectives

Abstract

High‐throughput droplet‐based digital PCR (ddPCR) is a refinement of the conventional polymerase chain reaction (PCR)‎ methods. In ddPCR, DNA/RNA is encapsulated stochastically inside the microdroplets as reaction chambers. A small percentage of the reaction chamber contains one or fewer copies of the DNA or RNA. After PCR amplification, concentrations are determined based on the proportion of nonfluorescent partitions through the Poisson distribution. Some of the main features of ddPCR include high sensitivity and specificity, absolute quantification without a standard curve, high reproducibility, good tolerance to PCR inhibitor, and high efficacy compared to conventional molecular methods. These advantages make ddPCR a valuable addition to the virologist's toolbox. The following review outlines the recent technological advances in ddPCR methods and their applications in viral identification.

Diagnostic, Prognostic, and Therapeutic Value of Droplet Digital PCR (ddPCR) in COVID-19 Patients: A Systematic Review

“…Of interest, ddPCR is an end-point system, where the droplets are simply counted as positive (fluorescent) or negative (non-fluorescent). In contrast to RT-qPCR, it provides absolute target quantification (which is based on the amount of positive and negative droplets), without requiring an external calibration curve or keeping the reference material needed to construct it [ 58 , 59 ]. RT-qPCR is susceptible to amplification inhibitors; however, ddPCR is more tolerant to them—due to its nature that involves partitioning of the initial mixture before amplification into multiple reactions, a process that dilutes the inhibitors and enhances the signal/noise proportion—resulting in higher SARS-CoV-2 detection rates and enhanced reproducibility [ 45 ].…”

“…For instance, Yin et al designed a rapid digital PCR providing robust results in less than 15 min [ 29 ]. Similar to other assays used for SARS-CoV-2 detection, another drawback of ddPCR includes the generation of false positive results, caused by the inaccurate interpretation of negative droplets as positive [ 58 ]. The presence of false positive droplets could even appear while testing no-template controls [ 58 ].…”

“…Similar to other assays used for SARS-CoV-2 detection, another drawback of ddPCR includes the generation of false positive results, caused by the inaccurate interpretation of negative droplets as positive [ 58 ]. The presence of false positive droplets could even appear while testing no-template controls [ 58 ]. To deal with this issue, a few of the included studies focused their investigation on symptomatic patients, for example during their hospitalization [ 21 , 22 , 34 , 48 ].…”

“…Even if less likely than RT-qPCR, ddPCR detection ability could also be impacted by inhibitors or non-target genetic material. Thus, optimization of the ddPCR assay and strict quality control measures are needed to achieve accurate and reproducible results [ 58 , 62 ]. Improved artificial intelligence algorithms could also help towards this direction [ 64 ].…”

Nucleic Acid Testing of SARS-CoV-2

“…In recent years, we have seen the advance of digital PCR (dPCR) as a complementary approach for measuring nucleic acids, a technique that is highly accurate and reproducible when targeting the viral genes of SARS-CoV-2 [ 114 , 115 , 116 , 117 ]. The advantages of digital PCR compared to quantitative PCR include quantification without the need for calibration curves, higher accuracy, and sensitivity that may arise from sub-optimal amplification efficacy because dPCR can detect low amounts of nucleic acid [ 118 , 119 ].…”