A roadmap to high-speed polymerase chain reaction (PCR): COVID-19 as a technology accelerator.

The limit of detection (LOD), speed, and cost of crucial COVID-19 diagnostic tools, including lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), and polymerase chain reactions (PCR), have all improved because of the financial and governmental support for the epidemic. The most notable improvement in overall efficiency among them has been seen with PCR. Its significance for human health increased during the COVID-19 pandemic, when it emerged as the commonly used approach for identifying the virus. However, because of problems with speed, complexity, and expense, PCR deployment in point-of-care settings continues to be difficult. Microfluidic platforms offer a promising solution by enabling the development of smaller, more affordable, and faster PCR systems. In this review, we delve into the engineering challenges associated with the advancement of high-speed microfluidic PCR equipment. We introduce criteria that facilitate the evaluation and comparison of factors such as speed, LOD, cycling efficiency, and multiplexing capacity, considering sample volume, fluidics, PCR reactor geometry and materials, as well as heating/cooling methods. We also provide a comprehensive list of commercially available PCR devices and conclude with projections and a discussion regarding the current obstacles that need to be addressed in order to progress further in this field.

Rapid Cycle and Extreme Polymerase Chain Reaction.

Rapid cycle polymerase chain reaction (PCR) amplifies DNA in 10-30 min, while extreme PCR is complete in less than 1 min. These methods do not sacrifice quality for speed; sensitivity, specificity, and yield are equivalent or better than conventional PCR. What is required (and not widely available) is rapid, accurate control of reaction temperature during cycling. Specificity improves with cycling speed, and efficiency can be maintained by increasing polymerase and primer concentrations. Speed is aided by simplicity, dyes that stain double-stranded DNA are less expensive than probes, and one of the simplest polymerases, the deletion mutant KlenTaq, is used throughout. Rapid amplification can be coupled with endpoint melting analysis to verify product identity. Instead of commercial master mixes, detailed formulations for reagents and master mixes compatible with rapid cycle and extreme PCR are described.

Rational PCR Reactor Design in Microfluidics.

Limit of detection (LOD), speed, and cost for some of the most important diagnostic tools, i.e., lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), and polymerase chain reaction (PCR), all benefited from both the financial and regulatory support brought about by the pandemic. From those three, PCR has gained the most in overall performance. However, implementing PCR in point of care (POC) settings remains challenging because of its stringent requirements for a low LOD, multiplexing, accuracy, selectivity, robustness, and cost. Moreover, from a clinical point of view, it has become very desirable to attain an overall sample-to-answer time (t) of 10 min or less. Based on those POC requirements, we introduce three parameters to guide the design towards the next generation of PCR reactors: the overall sample-to-answer time (t); lambda (λ), a measure that sets the minimum number of copies required per reactor volume; and gamma (γ), the system's thermal efficiency. These three parameters control the necessary sample volume, the number of reactors that are feasible (for multiplexing), the type of fluidics, the PCR reactor shape, the thermal conductivity, the diffusivity of the materials used, and the type of heating and cooling systems employed. Then, as an illustration, we carry out a numerical simulation of temperature changes in a PCR device, discuss the leading commercial and RT-qPCR contenders under development, and suggest approaches to achieve the PCR reactor for RT-qPCR of the future.

The kinetic requirements of extreme qPCR.

The kinetic requirements of quantitative PCR were experimentally dissected into the stages of DNA denaturation, primer annealing, and polymerase extension. The temperature/time conditions for 2 stages were kept optimal, while the other was limited until the amplification efficiency decreased as measured by an increase in quantification cycle (Cq). Extension was studied in a commercial capillary LightCycler®. Using a rapid deletion mutant of Taq (KlenTaq™), about 1 s was required for every 70 bp of product length. To study annealing and denaturation times of <1 s, a custom "extreme" PCR instrument with 3 temperatures was used along with increased primer and polymerase concentrations. Actual sample temperatures and times were measured rather than programmed or predicted. For denaturation, 200-500 ms above the denaturation threshold was necessary for maximal efficiency. For annealing, 300-1000 ms below the annealing threshold was required. Temperature thresholds were set at 98% primer annealing or PCR product denaturation as determined experimentally by melting curves. Progressing from rapid cycle PCR to extreme PCR decreased cycling times by 10-60 fold. If temperatures are controlled accurately and flexibility in reagents is allowed, PCR of short products can be performed in less than 15 s. We also put PCR in context to other emerging methods and consider its relevance to the evolution of molecular diagnostics.