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.

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.

Low Volume STR Amplification Options: Coupling with Standard or Fast PCR, Traditional or Normalized DNA Extraction, and/or Traditional or Alternative Capillary Electrophoresis.

STR amplification leads directly to profile development, which is also impacted by DNA extraction and capillary electrophoresis detection. Amplification for forensic human identification purposes is inherently a costly process; reduced volume reactions have long been an effective cost-savings measure. Processing known, high-quality, single-source DNA samples (i.e., buccal samples) allows for the use of even lower reaction volumes. This chapter provides examples of 3 µL and 6 µL reactions for a variety of commercial amplification kits for use with buccal samples, including standard and fast PCR using KAPA2G™ Multiplex Mix. These reactions can be utilized with traditional DNA extracts or those obtained from a normalized extraction with the ChargeSwitch® Forensic DNA Purification Kit. They can be detected via traditional capillary electrophoresis using POP-4™ polymer and a 36 cm array, or an alternative method using POP-6™ polymer and a 22 cm array on the 3130 series Genetic Analyzer instruments. This chapter also includes protocols for the normalized extraction and alternative detection method.

Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers.

A new approach for improved RT-PCR is described. It is based on primers designed to form controlled stem-loop and homodimer configurations, hence the name 'double-bubble' primers. The primers contain three main regions for efficient RT-PCR: a 3' short overhang to allow reverse transcription, a stem region for hot start and a template-specific region for PCR amplification. As proof of principle, GAPDH, SARS-CoV-2 synthetic RNA and SARS-CoV-2 virus-positive nasopharyngeal swabs were used as templates. Additionally, these primers were used to positively confirm the N501Y mutation from nasopharyngeal swabs. Evidence is presented that the double-bubble primers offer fast, specific, robust and cost-effective improvement in RT-PCR amplification for detection of gene expression in general and for diagnostic detection and genotyping of SARS-CoV-2 in particular.

Integrated, Automated, Fast PCR System for Point-Of-Care Molecular Diagnosis of Bacterial Infection.

We developed an integrated PCR system that performs automated sample preparation and fast polymerase chain reaction (PCR) for application in point-of care (POC) testing. This system is assembled from inexpensive 3D-printing parts, off-the-shelf electronics and motors. Molecular detection requires a series of procedures including sample preparation, amplification, and fluorescence intensity analysis. The system can perform automated DNA sample preparation (extraction, separation and purification) in ≤5 min. The variance of the automated sample preparation was clearly lower than that achieved using manual DNA extraction. Fast thermal ramp cycles were generated by a customized thermocycler designed to automatically transport samples between heating and cooling blocks. Despite the large sample volume (50 µL), rapid two-step PCR amplification completed 40 cycles in ≤13.8 min. Variations in fluorescence intensity were measured by analyzing fluorescence images. As proof of concept of this system, we demonstrated the rapid DNA detection of pathogenic bacteria. We also compared the sensitivity of this system with that of a commercial device during the automated extraction and fast PCR of Salmonella bacteria.

Fast identification of Escherichia coli in urinary tract infections using a virulence gene based PCR approach in a novel thermal cycler.

Uropathogenic Escherichia coli (UPEC) is the most common causal agent of urinary tract infections (UTIs) in humans. Currently, clinical detection methods take hours (dipsticks) to days (culturing methods), limiting rapid intervention. As an alternative, the use of molecular methods could improve speed and accuracy, but their applicability is complicated by high genomic variability within UPEC strains. Here, we describe a novel PCR-based method for the identification of E. coli in urine. Based on in silico screening of UPEC genomes, we selected three UPEC-specific genes predicted to be involved in pathogenesis (c3509, c3686 (yrbH) and chuA), and one E. coli-specific marker gene (uidA). We validated the method on 128 clinical (UTI) strains. Despite differential occurrences of these genes in uropathogenic E. coli, the method, when using multi-gene combinations, specifically detected the target organism across all samples. The lower detection limit, assessed with model UPEC strains, was approximately 104 CFU/ml. Additionally, the use of this method in a novel ultrafast PCR thermal cycler (Nextgen PCR) allowed a detection time from urine sampling to identification of only 52 min. This is the first study that uses such defined sets of marker genes for the detection of E. coli in UTIs. In addition, we are the first to demonstrate the potential of the Nextgen thermal cycler. Our E. coli identification method has the potential to be a rapid, reliable and inexpensive alternative for traditional methods.

A novel, integrated forensic microdevice on a rotation-driven platform: Buccal swab to STR product in less than 2 h.

This work describes the development of a novel microdevice for forensic DNA processing of reference swabs. This microdevice incorporates an enzyme-based assay for DNA preparation, which allows for faster processing times and reduced sample handling. Infrared-mediated PCR (IR-PCR) is used for STR amplification using a custom reaction mixture, allowing for amplification of STR loci in 45 min while circumventing the limitations of traditional block thermocyclers. Uniquely positioned valves coupled with a simple rotational platform are used to exert fluidic control, eliminating the need for bulky external equipment. All microdevices were fabricated using laser ablation and thermal bonding of PMMA layers. Using this microdevice, the enzyme-mediated DNA liberation module produced DNA yields similar to or higher than those produced using the traditional (tube-based) protocol. Initial microdevice IR-PCR experiments to test the amplification module and reaction (using Phusion Flash/SpeedSTAR) generated near-full profiles that suffered from interlocus peak imbalance and poor adenylation (significant -A). However, subsequent attempts using KAPA 2G and Pfu Ultra polymerases generated full STR profiles with improved interlocus balance and the expected adenylated product. A fully integrated run designed to test microfluidic control successfully generated CE-ready STR amplicons in less than 2 h (<1 h of hands-on time). Using this approach, high-quality STR profiles were developed that were consistent with those produced using conventional DNA purification and STR amplification methods. This method is a smaller, more elegant solution than current microdevice methods and offers a cheaper, hands-free, closed-system alternative to traditional forensic methods.

Development of a normalized extraction to further aid in fast, high-throughput processing of forensic DNA reference samples.

The goal of this project was to develop a "normalized" extraction procedure to be used in conjunction with previously validated 3µL fast PCR reactions (42-51min utilizing KAPA2G™ Fast Multiplex PCR Kit) and alternative capillary electrophoresis (24-28min injection using POP-6™ Polymer and a 22cm array). This was the final phase of a workflow overhaul for the database unit at Cellmark Forensics to achieve a substantial reduction in processing time for forensic DNA database samples without incurring significant added costs and/or the need for new instrumentation, while still generating high quality STR profiles. Extraction normalization aimed to consistently yield a small range of DNA concentrations, thereby eliminating the need for sample quantification and dilution. This was specifically achieved using the ChargeSwitch® Forensic DNA Purification Kit and a reduction in extraction bead quantity, thereby forcing an increase in bead binding efficiency. Following development of this extraction procedure, an evaluation ensued to assess the combination of normalized extraction, 3µL fast PCR (with PowerPlex 16 HS, Identifiler Plus and Identifiler primer sets), and alternative CE detection - further referred to as new "first pass" procedures. These modifications resulted in a 37% reduction in processing time and were evaluated via an in depth validation, from which nearly 2000 STR profiles were generated, of which 554 profiles from 77 swab donors and 210 profiles from 35 buccal collector donors specifically arose from the new first pass procedures. This validation demonstrates the robustness of these processes for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated and their ability to generate high quality STR profiles with 95-99% and 88-91% pass rates, respectively.

Rapid genotyping of 25 autosomal STRs in a Japanese population using fluorescent universal primers containing locked nucleic acids.

Amplification of fluorescently labeled products is one of the most popular methods for genotyping genetic variations. Two-step amplification using fluorescent universal primers simultaneously produces multiple targeted fragments labeled with fluorescent dyes, and this strategy is applicable to large-scale, cost-effective genotyping. In this study, we developed a fast PCR-based, multiple short tandem repeat (STR) genotyping method using fluorescent universal primers containing locked nucleic acids (LNAs). Four amplification reactions, each assaying six or seven markers and using 0.5-1.0 ng of genomic DNA, produced obvious Fam-labeled peaks in all 26 loci tested (25 autosomal STRs and amelogenin). The overall amplification time was 37 min. Moreover, fluorescent signals for the 25 STRs obtained from LNA-containing primers were 1.5-9.0 fold higher compared to those from non-LNA primers. Using genomic DNA from 120 Japanese individuals, 16 out of the 25 STRs had observed heterozygosity greater than 0.7. Some of these 25 STRs also had high discriminatory power, similar to that of the 13 core STRs in the Combined DNA Index System dataset. The probability of incorrectly assigning a match based on the accumulated matching probability for these 25 STRs is 1.2 × 10(-22), and their combined use can provide robust information for Japanese forensics.

Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.

Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd ) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.