Novel techniques, automated machines, sophisticated synthetic and bioprospected polymerases, and developments in symbiotic sequencing technologies have transformed PCR into a multifaceted, adaptable molecular tool.
Such advancements, however, have tethered PCR to expensive laboratories, requiring ever more specialized equipment and technicians. Looking to the future, improvements in PCR technologies will simplify and speed up the process, reduce costs, and enable miniaturization of PCR devices, taking diagnostics out of the lab and placing it firmly at the point of care (POC).
PCR is increasingly used to interrogate the genomes of humans, animals, bacteria, and viruses in different ways. Though the process has long been routinely used for detection of infectious diseases such as malaria and flu, quantitative PCR (qPCR) and digital PCR (dPCR) have significantly increased the sensitivity and precision of such tests.
The ability to identify single nucleotide polymorphisms (SNPs) or regions of DNA associated with infectious disease drug resistance is crucial to effectively treating diseases, and thereby preventing further spread within the population, or overprescribing therapeutics that will have no benefit.
The development of dPCR has seen advances in the precision and sensitivity of PCR for absolute quantification of nucleic acid targets, key capabilities for the detection and treatment of food-borne pathogens such as salmonella and E. coli, as well as rare mutations such as those seen in cancerous cells.
Neoplastic cells have been successfully detected in the sputum, urine, and stool of patients with lung, bladder, and colon cancer respectively, and circulating tumor cells have been shown to be a useful biomarker for metastatic breast cancer.
In addition, the development of dPCR as a noninvasive prenatal testing (NIPT) diagnostic using maternal blood is a promising focus of current research.
High-Quality Diagnostics
Though PCR is now used for a multitude of diagnostic applications, from infectious diseases to NIPT, these tests are costly, and it can take weeks to return results to the patient. As technology moves forward, and more complex automated devices allow for real-time results with higher specificity and resolution, affordability and accessibility are expected to improve. But such gains have yet to materialize.
As the cost of standard polymerases falls, new, high-priced PCR machines, which require costly reagents and polymerases, are incrementally introduced to maintain the market share of nextgen technologies.
Rather than allowing more accessibility, these new PCR machines require increasingly well-stocked labs and well-trained staff. The infrastructure and skill levels required prohibit the use of such tests outside the developed world. Reversing this trend requires new thinking and disruptive, game-changing technology.
Recent advances in microfluidics now allow for POC PCR. This technology will make PCR-based diagnostic testing accessible in various settings worldwide, especially in remote, underserved communities where the detection of drug resistance markers for infectious diseases could revolutionize healthcare provision.
Microfluidics and PCR
Microfluidic techniques have developed from the advent of dPCR, which relies upon the ability to generate small-reaction volumes in an accurate and repeatable manner. Single-molecule isolation technologies have shown promise of late, with Quanterix utilizing single-molecule array technology to produce an extremely sensitive assay.
Innovative researchers have been applying microfluidics techniques to PCR, with the recent production of thermal cyclers utilizing microwells, capillaries, and microdroplet formation. Companies such as RainDance Technologies and QuantaLife provide extremely high levels of sensitivity with their microdroplet PCR devices. Further microfluidic advancement is pivotal to future advances in amplification techniques and nucleic acid detection.
Microfluidics technology increases the speed of PCR by orders of magnitude. Speed is only theoretically limited by the processivity of the polymerase and the length of the region to be amplified.
In reality, the limiting factor is often the time it takes to cycle the reaction mixture through the reaction temperatures, so-called ramping. Ramping in conventional PCR machines is time- and energy-intensive due to the thermal mass of the block and is not readily made portable.
Improvements in buffer systems and engineered enzymes have increased the speed of standard benchtop thermal cyclers. Typically, however, these methods still take over 90 minutes, with 40 minutes being considered fast. This is far too long to be practical in a clinical POC setting.
Very few commercially available devices are capable of truly rapid PCR, none of which can be considered portable. BJS Technologies, though it doesn’t utilize true microfluidics technology, has developed the xxpress benchtop microwell thermal cycler. This device is capable of 40 cycles in just 10 minutes because it has a ramp rate of 10°C per second.
Microfluidic PCR systems fall into two categories—microwell reactors and continuous flow. Microwell thermal cyclers are essentially miniaturized versions of traditional PCR machines, reducing the input and chamber volumes such that the time it takes to heat and cool the reaction mix is likewise reduced.
Roche’s LightCycler holds PCR mix within capillaries, thereby increasing the surface-to-volume ratio and reducing the cycle time. Despite a runtime of approximately 30 minutes, the LightCycler technology has been commercially successful.
Continuous flow devices, on the other hand, require heating zones of different temperatures, over or between which the reaction mixture is passed in microfluidic channels. This lab-on-chip approach greatly reduces energy consumption, as once heat has been added to the system it only has to be maintained and not ramped. Provided there is good thermal contact between the flowing reaction mixture and the heaters, extremely rapid PCR can be achieved.
With microfluidic channels, surface area is increased and the time to reach thermal equilibrium at any given temperature zone becomes fractions of seconds. This equates to rapid ramping. An additional benefit of continuous flow devices is that they utilize disposable plastic microfluidics cassettes in lieu of large batch processing machines, lowering the cost per test and eliminating the possibility of cross-contamination between samples.
The increased surface-to-volume ratio in microfluidic PCR is not without its challenges. Polymerase can become denatured on the walls of microfluidic channels, and primer concentration often has to be increased for the same reasons. Inhibition can be overcome by careful choice of materials and utilizing surface chemistry to increase hydrophobicity.
Alternatively, active or passive passivation layers can be used to decrease the high adsorption rate of reagents by the materials. Pumping mechanisms and channel dimensions also require careful consideration because of their effect on laminar flow and dispersion. Research teams worldwide are optimizing continuous flow PCR, with advances in materials, heating and cooling systems, and connectors, all required to provide more efficient integrated systems.
Continuous flow PCR was born out of capillary electrophoresis in the early 1990s. It was pioneered by Andreas Manz, who has been central in the early development of microchip devices for chemical and biological analysis. However, during this time it was not commercialized.
More recently, academic labs, such as that of Niel Crews, have further developed the early iterations of continuous flow PCR, although the majority of these devices have only been used in the research setting. Thermal Gradient has produced a commercially available continuous flow PCR device capable of sub-10 minute runtime by pumping PCR reaction mix through a sandwich of two or three heating zones.
The single-use devices are intended for integration into POC devices. While rapid PCR would save time and money for researchers, it could also save lives if put into the hands of healthcare workers as part of a POC diagnostic test.
Continuous flow PCR is ideal for POC applications with amplification of DNA possible in just a few minutes, microfluidic PCR opens up the possibility for rapid diagnostic testing. When combined with automated sample preparation and DNA detection technologies, such as QuantuMDx’s Q-POC device, the development of which also benefits from microfluidics engineering, a portable, fully integrated POC diagnostic device could extend MDx to resource-limited settings.
Continuous flow PCR, with low power, sample size, and reagent volume requirements, is ideal for inclusion in a handheld device, which will be produced at much lower cost than traditional PCR machines due to the lack of robotics. Per-test costs also will be lower due to the much smaller volumes of reagents required.
Moreover, due to the speed of of amplification the speed of molecular analysis will be improved, and with assay times in the 10-minute range, this makes it relevant to in-field or in-clinic MDx testing.
The resulting sample-to-result device will not only be less costly than traditional PCR machines but significantly easier to operate, negating the need for well-stocked laboratories and highly skilled technicians. Such a technology leap could provide resource-scarce settings with access to the high-quality PCR-based diagnostic testing that is available in the developed world.
Providing diagnostic results in minutes rather than days, weeks, or months could revolutionize healthcare worldwide by surmounting many of the obstacles associated with traditional healthcare provision models in the developing world.
Lack of reliable electricity supply, clean water, transportation (particularly cold chain transportation for reagents), and highly skilled technicians are all hurdles that could be overcome by a simple POC test. Such a test would make complex diagnostics affordable and accessible, and could improve outcomes through early detection and treatment of disease.
Advancements in speed, accuracy, and cost control will open up developed and developing healthcare economies to the advantages of state-of-the-art PCR and related diagnostic technologies, including reduced test costs as healthcare costs skyrocket, reduced waiting times as hospitals overflow, and improved accuracy for priority diagnostics.
This will aid in diagnosis and treatment of patients worldwide, whether for a drug-resistant infectious disease, cancer, or a genetic condition. The development of such cutting-edge diagnostics is dependent on the creation of novel rapid microfluidic PCR methods, and the resulting technologies are now set to change the basis of genetic diagnosis.
Source: Genetic Engineering & Biotechnology News
