Honing Technology to Unveil New Opportunities

Thermal cyclers continue to take on increasingly diverse applications, expanding the horizons, thus, taking the technique out of the lab and into the clinics.

Thermal cyclers, in various forms have been a mainstay in the molecular biologist’s toolbox since their discovery more than three decades ago. The fundamental principle of exponential amplification of a sequence of DNA by reiterative use of a template-based polymerase has remained steady through vast improvements in reagents, protocols, and instrumentation, which have in turn given rise to corresponding leaps in speed, fidelity, and the ability to multiplex, quantitate, and more.

Thermal cyclers turn 34 this year, and life scientists use it as much as ever, especially the technologies that quantify the DNA, such as real-time or quantitative PCR (qPCR) and digital PCR (dPCR). Ongoing advancements in dPCR, continued expansion in distribution networks of emerging dPCR product manufacturers in global markets, and growing private funding to develop and commercialize innovative dPCR instruments are contributing to the growth of the dPCR technology.

The thermal cyclers industry has expanded and changed tremendously since its advent, from the initial labor-intensive and time-consuming methods. Technological improvements and novel method variations have simplified the process and made clinical implementation much easier. The use of thermal cyclers continues to augment into nonconventional applications, producing unprecedented growth in the global market. PCR allows DNA sequencing, as well as the production of millions of copies of a specific DNA sequence, within minutes. As a result, PCR technology has helped make molecular diagnosis easier and faster. It is used frequently to diagnose disease, identify bacteria and viruses, and aid forensic investigations.

Significant technological improvements include utilization of LED light sources, redesign of the heat-block elements to improve cycling times, additional fluorescent detectors, and the identification of alternative strategies to generate fluorescent signals. These enhancements have made it possible to perform a PCR assay in the traditional 96-well PCR plate in as less as 20–30 minutes. Miniaturization of the standard PCR tube and instrument to perform a single or small number of samples in a much smaller reaction vessel has the potential to significantly reduce the reagent use and cost, and in addition has further decreased the needed cycling times. The commercialization of many new instrument designs is ongoing and should have a significant impact on PCR-related testing, especially for utilization of PCR testing in smaller hospital settings and point-of-care testing applications.

Ultrafast thermal modules have pushed the speed limits of standard, well-based reaction blocks. More dramatic gains in speed are possible through reimagining how a sample may be thermally cycled. Improvements in thermal cycling are the next hurdle to be overcome to drive PCR technology forward. With speed and integration being the key steps forward, thermal cyclers will continue to be a central component for a variety of nucleic acid analysis solutions.

Indian Market Dynamics
The Indian thermal cyclers market is estimated at  83.12 crores.

Traditional PCRs in volume terms have a 73 percent share, whereas in value terms they have a 41 percent contribution. They may be segmented into two categories, with the high-end dominating with a 60 percent share in value terms. Thermo Fisher is a clear leader in this segment. Bio Rad and Eppendorf are also aggressive.

Real-time PCRs may also be segmented into two categories based on their unit price, functions, and capabilities. The top-of-the-line have a 40 percent share. Thermo Fisher leads the group, with an aggressive presence from Bio Rad. Agilent, Roche, and Hi-media also cater to this segment.

Global Market Dynamics

The global thermal cyclers market is expected to reach USD 9.8 billion by 2021 from USD 7.9 billion in 2016 at a CAGR of 4.4 percent, estimates BCC Research. With the advancements surging at a greater pace, the global dPCR and qPCR market is expected to grow at a higher CAGR in the next 5 years. Growth in the geriatric population, rising incidence of infectious diseases and genetic disorders, increasing investments and availability of funds for PCR-based research, and increasing use of biomarker profiling for disease diagnosis are driving the growth of the thermal cyclers market. Factors such as high cost of dPCR products and technical limitations associated with qPCR and dPCR are restraining the growth of this market.

Strides Made in Technology
Since the invention of the polymerase chain reaction in 1983, PCR has played a significant role in molecular diagnostics for genetic diseases, pathogens, oncogenes, and forensic identification. The simplicity of thermal cyclers as a molecular technique is, in some ways, responsible for the huge amount of innovation that surrounds it, as researchers continually think of new ways to tweak, adapt, and re-formulate concepts and applications. In the past three decades, thermal cyclers have evolved from end-point PCR, through real-time PCR, to its current version, which is the absolute quantitative dPCR. The manufacturers continue to seek ways to improve the speed and sensitivity of their thermal cyclers to gain a hold of the industry.

Digital PCR systems. With exceptional accuracy, sensitivity, reproducibility, direct quantification, and multiplexing, the dPCR systems allow for the detection of small-fold differences and can be a couple of orders of magnitude more sensitive in detecting rare targets in a complex background. This technique also provides day-to-day and lab-to-lab reproducibility, and the partitioning in dPCR allows for higher-level multiplexing compared to qPCR. Partitioning of a single sample into tens thousands of reaction modules, a capability demonstrated by dPCR, greatly improved robustness of nucleic acid detection, resulting in a massive improvement in precision and sensitivity.

Adaptive PCR. Biomedical engineers from Vanderbilt University, Tennessee developed a working prototype of an adaptive PCR machine that utilizes left-handed DNA (L-DNA) to monitor and control the molecular reactions that take place in the PCR process. The adaptive approach for controlling the PCR process promises to make the process simpler to operate, improve its reliability, reduce its sensitivity to environmental conditions, and shrink it from desktop to handheld size. Therefore, it could free PCR from the laboratory setting and allow it to work in the field or at the bedside where it could be used to identify different diseases by their DNA signatures.

Adaptive PCR sidesteps all the variables of normal PCR, by relying on the fluorescent L-DNA to determine the ideal cycle temperatures for annealing and denaturing. The researchers report that experiments with the prototype system have demonstrated that the technique duplicates the results of conventional PCR machines in controlled conditions and can efficiently amplify DNA under conditions that cause standard PCR to fail. These advantages have the potential to make PCR-based diagnostics more accessible outside of well-controlled laboratories, such as point-of-care and field settings that lack the resources to accurately control the reaction temperature or perform high-quality sample preparation.

Targeted multiplex amplification for next-generation sequencing.Next on the horizons for thermal cyclers is targeted multiplex amplification for next-generation sequencing (NGS), that is, the ability to perform multiplex PCR to amplify 10–1000 target regions of interest in a single reaction to prepare a library for sequencing. Barcoded libraries, that is, libraries with a different short sequence appended to one primer for each starting sample, can be pooled for sequencing. As a consequence, NGS can be affordable and provide more information than microarrays or multiple individual PCRs.

The Road Ahead
Thermal cyclers are considered one of the most significant biomedical advances of the 20th century and a fundamentally integral part of every molecular biology laboratory today. With the advent of PCR-based genomic screening assays, scientists can ask more questions and get faster answers than ever before. Concomitant advances in liquid handling and automation make it possible to boost sample throughput, as well as save time and increase reproducibility.

The next horizon for thermal cyclers will be improving detection and multiplexing capability for low-frequency SNVs from samples of limiting input quantity, such as liquid biopsy from circulating cell-free DNA (cfDNA). This can be achieved by NGS of multiplexed targeted amplicon panels. As the scientific community continues to demand greater data accuracy and more credible results, technologies such as dPCR, which can detect and quantify low-level nucleic acids, are becoming more and more commonplace. The future is ripe with opportunities to hone dPCR through improvements in reaction speed and integration.

Thermal cyclers continue to take on increasingly diverse applications, expanding the horizons, thus, taking the technique out of the lab and into the clinics. In the near future, the easiest-to-use technology for molecular diagnostics will continue to consist of PCR-based solutions. For point-of-care diagnostics, simplicity is the key for the wider adoption and growth of a broad clinical assay menu. Thermal cyclers will continue to play a leading role until another technology can achieve the simplicity and affordability that these systems offer today. Rapidly identifying the root cause responsible for a patient’s illness and accurately monitoring disease progression during treatment could influence clinical decisions and improve care. Point-of-care diagnostics will rely on PCR, most likely for pathogen detection for infectious disease, but perhaps also for screening of common resistance mutations to cancer therapies or other alleles relevant to drug response for a variety of conditions. New advances in the technology, including more sensitive assays and high-throughput instruments, may further revolutionize healthcare by enabling routine blood tests for early cancer detection and giving a momentous boost to the concept of personalized medicine in the coming years.

While technological improvements over the years have made thermal cyclers more precise and created equipment with smaller footprints, imagine a DNA photocopier small enough to hold in your hand that could identify the bacteria or virus causing an infection even before the symptoms appear. The future for thermal cyclers will be greater miniaturization while maintaining simplicity, sensitivity, and specificity. With the rise in demands for a faster, easier, and affordable technology, doing any one of the three may not be difficult, but achieving all three simultaneously requires real innovation.