Speeding up the PCR Process

The market is witnessing an emerging trend of digital and droplet digital PCR technology, which is much more sensitive and accurate than the traditional method.

Amazing scientific advancements have been made in the thermal cyclers, to expandknowledge from human evolution, to sequencing human and many other genomes, to understanding genetic variation and drug efficacy in human disease research. Since their introduction, there have been numerous and significant improvements in both instruments and reagents. These include the introduction of real-time PCR (qPCR) and digital PCR (dPCR) technologies, new fluorophores, specialist master mixes, and fast thermal cyclers, leading to an enormous expansion of practical uses, not least high-throughput applications. Over the decades various advances have also been made to improve the ability to control the speed and accuracy of this important instrument. Continuous technology advancements are making it more efficient and user-friendly.

Although polymerase chain reaction (PCR) has become a widely adopted technique in clinical laboratories, it has not become a commonly used point-of-care or field tool. Currently, the market players are involved in enhancing the capabilities of these systems, and increasing their specificity and sensitivity. One significant roadblock that prevents such applications is the need of thermal cyclers that are often too expensive and bulky. However, there have been significant efforts toward miniaturizing the instrument. The market is witnessing an emerging trend of digital and droplet digital PCR technology, which is much more sensitive and accurate compared to the traditional method.

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

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. The growing 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
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.

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 ten 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 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.

Outlook
As PCR technologies have evolved, so have their speed and thermal accuracy. This has opened the possibility of while you wait test, allowing doctors to perform diagnostic tests and prescribe the most appropriate medication all in one visit, particularly important in remote areas. Rapid testing also has a role in manufacturing, picking up suspected contamination quickly, and reducing the time that production lines are halted, or the amount of spoiled product that must be destroyed.

Rapid, simple, and reliable PCR tests can also have an important role in research, as shorter PCR cycle time, lower batch cost, and quicker turnaround between batches allow researchers to follow trains of thought much more intuitively. However, there is still room for greater speed, improved consistency and accuracy, and simpler workflows for researchers and technicians.