Pushing Forward The Capabilities Of Thermal Cyclers

The PCR machines now cover the entire range from very simple one-channel instruments to sophisticated high-throughput systems capable of multiplexing up to six channels, as well as portable devices enabling point-of-care diagnostics.

Thermal cyclers, also referred as polymerase chain reaction (PCR) machines and thermocyclers, are essential for any laboratory that relies on molecular biology. An indispensable tool for DNA amplification, the thermal cycler often becomes a nonstop workhorse that lab workers expect to function properly and efficiently. Now marking its 35th anniversary, PCR is a standard tool in research, analytical, and diagnostics laboratories, creating millions or even billions of copies of the target DNA or RNA in just a few hours or less. As the technologies have evolved, so have their speed and thermal accuracy. This opens up the possibility of PCR as a while you wait test, allowing doctors to diagnose disease and prescribe the most appropriate medication all in one visit, particularly important in remote areas. 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.

Like many scientific techniques, the applications drive improvements and the technology allows new applications. For example, using PCR with heterogeneous samples spawned the need for mixes based on inhibitor-resistant enzymes. Likewise, the interest in increasing PCR throughput drives an ongoing decrease in the size of samples. Scientists need 20–50 µL to run PCR in 96-well plates, but just a few microliters for 1536-well plates. Microfluidic-based PCR chips run the reaction on nanoliter-size samples. The platforms that run smaller samples also tend to run PCR faster. To broaden the applications of PCR, scientists often turn to significant changes in the technology. As an example, digital PCR is being used to partition samples for more accurate analysis. Getting the most from a technology today often depends crucially on the balance of reagents and bioinformatics.

The number of commercially available PCR thermal cyclers has increased remarkably since their introduction. The machines now cover the whole range from very simple one-channel instruments to sophisticated high-throughput systems capable of multiplexing up to six channels, as well as portable devices enabling point-of-care diagnostics. The cyclers are evolving at a rapid pace. Biologists who do field work can now accomplish more in less time by analyzing samples on site. Even if the work is confined to the lab rather than the field, one can find greater convenience and efficiency in the newest thermal cyclers that are simple to use and offer step-by-step instructions. Some even include wi-fi capability, which means that one can connect to them wirelessly through a mobile device like a tablet or smartphone. The convenience of having the data streamed directly into one’s hand cannot be overstated, and the ability to control the thermal cycler remotely can be a huge convenience.

Major players – an update

Thermo Fisher Scientific
In July 2017, Thermo Fisher announced the successful CE-IVD registration of its QuantStudio 5 Dx real-time PCR system for sale in European countries. The platform provides customers with a modern and user-friendly instrument that is designed to easily integrate into their existing diagnostic workflow. Additionally, Siemens Healthineers and Thermo Fisher announced a strategic agreement focused on incorporating the QuantStudio 5 system into Siemens’ Atellica MDX 160 molecular system to provide more flexibility and customization to its laboratory workflow. Moreover, Seegene Inc. and Thermo Fisher jointly announced a collaborative partnership to seek clearance from the USFDA for Seegene›s Allplex diagnostic assay portfolio on the QuantStudio 5 system, for which Thermo Fisher is seeking clearance in parallel.

Agilent Technologies
In July 2017, Agilent expanded its portfolio of instruments for molecular diagnostics with the introduction of the AriaDx real-time PCR system. AriaDx is the only modular real-time PCR instrument in the market intended for in vitro diagnostic use. Real-time PCR is routinely used in laboratories to help identify pathogens, genotype infectious agents, and for cancer diagnostics. Whereas the Stratagene qPCR products portfolio primarily catered to customers in basic research, the launch of AriaDx now extends the portfolio from its use in research to diagnostics.

Canon
In April 2018, Molecular Biology Systems (MBS) announced that it will launch its revolutionary NEXTGENPCR thermal cycler in the United States of America and Canada through Canon BioMedical, Inc. Described as the first real advance in thermal cycling for 15 years, the NEXTGENPCR dramatically slashes current time-consuming DNA amplification from hours to minutes.

Global market
Before PCR, the DNA segments were amplified by using vectors in bacteria. This process took weeks to get the amplified DNA. As PCR is faster than this technique, it was readily accepted on the global level. PCR is also more efficient than the conventional techniques used for amplification. The global PCR market was valued at approximately USD 7.41 billion in 2017 and is expected to generate revenue of around USD 10.62 billion by the end of 2023, growing at a CAGR of around 6.2 percent, predicts Zion Market Research.

Rise in R&D expenditure, developments in pharmacogenomics, increase in trend of self-diagnosis of diseases as a preventive means, and emergence of digital PCR technology highly benefiting cancer diagnosis are some factors that are expected to boost the global PCR market during the forecast period. Moreover, increasing research and development activities in advanced molecular biology, genetic engineering, and forensic science are expected to propel the global PCR market during the forecast period. However, the emergence of alternative technology such as next-generation sequencing and the high cost of some commercial PCR technologies are expected to hamper the PCR market.

In terms of product, the reagents segment held the leading market share in the year 2017 and is expected to expand at the highest CAGR. This is attributable to high consumption of reagents for the PCR technique. Introduction of advanced reagents specific to the type of test is expected to boost the global demand for PCR reagents in the near future. In the instruments segment, the digital PCR systems sub-segment is expected to witness growth at a significantly rapid pace that is attributable to advantages of digital PCR systems such as precision, sensitivity, accuracy, reproducibility, direct quantification and multiplexing, and speed of the analysis.

The PCR technique has been found to be useful in pharmaceutical and biotechnology research activities as well as microbial quality testing. The technique is also applied in genetic engineering. Genetic engineering is the key driver for the global PCR market. It is used to identify genes related to certain phenotypes including genetic disorders. Regular testing of the microbial load of raw materials and finished products is an important process in the pharmaceutical and biotechnology industry. Sophisticated analytical methods such as PCR have been widely applied for quality control analysis in the pharmaceutical sector.

North America is anticipated to remain the leading region. Molecular diagnosis has revolutionized the modern diagnosis technology. PCR has become a method of choice in early and accurate detection of diseases. Expansion by leading manufacturers of PCR products in the Asia-Pacific region by strengthening of the distribution network and new product launches in developing countries of Asia-Pacific are key factors likely to drive the PCR market in the region during the forecast period. Moreover, rise in the incidence of cancer and infectious diseases has resulted in increase in the demand for use of the PCR technique in clinical diagnosis of these diseases in Asia-Pacific. For instance, according to the Korea Central Cancer Registry published in 2016, there were 217,057 cancer cases in South Korea in 2014. Moreover, in 2016, the WHO estimated that the Asia-Pacific region has the second-highest number (i.e., 5.1 million) of people living with HIV across the world. Thus, Asia-Pacific is expected to be the most lucrative market for PCR by 2026.

Some of the key players in the global PCR market include Affymetrix, Agilent Technologies, Sigma Aldrich Corporation, F. Hoffmann-La Roche, Abbott Laboratories, Beckman Coulter, Bio-Rad Laboratories, GE Healthcare, Qiagen, Becton Dickinson & Company, Siemens Healthineers, bioMérieux, and Thermo Fischer Scientific. Key players are expanding their product portfolio through mergers and acquisitions and partnerships and collaborations with leading pharmaceutical and biotechnology companies and by offering technologically advanced products.

Technological advances
PCR technology has expanded and changed tremendously since its introduction. The initial method was extremely labor-intensive and time-consuming, but important technological improvements and novel variations in instruments have simplified the process and made clinical implementation much easier.

Changes in Peltier blocks. Using thicker metal blocks, including those made of silver, which is highly heat conductive, improves the conductivity of the heat exchange block. However, the increased thermal mass increases the time taken to raise the entire blocks to the same even temperature, and can lead to undershoots and overshoots of temperature. In a new approach, use of a hollow heat exchange block with a circulating conductive fluid improves temperature control and heat uniformity, for example, the current real-time PCR systems available in the market that take around 40 minutes for 40 cycles. Another step away from Peltier-based technology involves use of a ceramic heating plate in its cyclere, heating samples in disposable tubes, and then cooling using force-air cooling. These systems take 20–40 minutes for 40 cycles.

qPCR leading the way. In recent decades, the advert of the real-time quantitative PCR (qPCR) is gaining popularity for detection of pathogens in clinical microbiology. The majority of diagnostics is and will be based on real-time or qPCR technology that requires less than 5 hours for detection of pathogens and is simple, reproducible, and has improved quantitative capacity over conventional PCR. Recognizing this importance of molecular diagnostics for early diagnosis in life threatening infections, many manufacturers have recently launched qPCRs for invasive disease. Now, the qPCR has also been adapted to detect RNA viruses such as HIV and hepatitis C and the analysis of RNA transcripts associated with some cancers. A new ultra-rapid technology in development could promise a more efficient process, particularly in analyzing large numbers of samples. The technology utilizes a genetically engineered thermostable reverse-transcriptase so the PCR process can proceed directly from RNA.

Miniaturizing thermal cyclers. PCR is one of the most important (research) technologies, and yet it is one of the most limiting when one is outside of the lab because of the size and cost of the instrument. Most thermal cyclers control their temperatures using Peltier junctions, thermoelectric devices that can switch rapidly between heating and cooling. Unfortunately, Peltier junctions are inefficient, and the components required to operate them keep PCR machines heavy and greedy for electricity. However, this can be tackled by use of a different novel approach, wherein samples are heated with a thin-film resistive heater similar to the window defrosters found in cars and are cooled using a simple fan. A microcontroller drives the heating, cooling, and incubation cycles. This simpler design makes the machine much smaller and lighter than ordinary thermal cyclers, and has other benefits too such as low cost.

Improving instrumentation. Initial commercial instruments required laser light sources and/or heat-block elements that were relatively slow to cycle to the required temperatures, or utilized thin capillaries that were easily broken and hard to use. 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 little 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 may have a significant impact on its utilization in smaller hospital settings and point-of-care testing.

Droplet digital PCR. Researchers and instrument makers have also made more radical modifications to PCR by borrowing technologies from other fields. One such effort yielded droplet digital PCR (ddPCR), which combines aspects of fluorescence-activated cell sorting with conventional PCR. Though the protocol for ddPCR is somewhat complicated, it also becomes highly automated. In these systems, a sprayer separates a prepared PCR reaction into thousands of nanoliter-size droplets, and then keeps the droplets separate during the thermal cycling steps. Each droplet hosts its own series of amplifications, which the machine then sorts with fluorescent markers to detect target sequences. ddPCR has proven particularly useful for detecting scarce targets in samples with very high background levels of non-target DNA, such as DNA tumor markers circulating in patient blood samples.

Outlook
Clinical tests have always been a major focus for PCR developers, but sometimes the technique’s requirements have held it back. A number of companies and academic groups are looking at technology to improve PCR diagnostics. An alternative temperature cycling method is being developed by Luke Lee, Professor of Bioengineering at the University of California, Berkeley. He has devised a way of cycling the temperature faster – using plasmonics – the interaction of light with metal surface electrons which generates heat. LEDs heat a 120-nm gold film in contact with a DNA solution in a microfluidic well. The mechanism allowed cycling from 55°C to 95°C, 30 times in 5 minutes. Some developers are addressing some of the sensitivity issues by using some really novel physics called dielectrophoresis and are using clever microfluidics and nanowire field effect transistor technology to further improve sensitivity.

In large diagnostics laboratories, most PCR is being carried out by automated, robotic, high-throughput systems. But the standardization and quality control for PCR still require laborious human input. Innovative start-ups have come up with a solution using artificial intelligence. They effectively feed the learning machine a supervised dataset, a set of input data with a bunch of known results, and the learning machine automatically checks itself and ensures it can give the same results routinely – something that cannot be done without manual input in current systems because of the variability of real-time PCR across different platforms and assays. Once their system has been set up, results can be reported almost immediately, and it will
only flag-up unexpected results that require further analysis. The system is yet under trial in pathology laboratories.

PCR diagnosis is also set to have a big impact on the future growth of precision medicine – the customization of healthcare to the individual patient. Despite the fact that improving PCR technology has reduced the time for 40 PCR cycles down to as little as 20 minutes over the years, there is still room for greater speed, for improved consistency and accuracy, and for simpler workflows for researchers and technicians. Ultimately PCR diagnostics may be witnessed as something that will expand into primary care – one may soon see it in the doctor’s office and the pharmacy, and it is going to be a really exciting time for thermal cyclers. While the portable PCR machine is expanding the technique’s reach dramatically, researchers worldwide continue to push its capabilities forward in countless incremental ways as well. Some manufacturers are working on a hand-held PCR machine that one day might sit in the home medicine cabinet next to the thermometer, and there is no reason why that cannot happen within the next 10 years.