PCR, Emergence of the Digital Age

Each time a question is asked of PCR, researchers have found a way to make it work; from remote sub-Saharan Africa to onboard the International Space Station, no task seems too difficult.

Polymerase chain reaction (PCR) has become an indispensable tool for molecular biology over the past few decades. It was initially just an analytical laboratory tool, but it has now become a center point for diagnostics. However, it has taken nearly 30 years for PCR to make this transition, and there are several reasons it took that long.

Initially, there were some limitations to using PCR due to patent concerns. One of the tipping points was the advent of real-time PCR in the 1990s, which enabled quantitative analysis and eliminated the concerns around contamination, both of which are important for diagnostic applications. Real-time PCR allowed for an enclosed reaction so that the amplification product never left the reaction tube and minimized exposure to contaminants.

Melting analysis was introduced in the mid-1990s, and over the years it has developed into more powerful techniques, like high-resolution melting (HRM). HRM combines the convenience of real-time PCR with the elimination of contamination issues, making it more amenable to clinical laboratory adoption. Digital PCR and massively parallel sequencing are extensions of clonal amplification that are based on PCR, and they are all making their way into the clinic. There are other developments taking place right now that are also on the edge of laboratory research and clinical diagnostics.

What makes PCR more clinically important is a trend to move away from single-analyte testing toward running multiple tests that are all relevant to the sample being analyzed. This multiplex analysis is called syndromic analysis if the targets are all possible causes of a syndrome. Syndromic panel analysis has become popular and can help identify a specific causative agent from a spectrum of organisms. There are a number of FDA-approved syndromic kits now available that use multi-analyte PCR. The technical advances in PCR with multiplexing, along with cost reduction and increased understanding of diseases, have made it more relevant and suitable for clinical diagnostics. However, the central utility of PCR is really the common thread through all the reasons it succeeded in making the transition from the lab to the clinic.

Global market
The global digital PCR (dPCR) and real-time PCR (qPCR) market is highly competitive. The market is estimated to grow to USD 6270.9 million by 2024 from USD 4113.3 million in 2019, at a CAGR of 8.8 percent, predicts Research and Markets. Factors, such as the rising incidences of target infectious diseases and genetic disorders; continuous technological advancements in PCR technologies; increasing investments, funds, and grants; increasing use of biomarker profiling for disease diagnostics; and successful completion of the Human Genome Project, are driving the growth of this market. However, the high instrument costs – especially that of dPCR – and the technical limitations of PCR have restricted their greater use.

According to the World Health Organization (WHO), cancer was among the leading causes of death, responsible for 9.6 million deaths globally in 2018. The WHO estimates that 36.9 million individuals were infected with HIV in 2017. It also reported that infectious diseases were among the top causes of deaths worldwide in 2016. The rising cases of these diseases are anticipated to drive the market in near future.

Moreover, continued efforts by PCR manufacturers to develop novel systems and kits to use in molecular testing are likely to further propel growth of the qPCR and dPCR market. For instance, in January 2019, Qiagen N.V. announced its plans for the development of next-generation dPCR systems, anticipated to be launched in 2020. The new systems are expected to offer highly automated workflows and quicker turnaround time as compared to current digital PCR platforms, which are likely to provide significant competitive advantage to the company.

In addition, a broad range of applications of qPCR and dPCR technologies is expected to further boost the adoption in array of scientific disciplines. The technologies have wide-ranging applications in both basic and diagnostic research. They have been extensively used in areas, such as human genetic testing, forensic sciences, pathogen detection, and infectious disease testing. Expanding application in food microbiology and veterinary medicine, among other industries, is expected to further fuel the market.

Quantitative PCR held the largest market share in terms of value in 2018. Continued demand in areas, such as genetic variation analysis, gene-expression analysis, and genotyping, along with increasing focus on the development of point-of-care (PoC) platforms, is anticipated to drive the segment.

Digital PCR is a next-generation testing method, which helps precise quantification of nucleic acids. This system offers superior sensitivity and accuracy compared to other PCR-based approaches, which is anticipated to help the segment register robust growth during 2019 to 2024.

The consumables and reagents was the leading segment, accounting for the largest market share in terms of value in 2018. The segment is projected to witness strong growth rate as compared to other segments owing to launch of new PCR test kits.

Research application was the dominant segment in 2018. qPCR and dPCR systems are widely used for research applications, such as stem cell research and genetic disease and oncology research. The wide-spread use of these systems is attributed to features, such as enhanced specificity and quicker turnaround time. The clinical application segment is estimated to register the fastest CAGR of 8.7 percent with increasing adoption of automated processes.

North America led the regional segment in 2018, attributed to launch of new systems and test kits by PCR manufacturers. For instance, in September 2018, Thermo Fisher Scientific Inc. launched VetMAX MastiType – a qPCR-based test kit used for rapid detection of mastitis-causing pathogens in the dairy herds. Moreover, strong presence of major PCR manufacturers in this region, coupled with increasing demand for rapid diagnostic tests, is also expected to drive the regional growth over the next 5 years.

Asia-Pacific is likely to witness the fastest CAGR of 11.1 percent. Rise in the patient pool, owing to high prevalence of chronic and infectious diseases coupled with increasing awareness among patients about early diagnosis of these diseases, is anticipated to boost the demand for PCR products, in turn driving the growth of the market.

Major market players have been consistently focusing on deploying strategic business measures to maintain their competitive position. The key measures adopted by the players include new product launches, entering into collaborations and partnerships with institutes and companies, and mergers and acquisitions. For instance, in January 2019, Qiagen N.V. announced the acquisition of Formulatrix’s digital PCR assets in a deal valued at USD 260 million. The acquisition augments well with the company’s strategy of launching new digital PCR platforms in 2020 by incorporating Formulatrix’s advanced dPCR technology.
Some of the prominent players that dominate the market for qPCR and dPCR are Bio-Rad Laboratories; Thermo Fisher Scientific Inc.; Qiagen N.V.; Abbott Laboratories, Inc.; Agilent Technologies, Inc.; bioMerieux S.A.; Fluidigm Corporation.; and F. Hoffman La Roche.

Technology trends
Quantitative PCR (qPCR), or quantitative real-time PCR, is a technique that couples amplification of a target DNA sequence with quantification of the concentration of DNA species in the reaction. This also calculates the starting template concentration and is used as analytical tool in evaluating DNA copy number, viral load, allelic discrimination, and SNP detection. As compared with reverse-transcription PCR, qPCR is a more powerful tool to measure mRNA expression and is the gold standard for microarray gene expression data confirmation. One of the other uses of qPCR is that it has the ability to quantify DNA starting amount used in the reaction sample. This is important in order to evaluate the level of contamination in food and to check whether the food contamination is acceptable or is above the allowed threshold, especially for toxic or metal residues. At present, there are several molecular principles used for product detection by qPCR and use of reagents for multiple manufacturers. These molecular principles include hybridization probe-based FRET, hydrolysis probe TaqMan technology, and probe-less detection by dsDNA staining. The qPCR technology is one of the most commonly used methods for studying the expression of specific genes.

The real-time fluorescence-based quantitative polymerase chain reaction is used for detection of nucleic acids in the field of microbiology, biotechnology, biomedical research, and in forensic applications. Unlike other conventional PCR, qPCR allows accurate quantification of amplified DNA in real time during the exponential phase of the reaction. The increasing awareness for the need for standardization, quality control, and other significant problems associated with inadequate reporting of the assay, have resulted in the publication of guidelines for the publication of qPCR experiments (MIQE).

Homogenous fluorescent chemistries for real-time PCR. The development of fluorescent methods for the closed-tube PCR has simplified the process of quantification. Current approaches use fluorescent probes for interacting with the amplification products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to DNA quantification like fluorescent DNA binding dyes. There are also strand-specific probes that can be used as the phenomenon of fluorescent energy transfer.

Analysis of mRNA expression by real-time PCR. The last few years have seen the transformation of the real-time, fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR) from an experimental technology into a mainstream scientific tool for the quantification and detection of RNA, with a huge range of uses in basic research, biotechnology, and molecular medicine. The fluorescence-based reverse-transcription polymerase chain reaction has seen the continuous improvement of reagents and instruments, combined with the trend toward high throughput, which is likely to strengthen pre-eminence and continue to open up new application areas. Notwithstanding its principle, undoubtedly it is a straightforward technology with the reliability of RT-qPCR assays depending upon sequential steps that include careful experimental design, validation, and optimization.

Move over PCR and make way for L-TEAM
Scientists in Japan have developed a way of amplifying DNA on a scale suitable for use in the emerging fields of DNA-based computing and molecular robotics. By enabling highly sensitive nucleic acid detection, their method could improve disease diagnostics and accelerate the development of biosensors, for example, for food and environmental applications.
Researchers from Tokyo Institute of Technology (Tokyo Tech), Abbott Japan Co. Ltd., and the University of Electro-Communications, Japan, report a way to achieve million-fold DNA amplification and targeted hybridization that works at body temperature (37°C/98.6°F).

The method, named L-TEAM (Low-TEmperature AMplification), is the result of more than 5 years of research and offers several advantages over traditional PCR, the dominant technique used to amplify DNA segments of interest.

With its easy-to-use, one-pot design, L-TEAM avoids the need for heating and cooling steps and specialized equipment usually associated with PCR. That means it is an efficient, inexpensive method that can importantly prevent protein denaturation, thereby opening a new route to real-time analysis of living cells.

In their study published in Organic & Biomolecular Chemistry, the researchers introduced synthetic molecules called locked nucleic acids (LNAs) into the DNA strands, as these molecules are known to help achieve greater stability during hybridization. The addition of LNA led to an unexpected, but beneficial, outcome. The team observed a reduced level of leak amplification, a type of non-specific amplification that has long been an issue in DNA-amplification studies as it can lead to an error in disease diagnosis, that is, a false positive.

“We were surprised to discover the novel effect of LNA in overcoming the common leak problem in DNA amplification reactions,” says Ken Komiya, assistant professor at Tokyo Tech’s School of Computing, “We plan to investigate the mechanisms behind leak amplification in detail and further improve the sensitivity and speed of L-TEAM.”

In the near future, the method could be used to detect short nucleic acids such as microRNA for medical diagnostics. In particular, it could facilitate PoC testing and early disease detection. MicroRNAs are now increasingly recognized as promising biomarkers for cancer detection and may hold the key to uncovering many other aspects of human health and environmental science.

In addition, Komiya explains that L-TEAM paves the way for practical use of DNA computing and DNA-controlled molecular robotics. “The original motivation behind this work was the construction of a novel amplified module that is essential to build advanced molecular systems,” he says, “Such systems could provide insights into the operational principle behind living things.”

Outlook
More than 30 years after the invention of PCR, it is rare to find a molecular biologist that has not used this technology. Now, thanks to approaches such as ddPCR, miniaturized instruments and nucleic acid amplification and analysis, industry has a new insight into rapid and accurate quantitative diagnostics for future PoC applications.
Recent technological advances only highlight a fraction of the improvements made to this old technology. Each time a question is asked of PCR, researchers have found a way to make it work; from remote sub-Saharan Africa to onboard the International Space Station, no task seems too difficult.