August 1, 2014 (Vol. 34, No. 14)

MaryAnn Labant

The polymerase chain reaction (PCR), invented about three decades ago, soon entered mainstream use thanks to an ongoing series of refinements.

One particularly important refinement, introduced about two decades ago, is the “real time” quantification of DNA. The idea is to trace the rising level of DNA throughout the amplification step, and not just measure the final amount of amplified product. This idea turns standard PCR into real-time PCR, or quantitative PCR.

Real-time PCR has become the most widely used nucleic acid detection technology. It is routinely used in academic research, in applied testing settings such as food-safety or veterinary testing, and in molecular diagnostics. It continues to replace many older detection methods due to simple readouts, high sensitivity, and multiplex and quantification capabilities, as well as ease of use, cost effectiveness, and throughput flexibility with only moderate equipment investments.

According to Peter Urbitsch, Ph.D., head of the global assay technologies business at Qiagen, real-time PCR technology has evolved and diversified in multiple directions. Available formats include tubes, microarrays (96-, 384-, and 1,536-well plates), and capillary and rotor variants. Detection principles include SYBR green and probe-based detection, with the latter being increasingly diversified into FRET, Scorpions, TaqMan, and others. In addition, multiplex detection formats are being developed using different dyes and quencher molecules.

Innovations are driven by dissemination into new application areas and user profiles. Essentially, the technology is becoming easier to use and accessible to novices while providing more complex information faster and at lower costs. This development can be compared to computer technologies, which evolved from bulky “specialist equipment” to powerful and convenient end-user devices.

As real-time PCR continues to replace traditional technologies, less experienced users become routine users. While some instruments are small, simple, and accessible to almost anyone and deployable at nearly any bench, other platforms are targeting high-throughput data generation and require robust chemistry that allows automated handling and reaction set-up at room temperature.

Procedures include a higher degree of process controls (such as target controls via multiplexing) or operational controls (such as the visual pipetting control in Qiagen’s QuantiNova kits). This is particularly important in complex applications such as multiplex detection of various targets and integration or cross-linking of multiple scientific questions such as genotyping, mRNA and miRNA profiling, or copy number variation (CNV) analysis.

A common bottleneck for both real-time PCR and other technologies such as next-generation sequencing (NGS) is the consolidation and interpretation of data and results, increasingly requiring bioinformatics tools that support the interpretation of the biological meaning of gene expression data, such as Qiagen’s Ingenuity Pathway Analysis and Ingenuity iReport.

Qiagen’s QuantiNova kits have a built-in tracking system for visual identification of correct pipetting.

Beyond Instrumentation

Early real-time PCR was focused on perfecting instrumentation. Instrumentation is no longer a rate-limiting step in the production of quality data; instead, usability, software, and analysis are at the forefront.

“The big problem with real-time PCR is not the amount or complexity of data, but whether or not data are any good. Everyone can get traces, especially with poorly designed primers and probes,” explains Sam Ropp, Ph.D., senior business unit marketing manager, GXD Consumables, Bio-Rad Laboratories. “The focus has shifted from hardware improvements to tools that impact data quality. When users get better data, they can have confidence in the first run.”

A set of guidelines, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE), seeks to address a challenge with the widespread adoption of quantitative PCR, the lack of a universal standard to substantiate the quality of data.

Bio-Rad provides transcriptome-wide content for the human and mouse genomes—an assay for every protein-coding gene. (The company is close to providing similar content for the rat genome.) For over 20,000 assays per transcriptome, researchers receive validation data based on the MIQE Guidelines.

Assays are also prevalidated and arranged into subsets of biologically relevant information, leading to predesigned assay plates that correlate to biological signaling pathways and disease states, along with the ability to customize the plates to an individual’s liking. Run files supplied with plates allow users to import gene information and validation data into the instrument software, automating set-up and analysis.

Controls help determine if data reflect a biological event or an experimental artifact. Reference genes and controls are built into predesigned assay plates to check sample quality, reverse transcription, presence of PCR inhibitors, and genomic contamination.

“Every technique and application evolves over time; real-time PCR is no different,” continues Dr. Ropp. At present, real-time PCR is acquiring process improvements “from sample isolation to interpretation of results.”

The goal is to incorporate flexibility and ease of use so that users receive answers quickly and feel confident that their results are bulletproof. “It gets down to data quality and the experimental workflow—filling in the gaps to make [real-time PCR] easier, quicker, more efficient, and price effective,” concludes Dr. Ropp. “That is the future.”

Changing Industry Segments

In terms of adoption of the technology, the core academic segment is mature. Translational and applied market segments are growing as new applications are being enabled.

“Technology miniaturization means smaller components and instruments that are less expensive, smaller, lighter, and easier to deploy in the field. Integration from sample prep to answer, multiplexing to get more dimensions of information with extremely high specificity and sensitivity at a low cost point and efficient workflow, is making the technology attractive for emerging market segments to replace more laborious and time-consuming existing detection modalities,” remarks Chris Linthwaite, vice president, genetic, medical and applied sciences, for Thermo Fisher Scientific.

An example is microbiological applications where real-time PCR is replacing cell culture. Technicians used to have to plate, let the colonies grow, pick homologous colonies, and then type them. Real-time PCR allows detection with very few copies, reducing a once lengthy workflow to a few hours.

The fastest-growing segment of the real-time PCR market is enabling vast levels of multiplexing and density, running the same test with different samples or interrogating the same sample for different elements. Digital PCR, an absolute quantification, expands utility versus cannibalizing real-time PCR. The Life Technologies QuantStudio 3D Digital PCR System, for example, is a chip-based platform that generates up to 20,000 data points.

In personalized medicine, NGS and quantitative PCR are complementary tools. NGS platforms answer open-ended questions with lots of unknowns, but typically real-time PCR is the fastest and most cost-effective modality for detecting the same thing over and over again. PCR is translating to the clinic as more CE-IVD-labeled or 510K-cleared instruments are approved by regulatory bodies. Eventually, compact, personalized, highly decentralized, and push-button-answer instruments will be run bedside in the clinic.

The market will continue to mature and enter production environments, integrating with robotics and plate readers and allowing tens of millions of PCR reactions.

PCR is a detection tool; value gets added later, in data interpretation. To help users make the most of this stage, industry leaders hope to introduce the SaaS (software as a service) model and cloud-based software. Users may be able to access and share data (and update software) more easily. In addition, they may be able to purchase software modules as needed.

Absolute quantification by digital PCR is made possible by instruments that run thousands of individual reactions in parallel. One such instrument, the QuantStudio™ 3D System from Life Technologies, uses a sealed chip technology to streamline workflow. The instrument can generate up to 20,000 data points and enable more controlled sample loading and less reaction dropout than droplet-based approaches.

Innovation Drivers

PCR is not only the most sensitive way to do diagnostic testing, replacing ELISA as a method, but the technology also complements sequencing. Real-time PCR is a vital step in the sequencing reaction used to validate sequencing results or for target verification prior to sequencing.

Innovation drivers include lowering reaction volume and increasing throughput, without sacrificing accuracy. Regardless of market industry, research scientists continue to demand faster and inexpensive real-time PCR instruments and lower reagent costs.

Integrated with robotics and liquid handlers, Roche’s LightCycler® 1536 system runs 1,536 samples in a reduced reaction volume of 0.5–2 μL, in approximately an hour, greatly reducing reaction volume and cost.

“Master mixes are fairly optimized right off the bat; they may require a tweak here or there, but optimization is no longer a major issue. The challenge for the customer is always price. Reducing the cost of the individual reaction by reducing the required volume is key,” comments Joe Donnenhoffer, Ph.D., lead technical service scientist. “Additionally, temperature homogeneity across the plate is a challenge. Often outer and inner wells will get different results due to temperature fluctuation; temperature homogeneity across the plate allows use of all wells with accurate results.”

“Instrument calibration is another often overlooked laboratory chore that should be automated. When users have to periodically calibrate an instrument, it decreases their confidence in the consistency of the results,” continues Dr. Donnenhoffer. “The trend is toward instruments that require no calibration, enabling users to generate consistent results run after run.”

In the diagnostic world, PCR is more of a black box providing a yes or no answer. In the research field, users want to know quantity—“how much?” is a harder question to answer.

Training has expanded over time as research scientists incorporate more and more technologies into their toolbox, and dedicated PCR experts are not typically present in all laboratories. Basic and advanced training will teach users what is going on in the reaction, how to interpret the results, and how to determine their next steps.

The Roche LightCycler® 1536 instrument is a plate-based thermal block cycler with integrated real-time, online detection capabilities. When integrated with robotics and liquid handlers, the instrument can run 1,536 samples in a reduced reaction volume of 0.5–2 µL in approximately an hour.

Intellectual Property Concerns

According to Caroline Tsou, global marketing director for molecular and synthetic biology at Agilent, intellectual property and the associated licensing costs continue to weigh heavily on the market and constrain broader development and use of quantitative PCR. As some of that IP comes off patent in 2016 and beyond, opportunities for additional development are anticipated, especially in the diagnostics space. Though more advanced capabilities have been developed such as microarrays or NGS, there is still a use for faster, cheaper tools for quick diagnostics or validation.

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