Mike May earned an M.S. in biological engineering from the University of Connecticut and a Ph.D. in neurobiology and behavior from Cornell University. He worked as an associate editor at American Scientist, and he is the author of hundreds of articles for clients that include Nature, Science, Scientific American and many others.
Now 33 years old, the polymerase chain reaction (PCR) represents a standard process in molecular biology labs. Over the years, this technology has become increasingly easy to use, and that simplicity allows more scientists to make the most of PCR. Some of the key ongoing advances include automation and simpler real-time analysis. The evolving improvements to this proven technology are enabling researchers to perform many exciting applications and uncover new research findings.
As described by José Artur Chies of the immunogenetics laboratory at Brazil’s Universidade Federal do Rio Grande do Sul, “PCR is currently used by our research group in order to genotype patients, as well as to prepare samples for sequencing.” He adds, “We work with the typical approach of case-control gene association studies.” To evaluate gene expression or to genotype single nucleotide polymorphisms (SNPs), Chies and his colleagues use real-time PCR, also known as quantitative PCR (qPCR).
Chies has watched this technology advance. “As I started my studies in the late 1980s—when PCR was at its beginnings, at least here in Brazil—it became obvious that I experienced all the impact of the evolution of this methodology.” He adds, “I would say that the development of qPCR—including here the different probes and primer-specific approaches that allow these quantitative assays—represents the major impact in our research.”
Adding automation
To address researchers’ needs, tool providers are continually improving the features and the performance of PCR instrumentation. According to Alan Neo, product manager, IVD systems, at Luminex, “The ARIES Systems integrate all aspects of molecular testing, from sample preparation through analysis on a single sample.” He adds, “An integrated sample-processing control ensures that the assay run is successful from extraction through amplification.”
This PCR system also lets scientists run their own assays. As Neo explains, “Extraction Cassettes can be used with ASRs (analyte specific reagents) to create LDTs (laboratory developed tests) that can be run on ARIES.” To optimize an assay, says Neo, “Thermal cycling parameters can be uniquely defined by the assay developer through the SYNCT User-Defined Protocol application.”
From a scientist’s perspective, it’s the simplicity that really matters with this platform. “ARIES cassettes automate both extraction and amplification steps, which increases technician hands-off time, reduces user error and lowers the technical barrier for bringing molecular diagnostics into the testing laboratory,” Neo explains.
The system is being applied in various ways. “This technology is being used in a moderate-complexity clinical molecular setting, with cleared FDA IVD (in vitro diagnostic) assays for herpes simplex virus (HSV) 1 and 2, influenza A and B, and respiratory syncytial virus,” Neo says. “Additional assay launches are planned for 2017.”
Steve Miller, director of the clinical microbiology laboratory at the University of California, San Francisco, has been an early access user for applying the Luminex ARIES system to HSV PCR. He says, “We are currently evaluating the user-defined protocol to develop methods for detection of bacterial organisms.” He adds, “While the user-defined protocol project is at relatively early stages, the sample-to-answer capability of the system is attractive to enable quick turnaround time for PCR detection of various organisms for infection diagnosis.”
Miller says the system “has been very user-friendly, and we are planning to implement it for clinical HSV PCR testing on swab samples.” He adds, “It has the potential ability to accept a variety of sample types, and we are working on adapting it for plasma PCR to give a rapid and sensitive result for patients with suspected HSV sepsis.” This work aims to show that HSV PCR can work well with plasma samples.
Rocking in real time
At Thermo Fisher Scientific, some of the most recent advances are in qPCR instrumentation. As examples, Levente Egry, senior product manager of genetic analysis at Thermo Fisher Scientific, mentions “the development of the QuantStudio 3 and 5 Real-Time PCR systems, the latest addition to the QuantStudio family of instruments.” He notes that these are “the first in the market that can be seamlessly connected to the Thermo Fisher Connect cloud platform for online access to data, independent of time or location.”
These instruments include several sophisticated pieces of technology. According to Egry, “The new QuantStudio 3 and 5 systems utilize proven Optiflex technology and VeriFlex Blocks to offer improved speed, data accuracy, multiplexing capability and sensitivity for a broad range of genomic applications, such as analyses of gene expression, microRNAs and noncoding RNAs, SNP genotyping, copy number variation, mutation detection, drug metabolism enzymes and protein expression.”
For many qPCR users, the connectivity of these instruments is a key element.
As Egry says, “This allows users to quickly access fast and powerful secondary analysis software to extract and share results.” He also points out: “Applied Biosystems Analysis Modules offer innovative cloud-based data analysis applications that bring together multiple data sets in one convenient place and render them in stunning data visualizations for enhanced analysis and insights.”
That connectivity is even easier with the Instrument Connect mobile app, which lets scientists track their qPCR instruments from a smartphone. “With the Instrument Connect app,” says Egry, users can check the availability of their Thermo Fisher Connect networked instruments, monitor the progress of a run and view amplification plots in real time.”
Comparing PCRs
Today’s scientists can use various forms of PCR, such as qPCR and digital PCR (dPCR). Ruth Hall Sedlak, a research scientist in lab medicine at the University of Washington in Seattle, has used both in a clinical virology diagnostics lab. Digital PCR gives absolute quantitation and is more sensitive than qPCR, because it partitions a sample into tens of thousands of separate reactions and digitally counts how many reactions have the target DNA.
“Quantitative PCR, or qPCR, can be fairly high throughput, depending on how you set up your lab,” Sedlak says, “and it’s pretty hands-off, if you have the right robotics.” She adds that qPCR provides a large dynamic range that extends from very low to very high numbers of copies. The downside of qPCR is that scientists need to create a standard curve to get relative quantitation. “That can be laborious and be most of the work,” Sedlak explains.
For dPCR, Sedlak uses the Bio-Rad QX100 Droplet Digital PCR System—the predecessor of the current QX200. Although dPCR provides less throughput than qPCR, scientists don’t need to prepare a standard curve. “You just get the actual value or very close to it,” Sedlak says, “and that’s fantastic.”
Overall, says Sedlak, “When digital PCR is more accessible and less expensive, everyone will probably use it.”
Getting more done
The ultimate goal is to get the right answers, and quickly. Sometimes that means more collaboration. As an example, Egry mentions Eisei Noiri, associate professor at the University of Tokyo Hospital. Noiri studies tropical diseases, including kala-azar, which is caused by Leishmania. He has used the QuantStudio 5 to analyze blood from people infected with this disease, and the instrument’s connectivity enables him to work easily with collaborators in Bangladesh without needing to be on-site.
Today’s public-health issues and many other areas of basic and clinical science depend on collaborations around the world, and advances in PCR platforms simplify such interactions. In addition, all aspects of automation make the processes run more efficiently and can reduce errors by eliminating manual steps.