The biggest shift in molecular diagnostics in recent years has been toward the broad adoption of real-time qPCR testing for an array of applications. For infectious diseases and more, the ability to use this technology has introduced a faster and cheaper yet highly sensitive and accurate way to diagnose patients.
Real-time PCR increasingly adopted for pathogen detection
The traditional way to diagnose infectious diseases was through culture- or microscopy-based techniques. However, patient specimens often need to be cultured for days or even weeks to reliably identify a pathogen. Such a long wait time can severely limit the usefulness of these techniques for patients who need immediate care, for example, if they have sepsis or septic shock. Furthermore, not all targets can be cultured, which limits the scope of which pathogens can be identified.
Newer approaches include probe hybridization, serological tests, or further analysis after culturing the pathogen, but such approaches also have limited sensitivity for target detection. Compared to these technologies, “real-time qPCR is the best overall,” says Rui Yang, PhD, a staff scientist at Thermo Fisher Scientific.
Beyond just detecting SARS-CoV-2, real-time PCR can be used to detect pathogens underlying respiratory, vaginal, urinary, gastrointestinal, and sexually transmitted infections, as well as antibiotic resistance. With regards to this technology, Yang adds, “it’s reliable and highly sensitive, because PCR allows for amplification of the target, which is then detected by the specific probe. It’s very fast, providing quick turnaround time for labs and patients. It’s relatively affordable. And it’s versatile, ranging from single- or limited-target assays to comprehensive, multiplex assays.”
Limited-target real-time PCR panels for faster molecular diagnostics
Single- and limited-targeted real-time PCR assays can increase efficiency in your clinical lab, because they are faster and more affordable than comprehensive assays, without compromising accuracy. Examples of such panels include the Applied BiosystemsTM TaqPathTM or TrueMarkTM panels that can be used with Thermo Fisher Scientific’s real-time PCR systems like their QuantStudio™ systems. These panels enable rapid molecular detection that takes only a couple hours from sample collection to final results.
Importantly, even though they are targeted panels, the TrueMark Infectious Disease Panels enable molecular detection of a wide number of disease states from either a predefined panel in a 96-well plate (with eight targets) or a custom 96- or 384-well plate (with up to 24 targets), where you can choose from a list of more than 162 microbial assays and 62 antibiotic resistance assays. These plates include endogenous controls, where every well has an RNase P human internal control or Bacillus atrophaeus bacterial spike-in control, for added confidence in the results.
Larger real-time PCR panels for comprehensive, higher-throughput molecular diagnostics
While limited panels boost the efficiency for detecting a predefined set of targets, they may not include a wide enough range of targets for some research questions. In this case, clinical research labs have usually turned to next-generation sequencing (NGS) because it provides a comprehensive readout of genetic changes, but NGS is costly, more time-consuming, and can produce excessive data that needs more complex analysis than real-time PCR panels. With the introduction of technologies like Applied BiosystemsTM OpenArray™, you can test for significantly more targets and samples at a time. This can be used for high-throughput gene expression analysis, genotyping, microRNA (miRNA) analysis, and digital PCR applications, with same-day results and better affordability compared to NGS.
The OpenArray technology can help your lab significantly increase testing throughput. The technology uses a microscope slide-sized plate that can hold 3,072 assays of 33 nL volume, i.e., each plate can hold as many samples as eight traditional 384-well PCR plates. Due to hydrophilic and hydrophobic plate coatings, the liquid reagents are retained within the open well by surface tension. Up to four OpenArray® plates can be run simultaneously on the QuantStudio 12K Flex Real-Time PCR instrument, allowing your lab to process up to 1,728 genotyping samples and 2,304 gene expression samples within a typical eight-hour workday.
OpenArray plates are also customizable into 1 of 11 formats that best suit your lab’s testing needs. You can select from 17 million single nucleotide polymorphisms (SNP), 1.6 million copy number, 1.3 million TaqMan® Gene Expression, and 4.5 million predesigned drug-metabolizing enzyme (DME) genotyping assays, or your own custom TaqMan assays. The selected assays for your OpenArray panel are preloaded onto the plate during manufacturing, such that to use the panel, you simply mix the prepared samples with the master mix, pre-amplify samples if needed (for increased sensitivity), and load the samples onto the plate and into the PCR system. Overall, OpenArray helps simplify the workflow for lab personnel without compromising testing accuracy, thus boosting efficiency.
Real-time PCR for a diverse range of diagnostic applications
The broad range of available real-time PCR panels ultimately provide your lab with the flexibility to expand your molecular testing capabilities, not only for more pathogen detection than ever before, but also for oncology and pharmacogenomics applications. For example, TrueMark has a specific microsatellite instability analysis for Lynch syndrome-related cancers and for studying immunotherapy. Alongside panels specifically designed to look at the most common genes for pharmacogenomic research, you have the choice of 2,700 inventoried DME assays to create a custom OpenArray plate.
Compared to traditional infectious disease detection methods and other sequencing technologies like NGS, running real-time PCR panels is significantly faster, cost-effective, and less labor intensive, yet highly sensitive and accurate. Detecting variants with these panels can inform the diagnosis of infectious diseases and cancer, as well as assist clinicians in selecting the best course of action for an individual under their care, based on their genetic information.