Brandoch Cook, PhD
Although the polymerase chain reaction (PCR) is credited to the work of Kary Mullis, the history of its discovery and refinement is quite a bit more complicated. However, the combination over 30 years ago by his research team of repeated thermal cycling with a heat-stable polymerase unleashed the intrinsic power of the technique to exponentially replicate target DNA. Thus, it became a ubiquitous tool in genetics and molecular biology laboratories. It was nonetheless limited in that it could identify the presence of a gene or sequence, but not the extent to which it was expressed. This state of affairs continued until the advent of real-time PCR (or qPCR), which allows the detection of amplicons as they arise during the exponential phase of DNA replication, via probes that fluoresce in proportion to the total product of amplified DNA. This procedure allows demarcation of the exact moment a DNA sequence becomes detectable, making it a real-time, rather than an endpoint, assay. With this information, one can calculate relative gene expression using internal standards or quantify absolute levels in molecular weight or copy number. Additionally, the design of instrumentation and reagents make it naturally suited to automation and high throughput. Continual upgrades in materials that are available from major manufacturers, and improvements in complex data analysis software, have driven increased efficiency and sensitivity of detection of small amounts of nucleic acids within large samples. Therefore, it is potentially a powerful technique to diagnose and validate the presence of microbial infection or contamination in blood and tissue samples and in the food supply.
Fast detection, small samples
One major advantage of qPCR in microbial diagnostics is the speed with which samples can be assessed. For instance, bloodstream bacterial infections account for approximately 40 percent of sepsis, a systemic inflammatory response in which the risks of death and morbidity increase drastically with time. The current gold standard for positive detection requires culturing of blood samples, which can take two to four days. In contrast, qPCR analysis can be resolved in as little as six hours, so there is an inherent advantage to bypassing the culture step if possible. Procedures can also be executed in multiplex format so that the presence of multiple pathogenic species can be assessed simultaneously. Additionally, because of the large blood samples required for culturing analyses, qPCR is intrinsically well-suited to diagnostics in cases of neonatal and childhood infection, in which blood draws are necessarily low-volume. Because of the power of this technique, there is a burgeoning array of tests, protocols, and instrumentation tailored to different target genes of different microbial species. Detection typically proceeds in one of two ways: 1) amplification of the 16S ribosomal RNA gene, coupled with a species-specific fluorescent probe oligonucleotide; or 2) amplification of a unique region of a virulence or antibiotic resistance gene. Commercially available systems include Roche’s SeptiFast, which can identify 25 different pathogenic bacterial and fungal species; and Seegene’s MagicPlex, distributed through Thermo Fisher, which detects more than 80 species accounting for 90 percent of sepsis.
Infectious and emerging diseases
Equally important to detecting pathogenic bacteria in clinical cases of sepsis, the capabilities of qPCR extend to infectious and emerging diseases. A partial list of rapidly evolving research-based and clinical diagnostic approaches includes efforts to understand and detect urinary tract infections, respiratory infections such as pneumonia and influenza, agents of bio- and agroterrorism such as in the Amerithrax crisis following the 9/11 attacks, and outbreaks of emerging viral infectious diseases such as Zika and Ebola. In fact, qPCR techniques have become the most valuable tool in early identification of several outbreaks due to the ability to directly test urine samples, which are easy to collect from travelers before they leave or move throughout developing countries that lack the resources to maintain biocontainment facilities needed to isolate and culture virus-infected samples. Variations on Seegene’s detection platform include AnyPlex and AllPlex, which offer diagnostic panels for multiple respiratory, intestinal, and sexually transmitted pathogens. Additionally, Roche’s cobas analyzer series allows a high degree of automation in detection of emerging infectious diseases.
Another rapidly growing focus of qPCR-based diagnostics is food safety. There are approximately 250 different known vectors for foodborne illness, resulting annually in over 48 million individual cases, 128,000 hospitalizations, and more than 3,000 deaths from food poisoning. Wouldn’t it be nice to know whether the cruise ship buffet has norovirus before you decide to get on the boat? Again, qPCR has the potential to slash identification times from several days to a matter of hours. Similar to bloodstream infections, culture protocols are still the standard accepted method of detection for bacterial contamination such as salmonella or E. coli; however, in cases of viral or protozoan species, qPCR has become the method of choice. Among the reagents, kits, and instruments available are the SureTect system from Thermo Fisher and the DNeasy mericon kit series from Qiagen, which can be automated using the QIAcube.
Limitations relegate qPCR to a supportive technology
Generally, qPCR diagnostics bring a striking increase in sensitivity, especially in multiplex protocols for identification of multiple genes or species and for slow-growing or latent microbes that persist at low copy numbers. However, there is also a greater risk of false positives and less negative predictive value than in more established tests. The potential for contamination with extraneous nucleic acids is very high during sample collection and processing. Moreover, the presence of nucleic acids in dead cells and in species that remain at low levels after antimicrobial treatment regimes are in place can confound test results. Therefore, in sepsis and other diagnoses, qPCR is broadly considered a supportive technology that can shape understanding of, and response to, infection, rather than a diagnostic one reliable enough to replace blood culture-driven identification methods. Additionally, because the kits and reagents specific to each pathogen are often proprietary and unique to the instrumentation of the manufacturer, there is a great deal of cost, specialization, and expertise inherent in these techniques. As laboratories and facilities continue to elevate their technology and training, however, it is clear that qPCR will help push microbial diagnostics into new and fertile territory.
For additional resources on qPCR and protein biology, including useful articles and a list of manufacturers, visit https://www.labmanager.com/protein-biology