Laboratory diagnosis of respiratory viruses, including the novel SARS-CoV-2 coronavirus (COVID-19), requires rapid, specific diagnostic testing technology. For SARS-CoV-2, an accurate diagnosis is critical not only to direct clinical management, but to inform appropriate self-isolation and distancing behavior to prevent disease transmission. Currently, much of the diagnostic testing technology for SARS-CoV-2 is based on real-time polymerase chain reaction (RT-PCR), which detects viral RNA in a sample. Major limitations of RT-PCR diagnostic methods are specificity, and an inability to distinguish between multiple pathogens. PathogenDx’s novel DetectX-RV technology combines RT-PCR with powerful DNA microarray technology to improve specificity and creates new possibilities for multiplex testing. This is especially critical given the multitude of symptoms that cross-over in a patient with flu-like symptoms, including a bad cold, influenza A or B or COVID-19 itself. Going forward, having this level of diagnostic discrimination is key to properly triage and treat the patient.
For COVID-19 testing, RT-PCR is most widely used to detect viral RNA in samples obtained via nasopharyngeal swabs or from nasopharyngeal aspirate. The RNA is extracted, reverse transcribed, amplified, and the cDNA strand is labeled with a fluorescent dye for detection. The amplification curve is analyzed to obtain a cycle threshold (Ct) value, with lower Cts indicative of abundant target nucleic acid in the sample. Despite being widely used, RT-PCR offers limited sequence specificity, as it enables detection of only a few viral RNA target domains per reaction. This approach is also limited in its ability to accurately detect target sequences present in low copy numbers— which may occur among asymptomatic or mildly symptomatic individuals— as measurements may be obscured by a relatively low signal to noise for RT-PCR near the Lower Limit of Detection.
DetectX-RV combines RT-PCR with DNA microarray technology to increase specificity. The platform enables detection of multiple RNA target sequences simultaneously, and thus can identify specific signals relative to a large number of specificity controls. Following RNA extraction and PCR amplification, the resulting cDNA is labeled with a fluorophore and added to the DNA microarray containing 144 synthetic ssDNA probes, SARS-CoV-2, SARS-CoV-1, and an internal (RNase P) positive control arranged in a 12 x 12 configuration. These probes increase specificity by functioning as a large set of multiple tests, wherein the amplified COVID-19 cDNA can hybridize with all 144 probes in parallel. DetectX-RV supports rapid analysis, with results in 6-8 hours, and the microarray design enables 12 individual specimens per slide, and multiple slides are (up to 16) tested simultaneously for improved throughput.
The inclusion of SARS-CoV-1 and SARS-CoV-2 probes enables technicians and clinicians to distinguish between these two respiratory viruses. While the United States Food and Drug Administration is currently reviewing the approval of DetectX-RV as the first array platform under Emergency Use Authorization (EUA) for the detection and diagnosis of COVID-19, this technology creates new possibilities for the future of respiratory testing. In combining RT-PCR and DNA microarray technology, DetectX-RV enables multiplex diagnostic testing. Where traditional RT-PCR methods are confined to the identification of a single viral pathogen, DetectX-RV may be designed with a DNA microarray incorporating probes for multiple pathogens. As a result, a single test would be sufficient to ascertain whether a sample is positive for any number of respiratory viruses, such as SARS-CoV-1, SARS-CoV-2, or various influenza strains. DetectX-RV is an accurate, specific test to support COVID-19 diagnosis and management, and its multiplex capabilities may shape the future of respiratory virus testing.