When it comes to molecular diagnostics of infectious disease, the application of quantitative polymerase chain reaction (qPCR) continues to evolve, covering not only the need for rapid, accurate, and multipurpose diagnostic tools for pathogen detection in clinical settings, but also epidemiologic control.
Extraction of viral RNA obtained from nasopharyngeal swabs followed by real-time reverse transcription qPCR (RT-qPCR) enables the rapid and quantitative detection of viral nucleic acids after they are converted to cDNA. This technology was successfully applied in a multiplex assay format during the SARS-CoV-2 pandemic to reduce the processing time of individual tests. Specifically, multiplexed qPCR was used to amplify several viral genes encoding structural proteins, envelope and nucleocapsid, and non-structural proteins, including the RNA-dependent RdRp gene (1).
I believe the role of qPCR can only grow. And to support my point, I’ll share three exciting applications.
Syndromic panel testing
qPCR can enable the simultaneous detection of multiple pathogens in a single reaction and enable differentiation between them. Such multiplex assays can enable faster and more comprehensive diagnostics for infectious diseases and are used when co-infections are a concern between several pathogens with similar clinical symptoms at onset. For example, during the latter stages of the recent SARS-CoV-2 pandemic, clinicians noted that influenza strains (A and B) and respiratory syncytial virus (RSCV) shared similar initial symptoms with COVID-19. The ability to enable differentiation between these three infections was important because treatments differ for COVD-19 versus influenza versus RSCV infections. As a result, demand grew for testing of multiple pathogens during the 2021–2022 flu season.
Genotyping and variant analysis
qPCR can easily differentiate strains or subtypes of pathogens. This application is crucial in epidemiological studies to trace the foodborne sources of outbreaks and to understand the genetic diversity of pathogens present. A recent study of E. coli and other bacterial subtypes demonstrated how qPCR can be used to evaluate the different bacterial populations present in a patient population for rapid assessments (2). While whole genome sequencing (WGS) is employed for characterization of SARS-CoV-2 variants worldwide, RT-qPCR can also provide a rapid method for the identification of known VOCs geographically (3).
Emerging pathogen detection
The flexibility of qPCR facilitates the rapid development of assays for newly emerging pathogens, such as the human mpox virus (MPV). A recent study investigated the specificity and suitability of generic primers and probes used in commercially available real-time diagnostic assays for MPV and revealed that the current real-time generic assay may not be optimal for accurate detection. The reason? The researchers used qPCR to uncover sequence variation between presently circulating MPV strains and earlier MPV strains (4). A similar study demonstrated how modern real-time qPCR systems and reagents can be used to design an assay that ensures accurate detection of the DNA virus in human samples while distinguishing it from related viruses (5).
Though reliable qPCR instrumentation is required for pathogen detection, good PCR practices alongside high-quality qPCR reagents and well-designed assays are also essential. As noted by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, the correct primer and probe design ensures detection of the target sequence and, as demonstrated by a large study of toxoplasmosis in pregnant women, using more than one replicate for testing is useful to reduce the potential for false positives or low positive samples (6). In addition to implementing a well-designed assay, the inclusion of positive and negative controls, and internal controls for PCR inhibitors is also required.
Accurate diagnostics are fundamental for successful epidemic control and disease diagnosis; from this perspective, qPCR continues to excel in this regard, providing a rapid, reliable, and cost-effective tool for targeting a pathogen’s specific genome in human samples.
Newsletters
Receive the latest pathology news, personalities, education, and career development – weekly to your inbox.
References
- FRO Barros et al., “Performance of RT-qPCR detection of SARS-CoV-2 in unextracted nasopharyngeal samples using the Seegene Allplex™ 2019-nCoV protocol,” J Virol Methods, 300 (2022). PMID: 34919975.
- C Antoine et al., “Phage Targeting Neonatal Meningitis E. coli K1 In Vitro in the Intestinal Microbiota of Pregnant Donors and Impact on Bacterial Populations,” Int J Mol Sci, 24, 10580 (2023). PMID: 37445758.
- A Diotallevi et al., “Rapid monitoring of SARS-CoV-2 variants of concern through high-resolution melt analysis,” Sci Rep, 13, 21598 (2023). PMID: 38062105.
- F Wu et al., “Wide mismatches in the sequences of primers and probes for monkeypox virus diagnostic assays,” J Med Virol, 95, 1 (2022). PMID: 36504122.
- O Erster et al., “A Multi-Laboratory Evaluation of Commercial Monkeypox Virus Molecular Tests,” Clin Microbio, 11, 3 (2023). PMID: 37140382.
- MP Brenier-Pinchart et al., “Multicenter Evaluation of the Toxoplasma RealCycler Universal PCR Assay on 168 Characterized Human Samples,” J Mol Diagn, 24, 6 (2022). PMID: 35452843.
