qPCR Analysis: Key Applications in Infectious Disease Research

Applications of qPCR analysis have evolved in drug development, including pharmacokinetic testing and infectious disease research. With applications such as copy number qPCR analysis and format types such as ddPCR assays, PCR service providers not only offer rapid and accurate diagnostic tools in clinical pathogen analysis, but they also prove vital in epidemiological studies.

Today, researchers extract viral RNA from nasopharyngeal swabs and then perform real-time reverse transcription qPCR to detect and quantify viral nucleic acids. This approach has been massively successful through multiplex assays, such as recent viral detection during the COVID-19 pandemic. Specifically, researchers and clinicians used multiplexed qPCR to amplify genes encoding the nuclear capsid, envelope, and structural proteins. The applications and role of qPCR assays in infectious disease research will grow continuously. The current article reviews critical applications of qPCR analysis in supporting infectious disease research. 

The crucial role of qPCR analysis in infectious disease research 

qPCR assays can simultaneously detect multiple pathogens and differentiate between them in a single reaction volume. These characteristics can facilitate rapid and comprehensive evaluations in infectious disease research. Multiplex qPCR analysis is beneficial, particularly for coinfection where different pathogens have similar symptom onset. For example, in the latter stages of the COVID-19 pandemic, researchers observed that the respiratory syncytial virus and influenza strains A and B had similar primary symptoms of COVID-19. The ability to differentiate between them is critical for treating patients with different infections. Hence, the demand for multiplex qPCR analysis grew during the 2021-22 flu season. 

qPCR assays can readily differentiate between different pathogenic subtypes or strains. This feature has made qPCR analysis a primary tool in epidemiological research to trace sources of outbreaks and evaluate genetic diversity in pathogens. For example, one study with E. coli and different bacterial subtypes showed that qPCR successfully assessed different bacterial populations and helped in the rapid assessment of the patient population. While whole genome sequencing can characterize Sars-CoV2 variants, real-time qPCR assays can also offer rapid alternatives to identify known variants geographically. 

Must Read: Inside the World of GLP Labs: A Deep Dive into Their Impact on Research

qPCR assays are highly flexible. Researchers can rapidly develop qPCR assays for emerging new pathogens, for example, the human mpox virus. Recent research investigating the suitability and specificity of generic probes and primers used in commercial real-time assays for human mpox viruses shows that these generic assays may not be accurate or optimal for detecting human mpox viruses. Hence, after using qPCR assays, researchers successfully uncovered sequence variation among earlier and current circulating human mpox viral strains. 

Although reliable qPCR arrangement is crucial for pathogen analysis, researchers should also consider good PCR practices and high-quality reagents to design robust qPCR assays. Correct probe and primer design can ensure reliable detection and identification of target sequences. Incorporating multiple replicates while testing can be beneficial to minimize the generation of low-positive or false-positive samples. Additionally, including internal controls and positive and negative controls is crucial while designing accurate and reliable qPCR assays. 

In Conclusion

Accurate diagnosis is critical for successful disease diagnosis and epidemic control. As qPCR continues to evolve as a diagnostic tool, the reliability and cost-effectiveness of this assay will grow parallelly to target specific genomes of pathogens in human samples.