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Updated: Oct 11, 2025
Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
[Development of Multiplex RT-PCR with Immobilized Primers for Identification of Infectious Human Pneumonia Pathogens]
S A Lapa1,2, R A Miftakhov1, E S Klochikhina1
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia.
Abstract:
A prototype of a system for the detection of infectious human pneumonia pathogens based on multiplex solid-phase reverse transcription PCR (RT-PCR) was developed. Primers were designed to identify the DNA of six bacterial pneumonia pathogen strains, and the RNA of two viral pathogens of pneumonia: influenza A and SARS-CoV-2. The signal accumulation of elongated immobilized primers occurs due to the incorporation of fluorescently labeled nucleotides in the chain. The signal is detected after all the components of the mixture are removed, which significantly reduces the background signal and increases the sensitivity of the analysis. The use of a specialized detector makes it possible to read the signals of elongated primers directly through the transparent cover film of the reaction chamber. This solution is designed to prevent cross-contamination and is suitable for simultaneous testing of a large number of test samples. The proposed platform is able to detect the presence of several pathogens of pneumonia in a sample and has an open architecture that allows expansion of the range of pathogenic bacteria and viruses that can be detected.
Insights
A new multiplex real-time PCR system detects multiple pneumonia pathogens, including bacteria, influenza A, and SARS-CoV-2. This sensitive, contamination-resistant platform enhances infectious disease diagnostics.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- Infectious Disease Research
Background:
- Pneumonia is a significant global health concern caused by diverse bacterial and viral pathogens.
- Accurate and rapid detection of these pathogens is crucial for effective treatment and control.
Purpose of the Study:
- To develop a prototype multiplex system for simultaneous detection of common pneumonia pathogens.
- To enhance diagnostic sensitivity and prevent cross-contamination in pathogen identification.
Main Methods:
- Multiplex solid-phase reverse transcription PCR (RT-PCR) assay development.
- Design of primers for six bacterial and two viral (Influenza A, SARS-CoV-2) pneumonia pathogens.
- Detection of fluorescent signals from incorporated labeled nucleotides after background signal reduction.
Main Results:
- Successful development of a prototype system capable of detecting multiple pneumonia pathogens.
- Achieved high sensitivity through reduced background noise and direct signal detection.
- Demonstrated suitability for simultaneous testing of numerous samples with minimized cross-contamination risk.
Conclusions:
- The developed multiplex RT-PCR platform offers a sensitive and reliable method for detecting various pneumonia pathogens.
- The system's open architecture allows for future expansion to include a wider range of infectious agents.
- This technology has the potential to significantly improve infectious pneumonia diagnostics.
