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Updated: Oct 8, 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, R A Miftakhov1, E S Klochikhina1
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, 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 reverse transcription PCR (RT-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:
- Molecular Biology
- Infectious Disease Diagnostics
- Biotechnology
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 public health management.
- Existing diagnostic methods may lack multiplexing capabilities or suffer from sensitivity issues.
Purpose of the Study:
- To develop and prototype a novel multiplex system for the simultaneous detection of common pneumonia pathogens.
- To enhance diagnostic sensitivity and reduce background noise in pathogen detection.
- To create a contamination-resistant platform suitable for high-throughput sample analysis.
Main Methods:
- Development of a multiplex solid-phase reverse transcription PCR (RT-PCR) assay.
- Design of specific primers for six bacterial and two viral (Influenza A, SARS-CoV-2) pneumonia pathogens.
- Utilized fluorescently labeled nucleotides for signal accumulation and detection through a reaction chamber's cover film.
Main Results:
- The prototype system successfully detected multiple pneumonia pathogens in a single test.
- Signal detection through the cover film minimized background noise, increasing analytical sensitivity.
- The system demonstrated suitability for simultaneous testing of numerous samples with reduced cross-contamination risk.
Conclusions:
- The developed multiplex RT-PCR system offers a sensitive and efficient method for detecting infectious pneumonia pathogens.
- The platform's design facilitates high-throughput testing and minimizes contamination, improving diagnostic workflows.
- The open architecture allows for future expansion to detect a broader range of pathogenic microorganisms.
