[Development of Multiplex RT-PCR with Immobilized Primers for Identification of Infectious Human Pneumonia Pathogens].

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.

Specificities of multi-primer polymerase chain reaction optimization for the detection of infectious pneumonia agents in human

Objectives. The objectives of this work are the development of a multi-primer system based on the polymerase chain reaction (PCR) aimed at the simultaneous detection of six bacterial pathogens that cause human pneumonia and the determination of the parameters important for the optimization of this multi-primer system, including solid-phase PCR systems (biological microarrays). Methods. To determine the optimal parameters of the system, PCR methods were used in monoplex and multiplex formats. Results. Primers for Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenza, Legionella pneumophila, Klebsiella pneumoniae, and Streptococcus pneumoniae detection were designed, and the PCR cycling conditions were optimized. The patterns of primer design for solid-phase PCR were revealed. Conclusions. The developed prototype of a system specifically identifies six clinically significant bacterial pathogens. It could be expanded for the analysis of viral and fungal pathogens and used in clinical diagnostics. A prototype of a system for pathogenic agent detection in the immobilized phase (biological microarray) was created. © 2021, MIREA - Russian Technological University. All rights reserved.