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Vol 55(2021) N 6 p. 828-838; DOI 10.1134/S0026893321040063 Full Text

S.A. Lapa1*, R.A. Miftakhov1, E.S. Klochikhina1, Yu.I. Ammur2, S.A. Blagodatskikh3, V.E. Shershov1, A.S. Zasedatelev1, A.V. Chudinov1

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

1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia
2Mechnikov Institute of Vaccines and Serums, Moscow, 105064 Russia
3Scientific Center of Applied Microbiology and Biotechnology, Obolensk, 142279 Russia

*lapa@biochip.ru
Received - 2021-03-12; Revised - 2021-04-12; Accepted - 2021-04-13

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.

solid-phase RT-PCR, multiplex PCR, infectious pneumonia, influenza, SARS-CoV-2



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