One of the most common pathogens of bacterial pneumonia are representatives of several genera: Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenza, Legionella pneumophila, Klebsiella pneumonia and some others . For these five bacterial species, genetic targets were selected and primers were designed to perform multiplex PCR. When designing the primers, they were guided by the requirement of species specificity and intraspecific conservatism of selected regions of genetic targets, which is necessary for reliable differential diagnostics, for example, the ebpS gene for identification S. aureus  and the gene sidA for L. pneumophila . We took into account the need to obtain different lengths of PCR products for the convenience of identifying the pathogen during electrophoretic separation.
For each primer, a BLAST analysis procedure was performed, its physicochemical characteristics were determined, including testing for the presence of both intramolecular and intermolecular secondary structures.
The temperature-time profile of PCR was optimized using gradient PCR, as well as the composition of the buffer components and the concentration of each of the primers in the mixture. The specificity of primers to target and non-specific templates was determined experimentally (using the total genomic DNA of each of the tested bacterial strains), both in the mode of application of individual DNA templates and in the mode of DNA mixtures of several pathogens in a single tube. It was found that primers have high specificity in a mixture containing the DNA of various microorganisms and are able to identify solely their specific targets, without giving a false positive result in the presence of nonspecific DNA in the mixture.
The sensitivity of the test system was determined by DNA dilutions of each of the analyzed pathogens, which amounted to 102 to 103 copies of genomic DNA to the reaction tube, including the simultaneous introduction of several templates into the mixture.
The result of differential detection of the pathogen DNA in the sample by multiplex PCR is shown in Fig. 1.
The developed system can be expanded to identify pathogens of pneumonia of a viral and fungal nature. Currently, it is planned to conduct tests on clinical samples, use labeled dNTP derivatives for subsequent analysis on biochips, and also develop a mathematical algorithm for calculating amplification signals. The system is suitable for diagnostic laboratories specializing in clinical analysis using PCR.