Development of a PCR Test System for the Detection of Diagnostically and Prognostically Promising Clusters of Mycobacteriophages

Infections caused by Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM) are a significant medical and social problem. The insufficient informative value of microscopic and cultural methods in the diagnosis and evaluation of etiotropic treatment efficacy for mycobacterial infections, combined with the growing antibiotic resistance of mycobacteria and the increasing number of mycobacterial infections, necessitates the development of new laboratory diagnostic methods for these diseases. Currently, molecular genetic methods, including those based on PCR, open up additional opportunities for the detection of mycobacterioses and the assessment of their treatment. One such approach is the little-studied method of PCR detection of mycobacteriophages as promising diagnostic and prognostic markers of mycobacterial infections. The aim of this study was to develop and test primers for diagnostically and prognostically promising PCR detection of nucleotide sequences of clinically associated mycobacteriophage clusters. A multiple alignment of known complete genomic nucleotide sequences of mycobacteriophages was performed. Primers for subsequent PCR diagnostics were selected to identify conserved cluster-specific DNA regions of mycobacteriophages. The optimal annealing temperatures of the obtained primers were confirmed in experimental PCR with evaluation of the sensitivity and specificity of the reaction. Mycobacteriophage clusters (A, L, AB) were re-analyzed, and a new mycobacteriophage cluster MA, as well as previously undescribed subclusters AB1 and AB2 of the mycobacteriophage cluster AB, were proposed as promising targets for screening diagnostics and evaluation of etiotropic treatment efficacy for mycobacterioses. Primers were selected for amplification of clusteror subcluster-specific DNA regions of mycobacteriophages using synthetic controls. Based on the testing of clinical samples, the potential practical value of detecting nucleotide sequences of mycobacteriophage DNA regions specific for subclusters A1, A2, A3 of cluster A, for clusters L, MA, and for subclusters AB1, AB2 of cluster AB was established. PCR using the selected primers for the detection of mycobacteriophage DNA regions specific for subclusters A1, A2, A3 of cluster A, for clusters L, MA, and for subclusters AB1, AB2 of cluster AB is a promising laboratory method for screening diagnostics of a wide range of mycobacterial infections, as well as for confirming their eradication.