Development of a test system based on the quantitative polymer chain reaction for the detection of genetic material of the anthrax pathogen

Abstract

Goal. To develop a domestic test system for the detection of anthrax pathogen Bacillus anthracis DNA in biological material and environmental samples based on quantitative polymerase chain reaction (PCR) using recombinant control samples. Methods. The study was conducted in 2024–2025 on the basis of NSC «Institute of Experimental and Clinical Veterinary Medicine» of NAAS using molecular thymin-adenine cloning (for the manufacture of recombinant control samples), and real-time polymerase chain reaction (to determine the sensitivity and specificity of the test system). Results. A recombinant positive control sample containing a specific chromosomal marker dhp61 was designed, as well as an internal control sample, which made it possible to detect reaction inhibitors and prevent false negative results. Both components were integrated into the Anthrax DNA-test experimental test system, capable of detecting chromosomal and plasmid markers of the pathogen. The test system stably detected Bacillus anthracis DNA in a wide range of concentrations, did not respond to heterologous samples, and demonstrated complete reproducibility of the results in the case of repeated studies. According to the main analytical characteristics, it met international requirements for diagnostic tools. Conclusions. An experimental set of components for reliable detection of the anthrax pathogen by quantitative PCR using recombinant control samples was created and tested. The proposed test system is promising for implementation in the practice of laboratory diagnostics and provides an opportunity to increase the efficiency of epizootic surveillance in Ukraine.