Development of in-house diagnostic tool for the detection of Anthrax genetic material in real-time PCR

Abstract

This paper represents preliminary trials of the ‘Anthrax-DNA-test’, diagnostical tool for the detection of anthrax DNA. It includes recombinant positive controls p-pagA-TZ57R/T and p-capC-TZ57R/T for the detection of anthrax plasmid markers, as well as p-dhp61-CR2.1-TOPO, positive control for the detection of Bacillus anthracis chromosomal marker. Besides, three mixtures of primers and probes for the detection of each genetic marker (dhp61, pagA, and capC) and ready-to-use ‘RT-PCR МаsterМіx’ PCR diluent were also included. Concentrations of MgCl2 and Taq-polymerase obtained during qPCR validation procedure were considered when preparing the diluent. To determine specificity, qPCR was conducted with heterological panel of DNA of pathogenic bacteria and viruses causing diseases with similar to anthrax clinical signs. To determine repeatability of the results when using ‘Anthrax-DNA-test’ PCR test kit, samples were studied twice. The sensibility of the kit was analyzed by serial dilutions of p-dhp61-CR2.1-TOPO, p-pagA-TZ57R/T and p-capC-TZ57R/T plasmid DNAs containing fragments of anthrax chromosome and plasmids. To compare the tool’s ability to identify anthrax DNA, classical PCR was carried out using ANT-PA_F/R and ANT-CAP_F/R primers recommended by OIE for the detection of pXO1 and pXO2 plasmid DNA. Sensitivity testing has shown that the test kit is able to identify all positive samples. It has been found that the diagnostics tool detects anthrax DNA in recombinant positive control samples containing B. anthracis chromosomal and plasmid DNA fragments in serial dilutions from 1:100 to 1:1,000 with Ct values of 25.29–34.70. The specificity of this diagnostic tool is proved by the absence of Ct in heterological samples. Besides, repeatability of trial results has been found, which is proved by complete congruence in duplicates with each of the tested sample