Tularemia is a natural focal zoonotic infection that can cause epidemic manifestations of an emergency nature.
PURPOSE OF THE STUDY
Development of a method for detecting DNA strains of the tularemia pathogen Francisella tularensis by loop isothermal amplification (LAMP, loop isothermal amplification).
MATERIALS AND METHODS
Primers for the selected targets were calculated using the on-line program Primer Explorer 5 and tested for specificity using the BLAST program. Primers were synthesized by Synthol, Moscow. Isolation of DNA from vaccine and virulent strains of epidemically significant subspecies of the tularemia microbe, as well as pathogens of other infectious diseases, was performed using a commercial «DNA-sorb-B kit» (InterLabService, Russia). The amplification reaction was carried out at a temperature of 63 °C for 60 min (without loop primers) or 30 min (with loop primers) with preliminary heating at a temperature of 92 °C for 2 min on a Tertsik amplifier (DNA-Technology, Russia) with use of a thermostable SD polymerase.
RESULTS
Sequences of genes encoding acid phosphatase A (acpA), outer membrane protein (fopA), and the region of the iglC gene of the pathogenicity island were chosen as DNA targets for the detection of the causative agent of tularemia. Of the two sets of outer, inner, and loop original primers synthesized for each selected marker gene, the acpFt101, fopFt132, and iglCFt1 sets reproducibly and specifically detected the DNA of 100—1000 F. tularensis microbial cells. The opportunity to reduce the analysis time by half appeared due to the introduction of loop primers and visual detection of amplification products stained with the SYTO 82 intercalating dye without subsequent electrophoresis and visualization of the gel after staining with ethidium bromide.
CONCLUSION
An easy-to-use test that does not require sophisticated equipment is proposed for clinical and field diagnostics of the tularemia pathogen.
