AN ACCELERATED METHOD OF DIPHTHERIA GENE DIAGNOSTICS BASED ON ISOTHERMAL AMPLIFICATION TO DETECT DNA OF THE CAUSATIVE AGENT

Abstract

of C. from bacteriological the and sent to the of Epidemiology and Microbiology. Strain isolation was carried out in accordance with MI 4.2.698-98 and 4.2.3065-13. Chromosomal DNA was isolated by standard heating method, as well as using 3 commercial kits. Detection of the amplification results was carried out in horizontal electrophoresis in 1.5% agarose gel. Results. The developed method of gene diagnostics was established to allow detection of DNA of toxi­ genic C. diphtheriae strains of 2 biovars, as well as DNA of non-toxigenic tox-gene bearing strains (NTTB) of C. diphtheriae mitis biovar with mechanisms of lack of expression of diphtheria toxin gene due to the presence of deletion or mobile genetic IS element in the tox gene. Non-toxigenic tox-gene bearing C. diphtheriae strain with the mechanism of lack of diphtheria toxin gene expres­ sion due to the presence oftransposon in the tox gene are identified as non-toxigenic. Evaluation of the analytical sensitivity in comparative studies using 3 commercial kits for FNA isolation has shown that sensitivity reached 4.5x10' GE/ml using Ribo-prep kit. High specificity of the developed method is shown, it was evaluated in 18 strains of 16 other members of the Corynebacterium genus and 20 typical strains of microorganisms isolated from oropharynx or causing infections of the respiratory tract. Approbation of the developed method was carried out in model experiments in imitators of clinical samples by infection of single-use sterile dry tampons by consecutive dilutions of the bacterial cultures (with parallel seeding into dense nutrient media) and was 103 GE/ml. Conclusion. The developed method of accelerated gene diagnostics of the diphtheria infection has a high diagnostic effectiveness, specificity and sensitivity, allows to detect 103 — 4.5x10 GE/ml C. diphtheriae in clinical material with simultaneous verification of toxigenic and non-toxigenic strains. fused recombinant proteins of Pseudomonas that infection. P.