The development and application of reagents kit for detection RNA of the causative agents of melioidosis and glanders by transcription-based amplification (NASBA) in real-time.

Abstract

The polymorphism of clinical manifestations of melioidosis and glanders and their high mortality require improvement of diagnostics for detection of this agents. The perspectivity of development of transcription-based amplification real-time NASBA diagnostic kits is determined by high analytical sensitivity and the opportunity to perfom the verification of the results of other methods for pathogenic Burkholderia species detection. The fragment of 23S rRNA gene was selected as the target for development of real-time NASBA kit. The high specificity of the constructed oligonucleotides was confirmed during the analysis of wide range of heterological strains of microorganisms and sequencing of amplified fragments of 23S rRNA gene. The analytical sensitivity of the developed kit allowed to detect Burkholderia pseudomallei and Burkholderia mallei in concentration of 1×101 microbial cells per ml. The high functional characteristics of developed kit as well as the possibility to use it in case of appereance of discordant result during the detection of pathogenic Burkholderia species were demonstrated while studying biological samples.