DEVELOPMENT OF PARAMETER SETTING (LAMP) OF LOOP MEDIATED ISOTHERMAL AMPLIFICATION FOR ACCELERATED IDENTIFICATION OF BORDETELLA PETRII BACTERIA

Abstract

The article is devoted to development of loop mediated isothermal amplification (LAMP) method for accelerated identification of little-studied bacteria Bordetella petrii, which are isolated from animals and humans. LAMP is based on identification of a conserved gene in a target group of microorganisms against which a series of four to six primers should be designed. The study protocol takes less than one hour and includes amplification of the target gene using Bst polymerase in isothermal conditions. Species-specific primers were designed to identify B. petrii bacteria, transcription regulator gene LysR was used in the studies. The specificity of the selected primers was validated by BLAST at National Center of Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/). The researchers selected parameters for layout of the reaction, tested specificity of the developed protocol and appropriate amplification time, also they determined sensitivity of the developed LAMP protocol for identification of B. petrii bacteria. It was established that appropriate amplification result is recorded after 40-50 minutes; agarose gel electrophoresis indicates that sensitivity of the developed amplification protocol is 10 DNA copies. The developed method is specific, which is confirmed by a negative reaction on bacteria: Pseudomonas aeruginosa, Alcaligenes spp., Acinetobacter calcoaceticus, Aeromonas hydrophila, Citrobacter freundi, Escherichia coli, Klebsiella pneumoniae, Salmonella infantis, Yersinia enterocolitica, Staphylococcus aureus, Enterococcus faecalis, Bacillus sub.