OPTIMIZATION OF THE TECHNOLOGICAL SCHEME FOR THE PRODUCTION OF PREPARATIONS FOR GENE DIAGNOSTICS OF PARTICULARLY DANGEROUS INFECTIOUS DISEASES USING POLYMERASE CHAIN REACTION WITH HYBRIDIZATION-FLUORESCENT REGISTRATION OF RESULTS

Objective of the study was to optimize technological scheme for the production of preparation for gene diagnostics of plague with hybridization-fluorescent registration (HFR) of results, which is associated with the assessment and selection of synthesis conditions and method of oligonucleotide probe purification included into the kit and carrying fluorophore (6-carboxyfluorescein) and quencher (Black Hole Quencher-1) tags, as well as to evaluate their properties and implement this new technological line into manufacturing.

Materials and methods. The object of investigation was the reagent panel “Gene Yersinia pestis Identification-HFR” and its constituent elements, probes and primers, providing for hmsH gene amplification. Synthesis of the primers and probes was carried out in 8-well DNA synthesizer ASM-800 (Bioset, Russia), using solid phosphite amid method. For the experiments the probes obtained were purified either by means of electrophoresis in 20 % polyacrylamide gel (size 20×20) or (size 8×10) with further purification through RP-cartridges in semi-automated mode in OPS-201 system and manually, or on RP-cartridges only in manual mode; or applying highperformance liquid chromatography (HPLC). Specific activity of the produced probes was tested by polymerase chain reaction using the DNA isolated from bacterial suspensions of Yersinia pestis C-624 strain, in concentrations up to 1·10³ – 1·10⁶ mc/ml.

Results and conclusions. Optimized has been synthesis technology; selected has been the method for oligonucleotide probe purification, carrying phluorophore-6-carboxyphluorescin and Black Hole Quencher-1. Demonstrated is viability of applying the stated oligonucleotides or HPLC, or electrophoresis in 20 % polyacrylamide gel with 7 M urea and further purification through RP-cartridges in manual mode for purification purposes. Introduction of the optimized technological scheme in manufacturing of preparations for gene indication and identification of particularly dangerous pathogens with hybridization-fluorescent detection will allow for the reduction of lead time by 50 % and original cost – by 66 %. 

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