Veterynarna biotehnologija – Veterinary biotechnology, 2016, 29, 219-226 [in Ukrainian].
PUSTOVIT N.A., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.
State Scientific Control Institute of Biotechnology and Strains of Microorganisms
DEVELOPMENT AND IMPROVEMENT OF CAMPYLOBACTERIA INDENTIFICATION TECHNIQUE
Introductions. Polymerase chain reaction is an effective modern method of rapid diagnosis of campylobacteriosis. Using this method, it is possible not only carry out rapid identification of pathogens in acute forms of the disease, but also ensure the identification of excite in products and animals.
The goal of the work was to improve methods for the analysis of food products for the presence of pathogens of acute intestinal infections (AII), including bacteria of the genus Campylobacter spp. which is оne of the most pressing problems of food hygiene. This task is dictated by the need to include microbiological research in the prevention of acute intestinal infections, as well as the monitoring of pathogens in food. Study materials for the presence of Campylobacter microbiological method requires a significant investment of time, resources, and possession of certain skills of workers.
Materials and methods. The diagnostic kit based on PCR for rapid detection of microbial DNA genus Campylobacter spp., (conservative areas amplification gene (16S rRNC) in biological materials was used.
Results of research and discussion. The developed primers 16S-F1 and 16S-R2 were designed according to all requirements, have a high theoretical specificity to bind to the DNA matrix, and don’t have critical homology with other bacteria and viruses. The nucleotide sequence search was conducted using databases GeneBank, EMBL (European Molecular Biology Library), DDBJ (Japanese database of nucleotide sequences), and Entrez (National Center of Biotechnological information, USA). The PCR was performed using a four-channel thermocycler “Tertsik” (NVF “DNK-Tehnologiia”, Moscow, Russia). The developed PCR-based test kit is aimed at the rapid detection of DNA of the Campylobacter spp. microorganisms in biological materials by means of specific amplification of the conserved locus of 16S RNA gene. PCR system for Campylobacter diagnoses showed that all the requirements for the quality of the experiment. The results made it possible to create and provide for the implementation of test system «Campylobacter spp.-PCR-test" for detection and identification of the pathogens of the Campylobacter genus.
Conclusions and perspectives for further research: 1. The test-system is safe, have high specificity, compared to the test systems that are used nowadays. 2. Sensitivity of the developed test system is 10─11 g DNA. 3. Developed test system «Campylobacter spp. PCR-TEST" is proposed for implementation for use in veterinary practice at regional (regional and district veterinary laboratories) and national levels (State Research Institute of Laboratory Diagnostics and Veterinary Expertise) is.
Keywords: campylobacteriosis, test system, polymerase chain reaction, material, objects of the environment.
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