Acta Biomedica Scientifica (Nov 2013)
Diagnostic capacity of PCR test systems for epizootic study of the plague natural foci of Kazakhstan
Abstract
The aim of the research is to determine of the efficiency of developed uniplex, duplex, and multiplex variants of PCR test systems for the epizootic research of the plague natural foci. The research was carried out on the model of plague and its relative strains, and with the suspension of fleas and ticks. Selection of the primers used for detection of the genes cafl, pla and yopE is carried out by the program software Primer 3 and the complex of online program BLAST® (Basic Local Alignment Search Tool. Synthesis of primers is carried out by the traditional phosphoramidite method on the automatic synthesizer DNA/RNA H6 (K&A, Germany). Purification of the received solutions of primers is carried out by the gel filtration method on Centri-Pure N10 columns (emp Biotech GmbH, Germany). Extraction of genomic DNA of Y. pestis, Y. pseudotuberculosis and Y. enterocolitica is done by QIAamp DNA Mini Kit (QIAGEN, Germany). Testing of specific primers and test system was carried out by the standard PCR. Analysis of amplification's results is conducted by standard method of horizontal agarose gel electrophoresis. In the research, Y.pestis strains from the Alive cultures museum of M. Aikimbayev's Kazakh Scientific Center for Quarantine and Zoonotic Diseases, and suspensions of ticks and fleas collected from the Central Asian Desert Plague Focus were used. For the first time in Kazakhstan, for production, PCR test systems have been developed for detection of the plague microbe in the field and laboratory conditions. The PCR test systems developed on the base of specific primers to plague microbe's genes yopE, cafl and pla helped to detect the circulation of plague microbe without Fl (without fraction). Research of suspensions of fleas and ticks collected during the plague epizooty confirmed the specificity and high sensitivity of the developed test systems.
