Bulletin "Veterinary biotechnology"

Veterynarna biotehnologija – Veterinary biotechnology, 2018, 32(1), 44-49 [in Ukrainian]. https://doi.org/10.31073/vet_biotech32(1)-3

BILOIVAN O.V.e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., STEGNIY B.T.e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., SOLODIANKIN O.S.e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., GERILOVYCH A.P.e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

National Scientific Center "Institute of Experimental and Clinical Veterinary Medicine"

DEVELOPMENT OF POSITIVE CONTROL ASSAYS FOR THE DETECTION OF BACILLUS ANTHRACIS PLASMIDS PXO1 AND PXO2 VIA PCR

Introduction. For today, anthrax remains to be one of the most dangerous anthropozoonotic diseases, which pose a big threat to public and animal health. Despite the fact, that epizootic situation for anthrax in Ukraine remains stable, sporadic cases occur in all regions of the country. Capability of anthrax spores to remain viable in environment for decades is the one of factors affecting pathogenicity of this agent, and increases the possibility of its potential use as biological weapon. Generally, anthrax is diagnosed in cases, when amplicons with certain sizes are obtained after specific PCR for pag and cap genes, which encode pXO1 and pXO2 plasmids respectively. Positive controls prepared from standard B. anthracis strains are usually included into diagnostic PCR kits. However, using B. anthracis live strains requires BSL-3 laboratory to be available, as well as enhanced biosafety measures. Using of B. anthracis DNA as positive control for PCR also increases risk of cross-contamination and might negatively affect the results. To avoid such problems, it is reasonable to use recombinant control samples, and their production was a goal of our work.

Objective. To develop and prepare recombinant positive controls for the detection of pXO1 and pXO2 anthrax plasmids.

Materials and methods. B. anthracis DNA from Ames 3013 strain, as well as thermolysate of B. anthracis broth culture #625, obtained from Ukrainian Center of Diseases’ Monitoring and Control (Kyiv). Pag gene fragment of pXO1 plasmid was amplified using pagA_for and pagA_rev primers. As for cap gene fragment of pXO2 plasmid, it was amplified with capC_for and capC_rev primers. Both primer systems were developed at Bundeswehr Institute of Microbology (Munich, Germany). After gel-electrophoresis, obtained fragments were cut from agarose gel, purified and inserted into pTZ57R/T vector. E. coli DH5α competent cells were transformed with obtained inserts and seeded on LB-agar with 10 µg/ml of ampicillin. White colonies were transferred to LB-medium, centrifuged after 4 hours of growing and plasmids were extracted.

Results and discussion. Molecules were designed on the basis of pTZ57R/T cloning vector and inserts of pagA and capC with 201 and 171 bp length respectively. Molecules were constructed using Clone Manager software. Obtained pTZ_pagA and pTZ_capC molecules should have sizes 3087 and 3057 bp respectively. PagA fragments were obtained via conventional PCR from two samples of B. anthracis DNA: Ames 3013 and sample from broth culture thermolysate #625. CapC fragment was obtained from one DNA sample of B. anthracis Ames 3013 strain. All samples were successfully ligated with DNA of pTZ57R/T and E.coli DH5α competent cells were transformed with obtained plasmids. Obtained colonies were studied in real-time PCR. Plasmid DNA was extracted from each sample and diluted into concentrations of 1 thousand and 10 thousand copies per 5 µl. As the result of final conventional PCR, 201 bp (pTZ_pagA1,2) and 171 bp (pTZ_capC) fragments were obtained. These sizes are specific for pagA and capC genes of pXO1 and pXO2 plasmids respectively.

Conclusions and perspectives for further research. As the result of molecular cloning, positive DNA controls for the detection of pXO1 (pagA) and pXO2 (capC) anthrax plasmids were produced. As the result of gel-electrophoresis after PCR, all DNA fragments had specific length (201 and 171 bp respectively). These recombinant control samples are ready to use in conventional and real-time PCR with annealing temperatures from 57 to 62°С. In addition, these samples will be included to diagnostic kits for anthrax genome and plasmid detection.

Keywords: anthrax, plasmids, DNA, cloning

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