Development of in-house diagnostic tool for the detection of Anthrax genetic material in real-time PCR-Reference-Cited by-同舟云学术

Development of in-house diagnostic tool for the detection of Anthrax genetic material in real-time PCR

Published:2023-09-27 Issue:3 Volume:9 Page:18-22
ISSN:2411-0388
Container-title:Journal for Veterinary Medicine, Biotechnology and Biosafety
language:en
Short-container-title:J. Vet. Med. Biotechnol. Biosafety

Author:

Biloivan O. V.¹^ORCID,Popp C.²^ORCID,Schwarz J.²^ORCID

Affiliation:

1. National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine”, Kharkiv, Ukraine

2. Bundeswehr Institute of Microbiology

Abstract

This paper represents preliminary trials of the ‘Anthrax-DNA-test’, diagnostical tool for the detection of anthrax DNA. It includes recombinant positive controls p-pagA-TZ57R/T and p-capC-TZ57R/T for the detection of anthrax plasmid markers, as well as p-dhp61-CR2.1-TOPO, positive control for the detection of Bacillus anthracis chromosomal marker. Besides, three mixtures of primers and probes for the detection of each genetic marker (dhp61, pagA, and capC) and ready-to-use ‘RT-PCR МаsterМіx’ PCR diluent were also included. Concentrations of MgCl2 and Taq-polymerase obtained during qPCR validation procedure were considered when preparing the diluent. To determine specificity, qPCR was conducted with heterological panel of DNA of pathogenic bacteria and viruses causing diseases with similar to anthrax clinical signs. To determine repeatability of the results when using ‘Anthrax-DNA-test’ PCR test kit, samples were studied twice. The sensibility of the kit was analyzed by serial dilutions of p-dhp61-CR2.1-TOPO, p-pagA-TZ57R/T and p-capC-TZ57R/T plasmid DNAs containing fragments of anthrax chromosome and plasmids. To compare the tool’s ability to identify anthrax DNA, classical PCR was carried out using ANT-PA_F/R and ANT-CAP_F/R primers recommended by OIE for the detection of pXO1 and pXO2 plasmid DNA. Sensitivity testing has shown that the test kit is able to identify all positive samples. It has been found that the diagnostics tool detects anthrax DNA in recombinant positive control samples containing B. anthracis chromosomal and plasmid DNA fragments in serial dilutions from 1:100 to 1:1,000 with Ct values of 25.29–34.70. The specificity of this diagnostic tool is proved by the absence of Ct in heterological samples. Besides, repeatability of trial results has been found, which is proved by complete congruence in duplicates with each of the tested sample

Publisher

Kharkiv Entomological Society

Subject

General Medicine

Reference18 articles.

1. Antwerpen, M. H., Zimmermann, P., Bewley, K., Frangoulidis, D. and Meyer, H. (2008) ‘Real-time PCR system targeting a chromosomal marker specific for Bacillus anthracis’, Molecular and Cellular Probes, 22(5–6), pp. 313–315. doi: 10.1016/j.mcp.2008.06.001.

2. Beloyvan, A. V., Stegniy, B. T., Gerilovych, A. P., Solodiankin, A. S., Duerr, A., Schwarz, J. (2019) ‘Detection of specific marker CAPC of the Bacillus anthracis pXO2 plasmid via real-time PCR method’ [Detekciya specificheskogo markera CAPC plazmidy pXO2 Bacillus anthracis s pomoshchu metoda PTsR v realnom vremeni], Epizootology. Immunobiology. Pharmacology. Sanitary [Epizootologiya. Immunobiologiya. Farmakologiya. Sanitariya], 1, pp. 54–62. Available at: https://www.elibrary.ru/item.asp?id=38585181. [in Russian].

3. Beyer, W., Glöckner, P., Otto, J. and Böhm, R. (1995) ‘A nested PCR method for the detection of Bacillus anthracis in environmental samples collected from former tannery sites’, Microbiological Research, 150(2), pp. 179–186. doi: 10.1016/S0944-5013(11)80054-6.

4. Biloivan, O. V., Stegniy, B. T., Gerilovych, A. P., Solodiankin, O. S., Popp, C. and Schwarz, J. (2019) ‘Validation of Anthrax specific pagA quantitative PCR for detection of Bacillus anthracis pXO1 plasmid’, Journal for Veterinary Medicine, Biotechnology and Biosafety, 5(2), pp. 15–21. doi: 10.36016/JVMBBS-2019-5-2-3.

5. Biloivan, O. V., Stegniy, B. T., Solodiankin, O. S. and Gerilovych, A. P. (2018) ‘Development of positive control assays for the detection of Bacillus anthracis plasmids pXO1 and pXO2 via PCR’ [Rozrobka pozytyvnykh PLR-kontroliv dlia vyiavlennia plazmid Bacillus anthracis pXO1 ta pXO2], Veterinary Biotechnology [Veterynarna biotekhnolohiia], 32(1), pp. 44–49. doi: 10.31073/vet_biotech32(1)-3. [in Ukrainian].