Citation: Ukhovska T. (2026) Use of RAPD as a screening tool to detect B. Anthracis vaccine strain contamination. VeterinaryBiotechnology, 48, 222-237. https://doi.org/10.31073/vet_biotech48-16
UKHOVSKA T., Cand. Sci. (Vet. Med.), e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., https://orcid.org/0000-0002-1810-3804
Institute of Veterinary Medicine of the NAAS
USE OF RAPD AS A SCREENING TOOL TO DETECT B. ANTHRACIS VACCINE STRAIN CONTAMINATION
Introduction.The presence of a large number of anthrax burial sites across a significant portion of Ukraine is the main reason for the persistence of the infection, the formation of chronically high-risk areas, and the maintenance of their epizootic activity over the years; in this context, recurrent outbreaks can occur even after 26 years.
Molecular genetic methods are used to determine the genetic characteristics of microorganisms; RAPD fingerprint analysis is the most suitable for such tasks, as it allows for the generation of a specific profile of the microorganism that differs among related species. Certain regions of the genome exhibit high variability between species and even strains, making it possible to use them to identify specific markers for related species of the genus Bacillus and even among strains, which is important for monitoring the purity of vaccine and production strains.
The goal of the work. To evaluate the potential of RAPD as a screening tool for detecting contamination of the vaccine strain of B. anthracis by bacteria of the genus Bacillus, and to determine the sensitivity threshold of the method based on contamination modeling.
Materials and methods.The study utilized strains from the collection of the Institute of Microbiology of the National Academy of Agrarian Sciences: the vaccine strain B. anthracis UA-07 and B. cereus ATCC 10702. To simulate contamination, artificial mixtures of the vaccine strain B. anthracis UA-07 and B. cereus ATCC 10702 were prepared in ratios of 1%, 5%, and 10% (by volume of the cell suspension). Pure cultures of each strain served as controls. For each experimental variant, at least three independent replicates of the experiment were conducted.
DNA extraction was performed using the commercial QIAamp DNA Mini Kit (Qiagen, Germany) in accordance with the manufacturer’s instructions. DNA concentration and purity were determined spectrophotometrically (A260/A280).
Genotyping was performed by RAPD-PCR using two oligonucleotide primers 20–22 nucleotides in length. Amplification was performed on a CFX96 amplifier (Bio-Rad, USA). PCR conditions were optimized to ensure the reproducibility of amplification profiles. The amplification program included denaturation, annealing at a lower temperature, and extension with a number of cycles sufficient to generate reproducible RAPD profiles.
The reaction was set up using the commercial DreamTaq PCR Master Mix (2X) (Thermo Fisher Scientific, USA), which contained Taq DNA polymerase, dNTPs, and MgCl₂. The reaction mixture was prepared in a 25 μL volume by adding 12.5 μL of master mix; 1.0 μL of oligonucleotide primer (final concentration 0.4 μM), 2 μL of DNA template (20–50 ng), and sterile deionized water to a final volume of 25 μL.
Analysis of the amplification products was performed by electrophoresis in a 1.5% agarose gel in a TBE buffer system. Electrophoresis was carried out at a voltage of 10 V/cm until the dye front passed approximately 50% of the gel length. Visualization of DNA fragments was performed using a transilluminator (Bio-Rad) followed by photofixation.
Analysis of RAPD profile similarity was performed based on a binary matrix (presence/absence of fragments).
Results of research and discussion.As a result of our studies, we optimized the PCR protocol as follows: DNA denaturation (94°C – 1 min), annealing (38°C – 3 min), and amplification (72°C – 3 min), for a total of 40 cycles. At a lower annealing temperature, a greater accumulation of amplification products of the expected size is observed.
The primers used produced a set of DNA fragments of varying intensity characteristic of the genus, which differed from the corresponding set of amplificates from anthrax bacteria.
At a contamination level of 1%, the profile remained largely identical to the control, but in some replicates, weak additional fragments of 1.2 and 1.7 kbp, characteristic of B. cereus, were observed. These changes were inconsistent and did not allow for unequivocal identification of contamination.
When the proportion of B. cereus was increased to 5%, a stable formation of additional amplification products (0.8, 1.2, and 1.7 kbp) was observed, as well as a decrease in the intensity of individual fragments of the vaccine strain. These changes were reproduced in all replicates, allowing for a clear distinction between mixed cultures and pure cultures.
At 10% contamination, the RAPD profile underwent marked changes: a complete set of fragments characteristic of B. cereus appeared (0.8, 1.2, and 1.7 thousand base pairs), while the number of amplicons from the vaccine strain decreased (2.0 and 2.2 thousand base pairs). Overall, this resulted in the formation of a mixed profile that was clearly distinguishable from the reference profile.
Cluster analysis of RAPD profiles revealed a clear grouping of samples based on the degree of contamination.
Conclusions and prospects for further research. RAPD analysis revealed that the B. anthracis vaccine strain exhibits a stable and reproducible genetic profile, characterized by a distinctive set of amplified fragments.
Modeling of contamination with B. cereus bacteria showed that the threshold level for reliable detection of contamination is approximately ≥5%, whereas at 1%, the changes are unstable and cannot be unambiguously interpreted.
Cluster analysis of RAPD profiles based on the Dice coefficient and the UPGMA method confirmed the presence of a similarity gradient among the samples. Clustering confirms the results of visual and densitometric analysis and allows for a semi-quantitative assessment of the degree of contamination.
The proposed approach can be integrated into the quality control system for microbiological production as a rapid and cost-effective method for assessing the purity of cultures. However, to ensure the reliability of the results, it is essential to strictly adhere to standard reaction conditions and to use control samples.
Keywords: anthrax pathogen, polymerase chain reaction, molecular genetic properties, amplification, B. anthracis, RAPD.
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