The application of multiplex PCR and PCR-restriction fragment length polymorphism for differentiation of Bacillus anthracis from other Bacillus spp.

Abstract

Introduction: Bacillus anthracis causes an infectious disease called anthrax. Herbivores are more susceptible to the disease than omnivores, carnivores and humans. Grazing animals are the highest-risk group, and among them, anthrax outbreaks have extremely high fatality rates and impose heavy costs, besides posing a grave zoonotic risk. The aim of the study was the application and evaluation of simultaneous use of multiplex PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) allowing the differentiation of strains of the B. anthracis species from other species of the Bacillus genus.

Material and methods: The experiment involved 21 strains of B. anthracis. Strains of other species of the Bacillus genus were also included in the experiment. In the first part of the studies, two genes responsible for virulence - pag and cap, located on plasmids pXO1 and pXO2 - and the chromosomal sequence Ba813 were used for a multiplex PCR. In the next stage, PCR-RFLP, in which restriction analysis of the SG-749 sequence using the AluI enzyme was performed.

Results: The multiplex PCR allowed the identification of virulent B. anthracis strains, as well as the detection of the presence of the chromosomal sequence Ba813. Then, PCR-RFLP showed the restriction pattern characteristic of B. anthracis strains.

Conclusion: The simultaneous use of multiplex PCR and PCR-RFLP enables the distinction of B. anthracis strains with and without plasmids from other strains of the Bacillus genus, including those with the Ba813 chromosomal sequence.