[Evaluation of multiplex PCR to identify the species of microorganisms from Bacillus cereus group].

Abstract

Introduction: Bacillus genus comprises about 215 species. The most closely related are B. cereus, B. thuringiensis, B. anthracis, B. mycoides, B. pseudomycoides and B. weihenstephanensis. These bacterial species belong to the Bacillus cereus group. Identification and differentiation of Bacillus cereus group bacteria is difficult because of genetic and phenotypic similarity. Many molecular methods have already been suggested to differentiate B. cereus group members. However easier and more convenient methods are still sought. The aim of this study was to evaluate of multiplex PCR method to identify and distinguish strains of Bacillus cereus group, as proposed by Park et al. (J Microbiol Biotechnol 2007; 17: 1177-82).

Materials and method: Twenty four strains of Bacillus cereus group included B. cereus, B. anthracis, B. thuringiensis and B. weihestephanensis was examined. A multiplex-PCR assay for the differentiation of the species has been applied by using three pairs specific oligonucleotide primers based on sequences of gyrB genes which identify species from Bacillus cereus group and one pair specific primers based on sequences of groEL gene, which are used to identify Bacillus cereus group.

Results: Using a specific primers complementary to fragment of groEL gene, we received all PCR products and thus we identified Bacillus cereus group. We have not recived a specific products characteristic for each of the species. Oligonucleotide primers recognized by Park et al. as specific for each species were complementary (often 100%) for the gyrB gene sequence in almost all species of the B. cereus group.

Conclusions: The multiplex PCR method proposed by Park et al. multiplex PCR method for identification ofB. cereus group and individual bacterial species has been proved to be useful only for identification the entire group of B. cereus. This method does not provide specific identification of the individual species. Lack of specificity of the primers used in this study creates a risk of obtaining PCR product in more than one species of the entire examined group of bacteria and does not allow the precise identification to the species level.