DETECTION OF BACILLUS SPOROTHERMODURANS BY SEMI-NESTED PCR

Fernanda Cattani (PUCRS); Carlos Alexandre Sanchez Ferreira (PUCRS); Sílvia Dias de Oliveira (PUCRS)

Bacillus sporothermodurans are aerobic, Gram-positive bacteria, and has been characterized by the production of highly heat-resistant spores, which may survive ultra high temperature (UHT) processing of milk. Therefore, spores can germinate and grow in the stored milk. The B. sporothermodurans is considered non-pathogenic, do not affect the pH, stability nor sensory quality of the milk and rarely cause any characteristic spoilage. However, the B. sporothermodurans is a mesophilic microorganism and the growth of germinated spores can lead to an unacceptably level of the mesophilic bacteria according to the legislation of the different countries. The aim of this study was to standardize a semi-nested PCR for the specific detection of B. sporothermodurans. The standardization was performed with two strains of B. sporothermodurans, and other related strains: B. cereus, B. acidicola, B. lentus, B. firmus, B. circulans, B. coagulans, Geobacillus stearothermophilus and G. kaustophilus. The extraction of genomic DNA was performed with the kit NewGeneTM (Simbios Biotecnologia). The oligonucleotide primers for detection of B. sporothermodurans (5'-ACGGCTCAACCGTAGAG-3' and 5'-GTAACCTCGCGGTCTA-3') amplified a fragment of 664 bp of the 16S rRNA gene. After, was performed a semi-nested PCR with an oligonucleotide primer internal to the first amplicon (5'-AGAAGAGCGGAATTCCAC-3'). Among the bacteria tested in the PCR reaction, the two strains of B. sporothermodurans, five other species of Bacillus (B. cereus, B. acidicola, B. lentus, B. circulans, B. coagulans) and the G. stearothermophilus showed amplification products. However, in the reaction of semi-nested PCR, there was only the amplification of the fragment expected (613 bp) from the two strains of B. sporothermodurans. Thus, the protocol of semi-nested PCR displayed specificity for detection of B. sporothermodurans. Apoio financeiro: CNPq.