Detection of Bacillus cereus group bacteria from cardboard and paper with real-time PCR

The aim of this study was to develop a PCR-based rapid method to detect Bacillus cereus group cells from paper and cardboard. Primers targeting the 16S rDNA and real-time PCR with SYBR green I detection were used in order to be able to also quantify the target. Both autoclaved cardboard samples spiked with B. cereus vegetative cells or spores and naturally contaminated paper and cardboard samples were studied. Results were compared with culturing verified by commercial (API) tests. Several different methods were tested for DNA isolation from the paper and cardboard samples. Two commercial kits intended for soils, the UltraClean soil DNA kit and the FastDNA spin kit for soil, gave the most reproducible results. In spiked samples, the average yield was 50% of added vegetative cells, but spore yield was only about 10%. PCR results from adding vegetative cells correlated with added colony-forming unit (cfu) values ( r=0.93, P <0.001) in the range 100-10,000 cfu g(-1). Three out of nine studied paper and cardboard samples contained B. cereus group bacteria, based both on culturing and real-time PCR. The numbers were 10(2)-10(3) bacteria g(-1); and PCR gave somewhat higher results than culturing. Thus, real-time PCR can be used as a rapid semi-quantitative method to screen paper and cardboard samples for contamination with B. cereus group bacteria.