Molecular discrimination of <i>Bacillus cereus</i> group species in foods (lettuce, spinach, and kimbap) using quantitative real-time PCR targeting <i>groEL</i> and <i>gyrB</i>

다국어 초록 (Multilingual Abstract)

<P><B>Abstract</B></P> <P>The aim of the study was to identify and evaluate specific biomarkers to differentiate within <I>Bacillus cereus</I> group species from contaminated food samples with the use of real-time PCR. A total of 120 strains, comprising of 28 reference, 2 type, 78 wild strains of <I>B. cereus</I> and <I>B. thuringiensis</I> along with 12 strains representing 2 bacterial groups – <I>B. mycoides, B. pseudomycoides, B. weihenstephanensis</I> (<I>B. cereus</I> group); <I>B. amyloliquefaciens, B. subtilis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Micrococcus luteus, Salmonella enterica, Staphylococcus aureus, Streptococcus pyogenes</I> (non-<I>Bacillus</I> sp.) were identified by applying valid biomarkers (<I>groEL</I> and <I>gyrB</I>). In addition, the presence of <I>B. cereus</I> group was determined in three different artificially contaminated vegetable samples (lettuce, spinach, and kimbap), using prominent biomarkers targeting on chaperonin protein (<I>GroEL</I>) and topoisomerase enzyme protein (<I>gyrB</I>). Direct analysis of samples revealed the specificity towards identification and characterization of the <I>B. cereus</I> group among wild, reference and type strains and the type strain inoculated in vegetables. Our results demonstrated two existing biomarkers <I>groEL</I> and <I>gyrB</I> with a high specificity of 98% and 96% respectively to analyze the total <I>B. cereus</I> group. Further, we also reported the detection limit of <I>groEL</I> and <I>gyrB</I> in food samples was 3.5 and 3.7 log CFU/g respectively. Thus, the developed real-time PCR approach can be a reliable and effective tool for the identification of <I>B. cereus</I> group strains present in environment and food samples. This does not require band isolation, re-amplification, sequencing or sequence identification, thus reducing the time and cost of analysis.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Establishment of differentiating test method between species <I>B. cereus</I> group can be used as basic data. </LI> <LI> 120 types of <I>B. cereus</I> group strain were quantified using groEL and gyrB, qPCR specific primers for gene detectability. </LI> <LI> Applied as basic data for identification of contaminated sources and the degree of bacterial contamination in food samples. </LI> <LI> Further it is useful for distinguish <I>B. cereas</I> group based on the food safety management policy. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>