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Detection of Bacillus cereus containing voitoxin in food by loop-mediated isothermal amplification method

Liu, Zhiguo, Fang, Guoliang, Li, Qi, Zhou, Guoping, Li, Rui, Fu, Yunjie, Li, He, Qu, Shen
Journal of food safety 2011 v.31 no.3 pp. 313-319
Bacillus cereus, bacteria, detection limit, foods, milk, nucleic acids, rapid methods, temperature
Bacillus cereus strains containing voitoxin cereulide are pathogenic bacteria. The cereulide is produced by B. cereus strains containing plasmid pCER270. To establish a specific loop-mediated isothermal amplification (LAMP) method detecting pathogenic B. cereus, LAMP primer sets were designed based on 16SrDNA and pCER270 plasmid cesA sequence. The LAMP reaction was carried out under optimal conditions with 2 mM of Mg²⁺ at 65C for 1 h. Specificity of LAMP primers were validated by assaying 19 B. cereus strains with and without pCER270 plasmid and 42 non-B. cereus species. Detection limits of the LAMP assay were 1 cfu/mL of B. cereus without pCER270 plasmid and 11 cfu/mL of pathogenic B. cereus with pCER270 plasmid. Furthermore, liquid milk samples were detected by the LAMP method. The results showed that LAMP method is a reliable method used for rapid detection of B. cereus and its pathogenic strains of milk and other food stuff. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method. In this study LAMP method was specific and sensitive for detection of B. cereus which grown in liquid medium or on solid medium, or from liquid milk samples as well. In addition, the pathogenic strains expressed voitoxin cereulide could be readily identified after amplification using specific LAMP primer sets. The whole procedure is simple and rapid, the reaction carried out in tens of minutes at a constant temperature. LAMP could be routinely used for B. cereus especially pathogenic strains in food.