Patent Publication Number: US-6699679-B2

Title: Methods for rapid identification of bacillus cereus

Description:
FIELD OF THE INVENTION 
     This invention relates to a method for rapid identification of  Bacillus cereus  and the kit thereof. 
     BACKGROUND OF THE INVENTION 
       Bacillis cereus  is gram-positive, spore-forming, motile, aerobic rod which inhibits in soil and has been recognized as an opportunistic food poisoning pathogen.  Bacillis cereus  and the poison thereof usually result in the diarrheal syndrome and the emetic syndrome. Some food types that are preferentially contaminated with  B. cereus  are crude cereals, starchy foods, dairy products, meat, dehydrated foods, and spices. 
     Conventional procedures for the detection of  B. cereus  are based on morphologic observations, physiological and biological tests, such as plate count method and most probable number method. Harmon et al. utilized the hydrolysis of lecithin (egg yolk reaction) as a characteristic for isolation of suspect  B. cereus  on selection media such as mannitol-egg yolk-polymyxin (MYP) agar. However, it should take several days to complete the physiological and biological tests. (Harmon, S. M. et al., 1992, Compendium of methods for the Microbiological examination of foods. 3 rd  ed. American Public Health Association, Washington, D.C., pages 593-604) Schraft et al. developed polymerase chain reaction for detection of lecithin-hydrolyzing gene of  B. cereus . Since most strains of the  B. cereus  group (i.e.,  B. cereus, B. mycoides  and  B. thuringiensis ) possess lecithinase activity, the polymerase chain reaction method detects all species of the  B. cereus  group, but does not distinguish any single species. (Schraft, H. and M. W. Griffiths, 1995, Sepecific oligonucleotide primers for detection of lecithinase-positive Bacillus spp. by PCR. Appl. Environ. Microbiol. 61: 98-102). Thus, a rapid and specific method for identification of  B. cereus  is desired. 
     SUMMARY OF THE INVENTION 
     The objective of this invention relates to provide a method for rapid identification of  Bacillus cereus  in a sample comprising 
     (a) mixing an antibody or antisera specifically against the cell surface antigens of  B. cereus  with the sample, wherein the surface antigen is selected from the group of consisting of: surface antigens of  B. cereus  with molecular masses of 28.5, 26.5 and 20 kDa and the mixture thereof; and 
     (b) detecting the existence of  B. cereus  if the antibody-antigen binding reaction is positive. 
     Another objective of this invention is to provide a kit for rapid identification of  B. cereus , which comprises a solution of antibody or antisera against the cell surface antigens of  B. cereus , and the agents and apparatus required for detection of binding reaction between the said antibody or antisera and the bacteria antigen in a test sample; wherein the antigen is selected by at least one from the group consisting of: surface antigen of  B. cereus  with molecular masses of 28.5, 26.5 and 20 kDa and the mixture thereof. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 demonstrates the SDS-polyacryl amide gel electrophoresis (SDS-PAGE) of  Bacillus cereus  and Bacillus spp. FIG. 1A is the SDS-PAGE of cell surface antigens extracted with 1% sodium dodecyl sulfate. Left lane represents molecular weight markers. Lanes 1 through 6 represent  B. cereus  CCRC 10603, 11026, 15840, 15843, 15846 and 13481, respectively. FIG. 1B is the SDS-PAGE of cell surface antigens extracted with 1% SDS from Bacillus spp. Left lane represents molecular weight markers. Lanes 1 and 2 represent  B. cereus  CCRC 10603 and 11026, respectively. Lane 3 represents  B. mycoides  CCRC 12022. Lane 4 , B. licheniformis  CCRC 11556; Lane 5 , B. subtilis  CCRC 10029; and lane 6 , B. megaterium  CCRC 10608. 
     FIG. 2 demonstrates a dose-response curve between the concentration of total cell surface antigens of  B. cereus  CCRC 10603 and A 450  as determined by ELISA using the antibodies against the 28.5 kDa antigen. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     This invention is the first one by immunological methods for the identification of  B. cereus , which is characterized by the binding of special surface antigen of  B. cereus  and specific antibody to achieve the purpose of rapid identification. According to the invention, the identification method is very simple, fast, highly sensitive and specific. 
     The objective of this invention relates to providing a method for rapid identification of  Bacillus cereus  in a sample comprising 
     (a) mixing an antibody or antisera specifically against the cell surface antigen of  B. cereus  with the sample, wherein the surface antigen is selected from the group of consisting of: surface antigen of  B. cereus  with molecular masses of 28.5, 26.5 and 20 Kda and the mixture thereof; and 
     (b) detecting the existence of  B. cereus  if produce antibody-antigen binding reaction. 
     According to the invention, the term “antibody” is defined as any immunoglobulin against a specific antigen produced from animals. The term “antisera” refers to the animal serum rich in specific antibodies, obtained after immunization. In general, antibodies are produced by immunizing animals (such as rabbits, goats and mice) with an antigen which is emulsified with an adjuvant. The preparation of antibodies or antisera is well known to the persons skilled in the art. The surface protein of  B. cereus  used for production of an antibody from immunized animals is purified through SDS-PAGE. Although the protein is denatured, the antibody produced therefrom is able to recognize the surface protein of  B. cereus . The antibody can react with some antigenic epitopes of the native protein due to the polyclonal property. 
     The surface antigen of  B. cereus  used in the invention includes the surface antigens with molecular masses of 28.5 kDa, 36.5 kDa or 20 kDa or the mixture thereof. The antigen is preferably with molecular masses of 28.5 kDa and 20 kDa. More preferably, the molecular weight of the antigen is 28.5 kDa. The relevant preparation and purification of the surface antigen of the invention can be carried out by any conventional methods or techniques, for example, as described in the examples. 
     According to this invention, the antibodies against surface antigens of  B. cereus  possess high titers. Preferably, the titer is 1×10 5 -1×10 8 . More preferably, it is 1×10 6 -1×10 7 . The 28.5 kDa antigen has a stronger antigenicity than 26.5 kDa antigen and 20 kDa antigen. 
     According to this invention, any suitable method for detection of an antibody-antigen binding reaction can be used for detection of the binding of surface antigen of  B. cereus  and antibody of the invention. A preferred method is enzyme-linked immunosorbent assay (ELISA) or colony blot immunoassay. 
     In one preferred embodiment of the invention, the antigen-antibody reaction is combined with mannitol-egg yolk-polymyxin (MYP) selective agar. 
     Another objective of this invention is to provide a kit for rapid identification of  B. cereus , which comprises an antibody or antisera against the cell surface antigens of  B. cereus , and the agents and apparatus required for detection of binding reaction between the said antibody or antisera and the bacteria antigen in the test sample; wherein the antigen is selected by at least one from the group of consisting of: surface antigen of  B. cereus  with molecular masses of 28.5, 26.5 and 20 kDa and the mixture thereof. The agents and apparatus can be prepared or produced by the persons skilled in the art according to conventional technology and knowledge. 
     This invention provides a sensitive and specific method for rapid identification of  B. cereus  and the kit thereof, which completes the identification of  B. cereus  in short time with minimum labor and expense. 
     EXAMPLES 
     Example 1 
     Identification of  B. cereus    
     Bacteria and Culture Conditions 
     A total of 165 bacterial strains (Table 1) were tested in this study including 38 strains of  B. cereus , 79 strains of other Bacillus spp., and 48 non-Bacillus strains. Most cultures were obtained from the Culture Collection and Research Center (CCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan).  Bacillus anthracis  ATCC 8705 and ATCC 14578 were obtained from the American Type Culture Collection (Rockville, Md.). Cultures of Bacillus spp. were maintained on nutrient agar, whereas non-Bacillus bacteria were maintained on tryptic soy agar. For ELISA, Bacillus spp. were grown at 30° C. on MYP agar or nutrient agar, and other bacteria were grown at 37° C. on tryptic soy agar for 20 to 24 hours before analysis. 
     
       
         
           
               
               
             
               
                   
                 TABLE 1 
               
             
            
               
                   
                   
               
               
                   
                 No. of Strains 
               
            
           
           
               
               
               
            
               
                   
                 ELISA- 
                 ELISA- 
               
            
           
           
               
               
               
               
               
            
               
                 Microorganism 
                 CCRC No. 
                 Total 
                 positive 
                 negative 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 
                   Bacillus cereus 
                 
                 (CCRC no. is not 
                 38 
                 38 
                 0 
               
               
                   
                 specified for each 
               
               
                   
                 individual strain) 
               
               
                 
                   B. alvei 
                 
                 11840, 11842, 11220, 
                 6 
                 0 
                 6 
               
               
                   
                 11728, 11906, 11970 
               
               
                 
                   B. anthracis 
                   a 
                 
                 8705, 14578 
                 2 
                 2 
                 0 
               
               
                 
                   B. apiarius 
                 
                 11830 
                 1 
                 0 
                 1 
               
               
                 
                   B. badius 
                 
                 11699,11909 
                 2 
                 0 
                 2 
               
               
                 
                   B. brevis 
                 
                 10600, 11717, 11047, 
                 5 
                 0 
                 5 
               
               
                   
                 11841, 11912 
               
               
                 
                   B. circulans 
                 
                 13842, 13847, 10605, 
                 4 
                 0 
                 4 
               
               
                   
                 11027 
               
               
                 
                   B. coagulans 
                 
                 10606, 10272, 11592, 
                 6 
                 0 
                 6 
               
               
                   
                 12147, 12210, 11700 
               
               
                 
                   B. firmus 
                 
                 11729, 11730 
                 2 
                 0 
                 2 
               
               
                 
                   B. insolitus 
                 
                 11737 
                 1 
                 0 
                 1 
               
               
                 
                   B. larvae 
                 
                 14187 
                 1 
                 0 
                 1 
               
               
                 
                   B. laterosporus 
                 
                 10607, 11951 
                 2 
                 0 
                 2 
               
               
                 
                   B. lentus 
                 
                 11735, 12021 
                 2 
                 0 
                 2 
               
               
                 
                   B. licheniformis 
                 
                 11556, 12826, 11702 
                 3 
                 0 
                 3 
               
               
                 
                   B. macerans 
                 
                 12025, 13021, 14680 
                 3 
                 0 
                 3 
               
               
                 
                   B. maroccanus 
                 
                 14649 
                 1 
                 0 
                 1 
               
               
                 
                   B. megaterium 
                 
                 10608, 14706, 11962, 
                 4 
                 0 
                 4 
               
               
                   
                 11965 
               
               
                 
                   B. mycoides 
                 
                 10604, 11968, 12022, 
                 4 
                 4 
                 0 
               
               
                   
                 11716 
               
               
                 
                   B. pabuli 
                 
                 15857 
                 1 
                 0 
                 1 
               
               
                 
                   B. pantothenticus 
                 
                 14681 
                 1 
                 0 
                 1 
               
               
                 
                   B. polymyxa 
                 
                 14352, 12011, 12012 
                 3 
                 0 
                 3 
               
               
                 
                   B. popilliae 
                 
                 14650 
                 1 
                 0 
               
               
                 
                   B. pulvifaciens 
                 
                 15859 
                 1 
                 0 
                 1 
               
               
                 
                   B. pumilus 
                 
                 14688, 14700 
                 2 
                 0 
                 2 
               
               
                 
                   B. racemilacticus 
                 
                 12807 
                 1 
                 0 
                 1 
               
               
                 
                   B. sphaericus 
                 
                 14354, 12825, 11066, 
                 6 
                 0 
                 6 
               
               
                   
                 12006, 14702, 14703 
               
               
                 
                   B. stearothermophilus 
                 
                 10610, 11092 
                 2 
                 0 
                 2 
               
               
                 
                   B. subtilis 
                 
                 10029, 12815 
                 2 
                 0 
                 2 
               
               
                 
                   B. thuringiensis 
                 
                 14380, 14381, 14683, 
                 7 
                 7 
                 0 
               
               
                   
                 15853, 15854, 15855, 
               
               
                   
                 15856 
               
               
                 
                   B. thuringiensis 
                 
                 15860 
                 1 
                 1 
                 0 
               
               
                 subsp.  israelensis   
               
               
                 Bacillus spp. 
                 12276, 14642 
                 2 
                 0 
                 2 
               
               
                 
                   Corynebacterium 
                 
                 10367, 12469 
                 2 
                 0 
                 2 
               
               
                 
                   ammoniagenes 
                 
               
               
                 
                   C. glutamicus 
                 
                 10488 
                 1 
                 0 
                 1 
               
               
                 
                   C. glutamicum 
                 
                 11384 
                 1 
                 0 
                 1 
               
               
                 
                   Eschericia coli 
                 
                 10675, 10314, 10316, 
                 5 
                 0 
                 5 
               
               
                   
                 10324, 14824 
               
               
                 
                   Enterobacter 
                 
                 10370, 10401, 11507 
                 3 
                 0 
                 3 
               
               
                 
                   cloacae 
                 
               
               
                 
                   Methylobacterium 
                 
                 11048, 12234 
                 2 
                 0 
                 2 
               
               
                 
                   extorquens 
                 
               
               
                 
                   Microbacterium 
                 
                 11670, 12505 
                 2 
                 0 
                 2 
               
               
                 
                   ammoniaphilum 
                 
               
               
                 
                   Micrococcus luteus 
                 
                 11271, 15275, 15276 
                 3 
                 0 
                 3 
               
               
                 
                   M. pyogenes 
                 
                 11274, 11275 
                 2 
                 0 
                 2 
               
               
                 
                   M. varians 
                 
                 15216, 15217 
                 2 
                 0 
                 2 
               
               
                 
                   Proteus mirabilis 
                 
                 10725, 10726 
                 2 
                 0 
                 2 
               
               
                 
                   Pseudomonas 
                 
                 10944 
                 1 
                 0 
                 1 
               
               
                 
                   aeruginosa 
                 
               
               
                 
                   Rhodococcus equi 
                 
                 11367, 11368 
                 2 
                 0 
                 2 
               
               
                 
                   Salmonella Chester 
                 
                 15468 
                 1 
                 0 
                 1 
               
               
                 
                   Salmonella 
                 
                 15455 
                 1 
                 0 
                 1 
               
               
                 
                   Etterbeek 
                 
               
               
                 
                   Salmonella 
                 
                 15580 
                 1 
                 0 
                 1 
               
               
                 
                   Simsbury 
                 
               
               
                 
                   Salmonella 
                 
                 15581 
                 1 
                 0 
                 1 
               
               
                 
                   Tennessee 
                 
               
               
                 
                   Shigella boydii 
                 
                 10771 
                 1 
                 0 
                 1 
               
               
                 
                   S. sonnei 
                 
                 10773, 10774 
                 2 
                 0 
                 2 
               
               
                 
                   Sphingomonas 
                 
                 13954, 13955 
                 2 
                 0 
                 2 
               
               
                 
                   paucimobilis 
                 
               
               
                 
                   Sporosarcina urea 
                 
                 10766 
                 1 
                 0 
                 1 
               
               
                 
                   Staphylococcus 
                 
                 11551, 14941, 14943, 
                 4 
                 1 
                 3 
               
               
                 
                   aureus 
                 
                 14944 
               
               
                 
                   S. epidermidis 
                 
                 15245, 15246, 15247 
                 3 
                 0 
                 3 
               
               
                 
                   Streptococcus 
                 
                 10787 
                 1 
                 0 
                 1 
               
               
                 
                   agalactiae 
                 
               
               
                 
                   S. mutans 
                 
                 10793 
                 1 
                 0 
                 1 
               
               
                 
                   S. salivarius 
                 
                 12257 
                 1 
                 0 
                 1 
               
               
                   
               
               
                   a The two strains of  B. anthracis  were from ATCC.  
               
            
           
         
       
     
     Preparation of Cell Surface Antigens 
     The purification method was a modification of that described by Bhunia et al. (Bhunia et al., Infect. Immun. 59:3176-3184).  B. cereus  CCRC 10603 was grown on nutrient agar plates at 30° C. for 18 hours. Two milliliters of 70 mM phosphate buffer (pH 6.8) was added to each plate to harvest the bacteria. The cells were washed with the same phosphate buffer and centrifuged at 3,000×g for 15 min. The cell surface antigens were extracted with 2 ml of 70 mM phosphate buffer (pH 6.8) containing 1% sodium dodecyl sulfate (SDS) and 0.5% β-mercaptoethanol. After incubation at 70° C. for 20 min, the cell pellet was removed by centrifugation (10,000×g for 10 min), and the supernatant containing the SDS-extracted cell surface antigens was dialyzed for 18 to 24 hours. Cell-surface antigens of other strains were extracted with SDS in a similar manner. 
     Purification of Cell Surface Antigens 
     The extracted cell surface antigens were analyzed with SDS-polyacrylamide gel electorphoresis (SDS-PAGE). After electropohoresis, gels were stained with Coomassie brilliant blue. FIG. 1 demonstrates SDS-PAGE of strains  B. cereus  and Bacillus spp. as control. As indicated in FIG. 1A, the protein patterns of most strains of  B. cereus  had two major common bands with molecular masses of 26.5 and 20 kDa and a minor band with a molecular mass of 28.5 kDa (FIG. 1A, indicated by arrow heads). FIG. 2B indicates that there were more variations in the band patterns of other Bacillus spp. 
     Preparation of Antibodies 
     The protein bands corresponding to molecular masses of 28.5, 26.5 and 20 kDa were separately cut into small pieces, ground to fine particles with a hand homogenizer, and emulsified with incomplete Freund&#39;s adjuvant (Dicfo Laboratories, Detroit, Mich., USA). Two milliliters of the emulsified antigen were used to immunize New Zealand white rabbits (2.5 Kg in weight). Each rabbit was boosted four times at 3-week intervals. Ten days after the final injection, blood was collected from the rabbits&#39; ears. The antisera were decomplemented by heating at 56° C. for 30 min, and titers of the decomplemented antisera were determined by ELISA. Microtiter plates (Nunc, Denmark) were coated with SDS-extracted total cell surface antigens (10 μg/ml), and after the addition of serial 10-fold diluted antisera, protein A horseradish peroxidase (HRP) conjugate was used as the signal producer. The titers of the antisera against each of the 28.5 kDa, 26.5 kDa and 20 kDa antigens were around 1×10 6  to 1×10 7  as determined by ELISA. Although the abundance of the 28.5 kDa antigen was relatively small (FIG.  1 A), the titers of antibodies against this protein were comparable to those obtained against the 26.5 kDa and 20 kDa proteins. It was possible that the 28.5 kDa antigen had a stronger antigenicity than the other two antigens with molecular masses of 26.5 kDa and 20 kDa. 
     Purification of Antibodies and Preparation of Antibody-enzyme Conjugate 
     The immunoglobulin G (IgG) fraction of the antisera against each of the 28.5, 26.5 and 20 kDa antigens was purified by ammonium sulfate precipitation followed by diethylaminoethyl ion-exchange chromatography (Chen et al., J. Food Prot. 58: 873-878). Specific antibodies were further purified by affinity chromatography, using the SDS-extracted total cell surface antigens of  B. cereus  CCRC 10603 as ligands coupled to cyanogens bromide-activated Sepharose 4B gel (Amersham Pharmacia Biotech, Uppsala, Sweden). The affinity-purified antibodies against each antigen were conjugated with HRP (Boehringer GmbH, Mannheim, Germany) using the sodium peroxidase oxidation protocol (Hudson et al., 1989, Practical Immunology, 3 rd  ed., Blackwell Scientific Publication, London). 
     Identification of  B. cereus  by ELISA 
     The antibodies against each of the 28.5, 26.5 and 20 kDa antigens were used as capture and detection antibodies. Wells of microtiter plates were coated with 0.1 ml of affinity-purified antibodies (10 μg/ml) at 37° C. for 2 hours. The plates were washed with PBS containing 0.05% Tween 20 (PBST), blocked overnight with 1% bovine serum albumin (BSA) at 4° C., and washed again with PBST. One colony grown on MYP agar or tryptic soy agar was suspended in 0.2 ml of PBS containing 0.1% Teepol (primary alkyl [C 9 -C 13 ] sodium sulfates; Sigma, St. Louis, Mo.). The suspension was heated in a boiling water bath for 5 min and centrifuged (5,000×g for 10 min) to remove insoluble cells and debris. The resulting supernatant was diluted 1:1 with 2% BSA, and 0.1 ml of the diluted sample was added to each well of the microtiter plates. The plates were incubated at 37° C. for 1 hour and washed with PBST, and 0.1 ml of HRP-antibody conjugate diluted 1:10,000 in 1% BSA was added to each well. After 1-hour incubation at 37° C. and PBST washing, 0.1 ml of HRP substrate (3,3′,5,5′-tetramethylbenzidine and H 2 O 2 ; Kirkegarrd and Perry Laboratories, Gaithersburg, Md.) was added to each well. The reaction was stopped by the addition of 0.1 ml of 1 M H 3 PO 4  to each well, and absorbance at 450 nm (A 450 ) was recorded with an ELISA reader. A reading of A 450  higher than that of the negative control plus three standard deviations was considered a positive reaction. PBS was used as a negative control. FIG. 2 shows a dose-response curve between the concentration of total cell surface antigens of  B. cereus  CCRC 10603 and the ELISA signal. According to the invention, the sensitivity of the assay for the detection of total cell surface antigens of  B. cereus  is 10 ng/ml. 
     Table 2 shows that the antibodies against the 28.5, 26.5 and 20 kDa antigens are used to test  B. cereus  CCRC 10446, 10927 and 11026. All the antibodies could effectively detect their relevant antigens. The ELISA signals produced by the antibodies against the 28.5 and 20 kDa antigens were stronger than those produced by the antibodies against the 26.5 kDa antigen. 
     The ELISA results of individual species tested with the antibodies against the 28.5 kDa antigen are shown in Table 1. All 38 strains of  B. cereus  produced positive reactions. Most of the 127 strains of non- B. cereus  bacteria produced negative reactions. However, all strains belonging to the  B. cereus  group (i.e.,  B. anthracis, B. mycoides  and  B. thuringiensis ) produced false-positive results. Similar results were obtained by using antibodies against the 20 kDa antigen to test the bacteria. 
     Identification of  B. cereus  by Colony Blot Immunoassay 
     The colonies of  B. cereus  on NA plates were transferred and dotted to a paper membrane prior to being rinsed with PBS. The membrane with dotted colonies was placed in an oven for 10 min for drying and irradiated by UV light (1.2 J/cm×0.1 min). The membrane was incubated with 1:2000 diluted antibody-enzyme conjugate solution and gently agitated for 60 min at room temperature. The membrane was rinsed in PBST for 5 min three times. Finally, the membrane was rinsed with PBS and stained in 4-CN (4-chloro-1-naphthol) color development solution for about 45 min and stopped the reaction by washing the membrane with water. 
     A total of 62 strains of  B. cereus  were tested; 61 strains produced positive reactions and 1 of them produced negative reactions. Thirty-six of the total 38 non-Bacillus spp. (19 species) produced negative reactions. The results described above indicate that the identification method of the invention is accurate and rapid for  B. cereus  identification. 
     
       
         
           
               
               
             
               
                   
                 TABLE 2 
               
             
            
               
                   
                   
               
               
                   
                 ELISA signal a  (A450) using antibodies against antigen 
               
            
           
           
               
               
               
               
            
               
                 Microorganism 
                 28.5 KDa antigen 
                 26.5 KDa antigen 
                 20 KDa antigen 
               
               
                   
               
               
                   B. cereus  CCRC 
                 1.64 ± 0.03 
                 1.14 ± 0.03 
                 1.45 ± 0.02 
               
               
                 10446 
               
               
                   B. cereus  CCRC 
                 0.67 ± 0.04 
                 0.33 ± 0.01 
                 0.43 ± 0.03 
               
               
                 10927 
               
               
                   B. cereus  CCRC 
                 1.48 ± 0.07 
                 1.17 ± 0.01 
                 1.34 ± 0.00 
               
               
                 11026 
               
               
                 Negative control 
                  0.17 ± 0.001 
                 0.11 ± 0.01 
                 0.15 ± 0.01 
               
               
                   
               
               
                   a Mean ± SD of duplicate  
               
            
           
         
       
     
     Example 2 
     Detection of  B. cereus  in Foods 
     A total of 15 food samples, encompassing rice, beans, pudding, instant infant formula, pepper spice and ice cream were purchased from local supermarkets. The aerobic plate count of these samples was determined (Maturin et al., 1995, In Bacteriological Analytical Manual. 8th ed. Association of Official Analytical Chemists International, Arlington, Vir.). Serial 10-fold dilutions of the homogenized food samples were analyzed for  B. cereus  by inoculating three-tube MPN series in trypticase soy-polymyxin (TSP) broth. After selective enrichment, tubes that showed dense growth were subcultured onto separate MYP agar plates. One or more suspect colonies on each MYP agar plate were analyzed by ELISA for  B. cereus  identification; these colonies were also subcultured on nutrient agar slants followed by species identification with the conventional procedures. 
     Among the 15 food samples analyzed,  B. cereus  was detected in 11 samples by ELISA (see Table 3). However, only 10 samples were found to contain  B. cereus  as determined by the conventional method. The false-positive sample obtained by ELISA was mung bean, in which  B. thruingiensis  was isolated. Some strains of  B. thuringiensis  possess an interotoxin-like gene similar to that of  B. cereus  (Asano et al., 1997, Appl. Environ. Microbiol. 63:1054-1075). In addition, Carlson et al. strongly proposed that  B. cereus  and  B. thuringiensis  should be regarded as a single species (Carlson et al., Appl. Environ. Microbiol. 60: 1719-1725). The test results mentioned above exhibit relative accuracy of the method according to this invention for identification of  B. cereus , which is useful for rapid identification of  B. cereus . 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 3 
               
             
            
               
                   
                   
               
               
                   
                 Aerobic 
                 
                   B. cereus (MPN/g) 
                 
               
               
                   
                 plate count 
                  determined by: 
               
            
           
           
               
               
               
               
            
               
                 Food 
                 (CFU/g) 
                 Conventional method 
                 ELISA 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Millet 
                 1.2 × 10 3   
                 3.6 
                 3.6 
               
               
                 Oat 
                 4.8 × 10 2   
                 NDb 
                 ND 
               
               
                 Rice 
                 4.3 × 10 3   
                 9.1 
                 9.1 
               
               
                 Waxy rice 
                 2.1 × 10 3   
                 3.6 
                 3.6 
               
               
                 Black soybean 
                 4.5 × 10 3   
                 9.1 
                 9.1 
               
               
                 Butter bean 
                 1.5 × 10 3   
                 93 
                 93   
               
               
                 Mung bean 
                 2.9 × 10 3   
                 ND 
                  3.6c 
               
               
                 Kidney bean 
                 1.2 × 10 3   
                 3.6 
                  9.1d 
               
               
                 Adzuki bean 
                 8.8 × 10 3   
                 15 
                 15   
               
               
                 Soybean 
                 2.1 × 10 3   
                 23 
                 93c   
               
               
                 Spice 
                 4.9 × 10 5   
                 3 
                 3   
               
               
                 Ice cream 
                   1 × 10 2   
                 23 
                 23   
               
               
                 Instant infant 
                 ND 
                 ND 
                 ND 
               
               
                 formula 
               
               
                 Oak flake 
                 2.0 × 10 2   
                 ND 
                 ND 
               
               
                 Pudding 
                 ND 
                 ND 
                 ND 
               
               
                   
               
            
           
         
       
     
     The examples described above are offered by way of illustration of the method for rapid identification of  B. cereus  and the kit thereof claimed in this subject application and not by way of limitation.