Method for the detection of Bacillus cereus

Abstract

The present invention provides novel oligonucleotide primers for phosphotidyl inositol in B. cereus the primer comprising PI-1 (F) 5' AGTATGGGGAATGAG 3' and PI-2 (R) 5' ACAATTTTCCCACGA 3' and to a method for the detection of B. cereus in foods in a mixed microflora.

1 . A method for the detection of B. cereus in foods said method comprising using primers specific for phosphotidyl inositol gene in B. cereus in a mixed microflora, said primers comprising  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   PI-1 (F) 
                   5′ AGTATGGGGAATGAG 3′ 
                     
                 
                     
                     
                 
                     
                   PI-2 (R) 
                   5′ ACAATTTTCCCACGA 3′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         2 . A method as claimed in  claim 1 , wherein the food matrices for detecting B. cereus are milk and cooked rice.  
     
     
         3 . A method as claimed in  claim 1 , wherein template DNA from B. cereus in cooked rice is extracted using Triton X-100, 0.5-2%, boiling at 96-100.degree. C. for 3-8 min and treatment with phenol; chloroform in the ratio of 22:21-28:27.  
     
     
         4 . A method as claimed in  claim 1 , wherein the template DNA from B. cereus in milk is extracted using diethyl ether: chlorofomi in the ratio of 1:1-1:3, urea 1.5-3.5 M and sodium dodecyl sulphate in a range of 0.5-2%.  
     
     
         5 . A method as claimed in  claim 1 , wherein the PCR reaction mixture in a total volume of 25 .mu.l comprises of Tris HCl 8-12 mM; KCl: 45-55 mM; MgCl.sub.2:0.5-3.0 mM; gelatin: 0.005-0.02%; individual deoxynucleoside triphosphates: 150-300 .mu.M; each specific primer: 30-60 picomoles; Taq DNA polymerase: 0.5-2.0 units and template DNA: 1-3 .mu.1.  
     
     
         6 . A method as claimed in  claim 1 , wherein detection of B. cereus is effected by amplification profile of target gene from an initial denaturation at 90-98.degree. C. for 2-8 min, amplification cycles of 28-40, each cycle with a denaturation at 90-98.degree. C. for 40-70 seconds, annealing at 46-54.degree. C. for 40-80 seconds and extension at 68-76.degree. C. for 45-75 seconds and final extension at 68-76.degree. C. for 4-12 min.  
     
     
         7 . A method as claimed in  claim 1 , wherein analysis of the PCR product is done in 1.2 -1.8% agarose gel electrophoresis, visualization of the PCR product by staining with 0.5 (g/ml ethidium bromide and observation in a UV transilluminator.  
     
     
         8 . A method as claimed in  claim 1 , wherein detection of minimum number of cells of B. cereus is done in a food matrix by PCR.