Novel oligonucleotide primers for phosphotidyl inositol in Bacillus cereus and a 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′ PI - 2 (R) 5′ ACAATTTTCCCACGA 3′ and to a method for the detection of B. cereus in foods in a mixed microflora.

1 . A novel oligonucleotide primer for phosphotidyl inositol in  B. cereus  said primer comprising  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   PI - 1 (F) 5′ AGTATGGGGAATGAG 3′ 
                     
                 
                     
                     
                 
                     
                   PI - 2 (R) 5′ ACAATTTTCCCACGA 3′. 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         2 . 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′. 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         3 . A method as claimed in  claim 2 , wherein the food matrices for detecting  B. cereus  are milk and cooked rice.  
     
     
         4 . A method as claimed in  claim 2 , wherein template DNA from  B. cereus  in cooked rice is extracted using Triton X-100, 0.5-2%, boiling at 96-100° C. for 3-8 min and treatment with phenol; chloroform in the ratio of 22:21-28:27.  
     
     
         5 . A method as claimed in  claim 2 , 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%.  
     
     
         6 . A method as claimed in  claim 2 , wherein the PCR reaction mixture in a total volume of 25 μl comprises of Tris HCl 8-12 mM; KCI: 45-55 mM; MgCl 2 : 0.5-3.0 mM; gelatin: 0.005-0.02%; individual deoxynucleoside triphosphates: 150-300 μM; each specific primer: 30-60 picomoles; Taq DNA polymerase: 0.5-2.0 units and template DNA: 1-3 μl.  
     
     
         7 . A method as claimed in  claim 2 , wherein detection of  B. cereus  is effected by amplification profile of target gene from an initial denaturation at 90-98° C. for 2-8 min, amplification cycles of 28-40, each cycle with a denaturation at 90-98° C. for 40-70 seconds, annealing at 46-54° C. for 40-80 seconds and extension at 68-76° C. for 45-75 seconds and final extension at 68-76° C. for 4-12 min.  
     
     
         8 . A method as claimed in  claim 2 , 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.  
     
     
         9 . A method as claimed in  claim 2 , wherein detection of minimum number of cells of  B. cereus  is done in a food matrix by PCR.