Abstract:
B. anthracis  detection primers can be used to amplify the conserved regions of bclB alleles encoding the collagen-like proteins found in  B. anthracis  , as opposed to other  Bacillus  species, during PCR amplification of extracted  Bacillus  DNA or  Bacillus  spores. Additionally,  B. anthracis  strain fingerprinting primers amplify bclA-E polymorphic regions of collagen-like proteins found in  B. anthracis  strains. The  B. anthracis  strains differ in basepair size of these polymorphic coding regions of bclA-E so that strains can be discriminated based upon distinct PCR-band patterns that migrate differently in size while resolved on an agarose gel. 
     DRAWINGS

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority from U.S. provisional application No. 61/456,940 filed on Nov. 15, 2010 
     
    
     REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTING COMPACT DISC APPENDIX 
       [0002]    The application is filed with a computer readable form of a sequence listing. The listing is the same as described in the specification (Table 3). 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         [0003]      FIG. 1  is a schematic depiction of the location and orientation of the bclABCDE genes in  B. anthracis  strain Sterne. 
           [0004]      FIG. 2  is a gel electrophoresis of the products of PCR amplification using DNA templates from  B. anthracis  strains Sterne and Ames. 
           [0005]      FIG. 3  is a gel electrophoresis of the PCR amplification of the bcl genes from intact  Bacillus  spores. 
           [0006]      FIG. 4  is a gel electrophoresis of the PCR amplification of genomic DNAs from two  B. anthracis  strains, Sterne and Ames, using primers flanking the bclA-E collagen-like regions Seq. ID Nos. 3-12 loaded into individual wells. 
           [0007]      FIG. 5  is a gel electrophoresis of the PCR amplification of genomic DNAs from two B. anthracis strains, Sterne and Ames, using primers flanking the bclA-E collagen-like regions Seq. ID Nos. 3-12 loaded into single wells. 
           [0008]      FIG. 6  is a linear model that relates the theoretical fragment length to observed amplified fragment size of  B. anthracis  strains Sterne and Ames. 
           [0009]      FIG. 7  is a model of theoretical fragment length of strains Sterne and Ames where the normalized error was independent of the theoretical amplified fragment size. 
           [0010]      FIG. 8  is an annotated dendrogram depicting the ability to distinguish among the strains using the bclA-E genes. 
           [0011]      FIG. 9  is a gel electrophoresis of the PCR amplification of bclA-E genes of  B. anthracis, B. cereus , and one  B. thuringiensis  and  B. mycoides  strains 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0012]    The  Bacillus  cereus group organisms form a highly homogeneous subdivision of the genus  Bacillus  and include three main species of  B. anthracis , B. cereus , and  B. thuringiensis , as well as a closely related  B. mycoides . Sequence polymorphisms of the collagen-like protein bcl genes allow for specific detection of  B. anthracis  and the use of five bcl genes present within the  B. anthracis  genome can allow for fingerprinting and detection of specific  B. anthracis  strains. The bclABCDE sequences have been studied and a unique polymorphism in the bclB alleles of the  B. cereus  group organisms has allowed for designing of bclB-targeting primers that allow for the specific detection of  B. anthracis  strains by PCR, using both genomic DNA and purified  Bacillus  spores. Further, by exploiting the length variation of the collagen-like (CL) regions of bcl alleles, the combined bclABCDE PCR products generate markedly different fingerprints for the  B. anthracis  strains, which can be utilized for  B. anthracis  fingerprinting. 
         [0013]      Bacillus anthracis  contains five collagen-like bcl genes that are shown in  FIG. 1  and Table 1. The location and orientation of the bclABCDE genes in  B. anthracis  strain Sterne chromosome is depicted schematically in  FIG. 1 . Exact nucleotide positions, locus and gene designations, and gene lengths are listed in Table 1. Table 2 presents the overall BclA-E protein architectures, as well as main characteristics, including the CL-region length variation in BclABCDE variants found among  B. anthracis  strains (Sterne, A1055, Vollum, USA6153, CNEVA-9066, Kruger, Ames, and Australia 94) that allows for strain fingerprinting at the gene level. Table 3 identifies the primers used for  B. anthracis  detection and fingerprinting. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Gene 
                 Location 
                 Locus 
                 Clan 
                 Length (bp) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 bclA 
                 1,182,412-1,183,614 
                 BAS1130 
                 7 
                 1202 
               
               
                 bclB 
                 2,280,518-2,281,564 
                 BAS2281 
                 2a 
                 1046 
               
               
                 bclC 
                 3,515,305-3,516,750 
                 BAS3557 
                 4b 
                 1445 
               
               
                 bclD 
                 4,332,601-4,333,548 
                 BAS4423 
                 6a 
                 947 
               
               
                 bclE 
                 4,518,446-4,520,389 
                 BAS4623 
                 6a 
                 1943 
               
               
                   
               
             
          
         
       
     
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                 TABLE 2 
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
             
          
         
       
     
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                 TABLE 3 
               
               
                   
               
               
                 PCR target 
                 Primer name 
                 Sequence ID 
                 Sequence 
               
               
                   
               
             
             
               
                 bclB of B. anthracis 5′ 
                 bclB F2 
                 Seq. ID No 1 
                 AGGCCCAGAAAATATTGGAC 
               
               
                   
               
               
                 bclB of B. anthracis 3′ 
                 bclB R4 
                 Seq. ID No 2 
                 GAGTTCCTCCCACACCTGG 
               
               
                   
               
               
                 bclA gene 5′ 
                 bclA F1 
                 Seq. ID No. 3 
                 GAATCTTTATCAGCTAGTGCATTTG 
               
               
                   
               
               
                 bclA gene 3′ 
                 bclA R1 
                 Seq. ID No. 4 
                 AAGCAACTTTTTCAATAATAATGGATG 
               
               
                   
               
               
                 bclB gene 5′ 
                 bclB F1 
                 Seq. ID No. 5 
                 GGCCCAGAAAATATTGGACCTAC 
               
               
                   
               
               
                 bclB gene 3′ 
                 bclB R1 
                 Seq. ID No. 6 
                 ATTAGACGATATTAAGACCTGCGC 
               
               
                   
               
               
                 bclC gene 5′ 
                 bclC F1 
                 Seq. ID No. 7 
                 CCATGCTTTCCAAGTAGCGCTGG 
               
               
                   
               
               
                 bclC gene 3′ 
                 bclC R1 
                 Seq. ID No. 8 
                 ATTAAGCGATTCTAAATACAGTTAG 
               
               
                   
               
               
                 bclD gene 5′ 
                 bclD F1 
                 Seq. ID No. 9 
                 TGTAATAATCAAAATGGTGTACATC 
               
               
                   
               
               
                 bclD gene 3′ 
                 bclD R1 
                 Seq. ID No. 10 
                 ATCAACTTAACCTTATTATCGTTAAC 
               
               
                   
               
               
                 bclE gene 5′ 
                 bclE F1 
                 Seq. ID No. 11 
                 AGTCCATTAAATTCTAATTTCAAGAT 
               
               
                   
               
               
                 bclE gene 3′ 
                 bclE R1 
                 Seq. ID No. 12 
                 ATTAACTCAATCTAATAATCGTTAAAG 
               
               
                   
               
             
          
         
       
     
         [0014]    Each of the bcl genes potentially encodes a protein with an N region composed of 25 to 41 amino acids. The lengths of the collagen-like (CL) regions of each Bcl protein vary among  B. anthracis  strains. BclA-E proteins also contain the C-terminal domains (CTD) composed of 132-162 amino acids. In addition, only the BclC proteins contain a linker (L) region between the N and CL regions (Table 2). The genomes of  B. anthracis  strains contain five distinct bcl open reading frames encoding collagen-like proteins. While homologous bclA-E genes are also found in the genomes of other  B. cereus  group organisms, the genomes of  B. anthracis  strains contain a unique and differing bclB allele, which allows for specific detection of the  B. anthracis . The 5′- and 3′-bclB primers identified as Seq. ID Nos. 1 and 2 (Table 3) can be used to specifically detect the DNA of  B. anthracis.    
         [0015]    Example of PCR Detection 
         [0016]    PCR amplification using DNA templates from  B. anthracis  (Ba) strains Sterne and Ames yielded single products of expected sizes as shown in  FIG. 2 . Conversely, none of the PCRs that used as templates DNA from closely-related species of three  B. cereus  (Bc), two  B. thuringiensis  (Bt), and one  B. mycoides  (Bmy) strains resulted in bclB-product amplification. PCR was also negative for DNA templates from the control distant strains of  B. subtilis  (Bs, n=3) and  B. megaterium  (Bme, n=1) that do not harbor the bclB gene. To assess the feasibility of a bclB-based method of detecting  B. anthracis  directly in the field, PCR amplification of the bcl genes from intact Bacillus spores was tested with the results shown in  FIG. 3 . Purified  B. anthracis  Sterne spores, as well as control spores from  B. cereus, B. thuringiensis , and  B. mycoides , were obtained and adjusted for concentration. Equal amounts of spores from each  Bacillus  species were added to PCR mixtures and amplification was carried out with primers that were either specific for bclB allele of  B. anthracis  (Seq. ID No. 1 and 2) or with control primers specific for bclB allele of other  Bacillus cereus  group organisms.  B. anthracis  spores yielded DNA products with  B. anthracis  -specific primers, while neither  B. cereus , nor  B. thuringiensis  and  B. mycoides  spores yielded PCR products as shown in the upper panel of  FIG. 3 . Importantly, all of the latter spores amplified DNAs with the control specific primers, whereas  B. anthracis  spores did not. These data demonstrate that amplification of the bclB gene can specifically differentiate  B. anthracis  spores by direct PCR from spores of other  B. cereus  group members. 
         [0017]    Example 2 the fingerprinting of  B. anthracis  strains based on bclA-E-length polymorphism. The main sequence-length polymorphism was observed within the collagen-like regions of BclA-E proteins present in various  B. anthracis  strains. A simultaneous analysis of the collagen- like region lengths of all five bclA-E genes can be utilized to fingerprint the different  B. anthracis  strains. PCRs were performed with genomic DNAs from two  B. anthracis  strains, Sterne and Ames, using primers flanking the bclA-E collagen-like regions which are Seq. ID Nos. 3-12. The products were individually separated in 2% agarose gels and yielded single DNA bands of the predicted sizes with the results shown in  FIG. 4 . The combined bclA-E-gene products, obtained from each strain, were loaded into single wells and band patterns were resolved by agarose gel electrophoresis as shown in  FIG. 5  (left panel). The results in  FIGS. 4 and 5  show that significantly different fingerprints are generated from the  B. anthracis  strain Sterne and strain Ames. In both strains, bclB-, bclD-, and bclE-amplified fragments migrated as distinct bands that significantly differed in size in each strain. The bclA- and bclC-amplification products were of ˜0.8-kb in both strains (782 by and 752 by in Sterne; 728 by and 743 by in Ames) and therefore, were not well resolved by this method. Our data demonstrate that significant length variation in the collagen-like regions of the bclA-E genes that are all present in the genomes of all available  B. anthracis  strains can be a powerful tool in strain fingerprinting. Finally, multiplex PCR with all five primer pairs was attempted with DNA of the Sterne strain as the template by using a temperature gradient from 50 to 65° C. for primer annealing and an Mg +2  concentration range of 1.5 to 6.5 mM in the PCR buffer. The bcl ABCDE genes were all amplified with an annealing temperature of 50° C. and an Mg +2  concentration of 1.8 mM, although the intensities of the bclA and bclE bands were relatively low ( FIG. 5 , right panel). Together, these data demonstrate that significant length variation in the CL regions of the bcl ABCDE genes that are present in the genomes of all available B. anthracis strains can be a valuable tool in strain fingerprinting. 
         [0018]    Mathematical modeling of bclA-E-based fingerprinting of  B. anthracis  strains. A computational approach was used to establish the feasibility of discriminating among  B. anthracis  strains using multiplex measurement of the sequence polymorphisms within the bclA-E genes using PCR. The first step was to develop and to calibrate a quantitative relationship between experimentally measured amplified bclA-E gene products and theoretical amplified fragment length predicted from the nucleotide sequence. The profile of amplified bclA-E gene products from the Ames and Sterne strains of  B. anthracis  were measured in duplicate by PCR as shown in  FIGS. 4 and 5 . The combined results were used to calibrate a linear model that relates the theoretical fragment length to observed amplified fragment size as shown in  FIG. 6-8 . The slope and intercept of the linear model were determined to be 1.059 and −18.56. In  FIG. 7 , the normalized error (i.e., residuals) was independent of the theoretical amplified fragment size. The distribution in the residuals, marginalized across the theoretical amplified fragment size, exhibited a normal distribution with a standard distribution(s) of 0.0374 (compare the histogram against the solid line shown on the right vertical axis of  FIG. 7 . The calibrated model was used to predict the fragment sizes amplified by PCR for each of the bclA-E genes observed in the genomes of six additional  B. anthracis  strains. The uncertainty associated with strain fingerprinting using multiplex measurement of the amplified fragments derived from the bclA-E genes was estimated using Bootstrap resampling. Bootstrap resampling was used to create a population of synthetic replicates. Each synthetic replicate was generated using the following equation: 
         [0000]      Y syn (S i )=θ 1 ·Ŷ T  (S i )+θ 2 +Ŷ T  (S i )·N(0, 0.0374),
 
         [0000]    where N(0, 0.0374) represents a random number generated from a normal distribution with a mean of zero and standard deviation of 0.0374, Si represents the theoretical amplified fragment size for each gene, and θ i  represents the model parameters. The ability to distinguish among  B. anthracis  strains Sterne, A1055, Vollum, USA6153, CNEVA-9066, Kruger, Ames, and Australia 94 using the bclA-E genes was tested using this mathematical model and the levels of confidence associated with distinguishing among these strains is shown as an annotated dendrogram in  FIG. 8 . Hence, we predict that under the experimental conditions used here, we would be able to differentiate with confidence between two strains of  B. anthracis , with the exception of the Sterne and Australia 94 strains, using a multi-locus typing approach based upon bclA-E length polymorphism. 
         [0019]    bcl gene-based fingerprinting of the  B. cereus  group organisms. Determination of the origin of certain spores may also be important for non- anthracis Bacillus  sp in events of a hoax, blunder by the perpetrator, or psychological terrorism. Primer pairs (Seq. IDs Nos. 3-12) that were optimized for the bclA-E genes of  B. anthracis  were used to generate fingerprints using DNA templates from three  B. cereus , and one  B. thuringiensis  and  B. mycoides  strains as shown in  FIG. 9  and Table 4. 
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                 TABLE 4 
               
             
             
               
                   
               
               
                 Size of amplified DNA fragments (kb) 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 Total 
               
               
                   
                 bclA 
                 bclB 
                 bclC 
                 bclD 
                 bclE 
                 products 
               
               
                   
                   
               
             
          
           
               
                 Bc ATTC14579 
                 0.48 
                 0.65 
                 ND 
                 0.45 
                 ND 
                 3 
               
               
                 Bc ATTC4342 
                 0.48 
                 0.70 
                 ND 
                 0.78 
                 ND 
                 3 
               
               
                 Bc ATTC13061 
                 0.48 
                 ND 
                 1.25 
                 0.78 
                 3.50 
                 4 
               
               
                 Bt ATTC33879 
                 0.48 
                 0.70 
                 1.50 
                 ND 
                 ND 
                 3 
               
               
                 Bm ATTC6462 
                 ND 
                 0.65 
                 ND 
                 0.50 
                 2.50 
                 3 
               
               
                   
               
               
                 ND—Not detected 
               
             
          
         
       
     
         [0020]    Not all primer pairs yielded bclA-E-gene products for all DNA templates, however despite partial amplification of 3-4 bands the combined PCR samples generated unique fingerprint patterns for all strains analyzed. This test demonstrates that bcl-based fingerprinting could also be employed in forensic applications for strain differentiation of all members of the  Bacillus cereus  group organisms. 
         [0021]    These terms and specifications, including the examples, serve to describe the invention by example and not to limit the invention. It is expected that others will perceive differences, which, while differing from the forgoing, do not depart from the scope of the invention herein described and claimed. In particular, any of the function elements described herein may be replaced by any other known element having an equivalent function. The examples are illustrative only and show methods to produce the compounds, but are not meant to limit the production to those methods only as one skilled in the art could change the examples to produce the compounds without undue experimentation.