Molecular differences between species of the M. tuberculosis complex

Abstract

Specific genetic deletion are identified in mycobacteria isolates, including variations in the M. tuberculosis genome sequence between isolates, and numerous deletion present in BCG as compared to M. tb. These deletions are used as markers to distinguish between pathogenic and avirulent strains, and as a marker for particular M. tb isolates. Deletions specific to vaccine strains of BCG are useful in determining whether a positive tuberculin skin test is indicative of actual tuberculosis infection. The deleted sequences may be re-introduced into BCG to improve the efficacy of vaccination. Alternatively, the genetic sequence that corresponds to the deletion(s) are deleted from M. bovis or M. tuberculosis to attenuate the pathogenic bacteria.

What is claimed is:  
     
         1 . A nucleic acid comprising a junction of a deletion marker in Table 1.  
     
     
         2 . The nucleic acid of  claim 1 , wherein said nucleic acid hybridizes to a  M. tuberculosis  complex genome when the deletion is present, but not in an undeleted genome.  
     
     
         3 . The nucleic acid of  claim 1 , wherein said nucleic acid is from 15 to 25 nucleotides in length.  
     
     
         4 . The nucleic acid of  claim 1 , wherein said  M. tuberculosis  complex genome is BCG.  
     
     
         5 . The nucleic acid of  claim 1 , wherein said  M. tuberculosis  complex genome is a variant of  M. tuberculosis.    
     
     
         6 . The nucleic acid of  claim 1 , wherein said  M. tuberculosis  complex genome is  M. bovis.    
     
     
         7 . A pair of hybridization primers comprising the nucleic acid of  claim 1 , and a second nucleic acid that hybridizes to a second site in an  M. tuberculosis  complex genome.  
     
     
         8 . A genetically altered mycobacterium, comprising an exogenous nucleic acid sequence comprising one or more deletion markers as set forth in Table 1.  
     
     
         9 . The genetically altered mycobacterium of  claim 8 , wherein said mycobacterium is BCG, and wherein said deletion marker is deleted in BCG according to Table 1.  
     
     
         10 . The mycobacterium of  claim 8 , further comprising a physiologically acceptable carrier for injection.  
     
     
         11 . A genetically altered mycobacterium, comprising a deletion resulting from homologous recombination in a deletion marker as set forth in Table 1.  
     
     
         12 . The genetically altered mycobacterium according to  claim 11 , wherein said mycobacterium is  M. bovis.    
     
     
         13 . The mycobacterium of  claim 12 , further comprising a physiologically acceptable carrier for injection.  
     
     
         14 . The genetically altered mycobacterium according to  claim 11 , wherein said mycobacterium is  M. tuberculosis.    
     
     
         15 . The mycobacterium of  claim 14 , further comprising a physiologically acceptable carrier for injection.  
     
     
         16 . A method of distinguishing whether a patient has been exposed to BCG or to  M. tuberculosis,  the method comprising: 
 contacting said patient or a sample derived therefrom with a polypeptide encoded by a deletion marker of Table 1, wherein said deletion marker is present in  M. tuberculosis  and absent in BCG; and    determining the presence of an immune reaction to said polypeptide, wherein a positive response is indicative of exposure to  M. tuberculosis.      
     
     
         17 . The method of  claim 16 , wherein said contacting step comprises sub-cutaneous injection of said polypeptide.  
     
     
         18 . The method of  claim 16 , wherein said contacting step is performed in vitro and said sample comprises a blood sample or derivative thereof.  
     
     
         19 . A method of distinguishing a bacterial strain of the  M. tuberculosis  complex, the method comprising: 
 determining the presence of a deletion marker in Table 1, wherein said deletion is absent in at least one of said candidate strains;    wherein the presence of said deletion marker is indicative that said strain is not said candidate strain.    
     
     
         20 . The method according to  claim 19 , wherein said determining step comprises nucleic acid hybridization to said deletion marker.  
     
     
         21 . The method according to  claim 19 , wherein said determining step comprises antibody binding to a polypeptide encoded by said deletion marker.  
     
     
         22 . The method according to  claim 19 , wherein said determining step comprises PCR amplification across said deletion.  
     
     
         23 . The method according to  claim 19 , wherein said determining step comprises hybridization to a junction sequence associated with said deletion marker.