Method for isolating a polynucleotide of interest from the genome of a mycobacterium using a BAC-based DNA library: application to the detection of mycobacteria

Abstract

A method for isolating a polynucleotide of interest that is present in the genome of and/or expressed by a first mycobacterium strain that is also absent or altered in the genome of and/or not expressed by a second mycobacterium strain. The method includes contacting the genomic DNA of the first strain under hybridizing conditions with the DNA of at least one clone belonging to a bacterial artificial chromosome (BAC) genomic DNA library of the second strain and isolating the polynucleotide of interest that does not hybridize with the DNA of the second strain. This invention further pertains to a Mycobacterium tuberculosis strain H37Rv genomic DNA library, a Mycobacterium bovis BCG strain Pasteur genomic DNA library, and the recombinant BAC vectors belonging to those genomic DNA libraries. This invention also relates to mycobacterial nucleic acids, and methods and kits for using these nucleic acids to detect mycobacteria in a biological sample.

1 - 41 . (canceled)  
     
     
         42 . A method of detecting  Mycobacterium tuberculosis  in a sample, comprising: 
 (a) contacting the sample with a first nucleic acid, wherein the first nucleic acid is a polynucleotide sequence comprising SEQ ID NO:2, a fragment of SEQ ID NO:2 comprising at least 8 consecutive nucleotides of SEQ ID NO:2, or a polynucleotide whose sequence is complementary to SEQ ID NO:2 or to the fragment of SEQ ID NO:2; and    (b) detecting hybridization between the first nucleic acid and a second nucleic acid in the sample, wherein detecting hybridization between the first and second nucleic acids indicates the presence of  Mycobacterium tuberculosis  in the sample.    
     
     
         43 . A method of detecting  Mycobacterium tuberculosis  in a sample, comprising: 
 (a) contacting the sample with a first nucleic acid, wherein the first nucleic acid is a polynucleotide sequence comprising SEQ ID NO:3, a fragment of SEQ ID NO:3 comprising at least 8 consecutive nucleotides of SEQ ID NO:3, or a polynucleotide whose sequence is complementary to SEQ ID NO:3 or to the fragment of SEQ ID NO:3; and    (b) detecting hybridization between the first nucleic acid and a second nucleic acid in the sample, wherein detecting hybridization between the first and second nucleic acids indicates the presence of  Mycobacterium tuberculosis  in the sample.    
     
     
         44 . The method of any one of claims  42  or  43 , wherein the first nucleic acid hybridizes to the genome of  Mycobacterium tuberculosis  but not to the genome of  Mycobacterium bovis  under stringent hybridization conditions.  
     
     
         45 . The method of  claim 44 , wherein the stringent hybridization conditions comprise hybridizing at 65° C. in 6×SSC, followed by washing at 65° C. in 2×SSC.