Detection, identification and differentiation of eubacterial taxa using a hybridization assay

Abstract

The present invention relates to a method for the specific detection and/or identification of Enterococcus species, in particular Enterococcus faecalis and/or Enterococcus faecium , using new nucleic acid sequences derived from the ITS (Internal Transcribed Spacer) region. The present invention relates also to said new nucleic acid sequences derived from the ITS region, between the 16S and 23S ribosomal ribonucleic acid (rRNA) or rRNA genes, to be used for the specific detection and/or identification of Enterococcus species, in particular of Enterococcus faecalis and/or Enterococcus faecium , in a biological sample. It relates also to nucleic acid primers to be used for the amplification of said spacer region of Enterococcus species in a sample.

1 . An isolated nucleic acid molecule consisting of SEQ ID NO 1, its complementary form and the RNA form thereof. 
     
     
         2 . An isolated nucleic acid molecule consisting of SEQ ID NO 2, its complementary form and the RNA form thereof. 
     
     
         3 . An isolated nucleic acid molecule of more than 10 contiguous nucleotides that specifically hybridizes to SEQ ID NO 1 or 2, or to the RNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U, or to the complementary form of said SEQ ID NO 1 or 2, or to a fragment of at least 20 contiguous nucleotides thereof, or to any of their homologues, for the detection and/or identification of  Enterococcus  species. 
     
     
         4 . An isolated nucleic acid molecule according to  claim 3  consisting of a nucleic acid selected from the group consisting of SEQ ID NO 22 to 26, 28 to 43, 45 to 65 and 67 to 84. 
     
     
         5 . A set of two or three polynucleotide probes which hybridize to the same target sequence in adjacent locations on said target sequence, said probes hybridizing specifically to SEQ ID NO 1 or SEQ ID NO 2 or homologues, or to their RNA form wherein T is replaced by U, or to their complementary form, wherein there are no more than 25 nucleotides between said probes along said target sequence. 
     
     
         6 . A set of two or three polynucleotide probes according to  claim 5  consisting of any combinations of Table 3. 
     
     
         7 . A composition comprising at least one nucleic acid molecule according to  claim 1  and/or a set of two polynucleotide probes, said probes comprising more than 10 contiguous nucleotides. 
     
     
         8 . A method of detecting and/or identification of  Enterococcus  species in a sample comprising hybridizing a nucleic acid molecule of  claim 3  to nucleic acid sequences of said sample and detecting said hybridization. 
     
     
         9 . The method of  claim 8  wherein said  Enterococcus  species is at least one of  E. faecalis  and  E. faecium.    
     
     
         10 . A method according to  claim 8  for detection and/or identification of  Enterococcus  species in a sample comprising the steps of:
 (i) if need be releasing, isolating and/or concentrating the polynucleic acids in the sample;   (ii) if need be amplifying the 16S-23S rRNA spacer region, or a fragment comprising a  Enterococcus  species-specific polynucleic acid, with at least one suitable primer pair;   (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least one polynucleotide probe of  claim 3 ,   (iv) detecting the hybrids formed, and   (v) interpreting the signal(s) obtained and inferring the presence of  Enterococcus  species and/or identifying the  Enterococcus  species in the sample.   
     
     
         11 . A method according to  claim 10  wherein a suitable primer pair consists any combination of a forward primer polynucleotide selected from the group consisting of SEQ ID NO 3, 4, 5, 6, 7, 8, 9, 10 or 11 and their homologues, and a reverse primer polynucleotide selected from the group consisting of SEQ ID NO 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 and their homologues. 
     
     
         12 . A method according to  claim 10  wherein said at least one probe comprises two polynucleotide probes. 
     
     
         13 . A method according to  claim 12  wherein said at least one probe comprises a set of two polynucleotide probes which hybridize to the same target sequence in adjacent locations on said target sequence, wherein there are no more than 25 nucleotides between said probes along the hybridized polynucleic acid sequence. 
     
     
         14 . A method according to  claim 12  wherein the two polynucleotide probes consist of any combination of polynucleotides of Table 3. 
     
     
         15 . A kit for detection and/or identification of  Enterococcus  species comprising the following components:
 at least one nucleic acid molecule according to  claim 3 , and   a hybridization buffer, or components necessary for producing said buffer.