Nucleic acids and methods for the detection of enterobacter sakazakii (cronobacter spp.)

Abstract

Provided are detection means and method specific for the genus Cronobacter ( E. sakazakii ) which are sensitive by using a target molecule which is multiple existent in Cronobacter cells.

1 . A nucleic acid molecule suitable as a primer or probe in the detection of the genus  Cronobacter , wherein
 i) the nucleic acid molecule
 a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 1, which partial sequence is highly conserved or conserved in all species of the genus  Cronobacter , and 
 b) is between 10 and 50 nucleic acids long; 
   ii) the nucleic acid molecule
 a) is a sequence which hybridizes under specific conditions with the nucleic acid molecule of i) and 
 b) is between 10 and 50 nucleic acids long; 
   iii) the nucleic acid molecule
 a) is at least 70% identical to the nucleic acid molecule of i) or ii); and 
 b) is between 10 and 50 nucleic acids long; or 
   iv) the nucleic acid molecule is the complement of the nucleic acid molecule of any of i) to iii).   
     
     
         2 . The nucleic acid molecule of  claim 1 , which is
 a) a nucleic acid molecule selected from any of SEQ ID NOs: 2-18; or   b) a nucleic acid molecule which hybridizes under specific conditions with the nucleic acid molecule of a);   c) a nucleic acid molecule which is at least 70% identical to the nucleic acid molecule of a); or   d) the complement of the nucleic acid molecule of any of a) to c).   
     
     
         3 . A nucleic acid molecule suitable as a primer or probe in the detection of  Cronobacter turicensis , wherein
 i) the nucleic acid molecule
 a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 19, which partial sequence is highly conserved in a subgroup of the species  Cronobacter turicensis ; and 
 b) is between 10 and 50 nucleic acids long; 
   ii) the nucleic acid molecule
 a) is a sequence which hybridizes under specific conditions with the nucleic acid molecule of i) and 
 b) is between 10 and 50 nucleic acids long; or 
   iii) the nucleic acid molecule
 a) is at least 70% identical to the nucleic acid molecule of i) or ii); and 
 b) is between 10 and 50 nucleic acids long; or 
   iv) the nucleic acid molecule is the complement of the nucleic acid molecule of any of i) to iii).   
     
     
         4 . The nucleic acid molecule of  claim 3 , which is
 a) selected from SEQ ID NOs: 20-21; or   b) a sequence which hybridizes under specific conditions with the nucleic acid molecule of a);   c) at least 70% identical to the nucleic acid molecule of a); or   d) the complement of the nucleic acid molecule of any of a) to c).   
     
     
         5 . A method of using one or more nucleic acid molecules of  claim 1  in the detection of the genus  Cronobacter.    
     
     
         6 . A combination of two or more of the nucleic acid molecules of  claim 1  in combination with one or more nucleic acid molecules suitable as a primer or probe in the detection of  Cronobacter turicensis , wherein
 i) the nucleic acid molecule
 a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 19, which partial sequence is highly conserved in a subgroup of the species  Cronobacter turicensis ; and 
 b) is between 10 and 50 nucleic acids long; 
 
 ii) the nucleic acid molecule
 a) is a sequence which hybridizes under specific conditions with the nucleic acid molecule of i) and 
 b) is between 10 and 50 nucleic acids long; or 
 
 iii) the nucleic acid molecule
 a) is at least 70% identical to the nucleic acid molecule of i) or ii); and 
 b) is between 10 and 50 nucleic acids long; or 
 
 iv) the nucleic acid molecule is the complement of the nucleic acid molecule of any of i) to iii). 
 
     
     
         7 . A method of using of one or more nucleic acid molecules of  claim 1  together with one or more nucleic acid molecules suitable as a primer or probe in the detection of  Cronobacter turicensis , wherein
 i) the nucleic acid molecule
 a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 19, which partial sequence is highly conserved in a subgroup of the species  Cronobacter turicensis ; and 
 b) is between 10 and 50 nucleic acids long; 
 
 ii) the nucleic acid molecule
 a) is a sequence which hybridizes under specific conditions with the nucleic acid molecule of i) and 
 b) is between 10 and 50 nucleic acids long; or 
 
 iii) the nucleic acid molecule
 a) is at least 70% identical to the nucleic acid molecule of i) or ii); and 
 b) is between 10 and 50 nucleic acids long; or 
 
 iv) the nucleic acid molecule is the complement of the nucleic acid molecule of any of i) to iii), 
 in the detection of the genus  Cronobacter  and the discrimination of  Cronobacter turicensis  against other species. 
 
     
     
         8 . A method for amplifying bacterial DNA of the taxonomic unit genus  Cronobacter , using primers, in which
 a) in a first amplification step the DNA of the genus  Cronobacter  is amplified with conserved primers, wherein the primers used in the first amplification step comprise a nucleic acid molecule of  claim 1 .   
     
     
         9 . The method of  claim 8 , wherein
 b) in a further detection step, the DNA fragments obtained by amplification step a) which are specific for the genus  Cronobacter  are detected by means of probes, wherein the probes used in step b) comprise a nucleic acid molecule, wherein   i) the nucleic acid molecule
 a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 1 which partial sequence is highly conserved or conserved in all species of the genus  Cronobacter , and 
 b) is between 10 and 50 nucleic acids long; 
   ii) the nucleic acid molecule
 a) is a sequence which hybridizes under specific conditions with the nucleic acid molecule of i) and 
 b) is between 10 and 50 nucleic acids long; 
   iii) the nucleic acid molecule
 a) is at least 70% identical to the nucleic acid molecule of i) or ii); and 
 b) is between 10 and 50 nucleic acids long; or 
   iv) the nucleic acid molecule is the complement of the nucleic acid molecule of any of i) to iii).   
     
     
         10 . The method of  claim 9 , wherein
 c) in a further detection step, the DNA fragments obtained by amplification step a) which are specific for the species  Cronobacter turicensis  are detected by means of probes,   wherein the probes used in step c) comprise a nucleic acid molecule, wherein   i) the nucleic acid molecule
 a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 19, which partial sequence is highly conserved in a subgroup of the species  Cronobacter turicensis ; and 
 b) is between 10 and 50 nucleic acids long; 
   ii) the nucleic acid molecule
 a) is a sequence which hybridizes under specific conditions with the nucleic acid molecule of i) and 
 b) is between 10 and 50 nucleic acids long; or 
   iii) the nucleic acid molecule
 a) is at least 70% identical to the nucleic acid molecule of i) or ii); and 
 b) is between 10 and 50 nucleic acids long; or 
   iv) the nucleic acid molecule is the complement of the nucleic acid molecule of any of i) to iii).   
     
     
         11 . The method of  claim 10 , wherein
 d) in at least one further amplification step the DNA of the taxonomic unit Enterobacteriaceae is amplified with conserved primers.   
     
     
         12 . The method of  claim 11 , wherein
 e) in a further detection step, the DNA fragments obtained by amplification step d) which are specific for the genus Enterobacteriaceae are detected by means of probes.   
     
     
         13 . The method of  claim 12 , including a step of amplifying an amplification control nucleic acid, which is added during amplification step a), wherein, the amplification control nucleic acid is SEQ ID NO:22. 
     
     
         14 . The method of  claim 13 , further including a melting curve analysis, wherein  C. turicensis  is discriminated against other  Cronobacter  species. 
     
     
         15 . The method of  claim 14 , wherein viable cells of the genus  Cronobacter  or of other Enterobacteriaceae are discriminated against respective non-viable  Cronobacter  or other non-viable Enterobacteriaceae cells. 
     
     
         16 . The nucleic acid molecule of  claim 1 , wherein the nucleic acid molecule is at least 95% identical to the nucleic acid molecule of i) or ii). 
     
     
         17 . The nucleic acid molecule of  claim 2 , wherein the nucleic acid molecule is at least 95% identical to the nucleic acid molecule of a). 
     
     
         18 . The nucleic acid molecule of  claim 3 , wherein the nucleic acid molecule is at least 95% identical to the nucleic acid molecule of i) or ii). 
     
     
         19 . The nucleic acid molecule of  claim 4 , wherein the nucleic acid molecule is at least 95% identical to the nucleic acid molecule of a).