Detection of salmonella by real-time multiplex pcr

Abstract

The invention relates to the detection of Salmonella by nucleic acid amplification. The invention provides primer and probe oligonucleotides that can be used in multiplex to detect Salmonella in real-time amplification. The oligonucleotides of the invention detect all group I serovars, and have an increased Salmonella detection range: they enable to cover the seven Salmonella groups. They also have an increased sensitivity, without loss in specificity.

1 .- 57 . (canceled) 
     
     
         58 . A set of polynucleotides, which comprises the polynucleotides that are obtainable by nucleic acid amplification from at least one  Salmonella  strain by means of the primers of SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 1, wherein, for each one of said polynucleotides that are obtainable by said nucleic acid amplification:
 the 5′ end of a polynucleotide is at least 85% identical to SEQ ID NO: 1 or 2, and   the 3′ end of the complementary sequence of the same polynucleotide is at least 85% identical to SEQ ID NO: 3 or 4,   
       or
 the 3′ end of a polynucleotide is at least 85% identical to SEQ ID NO: 3 or 4, and 
 the 5′ end of the complementary sequence of the same polynucleotide is at least 85% identical to SEQ ID NO: 1 or 2. 
 
     
     
         59 . The set of  claim 58 , wherein said at least one  Salmonella  strain is at least one  Salmonella enterica  strain. 
     
     
         60 . The set of  claim 58 , wherein said at least one  Salmonella  strain is at least one of the following strains:
 i) at least one  S. enterica  ssp.  enterica  strain,   ii) at least one  S. enterica  ssp.  salamae  strain,   iii) at least one  S. enterica  ssp.  arizonae  strain,   iv) at least one  S. enterica  ssp.  diarizonae  strain,   v) at least one  S. enterica  ssp.  houtanae  strain, and   vi) at least one  S. enterica  ssp.  indica  strain.   
     
     
         61 . The set of  claim 58 , wherein said at least one  Salmonella  strain is at least one  Salmonella bongori  strain. 
     
     
         62 . The set of  claim 58 , wherein at least one of said polynucleotides hybridizes to at least one of
 i) the sequence of SEQ ID NO: 5 and   ii) the sequence that is complementary to SEQ ID NO: 5,   
       under stringent conditions, wherein said stringent conditions are:
 binding said at least one polynucleotide on a filter, 
 contacting said filter-bound polynucleotide with said sequence of SEQ ID NO: 5 or said sequence complementary to SEQ ID NO: 5, at 100 micrograms/mL in 5×SSC, 2% Sodium Dodecyl Sulfate (SDS), at 55-65° C. for 8 hours, 
 washing in 0.2×SSC and 0.2% SDS at 60-65° C. for thirty minutes. 
 
     
     
         63 . The set of  claim 58 , wherein at least one of said polynucleotides hybridizes to at least one of
 i) the sequence of SEQ ID NO: 6 and   ii) the sequence that is complementary to SEQ ID NO: 6,   
       under stringent conditions, wherein said stringent conditions are submitting an amplification reaction mix to cycles of nucleic acid amplification as follows:
 cycle 1: ×1; 
 cycle 2: ×50:
 15 seconds at 95° C.; 
 30 seconds at 58° C.; 
 30 seconds at 72° C., 
 
 
       wherein said amplification reaction mix is:
 5 μL of DNA extract of said at least one  S. enterica  or  S. bongori  strain, 
 Taq polymerase: 1 U, 
 polymerase buffer: 1×, 
 MgCl 2 : 6 mM, 
 dNTP: 100 mM each, 
 said primers of SEQ ID NOs: 1-4: 300 nM each, 
 said sequence of SEQ ID NO: 6 or said sequence that is complementary to SEQ ID NO: 6, with a FAM label: 300 nM, 
 in a final volume of 50 μL 
 
     
     
         64 . The set of  claim 58 , wherein at least one of said polynucleotides comprises at least one of the following sequences:
 i) the sequence of SEQ ID NO: 5,   ii) the sequence, which is complementary to SEQ ID NO: 5,   iii) a sequence of 20 to 29 nucleotides, which is at least 85% identical to SEQ ID NO: 5, and   iv) the sequence, which is complementary to said sequence of 20 to 29 nucleotides that is at least 85% identical to SEQ ID NO: 5.   
     
     
         65 . A composition, which comprises the set of  claim 58 . 
     
     
         66 . A composition, which comprises the set of  claim 58  and at least one DNA polymerase. 
     
     
         67 . A composition, which comprises the set of  claim 58  and the oligonucleotides of SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 1.