Compositions and methods for detection of salmonella species

Abstract

Described are compositions, methods and kits for detection and/or differential detection of serovars of Salmonella enterica subsp. enterica serovar such as S. Heidelberg and S. Hadar in samples. Some embodiments relate to multiplex amplification based molecular assays.

1 - 20 . (canceled) 
     
     
         21 . A composition comprising: at least one pair of synthetic forward and reverse PCR primer sets selected from a first primer set having SEQ ID NO: 1 and SEQ ID NO 2, and a second primer set having SEQ ID NO: 4 and SEQ ID NO: 5. 
     
     
         22 . The composition of  claim 21 , comprising both the first and second set of primers. 
     
     
         23 . The composition of  claim 21 , further comprising at least one labeled probe selected from SEQ ID NO: 3 and SEQ ID NO: 6, or full complements thereof. 
     
     
         24 . The composition of  claim 21  further comprising at least one pair of synthetic forward and reverse PCR primer sets selected from a third primer set having SEQ ID NO: 7 and SEQ ID NO: 8; and a fourth primer set having SEQ ID NO: 10 and SEQ ID NO 11, full complements thereof or a labeled derivative thereof. 
     
     
         25 . The composition of  claim 24  further comprising at least one labeled probe selected from SEQ ID NO: 9 and SEQ ID NO:12, or full complements thereof. 
     
     
         26 . A kit for the detection of a  Salmonella enterica  subsp.  enterica  serovar microorganism comprising:
 at least one pair of synthetic forward and reverse PCR primer sets selected from a first primer set having SEQ ID NO: 1 and SEQ ID NO 2, and a second primer set having SEQ ID NO: 4 and SEQ ID NO: 5,   at least one pair of synthetic forward and reverse PCR primer sets selected from a third primer set having SEQ ID NO: 7 and SEQ ID NO: 8; and a fourth primer set having SEQ ID NO: 10 and SEQ ID NO 11, full complements thereof or a labeled derivative thereof;   at least one labeled probe selected from SEQ ID NO: 9 and SEQ ID NO:12, or full complements thereof;   at least one labeled probe selected from SEQ ID NO: 3 and SEQ ID NO: 6, or full complements thereof; and   one or more components selected from a group consisting of: at least one enzyme, dNTPs, at least one buffer, at least one salt, at least one pair of synthetic primers specific to hybridize to and amplify an internal positive control, at least one control nucleic acid sample and an instruction protocol.   
     
     
         27 . A method for detection of a  Salmonella enterica  subsp.  enterica  serovar S. Hadar microorganism comprising:
 a) contacting a sample with one or more sets of synthetic oligonucleotide primers specific to hybridize to target nucleic acids specific to S. Hadar serovar specific target nucleic acid sequences selected from a first primer set having SEQ ID NO: 7 and SEQ ID NO: 8; and a second primer set having SEQ ID NO: 10 and SEQ ID NO: 11, full complements thereof, or a labeled derivative thereof;   b) contacting the sample with at least one set of oligonucleotide primers specific to hybridize to an internal positive control specific nucleic acid sequence;   c) optionally contacting the sample with one or more labeled probes specific to hybridize to at least one of the S. Hadar specific nucleic acids, fragments or complements thereof, the probe selected from SEQ ID NO:9 or SEQ ID NO: 12, full complements thereof, or sequences comprising at least 90% nucleic acid sequence identity thereto;   d) amplifying at least one S. Hadar specific target nucleic acid sequence and the internal positive control specific sequence by a multiplex amplification method;   e) detecting the one or more amplified S. Hadar specific nucleic acids or fragments or complements thereof; and   f) optionally identifying the S. Hadar specific nucleic acid;
 wherein detecting the one or more amplified S. Hadar specific nucleic acids, fragments or complements thereof is indicative of the presence of a  Salmonella enterica  subsp.  enterica  serovar S. Hadar microorganism in the sample. 
   
     
     
         28 . The method of  claim 27 , wherein the oligonucleotide primers have at least 90% nucleic acid sequence identity to the sequences of primer set one or primer set two and the probes have a 100% nucleic acid identity to the probe sequences of  claim 27 . 
     
     
         29 . The method of  claim 27 , wherein the labeled probes have at least a 90% nucleic acid identity to the probe sequences of  claim 27 , and the oligonucleotide primers have a 100% nucleic acid sequence identity to the sequences of primer set one or primer set two. 
     
     
         30 . The method of  claim 27 , wherein the sample is a food sample, a beverage sample, an agricultural sample, a produce sample, an animal sample, a clinical sample, an environmental sample, a biological sample, a water sample and an air sample. 
     
     
         31 . The method of  claim 27  further comprising steps of: nucleic acid extraction from the microorganism in the sample; and optional enrichment of the microorganisms in the sample prior to nucleic acid extraction. 
     
     
         32 . The method of  claim 27 , wherein identifying the S. Hadar specific nucleic acid comprises contacting the one or more amplified S. Hadar specific nucleic acids, fragments or complements thereof with a probe specific to hybridize to at least one of the amplified S. Hadar specific nucleic acids, fragments or complements thereof under conditions of hybridization and wherein detection of hybridization with the probe is indicative of the presence of a S. Hadar microorganism in the sample. 
     
     
         33 . The method of  claim 32 , wherein the probe specific to hybridize to at least one of the amplified S. Hadar specific nucleic acids, comprises SEQ ID NO:9 or SEQ ID NO: 12 or a labeled derivative thereof. 
     
     
         34 . The method of  claim 27 , further comprising contacting the sample with at least one primer set specific to hybridize to a least one S. Heidelberg specific target nucleic acid sequence, selected from a third primer set having SEQ ID NO: 1 and SEQ ID NO 2, a fourth primer set having SEQ ID NO: 4 and SEQ ID NO: 5, under conditions to amplify an S. Heidelberg specific sample target nucleic acid or a fragment or a complement thereof; and detecting at least one amplified S. Heidelberg specific nucleic acid sequence, wherein detection of the at least one amplified S. Heidelberg specific nucleic acid sequence indicates the presence of a S. Heidelberg organism in the sample. 
     
     
         35 . The method of  claim 27 , wherein the amplifying is quantitative. 
     
     
         36 . The method of  claim 35 , wherein the quantification is by a real-time polymerase chain reaction (PCR) assay. 
     
     
         37 . The method of  claim 27 , performed using a kit, the kit comprising:
 at least one pair of synthetic forward and reverse PCR primer sets selected from a first primer set having SEQ ID NO: 7 and SEQ ID NO: 8; and/or a second primer set having SEQ ID NO: 10 and SEQ ID NO 11, full complements thereof or a labeled derivative thereof;   at least one pair of primers specific to hybridize to and amplify the internal positive control;   at least one probe selected from probes selected from SEQ ID NO: 9 and SEQ ID NO:12, full complements thereof, or a labeled derivative thereof; and   one or more components selected from a group consisting of: at least one enzyme, dNTPs, at least one buffer, at least one salt, at least one control nucleic acid sample and an instruction protocol.   
     
     
         38 . The method of  claim 37 , the kit further comprising:
 at least one pair of forward and reverse PCR primers selected from SEQ ID NO: 1 and SEQ ID NO 2; and SEQ ID NO: 4 and SEQ ID NO 5, full complements thereof, or a labeled derivative thereof; and   optionally at least one labeled probe selected from SEQ ID NO: 3 and SEQ ID NO: 6 full complements thereof, or a labeled derivative thereof.