Serotype discrimination biomarkers to distinguish Infantis from Heidelberg and Newport from Hadar

Patent metadata

Field	Value
Publication number	US-10323286-B2
Application number	US-201615563838-A
Country	US
Kind code	B2
Filing date	Mar 30, 2016
Priority date	Mar 30, 2015
Publication date	Jun 18, 2019
Grant date	Jun 18, 2019

Abstract

The present invention provides a set of oligonucleotides to screen for the presence of targeted Salmonella serotypes in enrichment or to characterize presumptive colonies. The set of oligonucleotides includes at least one set of primers and probe for the detection of Salmonella serotype selected from Newport, Heidelberg, Infantis , and Hadar and to discriminate between. These new markers can be used after the initial screening assay described herein as a discrimination assay to differentiate S. Heidelberg from S. Infantis and S. Hadar from S. Newport.

First claim

The invention claimed is:

1. A kit for the identification of a Salmonella serotype comprising:
at least three oligonucleotides selected from:
a first primer set having SEQ ID NOS: 1 and 3, for the amplification of one or more sequences, and a first probe having SEQ ID NO: 2 attached to a dye, for discrimination with S. Infantis and identification of S. Heidelberg;
a second primer set having SEQ ID NOS: 4 and 6, for the amplification of one or more sequences, and a second probe having SEQ ID NO: 5 attached to a dye, for discrimination with S. Heidelberg and identification of S. Infantis;
a third primer set having SEQ ID NOS: 7 and 9, for the amplification of one or more sequences, and a third probe having SEQ ID NO: 8 attached to a dye, for discrimination with S. Newport and identification of S. Hadar ; or
a fourth primer set having SEQ ID NOS: 10 and 12, for the amplification of one or more sequences, and a fourth probe having SEQ ID NO: 11 attached to a dye, for discrimination with S. Hadar and identification of S. Newport ; and

at least one selected from a nucleic acid extraction solution; a recombinant DNA polymerase; at least one dNTPs; a buffer having a pH adapted to a DNA polymerase activity; a PCR Master Mix; and an instruction set for performing a real-time multiplex amplification to detect a Salmonella serotype.

2. The oligonucleotides of claim 1 , wherein the oligonucleotides include the first, second, third and fourth primer sets and the first, second, third and fourth probes, for discrimination with S. Infantis and identification of S. Heidelberg ; discrimination with S. Heidelberg and identification of S. Infantis ; discrimination with S. Newport and identification of S. Hadar ; or discrimination with S. Hadar and identification of S. Newport , respectively.

3. The oligonucleotides of claim 1 , wherein the Salmonella serotype is selected from Newport, Heidelberg, Infantis , and Hadar.

4. A kit for the identification of a Salmonella serotype comprising:
a pair of oligonucleotide primers comprising SEQ ID NOS: 1 and 3, SEQ ID NOS: 4 and 6, SEQ ID NOS: 7 and 9, or SEQ ID NOS: 10 and 12 for the amplification of one or more sequences of one or more Salmonella serotypes;
one or more probes selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 11, wherein each probe is attached to a dye, for the identification of one or more Salmonella serotypes; and
one or more vials comprising reagents for PCR amplification, including at least one selected from a nucleic acid extraction solution; a DNA polymerase; at least one dNTPs; a buffer having a pH adapted to a polymerase activity; a PCR Master Mix; and instructions for determining the Salmonella serotype based on the product of amplification using the primers and probes.

5. The kit of claim 4 , further comprising an instruction set for performing a multiplex amplification to detect a Salmonella serotype.

6. The kit of claim 5 , wherein the instructions provide for discrimination with S. Infantis and identification of S. Heidelberg ; discrimination with S. Heidelberg and identification of S. Infantis ; discrimination with S. Newport and identification of S. Hadar ; or discrimination with S. Hadar and identification of S. Newport , respectively.

7. The kit of claim 1 , wherein the instructions provide for discrimination with S. Infantis and identification of S. Heidelberg ; discrimination with S. Heidelberg and identification of S. Infantis ; discrimination with S. Newport and identification of S. Hadar ; or discrimination with S. Hadar and identification of S. Newport , respectively.

8. The kit of claim 1 , wherein the dye is a fluorescent dye.

9. The kit of claim 4 , wherein the dye is a fluorescent dye.

Assignees

• Univ Texas Tech System
• Texas Techn Univ System

Inventors

• Bugarel Marie
• Nightingale Kendra
• Loneragan Guy
• Brashears Mindy M

Classifications

• C12Q1/689 Primary

  for bacteria · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title