METHOD FOR SIMULTANEOUS DETECTION AND QUANTIFICATION OF Listeria monocytogenes, Salmonella spp., AND SHIGA TOXIN-PRODUCING Escherichia coli (STEC)

Patent metadata

Field	Value
Publication number	US-2025084490-A1
Application number	US-202118563954-A
Country	US
Kind code	A1
Filing date	May 26, 2021
Priority date	May 26, 2021
Publication date	Mar 13, 2025
Grant date	—

Abstract

The invention refers to a method for simultaneous detection and quantification of Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC), from any kind of sample related to food production, including complex food matrices such as fish, meat, or fruit; or simple matrices such as water, or food contact surfaces. The method of invention allows specific quantification of the above-mentioned pathogens simultaneously thanks to specificity of designed primers for the qPCR reaction. It is also highly reliable because the method includes a system to adequately control the quantification of pathogens within the matrix. This system comprises the inoculation of samples with a known concentration of a transformed microorganism (host) carrying a chimeric sequence, acting as an internal control host for the whole process.

First claim

1 . A method for simultaneous or independent detection and quantification of Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC) in a sample comprising the following steps:
(e) adding a known quantity of internal control host (I.C.) to the sample, wherein the internal control host carrying a polynucleotide comprising a chimeric polynucleotide sequence; (f) assessing the sample for the detection and quantification of nucleic acids of internal control (I.C.) and L. monocytogenes, Salmonella spp., Shiga toxin-producing Escherichia coli (STEC), or combinations thereof; (g) determining the pathogen concentration by standard curve analysis made for each of the pathogens; (h) validating the process with the results for I. C. which must give a signal according to the concentration used in (a).

2 . The method according to claim 1 , wherein the internal control host is selected from bacteria, archaea or yeast.

3 . The method of claim 2 , wherein the said host is Escherichia coli.

4 . The method of claim 1 , wherein said chimeric polynucleotide sequence comprises: a sequence of a forward primer selected from SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, and 17; a 20 to 70 bp central-linker region; and a complementary sequence to a reverse primer selected from SEQ ID No 2, 4, 6, 8, 10, 12, 14, 16 and 18.

5 . The method according to claim 4 wherein the complementary reverse sequence is selected from SEQ ID No. 36 to 44.

6 . The method according to claim 4 , wherein said central-linker region is SEQ ID No. 19.

7 . The method according to claim 4 , wherein said chimeric polynucleotide sequence is selected from the combinations of any of SEQ ID No 1, 3, 5 with any complementary of SEQ ID No 8, 10, 12, 14, 16, 18, wherein the central-linker region is SEQ ID No 19.

8 . The method according to claim 7 , wherein the full length sequence of said chimeric polynucleotide sequence is selected from SEQ ID No 21, 45 to 61.

9 . The method according to claim 4 , wherein said chimeric polynucleotide sequence is selected from the combinations of any of SEQ ID No 7, 9, 11 with any complementary of SEQ ID No 2, 4, 6, 14, 16, 18, wherein the central-linker region is SEQ ID No 19.

10 . The method according to claim 9 , wherein the full length sequence of said chimeric polynucleotide sequence is selected from SEQ ID No 62-79.

11 . The method according to claim 4 , wherein said chimeric polynucleotide sequence is selected from the combinations of any of SEQ ID No 13, 15, 17 with any complementary of SEQ ID No 2, 4, 6, 8, 10, 12, wherein the central-linker region is SEQ ID No 19.

12 . The method according to claim 11 , wherein the full length sequence of said chimeric polynucleotide sequence is selected from SEQ ID No 20, 80-96.

13 . The method of claim 2 , wherein said host is a transformed, edited or transfected host carrying the polynucleotide comprising the chimeric sequence selected from SEQ ID No. 20, 21, 44 to 96.

14 . The method of claim 1 , wherein said method further comprises a step to separate microorganisms out of the sample.

15 . The method of claim 14 , wherein said step of microorganism separation is performed by filtration, centrifugation, sorting, magnetic beads or combinations thereof.

16 . The method of claim 15 , wherein said method further comprises a step of nucleic acids extraction from the sample.

17 . The method of claim 1 , wherein said nucleic acids detection is performed by PCR, RT-PCR, qPCR, digital PCR, LinDA, DNA microarray, PCR coupled with high-throughput sequencing, PCR DGGE/TTGE, or combinations thereof.

18 . The method of claim 17 , wherein for said nucleic acids detection of L. monocytogenes is performed using primers comprising a sequence selected from SEQ ID No. 1 to 6, its derivatives, or combinations thereof.

19 . The method of claim 18 , wherein said nucleic acids detection of L. monocytogenes is performed by qPCR using probes comprising a sequence selected from SEQ ID No. 22 to 25, its derivatives, or combinations thereof.

20 . The method of claim 17 wherein for said nucleic acids detection of STEC is performed using primers comprising a sequence selected from SEQ ID No. 7 to 12, its derivatives, or combinations thereof.

21 . The method of claim 20 , wherein said nucleic acids detection of STEC is performed by qPCR using probes comprising a sequence selected from SEQ ID No. 26 to 29, its derivatives, or combinations thereof.

22 . The method of claim 17 , wherein for said nucleic acids detection of Salmonella -type bacteria is performed using primers comprising a sequence selected from SEQ ID No. 13 to 18, its derivatives, or combinations thereof.

23 . The method of claim 22 , wherein said nucleic acids of Salmonella spp. bacteria is performed by qPCR using probes comprising a sequence selected from SEQ ID No. 30 to 33, its derivatives, or combinations thereof.

24 . The method of claim 1 , wherein said c) step comprises a step of determining Listeria monocytogenes, Salmonella spp. bacteria and or Shiga toxin-producing Escherichia coli (STEC) concentration in a sample interpolating the Cq value, obtained by qPCR in step b), on a standard curve for each pathogen Listeria monocytogenes, Salmonella spp. bacteria and/or Shiga toxin producing Escherichia coli (STEC) concentration and validating it by the value for I.C. signal obtained in step d).

25 . The method of claim 24 , wherein said nucleic acids detection of IC is performed using as forward primer a sequence selected from SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, and 17, its derivatives, or combinations thereof; and as reverse primer a sequence selected from SEQ ID No 2, 4, 6, 8, 10, 12, 14, 16, and 18, its derivatives, or combinations thereof.

26 . The method of claim 25 , wherein said nucleic acids detection of IC is performed by qPCR using probes comprising a sequence selected from SEQ ID No. 34 to 35, its derivatives, or combinations thereof.

27 . The method of claim 1 , wherein said sample is a biological sample selected from meat, poultry, seafood, sausages, dairy, fruits, vegetables, ready to eat foods, beverages, water or surfaces.

28 . The method of claim 1 , wherein the I.C. host is added at a concentration of 10 2 -10 10 cells or CFU per mL.

29 . A polynucleotide to obtain an IC useful to detect and quantify Listeria monocytogenes, Salmonella spp., and/or Shiga toxin-producing Escherichia coli (STEC) in a sample, wherein said polynucleotide comprises:
a sequence of a forward primer selected from SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15 and 17; a 26 bp central-linker region; and a complementary sequence to a reverse primer selected from SEQ ID No 2, 4, 6, 8, 10, 12, 14, 16 and 18.

30 . The polynucleotide according to claim 29 wherein the complementary reverse sequence is selected from SEQ ID No. 36 to 44.

31 . The polynucleotide of claim 29 , wherein said central-linker region is SEQ ID No. 19.

32 . The polynucleotide of claim 29 wherein said polynucleotide is selected from the combinations of any of SEQ ID No 1, 3, 5 with any complementary of SEQ ID No 8, 10, 12, 14, 16, 18, wherein the central-linker region is SEQ ID No 19.

33 . The polynucleotide according to claim 32 wherein the full l

Assignees

• Univ Chile
• Fundacion Copec Univ Catolica

Inventors

• Reyes Angélica
• Navarrete Paola
• Chinnicci Gaétan
• Parra Angel

Classifications

• C12Q2600/166

  Oligonucleotides used as internal standards, controls or normalisation probes · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• C12Q1/689 Primary

  for bacteria · CPC title