Detection and typing of bacterial strains

• US 20060199190A1
• Filed: 04/27/2005
• Published: 09/07/2006
• Est. Priority Date: 04/28/2004
• Status: Active Grant

• Current Assignee: DuPont Nutrition Biosciences ApS (International Flavors & Fragrances Incorporated)
• Original Assignee: Danisco A/S (International Flavors & Fragrances Incorporated)
• Inventors: Russell, W. Michael, Barrangou, Rodolphe, Horvath, Philippe
• Granted Patent: US 7,919,277 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/142 Toxicological screening, e.g. expression profiles which identify toxicity

Abstract

Methods for the detection and typing of bacterial strains from food products and dietary supplements, environmental samples, in vivo/in vitro samples, and for studying the natural diversity of the species are disclosed. Potential applications also include product development and/or detection and differentiation of new bacterial strains.

Claims

1. An isolated nucleic acid molecule selected from the group consisting of:

a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO;

1, or a complement thereof;

b) a nucleic acid molecule comprising a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO;

1, or a complement thereof;

c) a nucleic acid molecule comprising the nucleotide sequence of any of SEQ ID NOS;

2-50, or a complement thereof;

d) a nucleic acid molecule comprising a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of any of SEQ ID NOS;

2-50, or a complement thereof;

e) a nucleic acid molecule comprising a fragment of any of SEQ ID NOS;

1-50; and

,

f) a nucleic acid molecule comprising 1-140 repeats of at least one of the nucleotide sequences set forth in SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, or a variant thereof.

2. A method for typing a Lactobacillus bacterial strain, comprising:

a) obtaining a sample;

b) amplifying a region of DNA comprising a nucleotide sequence having at least 75% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS;

1-7 and 37-48 in said sample to create amplified DNA; and

,

c) typing said bacterial strain based on said amplified DNA.

3. The method of claim 2, wherein said region of DNA to be amplified comprises SEQ ID NO:

1.

4. The method of claim 2, wherein said region of DNA to be amplified comprises at least one of the nucleotide sequences set forth in SEQ ID NOS:

1-7 and 37-48.

5. The method of claim 2, wherein said sample is selected from the group consisting of a food product, a dietary supplement, an animal feed and an animal feed supplement.

6. The method of claim 2, wherein said Lactobacillus is a L. acidophilus.

7. The method of claim 2, wherein said Lactobacillus is a L. brevis.

8. The method of claim 2, wherein said Lactobacillus is a L. casei.

9. The method of claim 2, wherein said Lactobacillus is a L. delbrueckii.

10. The method of claim 2, wherein said amplified DNA is obtained by PCR, and wherein said PCR is performed using at least one primer comprising SEQ ID NO:

49 or SEQ ID NO;

50.

11. The method of claim 2, further comprising:

a) adding to said amplified DNA at least one restriction enzyme that recognizes one or more sites in said amplified DNA;

b) incubating said restriction enzyme with said amplified DNA for a time sufficient to form restriction fragments;

c) determining the number of said restriction fragments and their size; and

,

d) typing said bacterial strain based on said number and size of said restriction fragments.

12. The method of claim 11, wherein said restriction enzyme is selected from the group consisting of AluI, MseI, and Tsp5091.

13. The method of claim 2, further comprising:

a) sequencing said amplified DNA to obtain sequencing results; and

,

b) typing said bacterial strain based on said sequencing results.

14. The method of claim 2, wherein said amplified DNA is obtained by:

a) providing a first primer that binds to any of SEQ ID NOS;

2-7 and 37-48;

b) providing a second primer that binds to DNA downstream or upstream of any of SEQ ID NOS;

2-7 and 37-48; and

,

c) using said first and second primers in a PCR reaction to create said amplified DNA.

15. The method of claim 2, wherein said amplified DNA is obtained by:

a) providing a first primer that binds to a region upstream of any of SEQ ID NOS;

2-7 and 37-48;

b) providing a second primer that binds to a region downstream of any of SEQ ID NOS;

2-7 and 37-48; and

,

c) using said first and second primers in a PCR reaction to create said amplified DNA.

16. A kit for detecting the presence of a Lactobacillus species in a sample, comprising polymerase chain reaction primers and instructions for use.

17. A kit for typing a Lactobacillus strain in a sample, comprising polymerase chain reaction primers for use in creating amplified DNA, at least one restriction enzyme that recognizes one or more sites in said amplified DNA, and instructions for use.

18. A method for detecting the presence of a Lactobacillus species in a sample, comprising:

a) obtaining a sample;

b) amplifying a region of DNA comprising at least one of SEQ ID NOS;

1-48, or a variant thereof, to create amplified DNA; and

,

c) detecting said amplified DNA.

19. The method of claim 18, wherein said amplifying is performed using at least one primer comprising SEQ ID NO:

49 or SEQ ID NO;

50, or a variant thereof.

20. A method for typing a bacterium having a CRISPR region, comprising:

a) obtaining a sample comprising said bacterium;

b) amplifying a region of DNA comprising said CRISPR region or a fragment thereof in said sample to create amplified DNA;

c) adding to said amplified DNA at least one restriction enzyme that recognizes one or more sites in said amplified DNA;

d) incubating said restriction enzyme with said amplified DNA for a time sufficient to form restriction fragments;

e) determining the number of said restriction fragments and their size; and

,

f) typing said bacterium based on said number and size of said restriction fragments.