BACTERIA DETECTION DEVICE, BACTERIA DETECTION METHOD AND BACTERIA DETECTION KIT

• US 20070212715A1
• Filed: 08/04/2005
• Published: 09/13/2007
• Est. Priority Date: 08/04/2004
• Status: Abandoned Application

• Current Assignee: Suntory Holdings Limited (Kotobuki Realty Co., Ltd.)
• Original Assignee: Suntory Holdings Limited (Kotobuki Realty Co., Ltd.)
• Inventors: Osano, Kaoru, Moriya, Shougo, Okegawa, Takashi, Yamanaka, Mikiko
• Application Number:
  US11/659,273
• Publication Number:
  US 20070212715A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

The present invention provides a bacteria detection device, bacteria detection method and bacteria detection kit enabling rapid and accurate determination of bacteria present in a test sample, and particularly spore-forming, aerobic bacteria, with a simple procedure. The bacteria detection device according to the present invention is a microarray type of device in which an oligonucleotide, which is based on a nucleotide sequence specific to a genus or species to which a target spore-forming, aerobic bacteria belongs, is immobilized on a substrate. Spore-forming, aerobic bacteria present in a test sample can be easily, rapidly and accurately detected and identified based on the presence or absence of successful hybridization between a probe prepared from the test sample and the oligonucleotide immobilized on the substrate.

Claims

1. A bacteria detection device for detecting and identifying bacteria in a test sample, wherein one or more oligonucleotides selected from the group of oligonucleotides comprising a nucleotide sequence of SEQ ID NO. 1 to 40, 43 to 56 or 57 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of bacteria targeted for detection, are immobilized on a substrate;

and bacteria in a test sample is detected and identified by hybridization between the oligonucleotide and a nucleic acid derived from the test sample.

2. The bacteria detection device according to claim 1, wherein at least one of the oligonucleotides comprising a nucleotide sequence of SEQ ID NO. 1, 2 or 3 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of bacteria belonging to the genus Bacillus is immobilized on the substrate.

3. The bacteria detection device according to claim 1, wherein an oligonucleotide comprising the nucleotide sequence of SEQ ID NO. 4 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of bacteria belonging to the genus Brevibacillus is immobilized on the substrate.

4. The bacteria detection device according to claim 1, wherein an oligonucleotide comprising the nucleotide sequence of SEQ ID NO. 5 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of bacteria belonging to the genus Alicyclobacillus is immobilized on the substrate.

5. The bacteria detection device according to claim 1, wherein an oligonucleotide comprising the nucleotide sequence of SEQ ID NO. 6 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of bacteria belonging to the genus Aneurinibacillus is immobilized on the substrate.

6. The bacteria detection device according to claim 1, wherein an oligonucleotide comprising the nucleotide sequence of SEQ ID NO. 7 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of bacteria belonging to the genus Geobacillus is immobilized on the substrate.

7. The bacteria detection device according to claim 1, wherein at least one of the oligonucleotides comprising a nucleotide sequence of SEQ ID NO. 3 or 8 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of bacteria belonging to the genus Paenibacillus is immobilized on the substrate.

8. The bacteria detection device according to claim 1, wherein at least one of the oligonucleotides comprising a nucleotide sequence of SEQ ID NO. 9, 10 or 11 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus cereus is immobilized on the substrate.

9. The bacteria detection device according to claim 1, wherein at least one of the oligonucleotides comprising a nucleotide sequence of SEQ ID NO. 12, 13 or 14 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus coagulans is immobilized on the substrate.

10. The bacteria detection device according to claim 1, wherein an oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 15, 43 or 44 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus sporothermodurans is immobilized on the substrate.

11. The bacteria detection device according to claim 1, wherein at least one of the oligonucleotides comprising a nucleotide sequence of SEQ ID NO. 16, 17 or 18 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus subtilis is immobilized on the substrate.

12. The bacteria detection device according to claim 1, wherein an oligonucleotide comprising the nucleotide sequence of SEQ ID NO. 19 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus licheniformis is immobilized on the substrate.

13. The bacteria detection device according to claim 1, wherein an oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 20 or 55 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillius pumilus is immobilized on the substrate.

14. The bacteria detection device according to claim 1, wherein at least one of the oligonucleotides comprising a nucleotide sequence of SEQ ID NO. 21 to 25 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus lentus is immobilized on the substrate.

15. The bacteria detection device according to claim 1, wherein an oligonucleotide comprising the nucleotide sequence of SEQ ID NO. 26 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus firmus is immobilized on the substrate.

16. The bacteria detection device according to claim 1, wherein an oligonucleotide comprising the nucleotide sequence of SEQ ID NO. 27 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus circulans is immobilized on the substrate.

17. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 29 or 30 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus benzoevorans is immobilized on the substrate.

18. The bacteria detection device according to claim 1, wherein an oligonucleotide comprising the nucleotide sequence of SEQ ID NO. 31 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus megaterium is immobilized on the substrate.

19. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 32 or 33 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus sphaericus is immobilized on the substrate.

20. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 34, 35 or 36 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Alicyclobacillus acidoterrestris is immobilized on the substrate.

21. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 28 or 37 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Alicyclobacillus acidocaldarius is immobilized on the substrate.

22. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 38, 39 or 40 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Paenibacillus validus is immobilized on the substrate.

23. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 45 or 46 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus smithii is immobilized on the substrate.

24. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 47, 48 or 49 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Alicyclobacillus acidiphilus is immobilized on the substrate.

25. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 50 or 51 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Alicyclobacillus hesperidum is immobilized on the substrate.

26. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 52 or 53 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus fumarioli is immobilized on the substrate.

27. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising the nucleotide sequence of SEQ ID NO. 54 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus acidogenesis is immobilized on the substrate.

28. The bacteria detection device according to claim 1, wherein at least one oligonucleotide comprising a nucleotide sequence of SEQ ID NO. 56 or 57 among nucleotide sequences corresponding to a 16S ribosomal RNA gene of Bacillus sonorensis is immobilized on the substrate.

29. The bacteria detection device according to claim 1, wherein all of the oligonucleotides comprising a nucleotide sequence of SEQ ID NO. 1 to 40, 43 to 56 or 57 are immobilized on the substrate.

30. The bacteria detection device according to claim 1, wherein the bacteria in the test sample is at least one type of bacteria selected from the group consisting of bacteria described in any of (a) to (f) below:

(a) bacteria belonging to the genus Bacillus;

(b) bacteria belonging to the genus Brevibacillus;

(c) bacteria belonging to the genus Alicyclobacillus;

(d) bacteria belonging to the genus Aneurinibacillus;

(e) bacteria belonging to the genus Geobacillus; and

,

(f) bacteria belonging to the genus Paenibacillus.

31. The bacteria detection device according to claim 1, wherein the device has a carbodiimide group or an isocyanate group on the substrate, and a covalent bond is formed as a result of a reaction between the carbodiimide group or the isocyanate group and the oligonucleotide or a linker added to a terminal of the oligonucleotide.

32. A bacteria detection method for detecting and identifying bacteria in a test sample, comprising the steps of:

preparing a nucleic acid of the bacteria in the test sample;

preparing a labeled probe using the nucleic acid as a template;

hybridizing the labeled probe with an oligonucleotide immobilized on a substrate using the bacteria detection device according to claim 1;

and

detecting a hybridization signal.

34. The bacteria detection method according to claim 32, wherein the test sample is a food.

35. The bacteria detection method according to claim 34, wherein the food is a beverage.

36. The bacteria detection method according to claim 35, wherein the beverage is a refreshing beverage.

37. A bacteria detection kit for carrying out the bacteria detection method according to claim 32.

38. A bacteria detection kit for carrying out the bacteria detection method according to claim 32, comprising a reagent used in the hybridization step and the signal detection step.

39. The bacteria detection kit according to claim 38, further comprising a reagent used in the probe preparation step and/or the nucleic acid preparation step.

33. A bacteria detection method for detecting and identifying bacteria in a test sample, comprising the steps of:

preparing a nucleic acid of the bacteria in the test sample;

preparing a labeled probe using the nucleic acid as a template;

using an oligonucleotide comprising a nucleotide sequence of any of SEQ ID NO. 1 to 40 and 43 to 57, to hybridize the labeled probe with the oligonucleotide; and

detecting a hybridization signal.