BACTERIA DETECTION DEVICE, BACTERIA DETECTION METHOD AND BACTERIA DETECTION KIT
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.
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.
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.
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.