JP2008200012A - Microarray for detecting heat-resistant bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and rapid means for detecting a bacterium group to cause food poisoning and identifying a bacterium species. <P>SOLUTION: The set of 12 kinds of oligonucleotide probes comprises the partial base sequences of 25-35 bases in a DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris or Staphylococcus epidermidis. The microarray is obtained by immobilizing the set of the oligonucleotide probes on a carrier. The method for detecting the bacteria is provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention provides a set of oligonucleotide probes for detecting a thermostable bacterium selected from the group consisting of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis, and the set of oligonucleotide probes is fixed. The present invention relates to a microarray that has been formed, and a method for detecting the thermostable bacteria using them.

  In recent years, food safety has been severely questioned. Above all, the occurrence of food poisoning is a big problem. The cause is mostly due to microorganisms. Therefore, microbial testing in production lines and products is mandatory. Current microbiological tests take samples from raw materials, processes, and products and incubate them, so it takes a very long time to obtain results, which is sufficient for the simplification and speedup of tests that have been required in recent years. It was not possible to cope with. In addition, it was difficult to identify the bacterial species by this method.

  Under such circumstances, a simple and rapid method using a bioluminescence method for measuring the amount of microorganisms based on the amount of microorganism-derived ATP has been developed. In addition, a method for detecting a gene of a pathogenic bacterium using a PCR method has been developed as a simple test method for highly dangerous microorganisms.

  However, each of these methods has its own challenges. In the bioluminescence method, it is difficult to detect a specific microorganism when a large amount of microorganism is present in the food to be examined. In addition, in the PCR method, since it is necessary to predict the type of microorganism to be detected in advance, it may take time to collect information for estimating the microorganism, and it may be difficult to measure quickly. .

  An object of the present invention is to provide a simple and rapid means capable of detecting a microbial group causing food poisoning and identifying a microbial species.

  As a result of intensive studies to solve the above problems, the present inventors have been able to detect Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris, and Staphylococcus epidermidis, and a set of oligonucleotide probes that can identify them. As a result, the present invention has been completed.

In one embodiment, the present invention includes at least one selected from the group consisting of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis, comprising the following oligonucleotide probes (1) to (12): A set of oligonucleotide probes for detecting species of microorganisms:
(1) a) an oligonucleotide probe having a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 190 to 219 of SEQ ID NO: 1 and / or b) 190 of SEQ ID NO: 1 Comprising a base sequence complementary to a continuous base sequence of 25 to 35 bases including base No. 219, consisting of a base sequence in which one or several bases are deleted, substituted, added or inserted, An oligonucleotide probe that hybridizes to the region to which the oligonucleotide a) of the DNA encoding rRNA hybridizes (2) a) 25 to 35 consecutive bases including bases 198 to 227 of SEQ ID NO: 2 An oligonucleotide probe having a base sequence complementary to the sequence, and / or b) a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 198 to 227 of SEQ ID NO: 2 An oligonucleotide which has a base sequence in which one or several bases have been deleted, substituted, added or inserted, and which hybridizes to the region to which the oligonucleotide a) of the DNA encoding the 16S rRNA of the microorganism hybridizes Nucleotide probe (3) a) an oligonucleotide probe comprising a base sequence of 25-35 bases comprising bases 167-196 in SEQ ID NO: 3, and / or b) 167-196 of SEQ ID NO: 3 Of the DNA sequence encoding a 16S rRNA of the microorganism, a) consisting of a base sequence in which one or several bases are deleted, substituted, added or inserted in a continuous base sequence of 25 to 35 bases including the base Oligonucleotide probe that hybridizes to the region to which the oligonucleotide hybridizes (4) a) contains bases 167 to 196 of SEQ ID NO: 3 An oligonucleotide probe having a base sequence complementary to a continuous base sequence of 25 to 35 bases, and / or b) complementary to a continuous base sequence of 25 to 35 bases including bases 167 to 196 of SEQ ID NO: 3 In a typical nucleotide sequence, it consists of a nucleotide sequence in which one or several bases have been deleted, substituted, added or inserted, and in the region where the oligonucleotide a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Hybridizing oligonucleotide probe (5) a) An oligonucleotide probe comprising a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 186 to 215 of SEQ ID NO: 4, and / or b) In the base sequence complementary to the continuous base sequence of 25 to 35 bases including the bases 186 to 215 in SEQ ID NO: 4, one or several bases are deleted, substituted, An oligonucleotide probe comprising an added or inserted base sequence and hybridizing to the region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes (6) a) 170 to An oligonucleotide probe comprising a base sequence complementary to a continuous base sequence of 25 to 35 bases including base number 199, and / or b) of 25 to 35 bases including bases 170 to 199 of SEQ ID NO: 5 The oligonucleotide of a) among the DNAs encoding the 16S rRNA of the microorganism, comprising a base sequence in which one or several bases are deleted, substituted, added or inserted in a base sequence complementary to a continuous base sequence Oligonucleotide probe that hybridizes to the region that hybridizes

(7) a) an oligonucleotide probe comprising a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 170 to 200 of SEQ ID NO: 6 and / or b) 170 of SEQ ID NO: 6 Comprising a base sequence complementary to a continuous base sequence of 25 to 35 bases including base No. 200 to base No. 1, consisting of a base sequence in which one or several bases are deleted, substituted, added or inserted, An oligonucleotide probe that hybridizes to the region to which the oligonucleotide a) hybridizes in the DNA encoding rRNA (8) a) 25 to 35 consecutive bases including bases 168 to 198 of SEQ ID NO: 7 An oligonucleotide probe having a base sequence complementary to the sequence, and / or b) a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 168 to 198 in SEQ ID NO: 7 An oligonucleotide which has a base sequence in which one or several bases have been deleted, substituted, added or inserted, and which hybridizes to the region to which the oligonucleotide a) of the DNA encoding the 16S rRNA of the microorganism hybridizes Nucleotide probe (9) a) an oligonucleotide probe comprising a base sequence of 25-35 bases comprising bases 196 to 225 of SEQ ID NO: 8, and / or b) nucleotides 196 to 225 of SEQ ID NO: 8 Of the DNA sequence encoding a 16S rRNA of the microorganism, a) consisting of a base sequence in which one or several bases are deleted, substituted, added or inserted in a continuous base sequence of 25 to 35 bases including the base Oligonucleotide probe (10) that hybridizes to the region where the oligonucleotide hybridizes a) Bases 196 to 225 of SEQ ID NO: 8 An oligonucleotide probe having a base sequence complementary to a continuous base sequence of 25 to 35 bases, and / or b) a continuous base sequence of 25 to 35 bases including bases 196 to 225 of SEQ ID NO: 8 Complementary nucleotide sequence consisting of a nucleotide sequence in which one or several bases are deleted, substituted, added or inserted, and a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes (11) a) an oligonucleotide probe comprising a base sequence of 25 to 35 bases comprising bases 191 to 220 of SEQ ID NO: 9, and / or b) of SEQ ID NO: 9 Is it a base sequence in which one or several bases are deleted, substituted, added or inserted in a continuous base sequence of 25 to 35 bases including bases 191 to 220? The oligonucleotide probe that hybridizes to the region to which the oligonucleotide a) of the DNA encoding the 16S rRNA of the microorganism hybridizes (12) a) a sequence comprising bases 191 to 220 of SEQ ID NO: 9 An oligonucleotide probe having a base sequence complementary to a base sequence of 25 to 35 bases, and / or b) complementary to a continuous base sequence of 25 to 35 bases including bases 191 to 220 of SEQ ID NO: 9 The base sequence consists of a base sequence in which one or several bases are deleted, substituted, added or inserted, and hybridizes to the region to which the oligonucleotide a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. To oligonucleotide probes.

  In one embodiment, the present invention provides at least one selected from the group consisting of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis, wherein the set of oligonucleotide probes is immobilized on a carrier. The present invention relates to a microarray for detecting species of microorganisms.

  In the microarray, the carrier preferably has a carbon layer and a chemical modification group on the surface.

  In one embodiment, the present invention is a method for detecting at least one microorganism selected from the group consisting of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis from a sample, Extracting RNA, using the extracted genomic DNA or RNA as a template, amplifying DNA encoding 16S rRNA of the microorganism, and detecting amplified DNA using the set of oligonucleotide probes or the microarray The method comprising the steps of:

  INDUSTRIAL APPLICABILITY According to the present invention, Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis can be detected, and a simple and quick means capable of identifying them is provided.

  In one embodiment, the present invention provides at least one selected from the group consisting of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis, comprising the following oligonucleotide probes (1) to (12): Relates to a set of oligonucleotide probes for detecting microorganisms.

The set of oligonucleotide probes of the present invention comprises (1) the following oligonucleotide probes as a) and / or b):
a) an oligonucleotide probe comprising a base sequence complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases, including bases 190 to 219 of SEQ ID NO: 1;
b) 1 or several, preferably 2 to 3 in a base sequence complementary to a continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 190 to 219 in SEQ ID NO: 1. And a oligonucleotide probe that hybridizes to a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes. Here, SEQ ID NO: 1 represents the base sequence of DNA encoding 16S rRNA of Geobacillus stearothermophilus.

  The set of oligonucleotide probes of the present invention is complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 190 to 219 of SEQ ID NO: 1, as the oligonucleotide probe of (1). An oligonucleotide probe that hybridizes to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Also good.

  The oligonucleotide probe (1) hybridizes to DNA encoding Bacillus caldotenax 16S rRNA. Therefore, in “b)“ of the DNA encoding the 16S rRNA of the microorganism hybridizes to the region where the oligonucleotide of a) hybridizes ”, in other words,“ of the DNA encoding the 16S rRNA of Bacillus caldotenax ” “It hybridizes to the region to which the oligonucleotide of a) hybridizes”.

  On the other hand, the oligonucleotide probe (1) does not specifically hybridize to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention comprises the following oligonucleotide probes of a) and / or b) as the oligonucleotide probes of (2):
a) Oligonucleotide probe comprising a base sequence complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 198 to 227 of SEQ ID NO: 2 b) 198 of SEQ ID NO: 2 In a base sequence complementary to a continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including base No. 227, 1 or several, preferably 2 to 3 bases are deleted, substituted, It comprises an oligonucleotide probe that hybridizes to a region to which the oligonucleotide a) of the DNA encoding the 16S rRNA of the microorganism hybridizes, comprising an added or inserted base sequence. Here, SEQ ID NO: 2 represents the base sequence of DNA encoding 16S rRNA of Geobacillus thermoglucosidasius.

  The oligonucleotide probe set of the present invention is complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 198 to 227 of SEQ ID NO: 2, as the oligonucleotide probe of (2). An oligonucleotide probe that hybridizes to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Also good.

  The oligonucleotide probe (2) hybridizes to DNA encoding 16S rRNA of Bacillus caldotenax, Bacillus cereus, Bacillus subtilis and Thermoactinomyces vulgaris. Therefore, in “b),“ hybridizes to the region in which the oligonucleotide of a) among the DNAs encoding 16S rRNA of the microorganism hybridizes ”means“ Bacillus caldotenax, Bacillus cereus, Bacillus subtilis and Thermoactinomyces vulgaris ”. Of the 16S rRNA-encoding DNA, it hybridizes with the region to which the oligonucleotide a) hybridizes.

  On the other hand, the oligonucleotide probe (2) does not specifically hybridize to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention comprises the following oligonucleotide probes of a) and / or b) as the oligonucleotide probes of (3):
a) an oligonucleotide probe comprising a base sequence of 25 to 35 bases, preferably 28 to 33 bases, comprising bases 167 to 196 of SEQ ID NO: 3;
b) In the continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 167 to 196 in SEQ ID NO: 3, one or several, preferably 2-3 bases are deleted, It comprises an oligonucleotide probe that consists of a substituted, added or inserted nucleotide sequence and hybridizes to a region to which the oligonucleotide of a) of the DNA encoding 16S rRNA of the microorganism hybridizes. Here, SEQ ID NO: 3 represents the nucleotide sequence of DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius.

  The set of oligonucleotide probes of the present invention comprises a base sequence of 25 to 35 bases, preferably 28 to 33 bases including the bases of 167 to 196 of SEQ ID NO: 3, and 95 as the oligonucleotide probe of (3). It may comprise an oligonucleotide probe that hybridizes to a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes.

  The oligonucleotide probe of (3) hybridizes to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius. Therefore, in “b)” “hybridizes to the region where the oligonucleotide of a) among the DNAs encoding 16S rRNA of the microorganism hybridizes” means, in other words, “of the DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius” “It hybridizes to the region to which the oligonucleotide of a) hybridizes”.

  On the other hand, the oligonucleotide probe (3) does not specifically hybridize to DNA encoding 16S rRNA of Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention includes the following oligonucleotide probes of a) and / or b) as the oligonucleotide probes of (4):
a) an oligonucleotide probe comprising a base sequence complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases, including bases 167 to 196 of SEQ ID NO: 3;
b) In the base sequence complementary to the continuous base sequence of 25 to 35 bases including bases 167 to 196 in SEQ ID NO: 3, one or several, preferably 2 to 3 bases are deleted or replaced And an oligonucleotide probe that hybridizes to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes.

  The oligonucleotide probe set of the present invention is complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases, including bases 167 to 196 of SEQ ID NO: 3, as the oligonucleotide probe of (4). An oligonucleotide probe that hybridizes to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Also good.

  The oligonucleotide probe (4) hybridizes to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius. Therefore, in “b)” “hybridizes to the region where the oligonucleotide of a) among the DNAs encoding 16S rRNA of the microorganism hybridizes” means, in other words, “of the DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius” “It hybridizes to the region to which the oligonucleotide of a) hybridizes”.

  On the other hand, the oligonucleotide probe (4) does not specifically hybridize to DNA encoding 16S rRNA of Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention comprises the following oligonucleotide probes of a) and / or b) as the oligonucleotide probes of (5):
a) an oligonucleotide probe comprising a base sequence complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases, including bases 186 to 215 of SEQ ID NO: 4;
b) 1 or several, preferably 2 to 3 in a base sequence complementary to a continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 186 to 215 in SEQ ID NO: 4 And a oligonucleotide probe that hybridizes to a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes. Here, SEQ ID NO: 4 represents the base sequence of DNA encoding 16S rRNA of Bacillus coagulans.

  The oligonucleotide probe set of the present invention is complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 186 to 215 of SEQ ID NO: 4, as the oligonucleotide probe of (5). An oligonucleotide probe that hybridizes to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Also good.

  The oligonucleotide probe of (5) hybridizes to DNA encoding Thermoactinomyces vulgaris 16S rRNA. Therefore, in b), “of the DNA encoding the 16S rRNA of the microorganism hybridizes to the region to which the oligonucleotide of a) hybridizes”, in other words, “of the DNA encoding the 16S rRNA of Thermoactinomyces vulgaris” “It hybridizes to the region to which the oligonucleotide of a) hybridizes”.

  On the other hand, the oligonucleotide probe (5) does not specifically hybridize to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius Bacillus caldotenax, Bacillus cereus, Bacillus subtilis and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention comprises the following oligonucleotide probes of a) and / or b) as the oligonucleotide probes of (6):
a) an oligonucleotide probe comprising a base sequence complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases, including bases 170 to 199 of SEQ ID NO: 5;
b) 1 or several, preferably 2 to 3 in a base sequence complementary to a continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 170 to 199 in SEQ ID NO: 5 And a oligonucleotide probe that hybridizes to a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes. Here, SEQ ID NO: 5 represents the base sequence of DNA encoding 16S rRNA of Bacillus caldotenax.

  The oligonucleotide probe set of the present invention is complementary to a continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 170 to 199 of SEQ ID NO: 5, as the oligonucleotide probe of (6). An oligonucleotide probe that hybridizes to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Also good.

  The oligonucleotide probe of (6) hybridizes to DNA encoding 16S rRNA of Bacillus caldotenax. Therefore, in “b)“ of the DNA encoding the 16S rRNA of the microorganism hybridizes to the region where the oligonucleotide of a) hybridizes ”, in other words,“ of the DNA encoding the 16S rRNA of Bacillus caldotenax ” “It hybridizes to the region to which the oligonucleotide of a) hybridizes”.

  On the other hand, the oligonucleotide probe (6) does not specifically hybridize to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention comprises the following oligonucleotide probes of a) and / or b) as the oligonucleotide probes of (7):
a) an oligonucleotide probe comprising a base sequence complementary to a base sequence of 25 to 35 bases, preferably 28 to 33 bases, including bases 170 to 200 of SEQ ID NO: 6;
b) 1 or several, preferably 2 to 3 in a base sequence complementary to a continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 170 to 200 of SEQ ID NO: 6 And a oligonucleotide probe that hybridizes to a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes. Here, SEQ ID NO: 6 represents the base sequence of DNA encoding 16S rRNA of Bacillus cereus.

  The set of oligonucleotide probes of the present invention is complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 170 to 200 of SEQ ID NO: 6, as the oligonucleotide probe of (7). An oligonucleotide probe that hybridizes to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Also good.

  The oligonucleotide probe of (7) hybridizes to DNA encoding Bacillus cereus 16S rRNA. Therefore, in “b)“ of the DNA encoding the 16S rRNA of the microorganism hybridizes to the region where the oligonucleotide of a) hybridizes ”, in other words,“ of the DNA encoding the 16S rRNA of Bacillus cereus ” “It hybridizes to the region to which the oligonucleotide of a) hybridizes”.

  On the other hand, the oligonucleotide probe (7) does not specifically hybridize to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention includes the following oligonucleotide probes of a) and / or b) as the oligonucleotide probes of (8):
a) an oligonucleotide probe comprising a base sequence complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases, including bases 168 to 198 of SEQ ID NO: 7;
b) 1 or several, preferably 2 to 3 in a base sequence complementary to a continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 168 to 198 in SEQ ID NO: 7 And a oligonucleotide probe that hybridizes to a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes. Here, SEQ ID NO: 7 represents the base sequence of DNA encoding 16S rRNA of Bacillus subtilis.

  The oligonucleotide probe set of the present invention is complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 168 to 198 of SEQ ID NO: 7, as the oligonucleotide probe of (8). An oligonucleotide probe that hybridizes to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Also good.

  The oligonucleotide probe of (8) hybridizes to DNA encoding Bacillus subtilis 16S rRNA. Therefore, in b), “of the DNA encoding 16S rRNA of the microorganism hybridizes to the region to which the oligonucleotide of a) hybridizes”, in other words, “of DNA encoding 16S rRNA of Bacillus subtilis” “It hybridizes to the region to which the oligonucleotide of a) hybridizes”.

  On the other hand, the oligonucleotide probe (8) does not specifically hybridize to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Thermoactinomyces vulgaris and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention comprises the following oligonucleotide probe of a) and / or b) as the oligonucleotide probe of (9):
a) an oligonucleotide probe comprising a base sequence of 25 to 35 bases, preferably 28 to 33 bases, comprising bases 196 to 225 of SEQ ID NO: 8;
b) In a continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 196 to 225 in SEQ ID NO: 8, one or several, preferably 2-3 bases are deleted, It comprises an oligonucleotide probe that consists of a substituted, added or inserted nucleotide sequence and hybridizes to a region to which the oligonucleotide of a) of the DNA encoding 16S rRNA of the microorganism hybridizes. Here, SEQ ID NO: 8 represents the base sequence of DNA encoding Thermoactinomyces vulgaris 16S rRNA.

  The set of oligonucleotide probes of the present invention comprises a base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 196 to 225 of SEQ ID NO. It may comprise an oligonucleotide probe that hybridizes to a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes.

  The oligonucleotide probe of (9) hybridizes to DNA encoding Thermoactinomyces vulgaris 16S rRNA. Therefore, in b), “of the DNA encoding the 16S rRNA of the microorganism hybridizes to the region to which the oligonucleotide of a) hybridizes”, in other words, “of the DNA encoding the 16S rRNA of Thermoactinomyces vulgaris” “It hybridizes to the region to which the oligonucleotide of a) hybridizes”.

  On the other hand, the oligonucleotide probe (9) does not specifically hybridize to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention comprises the following oligonucleotide probe of a) and / or b) as the oligonucleotide probe of (10):
a) an oligonucleotide probe comprising a base sequence complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases, including bases 196 to 225 of SEQ ID NO: 8;
b) 1 or several, preferably 2 to 3 in a base sequence complementary to a continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 196 to 225 in SEQ ID NO: 8 And a oligonucleotide probe that hybridizes to a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes.

  The oligonucleotide probe set of the present invention is complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 196 to 225 of SEQ ID NO: 8, as the oligonucleotide probe of (10). An oligonucleotide probe that hybridizes to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Also good.

  The oligonucleotide probe of (10) hybridizes to DNA encoding 16S rRNA of Bacillus caldotenax, Bacillus cereus, Bacillus subtilis and Thermoactinomyces vulgaris. Therefore, in “b),“ hybridizes to the region in which the oligonucleotide of a) among the DNAs encoding 16S rRNA of the microorganism hybridizes ”means“ Bacillus caldotenax, Bacillus cereus, Bacillus subtilis and Thermoactinomyces vulgaris ”. Of the 16S rRNA-encoding DNA, it hybridizes with the region to which the oligonucleotide a) hybridizes.

  On the other hand, the oligonucleotide probe of (10) does not specifically hybridize to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention comprises the following oligonucleotide probes of a) and / or b) as the oligonucleotide probes of (11):
a) an oligonucleotide probe comprising a base sequence of 25 to 35 bases, preferably 28 to 33 bases, comprising bases 191 to 220 of SEQ ID NO: 9;
b) In the continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 191 to 220 in SEQ ID NO: 9, one or several, preferably 2-3 bases are deleted, It comprises an oligonucleotide probe that consists of a substituted, added or inserted nucleotide sequence and does not hybridize to any DNA encoding 16S rRNA of the microorganism. Here, SEQ ID NO: 9 represents the base sequence of DNA encoding Staphylococcus epidermidis 16S rRNA.

  The set of oligonucleotide probes of the present invention comprises a base sequence of 25 to 35 bases, preferably 28 to 33 bases, including bases 191 to 220 of SEQ ID NO: 9, and the oligonucleotide probe of (11). It may contain an oligonucleotide probe which consists of a base sequence having at least% identity and does not hybridize to any DNA encoding 16S rRNA of the microorganism.

  The oligonucleotide probe (11) does not specifically hybridize to any DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris, and Staphylococcus epidermidis.

The set of oligonucleotide probes of the present invention comprises the following oligonucleotide probes of a) and / or b) as oligonucleotide probes of (12):
a) an oligonucleotide probe comprising a base sequence complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases, including bases 191 to 220 of SEQ ID NO: 9;
b) 1 or several, preferably 2 to 3 in a base sequence complementary to a continuous base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 191 to 220 of SEQ ID NO: 9 And a oligonucleotide probe that hybridizes to a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes.

  The oligonucleotide probe set of the present invention is complementary to the base sequence of 25 to 35 bases, preferably 28 to 33 bases including bases 191 to 220 of SEQ ID NO: 9, as the oligonucleotide probe of (12). An oligonucleotide probe that hybridizes to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Also good.

  The oligonucleotide probe of (12) hybridizes to DNA encoding Staphylococcus epidermidis 16S rRNA. Therefore, in b), “of the DNA encoding the 16S rRNA of the microorganism hybridizes to the region where the oligonucleotide of a) hybridizes”, in other words, “of the DNA encoding the 16S rRNA of Staphylococcus epidermidis” “It hybridizes to the region to which the oligonucleotide of a) hybridizes”.

  On the other hand, the oligonucleotide probe (12) does not specifically hybridize to DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis and Thermoactinomyces vulgaris.

  In the present specification, “DNA encoding 16S rRNA (ribosomal RNA)” refers to DNA amplified using microbial genomic DNA or RNA as a template, that is, a region encoding 16S rRNA using microbial genomic DNA as a template. DNA obtained by amplification and DNA obtained by amplifying this using 16S rRNA of a microorganism as a template are included. Therefore, DNA encoding 16S rRNA includes DNA encoding 16S rRNA and its complementary strand in the genomic DNA of microorganisms. Hereinafter, “DNA encoding 16S rRNA” may be referred to as “16S rDNA”. In addition, the base sequence of 16S rDNA of each bacterium can be obtained from the gene database GenBank of NCBI (National Center for Biotechnology Information), for example.

  In the present specification, “hybridize” preferably means to hybridize under stringent conditions. Stringent conditions refer to conditions in which specific hybrids are not formed and non-specific hybrids are not formed.For example, after hybridization at 50 ° C. for 16 hours, 2 × SSC / 0.2% SDS, room temperature This refers to the condition of washing for 5 minutes and 2 × SSC at room temperature for 5 minutes.

  The set of oligonucleotide probes of the present invention only needs to contain at least one kind of each of the oligonucleotide probes (1) to (12), and may contain a plurality of kinds of oligonucleotide probes. That is, the set of oligonucleotide probes of the present invention includes at least 12 types of oligonucleotide probes.

  DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius hybridizes with the oligonucleotide probes (3) and (4). Therefore, when genomic DNA or RNA extracted from a sample containing Alicyclobacillus acidocaldarius is used as a template, DNA encoding 16S rRNA is amplified and reacted with the set of oligonucleotide probes of the present invention, (3) and ( In 4), a specific hybridization reaction occurs.

  DNA encoding 16S rRNA of Bacillus caldotenax hybridizes with the oligonucleotide probes (1), (2), (6) and (10). Therefore, when genomic DNA or RNA extracted from a sample containing Bacillus caldotenax is used as a template, DNA encoding 16S rRNA is amplified and reacted with the set of oligonucleotide probes of the present invention, (1), ( Hybridization reaction occurs specifically in 2), (6) and (10).

  DNA encoding 16S rRNA of Bacillus cereus hybridizes with the oligonucleotide probes (2), (7) and (10). Accordingly, when genomic DNA or RNA extracted from a sample containing Bacillus cereus is used as a template, DNA encoding 16S rRNA is amplified and reacted with the set of oligonucleotide probes of the present invention, (2), ( Hybridization reaction occurs specifically in 7) and (10).

  DNA encoding 16S rRNA of Bacillus subtilis hybridizes with the oligonucleotide probes (2), (8) and (10). Accordingly, when genomic DNA or RNA extracted from a sample containing Bacillus subtilis is used as a template, DNA encoding 16S rRNA is amplified and reacted with the set of oligonucleotide probes of the present invention, the oligonucleotide probes (2), ( Hybridization reaction occurs specifically in 8) and (10).

  DNA encoding Thermoactinomyces vulgaris 16S rRNA hybridizes with the oligonucleotide probes (2), (5), (9) and (10). Accordingly, when genomic DNA or RNA extracted from a sample containing Thermoactinomyces vulgaris is used as a template, DNA encoding 16S rRNA is amplified and reacted with the set of oligonucleotide probes of the present invention, (2), ( 5), (9) and (10) specifically cause hybridization reaction.

  The DNA encoding Staphylococcus epidermidis 16S rRNA hybridizes with (12) of the oligonucleotide probe. Therefore, when genomic DNA or RNA extracted from a sample containing Staphylococcus epidermidis is used as a template, DNA encoding 16S rRNA is amplified and reacted with the set of oligonucleotide probes of the present invention, it is specific for oligonucleotide probe (12). Hybridization reaction occurs.

  The oligonucleotide probe of the present invention can be obtained, for example, by chemically synthesizing with a nucleic acid synthesizer. As the nucleic acid synthesizer, an apparatus called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer or the like can be used.

  The oligonucleotide probe set of the present invention is preferably used in the form of a microarray by being immobilized on a carrier. As the material for the carrier, those known in the art can be used and are not particularly limited. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductor materials such as graphite and carbon typified by carbon fiber; single crystal silicon, amorphous Silicon materials represented by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these silicon materials represented by SOI (silicon on insulator), etc .; glass, quartz glass, alumina, sapphire, ceramics, Inorganic materials such as stellite and photosensitive glass; polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol , Polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide And organic materials such as polysulfone. The shape of the carrier is not particularly limited, but is preferably a flat plate shape.

  In the present invention, a carrier having a carbon layer and a chemical modification group on the surface is preferably used as the carrier. Carriers having a carbon layer and a chemical modification group on the surface include those having a carbon layer and a chemical modification group on the surface of the substrate, and those having a chemical modification group on the surface of the substrate made of the carbon layer. As the material for the substrate, those known in the art can be used, and there are no particular restrictions, and the same materials as those described above can be used.

  The present invention is suitably used for a carrier having a fine flat plate structure. The shape is not limited to a rectangle, a square, or a round shape, but a shape of 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 3 to 5 mm square is usually used. Since it is easy to produce a carrier having a fine flat plate structure, it is preferable to use a substrate made of a silicon material or a resin material. In particular, a carrier having a carbon layer and a chemical modification group on the surface of a substrate made of single crystal silicon is more preferable. preferable. Single crystal silicon has a slightly different orientation of the crystal axis in some parts (sometimes called a mosaic crystal), or includes atomic scale disturbances (lattice defects) Are also included.

  In the present invention, the carbon layer formed on the substrate is not particularly limited, but synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene) , Carbon nanotubes), a mixture thereof, or a laminate of them is preferably used. Further, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide may be used. Here, the soft diamond is a generic term for an incomplete diamond structure that is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited. The carbon layer has excellent chemical stability, can withstand subsequent reactions in the introduction of chemical modification groups and binding to the analyte, and the binding is flexible because of the electrostatic binding to the analyte. It is advantageous in that it has the property of being transparent, it is transparent to the detection system UV because there is no UV absorption, and it can be energized during electroblotting. Further, it is advantageous in that nonspecific adsorption is small in the binding reaction with the analyte. As described above, a carrier whose substrate itself is made of a carbon layer may be used.

  In the present invention, the carbon layer can be formed by a known method. For example, microwave plasma CVD (Chemical vapor deposit) method, ECRCVD (Electric cyclotron resonance chemical vapor deposit) method, ICP (Inductive coupled plasma) method, DC sputtering method, ECR (Electric cyclotron resonance) sputtering method, ionization deposition method, arc Examples thereof include a vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.

  In the high-frequency plasma CVD method, a raw gas (methane) is decomposed by glow discharge generated between electrodes by a high frequency, and a DLC (diamond-like carbon) layer is synthesized on the substrate. In the ionization vapor deposition method, the source gas (benzene) is decomposed and ionized using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on the substrate by a bias voltage. The DLC layer may be formed by ionized vapor deposition in a mixed gas composed of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of the remaining methane gas.

  In the arc evaporation method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), and a plasma of carbon atoms is generated from the cathode to generate an evaporation source. Further, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.

  In the laser vapor deposition method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light and melting it, and depositing carbon atoms on a glass substrate.

  When the carbon layer is formed on the surface of the substrate, the thickness of the carbon layer is usually a monomolecular layer to about 100 μm. If it is too thin, the surface of the base substrate may be locally exposed, and conversely thick. In this case, the productivity is deteriorated, so that the thickness is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

  By introducing a chemical modification group on the surface of the substrate on which the carbon layer is formed, the oligonucleotide probe can be firmly immobilized on the carrier. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an active ester group.

  An amino group can be introduced, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment. Alternatively, the carbon layer can be chlorinated by irradiation with ultraviolet rays in chlorine gas, and further irradiated with ultraviolet rays in ammonia gas. Alternatively, it can also be carried out by reacting a polyvalent amine gas such as methylenediamine or ethylenediamine with a chlorinated carbon layer.

The introduction of the carboxyl group can be carried out, for example, by reacting an appropriate compound with the carbon layer aminated as described above. As a compound used for introducing a carboxyl group, for example, the formula: XR ¹ —COOH (wherein X represents a halogen atom and R ¹ represents a divalent hydrocarbon group having 1 to 12 carbon atoms). Halocarboxylic acids shown, such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R ² -COOH (Wherein R ² represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), for example, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid Polyvalent carboxylic acids such as polyacrylic acid, polymethacrylic acid, trimellitic acid and butanetetracarboxylic acid; Formula: R ³ —CO—R ⁴ —COOH (wherein R ³ is a hydrogen atom or having 1 to 12 carbon atoms) divalent hydrocarbon group, R ⁴ is a divalent hydrocarbon having 1 to 12 carbon atoms Keto acid or aldehyde acids represented by representing) a; ^{formula:. X-OC-R 5} -COOH ( wherein, X represents a divalent hydrocarbon group having a halogen atom, R ⁵ represents a single bond or 1 to 12 carbon atoms Dicarboxylic acid monohalides represented by, for example, succinic acid monochloride, malonic acid monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, butanetetracarboxylic anhydride, etc. Is mentioned.

  Introduction of an epoxy group can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Or it can obtain by making an organic peracid react with the carbon = carbon double bond which a carbon layer contains. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

  Introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.

  Introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.

  The active ester group means an ester group having an electron-withdrawing group having high acidity on the alcohol side of the ester group and activating the nucleophilic reaction, that is, an ester group having high reaction activity. The ester group has an electron-withdrawing group on the alcohol side of the ester group and is activated more than the alkyl ester. The active ester group has reactivity with groups such as amino group, thiol group, and hydroxyl group. More specifically, an active ester group in which phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. have much higher activity than alkyl esters etc. Known as. More specifically, examples of the active ester group include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, 5-norbornene-2,3-dicarboximide group and the like. In particular, an N-hydroxysuccinimide group is preferably used.

  The introduction of the active ester group is performed, for example, by converting the carboxyl group introduced as described above into a dehydrating condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N- It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the terminal of the hydrocarbon group via an amide bond can be formed (Japanese Patent Laid-Open No. 2001-139532).

  The oligonucleotide probe of the present invention is dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is spotted on a carrier by a spotter device or the like. By doing so, a microarray in which oligonucleotide probes are immobilized on a carrier can be produced. Alternatively, the spotting solution may be spotted manually with a micropipette.

  Incubation is preferably performed after spotting in order to advance the reaction of the oligonucleotide probe binding to the carrier. Incubation is usually performed at a temperature of −20 to 100 ° C., preferably 0 to 90 ° C., usually for 0.5 to 16 hours, preferably 1 to 2 hours. Incubation is preferably performed under a high humidity atmosphere, for example, under conditions of a humidity of 50 to 90%. Following the incubation, washing is preferably performed using a washing solution (for example, 50 mM TBS / 0.05% Tween 20) in order to remove DNA not bound to the carrier.

  The present invention is also selected from the group consisting of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis using the set of oligonucleotide probes of the present invention or a microarray on which this is immobilized on a carrier. The present invention relates to a method for detecting at least one microorganism.

  The microorganism detection method of the present invention includes a step of extracting genomic DNA or RNA from a sample, a step of amplifying DNA encoding 16S rRNA of the microorganism using the extracted genomic DNA or RNA as a template, Detecting the amplified DNA using a set of oligonucleotide probes or a microarray of the present invention.

  In the detection method of the present invention, first, genomic DNA or RNA is extracted from a sample. DNA extraction can be performed by the same method as in the case of conventional genomic DNA preparation. For example, a DNA extraction method using phenol / chloroform, ethanol, sodium hydroxide, CTAB or the like can be used. RNA extraction can also be performed by methods known to those skilled in the art, and is not particularly limited. For example, guanidine thiocyanate / cesium chloride ultracentrifugation method, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, guanidine thiocyanate / phenol / chloroform method (Chomczynski, P. and Sacchi, N., Anal. Biochem., 162 , 156-159 (1987)).

  The sample is not particularly limited as long as it can contain the target microorganism to be used. The detection method of the present invention is particularly preferably used for detection of microorganisms contained in food samples and samples derived from the production process. The food includes beverages, and the detection method of the present invention is, for example, vegetables, meat, fruits, cereals, eggs, seafood, spices (herbs, spices), potatoes, beans / nuts and seeds , Mushrooms, algae, milk, and processed products thereof can be widely targeted. Examples of the sample derived from the food production process include cleaning liquids for containers and plants.

  Next, in the detection method of the present invention, an amplification reaction is performed using the extracted genomic DNA or RNA as a template to amplify a region encoding 16S rRNA in genomic DNA or a region of 16S rRNA in ribosomal RNA. As the amplification reaction, polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), or the like can be applied.

  In the amplification reaction, it is desirable to add a label so that the amplified region can be identified. At this time, the method for labeling the amplified DNA is not particularly limited. For example, a method in which a primer used for the amplification reaction is labeled in advance may be used, or a modified nucleotide is used as a substrate for the amplification reaction. You may use the method to do. The labeling substance is not particularly limited, and radioisotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin can be used.

  In the amplification reaction, it is preferable to amplify the DNA region encoding the 16S rRNA of the microorganism to be detected or identified in one reaction system. That is, even if there are a plurality of types of microorganisms to be detected or identified, it is desirable to simultaneously amplify a plurality of types of microorganism-derived DNA in one reaction vessel. Such a reaction system is called “1-tube PCR” when PCR is applied as an amplification reaction.

  This reaction system also includes buffers necessary for amplification and labeling, heat-resistant DNA polymerase, amplification region-specific primers, modified nucleotide triphosphates (specifically, nucleotide triphosphates added with a DIG label, etc.), nucleotide triphosphates And a reaction system kit containing magnesium chloride and the like.

  When amplifying DNA encoding 16S rRNA of a microorganism to be detected or identified, a universal primer is preferably used as a primer. Specifically, a primer set consisting of the base sequence shown in SEQ ID NO: 10 and a primer set consisting of the base sequence shown in SEQ ID NO: 11 can be exemplified. According to the primer set, at least DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis can be amplified as a detection target region.

  By reacting the amplified DNA with the set of oligonucleotide probes of the present invention and detecting the hybridization reaction, the microorganisms contained in the sample can be identified and identified. The detection of the hybridization reaction includes the presence or absence of the hybridization reaction and the quantification of the amount of the hybridized DNA. The amount of microorganisms can also be quantified by measuring the amount of DNA hybridized to the oligonucleotide probe.

  According to the present invention, Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis can be detected in a sample. Furthermore, Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis can be distinguished from each other.

Example 1 Production of a microarray
Silicon carrier creation
A silicon support was prepared by coating a diamond-like carbon layer on a 3 mm square silicon substrate, modifying with a carboxyl group, and then active esterifying with N-hydroxysuccinimide.

Preparation of DNA-immobilized carrier Oligonucleotide (Hokkaido System Science Co., Ltd.) was adjusted to 100 μM with sterile water. It was dissolved in 4 times concentrated Sol.6 (manufactured by Toyo Kohan Co., Ltd.) to obtain a DNA solution with a final concentration of 5 μM. A heat-releasable double-sided tape (manufactured by Nitto Denko Corporation) was applied as a sheet coated with a heat-releasable adhesive on a slide glass, and a silicon carrier was placed using a transfer device (manufactured by Hitachi High-Technologies Corporation). The DNA solution was spotted with SPBIO2000 (manufactured by Hitachi Soft Engineering).

Cleaning DNA immobilization support
The slide glass on which the DNA-immobilized support was placed was placed in a baking chamber at 80 ° C. and left for 1 hour to be immobilized. Subsequently, washing was performed for 15 minutes with a washing solution at room temperature (2 × SSC / 0.2% SDS). Subsequently, washing was performed for 5 minutes with a boiling (about 90 ° C. to 100 ° C.) washing solution (2 × SSC / 0.2% SDS). Rinsing was performed 3 times with ultra pure water. Water was blown away with a centrifuge.

  In Table 1 below, the oligonucleotide probes immobilized on the carrier are numbered and the base sequences of the oligonucleotide probes and the microorganisms that are the origins of the base sequences are shown.

  Probe 2 corresponds to an oligonucleotide probe having a base sequence complementary to bases 190 to 219 in DNA base sequence (SEQ ID NO: 1) encoding Geobacillus steatothermophilus 16S rRNA.

  The probe 4 corresponds to an oligonucleotide probe having a base sequence complementary to the 198th to 227th bases in the base sequence (SEQ ID NO: 2) of DNA encoding 16S rRNA of Geobacillus thermoglucosidasius.

  The probe 5 corresponds to an oligonucleotide probe consisting of bases 167 to 196 in the base sequence (SEQ ID NO: 3) of DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius.

  Probe 6 corresponds to an oligonucleotide probe having a base sequence complementary to bases 167 to 196 in the base sequence (SEQ ID NO: 3) of DNA encoding 16S rRNA of Alicyclobacillus acidocaldarius.

  The probe 8 corresponds to an oligonucleotide probe having a base sequence complementary to the 186th to 215th bases in the DNA encoding 16S rRNA of Bacillus coagulans (SEQ ID NO: 4).

  The probe 10 corresponds to an oligonucleotide probe having a base sequence complementary to bases 170 to 199 of DNA (SEQ ID NO: 5) encoding Bacillus caldotenax 16S rRNA.

  The probe 12 corresponds to an oligonucleotide probe having a base sequence complementary to bases 170 to 200 of DNA (SEQ ID NO: 6) encoding Bacillus cereus 16S rRNA.

  The probe 14 corresponds to an oligonucleotide probe having a base sequence complementary to the 168th to 198th bases of DNA (SEQ ID NO: 7) encoding Bacillus subtilis 16S rRNA.

  The probe 17 corresponds to an oligonucleotide probe consisting of bases 196 to 225 in DNA (SEQ ID NO: 8) encoding Thermoactinomyces vulgaris 16S rRNA.

  The probe 18 corresponds to an oligonucleotide probe having a base sequence complementary to bases 196 to 225 in DNA (SEQ ID NO: 8) encoding Thermoactinomyces vulgaris 16S rRNA.

  The probe 19 corresponds to an oligonucleotide probe consisting of bases 191 to 220 in DNA (SEQ ID NO: 9) encoding 16S rRNA of Staphylococcus epidermidis group.

  The probe 20 corresponds to an oligonucleotide probe having a base sequence complementary to bases 191 to 220 in DNA (SEQ ID NO: 9) encoding Staphylococcus epidermidis group 16S rRNA.

  FIG. 1 shows the spot position of each probe on the microarray.

Example 2 Detection of microorganisms
DNA extraction:
Genomic DNA was extracted from six types of microorganisms (Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis). Specifically, a colony on an agar medium is scraped with a platinum loop, dissolved in 30 μl of ultrapure water, boiled at 100 ° C. for 10 minutes, centrifuged (10000 rpm, 1 minute), the supernatant was recovered, and a DNA sample It was.

Each obtained DNA sample was subjected to PCR to amplify the 16s rDNA region. At that time, a fluorescent substance (Cy5) was incorporated into DNA. The following primer pairs were used for amplification for all six microorganisms:
Forward primer: 5'-AGAGTTTGATCMTGGCTCAG-3 '(SEQ ID NO: 10)
Reverse primer: 5'-ATTACCGCGGCTGCTGGCA-3 '(SEQ ID NO: 11)
PCR conditions are as follows.

  The amplification reaction was confirmed using 0.5 μl of the PCR product for electrophoresis. The results are shown in FIG. From the results of FIG. 2, it can be seen that the 16S rDNA region was appropriately amplified in each microorganism by PCR.

  An equal amount of 10 × SSC / 1% SDS was added to the PCR product obtained for each of the six types of microorganisms to obtain a hybridization solution (final hybridization solution concentration: 5 × SSC / 0.5% SDS). 15 μl of the hybridization solution was added to each microarray prepared in Example 1, covered with a cover film, and reacted at 50 ° C. for 16 hours. The sample was washed once with 2 × SSC / 0.2% SDS and twice with 2 × SSC, and a fluorescent image was taken with a fluorescent scanner (Fuji Photo Film, FLA8000). The fluorescence images obtained for the PCR products derived from each microorganism are shown in FIG. The upper row is a fluorescence image obtained in the first experiment, and the lower row is a fluorescence image obtained in the second experiment.

  When a sample (PCR amplification product) derived from Alicyclobacillus acidocaldarius was reacted with the microarray, hybridization reaction was detected at spots 5 and 6.

  When a sample (PCR amplification product) derived from Bacillus caldotenax was reacted with the microarray, hybridization reaction was detected at spots 2, 4, 10 and 18.

  When a sample (PCR amplification product) derived from Bacillus cereus was reacted with the microarray, hybridization reaction was detected at spots 4, 12 and 18.

  When a sample (PCR amplification product) derived from Bacillus subtilis was reacted with a microarray, hybridization reaction was detected at spots 4, 14 and 18.

  When a sample (PCR amplification product) derived from Thermoactinomyces vulgaris was reacted with a microarray, hybridization reaction was detected at spots 4, 8 and 18.

  When a sample (PCR amplification product) derived from Staphylococcus epidermidis was reacted with the microarray, a hybridization reaction was detected at the spot 20.

  It turns out that said result has reproducibility in the 1st time and the 2nd time.

  From the above, six types of microorganisms (Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis) contained in the sample were detected by the combination (set) of the oligonucleotide probe of the present invention. It was shown that it could be identified.

The spot position on the microarray of each oligonucleotide probe is shown. In Example 2, the PCR product is subjected to electrophoresis to confirm the amplification reaction. The result of having made the microarray of this invention hybridize the sample derived from various microorganisms and image | photographed the fluorescence image is shown. The upper row is a fluorescence image obtained in the first experiment, and the lower row is a fluorescence image obtained in the second experiment.

Claims (4)

A method for detecting at least one microorganism selected from the group consisting of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis, comprising the following oligonucleotide probes (1) to (12): Set of oligonucleotide probes:
(1) a) an oligonucleotide probe having a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 190 to 219 of SEQ ID NO: 1 and / or b) 190 of SEQ ID NO: 1 Comprising a base sequence complementary to a continuous base sequence of 25 to 35 bases including base No. 219, consisting of a base sequence in which one or several bases are deleted, substituted, added or inserted, An oligonucleotide probe that hybridizes to the region to which the oligonucleotide a) of the DNA encoding rRNA hybridizes (2) a) 25 to 35 consecutive bases including bases 198 to 227 of SEQ ID NO: 2 An oligonucleotide probe having a base sequence complementary to the sequence, and / or b) a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 198 to 227 of SEQ ID NO: 2 An oligonucleotide which has a base sequence in which one or several bases have been deleted, substituted, added or inserted, and which hybridizes to the region to which the oligonucleotide a) of the DNA encoding the 16S rRNA of the microorganism hybridizes Nucleotide probe (3) a) an oligonucleotide probe comprising a base sequence of 25-35 bases comprising bases 167-196 in SEQ ID NO: 3, and / or b) 167-196 of SEQ ID NO: 3 Of the DNA sequence encoding a 16S rRNA of the microorganism, a) consisting of a base sequence in which one or several bases are deleted, substituted, added or inserted in a continuous base sequence of 25 to 35 bases including the base Oligonucleotide probe that hybridizes to the region to which the oligonucleotide hybridizes (4) a) contains bases 167 to 196 of SEQ ID NO: 3 An oligonucleotide probe having a base sequence complementary to a continuous base sequence of 25 to 35 bases, and / or b) complementary to a continuous base sequence of 25 to 35 bases including bases 167 to 196 of SEQ ID NO: 3 In a typical nucleotide sequence, it consists of a nucleotide sequence in which one or several bases have been deleted, substituted, added or inserted, and in the region where the oligonucleotide a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. Hybridizing oligonucleotide probe (5) a) An oligonucleotide probe comprising a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 186 to 215 of SEQ ID NO: 4, and / or b) In the base sequence complementary to the continuous base sequence of 25 to 35 bases including the bases 186 to 215 in SEQ ID NO: 4, one or several bases are deleted, substituted, An oligonucleotide probe comprising an added or inserted base sequence and hybridizing to the region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes (6) a) 170 to An oligonucleotide probe comprising a base sequence complementary to a continuous base sequence of 25 to 35 bases including base number 199, and / or b) of 25 to 35 bases including bases 170 to 199 of SEQ ID NO: 5 The oligonucleotide of a) among the DNAs encoding the 16S rRNA of the microorganism, comprising a base sequence in which one or several bases are deleted, substituted, added or inserted in a base sequence complementary to a continuous base sequence Oligonucleotide probe that hybridizes to the region that hybridizes to (7) a) a continuous 2 containing bases 170-200 of SEQ ID NO: 6 An oligonucleotide probe consisting of a base sequence complementary to a base sequence of 5 to 35 bases, and / or b) complementary to a continuous base sequence of 25 to 35 bases including bases 170 to 200 of SEQ ID NO: 6 The base sequence consists of a base sequence in which one or several bases are deleted, substituted, added or inserted, and hybridizes to the region to which the oligonucleotide a) of the DNA encoding the 16S rRNA of the microorganism hybridizes. An oligonucleotide probe (8) a) an oligonucleotide probe comprising a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 168 to 198 of SEQ ID NO: 7, and / or b) a sequence number In the base sequence complementary to the continuous base sequence of 25 to 35 bases including bases 168 to 198 out of 7, one or several bases may be deleted, substituted or added Consists of an inserted base sequence, and an oligonucleotide probe that hybridizes to the region to which the oligonucleotide of a) of the DNA encoding 16S rRNA of the microorganism hybridizes (9) a) 196 to 225 of SEQ ID NO: 8 1) In an oligonucleotide probe comprising a base sequence of 25 to 35 bases comprising the base number of No. 25, and / or b) a continuous base sequence of 25 to 35 bases comprising the bases of 196 to 225 of SEQ ID NO: 8, Alternatively, an oligonucleotide probe comprising a nucleotide sequence in which several bases are deleted, substituted, added or inserted, and hybridizing to a region to which the oligonucleotide of a) of the DNA encoding the 16S rRNA of the microorganism hybridizes ( 10) a) It consists of a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 196 to 225 of SEQ ID NO: 8. Oligonucleotide probe and / or b) deletion or substitution of one or several bases in a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 196 to 225 of SEQ ID NO: 8 An oligonucleotide probe comprising an added or inserted base sequence and hybridizing to a region to which the oligonucleotide of a) of the DNA encoding 16S rRNA of the microorganism hybridizes (11) a) 191 of SEQ ID NO: 9 An oligonucleotide probe comprising a base sequence of 25-35 bases comprising bases of ~ 220, and / or b) a continuous base sequence of 25-35 bases comprising bases 191-220 of SEQ ID NO: 9 Of a) of the DNA encoding 16S rRNA of the microorganism, comprising a nucleotide sequence in which one or several bases are deleted, substituted, added or inserted. Oligonucleotide probe (12) which hybridizes to the region to which the nucleotide is hybridized a) Oligo consisting of a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 191 to 220 A nucleotide probe, and / or b) one or several bases deleted or substituted in a base sequence complementary to a continuous base sequence of 25 to 35 bases including bases 191 to 220 of SEQ ID NO: 9, An oligonucleotide probe comprising an added or inserted base sequence and hybridizing to a region to which the oligonucleotide a) of the DNA encoding 16S rRNA of the microorganism hybridizes.   At least one microorganism selected from the group consisting of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis, wherein the set of oligonucleotide probes according to claim 1 is immobilized on a carrier. Microarray for detecting.   The microarray according to claim 2, wherein the carrier has a carbon layer and a chemical modification group on the surface. A method for detecting at least one microorganism selected from the group consisting of Alicyclobacillus acidocaldarius, Bacillus caldotenax, Bacillus cereus, Bacillus subtilis, Thermoactinomyces vulgaris and Staphylococcus epidermidis,
Extracting genomic DNA or RNA from the sample;
Using the extracted genomic DNA or RNA as a template, and amplifying DNA encoding 16S rRNA of the microorganism;
Detecting the amplified DNA using the set of oligonucleotide probes according to claim 1 or the microarray according to claim 2 or 3.

Images