# Patent

## Title
Infectious etiologic agent detection probe and probe set, carrier, and genetic screening method

## Abstract

An infectious etiologic agent detection probe set which detects an infectious etiologic agent gene, includes a plurality of kinds of probes including oligonucleotide having base sequences selected from each of a plurality of groups selected from a first group including base sequences of SEQ ID Nos. 1 to 14 and complementary sequences thereof, a second group including base sequences of SEQ ID Nos. 15 to 24 and complementary sequences thereof, a third group including base sequences of SEQ ID Nos. 25 to 36 and complementary sequences thereof, a fourth group including base sequences of SEQ ID Nos. 37 to 47 and complementary sequences thereof, a fifth group including base sequences of SEQ ID Nos. 48 to 57 and complementary sequences thereof, a sixth group including base sequences of SEQ ID Nos. 58 to 68 and complementary sequences thereof, a seventh group including base sequences of SEQ ID Nos. 69 to 77 and complementary sequences thereof, an eighth group including base sequences of SEQ ID Nos. 78 to 85 and complementary sequences thereof, a ninth group including base sequences of SEQ ID Nos. 86 to 97 and complementary sequences thereof, and a 10th group including base sequences of SEQ ID Nos. 98 to 106 and complementary sequences thereof.

## Background
FIELD OF THE INVENTION
The present invention relates to detection and/or identification of an infectious etiologic agent as an etiologic agent of an infectious disease and, more particularly, to a probe and probe set originated in an infectious etiologic agent, a carrier, and a genetic screening method, which are useful for detection and identification of the etiologic agent of an infectious disease.
The present invention also relates to a PCR amplification process of an infectious etiologic agent, which is suitable for detection and/or identification of an infectious etiologic agent.
BACKGROUND OF THE INVENTION
In recent years, gene expression analysis using DNA chips (also referred to as DNA microarrays hereinafter) is done in various fields including drug development. Different specimen DNAs are made to react with a DNA microarray in which various kinds of gene sets (probes) are arranged. Gene dosages which exist in the respective specimens are compared. Genes which are present in high dosages (the expression amounts are large) or inactive genes (the expression amounts are small) at each stage are classified and analyzed in association with functions.
An example is an infectious etiologic agent test. In Japanese Patent Laid-Open No. 2001-299396, Ezaki et al have proposed a microorganism identification method using, as a DNA probe, a DNA chip on which chromosome DNAs are immobilized. According to this method, a plurality of chromosome DNAs originated in a plurality of known microorganisms with different GC contents are made to react with chromosome DNAs originated in an unknown microorganism in a specimen. When the resultant hybridization complex is detected, the unknown microorganism in the specimen can be detected.
As probes used for DNA chips for infectious etiologic agent tests, Ono et al have proposed a bacterial detection probe using restriction enzyme fragments in Japanese Patent Laid-Open No. 6-133798, a Pseudomonas aeruginosa detection probe in Japanese Patent Laid-Open No. 10-304896, and a detection probe using restriction enzyme fragments of Escherichia coli, klebsiella pneumoniae, and Enterobacter cloacae in Japanese Patent Laid-Open No. 10-304897.
As a microarray, for example, a microarray using stamping called a Stanford method is known. For example, DNA chips on which cDNA fragments of known genes of human origin, which are related to cancers, are applied by spotting or stamping and chips prepared by bonding cDNA fragments of 1,000 kinds of known genes of human origin to slide glasses are commercially available from TAKARA SHUZO.
On the other hand, a chip available from Affymetrix is prepared by designing an oligonucleotide probe set on the basis of the known gene cDNAs, and probes are laid out by synthesis on a substrate. Oligoprobes are laid out on one chip at a high density so that the expression levels of 10,000 or more genes can be analyzed at once.
However, the DNA chips of the prior arts described above use DNA probes such as chromosome DNAs or restriction enzyme fragments. DNAs directly extracted from microorganisms are used as materials. For this reason, the chips can hardly be mass-prepared at a time and are not suitable for clinical diagnosis. For application to clinical diagnosis, mass production of inexpensive and uniform DNA chips is necessary. For this purpose, mass preparation of uniform DNAs as probe solutions is essential. However, mass preparation of DNA probes is impossible. Even for DNA probes, when PCR amplification reaction is used, the number of DNAs can gradually be increased. However, mass preparation at a time using the PCR reaction is difficult, and the DNA chips are difficult to use for clinical diagnosis.
In addition, since the base length of a DNA probe is large, it is difficult to identify one species in similar species. Such a DNA probe is therefore not suitable for, e.g., infection detection. In treating an infection, the species must be specified, and antibiotic drugs corresponding to it must be selected and administered. For this purpose, an infection detection probe is required to have a function capable of detecting a species while discriminating similar species, although bacteria belonging to the same species need not accurately be discriminated (that is, bacteria in the same species can be detected all together). However, in, e.g., the DNA chip using restriction enzyme fragments of Escherichia coli, klebsiella pneumoniae, and Enterobacter cloacae, which is disclosed in Japanese Patent Laid-Open No. 10-304897, cross reaction occurs between the three species because of the large base length of the probe. Since similar species cannot individually be discriminated, the DNA chip can hardly be used for infection detection.
As an application purpose of microarrays, infectious etiologic agent tests have received a great deal of attention. Some probe sets aiming at testing infectious etiologic agents have also been proposed.
As an important point of bacterial tests using microarrays, detection must be possible even when the number of infectious etiologic agents is small. To do this, it is effective to amplify specific parts in the base sequences of the DNAs of infectious etiologic agents by, e.g., PCR reaction using primers. For example, a 16s rRNA gene arrangement contains a sequence unique to the species in the information of about 1,700 base pairs. When the sequence is used, classification can be done to some extent. In detecting/identifying bacteria, 16s rRNA parts in the DNA base sequences of bacteria are preferably used. Hence, it is demanded to amplify the 16s rRNA parts.
For various kinds of bacteria, however, the gene arrangements are only partially clarified, and the 16s rRNAs are not totally known. For this reason, it is not easy to design primers for PCR amplification reaction.
SUMMARY OF THE INVENTION
The present invention has been made in consideration of the above situation, and has as its object to provide an infection detection probe which allows mass preparation at a time and identification of a species in similar species.
More specifically, it is an object of the present invention to provide an infection detection probe which can suitably be used to classify a plurality of kinds of etiologic agents of an infection on the basis of the species.
It is another object of the present invention to provide a probe set which also considers the stability of a hybrid body between an infection detection probe and a specimen so that the difference between similar species can accurately be evaluated on a DNA chip.
It is still another object of the present invention to provide a carrier on which the infection detection probe is immobilized to make the infection detection probe react with the specimen.
It is still another object of the present invention to provide a carrier on which the infection detection probes are chemically immobilized so that the infection detection probes are stably immobilized on the carrier, and a detection result with high reproducibility can be obtained in the process of reaction with a specimen solution.
It is still another object of the present invention to provide a PCR reaction primer which amplifies the 16s rRNAs of an etiologic agent in a specimen in order to detect and/or identify an infectious etiologic agent.
It is still another object of the present invention to provide a primer set which can commonly be used for a plurality of species and effectively amplify the 16s rRNAs of an etiologic agent even when the species is unknown.
It is still another object of the present invention to provide a primer set which can amplify the 16s rRNAs of a plurality of kinds of etiologic agents under the same PCR conditions.
The present invention provides a primer set characterized by amplifying all species without amplifying genes originated in human genomes by causing PCR reaction for a human blood specimen by using all the primer sets simultaneously. More specifically, a primer set having a sequence which is different from the base sequence of human genome genes by three or more bases is proposed.
Other features and advantages of the present invention will be apparent from the following description.


DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
A preferred embodiment of the present invention will now be described in.
In the following embodiment, an oligonucleotide probe used to identify the etiologic agent of an infection and, more specifically, a probe used to detect one or some of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Streptococcus pneumoniae, Haemophilus influenzae, Enterobacter cloacae, and Enterococcus faecalis will be described. That is, a nucleic acid probe or nucleic acid probe set, which is used to detect rRNA gene arrangements in the genes of the above 10 infectious etiologic agents in proper quantities, is disclosed.
According to this embodiment, the oligonucleotide probe to be reacted with a specimen solution containing the nucleic acid sequence of the genes of the infectious etiologic agents contains one base sequence which belongs to one of the first group (SEQ ID Nos. 1 to 14 in the attached sequence table) shown in Table 1, the second group (SEQ ID Nos. 15 to 24) shown in Table 2, the third group (SEQ ID Nos. 25 to 36) shown in Table 3, the fourth group (SEQ ID Nos. 37 to 47) shown in Table 4, the fifth group (SEQ ID Nos. 48 to 57) shown in Table 5, the sixth group (SEQ ID Nos. 58 to 68) shown in Table 6, the seventh group (SEQ ID Nos. 69 to 77) shown in Table 7, the eighth group (SEQ ID Nos. 78 to 85) shown in Table 8, the ninth group (SEQ ID Nos. 86 to 97) shown in Table 9, and the 10th group (SEQ ID Nos. 98 to 106) shown in Table 10 (to be described later). An oligonucleotide probe having a base sequence selected from the first group detects Staphylococcus aureus. An oligonucleotide probe having a base sequence selected from the second group detects Staphylococcus epidermidis. An oligonucleotide probe having a base sequence selected from the third group detects Escherichia coli. An oligonucleotide probe having a base sequence selected from the fourth group detects Klebsiella pneumoniae. An oligonucleotide probe having a base sequence selected from the fifth group detects Pseudomonas aeruginosa. An oligonucleotide probe having a base sequence selected from the sixth group detects Serratia marcescens. An oligonucleotide probe having a base sequence selected from the seventh group detects Streptococcus pneumoniae. An oligonucleotide probe having a base sequence selected from the eighth group detects Haemophilus influenzae. An oligonucleotide probe having a base sequence selected from the ninth group detects Enterobacter cloacae. An oligonucleotide probe having a base sequence selected from the 10th group detects Enterococcus faecalis. 
Complementary sequences of these probe sequences can also be used as effective probe sequences because they have the same functions as those of the above probe sequences (the complementary sequences of the first group are indicated by SEQ ID Nos. 113 to 126 in the attached sequence table, the complementary sequences of the second group are indicated by SEQ ID Nos. 127 to 136, the complementary sequences of the third group are indicated by SEQ ID Nos. 137 to 148, the complementary sequences of the fourth group are indicated by SEQ ID Nos. 149 to 159, the complementary sequences of the fifth group are indicated by SEQ ID Nos. 160 to 169, the complementary sequences of the sixth group are indicated by SEQ ID Nos. 170 to 180, the complementary sequences of the seventh group are indicated by SEQ ID Nos. 181 to 189, the complementary sequences of the eighth group are indicated by SEQ ID Nos. 190 to 197, the complementary sequences of the ninth group are indicated by SEQ ID Nos. 198 to 209, and the complementary sequences of the 10th group are indicated by SEQ ID Nos. 210 to 218).
The probes for the respective bacteria were designed from the genome parts coding the 16s rRNAs such that they could have a very high specificity with respect to the corresponding bacteria, any variation between the probe base sequences could be prevented, and a sufficient hybridization sensitivity could be expected.
These oligonucleotide probes are designed such that a stable hybrid body is formed by hybridization reaction between a specimen and two or more kinds of probes bonded onto a carrier, and a satisfactory result can be obtained.
As a characteristic feature, the carrier according to the present invention, on which the infection detection probe of the present invention is immobilized, is prepared by discharging oligonucleotide by using a BJ printer and chemically bonding it to the carrier. As compared to the prior arts, the probe hardly peels off. An additional effect for increasing the sensitivity is also obtained. When a DNA chip is produced by stamping called a Stanford method that is generally widely used (for example, TAKARA SHUZO produces DNA chips by applying cDNA fragments of known genes of human origin, which are related to cancers, by spotting or stamping), the applied DNA readily peels off. In addition, when a probe is laid out on a DNA chip by synthesis, as in the prior art (e.g., the DNA chip available from Affymetrix), accurate evaluation is impossible because the synthesis yield changes between probe sequences. The carrier according to the present invention is prepared also in consideration of these problems. As its characteristic features, the probe is stably immobilized and hardly peels off, as compared to the prior arts, and highly sensitive and accurate detection can be executed. The preferred embodiment of the present invention will be described below in detail.
The DNA chip of this embodiment can be applied to any specimen in which bacteria may be present, and for example, body fluids originated in animals such as human and livestock, including blood, spinal fluid, phlegm, stomach fluid, vaginal discharge, and intraoral mucus, and excretion such as urine and feces. All media which can be contaminated by bacteria can also be subjected to a test using the DNA chip, including food, drink water and hot spring water in the natural environment, which may cause food poisoning by contamination, filters from air and water cleaners, and so forth. Animals and plants which should be quarantined in import/export are also used as specimens.
The specimens used for the DNA chip of this embodiment include not only an extracted nucleic acid itself but also specimens prepared by various methods, such as an amplified specimen prepared by using an PCR reaction primer designed for 16s rRNA detection, a specimen prepared by causing PCR reaction on the basis of a PCR amplified product, a specimen prepared by an amplification method other than PCR, and a specimen labeled by various labeling methods for visualization.
The carrier used for the DNA chip of this embodiment includes all sorts of carriers including flat substrates such as a glass substrate, a plastic substrate, and a silicon wafer, a three-dimensional structure having a three-dimensional pattern, a spherical body such as a bead, and rod-, cord-, and thread-shaped structures. The carrier also includes a substrate whose surface is processed such that a probe DNA can be immobilized. Especially, a carrier prepared by introducing a functional group to its surface to make chemical reaction possible has a preferable form from the viewpoint of reproducibility because the probe is stably bonded in the process of hybridization reaction.
As an example of the immobilization method used in the present invention, a combination of a maleimide group and a thiol (—SH) group is used. More specifically, a thiol (—SH) group is bonded to the terminal of a nucleic acid probe, and a process is executed make the solid surface have a maleimide group. Accordingly, the thiol group of the nucleic acid probe supplied to the solid surface reacts with the maleimide group on the solid surface to immobilize the nucleic acid probe.
To introduce the maleimide group, first, an aminosilane coupling agent is caused to react on a glass substrate. Next, the maleimide group is introduced by reaction between the amino group and an EMCS reagent (N-(6-Maleimidocaproyloxy)succinimide: available from Dojin). Introduction of the SH group to a DNA can be done by using 5′-Thiol-ModifierC6 (available from Glen Research) when the DNA is synthesized by an automatic DNA synthesizer.
Instead of the above-described combination of a thiol group and a maleimide group, a combination of, e.g., an epoxy group (in the solid phase) and an amino group (nucleic acid probe terminal) can also be used as a combination of functional groups to be used for immobilization. Surface treatments using various kinds of silane coupling agents are also effective. Oligonucleotide in which a functional group which can react with a functional group introduced by a silane coupling agent is introduced is used. A method of applying a resin having a functional group can also be used.
The present invention will be described below in more detail on the basis of examples using the infectious etiologic agent detection probes to be used to detect the 10 etiologic agents described above.
Example 1
Microorganism Detection Using 1-Step PCR
1. Preparation of Probe DNAs
Nucleic acid sequences shown in Tables 1 to 10 were designed as probes to be used for detection of the 10 etiologic agents. More specifically, the following probe base sequences were selected from the genome parts coding the 16s rRNAs of the respective bacteria. These probe base sequences were designed such that they could have a very high specificity with respect to the corresponding bacteria, any variation between the probe base sequences could be prevented, and a sufficient hybridization sensitivity could be expected (The probe base sequences need not always completely match those shown in Tables 1 to 10. Probe base sequences having base lengths of 20 to 30, including the probe base sequences, are also included in the probe base sequence shown in the tables. As described above, complementary sequences (complementary strands) of the base sequences shown in the tables may also be used).
In the following tables, “Probe No.” is assigned for convenience. SEQ ID Nos. coincide with those in the attached sequence tables. As described above, the complementary strand sequences of the base sequences with SEQ ID Nos. 1 to 106 have SEQ ID Nos. 113 to 218.







TABLE 1







Probes for detecting Staphylococcus aureus strain











Name of
Probe
SEQ ID




microorganizm
No.
No.
Sequence














Staphylococcus
PA-1
1
5′ GAACCGCATGGTTCAAAAGTGAAAGA 3′



aureus


 



PA-2
2
5′ CACTTATAGATGGATCCGCGCTGC 3′


 



PA-3
3
5′ TGCACATCTTGACGGTACCTAATCAG 3′


 



PA-4
4
5′ CCCCTTAGTGCTGCAGCTAACG 3′


 



PA-5
5
5′ AATACAAAGGGCAGCGAAACCGC 3′


 



PA-6
6
5′ CCGGTGGAGTAACCTTTTAGGAGCT 3′


 



PA-7
7
5′ TAACCTTTTAGGAGCTAGCCGTCGA 3′


 



PA-8
8
5′ TTTAGGAGCTAGCCGTCGAAGGT 3′


 



PA-9
9
5′ TAGCCGTCGAAGGTGGGACAAAT 3′


 



PA-10
10
5′ ACGGACGAGAAGCTTGCTTCTCT 3′


 



PA-11
11
5′ TGTCACTTATAGATGGATCCGCGCT 3′


 



PA-12
12
5′ TGTAAGTAACTGTGCACATCTTGACG 3′


 



PA-13
13
5′ ACAACTCTAGAGATAGAGCCTTCCCC 3′


 



PA-14
14
5′ GTGGAGTAACCTTTTAGGAGCTAGCC 3′
















TABLE 2







Probes for detecting Staphylococcus epidermidis strain











Name of
Probe
SEQ ID




microorganizm
No.
No.
Sequence





Staphylococcus
PB-1
15
5′ GAACAGACGAGGAGCTTGCTCC 3′



epidermidis


 



PB-2
16
5′ TAGTGAAAGACGGTTTTGCTGTCACT 3′


 



PB-3
17
5′ TAAGTAACTATGCACGTCTTGACGGT 3′


 



PB-4
18
5′ GACCCCTCTAGAGATAGAGTTTTCCC 3′


 



PB-5
19
5′ AGTAACCATTTGGAGCTAGCCGTC 3′


 



PB-6
20
5′ GAGCTTGCTCCTCTGACGTTAGC 3′


 



PB-7
21
5′ AGCCGGTGGAGTAACCATTTGG 3′


 



PB-8
22
5′ AGACGAGGAGCTTGCTCCTCTG 3′


 



PB-9
23
5′ AGAACAAATGTGTAAGTAACTATGCACGT 3′


 



PB-10
24
5′ ACCATTTGGAGCTAGCCGTCGA 3′
















TABLE 3







Probes for detecting Escherichia coli strain











Name of
Probe
SEQ ID




microorganizm
No.
No.
Sequence





Escherichia
PC-1
25
5′ CTCTTGCCATCGGATGTGCCCA 3′



coli


 



PC-2
26
5′ ATACCTTTGCTCATTGACGTTACCCG 3′


 



PC-3
27
5′ TTTGCTCATTGACGTTACCCGCAG 3′


 



PC-4
28
5′ ACTGGCAAGCTTGAGTCTCGTAGA 3′


 



PC-5
29
5′ ATACAAAGAGAAGCGACCTCGCG 3′


 



PC-6
30
5′ CGGACCTCATAAAGTGCGTCGTAGT 3′


 



PC-7
31
5′ GCGGGGAGGAAGGGAGTAAAGTTAAT 3′


 



PC-8
32
5′ TAACAGGAAGAAGCTTGCTTCTTTGCTG 3′


 



PC-9
33
5′ TTGCCATCGGATGTGCCCAGAT 3′


 



PC-10
34
5′ GGAAGGGAGTAAAGTTAATACCTTTGCTC 3′


 



PC-11
35
5′ ATCTTTTGTTGCCAGCGGTCCG 3′


 



PC-12
36
5′ AAGGGAGTAAAGTTAATACCTTTGCTCATTG 3′
















TABLE 4







Probes for detecting Klebsiella pneumoniae strain











Name of
Probe
SEQ ID




microorganizm
No.
No.
Sequence





Klebsiella
PD-1
37
5′ TAGCACAGAGAGCTTGCTCTCGG 3′



pneumoniae


 



PD-2
38
5′ TCATGCCATCAGATGTGCCCAGA 3′


 



PD-3
39
5′ CGGGGAGGAAGGCGATAAGGTTAAT 3′


 



PD-4
40
5′ TTCGATTGACGTTACCCGCAGAAGA 3′


 



PD-5
41
5′ GGTCTGTCAAGTCGGATGTGAAATCC 3′


 



PD-6
42
5′ GCAGGCTAGAGTCTTGTAGAGGGG 3′


 



PD-7
43
5′ TCATGCCATCAGATGTGCCCAGAT 3′


 



PD-8
44
5′ CGGGGAGGAAGGCGATAAGGTTAA 3′


 



PD-9
45
5′ TTATCGATTGACGTTACCCGCAGAAGA 3′


 



PD-10
46
5′ CATTCGAAACTGGCAGGCTAGAGTC 3′


 



PD-11
47
5′ CCTTTGTTGCCAGCGGTTAGGC 3′
















TABLE 5







Probes for detecting Pseudomonas aeruginosa strain











Name of
Probe
SEQ ID




microorganizm
No.
No.
Sequence





Pseudomonas
PE-1
48
5′ TGAGGGAGAAAGTGGGGGATCTTC 3′



aeruginosa


 



PE-2
49
5′ TCAGATGAGCCTAGGTCGGATTAGC 3′


 



PE-3
50
5′ GAGCTAGAGTACGGTAGAGGGTGG 3′


 



PE-4
51
5′ GTACGGTAGAGGGTGGTGGAATTT 3′


 



PE-5
52
5′ GACCACCTGGACTGATACTGACAC 3′


 



PE-6
53
5′ TGGCCTTGACATGCTGAGAACTTTC 3′


 



PE-7
54
5′ TTAGTTACCAGCACCTCGGGTGG 3′


 



PE-8
55
5′ TAGTCTAACCGCAAGGGGGACG 3′


 



PE-9
56
5′ TGCATCCAAAACTACTGAGCTAGAGTAC 3′


 



PE-10
57
5′ GTCGACTAGCCGTTGGGATCCT 3′
















TABLE 6







Probes for detecting Serratia marcescens strain











Name of
Probe
SEQ ID




microorganizm
No.
No.
Sequence





Serratia
PF-1
58
5′ TAGCACAGGGAGCTTGCTCCCT 3′



marcescens


 



PF-2
59
5′ AGGTGGTGAGCTTAATACGCTCATC 3′


 



PF-3
60
5′ TCATCAATTGACGTTACTCGCAGAAG 3′


 



PF-4
61
5′ ACTGCATTTGAAACTGGCAAGCTAGA 3′


 



PF-5
62
5′ TTATCCTTTGTTGCAGCTTCGGCC 3′


 



PF-6
63
5′ ACTTTCAGCGAGGAGGAAGGTGG 3′


 



PF-7
64
5′ GGTAGCACAGGGGAGCTTGCTC 3′


 



PF-8
65
5′ CGAGGAGGAAGGTGGTGAGCTTAATA 3′


 



PF-9
66
5′ TACGCTCATCAATTGACGTTACTCGC 3′


 



PF-10
67
5′ GAAACTGGCAAGCTAGAGTCTCGTAGA 3′


 



PF-11
68
5′ TTATCCTTTGTTGCCAGCGGTTCG 3′
















TABLE 7







Probes for detecting Streptococcus pneumoniae strain











Name of
Probe
SEQ ID




microorganizm
No.
No.
Sequence





Streptococcus
PG-1
69
5′ AGTAGAACGCTGAAGGAGGAGCTTG 3′



pneumoniae


 



PG-2
70
5′ CTTGCATCACTACCAGATGGACCTG 3′


 



PG-3
71
5′ TGAGAGTGGAAAGTTCACACTGTGAC 3′


 



PG-4
72
5′ GCTGTGGCTTAACCATAGTAGGCTTT 3′


 



PG-5
73
5′ AAGCGGCTCTCTGGCTTGTAACT 3′


 



PG-6
74
5′ TAGACCCTTTCCGGGGTTTAGTGC 3′


 



PG-7
75
5′ GACGGCAAGCTAATCTCTTAAAGCCA 3′


 



PG-8
76
5′ GACATTTGCTTAAAAGGTGCACTTGCA 3′


 



PG-9
77
5′ GTTGTAAGAGAAGAACGAGTGTGAGAGTG 3′
















TABLE 8







Probes for detecting Haemophilus influenzae strain











Name of
Probe
SEQ ID




microorganizm
No.
No.
Sequence





Haemophilus
PH-1
78
5′ GCTTGGGAATCTGGCTTATGGAGG 3′



influenzae


 



PH-2
79
5′ TGCCATAGGATGAGCCCAAGTGG 3′


 



PH-3
80
5′ CTTGGGAATGTACTGACGCTCATGTG 3′


 



PH-4
81
5′ GGATTGGGCTTAGAGCTTGGTGC 3′


 



PH-5
82
5′ TACAGAGGGAAGCGAAGCTGCG 3′


 



PH-6
83
5′ GGCGTTTACCACGGTATGATTCATGA 3′


 



PH-7
84
5′ AATGCCTACCAAGCCTGCGATCT 3′


 



PH-8
85
5′ TATCGGAAGATGAAAGTGCGGGACT 3′
















TABLE 9







Probes for detecting Enterobacter Cloacae strain











Name of
Probe
SEQ ID




microorganizm
No.
No.
Sequence





Enterobacter
PI-1
86
5′ CAGAGAGCTTGCTCTCGGGTGA 3′



Cloacae


 



PI-2
87
5′ GGGAGGAAGGTGTTGTGGTTAATAAC 3′


 



PI-3
88
5′ GGTGTTGTGGTTAATAACCACAGCAA 3′


 



PI-4
89
5′ GCGGTCTGTCAAGTCGGATGTG 3′


 



PI-5
90
5′ ATTCGAAACTGGCAGGCTAGAGTCT 3′


 



PI-6
91
5′ TAACCACAGCAATTGACGTTACCCG 3′


 



PI-7
92
5′ GCAATTGACGTTACCCGCAGAAGA 3′


 



PI-8
93
5′ GTAGCACAGAGAGCTTGCTCTCG 3′


 



PI-9
94
5′ CGGGGAGGAAGGTGTTGTGGTTA 3′


 



PI-10
95
5′ ACCACAGCAATTGACGTTACCCG 3′


 



PI-11
96
5′ GAAACTGGCAGGCTAGAGTCTTGTAG 3′


 



PI-12
97
5′ AGGCGGTCTGTCAAGTCGGATG 3′
















TABLE 10







Probes for detecting Enterococcus faecalis strain











Name of
Probe
SEQ ID




microorganizm
No.
No.
Sequence














Enterococcus
PJ-1
98
5′ TTCTTTCCTCCCGAGTGCTTGCA 3′



faecalis


 



PJ-2
99
5′ AACACGTGGGTAACCTACCCATCAG 3′


 



PJ-3
100
5′ ATGGCATAAGAGTGAAAGGCGCTT 3′


 



PJ-4
101
5′ GACCCGCGGTGCATTAGCTAGT 3′


 



PJ-5
102
5′ GGACGTTAGTAACTGAACGTCCCCT 3′


 



PJ-6
103
5′ CTCAACCGGGGAGGGTCATTGG 3′


 



PJ-7
104
5′ TTGGAGGGTTTCCGCCCTTCAG 3′


 



PJ-8
105
5′ ATAGAGCTTTCCCTTCGGGGACAAA 3′


 



PJ-9
106
5′ CGAGGTCATGCAAATCTCTTAAAGCTTCT 3′









For each probe shown in the tables, as a functional group to immobilize the probe to a DNA microarray, a thiol group was introduced to the 5′ terminal of the nucleic acid after synthesis in accordance with a conventional method. After introduction of the functional group, purification and freeze-drying were executed. The freeze-dried probes were stored in a freezer at −30° C.
2. Preparation of Specimen Amplification PCR Primers
As 16s rRNA gene (target gene) amplification PCR primers for etiologic agent detection, nucleic acid sequences shown in Table 11 were designed. More specifically, probe sets which specifically amplify the genome parts coding the 16s rRNAs, i.e., primers for which the specific melting points were made uniform as much as possible at the two end portions of the 16s rRNA coding region of a base length of 1,400 to 1,700 were designed. In order to simultaneously amplify variants or a plurality of 16s rRNA coding regions on genomes, a plurality of kinds of primers were designed. Note that a primer set is not limited to primer sets shown in the table 11. A primer set which is available in common to a plural kinds of etiologic agents and amplify almost entire length of 16s rRNA coding region of the etiologic agents can also be employed.










TABLE 11






Primer
SEQ




No.
ID No.
Sequence







Forward
F-1
107
5′ GCGGCGTGCCTAATACATGCAAG 3′


Primer





 



F-2
108
5′ GCGGCAGGCCTAACACATGCAAG 3′


 



F-3
109
5′ GCGGCAGGCTTAACACATGCAAG 3′


 


Reverse
R-1
110
5′ ATCCAGCCGCACCTTCCGATAC 3′


Primer





 



R-2
111
5′ ATCCAACCGCAGGTTCCCCTAC 3′


 



R-3
112
5′ ATCCAGCCGCAGGTTCCCCTAC 3′









The primers shown in Table 11 were purified by HPLC (High Performance Liquid Chromatography) after synthesis. Three forward primers and three reverse primers were mixed and dissolved in a TE buffer solution such that each primer concentration had an ultimate concentration of 10 pmol/μl.
3. Extraction of Genome DNAs (Model Specimens) of Etiologic Agents
[3-1] Microbial Culture & Preprocess for Genome DNA Extraction
First, microbial culture media were produced by culturing type strains of the etiologic agents (Staphylococcus aureus type strain (ATCC12600), Staphylococcus epidermidis type strain (ATCC14990), Escherichia coli type strain (ATCC11775), Klebsiella pneumoniae type strain (ATCC13883), Pseudomonas aeruginosa type strain (ATCC10145), Serratia marcescens strain, Streptococcus pneumoniae type strain, Haemophilus influenzaestrain, Enterobacter Cloacae type strain (ATCC13047), and Enterococcus faecalis type strain (ATCC19433)) in accordance with a conventional method. Each of the microbial culture media was sampled 1.0 ml (0D600=0.7) into a 1.5-ml microtube. The cells were collected by centrifuge (8,500 rpm, 5 min, 4° C.). After the supernatant was removed, a 300-μl enzyme buffer (50 mM Tris-HCl: p.H. 8.0, 25 mM EDTA) was added, and the broth was re-suspended by using a mixer. From the re-suspended broth, the cells were collected again by centrifuge (8,500 rpm, 5 min, 4° C.). After the supernatant was removed, the following enzyme solution was added to the collected cells, and the broth was re-suspended by using the mixer.
Lysozyme 50 μl (20 mg/ml in Enzyme Buffer)
N-Acetylmuramidase SG 50 μl (0.2 mg/ml in Enzyme Buffer
The broth added with the enzyme solution and re-suspended was left stand still in an incubator at 37° C. for 30 min to melt cell walls.
[3-2] Genome Extraction
Microbial genome DNA extraction to be described below was done by using a nucleic acid purification kit (MagExtractor-Genome: available from TOYOBO).
More specifically, a 750-μl melting/absorption solution and a 40-μl magnetic beads were added to the preprocessed microbial suspension. The suspension was intensely stirred for 10 min by using a tube mixer (step 1).
Next, the microtube was set in a separation stand (Magical Trapper) and left stand still for 30 sec to gather magnetic particles to the wall surface of the tube. The supernatant was removed while the microtube was kept set in the stand (step 2).
Next, a 900-μl cleaning solution was added. The solution was re-suspended by stirring it for about 5 sec by a mixer (step 3).
The microtube was set in the separation stand (Magical Trapper) and left stand still for 30 sec to gather magnetic particles to the wall surface of the tube. The supernatant was removed while the microtube was kept set in the stand (step 4).
Steps 3 and 4 were repeated to execute the second cleaning process (step 5). After that, 900-μl 70% ethanol was added. The solution was re-suspended by stirring it for about 5 sec by a mixer (step 6).
The microtube was set in the separation stand (Magical Trapper) and left stand still for 30 sec to gather magnetic particles to the wall surface of the tube. The supernatant was removed while the microtube was kept set in the stand (step 7).
Steps 6 and 7 were repeated to execute the second cleaning process by using 70% ethanol (step 8). After that, 100-μl pure water was added to the collected magnetic particles. The solution was stirred for 10 min by a tube mixer (step 9).
The microtube was set in the separation stand (Magical Trapper) and left stand still for 30 sec to gather magnetic particles to the wall surface of the tube. The supernatant was collected to a new tube while the microtube was kept set in the stand.
[3-3] Test of Collected Genome DNAs
The collected genome DNAs of microorganisms (etiologic agent strain) underwent agarose electrophoresis and 260/280-nm absorbance determination in accordance with the conventional method so that the quality (the admixture amount of low molecular nucleic acid and the degree of decomposition) and collection amount were tested. In this embodiment, about 9 to 10-μg genome DNAs were collected in each bacterium. No degradation of genome DNAs or admixture of rRNA was observed. The collected genome DNAs were dissolved in a TE buffer solution at an ultimate concentration of 50 ng/μl and used in the following examples.
4. Preparation of DNA Microarray
The DNA Microarray was prepared according to Japanese Patent Application Laid-Open No. 11-187900.
[4-1] Cleaning of Glass Substrate
A glass substrate (size: 25 mm×75 mm×1 mm, available from Iiyama Tokushu Glass) made of synthetic silica was placed in a heat- and alkali-resisting rack and dipped in a cleaning solution for ultrasonic cleaning, which was prepared to a predetermined concentration. The glass substrate was kept dipped in the cleaning solution for a night and cleaned by ultrasonic cleaning for 20 min. The substrate was picked up, lightly rinsed by pure water, and cleaned by ultrasonic cleaning in ultrapure water for 20 min. The substrate was dipped in a 1N aqueous sodium hydroxide solution heated to 80° C. for 10 min. Pure water cleaning and ultrapure water cleaning were executed again. A silica glass substrate for a DNA microchip was thus prepared.
[4-2] Surface Treatment
A silane coupling agent KBM-603 (available from Shinetsu Silicone) was dissolved in pure water at a concentration of 1% and stirred at room temperature for 2 hrs. The cleaned glass substrate was dipped in the aqueous solution of the silane coupling agent and left stand still at room temperature for 20 min. The glass substrate was picked up. The surface was lightly rinsed by pure water and dried by spraying nitrogen gas to both surfaces of the substrate. The dried substrate was baked in an oven at 120° C. for 1 hr to complete the coupling agent treatment, thereby introducing an amino group to the substrate surface. Next, N-(6-Maleimidocaproyloxy)succinimido) (to be abbreviated as EMCS hereinafter) available from Dojindo Laboratories was dissolved in a 1:1 medium mixture of dimethyl sulfoxide and ethanol such that an ultimate concentration of 0.3 mg/ml was obtained, thereby preparing an EMCS solution.
The baked glass substrate was left stand and cooled and dipped in the prepared EMCS solution at room temperature for 2 hrs. With this process, the amino group introduced to the surface by the silane coupling agent reacted with the succinimide group in the EMCS to introduce the maleimide group to the surface of the glass substrate. The glass substrate picked up from the EMCS solution was cleaned by using the above-described medium mixture in which the EMCS was dissolved. The glass substrate was further cleaned by ethanol and dried in a nitrogen gas atmosphere.
[4-3] Probe DNA
The microorganism detection probe prepared in Example 1 was dissolved in pure water. The solution was dispensed such that the ultimate concentration (at ink dissolution) became 10 μM. Then, the solution was freeze-dried to remove water.
[4-4] DNA Discharge by BJ Printer and Bonding to Substrate
An aqueous solution containing 7.5-wt % glycerin, 7.5-wt % thioglycol, 7.5-wt % urea, and 1.0-wt % Acetylenol EH (available from Kawaken Fine Chemicals) was prepared. Each of the seven probes (Table 1) prepared in advance was dissolved in the medium mixture at a specific concentration. An ink tank for an inkjet printer (tradename: BJF-850, available from Canon) is filled with the resultant DNA solution and attached to the printhead.
The inkjet printer used here was modified in advance to allow printing on a flat plate. When the inkjet printer inputs a printing pattern in accordance with a predetermined file creation method, an about 5-picoliter DNA solution can be spotted at a pitch of about 120 μm.
The printing operation was executed for one glass substrate by using the modified inkjet printer, thereby preparing a DNA microarray. After confirming that printing was reliably executed, the glass substrate was left stand still in a humidified chamber for 30 min to make the maleimide group on the glass substrate surface react with the thiol group at the nucleic acid probe terminal.
[4-5] Cleaning
After reaction for 30 min, the DNA solution remaining on the surface was cleaned by using a 10-mM phosphate buffer (pH 7.0) containing 100-mM NaCl, thereby obtaining a gene chip (DNA microarray) in which single-stranded DNAs were immobilized on the glass substrate surface.
5. Amplification and Labeling of Specimens (PCR Amplification & Fluorescent Labeling)
Amplification of microbial genes as specimens and labeling reaction will be described below

















Premix PCR reagent
25 μl 




(TAKARA ExTaq)



Template Genome DNA
2 μl
(100 ng)



Forward Primer mix
2 μl
(20 pmol/tube each)



Reverse Primer mix
2 μl
(20 pmol/tube each)



Cy-3 dUTP (1 mM)
2 μl
(2 nmol/tube)



H20
17 μl 



Total
50 μl 










Amplification reaction of the reaction solution having the above composition was caused by using a commercially available thermal cycler in accordance with the following protocol.
















95° C.
10 min.



 



92° C. 55° C.







 



72° C.
45 sec.



72° C.
10 min.










After the end of reaction, the primers were removed (purified) by using a purification column (QIAquick PCR Purification Kit available from QIAGEN). Then, determination of the amplified products was executed to obtain labeled specimens.
6. Hybridization
Detection reaction was performed by using the gene chips prepared by [4. Preparation of DNA Microarray] and the labeled specimen prepared by [5. Amplification and Labeling of Specimen (PCR Amplification & Fluorescent Labeling)].
[6-1] Blocking of DNA Microarrays
BSA (fetal bovine serum albumin, Fraction V: available from Sigma) was dissolved in a 100-mM NaCl/10-mM phosphate buffer such that a 1 wt % solution was obtained. The gene chips prepared by [4. Preparation of DNA Microarray] were dipped in the solution at room temperature for 2 hrs to execute blocking. After the end of blocking, the chips were cleaned by using a 2×SSC solution (NaCl 300 mM, Sodium Citrate (trisodium citrate dihydrate, C6H5Na3.2H2O) 30 mM, pH 7.0) containing 0.1-wt % SDS (Sodium Dodecyl Sulfate), rinsed by pure water, and hydro-extracted by a spin dryer.
[6-2] Hybridization
The hydro-extracted gene chips were set in a hybridization apparatus (Hybridization Station available from Genomic Solutions Inc). Hybridization reaction was caused in a hybridization solution under conditions to be described below.
[6-3] Hybridization Solution
6×SSPE/10% Form amide/Target (all 2nd PCR Products)
6×SSPE: NaCl 900 mM, NaH2PO4.H2O 60 mM, EDTA 6 mM, pH, 7.4)
[6-4] Hybridization Conditions
65° C. 3 min 92° C. 2 min 45° C. 3 hrs Wash 2×SSC/0.1% SDS at 25° C. Wash 2×SSC at 20° C. (Rinse with H2O: Manual) Spin dry (The hybridization reaction was caused at 65° C. for 3 min, at 92° C. for 2 min, and at 45° C. for 3 hrs. The gene chips were cleaned by using 2×SSC/0.1% SDS at 25° C. and 2×SSC at 20° C., rinsed by pure water, and spin-dried).
7. Microorganism Detection (Fluorometry)
The gene chips after the end of hybridization reaction were subjected to fluorometry by using a gene chip fluorescent detector (GenePix 4000B available from Axon). As a result, the respective bacteria could be detected with sufficient signals at a high reproducibility, as shown in Tables 12 to 21. No hybrid bodies for other bacteria were detected.
In this example, fluorometry was executed twice for each gene chip. The results are shown below.







TABLE 12







Staphylococcus aureus










First
Second














SEQ.

Fluores-

Fluores-



Probe
ID.

cence

cence



No.
No.
Sequence
luminance
S/N
luminance
S/N
















PA-1
1
5′ GAACCGCATGGTTCAAAAGTGAAAGA 3′
3000
42.9
2900
40.8


 


PA-2
2
5′ CACTTATAGATGGATCCGCGCTGC 3′
7700
110.0
7700
108.5


 


PA-3
3
5′ TGCACATCTTGACGGTACCTAATCAG 3′
6400
91.4
6400
90.1


 


PA-4
4
5′ CCCCTTAGTGCTGCAGCTAACG 3′
2500
35.7
2500
35.2


 


PA-5
5
5′ AATACAAAGGGCAGCGAAACCGC 3′
7800
111.4
7800
109.9


 


PA-6
6
5′ CCGGTGGAGTAACCTTTTAGGAGCT 3′
4800
68.6
4800
67.6


 


PA-7
7
5′ TAACCTTTTAGGAGCTAGCCGTCGA 3′
4500
64.3
4300
60.6


 


PA-8
8
5′ TTTAGGAGCTAGCCGTCGAAGGT 3′
4800
68.6
4800
67.6


 


PA-9
9
5′ TAGCCGTCGAAGGTGGGACAAAT 3′
5300
75.7
5200
73.2
















TABLE 13







Staphylococcus epidermidis










First
Second














SEQ.

Fluores-

Fluores-



Probe
ID.

cence

cence



No.
No.
Sequence
luminance
S/N
luminance
S/N
















PB-1
15
5′ GAACAGACGAGGAGCTTGCTCC 3′
1000
14.5
1100
15.7


 


PB-2
16
5′ TAGTGAAAGACGGTTTTGCTGTCACT 3′
1800
26.1
1800
25.7


 


PB-3
17
5′ TAAGTAACTATGCACGTCTTGACGGT 3′
1400
20.3
1400
20


 


PB-4
18
5′ GACCCCTCTAGAGATAGAGTTTTCCC 3′
1000
14.5
1100
15.7


 


PB-5
19
5′ AGTAACCATTTGGAGCTAGCCGTC 3′
1800
26.1
2000
28.6


 


PB-6
20
5′ GAGCTTGCTCCTCTGACGTTAGC 3′
1200
17.4
1300
18.6


 


PB-7
21
5′ AGCCGGTGGAGTAACCATTTGG 3′
1100
15.9
1100
15.7
















TABLE 14







Escherichia coli










First
Second














SEQ.

Fluores-

Fluores-



Probe
ID.

cence

cence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PC-1
25
5′ CTCTTGCCATCGGATGTGCCCA 3′
1200
17.6
1200
17.9


 


PC-2
26
5′ ATACCTTTGCTCATTGACGTTACCCG 3′
1500
22.1
1600
23.9


 


PC-3
27
5′ TTTGCTCATTGACGTTACCCGCAG 3′
1100
16.2
1200
17.9


 


PC-4
28
5′ ACTGGCAAGCTTGAGTCTCGTAGA 3′
2000
29.4
2100
31.3


 


PC-5
29
5′ ATACAAAGAGAAGCGACCTCGCG 3′
1500
22.1
1500
22.4


 


PC-6
30
5′ CGGACCTCATAAAGTGCGTCGTAGT 3′
2400
35.3
2600
38.8


 


PC-7
31
5′ GCGGGGAGGAAGGGAGTAAAGTTAAT 3′
1200
17.6
1200
17.9
















TABLE 15







Klebsiella pneumoniae










First
Second














SEQ.

Fluores-

Fluores-



Probe
ID.

cence

cence



No.
No.
Sequence
luminance
S/N
luminance
S/N
















PD-1
37
5′ TAGCACAGAGAGCTTGCTCTCGG 3′
500
7.6
600
9


 


PD-2
38
5′ TCATGCCATCAGATGTGCCCAGA 3′
600
9.1
600
9


 


PD-3
39
5′ CGGGGAGGAAGGCGATAAGGTTAAT 3′
700
10.6
700
10.4


 


PD-4
40
5′ TTCGATTGACGTTACCCGCAGAAGA 3′
1000
15.2
1200
17.9


 


PD-5
41
5′ GGTCTGTCAAGTCGGATGTGAAATCC 3′
2700
40.9
2700
40.3


 


PD-6
42
5′ GCAGGCTAGAGTCTTGTAGAGGGG 3′
3400
51.5
3300
49.3
















TABLE 16







Pseudomonas aeruginosa










First
Second














SEQ.

Fluores-

Fluores-



Probe
ID.

cence

cence



No.
No.
Sequence
luminance
S/N
luminance
S/N
















PE-1
48
5′ TGAGGGAGAAAGTGGGGGATCTTC 3′
3500
50.0
3600
50


 


PE-2
49
5′ TCAGATGAGCCTAGGTCGGATTAGC 3′
1600
22.9
1400
19.4


 


PE-3
50
5′ GAGCTAGAGTACGGTAGAGGGTGG 3′
3500
50.0
3400
47.2


 


PE-4
51
5′ GTACGGTAGAGGGTGGTGGAATTT 3′
3100
44.3
3100
43.1


 


PE-5
52
5′ GACCACCTGGACTGATACTGACAC 3′
1600
22.9
1600
22.2


 


PE-6
53
5′ TGGCCTTGACATGCTGAGAACTTTC 3′
1200
17.1
1200
16.7


 


PE-7
54
5′ TTAGTTACCAGCACCTCGGGTGG 3′
1000
14.3
1200
16.7


 


PE-8
55
5′ TAGTCTAACCGCAAGGGGGACG 3′
1100
15.7
1100
15.3
















TABLE 17







Serratia marcescens










First
Second














SEQ.

Fluores-

Fluores-



Probe
ID.

cence

cence



No.
No.
Sequence
luminance
S/N
luminance
S/N
















PF-1
58
5′ TAGCACAGGGAGCTTGCTCCCT 3′
600
8.8
600
8.7


 


PF-2
59
5′ AGGTGGTGAGCTTAATACGCTCATC 3′
700
10.3
600
8.7


 


PF-3
60
5′ TCATCAATTGACGTTACTCGCAGAAG 3′
2000
29.4
2200
31.9


 


PF-4
61
5′ ACTGCATTTGAAACTGGCAAGCTAGA 3′
2800
41.2
2700
39.1


 


PF-5
62
5′ TTATCCTTTGTTGCAGCTTCGGCC 3′
700
10.3
700
10.1


 


PF-6
63
5′ ACTTTCAGCGAGGAGGAAGGTGG 3′
3400
50.0
3300
47.8
















TABLE 18







Streptococcus pneumoniae










First
Second














SEQ.

Fluores-

Fluores-



Probe
ID.

cence

cence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PG-1
69
5′ AGTAGAACGCTGAAGGAGGAGCTTG 3′
1000
14.9
1100
16.2


 


PG-2
70
5′ CTTGCATCACTACCAGATGGACCTG 3′
1200
17.9
1300
19.1


 


PG-3
71
5′ TGAGAGTGGAAAGTTCACACTGTGAC 3′
1000
14.9
1100
16.2


 


PG-4
72
5′ GCTGTGGCTTAACCATAGTAGGCTTT 3′
1800
26.9
1900
27.9


 


PG-5
73
5′ AAGCGGCTCTCTGGCTTGTAACT 3′
1300
19.4
1500
22.1


 


PG-6
74
5′ TAGACCCTTTCCGGGGTTTAGTGC 3′
1300
19.4
1300
19.1


 


PG-7
75
5′ GACGGCAAGCTAATCTCTTAAAGCCA 3′
2000
29.9
2100
30.9
















TABLE 19







Haemophilus influenzae










First
Second














SEQ.

Fluores-

Fluores-



Probe
ID.

cence

cence



No.
No.
Sequence
luminance
S/N
luminance
S/N
















PH-1
78
5′ GCTTGGGAATCTGGCTTATGGAGG 3′
3500
50.0
3600
50


 


PH-2
79
5′ TGCCATAGGATGAGCCCAAGTGG 3′
600
8.8
700
10.1


 


PH-3
80
5′ CTTGGGAATGTACTGACGCTCATGTG 3′
600
8.8
600
8.7


 


PH-4
81
5′ GGATTGGGCTTAGAGCTTGGTGC 3′
1100
16.2
1200
17.4


 


PH-5
82
5′ TACAGAGGGAAGCGAAGCTGCG 3′
700
10.3
600
8.7


 


PH-6
83
5′ GGCGTTTACCACGGTATGATTCATGA 3′
1300
19.1
1300
18.8


 


PH-7
84
5′ AATGCCTACCAAGCCTGCGATCT 3′
2100
30.9
2200
31.9


 


PH-8
85
5′ TATCGGAAGATGAAAGTGCGGGACT 3′
700
10.3
600
8.7
















TABLE 20







Enterobacter cloacae










First
Second














SEQ.

Fluores-

Fluores-



Probe
ID.

cence

cence



No.
No.
Sequence
luminance
S/N
luminance
S/N
















PI-1
86
5′ CAGAGAGCTTGCTCTCGGGTGA 3′
2100
29.2
2200
.31


 


PI-2
87
5′ GGGAGGAAGGTGTTGTGGTTAATAAC 3′
7900
109.7
7900
111.3


 


PI-3
88
5′ GGTGTTGTGGTTAATAACCACAGCAA 3′
1000
13.9
1300
18.3


 


PI-4
89
5′ GCGGTCTGTCAAGTCGGATGTG 3′
6400
88.9
6400
90.1


 


PI-5
90
5′ ATTCGAAACTGGCAGGCTAGAGTCT 3′
9400
130.6
9200
129.6


 


PI-6
91
5′ TAACCACAGCAATTGACGTTACCCG 3′
4700
65.3
4800
67.6


 


PI-7
92
5′ GCAATTGACGTTACCCGCAGAAGA 3′
4600
63.9
4500
63.6
















TABLE 21







Enterococcus faecalis










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N
















PJ-1
98
5′TTCTTTCCTCCCGAGTGCTTGCA 3′
1500
22.1
1500
20.8


 


PJ-2
99
5′AACACGTGGGTAACCTACCCATCAG 3′
2400
35.3
2700
37.5


 


PJ-3
100
5′ATGGCATAAGAGTGAAAGGCGCGTT 3′
5600
82.4
5600
77.8


 


PJ-4
101
5′GACCCGCGGTGCATTAGCTAGT 3′
2300
33.8
2300
31.9


 


PJ-5
102
5′GGACGTTAGTAACTGAACGTCCCCT 3′
1000
14.7
1400
19.4


 


PJ-6
103
5′CTCAACCGGGGAGGGTCATTGG 3′
4400
64.7
4400
61.1


 


PJ-7
104
5′TTGGAGGGTTTCCGCCCTTCAG 3′
1700
25  
1800
25  









The numerical values of florescent luminances (photomultiplier voltage: 400 V) in Tables 12 to 21 indicate average pixel luminances (resolution: 5 μm). The S/N ratios indicate values obtained by dividing the fluorescent luminances by background average values measured by analysis software (GenePix Pro Ver.3.0 available from Axon) attached to the measuring device.
As is apparent from Tables 12 to 21, the respective etiologic agents can be detected with sufficient signals at a high reproducibility.
Example 2
Microorganism Detection Using 2-Step PCR
As in Example 1, probe DNAs, specimen amplification PCR primers, the genome DNAs of etiologic agents, and DNA microarrays were prepared, and the following experiments were conducted.
1. Amplification and Labeling of Specimens (PCR Amplification & Fluorescent Labeling)
Amplification of microbial genes as specimens (1st PCR) and labeling (2nd PCR) reaction will be described below.
2. Amplification Reaction Solution Composition: 1st PCR
Premix PCR reagent (TAKARA 25 μl ExTaq)

















Template Genome DNA
2 μl
(10 ng)



Forward Primer mix
2 μl
(20 pmol/tube each)



Reverse Primer mix
2 μl
(20 pmol/tube each)



H20
19 μl 



Total
50 μl 










Amplification reaction of the reaction solution having the above composition was caused by using a commercially available thermal cycler in accordance with the following protocol.
















95° C.
10 min.



 



92° C. 55° C.







 



72° C.
45 sec.



72° C.
10 min.










After the end of reaction, purification was performed by using a purification column (QIAquick PCR Purification Kit available from QIAGEN). Then, determination of the amplified products was executed.
3. Labeling Reaction Solution Composition: 2nd PCR Enzyme














(QIAGEN Hotstar Taq
 0.5 μl
(2.5 u)


Polymerase)


Template DNA (1st PCR Product)
  10 μl
(30 ng)


dNTP mix (Low dTTP)*
  2 μl


Cy-3 dUTP (1 mM)
  2 μl
(2 nmol/tube)


Reverse Primer mix
  5 μl
(50 pmol/tube each)


10 × Buffer
  5 μl


H20
25.5 μl


Total
  50 μl





*dNTP mix (Low dTTP): dATP, dCTP, dGTP/5 mM(final: 10 nmol/tube) dTTP/4 mM (final: 8 nmol/tube)






Amplification reaction of the reaction solution having the above composition was caused by using a commercially available thermal cycler in accordance with the following protocol.
















95° C.
10 min.



 



92° C. 55° C.







 



72° C.
45 sec.



72° C.
10 min.










After the end of reaction, purification was performed by using a purification column (QIAquick PCR Purification Kit available from QIAGEN) to obtain labeled specimens.
4. Hybridization
Hybridization was done in accordance with the same procedures as in Example 1.
5. Microorganism Detection (Fluorometry)
Fluorometry was executed for the DNA microarrays after the end of hybridization reaction by using a DNA microarray fluorescent detector (GenePix 4000B available from Axon). Tables 22 to 31 show the measurement results.
Even in this example, fluorometry was executed twice for each DNA microarray. The results are shown in Tables 22 to 31.







TABLE 22







Staphylococcus aureus










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PA-1
1
5′ GAACCGCATGGTTCAAAAGTGAAAGA 3′
14000
186.7
13000
173.3


 


PA-2
2
5′ CACTTATAGATGGATCCGCGCTGC 3′
36000
480  
35000
466.7


 


PA-3
3
5′ TGCACATCTTGACGGTACCTAATCAG 3′
31000
413.3
29000
386.7


 


PA-4
4
5′ CCCCTTAGTGCTGCAGCTAACG 3′
10000
133.3
10000
133.3


 


PA-5
5
5′ AATACAAAGGGCAGCGAAACCGC 3′
39000
520  
38500
513.3


 


PA-6
6
5′ CCGGTGGAGTAACCTTTTAGGAGCT 3′
22000
293.3
22100
294.7


 


PA-7
7
5′ TAACCTTTTAGGAGCTAGCCGTCGA 3′
22000
293.3
21800
290.7


 


PA-8
8
5′ TTTAGGAGCTAGCCGTCGAAGGT 3′
25000
333.3
24000
320  


 


PA-9
9
5′ TAGCCGTCGAAGGTGGGACAAAT 3′
26000
346.7
25500
340  
















TABLE 23







Staphylococcus epidermidis










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PB-1
15
5′ GAACAGACGAGGAGCTTGCTCC 3′
4500
 62.5
4700
 67.1


 


PB-2
16
5′ TAGTGAAAGACGGTTTTGCTGTCACT 3′
9000
125  
8900
127.1


 


PB-3
17
5′ TAAGTAACTATGCACGTCTTGACGGT 3′
7100
 98.6
7300
104.3


 


PB-4
18
5′ GACCCCTCTAGAGATAGAGTTTTCCC 3′
4800
 66.7
5200
 74.3


 


PB-5
19
5′ AGTAACCATTTGGAGCTAGCCGTC 3′
9100
126.4
9300
132.9


 


PB-6
20
5′ GAGCTTGCTCCTCTGACGTTAGC 3′
5800
 80.6
6300
90 


 


PB-7
21
5′ AGCCGGTGGAGTAACCATTTGG 3′
5400
75 
5500
 78.6
















TABLE 24







Escherichia coli










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PC-1
25
5′ CTCTTGCCATCGGATGTGCCCA 3′
 5600
 76.7
 6200
 83.8


 


PC-2
26
5′ ATACCTTTGCTCATTGACGTTACCCG 3′
 7600
104.1
 7500
101.4


 


PC-3
27
5′ TTTGCTCATTGACGTTACCCGCAG 3′
 5600
 76.7
 5700
77 


 


PC-4
28
5′ ACTGGCAAGCTTGAGTCTCGTAGA 3′
 9400
128.8
 9300
125.7


 


PC-5
29
5′ ATACAAAGAGAAGCGACCTCGCG 3′
 7200
 98.6
 7200
 97.3


 


PC-6
30
5′ CGGACCTCATAAAGTGCGTCGTAGT 3′
11500
157.5
11500
155.4


 


PC-7
31
5′ GCGGGGAGGAAGGGAGTAAAGTTAAT 3′
 5600
 76.7
 5500
 74.3
















TABLE 25







Klebsiella pneumoniae










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PD-1
37
5′ TAGCACAGAGAGCTTGCTCTCGG 3′
 2000
 28.6
 2100
30 


 


PD-2
38
5′ TCATGCCATCAGATGTGCCCAGA 3′
 2500
 35.7
 2600
 37.1


 


PD-3
39
5′ CGGGGAGGAAGGCGATAAGGTTAAT 3′
 2900
 41.4
 2900
 41.4


 


PD-4
40
5′ TTCGATTGACGTTACCCGCAGAAGA 3′
 4500
 64.3
 4700
 67.1


 


PD-5
41
5′ GGTCTGTCAAGTCGGATGTGAAATCC 3′
 9900
141.4
10100
144.3


 


PD-6
42
5′ GCAGGCTAGAGTCTTGTAGAGGGG 3′
13000
185.7
13400
191.4
















TABLE 26







Pseudomonas aeruginosa










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PE-1
48
5′ TGAGGGAGAAAGTGGGGGATCTTC 3′
17000
239.4
17300
240.3


 


PE-2
49
5′ TCAGATGAGCCTAGGTCGGATTAGC 3′
 8300
116.9
 8600
119.4


 


PE-3
50
5′ GAGCTAGAGTACGGTAGAGGGTGG 3′
17400
245.1
17000
236.1


 


PE-4
51
5′ GTACGGTAGAGGGTGGTGGAATTTC 3′
15000
211.3
16000
222.2


 


PE-5
52
5′ GACCACCTGGACTGATACTGACAC 3′
 8000
112.7
 8300
115.3


 


PE-6
53
5′ TGGCCTTGACATGCTGAGAACTTC 3′
 5400
 76.1
 5800
 80.6


 


PE-7
54
5′ TTAGTTACCAGCACCTCGGGTGG 3′
 5300
 74.6
 5100
 70.8


 


PE-8
55
5′ TAGTCTAACCGCAAGGGGGACG 3′
 5400
 76.1
 5000
 69.4
















TABLE 27







Serratia marcescens










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PF-1
58
5′ TAGCACAGGGAGCTTGCTCCCT 3′
 3100
 43.7
 3300
 45.2


 


PF-2
59
5′ AGGTGGTGAGCTTAATACGCTCATC 3′
 3300
 46.5
 3200
 43.8


 


PF-3
60
5′ TCATCAATTGACGTTACTCGCAGAAG 3′
10100
142.3
10000
137  


 


PF-4
61
5′ ACTGCATTTGAAACTGGCAAGCTAGA 3′
12000
169  
11800
161.6


 


PF-5
62
5′ TTATCCTTTGTTGCAGCTTCGGCC 3′
 4100
 57.7
 4200
 57.5


 


PF-6
63
5′ ACTTTCAGCGAGGAGGAAGGTGG 3′
14300
201.4
14300
195.9
















TABLE 28







Streptococcus pneumoniae










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PG-1
69
5′ AGTAGAACGCTGAAGGAGGAGCTTG 3′
 4500
 63.4
4300
 60.6


 


PG-2
70
5′ CTTGCATCACTACCAGATGGACCTG 3′
 5800
 81.7
5600
 78.9


 


PG-3
71
5′ TGAGAGTGGAAAGTTCACACTGTGAC 3′
 5000
 70.4
4900
69 


 


PG-4
72
5′ GCTGTGGCTTAACCATAGTAGGCTTT 3′
 8700
122.5
8800
123.9


 


PG-5
73
5′ AAGCGGCTCTCTGGCTTGTAACT 3′
 7200
101.4
7300
102.8


 


PG-6
74
5′ TAGACCCTTTCCGGGGTTTAGTGC 3′
 6700
 94.4
7000
 98.6


 


PG-7
75
5′ GACGGCAAGCTAATCTCTTAAAGCCA 3′
10200
143.7
9900
139.4
















TABLE 29







Haemophilus influenzae










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PH-1
78
5′GCTTGGGAATCTGGCTTATGGAGG 3′
 3100
 44.3
 3200
 45.1


 


PH-2
79
5′TGCCATAGGATGAGCCCAAGTGG 3′
 3200
 45.7
 3200
 45.1


 


PH-3
80
5′CTTGGGAATGTACTGACGCTCATGTG 3′
 4900
70 
 3600
 78.9


 


PH-4
81
5′GGATTGGGCTTAGAGCTTGGTGC 3′
 3900
 55.7
 3800
 53.5


 


PH-5
82
5′TACAGAGGGAAGCGAAGCTGCG 3′
 6700
 95.7
 6500
 91.5


 


PH-6
83
5′GGCGTTTACCACGGTATGATTCATGA 3′
10200
145.7
11000
154.9


 


PH-7
84
5′AATGCCTACCAAGCCTGCGATCT 3′
 4200
60 
 4100
 57.7


 


PH-8
85
5′TATCGGAAGATGAAAGTGCGGGACT 3′
 3200
 45.7
 3500
 49.3
















TABLE 30







Enterobacter cloacae










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PI-1
86
5′ CAGAGAGCTTGCTCTCGGGTGA 3′
10000
133.3
 9900
133.8


 


PI-2
87
5′GGGAGGAAGGTGTTGTGGTTAATAAC 3′
38000
506.7
38000
513.5


 


PI-3
88
5′GGTGTTGTGGTTAATAACCACAGCAA 3′
 4700
 62.7
 4700
 63.5


 


PI-4
89
5′GCGGTCTGTCAAGTCGGATGTG 3′
31000
413.3
32000
432.4


 


PI-5
90
5′ATTCGAAACTGGCAGGCTAGAGTCT 3′
47500
633.3
45000
608.1


 


PI-6
91
5′TAACCACAGCAATTGACGTTACCCG 3′
23600
314.7
24000
324.3


 


PI-7
92
5′GCAATTGACGTTACCCGCAGAAGA 3′
21500
286.7
22700
306.8
















TABLE 31







Enterococcus faecalis










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N
















PJ-1
98
5′TTCTTTCCTCCCGAGTGCTTGCA 3′
 7000
 98.6
 7300
101.4


 


PJ-2
99
5′AACACGTGGGTAACCTACCCATCAG 3′
12300
173.2
12000
166.7


 


PJ-3
100
5′ATGGCATAAGAGTGAAAGGCGGCTT 3′
25000
352.1
27400
380.6


 


PJ-4
101
5′GACCCGCGGTGCATTAGCTAGT 3′
10000
140.8
11000
152.8


 


PJ-5
102
5′GGACGTTAGTAACTGAACGTCCCCT 3′
 5600
 78.9
 5200
 72.2


 


PJ-6
103
5′CTCAACCGGGGAGGGTCATTGG 3′
22100
311.3
22200
308.3


 


PJ-7
104
5′TTGGAGGGTTTCCGCCCTTCAG 3′
 8800
123.9
 9000
125  









The numerical values of florescent luminances (photomultiplier voltage: 400 V) in Tables 22 to 31 indicate average pixel luminances (resolution: 5 μm). The S/N ratios indicate values obtained by dividing the fluorescent luminances by background average values measured by analysis software (GenePix Pro Ver.3.0 available from Axon) attached to the measuring device.
As is apparent from Tables 22 to 31, the respective etiologic agents can be detected with sufficient signals at a high reproducibility.
Example 3
Microorganism Detection Using 2-Step PCR
As in Examples 1 and 2, probe DNAs, specimen amplification PCR primers, the genome DNAs of etiologic agents, and DNA microarrays were prepared, and the following experiments were conducted.
1. Amplification and Labeling of Specimens (Utilization of PCR Amplification with Fluorescent Labeling)
Amplification of microbial genes as specimens (1st PCR) and labeling (2nd PCR) reaction will be described below.
2. Amplification Reaction Solution Composition: 1st PCR
















AmpliTaq Gold LD(5U/μL)
0.5 μL



Template DNA
variable



dNTP mis(2.5 mM/each)
4.0 μL



×10 PCR buffer
5.0 μL



25 mM MgCl2
7.0 μL



Forward Primer
0.25 μL 



Mix(10 μM/each)



Reverse Primer
0.25 μL 



Mix(10 μM/each)



H2O
variable



Total
 50 μL










Amplification reaction of the reaction solution having the above composition was caused by using a commercially available thermal cycler in accordance with the following protocol.
















95° C.
10 min.



 



92° C. 67° C.







 



72° C.
45 sec.



72° C.
10 min.










After the end of reaction, purification was performed by using a purification column (QIAquick PCR Purification Kit available from QIAGEN). Then, determination of the amplified products was executed.
3. Labeling Reaction Composition: 2nd PCR

















Premix PCR reagent(TAKARA
25 μl




ExTaq)



Template DNA (1st PCR Product)
Variable
(30 ng/tube)



Cy3 Labeled Reverse primer Mix
 5 μl



H20
Variable



Total
50 μl










Amplification reaction of the reaction solution having the above composition was caused by using a commercially available thermal cycler in accordance with the following protocol.
















95° C.
10 min.



 



92° C. 65° C.







 



72° C.
45 sec.



72° C.
10 min.










After the end of reaction, purification was performed by using a purification column (QIAquick PCR Purification Kit available from QIAGEN) to obtain labeled specimens.
4. Hybridization
Hybridization was done in accordance with the same procedures as in Example 1.
5. Microorganism Detection (Fluorometry)
Fluorometry was executed for the DNA microarrays after the end of hybridization reaction by using a DNA microarray fluorescent detector (GenePix 4000B available from Axon). Tables 32 to 41 show the measurement results.
Note that, in this example, fluorometry was executed once twice for each DNA microarray. The results are shown in Tables 32 to 41.







TABLE 32







Staphylococcus aureus










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PA-10
10
5′ ACGGACGAGAAGCTTGCTTCTCT 3′
  247
  3.4
  146
  2.1


 


PA-11
11
5′ TGTCACTTATAGATGGATCCGCGCT 3′
 4177
 57.9
 3083
 43.4


 


PA-12
12
5′ TGTAAGTAACTGTGCACATCTTGACG 3′
 4686
 64.9
 3768
 53.1


 


PA-13
13
5′ ACAACTCTAGAGATAGAGCCTTCCCC 3′
 2612
 36.2
 2709
 38.2


 


PA-14
14
5′ GTGGAGTAACCTTTTAGGAGCTAGCC 3′
26505
367.2
17560
247.3
















TABLE 33







Staphylococcus epidermidis










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PB-2
16
5′ TAGTGAAAGACGGTTTTGCTGTCACT 3′
 7000
 94.1
 1800
 25.7


 


PB-4
18
5′ GACCCCTCTAGAGATAGAGTTTTCCC 3′
 3274
 44.0
 1100
 15.7


 


PB-8
22
5′ AGACGAGGAGCTTGCTCCTCTG 3′
  111
  1.5
  59
  0.8


 


PB-9
23
5′ AGAACAAATGTGTAAGTAACTATGCACGT 3′
 6920
 93.0
 4910
 70.1


 


Pb-10
24
5′ ACCATTTGGAGCTAGCCGTCGA 3′
15244
205.0
18136
259.1
















TABLE 34







Escherichia coli










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PC-4
28
5′ ACTGGCAAGCTTGAGTCTCGTAGA 3′
5416
74.7
2100
31.3


 


PC-8
32
5′ TAACAGGAAGAAGCTTGCTTCTTTGCTG 3′
 160
 2.2
 112
 1.7


 


PC-9
33
5′ TTGCCATCGGATGTGCCCAGAT 3′
4133
57.0
4581
68.4


 


PC-10
34
5′ GGAAGGGAGTAAAGTTAATACCTTTGCTC 3′
4194
57.8
5349
79.8


 


PC-11
35
5′ ATCTTTTGTTGCCAGCGGTCCG 3′
6719
92.7
2594
38.7


 


PC-12
36
5′ AAGGGAGTAAAGTTAATACCTTTGCTCATTG 3′
3984
58.6
4021
60.0
















TABLE 35







Klebsiella pneumoniae










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PD-7
43
5′ TCATGCCATCAGATGTGCCCAGAT 3′
 5414
40.0
4171
62.3


 


PD-8
44
5′ CGGGGAGGAAGGCGATAAGGTTAA 3′
 4096
30.2
6227
93.0


 


PD-9
45
5′ TTATCGATTGACGTTACCCGCAGAAGA 3′
 4122
30.4
3269
48.8


 


PD-10
46
5′ CATTCGAAACTGGCAGGCTAGAGTC 3′
 9474
70.0
6486
96.9


 


PD-11
47
5′ CCTTTGTTGCCAGCGGTTAGGC 3′
10648
78.6
2754
41.1
















TABLE 36







Pseudomonas aeruginosa










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PE-1
48
5′ TGAGGGAGAAAGTGGGGGATCTTC 3′
6175
 82.2
3600
 50.0


 


PE-6
53
5′ TGGCCTTGACATGCTGAGAACTTTC 3′
8159
108.6
1200
 16.7


 


PE-7
54
5′ TTAGTTACCAGCACCTCGGGTGG 3′
3277
 43.6
1200
 16.7


 


PE-8
55
5′ TGCATCCAAAACTACTGAGCTAGAGTAC 3′
6626
 88.2
7432
103.4


 


PE-9
56
5′ GTCGACTAGCCGTTGGGATCCT 3′
5734
 76.3
3365
 46.8
















TABLE 37







Serratia marcescens










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



 No.
No.
Sequence
luminance
S/N
luminance
S/N





PF-7
64
5′ GGTAGCACAGGGGAGCTTGCTC 3′
 4482
 66.4
1040
15.1


 


PF-8
65
5′ CGAGGAGGAAGGTGGTGAGCTTAATA 3′
 6362
 94.2
3199
46.3


 


PF-9
66
5′ TACGCTCATCAATTGACGTTACTCGC 3′
 4569
 67.7
2884
41.8


 


PF-10
67
5′ GAAACTGGCAAGCTAGAGTCTCGTAGA 3′
 7905
117.1
6786
98.3


 


PF-11
68
5′ TTATCCTTTGTTGCCAGCGGTTCG 3′
12787
189.4
4849
55.7
















TABLE 38







Streptococcus pneumoniae










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PG-1
69
5′ AGTAGAACGCTGAAGGAGGAGCTTG 3′
10078
70.3
1100
 16.2


 


PG-5
73
5′ AAGCGGCTCTCTGGCTTGTAACT 3′
 4331
30.2
1500
 22.1


 


PG-6
74
5′ TAGACCCTTTCCGGGGTTTAGTGC 3′
 4730
33.0
1300
 19.1


 


PG-8
76
5′ GACATTTGCTTAAAAGGTGCACTTGCA 3′
 7128
49.7
7720
113.6


 


PG-9
77
5′ GTTGTAAGAGAAGAACGAGTGTGAGAGTG 3′
 6665
46.5
3297
 48.5
















TABLE 39







Haemophilus influenzae










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PH-1
78
5′ GCTTGGGAATCTGGCTTATGGAGG 3′
11106
150.3
3600
50.0


 


PH-2
79
5′ TGCCATAGGATGAGCCCAAGTGG 3′
 7056
 95.5
 700
10.1


 


PH-4
81
5′ GGATTGGGCTTAGAGCTTGGTGC 3′
  100
  1.4
1200
17.4


 


PH-5
82
5′ TACAGAGGGAAGCGAAGCTGCG 3′
11237
152.1
 600
 8.7


 


PH-7
84
5′ AATGCCTACCAAGCCTGCGATCT 3′
 5054
 68.4
2200
31.9
















TABLE 40







Enterobacter cloacae










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Sequence
luminance
S/N
luminance
S/N





PI-8
93
5′ GTAGCACAGAGAGCTTGCTCTCG 3′
2221
 30.1
 582
 8.2


 


PI-9
94
5′ CGGGGAGGAAGGTGTTGTGGTTA 3′
5484
 74.2
2193
30.9


 


PI-10
95
5′ ACCACAGCAATTGACGTTACCCG 3′
3325
 45.0
 646
 9.1


 


PI-11
96
5′ GAAACTGGCAGGCTAGAGTCTTGTAG 3′
7574
102.5
3039
42.8


 


PI-12
97
5′ AGGCGGTCTGTCAAGTCGGATG 3′
5768
 78.0
5701
80.3
















TABLE 41







Enterococcus faecalis










First
Second














SEQ.

Fluo-

Fluo-



Probe
ID.

rescence

rescence



No.
No.
Fluorescence
luminance
S/N
luminance
S/N
















PJ-1
98
5′ TTCTTTCCTCCCGAGTGCTTGCA 3′
1012
14.9
1500
20.8


 


PJ-3
100
5′ ATGGCATAAGAGTGAAAGGCGCTT 3′
4266
62.6
5600
77.8


 


PJ-5
102
5′ GGACGTTAGTAACTGAACGTCCCCT 3′
652
9.6
1400
19.4


 


PJ-8
105
5′ ATAGAGCTTTCCCTTCGGGGACAAA 3′
3232
47.5
810
11.2


 


PJ-9
106
5′ CGAGGTCATGCAAATCTCTTAAAGCTTCT 3′
11411
167.6
18776
260.7









As is apparent from Tables 32 to 41, the respective etiologic agents can be detected with sufficient signals at a high reproducibility.
As described above, according to the examples, an infectious etiologic agent can be identified by using microarrays on which probe sets capable of detecting the 10 bacteria, i.e., Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Streptococcus pneumoniae, Haemophilus influenzae, Enterobacter cloacae, and Enterococcus faecalis are separately immobilized or combined. Hence, the problems of the DNA probe of microbial origin are solved. More specifically, because of the small number of bases, oligonucleotide probes can chemically be mass-produced, and purification and concentration control are possible. In addition, a probe set can be provided, which allows to detect bacteria in the same species all together and discriminatingly detect bacteria in other species for the purpose of classifying the bacteria depending on the species.
Furthermore, a probe set can be provided, which also considers the stability of a hybrid body between a probe and a specimen so that the difference between the species can accurately be evaluated on a DNA microarray. A carrier on which the probe DNAs are immobilized to make the probe DNAs react with specimens can also be provided. Also, a carrier can be provided, on which the probe DNAs are chemically immobilized so that the probe DNAs are stably immobilized on the carrier, and a detection result with high reproducibility can be obtained in the process of reaction between a specimen solution and the probes and probe sets.
According to the above examples, 16s rRNA gene arrangements in the genes of infectious etiologic agents can be detected in proper quantities. Hence, the presence of an infectious etiologic agent can efficiently and accurately be determined.
Example 4
Primer Set
The primer sets (Table 11) used in the above examples to amplify the 16s rRNA gene arrangements of one or some of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Streptococcus pneumoniae, Haemophilus influenzae, Enterobacter cloacae, and Enterococcus faecalis will be described.
The primer sets of this example are designed to give a satisfactory amplification result in PCR reaction executed to identify an infectious etiologic agent. “Satisfactory” means not only that the target 16s rRNAs are sufficiently amplified but also that no products other than the 16s rRNAs are generated.
“Satisfactory” also means that only the 16s rRNAs of an infectious etiologic agent are amplified without amplifying human genome genes originated in a specimen, which are contained in the specimen.
Any specimen in which bacteria may be present, and for example, body fluids originated in animals such as human and livestock, including blood, spinal fluid, phlegm, stomach fluid, vaginal discharge, and intraoral mucus, and excretion such as urine and feces are used in this example. All media which can be contaminated by bacteria can also be used in this example, including food, drink water and hot spring water in the natulal environment, which may cause food poisoning by contamination, filters from air and water cleaners, and so forth. Animals and plants which should be quarantined in import/export are also used as specimens.
The PCR reaction used in this example includes PCR reaction which uses an extracted nucleic acid itself as a template, asymmetrical PCR reaction which uses primers on one side of SEQ ID Nos. 107 to 109 (F1 to F3 in Table 11) or SEQ ID Nos. 110 to 112 (R1 to R3 in Table 11), and PCR which executes labeling for visualization.
1. Preparation of Specimen Amplification PCR Primers
As 16s rRNA gene (target gene) amplification PCR primers for etiologic agent detection, nucleic acid sequences shown in Table 11 were designed.
More specifically, probe sets which specifically amplify the genome parts coding the 16s rRNAs, i.e., primers for which the specific melting points were made uniform as much as possible at the two end portions of the 16s rRNA coding region of a base length of 1,500 were designed. In order to simultaneously amplify variants or a plurality of 16s rRNA coding regions on genomes, a plurality of kinds of primers were designed.
The primers shown in Table 11 were purified by HPLC (High Performance Liquid Chromatography) after synthesis. All of three forward primers and three reverse primers were mixed and dissolved in a TE buffer solution such that each primer concentration had an ultimate concentration of 10 pmol/μl. In this example, all the forward primers and reverse primers were used. Alternatively, one to three forward primers and one to three reverse primers may be used.
By using a thus prepared solution of forward primers and reverse primers (forward primer mix and reverse primer mix), genome DNAs extracted by the method described in [3. Extraction of Genome DNAs (Model Specimens) of Etiologic Agents] were amplified by the method described in [5. Amplification and Labeling of Specimens (PCR Amplification & Fluorescent Labeling)].
After the end of reaction, the primers were removed by using a purification column (QIAquick PCR Purification Kit available from QIAGEN). Then, the amplified products were examined by gel electrophoresis. One band was detected in 1,500 base pair regions, and it was confirmed that satisfactory PCR reaction was executed. No byproducts were generated.
When the primers shown in Table 11 were used, satisfactory PCR amplification results were obtained in, e.g., all of the above-described 10 infectious etiologic agents (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia Coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Streptococcus pneumoniae, Haemophilus influenzae, Enterobacter cloacae, and Enterococcus faecalis).
Example 5
Amplification of 16s rRNA Genes from Mixture of Blood and Broth
Bacteremia model systems were prepared by adding 103, 104, and 105 Enterobacter cloacae, which was cultured in accordance with the procedures described in Example 1, to 200-μl human blood (collected EDTA blood). An N-acetyl muramidase solution (0.2 mg/ml in Enzyme Buffer) was added to each solution. The solutions were heated to 37° C. for 30 min. After that, DNAs were extracted by using Qiamp Blood mini Kit (available from QIAGEN) to prepare templates for PCR reaction.
PCR reaction was caused for these DNAs by using the primers shown in Table 11, as in Example 4.
As a result, as in Example 4, one band was detected in 1,500 base pair regions, and it was confirmed that satisfactory PCR reaction was executed. No byproducts were generated. The amount of PCR amplified products obtained from the band was proportional to the added cell amount. This indicates that when the primer sets were used, only the 16s rRNAs of Enterobacter cloacae were amplified without generating any PCR byproduct of human genome.
As described above, according to this example, the 16s rRNA parts in the genes of a plurality of kinds of infectious etiologic agents can efficiently be amplified at a high purity. In addition, even when human genome DNAs are present, only the 16s rRNAs of an infectious etiologic agent can efficiently be amplified.
As has been described above, according to the present invention, an infection detection probe which allows mass preparation at a time and identification of a species in similar species can be provided. More specifically, an infection detection probe which can suitably be used to classify a plurality of kinds of etiologic agents of an infection on the basis of the species can be provided.
Alternatively, an infection detection probe suitable for detection of, e.g., the above-described 10 bacteria as the etiologic agents of infections can be provided.
A probe set can also be provided, which also considers the stability of a hybrid body between an infection detection probe and a specimen so that the difference between similar species can accurately be evaluated on a DNA chip.
In addition, a carrier on which the infection detection probe is immobilized to make the infection detection probe react with the specimen can be provided.
Furthermore, a carrier can be provided, on which the infection detection probes are chemically immobilized so that the infection detection probes are stably immobilized on the carrier, and a detection result with high reproducibility can be obtained in the process of reaction with a specimen solution.
According to the present invention, there is also provided a PCR reaction primer which amplifies the 16s rRNAs of an etiologic agent in a specimen in order to detect and/or identify an infectious etiologic agent.
According to the present invention, there is also provided a primer set which can commonly be used for a plurality of species and effectively amplify the 16s rRNAs of an etiologic agent even when the species is unknown.
According to the present invention, there is also provided a primer set which can amplify the 16s rRNAs of a plurality of kinds of etiologic agents under the same PCR conditions.
As many apparently widely different embodiments of the present invention can be made without departing from the spirit and scope thereof, it is to be understood that the invention is not limited to the specific embodiments thereof except as defined in the appended claims.

## Claims

1. A probe set of infection detection probes each of which can detect an existence of a 16s rRNA gene originating in Staphylococcus aureus in a sample in which one or more of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Streptococcus pneumoniae, Haemophilus influenzae, Enterobacter cloacae and Enterococcus faecalis possibly exists, wherein the probe set comprises nine oligonucleotides, each consisting of a different one of the following base sequences (A) to (I):
(A) SEQ ID No. 1 or the complementary sequence thereof;
(B) SEQ ID No. 2 or the complementary sequence thereof;
(C) SEQ ID No. 3 or the complementary sequence thereof;
(D) SEQ ID No. 4 or the complementary sequence thereof;
(E) SEQ ID No. 5 or the complementary sequence thereof;
(F) SEQ ID No. 6 or the complementary sequence thereof;
(G) SEQ ID No. 7 or the complementary sequence thereof;
(H) SEQ ID No. 8 or the complementary sequence thereof; and
(I) SEQ ID No. 9 or the complementary sequence thereof.

2. A probe set which can detect an existence of a 16s rRNA gene originating in Staphylococcus aureus, the probe set consisting of different types of oligonucleotides, wherein the different types of oligonucleotides consist of nine oligonucleotides, each consisting of a different one of the following base sequences (A) to (I):
(A) SEQ ID No. 1 or the complementary sequence thereof;
(B) SEQ ID No. 2 or the complementary sequence thereof;
(C) SEQ ID No. 3 or the complementary sequence thereof;
(D) SEQ ID No. 4 or the complementary sequence thereof;
(E) SEQ ID No. 5 or the complementary sequence thereof;
(F) SEQ ID No. 6 or the complementary sequence thereof;
(G) SEQ ID No. 7 or the complementary sequence thereof;
(H) SEQ ID No. 8 or the complementary sequence thereof; and
(I) SEQ ID No. 9 or the complementary sequence thereof.

3. A carrier on which the probe set according to claim 1 or 2 is chemically immobilized.

4. A method of detecting a gene originating in Staphylococcus aureus in a specimen in which bacteria may be present comprising reacting the carrier of claim 3 with the specimen and detecting the gene originating in Staphylococcus aureus based on hybridization of the probe set on the carrier with the specimen.

5. The probe set according to claim 1, wherein the probe set comprises fourteen oligonucleotides, each consisting of a different one of the following base sequences (A) to (N):
(A) SEQ ID No. 1 or the complementary sequence thereof;
(B) SEQ ID No. 2 or the complementary sequence thereof;
(C) SEQ ID No. 3 or the complementary sequence thereof;
(D) SEQ ID No. 4 or the complementary sequence thereof;
(E) SEQ ID No. 5 or the complementary sequence thereof;
(F) SEQ ID No. 6 or the complementary sequence thereof;
(G) SEQ ID No. 7 or the complementary sequence thereof;
(H) SEQ ID No. 8 or the complementary sequence thereof;
(I) SEQ ID No. 9 or the complementary sequence thereof
(J) SEQ ID No. 10 or the complementary sequence thereof;
(K) SEQ ID No. 11 or the complementary sequence thereof;
(L) SEQ ID No. 12 or the complementary sequence thereof;
(M) SEQ ID No. 13 or the complementary sequence thereof; and
(N) SEQ ID No. 14 or the complementary sequence thereof.

6. An array on which the probe set according to claim 1 is immobilized, wherein the probe set includes the only probes on the array for detecting Staphylococcus aureus.