JPH0690798A - Probe for detecting staphylococcus aureus and method for detecting the same - Google Patents

Abstract

PURPOSE:To conduct the detection quickly and accurately in clinical examinations, esp. those involving food poisoning or food examinations. CONSTITUTION:The objective probe consisting of a probe for a DNA or RNA nucleic acid having a specific sequence. The second objective method for detecting Staphylococcus aureus using this probe. With this probe, Staphylococcus aureus can be detected quickly and accurately; besides, there is no need for any medium for specimen transport.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the detection of Staphylococcus aureus in clinical tests, particularly food poisoning tests or food tests.

[0002]

2. Description of the Related Art In the detection of Staphylococcus aureus, when the test material is a patient's vomiting substance, feces, food or a wiping material, it can be identified as Staphylococcus aureus by enrichment culture, isolation culture, pure culture, Although operations for confirmation culture are performed, the time required for each culture time is 18 to 24 hours, and when the total required time is as long as about 4 days. In addition, biochemical tests in confirmation culture,
It is necessary to examine aerobic confirmation, VP reaction, nitrate reduction, Tween80 hydrolysis, hyaluronidase, and glycolysis, which is a complicated method and is a problematic method in terms of time and cost.

In order to solve the above problems, a DNA probe method or a hybridization method using an oligonucleotide has been tried. For example, Kohne [Biochemical Journal 8 : 1104-1118 (1968)]
Et al. Discuss one method for preparing a probe of rRNA sequences, see Pace and Campbell [Journal of
Bacteriology 107 : 543-547 (1971)] discusses the homology of rRNAs from different bacterial species and hybridization methods for quantifying the extent of these homologies. In addition, Sogin [Journal of Molecular Evolu
tion 1 : 173-184 (1972)] et al. discuss the logical and practical aspects of using the primary structural properties of different ribosomal RNA molecules for the evaluation of phylogenetic relationships, Fox [International Journal of Systematic Bacter
Biology 27 : 44-57 (1977)] et al. discuss a comparative catalog method for prokaryotic classification. Also, Kohne et al.
Ribosomal RNA in the patent application (1983) of Gen-Probe
A strategy for obtaining nucleic acid fragments for use as a probe for a molecule is described.

[0004]

DISCLOSURE OF THE INVENTION The present invention is used for detecting DNA or RNA which can be used in a DNA probe method or a hybridization method using an oligonucleotide, particularly a 16S rRNA gene derived from Staphylococcus aureus. Complementary D belonging to the strain to be identified which has been previously denatured, if necessary if it is first double-stranded.
It is an object of the present invention to provide an oligonucleotide probe for detecting Staphylococcus aureus that hybridizes with an NA or RNA sequence, a food poisoning bacteria test, and a simple and highly sensitive test method for Staphylococcus aureus when testing bacterial infection or contamination. is there.

[0005]

The present inventors extensively searched for DNA or RNA that can be used in a DNA probe method or a hybridization method using an oligonucleotide, and selected the 16S rRNA gene of Staphylococcus aureus. The present invention has been completed by successfully producing an oligonucleotide that hybridizes with each other.

That is, the present invention comprises the following three inventions. 1. D having the following sequence or its complementary sequence
An invention relating to a probe for detecting Staphylococcus aureus , which is an NA or RNA nucleic acid probe. 10 20 30 40 50 XGAGXAACAC GXGGAXAACC XACCXAXAAG ACXGGGAXAA CXXCGGGAAA 60 70 80 90 100 CCGGAGCXAA XACCGGAXAA XAXXXXGAAC CGCAXGGXXC AAAAGXGAAA 110 120 130 140 150 GACGGXCXXG CXGXCACXXA XAGAXGGGAX CCGCGCXGCA XXAGCXAGXX GGXA 10 20 30 40 50 GAGXAACACG XGGAXAACCX ACCXAXAAGA CXGGGAXAAC XXCGGGAAAC 60 70 80 90 100 CGGAGCXAAX ACCGGAXAAX AXXXXGAACC GCAXGGXXCA AAAGXGAAAG 110 120 130 140 CAGGXCXXCG XGXCACXXAX AGAXGGAXCC CGCGXCGAXX AGC 10 20 30 40 50 GGXCXXCGGA XCGXAAAACX CXGXXAXXAG GGAAGAACAX AXGXGXAAGX 60 70 80 AACXGXGCAC AXCXXGACGG XACCXAAXCA GAAAGC 10 20 30 40 50 AXGGAGGAAC ACCAGXGGCG AAGGGCACXX XCXGGXCXGX AACXGACGCX 10 20 30 40 AGXGXXAGGG GGXXXCCGCC CCXXAGXCXG CAGCXAACGC AXXAAGCA 10 20 30 40 AGCAACGCAA AGAACCXXAC CAAAXCXXGA CAXCCXXXGA CAACXCXA 10 20 30 CXXAAGCXXA GXXGCCAXCA XXAAGXXGGG 10 20 30 40 XCAAAXCAXC AXGCCCCXXA XGAXXXGGGC XACACACGXG CXACA 10 20 30 40 50 GXGAAXACGX XCCCGGCCXX XCXGAXXCAG CGXCCCGCCA XGCACGCGCA 60 70 80 90 100 CGAGXXXGXA ACACCCGAAG CCGGXGGAGX AACCXXXXAG GAGCXAGCCG 110 120 XC GAAGGXGG GACAAAXGAX (when both sequences are DNA sequences, X = T: when RNA sequences are X = U) 2. 15 derived from the sequence described in Invention 1 above
Staphylococc which is a DNA or RNA nucleic acid probe having a sequence of 35, preferably 18-24 nucleotides or its complementary sequence.
invention relates to Bed - us aureus) detection for the professional. 3. The probe for detecting Staphylococcus aureus described in the above invention 1 or 2 is hybridized with a nucleic acid belonging to a strain to be identified which is denatured if necessary in the case of the first double strand. Staphylococcus aureus characterized by selective detection or identification
coccus aureus) invention relates to a method for detecting.

To understand the present invention, the present invention will be described in more detail below. The probe in the present invention can be obtained from Staphylococcus aureus DNA or ribosome, or can be synthesized in vitro. A probe having a length of 15 bases or more is preferable for effective use in a detection assay. Generally, the probe is preferably labeled by a publicly known method that enables detection thereof, and the preferred DNA probe is, for example, radiolabeled with ³² P or the like, or is detected. It is non-radioactively labeled with a possible compound or a compound capable of selectively binding to an indicator substance forming a complex. The complex includes avidin and biotin, an antigen and an antibody,
And compounds such as enzymes and their corresponding substrates. Other non-isotopic labels include fluorescent compounds, electron-dense compounds, acridine esters and luminescent lanthanides.

The probe of the present invention is a nucleic acid probe for use in the detection and identification of bacterial microorganisms of Staphylococcus aureus. More precisely, the probe of the present invention is a nucleic acid probe of the present invention. Respectively labeled DNA or RNA useful for specifically detecting and identifying various bacterial species
DNA or RNA nucleic acid probe having a specific sequence of
That is, it is a specific probe of Staphylococcus aureus. The specific probe of Staphylococcus aureus of the present invention is characterized by having the following sequence or its complementary sequence, that is, a sequence in which X and A are replaced with C and G in the following sequences. It is a thing. 10 20 30 40 50 XGAGXAACAC GXGGAXAACC XACCXAXAAG ACXGGGAXAA CXXCGGGAAA 60 70 80 90 100 CCGGAGCXAA XACCGGAXAA XAXXXXGAAC CGCAXGGXXC AAAAGXGAAA 110 120 130 140 150 GACGGXCXXG CXGXCACXXA XAGAXGGGAX CCGCGCXGCA XXAGCXAGXX GGXA 10 20 30 40 50 GAGXAACACG XGGAXAACCX ACCXAXAAGA CXGGGAXAAC XXCGGGAAAC 60 70 80 90 100 CGGAGCXAAX ACCGGAXAAX AXXXXGAACC GCAXGGXXCA AAAGXGAAAG 110 120 130 140 CAGGXCXXCG XGXCACXXAX AGAXGGAXCC CGCGXCGAXX AGC 10 20 30 40 50 GGXCXXCGGA XCGXAAAACX CXGXXAXXAG GGAAGAACAX AXGXGXAAGX 60 70 80 AACXGXGCAC AXCXXGACGG XACCXAAXCA GAAAGC 10 20 30 40 50 AXGGAGGAAC ACCAGXGGCG AAGGGCACXX XCXGGXCXGX AACXGACGCX 10 20 30 40 AGXGXXAGGG GGXXXCCGCC CCXXAGXCXG CAGCXAACGC AXXAAGCA 10 20 30 40 AGCAACGCAA AGAACCXXAC CAAAXCXXGA CAXCCXXXGA CAACXCXA 10 20 30 CXXAAGCXXA GXXGCCAXCA XXAAGXXGGG 10 20 30 40 XCAAAXCAXC AXGCCCCXXA XGAXXXGGGC XACACACGXG CXACA 10 20 30 40 50 GXGAAXACGX XCCCGGCCXX XCXGAXXCAG CGXCCCGCCA XGCACGCGCA 60 70 80 90 100 CGAGXXXGXA ACACCCGAAG CCGGXGGAGX AACCXXXXAG GAGCXAGCCG 110 120 XC GAAGGXGG GACAAAXGAX (when all sequences are DNA sequences X = T: when RNA sequences are X = U) Furthermore, as a specific probe of S. aureus of the present invention, it is derived from the sequences described above 15 ~ 35,
Preferred are those having a sequence of 18 to 24 nucleotides or a sequence complementary thereto. 15-35, preferably 18 derived from the sequences described above
A probe composed of a labeled oligomer of ˜24 nucleotides or a complementary oligomer is a preferable probe for the present invention, and specific examples thereof include the following sequences. As from an array of:

These nucleic acid probes can be obtained by well-known state-of-the-art techniques and can be obtained by various routes, especially genetic engineering or direct manual or automatic synthesis.

Obtaining and synthesizing the probe The present inventors first used reverse transcriptase to obtain a DNA sequence complementary to rRNA of Staphylococcus aureus, which has no homology with rRNA of other organisms. , Isolated and identified. Reverse transcriptase is a method in which RNA is used as a template and is transcribed to form a complementary DNA chain (cDNA). By using this enzyme, rRNA of Staphylococcus aureus is converted to cD
I was able to synthesize NA. After identifying the DNA fragments encoding 16S rRNA by hybridization, the nucleotide sequences of those DNAs were standardized (see, eg, Sanger et al, 74 : 5453 (1977), Proc. Natl.
Acad.Sci). This nucleotide sequence was then compared to various other published rRNA sequences to determine regions of homology and non-homology. A region not homologous to other published sequences was subcloned or an oligonucleotide automatic synthesizer was used to obtain a Staphylococcus aureus DNA fragment. These characteristic S. aureus DNA fragments were labeled with ³² P or ³⁵ S
Can be used as a probe for testing against both S. aureus and non-S. Aureus. If necessary, vector the probe sequence.
It is also possible to use the obtained vector by incorporating it into a probe. The vector used for this should have no homology to any non-staphylococcus aureus that may be present in the sample to be tested.

The sequence of the probe specific for S. aureus is described below. The sequences of SEQ ID NOs: 1 to 9 in (Sequence Listing) show the nucleotide sequences of 9 parts of S. aureus 16S rRNA. This sequence is obtained by using computer program genetics (GENETYX).
tment of Health and Human Services) and EMBL (Europea
n Molecular Biology Laboratory), it is a part that was confirmed to be surely different from the non-staphylococcus aureus in the region of several bases when compared with the nucleic acid sequence.

The probe of the present invention has the above-mentioned sequences, but is not limited thereto. In addition to the sequences shown in the sequence listing, 15 to 35, preferably derived from those sequences are preferable. It also includes a probe having a sequence composed of 18-24 nucleotide oligomers or complementary oligomers. These probes can also be used in combination of two or more kinds of probes.

A method for synthesizing a polynucleotide probe using an automatic oligonucleotide synthesizer will be described below. A 37-nucleotide-long polynucleotide probe having the following defined sequence was synthesized by the thiophosphite method (Japanese Patent Laid-Open No. 61-180002). When the synthesis was completed, the polynucleotide was separated from the carrier, and the target substance was fractionated by HPLC using a C ₁₈ column by a known chromatography method, whereby a polynucleotide having a purity of about 99% could be obtained. The produced polynucleotide was compared with a 37-base standard polynucleotide by electrophoresis to confirm the production. 5'- CACCTATTGG ATGGATATTC TGACCCTATT GAAGCCC-3 'After confirming by the known dideoxy method that the nucleotide sequence of the polynucleotide prepared by the above method is correct, this polynucleotide was identified as 16S of Staphylococcus aureus.
It was investigated whether it binds to rRNA. First, 16S rRNA was extracted from Staphylococcus aureus by carrying out operations such as phenol / chloroform extraction and ethanol precipitation. This is mixed with the created probe, and α
-A reverse transcriptase was allowed to act in the presence of ³⁵ S-dATP, and the fact that this probe became a primer and the cDNA elongation reaction was proceeding was confirmed by methods such as electrophoresis and autoradiography. Confirmed using. From this result, the prepared polynucleotide probe was targeted to 16
SrRNA was confirmed and confirmed to be able to bind.

These nucleic acid sequences may be denatured complementary DNA or RN, if necessary in the case of the first duplex.
It has the property of forming a hybrid with the A sequence. The denaturation of nucleic acid can be carried out by incubating in a basic medium, raising the medium temperature, a combination of these two processes, or again by the action of microwaves. Hybrid formation can be accomplished by a variety of methods.

The probe of the present invention is labeled by one of the methods known to those skilled in the art for labeling nucleic acid probes. For example, it is labeled with a radioactive isotope such as ³² P or a non-radioactive isotope such as an enzyme, which is also known. In some cases, the probe is not enzymatically labeled, but is chemically modified with, for example, biotin, which may be present after hybridization.

[0016]

The probe of the present invention comprises contacting the probe with a bacterium in a sample under conditions that allow the probe to hybridize with any S. aureus rRNA present in the sample. The formation of a nucleic acid hybrid complex can detect the presence of Staphylococcus aureus in a sample by detecting the complex, and the presence of Staphylococcus aureus in a sample can be proved. In particular, most genes consist of rRNA [a complex mixture of protein and RNA and are involved in the translation of genetic information in all organisms, and there are three different ribosomal RNA molecules in bacteria ( 5S, 16S, 23S)]
(Noller (1984) Ann. Rev. Biochem.
53 : 119), since various bacterial cells contain about 1.2 × 10 ⁴ ribosomes, at least 1.2 × 10 ^{4 of} all cells are contained.
^While each gene contains ^four molecules of rRNA, other genes in bacteria are only present in 1-2 copies per cell, and their mRNA products are not stable and are not always transcribed. Therefore, rR by hybridization
The detection of NA will be about 10 ⁴ times more sensitive to hybridization of other genes to DNA.

[0017]

[Example] Nine kinds of probes (SA31, SA32, SA33, SA41, SA5) having the following sequences homologous to the sequences shown in the sequence listing
1, SA81, SA9, SA101, SA111) were synthesized by the above thiophosphite method, and identification tests were performed on Staphylococcus aureus and non-Staphylococcus aureus by the hybridization assay. The test results are shown in Table 1. As shown in Table 1, among these probes, SA31, SA32, SA33, SA41, SA51, SA9, SA111
Was shown to be specific to Staphylococcus aureus and showed almost no hybridization with DNA or RNA derived from non-staphylococcus aureus. However, some false positives were found for SA81 and SA101. In addition, the non-staphylococcus aureus shown in Table 1 is a part of the bacterial species that can be an inspection target in the Staphylococcus aureus test.

[0018]

[Table 1]

[0019]

[Effect] The detection of Staphylococcus aureus using the probe of the present invention gives a quick and accurate result, and the presence of Staphylococcus aureus in a sample can be examined in a short time in a clinical or food laboratory. You can Also, the detection method of the present invention relies on the nucleic acid content of the bacterium rather than on a variety of other variable biological properties, so that it is not only a biologically typical species of Staphylococcus aureus but also atypical. It is also possible to detect various species, and furthermore, since the detection method does not necessarily require living cells for detection, it has an excellent effect that the need for a transport medium for transporting a specimen to a laboratory is eliminated. Is played.

[0020]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 154 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: 16S rRNA Sequence XGAGXAACAC GXGGAXAACC XACCXAXAAG ACXGGGAXAA CXXCGGGAAA CCGGAGCXAGA CGCAXXGACACGAGACAAGCACXGACAAGACGAXGACAGACGAXGACAAGACGAXGAA 120 XAGAXGGGAX CCGCGCXGCA XXAGCXAGXX GGXA 154 Sequence number: 2 Sequence length: 143 Sequence type: Nucleic acid Number of strands: Single-stranded topology: Linear Sequence type: 16SrRNA sequence GAGXAACACG XGGAXAGCXGAGCAXACACGGACGACCACACGGACGACCAACXXGCGGCAAAC 60XXCGGGAAA 60XXCGGGAAAC 60XXCGGGAA CAGGXCXXCG XGXCACXXAX 120 AGAXGGAXCC CGCGXCGAXX AGC 143 SEQ ID NO: 3 Sequence length: 86 Sequence type: Nucleic acid Strand number: Single strand Topology: Linear Sequence type: 16SrRNA sequence GGXCXXCGAX GACGXAAAXACCXGXXAXXCGAXCGXAAAGACGAX 86 SEQ ID NO: 4 Sequence length: 0 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: 16S rRNA sequence AXGGAGGAAC ACCAGXGGCG AAGGGCACXX XCXGGXCXGX AACXGACGCX 50 SEQ ID NO: 5 Sequence length: 48 Sequence type: Nucleic acid Number of strands: 1 Single-stranded topology: Linear Sequence type: 16S rRNA sequence AGXGXXAGGG GGXXXCCGCC CCXXAGXCXG CAGCXAACGC AXXAAGCA 48 SEQ ID NO: 6 Sequence length: 48 Sequence type: Nucleic acid Number of strands: Single-stranded topology: Linear Sequence type: 16S rRNA sequence AGCAACGCAA AGAACCXXAC CAAAXCXXGA CAXCCXXXGA CAACXCXA 48 Sequence number: 7 Sequence length: 30 Sequence type: Nucleic acid Strand number: Single strand Topology: Linear Sequence type: 16S rRNA sequence CXXAAGCXXA GXXGCCAXCA XXAAGXXGGG sequence Length: 45 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: 16S rRNA Sequence XCAAAXCAXC AXGCCCC XXA XGAXXXGGGC XACACACGXG CXACA 45 SEQ ID NO: 9 Sequence length: 120 Sequence type: Nucleic acid Number of strands: Single-stranded topology: Linear sequence type: 16SrRNA sequence GXGAAXACGX XCCCGGCCGG XCXGAXCAG CGA ACGAGGACGACGACGCGACCGCGCACGCGAGCAGCGCGCGCGCGCGCGCGCGCGCGCGCGCG GACAAAXGAX 120

Claims (3)

[Claims] 1. A Staphylococcus aureus , which is a DNA or RNA nucleic acid probe having the following sequence or a sequence complementary thereto.
) A probe for detection. 10 20 30 40 50 XGAGXAACAC GXGGAXAACC XACCXAXAAG ACXGGGAXAA CXXCGGGAAA 60 70 80 90 100 CCGGAGCXAA XACCGGAXAA XAXXXXGAAC CGCAXGGXXC AAAAGXGAAA 110 120 130 140 150 GACGGXCXXG CXGXCACXXA XAGAXGGGAX CCGCGCXGCA XXAGCXAGXX GGXA 10 20 30 40 50 GAGXAACACG XGGAXAACCX ACCXAXAAGA CXGGGAXAAC XXCGGGAAAC 60 70 80 90 100 CGGAGCXAAX ACCGGAXAAX AXXXXGAACC GCAXGGXXCA AAAGXGAAAG 110 120 130 140 CAGGXCXXCG XGXCACXXAX AGAXGGAXCC CGCGXCGAXX AGC 10 20 30 40 50 GGXCXXCGGA XCGXAAAACX CXGXXAXXAG GGAAGAACAX AXGXGXAAGX 60 70 80 AACXGXGCAC AXCXXGACGG XACCXAAXCA GAAAGC 10 20 30 40 50 AXGGAGGAAC ACCAGXGGCG AAGGGCACXX XCXGGXCXGX AACXGACGCX 10 20 30 40 AGXGXXAGGG GGXXXCCGCC CCXXAGXCXG CAGCXAACGC AXXAAGCA 10 20 30 40 AGCAACGCAA AGAACCXXAC CAAAXCXXGA CAXCCXXXGA CAACXCXA 10 20 30 CXXAAGCXXA GXXGCCAXCA XXAAGXXGGG 10 20 30 40 XCAAAXCAXC AXGCCCCXXA XGAXXXGGGC XACACACGXG CXACA 10 20 30 40 50 GXGAAXACGX XCCCGGCCXX XCXGAXXCAG CGXCCCGCCA XGCACGCGCA 60 70 80 90 100 CGAGXXXGXA ACACCCGAAG CCGGXGGAGX AACCXXXXAG GAGCXAGCCG 110 120 XC GAAGGXGG GACAAAXGAX (when all sequences are DNA sequences, X = T: when RNA sequences, X = U) 2. A DNA or RNA nucleic acid probe having a sequence of 15 to 35, preferably 18 to 24 nucleotides derived from the sequence of claim 1 or its complementary sequence. Staphylococcus aureus ( St
aphylococcus aureus ) detection probe. 3. A nucleic acid belonging to a strain to be identified, wherein the probe for detecting Staphylococcus aureus according to claim 1 or 2 is first denatured if necessary in the case of a double strand. A method for detecting Staphylococcus aureus, which comprises hybridizing with and performing selective detection or identification.