JP2000342261A - Method and kit for detecting burkholderia cepacia - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an oligonucleotide capable of directly, rapidly and surely detecting Burkholderia cepacia, useful for detecting opportunistic infectious disease, etc., by making the oligonucleotide include a specific base sequence or a sequence complementary to the sequence. SOLUTION: This oligonucleotide comprises an antisense chain sequence of the formula cccgrctata ttagarccaa gg (r is a or g) from base no.451 to 462 of 16 SrRNA gene of Burkholderia cepacia strain 6 or a sequence complementary to the sequence. Preferably the oligonucleotide is used as a probe or is contained as a primer for gene amplification reaction to detect Burkholderia cepacia. Preferably a kit for detecting Burkholderia cepacia is prepared by making the kit include the oligonucleotide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a Burkholderia cepacia (hereinafter referred to as Burkholderia cepacia or B. cepaci).
a)) and a kit for detection, and an oligonucleotide used in the kit.

[0002]

2. Description of the Related Art Burkholderia cepacia is a gram-negative bacillus widely present in the environment mainly in water and soil, and is known as one of the causative bacteria of opportunistic infections. When contaminated and proliferated in foods, medicines, cosmetics, and the like, they may cause quality deterioration such as an unpleasant odor, and are important bacteria in quality control. Usually, the detection and identification of Burkholderia cepacia is performed by a combination of a selective medium and an examination of physiological and biochemical properties by a culture method. However, in principle, the culture method requires at least one day or more for bacteria to grow to a detectable level, and lacks rapidity. Also Burkholderia cepacia
Physiological and biochemical properties are similar to other Burkholderia bacteria and related Pseudomonas bacteria, and it is difficult to make an accurate determination. The PCR method has a 20-30 bp sequence having a sequence complementary to a specific gene sequence to be targeted.
This is a technique of amplifying and synthesizing a gene sequence sandwiched between oligonucleotides (hereinafter referred to as primers) of a certain degree using a heat-resistant DNA synthase. It is a technique that is routinely used in the field of gene analysis, and is useful as a means for rapidly and highly sensitively detecting and identifying genes. It can also be used for highly sensitive detection and simple identification of microorganisms by targeting genes specific to certain microorganisms,
Detection kits using this principle are also commercially available.

[0003] Burkholderia c reported so far
The following can be considered as primers for epacia detection. Targeting a gene specifically present in Burkholderia cepacia Targeting a gene that is present in all bacteria, but each with a slightly different sequence At present, the gene region specifically present in Burkholderia cepacia is ,unknown. Examples include the 23S rRNA gene by Tyler et al., And Cal.
A report targeting 16S rRNA gene was reported by lagham et al. For the former, there is a problem in that it is not possible to verify that the target gene is specifically present in Burkholderia cepacia, because the gene information that has been clarified is small.

[0004] Regarding the latter, many researchers around the world have analyzed the 16S rRNA gene sequences of various fungi,
The results are easily available via the Internet, and there seems to be no problem with verification. Until now, O'Callagham et al. (J. Clin. Pathol. 47, 222-226, 199
4), Campbell et al. (Pediatr. Pulmonol. 1995, 20, 44-4
9), Karpati et al. (Mol.Cell.Probes, 10, 397-403, 1996)
Report primers for detecting Burkholderia cepacia. However, our NCBI BLAS
As a result of homology search by T search, it was found that these primers had a problem in specificity.

[0005] First, the primer of O'Callagham et al. Is forward primer: 5'-GGTACCGGAA GAATAAGC-3 'reverse primer: 5'-CTGTTCCGACCATTGTAT-3'. No. AF042161, hereinafter abbreviated as AF042161), B. cepacia strain (MEDLINE ACCESSION No. U9692)
7, hereinafter abbreviated as U96927), B. cepacia strain (MEDLINE A
CCESSION No.AB015606.1, hereinafter abbreviated as AB015606.1), B.cepacia strain (MEDLINE ACCESSION No.M22518, hereinafter abbreviated as M22518), B.cepacia strain (MEDLINE ACCESSI
ON No.L28675, hereinafter abbreviated as L28675) and B.gladio
li, B.mallei, B.pickettii, B.solanacearum, B.caryo
Homology was also observed in phylli, B. cocovenenans and the like.
On the other hand, B. cepacia strain (MED
LINE ACCESSION No.X80284, hereafter abbreviated as X80284),
B.cepacia strain (MEDLINE ACCESSION No.X80286, hereafter X802
86), B. cepacia strain (MEDLINE ACCESSION No.
X87275, hereinafter abbreviated as X87275), B. cepacia strain (MEDLI
NE ACCESSION No.AF107072, hereinafter abbreviated as AF107072), B.cepacia strain U96927, B.cepacia strain AB015606.1, B.gladioli, B.mallei, B.glathei, B.caribiens
is, B.caryophylli, B.ocovenenans, B.glumae, B.pseu
Homology is also found in domallei and the like, and with the combination of these primers, bacteria other than B. cepacia are also detected, so there may be no specificity. (Table 2)

The primer of Campbell et al. Is a forward primer: 5′-AACTAGTTGT TGGGGATTCATTC-
3 'reverse primer: 5'-TTTCGAGCAC TCCCGCCTCTCAG-
3 ', but B. cepacia strain
X80284, B. cepacia strain X80286, B. cepacia strain X87275, B.
cepacia strain AF107072, B. cepacia strain U96927, B. cepacia
AB015606.1 strain, B.cepacia strain (MEDLINE ACCESSION No.M
22518, hereinafter abbreviated as M22518), B. cepacia strain L2867
5. B. cepacia strain (MEDLINE ACCESSION No.D13046; D
13046), B.gladioli, B.mallei, Bg
lathei, B. caryophylli, B. cocovenenans, B. thailande
Homology is found in nsis etc. On the other hand, homology is observed in the reverse primers in B.cepacia strains U96297 and B.cepacia.
pacia strain M22518 and B. thailandensis strain U91838. In these combinations, besides B.cepacia, B.thailan
densis may be detected. (Table 3)

The primers of Karpati et al. Include forward primer: 5'-GTGCCTGCAG CCGCGGTAAT-3 'reverse primer: 5'-TCCCGCCTCT CAGCAAGGATTCC-
3 ', but the forward primer is universal and the reverse primer is B. cepacia strain M2251
8.In addition to B. cepacia strain U96927, B. thailandensis strain U918
Since there is homology in 38 as well, this combination may have low specificity. (Table 4)

Thus, O'Callagham et al., Campbell
The primers of Karpati et al. Also used B.gladioli, B.mallei, B.glathei, B.caryophyl in addition to Burkholderia cepacia.
li, B. cocovenenans, B. thailandensis, etc. are detected, and the specificity is low.

[0009]

Accordingly, an object of the present invention is to enable detection of Burkholderia cepacia with high specificity.

[0010]

Means for Solving the Problems The present inventors have proposed Burkhold
By comparing the gene sequences of closely related species of Burkholderia cepacia and Burkholderia cepacia,
A primer sequence capable of detecting only ia was found. That is, the present invention provides an oligonucleotide having the sequence of SEQ ID NO: 1 or its complementary sequence. The present invention also provides a method for detecting Burkholderia cepacia, which comprises using an oligonucleotide having the sequence of SEQ ID NO: 1 or its complementary sequence as a probe. Also provided is a method for detecting Burkholderia cepacia, which comprises using an oligonucleotide having the sequence of SEQ ID NO: 1 or a complementary sequence thereof as a primer for gene amplification reaction.
Furthermore, Burkho, characterized by containing an oligonucleotide having the sequence of SEQ ID NO: 1 or a complementary sequence thereof.
Provide a kit for detecting lderia cepacia.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The 16S rRNA gene sequence of Burkholderia cepacia currently registered in the database is the Burkholderia cepacia D registered by Ludwig et al.
SM50181 strain (MEDLINE ACCESSION No.X87275), Tabacchion
Burkholderia cepacia strain (MEDLINE
ACCESSION No.X80286), a Burkholderia cepacia strain registered by Tabacchioni et al. (MEDLINE ACCESSION No.X802
84), Burkholderia cepaci registered by Dewhirst et al.
a ATCC25416 strain (MEDLINE ACCESSION No.M22518), Zhou
Burkholderia cepacia G4 strain (MEDLIN
E ACCESSION NO.L28675), registered by Hanada et al.
rkholderia cepacia KP24 strain (MEDLINE ACCESSION No.AB0
15606, abbreviated as AB015606), Burkholderiacepacia PVFi5A strain (MEDLINE ACCES) registered by Di Cello et al.
SION No. AF042161), Burkh registered by Viallard et al.
olderia cepacia ATCC25416T strain (MEDLINE ACCESSION No.
U96927), Burkholderi registered in NCBIBLAST Research
a cepacia strain (MEDLINE ACCESSION No. U91567, U91568; hereinafter abbreviated as U91567, U91568). The present inventors have developed Burkholdia cepacia, a related species of Burkholderia cepacia.
The 16S rRNA gene sequences of other bacteria of the genus eria were obtained from various gene databases and compared in detail. as a result,
In addition to Burkholderia cepacia, it was found that the sequence of SEQ ID NO: 1 was conserved only in a very small number of other species.

That is, as shown in Table 1, Burkholder
ia cepacia strain AB015606, Burkholderia cepacia strain AF04
2161, Burkholderia cepacia strain X80286, Burkholderia
cepacia strain X80284, Burkholderia cepacia strain U91567, B
urkholderia cepacia strain 16 strains of U91568 16S rRNA
In the gene sequence, a sequence complementary to SEQ ID NO: 1 is found at the positions of base numbers 451 to 472 (SEQ ID NO: 1), but no other bacteria in which SEQ ID NO: 1 is conserved are found. In SEQ ID NO: 1, r at the 5th and 16th positions represents a or g. That is, the oligonucleotide of the present invention may be any combination of four kinds of sequences whose fifth and 16th bases are a and a, a and g, g and g, or g and a. A mixture of any two or more of them may be used.

The oligonucleotide of the present invention can be prepared by any suitable method. For example, the whole or a part of the sequence may be chemically synthesized according to the method described in Examples below. For example, it can be chemically synthesized using an automatic DNA synthesizer. Or Burkholderia
It is also possible to cut out directly from the cepacia gene using restriction enzymes, etc.
It is also possible to clone the plasmid into such a plasmid, collect the plasmid after bacterial growth, cut out the plasmid, and use it.

The oligonucleotide thus prepared can be used as a primer and Burk.
It can also be used as a probe for detecting holderia cepacia. However, it is preferable to use it as a primer because the specificity of detection of Burkholderia cepacia is enhanced. Such an oligonucleotide or a partial base sequence that can be used as a primer or a probe may have a site capable of binding to a label or a solid phase carrier, if necessary. Here, in the case of performing detection using a primer, a site where a site capable of binding to a label or a solid phase carrier may be introduced may be any site which does not hinder the extension reaction of the primer. Terminals are most preferred. In the case of performing detection using a probe, the position at which a site capable of binding to a label or a solid phase carrier may be introduced may be a 3′-terminal or 5′-terminal hydroxyl group portion, or a base portion or a phosphate diester portion. It is possible,
It is desirable to consider the base sequence and length of the probe so as not to hinder hybridization.

As the label, either a radioactive substance or a non-radioactive substance may be used. Examples of the non-radioactive label include those that can be directly labeled, such as fluorescent substances such as fluorescein derivatives, rhodamine and its derivatives, chemiluminescent substances, and delayed fluorescent substances. It is also possible to indirectly detect a label using a substance that specifically binds to the label. Examples of such a label include biotin and hapten. For biotin, avidin or streptavidin is used, and for hapten, an antibody that specifically binds to avidin or streptavidin is used. As the hapten, a compound having a 2,4-dinitrophenyl group, digoxigenin, or the like can be used. All of these labels may be used alone or in combination of two or more as necessary.
It can be introduced into a probe or primer.

When it is necessary to specifically bind a specific fragment of a nucleic acid to a solid phase carrier such as sandwich hybridization, the site capable of binding to the solid phase carrier may be any site that can selectively bind to the carrier. Anything is fine. For example, biotin, fluorescein, compounds having a 2,4-dinitrophenyl group, and haptens such as digoxigenin can be mentioned, and any of these can be introduced into a probe or a primer alone or in combination of two or more as needed.

As the solid phase carrier, a solid phase material such as polystyrene balls, agarose beads, polyacryl beads, latex, microtiter wells, etc. capable of capturing the binding site introduced into the primer, for example, streptoa Pididine, antibodies and the like are introduced. For example, to capture a PCR product from a biotin-introduced primer, a carrier having streptavidin bound to a solid phase, and to capture an elongation reaction product from a primer into which fluorescein or the like has been introduced, use an antibody against each. Is used.

Furthermore, by making the solid phase carrier into fine particles, the target nucleic acid can be easily determined by the presence or absence of aggregation or precipitation. In addition, one of the primers has a site capable of binding to the solid phase carrier, and the other primer has a label introduced therein. There is also a method of washing away with a suitable solvent, and the target nucleic acid is immobilized on the solid support in a form having a label, and is specifically detected.

Next, a method for detecting Burkholderia cepacia using the oligonucleotide of the present invention as a primer will be described. According to the present invention, base numbers 451 to 47
Using an oligonucleotide homologous to a sequence upstream of No. 2 as an upstream forward primer and an oligonucleotide represented by SEQ ID NO: 1 as a downstream reverse primer, preferably by Burckholderia PCR (Polymerase Chain Reaction) method cepacia can be detected.

Tables 1 to 4 show the bacteria that can be detected using the primer of the present invention and the conventional primer. In Tables 1 to 4, Burkholderia is abbreviated as B. It can be seen that the primer of SEQ ID NO: 1 has higher specificity for six Burkholderiacepacia strains than the conventional reverse primer.

[0021]

[Table 1]

[0022]

[Table 2]

[0023]

[Table 3]

[0024]

[Table 4]

Therefore, the oligonucleotide represented by SEQ ID NO: 1 was used as a reverse primer, which was combined with a known upstream primer such as Campbell et al.
Burkho
lderia cepacia can be specifically detected. That is, if the oligonucleotide represented by SEQ ID NO: 1 is used as the reverse primer on the downstream side, the forward primer on the upstream side is not particularly limited, and even if a universal primer common to all the bacteria is used, the specificity of detection is low. It is not spoiled. Alternatively, an oligonucleotide having a complementary sequence to the sequence represented by SEQ ID NO: 1 may be used as an upstream forward primer, and an oligonucleotide complementary to a sequence downstream of base numbers 451 to 472 may be used as a downstream reverse primer. PCR (Polymeras
e Chain Reaction) method specifically to Burkholderia cepa
cia can be detected.

Here, the basic form of the primer of the present invention may be unmodified, or a primer capable of introducing a detectable label or a portion capable of binding to a solid phase carrier as described above. And primers into which a moiety capable of binding to a solid phase carrier has been introduced.

Although various methods have been devised for the enzymes and reaction conditions used for the gene amplification reaction, the oligonucleotides of the present invention have a sufficient length and base sequence to be used in various PCR methods. It can be used to reliably and easily detect Burkholderia cepacia.

Hereinafter, the detection method of the present invention will be described according to a general PCR method. That is, the method is carried out by adding the primers and the DNA polymerase to the sample, repeating denaturation treatment to single-stranded DNA, annealing and extension reaction several tens of times, and detecting the amplified gene.

The sample used in the detection method of the present invention is not particularly limited, and various samples in which it is desired to determine the presence or absence of Burkholderia cepacia in fields such as research, medical care, environment, and quality control are used. For the test, it is necessary to extract the nucleic acid component of Burkholderia cepacia as a PCR sample from these materials, but since Burkholderia cepacia is a gram-negative bacterium with low cell wall strength, no special extraction operation is required. Can be detected by the present detection method if the number of bacteria contained in the sample is large. Further, by using a method by a physical operation such as heat treatment, pressure treatment, or grinding, a method using an alkali or a surfactant, a method using a protease such as proteinase K, etc., more efficient D
NA can be extracted, and a highly sensitive test can be expected. 16SrRN, which has many copies in the cells
A is DNated by reverse transcriptase
By transferring to A, the sensitivity can be further improved.

A part of these nucleic acid extraction solutions can be directly subjected to PCR, but the sensitivity of PCR can be increased by adding nucleic acid purification and concentration operations. As a method for purification or concentration, a method using an organic solvent such as phenol as a protein denaturant, or a method using other specific protein denaturants [Beutler et al., Bi
oTechniques 9, P. 166 (1990)], a method using a carrier such as latex particles having a capture and concentration probe immobilized thereon (JP-A-63-117262), and the like.
Other commercially available DNA extraction kits and the like may be used.

As the polymerase used for PCR, it is preferable to use a heat-resistant DNA polymerase, for example, Taq DNA polymerase. More preferably 3 '→
By using a thermostable DNA polymerase having 5 'exonuclease activity, an improvement in detection sensitivity can be expected. As preferable PCR conditions, for example, when a heat-resistant DNA polymerase is used and the temperature cycle is performed in three steps of denaturation, annealing, and extension, the temperature is 90 ° C. to 96 ° C. for 1 second to 60 seconds, and 50 ° C. to 70 ° C., respectively.
For 1 second to 120 seconds, and 65 to 75 ° C. for 1 second to 120 seconds. When the reaction is carried out on a scale using a 0.2 mL tube which is usually used, the reaction is performed at 94 ° C. for 30 seconds.
The conditions of 55 ° C. for 60 seconds and 72 ° C. for 60 seconds are preferred. When annealing and elongation are performed in two steps in one step, the steps are performed at 90 to 96 ° C for 1 to 60 seconds and 65 to 65 ° C, respectively.
It is preferably performed at 72 ° C. for 1 to 120 seconds. When performing the reaction on a scale using a 0.2 mL tube, the conditions of 94 ° C. for 30 seconds and 68 ° C. for 60 seconds are preferred.

PCR using primers of the present invention
DNA amplification is determined by a method in which the size of the generated amplified DNA is confirmed by electrophoresis, a method in which PCR is further performed on the amplified product, or a method in which the amplified product is confirmed using an internal probe and the sensitivity is simultaneously improved. , A method of confirming PCR amplification in real time by fluorescently labeling the internal probe, and using a labeled primer for identification.
All commonly used methods such as a method of performing CR and capturing and identifying a labeled amplified DNA on a solid phase, and an in situ PCR method can be used.

As a method for capturing and identifying amplified DNA, a method using a solid phase in which a substance specifically binding to a label is immobilized in advance (Japanese Patent Laid-Open No. 1-252300),
There is a method in which a capture probe having a sequence complementary to the sequence of the target amplified DNA is immobilized on a solid phase in advance, and specific binding is performed by hybridization. For such a solid phase, a microtiter well is used. Alternatively, beads or the like can be used, but it is advantageous to use microtiter wells for processing multiple samples at once.

When a clinical sample is used as a direct sample for testing, even if Burkholderia cepacia is present in the sample, its number is small and DNA
It is conceivable that it is necessary to repeat a considerable number of temperature cycles for amplification. In such a case, even with the use of carefully studied primers, the danger of non-specific amplified DNA cannot be completely eliminated, and amplification D
For capture of NA, it is desirable to use hybridization with a capture probe. Kawai et al.'S method for fixing capture probes to microtiter wells [Analytical
Biochemistry 209. P.63-69] can be used.

When PCR is carried out using a primer to which an identifiable label has been given and the labeled amplified DNA is detected, a general method may be used according to the labeling substance to be used. For example, if the labeling substance is a radioisotope, the activity may be measured as it is. For example, if biotin is used, an avidin-enzyme conjugate may be used. And detection may be performed by optical or fluorescent means.

The present invention also relates to Burk,
Provide a detection kit for performing the holderia cepacia detection method. The detection kit of the present invention comprises the above-described SEQ ID NO: 1
A kit comprising an oligonucleotide having a base sequence of: and a reagent necessary for PCR. The reagents included in the detection kit include DNA polymerase, primers, buffers (concentration buffer solution if necessary),
Each solution of dATP, dCTP, dTTP, dGTP and, if necessary, control primers, and the like. Preferably, these components are dispensed into a PCR tube for each use of one sample in a PCR tube, or a tablet. It may be.

For example, the composition of the final reaction solution is 0.1 to 5
mM Tris-HCl, pH 7.5-8.5,0-0.
5 mM MgCl ₂ , 1-500 mM KCl, 1-5
00 μM dATP, 1 to 500 μM dCTP, 1
500 μM dGTP, 1-500 μM dTTP,
0.1 to 1000 μM primer, 0.1 to 2 U / 5
A kit set to be 0 μl Taq DNA polymerase can be exemplified, but the concentration of each component may be any range as long as a normal PCR reaction is possible, and is not limited thereto. Other components can be added as long as they do not interfere with the PCR reaction.

Although the oligonucleotide of the present invention can be used as a probe for detecting Burkholderia cepacia, the detection method used as a primer is more effective in detecting Burkholderia cepacia.
This is preferred because the specificity of eriacepacia detection is increased. In general, detection methods using probes include dot hybridization, southern hybridization, and in hybridization.
There are in situ hybridizations, and any of these can be used to carry out hybridization in the usual manner using the above labeled oligonucleotide.

In recent years, in order to increase the sensitivity of hybridization, rRN has been used in many copies per cell.
Although a method using A as a detection target has been developed, the oligonucleotide in the present invention is an oligonucleotide containing a region encoding 16S rRNA, and can be used for rRNA detection by a similar method. Also in
In situ hybridization, generally, rR
By using NA as a detection target, a good S / N ratio can be detected, but the oligonucleotide of the present invention can be used for detection as described above. In addition, methods based on sandwich hybridization have been developed for the simplification of the operation, and these methods also utilize the oligonucleotide of the present invention to produce Bu.
rkholderia cepacia can be detected.

[0040]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1] Selection of primers (1) DNA was extracted and prepared from the culture of the following strains by a heat treatment method. Burkholderia cepacia ATCC25416 Burkholderia cepacia GIFU725 Burkholderia cepacia GIFU2427 Burkholderia gladioli IFO13700 Burkholderia andropogonis ATCC23061 Burkholderia pickettii ATCC27511 Burkholderia caryophylli ATCC25418 Burkholderia (Ralstona)

(2) Synthesis and Labeling of Primers Oligonucleotides as primers were synthesized by a phosphoramidite method using a DNA synthesizer manufactured by Applied Biosystems. An amino group was introduced into the 5'-terminal base using Aminolink II (trade name: Applied Biosystems), and then purified by gel filtration using Sephadex G-50. The oligonucleotide represented by SEQ ID NO: 1 (where the 5th and 16th bases are a and a, a
, G, g and g, and a mixture containing equal amounts of four types of oligonucleotides, g and a), as the reverse primer. On the other hand, the forward primer includes the oligonucleotide 5′-TTGGTGGGGT designed by the present inventors.
The combination of AAAGGCCTAC-3 'or a known primer such as the forward primer of O'Callagham et al. Or Karpati et al.
Can be specifically detected. In the example,
Oligonucleotides designed by the present inventors are used as forward primers.

(3) PCR 20 pmol / μl was added to a 0.2 ml reaction tube.
2.5 μl each of the above primers, 1 μl each of the above samples, 25 μl of Premix Taq (TAKARA), and 19 μl of sterilized purified water, were added to a PCR reaction tank TP-3200 (TAK
ARA), set 94 ° C, 30 seconds, 68 ° C, 60 seconds to 1
30 cycles of the reaction were performed. After the reaction,
Take 10 μl from the reaction solution, agarose gel electrophoresis,
Ethidium bromide staining or Gel Star (r) staining was performed to detect amplified nucleic acid chains. As a result, Burkholder
One clear band was confirmed at a length of about 200 base pairs expected only for the three strains of ia cepacia. As a result, Burkholderia cep, which conventionally required culturing for one day to one week,
Detection of acia could be performed in one hour to one day.

Therefore, Burkholder can be obtained by using the oligonucleotide of the present invention as a primer or a probe.
It was found that ia cepacia could be specifically detected. Further, P
Since high sensitivity can be expected from nucleic acid amplification such as CR, it is possible that Burkholderia cepacia can be directly detected from products, etc. without isolating and culturing, and if the number of days required for conventional culturing can be reduced. Conceivable.

Example 2 Burkholder from liquid medium
Detection of ia cepacia (1) Recovery of bacterial cells by centrifugation Soybean, casein, digest, polysorbate, and lecithin medium (hereinafter referred to as SCDLP medium) (trade name: Nippon Pharmaceutical "Digo") And then suspended in sterile saline
Inoculate holderia cepacia and stir. Take 1 ml of the mixture in a 1.5 ml micro tube, rotate 9500 times,
The precipitate is obtained by centrifugation for 30 minutes or more, and washed several times with sterilized purified water or TE buffer. The products used this time are liquid detergents such as shampoo and rinse, cosmetics such as cream, and other raw materials.

(2) Bacterial Cell Recovery by Filter Filtration Burkholderia cepacia was added to SCDLP medium (trade name: Nippon Pharmaceutical “Digo”) at a concentration of 10% and suspended in sterile physiological saline. Inoculate in 1g,
Stir. A part thereof is filtered with a polycarbonate filter and washed with 70% ethanol, and then the filter is placed in a microtube and washed with sterilized purified water or an active agent aqueous solution. The washing solution is centrifuged and the precipitate is collected. Alternatively, the polycarbonate filter is placed in a microtube as it is, and subjected to the phenol / chloroform treatment described in (3).

(3) DNA extraction method-Heat treatment The precipitate obtained in (1) or (2) is suspended in a small amount of sterilized purified water or TE buffer and heat-treated at 100 ° C for 10 minutes.・ Instagene (r) Matrix treatment 0.2 ml of In precipitate is added to the precipitate obtained in (1) or (2).
Add stagene (r) Matrix (TAKARA), 56 ° C, 30 minutes, 100
Heat treatment at 8 ° C for 8 minutes, and use the supernatant obtained by centrifugation as a sample. -Phenol-chloroform treatment 0.5 ml of sterile purified water or TE buffer is added to the precipitate obtained in (1) or (2) to suspend, or (2)
Is placed in a microtube as it is, and treated using a phenol / chloroform / isoamyl alcohol (50: 48: 2) solution (hereinafter abbreviated as PCI) according to a conventional method. DNA obtained by alcohol precipitation is dissolved in about 10 μl of sterilized purified water or TE buffer to prepare a sample for PCR.

(4) PCR The DNA obtained in (3) in the same manner as in Example 1 according to a conventional method.
The PCR reaction was performed for 1 hour using 1 μl of the solution. As a result, Burkholderiacepacia
One clear band was confirmed at a length of about 200 base pairs indicating the specificity of the DNA. As described above, the time required for all detection work is 2
~ 3 hours.

The above results indicate that various products in the present invention
This suggests that Burkholderia cepacia can be rapidly detected after enrichment in a liquid medium, regardless of the type of surfactant contained therein.

Example 3 Direct Detection of Burkholderia cepacia in Cosmetics, Pharmaceuticals and Miscellaneous Goods In Example 2, it was shown that bacteria in a product enriched in a liquid medium can be rapidly detected. However, when quickness is required, direct detection from a product can be considered.
Therefore, a method for detecting each product tested in Example 2 without directly culturing the product is described below. First, 0.5 g of the product is placed in a 1.5 ml microtube, and if necessary, the pH is adjusted to about 8 with a TE buffer solution.
Heat treatment at ℃ for 10 minutes. In the case of a sample containing a cationic surfactant, the alkali-SDS method may be used at the time of heating. After heating, 0.5 ml of a PCI solution is added, and the aqueous layer is subjected to PCI treatment several times according to a conventional method. The DNA obtained by alcohol precipitation was dissolved in a small amount of sterile purified water or TE buffer, and QIAquick® DNA purification kit (QIAG
The DNA is purified using EN). A PCR reaction was carried out in the same manner as in Examples 1 and 2, using 1 μl of the obtained DNA solution. As a result, as shown in Table 5, a clear single band having a length of about 200 base pairs showing the specificity of Burkholderia cepacia was confirmed in all samples.

[0051]

[Table 5]

The above results show that the present invention has
This suggests that Burkholderia cepacia can be directly detected without culturing such as isolating bacteria. Further, the present invention can be used for detection of other microorganisms, and is not limited to detection of Burkholderia cepacia.

Hereinafter, embodiments included in the present invention will be described. (1) An oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence. (2) a primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence. (3) A probe containing an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence. (4) A primer having the nucleotide sequence of SEQ ID NO: 1 into which a site capable of binding to a label and / or a solid phase carrier has been introduced, or a complementary sequence thereof. (5) A primer having the base sequence of SEQ ID NO: 1 in which a part of the base sequence of the oligonucleotide is deleted or substituted or added with another base or base sequence or a complementary sequence thereof. (6) A primer or probe having a base sequence of SEQ ID NO: 1 or a complementary sequence thereof, wherein a site capable of binding to a label or a solid phase carrier is introduced on the 5′-terminal side of an oligonucleotide. (7) A method for detecting Burkholderia cepacia, wherein an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 or a complementary sequence thereof is used as a probe. (8) A method for detecting Burkholderia cepacia, which comprises using an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence as a gene amplification reaction primer. (9) Prepare a sample for which the presence or absence of Burkholderia cepacia is to be detected, and In accordance with, the cells in the sample are crushed, a primer having the nucleotide sequence of SEQ ID NO: 1 or a complementary sequence thereof is added to the sample, a primer extension reaction is performed, and a detection operation is performed on the obtained extension reaction product. A method for detecting Burkholderia cepacia, comprising: (10)
In (9), the extension reaction of the primer is PCR.
Burkholderia cepacia detection method.

[0054]

As described above, the oligonucleotide of the present invention has an antisense strand sequence (SEQ ID NO: 1) from base numbers 451 to 462 of the 16S rRNA gene of Burkholderia cepacia or a sequence complementary thereto. As a probe using an oligonucleotide,
Alternatively, by using it as a primer in combination with a known primer, direct, simple, rapid and reliable
Burkholderia cepacia can now be detected.

[0055]

[Sequence List] SEQUENCE LISTING <110> Lion Corporation <120> Detection Method of Burkholderia cepacia and
Kit therefor <130> J78863A1 <160> 1 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220><223> Designed based on a part of 16S rRNA gene <400> 1 cccgrctata ttagarccaa gg

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[Procedure amendment]

[Submission date] July 29, 1999 (1999.7.2
9)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction contents]

[0005] First, the primer of O'Callagham et al. Is forward primer: 5'-GGTACCGGAA GAATAAGC-3 'reverse primer: 5'-CTGTTCCGACCATTGTAT-3'.
(A CCESSION No.AF042161, hereinafter abbreviated as AF042161), B.cepacia strain (A CCESSION No.U96927, hereafter U96927)
B. cepacia strain (A CCESSION No. AB015606.
1, hereinafter abbreviated as AB015606.1), B. cepacia strain (A CCESS
ION No. M22518, hereinafter abbreviated as M22518), B. cepacia strain
(A CCESSION No.L28675; hereinafter abbreviated as L28675), B.gladioli, B.mallei, B.pickettii, B.solanacea
Homology was also observed in rum, B. caryophylli, B. cocovenenans and the like. On the other hand, for the reverse primer, B.ce
pacia strain (A CCESSION No.X80284, hereinafter abbreviated as X80284), B.cepacia strain (A CCESSION No.X80286, hereinafter X80286)
B. cepacia strain (A CCESSION No. X87275; hereinafter abbreviated as X87275), B. cepacia strain (A CCESSION No. AF)
107072, hereinafter abbreviated as AF107072), B. cepacia strain U969
27, In addition to B.cepacia strain AB015606.1, B.gladioli, B.ma
llei, B.glathei, B.caribiensis, B.caryophylli, Bo
covenenans, B.glumae, B.pseudomallei, etc. are also found to be homologous.
Bacteria other than pacia may be detected and may not have specificity. (Table 2)

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0006

[Correction method] Change

[Correction contents]

The primer of Campbell et al. Is a forward primer: 5′-AACTAGTTGT TGGGGATTCATTC-
3 'reverse primer: 5'-TTTCGAGCAC TCCCGCCTCTCAG-
3 ', but B. cepacia strain
X80284, B. cepacia strain X80286, B. cepacia strain X87275, B.
cepacia strain AF107072, B. cepacia strain U96927, B. cepacia
AB015606.1 strain, B.cepacia strain (A CCESSION No.M22518,
M22518), B.cepacia strain L28675, B.cepa
cia strain (A CCESSION No. D13046, abbreviated as D13046 below)
Besides, B.gladioli, B.mallei, B.glathei, B.caryoph
Homology is found in ylli, B. cocovenenans, B. thailandensis, etc. On the other hand, homology was observed in the reverse primers in B. cepacia strain U96297 and B. cepacia strain M2251.
8, B.thailandensis strain U91838. In the case of these combinations, B. thailandensis may be detected in addition to B. cepacia. (Table 3)

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[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

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[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The 16S rRNA gene sequence of Burkholderia cepacia currently registered in the database is the Burkholderia cepacia D registered by Ludwig et al.
SM50181 strain (A CCESSION No.X87275), Burkholderia cepacia strain registered by Tabacchioni et al. (A CCESSION No.X8
0286), Burkholderia c registered by Tabacchioni et al.
epacia strain (A CCESSION No.X80284), Burkholderia cepacia ATCC25416 strain (A CCESSION No.
No.M22518), Burkholderia c registered by Zhou et al.
epacia G4 strain (A CCESSION NO.L28675), Burkholderia cepacia KP24 strain registered by Hanada et al. (A CCESSION No.A
B015606, hereinafter abbreviated as AB015606), Burkholderia cepacia PVFi5A strain registered by Di Cello et al. (A CCESSION
No.AF042161), Burkholder registered by Viallard et al.
ia cepacia ATCC25416T strain (A CCESSION No.U96927), NCBI
Burkholderia cepacia strain registered with BLAST Research
(A CCESSION No. U91567, U91568, hereinafter abbreviated as U91567, U91568). The present inventors, Burkholderi
16S rRNA gene sequences of other bacteria belonging to the genus Burkholderia, which are related to a cepacia, were obtained from various gene databases and compared in detail. As a result, Burkholderia cepac
It has been found that the sequence of SEQ ID NO: 1 is conserved only in a very small number of other species of bacteria in addition to ia.

[Procedure amendment 4]

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[Correction target item name] 0013

[Correction method] Change

[Correction contents]

[0013] Oligonucleotides of the present invention can be prepared by any method purposive, according to the method described in below Example For example, all or part sequences may be chemically synthesized. For example, it can be chemically synthesized using an automatic DNA synthesizer. Or Burkholderia
It is also possible to cut out directly from the cepacia gene using restriction enzymes, etc.
It is also possible to clone the plasmid into such a plasmid, collect the plasmid after bacterial growth, cut out the plasmid, and use it.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0043

[Correction method] Change

[Correction contents]

(3) PCR 20 pmol / μl was added to a 0.2 ml reaction tube.
2.5 μl each of the above primers, 1 μl each of the above samples, 25 μl of Premix Taq (TAKARA), and 19 μl of sterilized purified water, were added to a PCR reaction tank TP-3200 (TAK
ARA), set 94 ° C, 30 seconds, 68 ° C, 60 seconds to 1
30 cycles of the reaction were performed. After the reaction,
Take 10 μl from the reaction solution, agarose gel electrophoresis,
Ethidium bromide staining or Gel Star ( R ) staining was performed to detect amplified nucleic acid chains. As a result, Burkholder
One clear band was confirmed at a length of about 200 base pairs expected only for the three strains of ia cepacia. As a result, Burkholderia cep, which conventionally required culturing for one day to one week,
Detection of acia could be performed in one hour to one day.

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0047

[Correction method] Change

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(3) DNA extraction method-Heat treatment The precipitate obtained in (1) or (2) is suspended in a small amount of sterilized purified water or TE buffer and heat-treated at 100 ° C for 10 minutes. -Instagene ( R ) Matrix treatment 0.2 ml of Indium was added to the sediment obtained in (1) or (2).
Add stagene ( R ) Matrix (TAKARA), 56 ° C, 30 minutes, 100
Heat treatment at 8 ° C for 8 minutes, and use the supernatant obtained by centrifugation as a sample. -Phenol-chloroform treatment 0.5 ml of sterile purified water or TE buffer is added to the precipitate obtained in (1) or (2) to suspend, or (2)
Is placed in a microtube as it is, and treated using a phenol / chloroform / isoamyl alcohol (50: 48: 2) solution (hereinafter abbreviated as PCI) according to a conventional method. DNA obtained by alcohol precipitation is dissolved in about 10 μl of sterilized purified water or TE buffer to prepare a sample for PCR.

 ──────────────────────────────────────────────────続 き Continued on front page (72) Inventor Kenji Takahashi 1-3-7, Honjo, Sumida-ku, Tokyo Inside Lion Corporation (72) Inventor Kensuke Tanaka 1-3-7, Honjo, Sumida-ku, Tokyo F term in Lion Corporation (reference) 4B024 AA13 CA09 GA19 GA27 HA14 4B063 QA01 QA18 QA19 QQ06 QQ16 QQ17 QR08 QR56 QR62 QS03 QS13 QS25 QS34 QS36 QX01 QX05

Claims (4)

[Claims] 1. An oligonucleotide comprising the following sequence of SEQ ID NO: 1 or its complementary sequence. SEQ ID NO: 1 cccgrctata ttagarccaa gg (r is a or
g) 2. The method according to claim 1, wherein the oligonucleotide according to claim 1 is used as a probe.
Sepacia detection method. 3. A method for detecting Burkholderia cepacia, comprising the oligonucleotide according to claim 1 as a primer for gene amplification reaction. 4. A kit for detecting Burkholderia cepacia, comprising the oligonucleotide according to claim 1.