JPH06253847A - Polynucleotide for detecting bacillus anthracis and method for detection using the same - Google Patents

Abstract

PURPOSE:To obtain a new oligonucleotide having high specificity as a primer for detecting Bacillus anthracis. CONSTITUTION:This oligonucleotide is complementary to a nucleotide sequence capable of coding a capA gene of a plasmid of Bacillus anthracis and has a sequence group expressed by the formula or a complementary sequence corresponding thereto. The polynucleotide is obtained by chemically synthesizing two kinds of synthetic oligonucleotides (MO1 and M02) with high contents of guanine and cytosine residues from 288 bases of the base sequence in the capA gene of the Bacillus anthracis according to a triester method using a DNA synthesizer manufactured by Applied Biosystems, Inc. and purifying the resultant oligonucleotides with a C18 reversed phase column.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an oligonucleotide used for detecting anthrax in clinical tests, veterinary clinical tests, or food inspection, and a method for detecting anthrax using the same.

[0002]

BACKGROUND OF THE INVENTION Although anthrax is an infectious disease in herbivores,
It occurs in humans and is an extremely zoonotic disease that is extremely important for public health. In Japan, this disease is designated as a legal infectious disease of livestock, and it is a reported infectious disease in humans. Although the incidence of anthrax has decreased in recent years,
In areas with soil contaminated with B. anthracis spores, it is always necessary to monitor human and livestock contamination by B. anthracis. Previously, Bacillus c
ereus ) detection method. In other words, 9 times the amount of dilution water is added to the sample and homogenized with a stomacher or blender. The produced homogenate was treated with polymyxin-containing tryptocase soy broth medium at 32 to 3%.
Selective enrichment culture is performed at 5 ° C. for 18 to 24 hours. After culturing, 1 platinum loop amount of the solution is spread on a Bacillus cereus selective separation medium such as mannitol egg yolk polymyxin agar medium or Kim Geppet agar medium. 32-35 more
After culturing at 18 ° C for 18 to 24 hours, non-degraded mannitol-degraded, yolk-reactive positive, grayish-white waxy, and large-sized colonies with rough surfaces are picked up on agar. This is further added to normal agar medium or heart infusion agar medium by 32 to 3
Regarding the bacterial cells grown by culturing at 5 ° C for 18 to 24 hours,
Gram stain, capsule stain and immunological examination. Alternatively, the sensitivity of gamma phage is tested.

[0003]

However, Gram's stain and capsular stain are used for other related bacteria such as Bacillus megaterium.
Potassium (Bacillus megaterium), Bacillus Rikenifu
Orumisu (Bacillus licheniformis), Bacillus dip
Chiris (Bacillus subtilis ) and the like cause similar reactions to B. anthracis, and the gamma-phage susceptibility test requires a large amount of cells and several days for the test, so it is not an effective means for monitoring B. anthracis . From a public health point of view, the reality is that a highly sensitive, highly reliable, rapid and simple method for detecting B. anthracis is required.

[0004]

The present invention has the following constitution. (1) Bacillus anthracis present in the sample
An oligonucleotide for selectively detecting, or an oligonucleotide chemically synthesized so as to be complementary to the nucleotide sequence that targets the nucleotide sequence encoding the capA gene of the anthrax plasmid. , An oligonucleotide in which the synthetic nucleotide consists of the following sequence group or a complementary sequence corresponding thereto. 5 '> GCTGATCTTTGAACTATGTGGGGTG <3' ... MO1 5 '> GGCTCAGGATCTGTCCTTCGGG <3' ... MO2 (2) Having at least one of the sequences described in the above item 1. A method of causing an oligonucleotide to function as a primer for a chain length reaction to selectively amplify a target nucleotide sequence, comprising: (a) adding a primer to a target nucleotide sequence in a single-stranded state in a sample. When the resulting double-stranded nucleotide sequence is hybridized to cause a chain length reaction by a polymerization reaction of four kinds of nucleotides and the obtained double-stranded nucleotide sequence is separated into single strands, the complementary strand is a chain length reaction by the other primer. (C) Simultaneous chain length reaction by these two types of primers, separation of chain length product from template, New primer Te - by hybridization - specific nucleotide sequence by repeating Deployment is amplified, electrophoresis, chromatography -
The method for detecting B. anthracis characterized by detecting the nucleotide fragment amplified in step (d), and determining (d) whether or not a sequence to be recognized is present in the sample. (3) A method for detecting B. anthracis by performing electrophoresis and nucleic acid staining using the reaction product of the method described in the above item 2.

The present invention is characterized by selectively detecting B. anthracis by an oligonucleotide and a gene amplification method in which the oligonucleotide functions as a primer. Although various methods have been reported as gene amplification methods, the most common one is the polymerase chain reaction developed by Saiki et al. (Hereinafter abbreviated as PCR method. Science.
230: 1350-1354 (1985)). In this method, when detecting a sequence of a specific nucleotide, synthetic nucleotides having sequences complementary to one of the nucleic acids at both ends of the sequence are prepared and used as primers. On the other hand, 90-
The nucleic acid of the sample which was heat-denatured at 95 ° C. to be single-stranded was
Hybridize the primers at 65 ° C. Then D
NA synthase and a substrate are added and a nucleotide polymerization reaction is carried out at 50 to 75 ° C. When the produced double-stranded DNA is again made into a single strand by heat denaturation and the same reaction is carried out, a DNA having a sequence complementary to the single strand is obtained and becomes a double strand. When this cycle is repeated about 20 to 40 times, the nucleic acid having the target nucleotide sequence is amplified, and this is detected by means such as electrophoresis.

The specimen may be clinical materials such as urine, blood, feces and tissue slices, veterinary clinical materials, or food materials such as meat. Alternatively, it may be a microbial cell or a culture solution separated from these materials. By treating these specimens with a lytic enzyme, a surfactant, an alkali, etc. for a short time, a sample liquid containing a nucleic acid necessary for PCR is obtained.

The primer may be either a chemically synthesized product or a natural product, as long as it is a nucleic acid fragment having a length of 10 bases or more, but it is complementary to a specific gene of the anthrax chromosome or plasmid. It is preferable to have a sequence that is not complementary to a gene of a species other than B. anthracis, including B. anthracis related bacteria. More preferably, it has a sequence complementary to any part of the base sequence of the capsulation gene capA present in the pathogenic plasmid pTE702 of the wild strain of B. anthracis.

A thermostable DNA polymer is used for the PCR reaction.
There is a need. This enzyme may be derived from any biological species as long as it retains thermostability at a temperature of 90 to 95 ° C.

The nucleic acid amplified by the PCR reaction may be detected by any means, but preferably it is detected by electrophoresis using polyacrylamide or agarose as a carrier and ethidium bromide as a nucleic acid stain. preferable.

[0010]

EXAMPLES The present invention will be described in more detail below with reference to examples.

[0011] Example 1 anthrax as Bacillus Antorakisu Davis (Baci
llus anthracis Davis) and Bacillus anthracis
Pasteur II ( Bacillus anthracis PasteurII) was used. In addition, as shown in Table 1, 16 as Gram-positive bacteria
22 strains were used as seeds and Gram-negative bacteria. Each of these strains was cultured in broth medium at 37 ° C overnight,
The produced bacterial cells were collected by centrifugation. Rhizochi-
TES buffer (pH 8.
0), that is, tris hydrochloride 50 mM, EDTA
Suspended in 5 mM sodium chloride and 50 mM sodium chloride to give sodium dodecyl sulfate and proteinase K.
Was added. After keeping the temperature at 55 ° C., RNA was removed by ribonuclease treatment. The DNA was deproteinized by the phenol-chloroform method, extracted three times, precipitated with ethanol, and the collected DNA was treated with TE buffer (pH 8.0).
), That is, tris hydrochloride 10 mM, EDTA
Suspended to a concentration of 1 mmol.

[0012] The primer synthesis is based on the sequence of 288 nucleotides within the capA gene of Bacillus anthracis (Ma
kino, S. et al .: J. Bacteriol . 171, 722-730 (1989))
To the above-mentioned two synthetic oligonucleotides (M01 and M02) having a high content of guanine and cytosine residues
Was chemically synthesized by the triester method using a DNA synthesizer manufactured by Applied Biosystems. The synthesized nucleotides were purified by C18 reverse phase column.

The PCR reaction was carried out in 100 μL of 10 mM tris hydrochloride (pH 8.3), 60 mM potassium chloride, 1.5 mM magnesium chloride, 1 mg gelatin, 200 mM each dATP, dTTP, dCTP and dGTP. Primer-50 picomolar, Taq polymerase 2.5 units, and 0.01-0.1 μ
Reactions containing g of each DNA were prepared and performed.
Thermal denaturation was carried out at 95 ° C for 1 minute, annealing at 65 ° C for 2 minutes, and polymerization at 72 ° C for 1.5 minutes. One cycle from the start of heat denaturation to the end of the polymerization reaction, 30
A cycle of reactions was performed. 6% amplified reaction product
Separation was performed by (W / V) polyacrylamide gel electrophoresis, and a band shining upon irradiation with ultraviolet rays was detected by staining with ethidium bromide.

[0014] The results of electrophoresis, Bacillus is Bacillus anthracis
Anthracis Pasteur II (Bacillus anthracis Dav
is) and Bacillus Anthracis Pasteur II ( Bacil
A band corresponding to 288 base pairs was detected only in the lane of lusanthracis Pasteur II). No band was detected in the strains shown in Table 1.

[0015]

[Table 1]

As a result, it is difficult to distinguish it from B. anthracis by the conventional method.
It was difficultBacillus cereus(Bacillus cereus),
Bacillus licheniformis(Bacillus licheniformi
s),Bacillus megaterium(Bacillus megaterium),
 Bacillus subtilis(Bacillus subtilis),Bacil
Thuringia (Bacillus) thuringiensi
s), A band corresponding to 288 base pairs was not detected.
It was found that B. anthracis can be specifically detected by this method.

Example 2 100 μL of sterilized water was added to 100 mg of mouse spleen tissue, and the mixture was pulverized into a homogenate. To this homogenate, suspensions of spores of Bacillus anthracis Pasteur II at various concentrations were added. To this homogenate, use the protein
100 μL of TES buffer-2 times concentrated solution (pH 8.0) containing zeK at a concentration of 200 μg / mL, that is, tris hydrochloride 100
Mix a solution of millimolar concentration, 10 millimolar concentration of EDTA and 100 millimolar concentration of sodium chloride, and add 20% to it.
(W / v) 50 μL of sodium dodecyl sulfate and 20%
(W / v) 50 μL of sarcosyl was added. added. This was heated at 55 ° C. for about 3 hours until the liquid became clear.
DNA was extracted from this solution by deproteinizing three times by the phenol-chloroform method. The DNA was collected by ethanol precipitation and lysed with 500 μL TE buffer. This 1 μL was used for PCR reaction.

The same primer as in Example 1 was used. As the sample DNA, 1 μg was used in the PCR reaction. PC
The R reaction was also performed under the same conditions as in Example 1.

As a result, when the spores were contained in the DNA of the sample in an amount corresponding to about 1,10 ³ and 10 ⁶ , respectively, P
Of the samples that underwent CR reaction, about 10 ³ and 10 ⁶ spores were added to the electrophoretic lane, and the ca.
A clear band corresponding to 288 base pairs of the pA gene was observed. A faint band corresponding to 288 base pairs of the capA gene of Bacillus anthracis was also observed in the sample treated with about 1 spore.

Example 3 Mice were inoculated intraperitoneally with B. anthracis. After 48 hours, spleen tissue was collected. At this time, the number of B. anthracis was measured by a conventional method and was 1.8 × 10 ⁸ cells / g. DNA was extracted from this spleen tissue and PCR reaction was carried out in the same manner as in Example 2.

As a result of electrophoresis, 28 of the capA gene of Bacillus anthracis
A clear band corresponding to 8 base pairs was observed.

Example 4 Anthracis (Bacillus anthracis Pasteur) was intraperitoneally injected into mice.
The spore suspension of II) was inoculated, and 48 hours later, spleen tissues were collected. At this time, when the number of B. anthracis was measured by a conventional method, it was 1.8 × 10 ⁸ cells / g. This spleen tissue 10
To 0 mg, 100 μL of sterilized water was added, and the mixture was crushed and homogenized. This homogenate was boiled in boiling water for 15 minutes, cooled, and then centrifuged at 4 ° C for 10 minutes at 10,000 x g . 1 μL of this supernatant was used for PCR reaction. The PCR reaction was performed according to Example 1.

As a result of electrophoresis, 28 of the capA gene of Bacillus anthracis was detected.
A clear band corresponding to 8 base pairs was observed.

Example 5 Anthracis (Bacillus anthracis Pasteur) was intraperitoneally injected into mice.
The spore suspension of II) was inoculated and blood was collected 48 hours later. At this time, the number of B. anthracis was measured by a conventional method, and it was 7 × 10 ⁶ cells / g. 100 μL of TES buffer-2 times concentrated solution (pH 8.0) containing 100 μL of blood and proteinase K at a concentration of 200 μg / mL, that is, 100 mM tris hydrochloride, 10 mM EDTA,
Mixing a 100 mM sodium chloride solution,
Add 20% (w / v) sodium dodecyl sulfate to it.
μL was added. This was heat-treated at 55 ° C. for 60 minutes, and then deproteinized three times by the phenol-chloroform method to extract DNA. The DNA was collected by ethanol precipitation and lysed with 100 μL TE buffer. This 1 μL was used for PCR reaction. The PCR reaction was performed according to Example 1.

[0025] As a result of electrophoresis, 28 of the capA gene of Bacillus anthracis
A clear band corresponding to 8 base pairs was observed.

Example 6 Intraperitoneal administration of Bacillus anthracis Pasteur to mice
The spore suspension of II) was inoculated and blood was collected 48 hours later. At this time, the number of B. anthracis was measured by a conventional method, and it was 7 × 10 ⁶ cells / g. 100 μL of this blood
Boil in boiling water for 5 minutes, cool, and then at 4 ℃ 10,000 × g 10
Centrifuge for minutes. 1 μL of this supernatant was used for PCR reaction. The PCR reaction was performed according to Example 1.

As a result of electrophoresis, 28 of the capA gene of Bacillus anthracis
A clear band corresponding to 8 base pairs was observed.

[0028]

The oligonucleotide of the present invention has high specificity as a primer for detecting B. anthracis, and the detection method using the oligonucleotide is an excellent detection method capable of specifically detecting B. anthracis. is there.

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Claims (3)

[Claims] 1. A Bacillus anthr which is present in a sample.
acis ) for the selective detection of
Alternatively, targeting a nucleotide sequence encoding the capA gene of the anthrax plasmid, an oligonucleotide chemically synthesized so as to be complementary to the nucleotide sequence, the synthetic nucleotide is An oligonucleotide comprising a corresponding complementary sequence. 5 '> GCTGATCTTGAACTATGTGGGGTG <3' ... MO1 5 '> GGCTCAGGATCTGTCCTCTCGG <3' ... MO2 2. A method which comprises causing an oligonucleotide having at least one of the sequences described in claim 1 to function as a primer for chain reaction and selectively amplifying a target nucleotide sequence. Therefore, (a) a primer is attached to the target nucleotide sequence in the single-stranded state in the sample.
When the resulting double-stranded nucleotide sequence is separated into single strands, the complementary strand has a chain length of the other primer. Functions as a template for the reaction, (c)
A specific nucleotide sequence is amplified by repeating simultaneous chain length reaction with these two types of primers, separation of the chain length product from the template, and hybridization with a new primer, and electrophoresis and chromatography. A method for detecting B. anthracis, which comprises detecting the amplified nucleotide fragment and (d) determining whether or not a sequence to be recognized is present in the sample. 3. A method for detecting B. anthracis by performing electrophoresis and nucleic acid staining using the reaction product of the method according to claim 2.