JP2003061665A - Method for detecting spore forming bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which is useful for detecting a spore forming bacterium having strong toxicity such as Bacillus anthracis because accurate detection can be carried out if the one spore forming bacterium is present in a sample such as soil. SOLUTION: This method for isolating a spore forming bacterium-derived nucleic acid from a spore forming bacterium-containing composition is characterized by subjecting the spore forming bacterium-containing composition to the following steps (a) to (c). (a) a step of treating the spore forming bacterium- containing composition with an alcohol, (b) a step of culturing the spore forming bacterium in the resultant composition and (c) a step of extracting the nucleic acid from the resultant cultured product by a physical crushing means.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently isolating a nucleic acid sample suitable for a PCR reaction from a composition containing spore-forming bacteria such as soil, and accurately detecting the spore-forming bacteria.

[0002]

BACKGROUND OF THE INVENTION Anthrax, which is a type of spore-forming bacterium, is a causative bacterium of anthrax, an infectious disease feared all over the world. Since Bacillus anthracis is a spore-forming bacterium, it can be easily purified as a large amount of spore-forming cells, and thus has always been feared as a pathogen for biological weapons when a conflict occurs. Therefore, there is a demand for a highly accurate detection method for spore-forming bacteria such as soil and food.

The PCR method is a highly sensitive bacterial detection method. When the detection method by the PCR method was applied to a spore-forming bacterium-containing composition represented by soil, accurate detection could not be performed. Therefore, it has been desired to establish a method for detecting spore-forming bacteria in a composition containing spore-forming bacteria such as soil with high accuracy.

[0004]

The inventors of the present invention have investigated the reason why the detection by the PCR method is not sufficient when soil or the like is used as a sample, and as a result, isolation of nucleic acid from a spore-forming bacterium-containing composition is confirmed. It turned out to be a problem with the means. Then, further study, if the composition containing spore-forming bacteria is first treated with alcohol and then cultured, and then physically disrupted to extract the nucleic acid, the nucleic acid derived from the spore-forming bacteria can be isolated very efficiently. It was found that PCR can detect spore-forming bacteria with extremely high accuracy by using
The present invention has been completed.

That is, the present invention provides the spore-forming bacterium-containing composition with the following steps (a) to (c): (a) a step of treating the spore-forming bacterium-containing composition with alcohol, (b) the resulting composition A spore-forming bacterium-derived nucleic acid from a spore-forming bacterium-containing composition, comprising the steps of: culturing the spore-forming bacterium in the medium; and (c) extracting the nucleic acid from the obtained culture by physical disruption An isolation method is provided. The present invention also provides a method for detecting a spore-forming bacterium in a spore-forming bacterium-containing composition, characterized by amplifying and identifying the nucleic acid isolated by the above method by PCR using a specific primer. It is provided.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of spore-forming bacteria to be measured according to the present invention include Bacillus anthracis and Bacillus
Bacillus brevis, Bacillus subtilis
cillus subtilis), Bacillus cereus
us), Bacillus pumilus, Bacillus lentus, Bacillus circulans, Bacillus sphaericus and other Bacillus bacteria; Clostridium tetani, Clostridium tetani (Clostridiu
Clostridium bacteria such as mbotulinum).
Of these, Bacillus bacteria, particularly Bacillus anthracis, are preferred.

The spore-forming bacterium-containing composition is a spore-forming bacterium-containing test sample for performing gene detection (mainly PCR) of the spore-forming bacterium, and examples thereof include soil and air. Above all, it is particularly preferable to apply the method of the present invention to the soil which has not been able to be detected accurately in the past.

In the present invention, the sample (spore-forming bacterium-containing composition) is first treated with alcohol [step (a)]. Examples of the alcohol used here include ethanol and isopropanol, and ethanol is preferable. As the alcohol used for the treatment, 70% by weight to 90% by weight of an aqueous alcohol solution is preferable in order to enhance the detection sensitivity of spore-forming bacteria, and particularly preferably 85% by weight to 90% by weight of an aqueous ethanol solution. For example, the sample 1 is treated with alcohol.
It may be washed with 1 to 100 mL of alcohol based on g, for example, 70% by weight or more of an aqueous alcohol solution.

Next, the spore-forming bacterium in the obtained sample is cultured [step (b)]. The culture conditions may be such that the spore-forming bacteria grow, and for example, 30 to 30 using various liquid media.
It is preferable to culture at a temperature of 40 ° C., particularly around 37 ° C. for 12 to 16 hours. When targeting B. anthracis, it is preferable to culture in a liquid medium such as tryptocase soy liquid medium at 30 to 40 ° C., particularly at 37 ° C. for 12 to 16 hours. In addition, it is preferable that the culture is performed twice or more, that is, after the culture is performed once, the culture solution is diluted with a fresh liquid medium or the like, and then the culture is performed again, so that the sensitivity of PCR as a subsequent step can be increased. The second and subsequent cultures are performed at 30 to 40 ° C., especially 37
It is preferable to carry out shaking culture for 3 to 6 hours at a temperature in the vicinity of ° C in order to suppress the formation of spores as much as possible, and it is preferable to set the shaking speed as fast as possible. Further, the medium may be diluted by about 100 times, and the culture is preferably performed twice.

Next, the obtained culture is subjected to physical disruption to extract nucleic acids [step (c)]. In step (c), specifically, the cells are crushed by stirring in the presence of beads to extract nucleic acids from the cells, and contaminants other than the nucleic acids are transferred to the organic solvent layer. The material of the beads used at this time is not particularly limited as long as it has a hardness enough to crush the bacterial cells, and glass, silicon, zirconium (zi
rconium) and the like can be exemplified, but it is preferable to use glass beads from the viewpoint of easy availability and cost. Further, the shape of the beads is preferably spherical or ellipsoidal from the viewpoint of crushing efficiency, and the particle diameter is such that the major axis is 0.
From the standpoint of cell crushing efficiency, it is preferable that the thickness is from 01 mm to 1 mm, particularly from 0.08 mm to 0.5 mm.

For the physical crushing treatment, known methods and devices such as FastPrep FP120 (manufactured by BIO101) and Mini Be
For example, ad Beater (manufactured by Biospec Products) may be used.
This physical disruption requires optimization depending on the sample, because if the agitation is too weak, the bacterial cells are not sufficiently disrupted, and if the agitation is too strong, DNA fragmentation may occur and PCR may not work well. Yes, but generally 4000-6000rp
m is 10 seconds to 3 minutes, and particularly 20 seconds to 1 minute is a preferable condition.

Specifically, if Fast Prep is used, the power level is 4.0 to 6.0 and 20 seconds to 3 seconds.
It is desirable to react for about 20 to 30 seconds at a power level of 5.0 when a minute, particularly a soil sample is used as a sample. When using the Mini Bead Beater, 4200 rpm to 5
It is desirable to react at 000 rpm for 20 seconds to 5 minutes, especially when a soil sample is used as a sample, for about 30 seconds to 3 minutes at 5000 rpm.

More specific conditions include, for example, suspending a sample in about 0.5 mL of Tris-EDTA buffer and stirring in the presence of about 0.3 g of beads and about 0.5 mL of an organic solvent, A method of releasing the nucleic acid from the bacterial cells can be mentioned.

The organic solvent is not particularly limited as long as it has a protein denaturing action, but TE (or water) saturated phenol is preferable, and phenol is particularly preferable from the viewpoint of extraction efficiency and the like.

After crushing the cells, the aqueous layer and the organic solvent layer are separated by a means such as centrifugation to recover the aqueous layer containing nucleic acids such as DNA. At this time, it is preferable that the recovered substance is further treated with the organic solvent having the protein denaturing property, because most of the impurities other than the nucleic acid are eluted in the organic solvent layer in the organic solvent layer. The organic solvent used is phenol, phenol / chloroform / isoamyl alcohol (25:24:
1), benzyl chloride and the like, particularly phenol and phenol / chloroform / isoamyl alcohol (25:
It is preferable to carry out the combination of 24: 1) treatments. The obtained nucleic acid is finally recovered by ethanol precipitation or the like.

Next, the recovered nucleic acid is purified using a gel filtration column, if necessary. The column used is preferably Micro Spin Column S-400 (manufactured by Pharmacia) or the like, but is not particularly limited to these as long as it is a similar packing material.

The nucleic acid thus obtained, for example DN
A can detect whether or not the target spore-forming bacterium was present in the sample by a PCR reaction using a specific primer. To increase the sensitivity of detection and enable detection even when the number of bacteria in the sample is extremely small, PC
It is preferable to use capillary PCR or nested PCR as R, and capillary PCR is particularly preferable because rapid detection is possible.

When the target spore-forming bacterium is anthrax, Table 1
It is preferable to use a primer as shown in. That is, the combination of SL-U1 and SL-D1, the combination of SL-U2 and SL-D2, the combination of PA8 and PA5, P
The combination of A7 and PA6, the combination of MO11 and MO12, and the combination of BA547 and BA546 are preferable. Table 1 shows the primers for nested-PCR. In Table 1, the target site S-layer is a chromosome part and PA
Is the toxin gene part in the plasmid and CAP is the cap gene part in another plasmid. These are particularly excellent primers that can be detected with high sensitivity and non-specific reaction even when used for samples containing many contaminants such as soil.

[0019]

[Table 1]

In Table 1, the primers SL-U2 (SEQ ID NO: 3), SL-D2 (SEQ ID NO: 4), PA7 (SEQ ID NO: 7), PA6 (SEQ ID NO: 8), MO11 (SEQ ID NO: 9).
And MO12 (SEQ ID NO: 10) are new. If these primers, that is, primers specific to three kinds of genes including a chromosomal gene, a plasmid containing a toxin gene and a plasmid containing a capsular membrane, are used in combination, even a bacterium that has lost the plasmid can be detected with high sensitivity. It is possible to

In addition, Light Cyclar (manufactured by Roche) and ABI Pri
zm 7700, 7900 (PE Biosystems), I-Cycler (BIO-
Quantitative analysis of whether the target spore-forming bacteria were present in the sample by detecting the amount of amplified product as the fluorescence amount of Cyber Green, Taqman probe, molecular beacon, etc. using RAD) can do.

Further, the nucleic acid obtained by the method of the present invention can be used for hybridization by a DNA chip, clone library method, denaturing gradient gel electrophoresis method (DGGE method), temperature gradient gel electrophoresis method (TGG).
By the E) method and the SSCP method, it is possible to specifically detect and identify the spore-forming bacterium in the composition containing the spore-forming bacterium.

[0023]

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

Example 1 (1) Cell and primer setting and PCR conditions As a novel primer for detecting B. anthracis, a capsular (Cap), toxin (PA), which is a causative agent of B. anthracis, and a sequence on the chromosome A primer specific to (S-layer gene) was designed.
The sequences are shown in Table 1. (Design method) M011, M012, BA547, BA5
Each of the 46 primers was designed after the nucleotide sequence of the capsular gene of B. anthracis was determined. The base sequences of the following bacterial species such as the base sequences downloaded from the data bank (National Institute of Genetics) were used for designing the primers. The specificity of the designed primer is the chromosome D obtained by the conventional method from the following bacterial species.
After NA was separated and purified, these were confirmed as template DNAs. Strains are Lister for Gram-positive bacteria other than Bacillus
ia monocytogenes (serotype 1 / 2a, 1 / 2b, 1 / 2c, 3a, 3b, 3c, 4a,
4ab, 4b, 4c, 4d, 4e, 5,6,7), L.innocua, L.seeligeri, L.
welshimeri, L.ivanovii, L.grayi, L.murrayi, Clostr
idium botulinum 003-9, Clostridium difficile7626,
Clostridium perfringens 5256, Corynebacterium diph
theriae 3182, Corynebacterium pyogenes, Erysipelot
hrix rhusiopathiae, Enterococcus faecalis 8357, St
reptococcus suis, Lactococcus lactis 8591, Mycobac
terium tuberculosis 27874, Staphylococcus aureus,
Streptococcus pneumoniae, the Gram-negative bacterium is Actinoba
cillus pleuropneumoniae Ngl, Aeromonas sp. ATCC 90
71, Campylobacter fetus 8746, Campylobacter jejuni
91-569, Enterohemorrhagic Escherichia coli, Enter
opathogenic Escherichia coli, EnterotoxigenicEsche
richia coli, Klebsiella pneumoniae IID 5209, Legio
nella pneumophila 10260, Pasteurella multocida 87-3
7, Proteus vulgaris IID 874, Pseudomonas acidovora
ns 11501, Pseudomonas aeruginosa P 13, Salmonella
choleraesuis SB 242, Salmonella enteritidis, Salmo
nella typhimurium LT 2, Serratia marcescens IID 521
8, Shigella flexneri YSH 6000, Vibrio cholerae IID
936, Vibrio parahaemolyticus 91-572, Yersinia ent
erocolitica and Yersinia pseudotuberculosis. B. cereus HSCC 187, B. cer, except Bacillus anthracis
eus HSCC 1049, B. cereus HSCC 1207, B. thuringieusis
HSCC 345, B.mycoides HSCC 395, B.pseudomycodes HS
CC 1477, B.weihenstephanensis HSCC 1480, B.amyloli
quefaciens IAM 1521, B. badius IAM 11059, B. cereus
IAM 12605, B.circulans IAM 12462, B.firmus IAM 124
64, B. licheniformis IAM 13417, B. megaterium IAM 13
418, B.mycoides IAM 1190, B.pumilus IAM 12469, Bs
phaericus IAM 13420, B. subtilis IAM 12118, B. thuri
ngiensis IAM 12077, B.subtilis UOTO 277, B.subtili
s ISW 1214, B.megaterium # 210, B.cereus # 211, B.th
uringensis # 212, B.nato # 213, B.subtilis # 360,
B. brevis HPD 31, B. anthracis Shikan, Ba
nthracis Morioka, B.anthracis No.1, B.anthracis N
o.2, B.anthracis Nakakawa, B.anthracis Ryugasaki,
B.anthracis 52, B.anthracis PI, B.anthracis 34-F2
Was used. PCR was carried out using a primer set using the three kinds of primers thus obtained. PCR conditions were 95 ° C. for 15 seconds, 55 ° C. for 30 seconds, 72 ° C. for 60 seconds for 35 cycles, and the amplification product was confirmed by agarose gel electrophoresis.

Pasteur 2 seedlings (toxin, capsular strain) were used as the original strain for producing anthrax spores. The spore fluid was prepared by culturing Pasteur 2 seedlings in L broth overnight at 37 ° C., centrifuging, washing the cells once with physiological saline, and resuspending in physiological saline.
It was prepared by shaking culture at 7 ° C for 3 days. After heating at 80 ° C for 15 minutes, centrifuge and wash with PBS three times, and then measure the number of spores with L.
It was performed on an agar plate and used.

(2) Medium Selection medium for B. anthracis PLET medium (heart infusion agar medium containing thallium acetate;
04g / l, Polymyxin B; 30000units / l, lyzosyme; 3000
00units / l, EDTA; 0.3g / l added) together with Bacillus ce
Reusselective agar (BCA; Oxoid) plates and non-selective medium Trypticases oy agar (TSA) were used. Trypticase soy broth (TSB) was used as the liquid medium.

(3) (Establishment of Method for Separation of B. anthracis Spores from Soil) Various soils were collected to artificially remove B. anthracis spores from 0, 1, 1
The experiment was conducted by adding 0, 100 and 1000 pieces.

(A) Separation of Bacillus anthracis from soil: In accordance with WHO guidelines, each soil was diluted 10-fold and 100-fold with saline and the diluted solution was treated with 70% by weight alcohol aqueous solution to remove spores Excluded. Direct PCR using this sample could not be detected unless 1000 or more anthrax bacteria per 10 g were present in the soil.

(B) Detection by enrichment method: P directly from soil
CR was also unsuccessful with high B. anthracis spores, so the enrichment method was used. First, 10 g of the spore-added soil was washed twice with 100 mL of 70% ethanol by centrifugation. Then 2 with 100 mL of sterile distilled water
The cells were washed by centrifugation once, finally mixed with 100 mL of tryptocase soy liquid medium (TSB), and shake-cultured at 37 ° C. for 12 hours. The next morning, 10 mL of the primary enrichment solution 0.1 mL
In addition to TSB, the cells were further cultured with shaking at 37 ° C. for 4 hours. After centrifuging 1.0 mL of the secondary enrichment solution, washed twice with sterile water,
Fast Prep TMFP120 instrument (Bio
101, Inc. CA., USA) was used to purify DNA for PCR (physical disruption treatment at 5000 rpm for 30 seconds). DNA
After adjusting the concentration, 100 ng was used for nested-PCR.

(4) Detection of spore-forming bacteria from soil In the direct method of (3) (a) above, 10 per 10 g was calculated.
It could not be detected unless more than 00 anthrax was present in the soil. Therefore, it was concluded that it is impossible to separate the cells from the soil sample containing a small amount of Bacillus anthracis spores by conventional means.

(5) Then, DNA is isolated by the method of (3) (b) (alcohol treatment and enrichment method) and nested-P
CR was performed. The result of electrophoresis of the obtained amplification product is shown in FIG. In FIG. 1, positive purified DNA, spore number 0, spore number 1, spore number 10, spore number 100, and DNA marker are shown from the left lane of each photograph. As a result, when the nucleic acid isolated by the method of the present invention is used, 1 spore is contained in the sample.
It can be seen that even if there are individual pieces, they can be detected accurately.

Comparative Example 1 A nucleic acid was isolated in the same manner as in Example 1 (3) (b) except that the operation of washing the soil with 100 mL of 70% ethanol was changed by heating at 75 ° C. for 30 minutes. , Nested-PCR was performed. As a result, when the alcohol treatment was changed to the heat treatment, there were cases where the spores could not be detected for the first time with 10 or more spores, and cases where the spores could not be detected. Moreover, it could not be detected in the case of one spore.

Example 2 (1) 0, 1, 10, 10 of B. anthracis spores was added to 100 g of soil.
Add 0 or 1000 pieces and add distilled water to each 200
mL was added to obtain a diluted solution. 100 mL of 90% ethanol was added to the diluted solution, mixed well, and shaken at room temperature for about 15 minutes. After centrifugation at 500 rpm for 15 minutes, the supernatant was discarded, and these operations were repeated once again to remove other bacteria. After washing the precipitate twice with 100 mL of sterile distilled water,
100 mL of TSB was added, and the mixture was cultured at 37 ° C for 12 hours with shaking to obtain a culture. Secondary enrichment solution 1.0 obtained by adding 0.1 mL of culture to 10 mL of TAB and culturing at 37 ° C. for 4 hours
After centrifuging mL, wash twice with sterilized water and fast as per manual.
The DNA for PCR was purified using Prep ™ FP120 instrument (Bio 101, Inc. CA., USA). After adjusting the concentration of DNA, it was used for capillary PCR.

(2) In addition, the results of using Light Cycler with M011 and M012 as primers are shown in FIG.
And 3 are shown. Figure 2 shows the real-time amplification curve,
FIG. 3 is an electrophoretic image of the amplified product. The amplification conditions at this time were 95 ° C for 10 minutes, then 95 ° C for 15 seconds, and 62 ° C.
40 cycles of 10 seconds and 72 ° C. for 25 seconds were performed. As a result, it can be seen that the nucleic acid isolated by the method of the present invention can be accurately detected even if there is even one spore in the sample.

Comparative Example 2 A culture was obtained in the same manner as in Example 1 except that the diluted solution of soil was heated at 70 ° C. for 30 minutes to remove spores. In the case of heat sterilization, detection is not stable,
Even if the number of spores was 10 or more, the detection could not be successful in some cases.

[0036]

EFFECTS OF THE INVENTION According to the method of the present invention, one spore-forming bacterium can be accurately detected in a sample such as soil, and therefore it is useful for detecting highly virulent spore-forming bacterium such as anthrax.

[0037]

[Sequence list] SEQUENCE LISTING <110> ZAIDAN HOJIN YAKULT ・ BIO SCIENCE KENKYU ZAIDAN <120> DETECTING METHOD FOR SPORE FORMATION BACTERIA <130> P03571308 <140> <141> <160> 12 <170> PatentIn Ver. 2.1 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis S-layer <400> 1 cgcgtttcta tggcatctct tct 23 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis S-layer <400> 2 ttctgaagct ggcgttacaa at 22 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis S-layer <400> 3 cggtacagaa gcagcaaaa 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis S-layer <400> 4 gctgttggct catcagcta 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis S-layer <400> 5 gaggtagaag gatatacggt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis PA <400> 6 tcctaacact aacgaagtcg 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis PA <400> 7 atcaccagag gcaagacacc c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis PA <400> 8 accaatatca aagaacgacg c 21 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis CAP <400> 9 gacggattat ggtgctaag 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis CAP <400> 10 gcactggcaa ctggttttg 19 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis CAP <400> 11 gctgatcttg actatgtggg tg 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed DNA       based on Bacillus brevis CAP <400> 12 ggcttcctgt ctaggactcg g 21

[Brief description of drawings]

FIG. 1 CAP, PA and S- as specific primers.
1 is an electrophoretic image showing the results of nested-PCR using the sequence on the layer (Table 1) (lanes of each electrophoretic image are from the left, positive special DNA, spore number 0, spore number 1, spore number 1
0, 100 spores, is a DNA marker).

FIG. 2 is a real-time amplification curve showing the results using Light Cycler.

FIG. 3 is an electrophoretic image of the amplified product showing the results using Light Cycler (lanes are from left to right: DNA marker, spore number)
0, positive purified DNA, spore number 1, spore number 10, spore number 100).

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B024 AA01 AA13 CA09 GA27 HA12                 4B063 QA01 QA18 QA19 QQ05 QQ19                       QQ42 QR08 QR32 QR42 QR55                       QR62 QS25 QS32 QX01                 4B065 AA15X AA15Y AC20 BD01                       CA23 CA46

Claims (8)

[Claims] 1. A composition containing a spore-forming bacterium is subjected to the following step (a):
-(C): (a) a step of treating the spore-forming bacterium-containing composition with alcohol, (b) a step of culturing the spore-forming bacterium in the obtained composition, and (c) a physical disruption of the obtained culture. A method of isolating a spore-forming nucleic acid from a composition containing a spore-forming bacterium, which comprises the step of extracting the nucleic acid by means. 2. The isolation method according to claim 1, wherein the spore-forming bacterium-containing composition is soil. 3. The isolation method according to claim 1 or 2, wherein the alcohol treatment is treatment with an aqueous 70% by weight to 90% by weight ethanol solution. 4. A nucleic acid isolated by the method according to any one of claims 1 to 3 is prepared by using P using a specific primer.
A method for detecting a spore-forming bacterium in a composition containing a spore-forming bacterium, which is characterized by being amplified and identified by CR. 5. The method for detecting a spore-forming bacterium according to claim 4, wherein the PCR is a capillary PCR. 6. An anthrax primer comprising a DNA fragment having the base sequence represented by SEQ ID NO: 7 and a DNA fragment having the base sequence represented by SEQ ID NO: 8. 7. A primer for anthrax comprising a DNA fragment having the base sequence represented by SEQ ID NO: 11 and a DNA fragment having the base sequence represented by SEQ ID NO: 12. 8. A primer for anthrax comprising a DNA fragment having the base sequence represented by SEQ ID NO: 13 and a DNA fragment having the base sequence represented by SEQ ID NO: 14.