JP3016395B2 - Mycoplasma detection method - Google Patents

Description

The present invention relates to a method for detecting mycoplasma, and more particularly, to the detection of a specific DNA region of the mycoplasma. The invention also relates to a detection kit for such a detection.

[Conventional technology]

Mycoplasma, which was discovered as a causative agent of bovine respiratory disease in the late 19th century, widely inhabits the animal and plant kingdoms, some of which cause host disease. For example, primary atypical pneumonia in humans, respiratory mycoplasmosis in chickens, mycoplasma pneumonia in pigs, etc.
Research on prevention is being actively conducted. In addition, mycoplasma contamination during tissue culture was also discovered, and research on a method for detecting and removing contaminated mycoplasma is ongoing. In addition, the importance of laboratory animals in medical and biological research has been increasing in recent years, and their quality has been required to be high. In maintaining and managing colonies of laboratory animals such as mice and rats, etc. But diagnosis and prevention of mycoplasma contamination has become an important issue.

As a method for detecting mycoplasma, a separation culture method,
DNA fluorescent staining methods, biochemical methods, immunological methods and the like have been developed.

[Problems to be solved by the invention]

Each of the above methods is generally used as a method for detecting mycoplasma, but the operation is complicated and not a simple method. On the other hand, various gene diagnosis methods have recently been developed. The characteristics of mycoplasmas are defined by their genes. If the gene sequence characteristic of this mycoplasma can be detected with high sensitivity and speed, the presence of mycoplasma can be determined simply and easily, and the management of livestock, experimental animals, tissue culture and the like becomes easy.

That is, an object of the present invention is to clarify a gene sequence common to mycoplasmas in general, and to provide a detection method therefor and a kit used therefor.

[Means for solving the problem]

To summarize the present invention, the first invention of the present invention relates to a method for detecting mycoplasma, and among the DNA sequences of mycoplasma represented by 16SrRNA-spacer region-23SrRNA,
The DNA sequence of the spacer region, or a part thereof, and at least the following formula (1): A second invention relates to a mycoplasma detection kit for performing detection by using the method of the first invention, wherein the kit comprises the steps of: DNA region of the spacer region,
Or a specific primer for amplifying a DNA region which is a part thereof and contains at least the base sequence represented by the above formula (1).

DNA encoding mycoplasma rRNA is 16S rRNA-
Spacer region-23S rRNA-Spector region-5S rRNA each D
Consists of an NA sequence. The present inventors have solved a part of a DNA sequence encoding rRNA of 11 types of mycoplasma, that is, 16S rRNA-spacer region-
Identify part of the DNA sequence encoding 23S rRNA,
Next, we found a common DNA sequence for mycoplasma.

Next, the most sensitive and simple gene detection method
PCR (Polymerase Chain Reaction) method [Saik
i) et al., Science, Vol. 230, 1350-135
On page 4, (1985)], oligonucleotide primers for amplifying a specific region DNA of a DNA sequence common to mycoplasma by PCR were synthesized. Probes for detecting the amplified mycoplasma DNA were also synthesized. Next, the RCR method was performed using each mycoplasma DNA as a template, and it was found that specific regions of all mycoplasma DNAs were efficiently amplified and detected.

 Hereinafter, the present invention will be specifically described in order.

Encodes rRNA used for mycoplasma detection
The DNA sequence may be any sequence that is common to mycoplasma. For example, a common sequence may be found from the DNA sequence encoding 16S rRNA-spacer region-23S rRNA. The DNA sequence encoding the 16S rRNA-spacer region-23S rRNA of mycoplasma is determined as follows.

For example, Mycoplasma fermentans (Mycoplas
ma fermentans) PG18 strain, Mycoplasma hyorhinis BTS7 strain, Mycoplasma aurere (M.orale) CH19299 strain, Mycoplasma hominis (M.
hominis) PG21 strain, Mycoplasma salivaryum (Ms
alivalium) PG20 strain, Mycoplasma arginini (Ma
rginini) G230 strain, Ureaplasma urealyticum T960 strain, pathogenic mycoplasma of mice and rats, mycoplasma M. pulmonis m53 strain, mycoplasma M. arthritidis PG6 strain, mycoplasma
M. neurolyticum PG28 strain, swine pathogenic mycoplasma mycoplasma M. hyopneumoniae VPP11 strain, goat pathogenic mycoplasma mycoplasma M. capricolum CALIF, KID strain And then prepare DNA. On the other hand, the 16SrRNA-spacer region of Mycoplasma capricolumn-DN encoding 23SrRNA
A sequence [Molecular and General Genetics No. 196
Vol., Pp. 317-322 (1984)]
From the DNA sequence shown in the figure, a pair of primer pairs that can amplify this region and other mycoplasma DNA by the PCR method, for example, a primer pair shown in Table 1 below are selected.

Fig. 1 shows the structure of the Mycoplasma Capri Column.
It is a figure which shows the base sequence of 22 bp.

This primer pair can be synthesized using a DNA synthesizer, HPLC
Can be purified. Using this primer pair, the above 11 types of DNA other than Mycoplasma
A CR reaction is performed to amplify DNA for DNA sequencing.

For the PCR method, tack-polymerase (Taq-polyme
A gene amplification kit and an automatic gene amplifying apparatus containing the above-mentioned primer pair are commercially available from Takara Shuzo Co., Ltd., and an amplification reaction of mycoplasma DNA is carried out using the primer pair described above.

As a PCR method, as an enzyme, for example, a heat-resistant tack-
Using a polymerase, a temperature cycle consisting of DNA denaturation (95 ° C), primer DNA annealing (37 ° C), and enzymatic synthesis of DNA complementary strands (72 ° C) is repeated 50 times to obtain the target gene. Amplify. In this case, the annealing temperature and the number of temperature cycles depend on the template DNA and primer DNA.
It is appropriately selected in consideration of the homology between the Tm template and the primer.

 Next, the base sequence of the amplified DNA is determined.

Once the nucleotide sequence of the DNA amplified by the PCR method has been determined,
The method can be determined by a method of cloning DNA into a phage or plasmid vector such as M13 or pUC, or a method of omitting this cloning step and directly using PCR-amplified DNA (Direct Sequence method). To prevent misreading of DNA sequences due to misincorporation that can occur during in vitro DNA synthesis using tack-polymerase, and to save time and money, the direct sequencing method is used. It is advantageous.

Conventionally, in the direct sequencing method, the ratio of two primers is set to about 1: 100 during PCR amplification, and one strand of DNA is generated in excess of the other strand (Asymmetric PCR). , The resulting single-stranded D
The mainstream was to use NA as a template. However, in this method, the priming efficiency greatly differs depending on the primer used. Therefore, in order to obtain a sufficient amount of single-stranded DNA for the sequencing reaction, it was necessary to carry out an experiment in advance to determine the primer concentration ratio. This is not practical if you need to sequence a large number of samples as in this case. The present inventors have studied a method for determining a base sequence using double-stranded DNA as it is without generating single-stranded DNA. As a result, when the base sequence determination primer is annealed to the linear template DNA, the template DNA is denatured by heat and then rapidly cooled rather than gradually cooled as conventionally used exclusively. Is found to occur more preferentially due to the association between templates, and by applying this quenching method, the dideoxy method can be easily performed even when double-stranded and chain-PCR amplified DNA is used as a template. As a result, the base sequence of mycoplasma could be determined. In addition, when using this method, if a primer labeled at the end with a radioisotope or the like is used, the method for purifying the template DNA required to obtain a readable sequence ladder is PCR amplification by a fractional precipitation method using PEG. A simple operation of removing the primer from the product is sufficient.

16S rRNA-spacer region of each mycoplasma-2
The DNA sequence encoding 3S rRNA is as shown in FIG. 2 to FIG. Fig. 2 shows Mycoplasma hyorinis (480bp), Fig. 3 shows Mycoplasma aurele (460bp), Fig. 4 shows Mycoplasma salivaryum (438bp), Fig. 5 shows Mycoplasma arginini (400bp), and Fig. 6 shows Figures 7 and 8 of Mycoplasma purmonis (513 bp), Figure 7 of Mycoplasma neuroliticam (539 bp), Figure 8 of Mycoplasma arthritisdis (444 bp), and Figure 9 of Mycoplasma Hio pneumoniae (731 bp). Figure 11 shows the first of Mycoplasma fermentans (522 bp).
Figures 2 and 13 show Mycoplasma Hominis (4
FIG. 14 and FIG. 15 show ureaplasma urearicum (517 bp and 516 bp, respectively).
Shows the respective DNA sequences.

If the homology of the mycoplasma DNA sequences shown in FIGS. 1 to 15 is examined, and a common sequence specific to these mycoplasmas is selected and detected, mycoplasmas in general can be detected. As a method for detecting these sequences, a genetic engineering detection method may be used, but the aforementioned PCR method is currently the most sensitive detection method. As a primer for amplifying all mycoplasma DNA by PCR, primer DNA that can anneal to all mycoplasmas in the region
Any pair may be used, and a plurality of such primer pairs may be prepared and mixed for use.

As the primer pair, for example, the primer pairs in Table 2 can be synthesized using a DNA synthesizer and purified by HPLC.

As a PCR method, a primer pair to be used is selected, and then, for example, under the conditions of an annealing temperature of 55 ° C. and a cycle number of 30 times
A PCR reaction may be performed.

Detection of mycoplasma DNA after amplification can be performed, for example, by agarose gel electrophoresis, polyacrylamide gel electrophoresis,
It can be performed using a spot method and a Southern blot method. In the spot method and the Southern blot method, a probe DNA may be selected in the amplification region. As an example, probe 1 in Table 3 may be used as a probe for detecting PCR-amplified DNA when primers 2-1 and 2-2 are used (Table 3).

The probe DNA can be synthesized and purified in the same manner as the primer DNA. By labeling the probe DNA, highly sensitive detection becomes possible.

The labeling method is not limited to the radioisotope labeling method, but may be any known method such as enzyme labeling, fluorescent labeling, biotin, avidin-based labeling, and chemiprobe labeling.

Using the primers actually selected, each Mycoplasma
A specific region of DNA can be amplified by a PCR reaction, and can be detected with high sensitivity by electrophoresis, Southern hybridization, or the like.

On the other hand, in order to specifically amplify each individual mycoplasma DNA, at least one primer may be selected so that a DNA sequence specific to each mycoplasma is obtained in the primer region. As an example, a primer pair as shown in Table 4 can be selected as a primer pair that specifically amplifies only Mycoplasma hyopneumoniae.

By clarifying the DNA sequence common to mycoplasmas in general and the DNA sequence specific to each mycoplasma, detection of mycoplasma in a sample, identification of contaminated mycoplasma, detection of pathogenic mycoplasma, etc. can be performed with high sensitivity. Can be.

The Mycoplasma Capri Column shown in Table 1
The primer pair selected from the DNA sequence can amplify the other 11 types of mycoplasma DNA by PCR, but the sequence does not completely match the DNA sequence of other mycoplasmas, and in the PCR reaction, 37 C. Annealing is required, and amplification can be performed by performing 50 reaction cycles. For this reason, DNA of other bacteria, for example, DNA of E. coli K-12 strain and Bacillus subtilis DNA were also amplified.

On the other hand, the primer pairs shown in Table 2 are unique to mycoplasma and have the same DNA sequence. In the PCR reaction, annealing at 55 ° C. and 30 cycles of mycoplasma DNA were performed.
Was amplified specifically, and amplification of the bacterial DNA was not observed.

In the case of the PCR method, detection can be performed from only one mycoplasma-infected bacterium, early detection of mycoplasma becomes possible, and management of contaminated cells, contaminated animals, and the like becomes easy.

In addition, according to the present invention, detection of mycoplasma can be easily performed by preparing a kit with primer pairs for amplifying the mycoplasma specific DNA region. The reagent used in the kit may be in the form of a solution or a lyophilized product.

The high-sensitivity detection method for mycoplasma using the PCR method has been described in detail above.However, the present invention is not limited to the PCR method, and all methods for detecting a specific DNA and its complementary strand with high sensitivity are described in the present invention. Included in the invention,
For example, Qβ-Replicase amplification
System [Bio / technology,
6, 1197 (1988)].

〔Example〕

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

Example 1 Determination of base sequence of DNA region coding for 16S rRNA-spacer region-23S rRNA of mycoplasma (1) Preparation of mycoplasma DNA As mycoplasma that frequently contaminates cultured bacteria,
Mycoplasma Fermentans PG18, Mycoplasma Hiorinis BTS7, Mycoplasma Aurare
CH19299 strain, Mycoplasma Hominis PG21 strain, Mycoplasma saliva barium PG20 strain, Mycoplasma arginini G230 strain, Ureaplasma Ureariticam T960
Strains, mouse pathogenic mycoplasmas, Mycoplasma plumonis m53 strain, Mycoplasma arsitidis PG6 strain, Mycoplasma neuroliticam PG2
8 strains, as swine pathogenic mycoplasma, Mycoplasma H. pneumoniae VPP11 strain, as goat pathogenic mycoplasma, Mycoplasma Capricolum CALIF, K
ID strains (both are distributed from the University of Tokyo Animal Research Facility)
Was cultured at 37 ° C. in a known and improved Edward medium 3 and centrifuged at 10,000 g for 1 hour to remove the medium. TE buffer [10 mM Tris-HCl, pH 7.5, 1 mM
After washing the cells with EDTA], the method of Razin et al. [International Journal of Systematic Bacteriology (Int. J. Syst. Bacteriol.), No. 33]
Vol., Pp. 201-206 (1983)].

(2) Determination of base sequence of individual 16S rRNA-spacer region of individual Mycoplasma-DNA region encoding 23S rRNA Known 16S rRNA and 2S of Mycoplasma capricolum
A pair of oligonucleotides for amplifying the region by the PCR method from the DNA sequence encoding 3S rRNA A primer 1-1 and a primer 1-2 in Table 1 were selected, and a DNA synthesizer manufactured by Applied Biosystems was used. After synthesis, deprotection and ion exchange HPLC (TSK gel, DEAE-2SW column)
And desalted with SEP-PAK C ₁₈ (Waters) to obtain about 50 μg of each DNA.

DNA50n of mycoplasma prepared in Example 1- (1)
g in a 0.5 ml tube, and 10 μl of 10 × amplification buffer, 16 μl of 1.25 mM dNTP mixture (dATP, dGTP, d
CTP, dTTP), 1μ of 20μM primer 1-1, 1μ
20 μM primer 1-2, 5 units of 0.5 μ /
Add μ-tack polymerase and add sterile water
A 100 μl solution was obtained.

After adding 100 μm of mineral oil (Sigma) to the upper layer of this reaction solution, an amplification reaction was carried out by an automatic gene amplifier Thermal-Cycler (Takara Shuzo). The reaction conditions were denaturation at 94 ° C for 30 seconds → 37
50 ° C. for 2 minutes of primer annealing at 72 ° C. for 2 minutes.

After the reaction, the upper layer of mineral oil was removed, 2 μl of the reaction solution was taken, 1% agarose (Takara Shuzo) gel electrophoresis was performed, and the DNA was stained with ethidium bromide.
NA amplification was confirmed.

As a result, the Ureaplasma
550 bp and 150 bp bands were detected, and other mycoplasmas detected a single band of about 450 bp to 700 bp.

Next, 98 µ of the reaction solution was transferred to another 1.5-ml Eppendorf tube, and TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was transferred.
After extraction with an equal volume mixture of phenol and chloroform saturated with a buffer solution, the upper layer is transferred to another Eppendorf tube, 10 μm of a 3 M sodium acetate solution and 250 μl of ethanol are added, and the mixture is stirred and stirred at −80 ° C. for 15 minutes. After standing for 1 minute, the mixture was centrifuged at 16000 rpm for 10 minutes to remove the supernatant, and the DNA precipitate was recovered. Next, 200 μl of 80% ethanol was added, and 16000
After centrifugation at rpm for 3 minutes, the supernatant was removed, followed by vacuum drying (ethanol precipitation). Eleven mycoplasma DNAs with a single band were dissolved in 100 μl of TE buffer.
Add 60% of 20% polyethylene glycol and 2.5M sodium chloride solution, stir and leave at 0 ° C for 1 hour.
The supernatant was removed by centrifugation at 16000 rpm for 10 minutes. Obtained DN
A precipitate was washed with 80% ethanol, vacuum dried, and then
Dissolved in water. Two types of bands were obtained.

For Ureaplasma Ureariticam, ethanol precipitated DNA was dissolved in 10 μl of TE buffer and 1% low melting point agarose [Sea Plaq Agarose (Sea Plaq
ue Agarose), electrophoresed on a Takara Shuzo gel, and cut out a band of about 550 bp in size, using Gene Clean (GENE CLE).
AN) 100 DNA samples using II kit [Funakoshi Pharmaceutical Co., Ltd.]
Extracted into μ of water. After treatment with a mixed solution of an equal volume of phenol and chloroform, ethanol precipitation was performed, and the resultant was dissolved in 20 µ of water. Using the DNA thus obtained as a template, DNA sequencing was directly performed by the dideoxy method as follows. Primers 1-1 and 1-2 used for DNA amplification by PCR were labeled with ³² P at the 5 'end using a MEGALABEL kit (Takara Shuzo). Labeled primer 1-1 or primer 1-2
1 pmol, about 0.8 pmol of template DNA, 1.5 μm of × 10 buffer solution (70 mM Tris-HCl, pH 7.5, 200 mM sodium chloride, 70 mM magnesium chloride, 1 mM EDTA), water was added to 14 μm, and then 14 μm, followed by 94 ° C. for 3 minutes Heat and quench in ice. After adding and mixing 1 μl of Klenow (2 units) (Takara Shuzo), 3 μl was dispensed into four tubes containing 2 μm of a mixture of four types of dNTP-ddNTPs and mixed.

The composition of a mixture of four types of dNTP-ddNTP is as follows. (G) 83μM dATP, dTTP, 4.2μM dc 7 GTP (7-dea
za2'dGTP), 2.5 μM dCTP, 58 μM ddGTP, (A) 83 μM
dc ⁷ GTP, dTTP, 2.5 μM dCTP, 4.2 μM dATP, 100 μM dd
ATP, (T) 83 μM dc ⁷ GTP, dATP, 2.5 μM dCTP, 4.2 μM
dTTP, 200 μM ddTTP, (C) 62 μM dc ⁷ GTP, dATP, dTT
P, 2.5 μM dCTP, 50 μM ddCTP.

The mixed reaction solution was kept at 42 ° C. for 20 minutes, and 1 μm of a chase mixed solution (1 mM, dGTP, dATP, dTTP, dCTP) was added thereto, and further 2 μm was added.
After holding for 0 minutes, 4 μl of a reaction stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.0%
5% xylene cyanol FF) was added. After heating at 94 ° C. for 3 minutes, the mixture was rapidly cooled on ice, electrophoresed on a denaturing polyacrylamide gel, and after autoradiography, the ladder was read and DNA sequencing was performed.

The DNA sequences of Mycoplasma hyorinis, Mycoplasma aurare, Mycoplasma salivaryum, Mycoplasma arginini, Mycoplasma plumonis, Mycoplasma aristilidis, Mycoplasma neuroliticam, and Mycoplasma hyopneumoniae are as shown in FIG. 2 to FIG.

On the other hand, there were places where two ladders were continuously generated for Mycoplasma Fermentans, Mycoplasma Hominis, and Ureaplasma Ureariticam.
For these mycoplasmas, DNA amplified by PCR
Approximately 250ng of the fragment is used for DNA blunting kit (Blunting k
It) was blunt-ended using (Takara Shuzo). Smoothed
50 ng of the DNA fragment and 100 ng of the DNA fragment obtained by cutting pUC18 with the restriction enzyme Hinc II were ligated using a DNA ligation kit (Takara Shuzo). The reaction solution was transferred to Cohen
n) were used for transformation of Escherichia coli JM109, and the transformants were selected by the method of J. Vieira et al.
The white colonies obtained for the three mycoplasmas were
Suspended in 50μ sterile water to 0.5ml tube chosen by number, was heated for 5 min, are included in the GeneAmp ^TM kit (Gene Amp ^TM Kit) (Takara Shuzo Co. sold) 1
0 μ of 10 × amplification buffer (100 mM Tris-HCl, pH 8.3,
500 mM KCl, 15 mM MgCl ₂ , 0.1% (W / V) gelatin], 16
μ of 1.25 mM dNTP mixture (dATP, dGTP, dCTP, dTT
P) 1 μM M13 primer M4 (manufactured by Takara Shuzo), 1 μM 10 mM M13 primer RV (manufactured by Takara Shuzo),
0.5 μ of 5 units / μ tack-polymerase was added and sterile water was added to make a 100 μ solution.

After adding 100 μm of mineral oil to the upper layer of this reaction solution, an amplification reaction was carried out by a thermal cycler of an automatic gene amplifying apparatus. The reaction conditions were: denaturation at 94 ° C for 1 minute → 55 ° C for 2 minutes, annealing of primer for 2 minutes → 72 ° C,
A 25 minute synthesis reaction cycle was performed.

After the reaction, after removing the upper layer of mineral oil, 10 µ of the reaction solution was taken, 1% agarose (Takara Shuzo) gel electrophoresis was performed, and the DNA was stained with ethidium bromide.
The lengths of the amplified DNAs were confirmed, and the DNAs amplified for the three types of mycoplasmas were directly subjected to DNA sequencing in the same manner using primers 1-1 and 1-2 for each of the three clones. As a result, two types of DNA sequences were obtained for each of the three types of mycoplasmas. Fig. 10
As shown in FIG.

Example 2 Amplification of Mycoplasma DNA by PCR (1) Synthesis and Purification of Oligonucleotide Primer DNA Two primer DNA pairs shown in Table 2 selected to amplify the common sequence of mycoplasma DNA shown in the drawing were applied to Applied Bio. It is synthesized using Systems DNA Synthesizer, deprotected, and ion-exchange HPLC (TSK gel, DEAE
-2SW column) and desalted with Sep-Pak _C18 Waters) to obtain about 50 ng of each DNA.

(2) Amplification of Mycoplasma common sequence DNA by PCR 5 ng of each of the 12 mycoplasma DNAs prepared in Example 1- (1) and 5 ng of mouse DNA prepared as a control,
Escherichia coli K12 strain DNA 5ng, Bacillus subtilis ISW1214 strain DNA 5ng as template DNA
2 in 0.5 ml Eppendorf tubes
Take 10 tubes each, 10μ of 10 × amplification buffer, 16μ of 1.25
A mM dNTP mixture (dATP, dGTP, dCTP, dTTP) was added, and 1 μm of 20 μM primer 2-1 and 1 μm of 20 μM primer 2-2 were added to one tube of each template DNA, and 1 μm of 20 μM primer 3 was added to the other tube. -1 and 1μ
20 μM primer 3-2 was added, 0.5 μ of 5 units / μ tack-polymerase was added, and sterile water was further added to make a 100 μ solution.

After adding 100 μm of mineral oil to the upper layer of this reaction solution, an amplification reaction was carried out using a mobile gene amplifier thermal cycler. The reaction conditions are denaturation at 94 ° C for 30 seconds →
Primer annealing at 55 ° C. for 2 minutes → Synthesis reaction cycle at 72 ° C. for 1 minute was performed 30 times.

After the reaction, after removing the upper layer of mineral oil, 5 μl of the reaction solution was taken, 1% agarose (manufactured by Takara Shuzo) gel electrophoresis was performed, and the DNA was stained with ethidium bromide.
NA amplification was confirmed.

As a result, a band having a length expected from each DNA sequence was detected for each mycoplasma using any of the primer pairs. But mouse DN
A, no band was detected when Escherichia coli K-12 DNA and Bacillus subtilis ISW1214 DNA were used as templates, and each primer pair was specific to DNA encoding mycoplasma rRNA.

Example 3 P of mycoplasma porcine pneumonia H. pneumoniae DNA
Amplification by CR (1) Synthesis and purification of oligonucleotide primer DNA Mycoplasma A primer pair shown in Table 3 for amplifying a DNA sequence specific to the mycoplasma selected from a DNA sequence encoding rRNA of H. pneumoniae was implemented. It was synthesized and purified in the same manner as in Example 2- (1).
50 ng of DNA was obtained.

(2) Mycoplasma H. pneumoniae DNA PCR
5 ng of each mycoplasma DNA prepared in Example 1- (1) as template DNA, 5 ng of the mouse DNA described above, and 5 ng of E. coli DNA
g, 5 ng of Bacillus subtilis DNA, and the above 3- (1) as a primer pair
PCR as in Example 2- (2) using the primers obtained in
The reaction was performed to amplify and detect DNA. as a result,
Only for Mycoplasma hyopneumoniae DNA, a band of the expected length was detected from its DNA sequence, and other Mycoplasma DNA, mouse DNA, E. coli DNA,
No band was detected when B. subtilis DNA was used as a template,
This primer pair specifically amplified Mycoplasma hyopneumoniae.

Example 4 Mycoplasma by Southern Hybridization
Detection of DNA (1) Detection of Mycoplasma DNA 2 μm of a reaction solution obtained by performing a PCR reaction using primer 2-1 and primer 2-2 as a primer pair in Example 2- (2) was electrophoresed on a 1% agarose gel. After denaturation with alkali, Southern blotting was performed overnight on a nylon membrane (Amersham Hybond-N). Ultraviolet transilluminator (254nm)
For 10 minutes to immobilize the DNA on the membrane.

This membrane was prepared using a prehybridization buffer (5 × Denhardt's solution, 5 × SSC, 0.1% SDS, 100 μg / ml).
Prehybridization was performed at 50 ° C. for 2 hours in 5 ml of salmon sperm DNA). Next, as a probe, Example 2-
Using the primer 3-1 synthesized in (1), a probe labeled with ³² P was added thereto, and hybridization was carried out overnight.

³² P- labeled probes was performed as follows using the Megaraberu kit (Takara Shuzo). 10pmol probe,
1 μ of 10 × phosphorylation buffer, 50 μCi of [γ- ³² P] ATP
(Amersham), sterilized water was added to the reaction solution containing 10 units of T4-polynucleotide kinase to make 10 μm,
The reaction was performed at 37 ° C. for 30 minutes. After the reaction, the mixture was treated at 94 ° C. for 5 minutes, and the entire amount of the reaction solution (about 10 ⁸ cpm) was used for hybridization.

After hybridization, the membrane was replaced with 2xSSC,
Washing was performed four times at room temperature for 10 minutes with a washing solution 1 containing 0.1% SDS, and then twice with washing solution 2 containing 1 × SSC and 0.1% SDS at 55 ° C. for 50 minutes. After drying the membrane, the membrane was exposed overnight at -70 ° C in a cassette containing an X-ray film (Fuji Film), and an autoradiograph was taken.

As a result, bands were detected in all 12 amplified mycoplasma DNA products, but no bands were observed in the reaction solution using mouse DNA, Escherichia coli DNA and Bacillus subtilis DNA as templates.

Example 5 Preparation of Mycoplasma DNA Amplification / Detection Kit A kit for amplifying / detecting mycoplasma DNA in a sample was prepared.

-Mycoplasma common sequence amplification / detection kit As primers for amplifying the common sequence DNA, the primers 2-1 and 2-2 in Table 2 were dissolved in 20 µM of TE buffer so that each solution became 20 µM, and the mycoplasma primer solution (agent A ). In addition, primer 3-1 in Table 2
And TE buffer solution (20 μM) so that each of 3 and 2 becomes a 20 μM solution.
To give a Mycoplasma primer solution (agent B).

D when selecting A agent and amplifying mycoplasma DNA
As a probe for NA detection, 2 μg of probe 1 in Table 3 was used.
It was dissolved in 20 μ of TE buffer to obtain a mycoplasma probe solution (agent C).

Kits I-II with agent A, agent B and a combination of agent A and agent C
I was created (Table 5).

゜ F Mycoplasma H. pneumoniae DNA amplification / detection kit As primers for Mycoplasma H. pneumoniae DNA amplification, primers 4-1 and 4-2 in Table 4 were dissolved in 20 μM TE buffer so that each would be a 20 μM solution. Kit IV was prepared as the liquid (D agent) (Table 6).

[Effects of the Invention] As described above in detail, according to the present invention, a specific gene region common to mycoplasma is generally revealed, and by detecting this region, a method and a kit for highly sensitive detection of mycoplasma in a sample Was provided.

In addition, a part of the DNA sequence encoding rRNA of 11 types of mycoplasma has been newly clarified, and it is possible to detect the characteristic region of each of these mycoplasma DNAs, and a kit for specifically detecting porcine pneumoniae mycoplasma hyopneumoniae Was also provided.

[Brief description of the drawings]

FIG. 1 is a diagram showing the 1992 bp nucleotide sequence of Mycoplasma capricolumn, FIG. 2 is a diagram showing the 480 bp nucleotide sequence of Mycoplasma hyorinis, FIG. 3 is a diagram showing the 460 bp nucleotide sequence of Mycoplasma aurele, FIG. FIG. 5 shows a 400 bp nucleotide sequence of Mycoplasma arginini, FIG. 6 shows a 513 bp nucleotide sequence of Mycoplasma plumonis, and FIG. 7 shows a nucleotide sequence of Mycoplasma neurolitica. FIG. 8 shows the base sequence of 539 bp, FIG. 8 shows the base sequence of 440 bp of Mycoplasma altidisdis, FIG. 9 shows the base sequence of 731 bp of Mycoplasma hyopneumoniae, FIG. 10 and FIG.
522 each of the mycoplasma fermentances in Figure 1
FIGS. 12 and 13 show the nucleotide sequences of 406 bp and 405 bp, respectively, of Mycoplasma hominis, and FIGS. 14 and 15 show the nucleotide sequences of 517 bp and 516 bp, respectively, of Ureaplasma urealyticum. It is a figure showing an arrangement.

Continuation of the front page (72) Inventor Ryo Harasawa 3-9-12 June, Adachi-ku, Tokyo (56) References Table 1-503356 (JP, A) European publication 250662 (EP, A1) European patent 305145 ( EP, B1) Bacteriol, 171 (1989) p. 6455-6467 Gen. Microbiol. , 133 (1987) p. 1969-1974 Mol. Gen. Genet. , 205 (1986) p. 434-441 Journal of Clinical Microbiology, 25 (1987) p. 726-728 Mol. Gen. Genet. , 196 (1984) p. 311-316 Mol. Gen. Genet. , 208 (1987) p. 23-29 Nucleic Acids Research, 15 (1987) p. 1327 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12Q 1/68 Genbank

Claims (2)

(57) [Claims] 1. A method for detecting mycoplasma, comprising the steps of:
Among the DNA sequences of mycoplasma represented by RNA-spacer region-23SrRNA, the DNA sequence of the spacer region, or a part thereof, at least the following formula (1): A method for detecting mycoplasma, comprising detecting a DNA sequence containing a base sequence represented by: 2. A detection kit for performing detection using the method according to claim 1, wherein the DNA is a DNA region of the spacer region according to claim 1 or a part thereof, and is at least represented by the formula (1). A mycoplasma detection kit, comprising a specific primer for amplifying a DNA region containing the represented nucleotide sequence.