JP2000135084A - Base sequence specific to babesia ovata and detection of babesia ovata by utilizing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new base sequence comprising a base sequence equal to or complementary to a specific base sequence, capable of specifically detecting a genomic DNA of pathogen Babesia ovata of bovine babesiosis, and usable for discrimination or the like of the infection by the microorganism. SOLUTION: This DNA is a new one having a base sequence equal to or complementary to the base sequence of the formula, and a nucleic acid capable of hybridizing with a specific base sequence part of Babesia ovata. The DNA can specifically detect the DNA of the Babesia ovata as a pathogen of bovine hemocytozoon disease having cardinal symptoms of anemia, jaundice and hemoglobin urine, and can be used for a discrimination method of the Babesia ovata infection, or the like. The DNA is obtained by separating genomic DNA of the Babesia ovata, forming a genomic DNA library by using the genomic DNA, hybridizing the library by using a genomic DNA of another microorganism of the genus Babesia as a probe, and selecting a clone not reacting with the genome DNA but reacting with the Babesia ovata.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a DNA comprising a nucleotide sequence specific to Babesia obata, a nucleic acid capable of hybridizing to the DNA, and a method for determining Babesia obata infection using the nucleic acid.

[0002]

BACKGROUND OF THE INVENTION Babesia disease in cattle is a schistosomiasis characterized by anemia, jaundice and excretion of hemoglobinuria. Its pathogen is classified as Babesia of the order Piroplasma and transmitted by ticks. By around 1992, in Japan, Babesia obata (Babesia obata)
sia ovata), Babesia bigemin
a) and three distributions of Babesia bovis were confirmed.

[0003] Of these three species, Babesia vigemina and Babesia bovis have strong pathogenicity and are designated as statutory infectious diseases. It is necessary to determine that it is Obata. Babesia Obata is said to be slightly pathogenic, but Babesia majo
r) It is important to discriminate between Babesia and Obata infections in the livestock industry, as they are often stronger and are mixedly infected with Thyleria protozoa.

In particular, recently, domestic and overseas transactions of livestock products, including the import of cattle, have become active, and the necessity for a method for determining Babesia obata infection has been increasing. As a method of discriminating the infection of Babesia parasite, it is common to observe the Giemsa stained specimen image of blood smear using an optical microscope, but to discriminate the species of Babesia parasite based only on the observation of the blood smear image It is difficult. In addition, there is also a method of discriminating infection with Babesia genus protozoa by a serum reaction, but this method requires preparation of antigen from experimentally infected cattle, so the operation is complicated and the antibody titer is increased. No protozoal infection can be detected early in the infection.

On the other hand, recently, it has become possible to determine the infection of Babesia vigemina or Babesia bovis by some protozoan detection methods utilizing genetic engineering techniques. Protozoan detection methods using these genetic engineering techniques have high detection sensitivity and can detect protozoa in the early stage of infection with a low parasite infestation rate, analyze multiple sample samples, analyze infected host tissues and vectors. It is also applicable to protozoan detection in Nakauchi, epidemiological surveys, etc.

For Babesia obata, a DNA-DNA hybridization method has been developed (Ohta et al.
al., J. Protozool. Res. 5, 58-65 (1995)), and it is difficult to process a large number of samples using this method.
Special facilities and techniques are required to utilize radioisotopes, and a series of operations usually require about 2 to 7 days. Further, sufficient sensitivity cannot be obtained when Babesia obata is detected in the body of a protozoan vector, an animal at an early stage of infection, an animal serving as a carrier, and the like.

[0007] Babesia parasites morphologically similar to Babesia obata isolated from Japanese brown cattle in the Oshima area of Hokkaido in 1993 are classified as Babesia obata varieties based on their properties and named Babesia ovata var. Oshimensis. Was done. For this variant, a specific detection method using its nucleotide sequence has been developed, and protozoa in the vector mite have also been detected. Under these circumstances, Babesia is suitable for multi-sample processing, versatile, simple and accurate.
There is a strong need for the development of a method for determining Obata infection.

[0008]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for judging Babesia obata infection which is suitable for multi-sample processing, is versatile, simple and accurate.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, out of the genomic DNA of Babesia obata, a base sequence specific to Babesia obata (sequence) No. 1) was successfully identified. Then, an oligonucleotide (SEQ ID NOs: 2 to 5) capable of hybridizing to the nucleotide sequence is prepared, and the presence of the Babesia obata genomic DNA in the sample is determined by using the oligonucleotide as a probe or a primer for PCR. They have found that they can be specifically detected, and have completed the present invention.

That is, the present invention includes the following inventions. (1) DNA comprising a nucleotide sequence identical or complementary to the nucleotide sequence of SEQ ID NO: 1. (2) A nucleic acid capable of hybridizing to the DNA according to (1). (3) The nucleic acid according to (2), wherein the nucleic acid has 14 to 855 bases.

(4) A nucleic acid comprising the following nucleotide sequences (a) to (d): (A) a base sequence identical or complementary to the base sequence of SEQ ID NOs: 2 to 5 (b) In the base sequence identical or complementary to the base sequence of SEQ ID NOs: 2 to 5, all thymines are substituted with uracil (C) a base sequence in which one or more bases are deleted, substituted or added in the base sequence (a), and which is capable of hybridizing to the DNA according to claim 1; A base sequence in which one or more bases are deleted, substituted or added in the sequence (b), and which can hybridize to the DNA according to claim 1.

(5) the following steps: (a) a step of preparing a DNA-containing sample from a target animal; and (b) a nucleic acid according to any one of the above (2) to (4) as a probe. Confirming whether or not the genomic DNA of Babesia obata is present in the sample, and determining, based on the result, whether the target animal is infected with Babesia obata. A method for distinguishing Babesia obata infection, comprising:

(6) The following steps: (a) a step of preparing a sample containing DNA from a target animal; and (b) The above-mentioned sample is described in any one of (2) to (4) above. PCR using a nucleic acid as a primer
And (c) whether or not the genomic DNA of Babesia obata is present in the sample based on the presence or absence of the amplified fragment obtained in the step (b). A method of determining whether or not Babesia obata is infected. A method for determining Babesia obata infection.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
A first aspect of the present invention is a DNA consisting of a nucleotide sequence identical or complementary to the nucleotide sequence of SEQ ID NO: 1
A ”). The DNA of the first invention is, for example, a primer consisting of the same nucleotide sequence as the 5'-terminal side of the nucleotide sequence of SEQ ID NO: 1 and a genomic DNA of Babesia obata as a template, and 3 of the nucleotide sequence of SEQ ID NO: 1. 'It can be obtained by performing PCR using a primer consisting of a nucleotide sequence complementary to the terminal side.

[0015] The DNA of the first invention is a base sequence portion specific to Babesia obata in the genomic DNA of Babesia obata. Therefore, the DNA of the first invention can be used as a probe for specifically detecting genomic DNA of Babesia obata. Also, the genomic DNA of Babesia Obata
When preparing a probe or a primer for PCR for specifically detecting DNA, their base sequences can be determined based on the base sequence of the DNA of the first invention.

A second aspect of the present invention is a nucleic acid capable of hybridizing to the DNA of the first aspect (hereinafter, referred to as “nucleic acid of the second aspect”). The nucleic acid of the second invention may be a DNA or an RNA as long as it can hybridize to the DNA of the first invention, and its base number and base sequence are not particularly limited. Examples of the nucleic acid of the second invention include a DNA comprising a base sequence identical or complementary to a part of the base sequence shown in SEQ ID NO: 1. Examples of the nucleic acid of the second invention include an RNA having a base sequence identical or complementary to a part of the base sequence described in SEQ ID NO: 1 and including a base sequence in which all thymines are substituted with uracil. .

Here, the part of the nucleotide sequence of SEQ ID NO: 1 contained in the nucleic acid of the second invention may be any part of the nucleotide sequence of SEQ ID NO: 1. The number of the bases is not particularly limited, but when the nucleic acid of the second invention is used as a probe, it is preferably at least 14 bases or more, more preferably 17 bases or more, and 90 bases or more. Most preferably, when the nucleic acid of the second invention is used as a primer, it is preferably at least 16 bases or more, more preferably 18 to 30 bases, and more preferably 20 to 24 bases. Is most preferred. In addition, the nucleotide sequence identical or complementary to the part may be included in any part of the nucleic acid of the second invention, and, in the nucleic acid of the second invention, the nucleotide sequence part identical or complementary to the part. The nucleotide sequence of the other part is not particularly limited. Further, the number of bases of the nucleic acid of the second invention is not particularly limited, but when the nucleic acid of the second invention is used as a probe, it is preferably at least 14 bases, more preferably at least 17 bases, Most preferably 90 bases or more, when using the nucleic acid of the second invention as a primer, preferably 16 bases or more, more preferably 18-30 bases, more preferably 20-24 bases Is most preferred.

[0018] The nucleic acid of the second invention includes a DNA consisting of a base sequence identical or complementary to the base sequence of SEQ ID NOs: 2 to 5 (hereinafter referred to as "base sequence (A)") or SEQ ID NO: 2 RNA consisting of a nucleotide sequence identical to or complementary to the nucleotide sequence of any one of (1) to (5) in which all thymines are substituted with uracil (hereinafter, referred to as “base sequence (a)”)
Can be exemplified as preferable ones.

The nucleotide sequence described in SEQ ID NO: 2 is
It is the same as the portion from the 21st base to the 42nd base in the described base sequence. Further, the base sequence described in SEQ ID NO: 3 is complementary to the portion from the 855th base to the 834th base in the base sequence described in SEQ ID NO: 1. In addition, the base sequence described in SEQ ID NO: 4 is the same as the portion from the 63rd base to the 84th base in the base sequence described in SEQ ID NO: 1.

The nucleotide sequence of SEQ ID NO: 5 is complementary to the portion of the nucleotide sequence of SEQ ID NO: 1 from the 647th base to the 626th base. Therefore, the DNA consisting of the base sequence (A) and the RNA consisting of the base sequence (A)
It can hybridize to a DNA consisting of a nucleotide sequence identical or complementary to the nucleotide sequence of SEQ ID NO: 1.

In addition, the nucleotide sequence of the nucleic acid of the second invention is a nucleotide sequence which can hybridize to DNA consisting of a nucleotide sequence identical to or complementary to the nucleotide sequence of SEQ ID NO: 1.
In the base sequence (A) or the base sequence (A), one or more bases may be deleted, substituted or added. Here, the "one or more bases" is not particularly limited as long as it can hybridize to a DNA consisting of a base sequence identical or complementary to the base sequence of SEQ ID NO: 1.

The nucleic acid of the second invention can be synthesized by a conventional method according to its nucleotide sequence. The nucleic acid of the second invention,
It can be used as a probe for specifically detecting Babesia obata genomic DNA or a primer for PCR. When using the nucleic acid of the second invention as a primer for PCR,
The GC content of the nucleic acid of the second invention is preferably from 40 to 70%, more preferably from 50 to 60%. Further, the Tm value is preferably from 55 to 80 ° C, more preferably from 60 to 78 ° C.

The third step of the present invention is to provide the following steps: (a) a step of preparing a sample containing DNA from a target animal; and (b) a step of using the nucleic acid of the second invention as a probe, Babesia Obata Genome DN
Confirming whether or not A is present, and determining, based on the result, whether or not the target animal is infected with Babesia obata.
This is a method of determining Obata infection. Hereinafter, each step will be described.

(1) Step (a) Step (a) is a step of preparing a sample containing DNA from a target animal. Here, the “subject animal” means an animal whose presence or absence of Babesia obata infection is to be determined. The target animal is not particularly limited as long as it is an animal that can be infected with Babesia obata, and may be any animal. Examples of such target animals include cattle, ticks, mice having bovine red blood cells, and the like.

A sample containing DNA can be prepared from erythrocytes, spleen and the like of a subject animal by a conventional method. When the target animal is a tick, a sample containing DNA can be prepared from the digestive tract, eggs, salivary glands, and the like according to a conventional method. For example, red blood cells and the like of a target animal are frozen and crushed in the presence of liquid nitrogen, and then treated in a lysate containing sodium dodecyl sulfate and proteinase K. Next, the DNA is extracted and purified using phenol, a solvent containing an equal amount of phenol and chloroform, and chloroform, and further purified and concentrated by ethanol precipitation. As a result, a sample containing DNA can be prepared from the target animal. The sample containing the prepared DNA includes various RNAs, DNAs, proteins, etc. in addition to the genomic DNA of Babesia obata. Therefore, the sample may be purified according to a conventional method.

(2) Step (b) In the step (b), the genomic DN of Babesia obata is contained in the sample by using the nucleic acid of the second invention as a probe.
This is a step of confirming whether or not A is present, and determining whether or not the target animal is infected with Babesia obata based on the result. Whether or not the genomic DNA of Babesia obata is present in the sample may be confirmed by any method as long as the nucleic acid of the second invention is used as a probe.

For example, after the DNA fragment contained in the sample is developed by electrophoresis, it is immobilized on the main membrane by Southern blotting, and immobilized on the membrane using the labeled nucleic acid of the second invention as a probe.
It is hybridized with the DNA fragment, and it can be confirmed whether or not genomic DNA of Babesia obata is present in the sample using the label as an index. At this time, electrophoresis and Southern blotting can be performed according to a conventional method. Examples of the label of the nucleic acid of the second invention include, for example, radiolabeling with a radioisotope such as ³² P, non-radioactive labeling with biotin, digoxigenin, and the like.These labeling can be performed according to a conventional method. it can.

The nucleic acid of the second invention can hybridize to a base sequence (SEQ ID NO: 1) specific to Babesia obata in genomic DNA of Babesia obata. Therefore,
By using the nucleic acid of the second invention as a probe, it is possible to specifically detect whether or not genomic DNA of Babesia obata is present in the sample. If the genomic DNA of Babesia obata is present in the sample, it can be determined that the target animal is infected with Babesia obata, while if the genomic DNA of Babesia obata is not present in the sample, It can be determined that the target animal is not infected with Babesia obata.

The fourth step of the present invention is as follows: (a) a step of preparing a sample containing DNA from a subject animal; and (b) PCR using the nucleic acid of the second invention as a primer with respect to the above sample. And (c) whether or not the genomic DNA of Babesia obata is present in the sample based on the presence or absence of the amplified fragment obtained in the step (b). A step of determining whether or not the bacterium is infected with Babesia obata. Hereinafter, each step will be described.

(1) Step (a) Step (a) is a step of preparing a DNA-containing sample from a target animal. Step (a) can be performed in the same manner as described above. (2) Step (b) Step (b) is a step of performing PCR on the sample using the nucleic acid of the second invention as a primer. Other conditions are not particularly limited as long as the nucleic acid of the second invention is used as a primer, and PCR can be performed according to a conventional method.

As a primer set used for PCR,
For example, a primer set consisting of a primer consisting of the base sequence of SEQ ID NO: 2 and a primer consisting of the base sequence of SEQ ID NO: 3, a primer consisting of the base sequence of SEQ ID NO: 4 and the base sequence of SEQ ID NO: 5 A primer set composed of primers can be exemplified.

The nucleic acid of the second invention can hybridize to a base sequence specific to Babesia obata (SEQ ID NO: 1) in Babesia obata genomic DNA. Therefore,
When the genomic DNA of Babesia obata is contained in the sample, an amplified fragment derived from the genomic DNA of Babesia obata is obtained by PCR using the nucleic acid of the second invention as a primer. If the genomic DNA of Babesia obata is not contained therein, an amplified fragment derived from the genomic DNA of Babesia obata cannot be obtained. In order to more efficiently obtain only an amplified fragment derived from Babesia obata genomic DNA, it is preferable to perform nested PCR on the amplified fragment obtained in step (b).

The nested PCR can be performed according to a conventional method, and the primer set used in the nested PCR can be appropriately determined depending on the amplified fragment to be obtained. For example, in the step (b), when a primer consisting of the nucleotide sequence of SEQ ID NO: 2 and a primer consisting of the nucleotide sequence of SEQ ID NO: 3 are used, in the nested PCR, a primer consisting of the nucleotide sequence of SEQ ID NO: 4 And SEQ ID NO: 5
Primers consisting of the described nucleotide sequences can be used.

(3) Step (c) In step (c), the presence or absence of the amplified fragment obtained in step (b) is used to confirm whether or not the genomic DNA of Babesia obata is present in the sample. This is a step of determining whether or not the target animal is infected with Babesia obata based on the result. Confirmation of the presence or absence of the amplified fragment can be performed according to a conventional method. For example, the presence or absence of the amplified fragment can be confirmed by electrophoresis. As a result of confirming the presence or absence of the amplified fragment, when the amplified fragment is present, it can be confirmed that the genomic DNA of Babesia obata is present in the sample,
As a result of confirming the presence or absence of the amplified fragment, when the amplified fragment does not exist, it can be confirmed that the genomic DNA of Babesia obata does not exist in the sample.

The above sample contains the genome DN of Babesia obata
In order to check with more accuracy whether or not A exists,
It is preferable to confirm that the amplified fragment is derived from Babesia obata genomic DNA. As a method for confirming that the amplified fragment is derived from Babesia obata genomic DNA, for example, there is a method of performing a restriction fragment length analysis obtained by treating the amplified fragment with a restriction enzyme. The restriction enzyme used at this time is not particularly limited as long as it can cleave the amplified fragment. The restriction enzymes may be used alone, but it is desirable to use two or more restriction enzymes in combination.
If the genomic DNA of Babesia obata is present in the sample, it can be determined that the target animal is infected with Babesia obata, while if the genomic DNA of Babesia obata is not present in the sample, The target animal is Babesia
You can determine that you are not infected with Obata.

[0036]

[Example 1] Genomic DNA of Babesia obata
Isolation and identification of fragments (1) Isolation of genomic DNA fragment of Babesia obata Genomic DNA of Babesia obata was treated with restriction enzyme EcoRI, and then incorporated into λZAPII phage to produce a subgenomic DNA library of Babesia obata. did. And
By performing plaque hybridization using genomic DNA of Babesia obata and genomic DNA of Babesia bovis as probes, from this library,
Clone BOZ, which reacts with Babesia obata genomic DNA but does not cross-react with Babesia bovis genomic DNA
AP6 was obtained (Kawazu et al., J. Protozool. Res. 3, 64-68 (199
3)).

On the other hand, a variant of Babesia obata (Babesia
ovamen var. oshimensis) genomic DNA with EcoRI restriction enzyme
After that, the resultant was integrated into λZAPII phage to prepare a subgenomic DNA library of a Babesia obata variant. By performing plaque hybridization using BOZAP6 as a probe, a clone pBU cross-reacting with BOZAP6 was obtained from this library.

BOZAP6 is converted to a restriction enzyme (MluI, DraII, EcoRI)
Hybridization was performed using the pBU-inserted DNA fragment as a probe to identify the crossover region and the non-crossover region in BOZAP6. Using a 0.9 kbp DraII / EcoRI DNA fragment belonging to the non-crossing region as a probe, the cross-reactivity of this DNA fragment with the genomic DNA of a Babesia obata variant was examined. As a result, this DNA fragment is the genomic DNA of a Babesia obata variant.
Did not have cross-reactivity. Hereafter, this DN
The A fragment is called OvS9.

(2) Identification of genomic DNA fragment of Babesia obata OvS9 and pBluescriptII were digested with 10 mM magnesium chloride, 10 mM dithiothreitol, 0.5 mM adenosine 3-phosphate, and 50 μg / ml bovine serum albumin using T4 ligase. In 40 mM Tris-HCl buffer (pH 7.5) containing After introducing this recombinant plasmid into Escherichia coli by DNA electroporation, the Escherichia coli was cultured and grown at 37 ° C.

Next, the plasmid DN present in the above E. coli was used.
A was purified by ethidium bromide-cesium chloride equilibrium density gradient centrifugation. One-tenth volume of 3M sodium acetate (pH 7.5) was added to this purified plasmid DNA and mixed, and then 2-3 volumes of 99.5 (v / v)% ethanol was added and mixed. After cooling at −20 ° C., the supernatant was removed by centrifugation (1,500 rpm for 10 minutes), the precipitated DNA was washed with 70% (v / v) ethanol, and 10 mM Tris containing 1 mM disodium ethylenediaminetetraacetate was removed. It was dissolved in hydrochloric acid buffer (pH 7.6). After measuring the amount of DNA using the ultraviolet absorbance method, this D
The nucleotide sequence of NA was determined by the dideoxy method of Sanger et al.
al., Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977))
Analyzed by

As a result, the analysis results from both directions of the plasmid were consistent, and the total length of OvS9 was 865 bp (SEQ ID NO: 1). OvS using GENETYX (software development)
When the nucleotide sequence of 9 was compared with the nucleotide sequence contained in GenBank, no sequence with high similarity was found. Therefore, the nucleotide sequence described in SEQ ID NO: 1 was found to be a nucleotide sequence portion specific to Babesia obata in genomic DNA of Babesia obata.

[Example 2] Preparation of primer OvS9 (total length of 855 b
p) The region from the 21st base to the 855th base (full length)
One set of primers was designed to amplify 835 bp) (FIG. 1). In this primer set, the 5 ′ primer was B5OV1 (SEQ ID NO: 2), and the 3 ′ primer was B3OV1
(SEQ ID NO: 3). Another primer set was designed so as to amplify the region inside the amplification region by the above primer set, that is, the region (base length 585 bp) from the 63rd base to the 647th base of OvS9 (FIG. 1). . In this primer set, the 5 'primer is B5OV2
(SEQ ID NO: 4) and the 3 ′ primer is B3OV2 (SEQ ID NO: 5).

Each primer of the two primer sets is a 22 mer, and was designed with attention to the following points in order to increase the efficiency and specificity of PCR. In order that each primer and OvS9 hybridize in one place, the OvS9 portion other than the portion where each primer hybridizes to OvS9 is made as less complementary as possible to each primer. In order to prevent hybridization between primers,
If possible, make sure that there is no complementary portion between primers (especially on the 3 'side).

The melting temperature (T _m ) of the primer is 55 to 80
° C, preferably 64 ° C. Normally, the length of the amplified fragment is confirmed to be the target by confirming the length of the amplified fragment.However, the amplified fragment should contain a restriction enzyme cleavage site so that it can be more accurately confirmed by restriction fragment length analysis. To Each primer was prepared according to Arnole et al., Nucleic Acids Symp
Ser, 18: 181-184 (1987)).

[Example 3] Detection of Babesia obata genomic DNA by PCR (1) Using 100 pg each of genomic DNA of Babesia obata, genomic DNA of a typical stomach microorganism and bovine genomic DNA as templates, B5OV1 and PCR was performed using B3OV1 as a primer. The schistem microorganisms used are as follows.

Theileria sergenti Anaplasma marginal
e) Anaplasma centrale (Eperythrozoon weny)
oni) Babesia bovis Babesia bigemina Babesia ovata var. oshi
mensis)

The genomic DNA of Babesia obata, the genomic DNA of each schismic microorganism, and the genomic DNA of cattle were prepared as follows. First, leukocytes were removed from the blood of bovine infected with Babesia obata, and erythrocytes were separated and purified. Next, the red blood cells were crushed using high-pressure nitrogen gas, and the red blood cell membrane fraction and the microorganism fraction were separated by centrifugation. The obtained microbial fraction was treated in a lysate containing sodium dodecyl sulfate and proteinase K. Next, phenol, an equal amount of phenol and chloroform, and
A was extracted and purified, and further precipitated with ethanol, and purified and concentrated. Thereby, genomic DNA of Babesia obata was prepared. In addition, bovine blood infected with each of the stomach microorganisms was treated in the same manner as described above to prepare genomic DNA of each of the scioblood microorganisms. Furthermore, a leukocyte fraction was collected by the specific gravity centrifugation method using uninfected bovine blood, and treated in the same manner as described above to prepare bovine genomic DNA.

The PCR was carried out using 50 mM potassium chloride, 15 mM magnesium chloride, 200 μM dNTP (dATP, dGTP, dCTP and dTTP).
TP), 2.5 μM of each primer (B5OV1 and B3OV1), and
Performed in 100 μl of 10 mM Tris-HCl buffer (pH 8.3) containing 2.5 units of Taq polymerase. One cycle of PCR is DNA
Denaturation at 94 ° C for 2 minutes, primer annealing at 60 ° C for 2 minutes
Min and DNA elongation at 72 ° C. for 3 min, and this was performed for 35 cycles. However, only the first DNA denaturation was performed at 94 ° C for 5 minutes, and only the final DNA extension was performed at 72 ° C for 7 minutes.

After PCR, take one-tenth volume (10 μl) of the reaction solution and contain 1 mM disodium ethylenediaminetetraacetate.
Electrophoresis was performed using a 2% agarose gel in a 40 mM Tris-acetate buffer, and the separated PCR product was detected by staining with 0.5 μg / ml ethidium bromide. The result is shown in FIG.
As shown in FIG. 2, Tyrelia Sergenti, Anaplasma Marginale, Anaplasma Mascentrale,
No amplified fragment was detected when genomic DNA of Eperithrozone wenyoni, bovine, Babesia bovis, Babesia bigemina and Babesia obata variants was used as a template. On the other hand, the genome DN of Babesia Obata
When A was used as a template, the target amplified fragment of 835 bp was detected.

From these results, it was found that only DNA fragments derived from Babesia obata genomic DNA were amplified by PCR using B5OV1 and B3OV1 as primers. Therefore, it was found that by performing PCR using B5OV1 and B3OV1 as primers and using the presence or absence of the obtained amplified fragment as an index, the genomic DNA of Babesia obata can be specifically detected.

(2) Next, the genome DN of Babesia obata
A100pg is serially diluted 10-fold (100pg, 10pg, 1pg, 100f
g, 10fg), PCR was performed using the DNA at each concentration as a template.
The detection limit by CR was determined. PCR and electrophoresis of the PCR product were performed as described above. The result is shown in FIG. As shown in FIG. 3, it was found that an amplified fragment could be detected when 1 pg or more of DNA was used as a template. This detection limit is determined by using a previously reported method using a DNA probe (Ohta et al.
l., J. Protozool. Res. 5, 58-65 (1995))
It was twice as sensitive.

(3) In PCR, as the amount of template DNA decreases, the staining intensity of the amplified fragment decreases, and it becomes difficult to confirm the amplified fragment near the detection limit. Therefore, after PCR using B5OV1 and B3OV1, 100% of the reaction solution containing the amplified PCR product was used.
One-half the volume (1 μl) was again subjected to PCR (nested PC
R). In the case of nested PCR, B5OV2 and
B3OV2 was used.

As described above, the genomic DN of Babesia obata
A100pg was serially diluted 10-fold, and the detection limit was determined by nested PCR using the DNA at each concentration as a template. PCR and PCR
Electrophoresis of the product was performed as described above. FIG. 4 shows the results. As shown in FIG. 4, it was found that an amplified fragment could be detected when DNA of 100 fg or more was used as a template.

In the nested PCR, the decrease in the staining intensity of the amplified fragment due to the decrease in the template DNA was not so remarkable as in the ordinary PCR, but only slightly weakened, and a clear staining image was obtained up to the detection limit. It was easy to confirm the positive band. In addition, the fact that detection was possible if there was 100 fg or more of DNA indicates that nested PCR is 10 times more sensitive than normal PCR, and that the genome size of Babesia obata is the same as that of Theileria parva. 1 × 10 ⁷
Assuming bp, infected blood 10 with a parasitic rate of 0.00002%
It is considered that detection of Babesia obata from μl or detection of 1 to 10 Babesia obata is possible.
In nested PCR, it takes longer than usual PCR to repeat the PCR, but if the preparation of the DNA sample to be a template has been completed, all the operations can be completed in one day. In addition, the second PCR using the second primer set
Even if the number of reactions is reduced by 20 to 15 times, a result similar to that of 35 times is obtained, so that the operation can be completed in less than twice the time required for normal PCR.

[0055]

According to the present invention, there is provided a DNA comprising a nucleotide sequence identical or complementary to the nucleotide sequence of SEQ ID NO: 1. The DNA of the present invention is a base sequence portion specific to Babesia obata in genomic DNA of Babesia obata.
Therefore, the DNA of the present invention is a genomic DNA of Babesia obata.
Is useful as a probe capable of specifically detecting. Further, when a probe or a primer for PCR capable of specifically detecting the genomic DNA of Babesia obata is prepared, its base sequence can be determined based on the base sequence of the DNA of the present invention.

According to the present invention, there is provided a nucleic acid capable of hybridizing to a DNA having the same or complementary nucleotide sequence as the nucleotide sequence of SEQ ID NO: 1. The nucleic acid of the present invention
It can hybridize to a base sequence specific to Babesia obata in the genomic DNA of Babesia obata. Therefore, by using the nucleic acid of the present invention as a probe or primer for PCR, genomic DNA of Babesia obata
Can be specifically detected.

In general, in the case of protozoan infections, the rate of parasitism decreases when the infection is changed to chronic infection, and the protozoa disappear from peripheral blood. The Babesia genus protozoa are often co-infected with other squid microorganisms, and in many cases the parasitic rate is kept low by interference of the Tyrelia genus protozoa. Therefore, it is not easy to determine Babesia obata infection in the target animal. However, by using the nucleic acid of the present invention as a probe or a primer for PCR, it is possible to specifically detect Babesia obata genomic DNA, thereby accurately and quickly detecting Babesia obata infection in a target animal. can do.

In particular, by performing nested PCR using the nucleic acid of the present invention as a primer for PCR, Babesia obata infection of a target animal can be detected with higher accuracy. Furthermore, by performing PCR using the DNA of the present invention as a primer for PCR, Babesia obata in the early stage or chronic stage of infection with a low infestation rate, and Babesia in the tissues of the infected host or in the mediator (eg, mites). -Observers can be detected, and multi-sample samples can be easily analyzed.

[0059]

[Sequence List] SEQUENCE LISTING <110> National Institute of Animal Health <120> a base sequence specific to Babesia ovata and a method of detection for Babesia ovata by use of the sequence <130> Babesia ovata <160> 5 <170> PatentIn ver. 2.0 <210> 1 <211> 855 <212> DNA <213> Babesia ovata <400> 1 acctagatga aatggagaaa caaataaggg ggacgtttga ctttttaaaa acaggtgtga 60 atgcagtaaa cacatcgcag gtaaatgagt tggtggagaa gttgaaatgg aaagttgctg 120 ctataagggt tcagattgaa ggcatcggtg taatacttga gaatcgtatt aaggagttag 180 agaactggaa ggaggaggca gcaaaacttg tggatggtac cgccaaagtg gcgggtgaaa 240 tcgagagaaa ggatcccgga aaaattaatt acgatgagat tttgggtaaa gagaaggagt 300 tggaagatat tcttgctgcg ctagatgaca acgtaaatag gttcaagggt gatgtgaaag 360 ctgctgctac caagggtgag gagcttgtta aaggttttga caaagtgctt caaacggatt 420 tgaaggtatt ggagggttat attgaaggcg cgatcaagga gtatgttgag aagttgaaga 480 gtgggcactt tggaaagatt aaaaagggcg ttggaaacag agagcgccca ctagagggga 540 atgatgagag catagatgct tgttgggaaa agcttaagca ggagattacg ggccttgttg 600 gtg aggttat tggcaaagag ccggggccga agaaatatga tggccttaag ggaattaaac 660 aaaaggtgaa ggaatatgct cagtttttca ctcaggagag ttttgagagt acagttcaag 720 gctggataaa aacaattctg gagaagaatg agattgttat tgagcgtatt gggtactata 780 tcagctatga ccataacaag acgcattttg tgcacgagaa agttaagcaa caaggtaaag 840 acgctattga tgacg 855 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: B5OV1 <400> 2 caaataaggg ggacgtttga ct 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: B3OV1 <400> 3 cgtcatcaat agcgtcttta cc 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: B5OV2 <400> 4 gcagtaaaca catcgcaggt aa 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: B3OV2 <400> 5 aaggccatca tatttcttcg gc 22

[Brief description of the drawings]

FIG. 1 is a schematic diagram of PCR and nested PCR.

FIG. 2 is a photograph showing the results of electrophoresis of a PCR product.

FIG. 3 is a photograph showing the results of electrophoresis of a PCR product.

FIG. 4 is a photograph showing the results of electrophoresis of a PCR product.

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[Submission date] November 2, 1998 (1998.11.
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──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:90) (C12Q 1/68 C12R 1:90) (72) Inventor Hiroshi Terada Matsushiro, Tsukuba, Ibaraki Prefecture 5-chome, 2-637-2 (72) Inventor Shinichiro Kawazu 5-15-1, Matsushiro, Tsukuba-shi, Ibaraki Prefecture 503-301 (72) Inventor Naotoshi Tsuji 5--16, Matsushiro, Tsukuba-shi, Ibaraki 527-206 F-term ( Reference) 2G045 AA25 AA35 CB17 DA12 DA13 DA14 FB01 FB02 4B024 AA13 BA80 CA03 DA06 EA04 GA11 GA30 HA12 4B063 QA01 QQ03 QQ43 QR55 QR66 QS16 QS25 QS34 QX01

Claims (6)

[Claims] 1. A DNA comprising a nucleotide sequence identical or complementary to the nucleotide sequence of SEQ ID NO: 1. 2. A nucleic acid capable of hybridizing to the DNA according to claim 1. 3. The nucleic acid according to claim 2, which has 14 to 855 bases. 4. A nucleic acid comprising a base sequence shown in the following (a) to (d). (A) a base sequence identical or complementary to the base sequence of SEQ ID NOs: 2 to 5 (b) In the base sequence identical or complementary to the base sequence of SEQ ID NOs: 2 to 5, all thymines are substituted with uracil (C) a base sequence in which one or more bases are deleted, substituted or added in the base sequence (a), and which is capable of hybridizing to the DNA according to claim 1; A base sequence in which one or more bases are deleted, substituted or added in the sequence (b), and which can hybridize to the DNA according to claim 1. 5. The following steps: (a) preparing a DNA-containing sample from a target animal; and (b) using the nucleic acid according to any one of claims 2 to 4 as a probe, Babesia in the above sample
Confirming whether or not genomic DNA of Obata is present, and determining, based on the result, whether or not the target animal is infected with Babesia obata. How to determine Obata infection. 6. The following steps: (a) a step of preparing a sample containing DNA from a target animal; and (b) a primer comprising the nucleic acid according to claim 2 and a primer for the sample. And (c) confirming whether or not the genomic DNA of Babesia obata is present in the sample based on the presence or absence of the amplified fragment obtained in step (b). Judging whether or not the target animal is infected with Babesia obata. A method for judging Babesia obata infection.