JPH11137299A - New dna fragment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA fragment composed of a DNA fragment specific to patients with Kawasaki disease, having a specific base sequence and capable of simple and certain diagnosis, and pre-onset and early diagnoses of Kawasaki disease, therefore useful as, e.g. a primer (pair) for diagnosis of Kawasaki disease. SOLUTION: This new DNA fragment is expressed by a base sequence described in the formula, specific to patients with Kawasaki diseases and useful as e.g. a primer (primer pair) enabling simple and certain diagnosis, and pre- onset and early diagnoses of Kawasaki disease which have been impossible with the conventional diagnostic method based on symptomatic diagnosis. This DNA fragment is obtained by conducting gene amplification using 5 kinds of primers with modified PCR technique capable of amplifying genes whose base sequences are not known, using a nucleic acid fraction derived from the blood plasma collected from child patients with Kawasaki disease as a material, further, conducting gene amplification with 15 kinds of primer pairs using the 5 kinds of primers as materials, and analysing the amplified gene according to Southern blot technique using a gene derived from the blood plasma collected for the patients in the acute period as a probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel DNA fragment, a method for diagnosing Kawasaki disease using the DNA fragment, and a method for diagnosing Kawasaki disease comprising a primer having a nucleotide sequence complementary to the DNA fragment and a primer pair. For diagnostic kits.

[0002]

2. Description of the Related Art Kawasaki disease is one of the vasculitis syndromes particularly prevalent in infants under 4 years old, and was first reported in 1967 by Dr. Tosaku Kawasaki. Pathologies such as high fever persistence, cervical lymphadenopathy, leukocytosis with left nucleus migration, accelerated blood sedimentation, and CRP-positive strongly suggest that this disease is an infectious disease. No specific virus, bacterium, rich care, or other microorganism associated with the virus has been identified, and there is no increase in serum antibodies such as known viruses. Kawasaki disease began to appear in Japan in 1960, and has been increasing year by year since 1968. Since it is still occurring frequently, it is speculated that it is related to rapid economic growth in Japan. There are various theories such as detergent allergy theory, mercury theory, pesticide theory, and antibiotics theory, but none of them have been proven yet and the etiology is still unknown at all.

[0003] The main symptoms of Kawasaki disease consist of the following six. Unexplained fever for more than 5 days Changes in extremities (acute phase: hard edema of limbs, erythema of palm or toe tip, recovery phase: film-like exfoliation from fingertips) Irregular rash that does not form blisters and crusts Bilateral conjunctival congestion Lip / oral findings (blushing of the lips, strawberry tongue, diffuse redness of the oropharyngeal mucosa) Non-suppurative cervical lymphadenopathy in the acute phase

[0004] Because the etiology is unknown, the diagnosis of Kawasaki disease is currently made by the diagnosis of major symptoms. In general, "Kawasaki Disease Diagnosis Handbook,"
Revised 4th Edition (1984) ". That is, “5 out of the 6 main symptoms
This disease is associated with one or more symptoms. However, the above 6
Even if only four of the main symptoms are observed, if a coronary aneurysm (including so-called enlargement) is confirmed during the course by tomographic echocardiography or cardioangiography and other diseases are excluded , And this disease. This is a diagnosis based on the criterion. Therefore, the current diagnosis method of Kawasaki disease requires diagnosis of as many as six items, so there is a problem in the simplicity and certainty of the diagnosis. There is also a very serious problem that an early diagnosis is impossible, and thus a treatment method cannot be determined early.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have studied the onset of Kawasaki disease from the genetic field in order to solve the above problems in the diagnosis of Kawasaki disease, and as a result, have found a DNA fragment specific to Kawasaki disease patients. Was. By using this DNA fragment as an index, a simple and reliable diagnosis of Kawasaki disease, which was impossible with a conventional diagnostic method based on symptom diagnosis, and also a pre-onset and early diagnosis are possible. In addition, two different types of primers (primer pairs) having a base sequence complementary to the base sequence of this DNA fragment can be used in a kit for diagnosing Kawasaki disease.

[0006]

An object of the present invention is to provide a novel DNA fragment, a method for diagnosing Kawasaki disease using the DNA fragment, a primer having a nucleotide sequence complementary to the DNA fragment, and a primer pair. An object of the present invention is to provide a kit for diagnosing Kawasaki disease.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a DNA fragment represented by SEQ ID NO: 1 which is useful for diagnosing Kawasaki disease. Preferred embodiments of the present invention are described below. (1) A DNA fragment represented by the nucleotide sequence of SEQ ID NO: 1. (2) A DNA fragment in which one or more bases have been deleted, substituted or added in the base sequence described in SEQ ID NO: 1. (3) The DNA fragment according to (2), which has a homology of 60% or more with the nucleotide sequence described in SEQ ID NO: 1. (4) D according to any one of (1) to (3) above
A DNA fragment containing the base sequence of the NA fragment. (5) D according to any one of the above (1) to (4)
A method for diagnosing Kawasaki disease using NA fragments. (6) The method for early diagnosis of Kawasaki disease according to the above (5). (7) The pre-onset diagnosis method of Kawasaki disease according to the above (5). (8) D according to any one of (1) to (4) above
A primer having a nucleotide sequence complementary to a part of the NA fragment. (9) A kit for diagnosing Kawasaki disease, comprising two different primers according to (8).

[0008] Diagnosis of Kawasaki disease using the DNA fragment of the present invention can be performed, for example, by the following method. The nucleic acid fraction is extracted from the plasma of a patient suspected of having Kawasaki disease, and if necessary, the RNA component is converted into DNA by RT-PCR, and then the gene of the DNA fragment of the present invention is amplified by PCR. By analyzing the amplification product and examining the increased level of the transcript of the DNA fragment of the present invention, it is possible to diagnose whether the patient has Kawasaki disease. Further, two different types of primers (primer pairs) having an appropriate base sequence complementary to the base sequence of the DNA fragment of the present invention can be used in a kit for diagnosing Kawasaki disease. The gene of the DNA fragment of the present invention is amplified by the PCR method using the primer pair, and the amplification product is analyzed to examine the level of increase in the transcript of the DNA fragment of the present invention, thereby diagnosing whether the patient has Kawasaki disease. It is possible to

[0009]

EXAMPLES Examples of the present invention will be shown below, but the present invention is not limited by these examples. Example 1 Identification of DNA fragments specific to acute phase of Kawasaki disease The nucleotide sequence is unknown using the nucleic acid fraction derived from plasma of children with Kawasaki disease (the acute phase plasma of patient A and the acute phase plasma and convalescent phase plasma of patient B). The gene was amplified using a PCR modification method (a method described in JP-A-8-242897) capable of amplifying the gene (5 types of primers were used). Further, using these five types of primers as materials, gene amplification was performed using 15 types of primer pairs. The DNA fragment from the acute phase plasma and the DNA fragment from the convalescent phase of patient B were separated using polyacrylamide electrophoresis, and further copied onto a nylon membrane. The mixture of the gene amplification products derived from the acute phase plasma of patient A was labeled with ³² P, and this was used as a probe for analysis by Southern blotting. Autoradiogram detected only in acute plasma of patient B,
A band that was not detected in convalescent plasma was selected. Further, the same analysis was performed using a gene amplification product of a sample containing no nucleic acid material to eliminate the background. A band (DNA fragment) was cut out from the gel, gene amplification, treatment with a restriction enzyme, and the like were repeated, the target band was purified, and the nucleotide sequence of the purified DNA fragment was determined. D
The data was analyzed using NA analysis software (manufactured by SDC Software) and a database, and 11 types of DNA fragments specific to the acute stage of Kawasaki disease were found. Eight types of gene sequences were identified among the DNA fragments. The results are shown in SEQ ID NO: 1 to SEQ ID NO: 8. The remaining three sequences were similar to Alu, a human repeat sequence similar to SEQ ID NO: 2.

[0010] 2. Determination of DNA fragments showing a clear association with Kawasaki disease Plasma (8 samples) of plasma of children with Kawasaki disease, plasma (4 samples of non-Kawasaki disease children (with inflammatory symptoms, suspected streptococcal infection, etc.)) ), The nucleic acid fraction was extracted. Using the extracted nucleic acid fraction as a material, RT-PCR was performed according to a conventional method using a primer pair prepared based on the gene sequence of the DNA fragment identified in Example 1. The reverse transcription reaction was performed at 37
C. for 60 minutes. PCR was performed at 94 ° C for 1 minute and at 55 ° C
1 minute, 72-2 minutes are 1 cycle, and 30 cycles are 2
Set and one set of nested PCR. The amplification products were analyzed by electrophoresis using polyacrylamide gel and Southern blotting, and homology and the like were determined using DNA analysis software. As a result, the 11 types of D
From the NA fragment, a transcript was significantly increased in children with Kawasaki disease, and one kind of DNA fragment described in SEQ ID NO: 1 was found, which was hardly found in children with non-Kawasaki disease. In the plasma of the above non-Kawasaki disease children and Kawasaki disease children, the expression level of the transcript of the gene of the present invention was measured by the degree of blackening on an autoradiogram, and the expression level of β-globin was similarly measured as a control. The results are shown in the figure below.

[0011]

FIG.

[0012]

In the above Examples, the control β-globin was detected in both non-Kawasaki disease children and children with Kawasaki disease, whereas the transcript of the gene of the present invention was specifically detected in children with Kawasaki disease. Detected high. Therefore, the present DN
The use of the A fragment as an index strongly suggested the possibility of a simple and reliable diagnosis of Kawasaki disease, which was impossible with a conventional diagnostic method based on symptom diagnosis, and the possibility of pre-onset and early diagnosis.

[0013]

[Sequence list]

SEQ ID NO: 1 Sequence length: 120 Sequence type: Nucleic acid number of strands: Unknown Topology: Unknown Antisense: No Direct origin Library name: RNA or DNA sequence derived from plasma of Kawasaki disease onset patient TGGTCCATTT ACATGCGGTG GGATATCTGG GCTGCCAAGA CCAGGAAAGA GGCTTCTCCA 60 TATTATTCCA GGTTNTTGGT GTCTTTTACA GCTTCCTATA CCCTCTTTTT CCCCCCCCCG 120

SEQ ID NO: 2 Sequence length: 257 Sequence type: Nucleic acid number of strands: Unknown Topology: Unknown Antisense: No Direct origin Library name: RNA or DNA sequence derived from plasma of a patient with Kawasaki disease ATGTTGANAC CCTATTTTTA CTAAAAATAC ATTTTTTAGC TGGGCATGGT GGTGCATGCC 60 TGTAATNTNA GNTANTTGGG AGGCTGNGGT AGGTGATTNN NTTGAACCGA GGTGGTGGAG 120 GAGCGAGTTG CCCTGAGCCG AGATTGCACC ACTGCACTTT CAGCAGCATCAAGT GGCACAAGAACA GAAGCACAAGAAGA

SEQ ID NO: 3 Sequence length: 62 Sequence type: number of nucleic acid chains: unknown Topology: unknown Antisense: No Direct origin Library name: RNA or DNA sequence derived from plasma of a patient with Kawasaki disease onset TNNAGCTGAC TTGATCGTGG CGGATNGCTT TTTGATGTGC TACTGGATTT GGTTTGCCAG 60 TA 62

Sequence number: 4 Sequence length: 134 Sequence type: Number of nucleic acid chains: Unknown Topology: Unknown Antisense: No Direct origin Library name: RNA or DNA sequence derived from plasma of a patient with Kawasaki disease TCATGCACTG GAGTAAAATA CCTGCAGCTC TCCNAGGCTG AAGTTGCACG CTAATGGCCC 60 TACCAGGCTG GAGTCTCAGA AGCAACCTTC CCCTGACAAC TCCATTGAGC ATTGCCCTAA 120 TTGGGACTCA CTGG 134

SEQ ID NO: 5 Sequence length: 123 Sequence type: number of nucleic acid chains: unknown Topology: unknown Antisense: No Direct origin Library name: RNA or DNA sequence derived from plasma of a patient with Kawasaki disease onset TAGGAATTGC AGCCCTAGAT NGGGCAAGAA TGATGACAAG CCATGGTGAC CCAGTGGAAT 60 GAGGGAGGNN NNGGGTAAAT ACCTGACTTA TCTCCTTCNT CCCTGCCAGC CTTGCCCNAT 120 CAA 123

SEQ ID NO: 6 Sequence length: 127 Sequence type: number of nucleic acid chains: unknown Topology: unknown Antisense: No Direct origin Library name: RNA or DNA sequence derived from plasma of Kawasaki disease onset patient CCTCTCCCCN GAGGTGTTTT CTCACTGGGT TTCTAGAACA TTCTGCCATT GCTTTTCTCC 60 CATCTCCCAG GACTNCNNGG CTCTACCAAC TATTGCTTCA GCTCACCAAC TATTGCTTCA 120 GCTCCAT 127

SEQ ID NO: 7 Sequence length: 98 Sequence type: Number of nucleic acid chains: Unknown Topology: Unknown Antisense: No Direct origin Library name: RNA or DNA sequence derived from plasma of a patient with Kawasaki disease GANNNAGCGA GGCGGCAGAG CTCCATGCTC CACCTCCNGG TTCTCAGTGC CTTCTTTCCT 60 NTGCAAAGTC GATATCCGAC TGNGAGACTT CAGCGTTT 98

Sequence number: 8 Sequence length: 98 Sequence type: Number of nucleic acid chains: Unknown Topology: Unknown Antisense: No Direct origin Library name: RNA or DNA sequence derived from plasma of a patient with Kawasaki disease CAGTAATGCT CTGCAGGGGG CAGAGCTAAC AGAGTNNTGC TCTGGCAGTG GGTCCCNGGN 60 CTTNNNGGCT CTNGTCCCNG GCTTNNNGGC TCTCCNNG 98

[Brief description of the drawings]

FIG. 1. Invention D in non-Kawasaki disease children and children with Kawasaki disease
It is an example of the result of measuring the expression level of the transcript of the NA fragment based on the degree of blackening on the autoradiogram and the result of similarly measuring the expression level of β-globin as a control.

Claims (5)

[Reference number] PC-271 [Claims] 1. D represented by the nucleotide sequence of SEQ ID NO: 1.
NA fragment. 2. A DNA comprising the nucleotide sequence of SEQ ID NO: 1.
fragment. 3. A method for diagnosing Kawasaki disease using the DNA fragment according to claim 1 or 2. 4. A primer comprising a base sequence complementary to a part of the base sequence of the DNA fragment according to claim 1 or 2. A diagnostic kit for Kawasaki disease, comprising the two different primers according to claim 4.