JP2012179066A - Oligonucleotide and primer for detecting poisonous mushroom, and diagnostic kit, detecting kit and method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a quick and specific method for detecting poisonous mushrooms (Entoloma rhodopolius and Clitocybe acromelalga) not based on the forms of fruit bodies.SOLUTION: The quick and specific method for detecting the poisonous mushrooms not based on the forms of the fruit bodies is provided by using gene-related technologies such as DNAs containing a part of ribosome RNA genes of the poisonous mushrooms (Entoloma rhodopolius and Clitocybe acromelalga), a DNA primer and probe having specific sequences for the Entoloma rhodopolius and Clitocybe acromelalga, a PCR method by using them, a DNA microarray, etc.

Description

  The present invention relates to oligonucleotides and primers for detection of poisonous mushrooms, and diagnostic kits, detection kits and methods using the same.

Although it is said that there are 4000-5000 species of mushrooms in Japan, the exact number is unknown, and most of them are unknown to poison. Of the mushrooms that exist in Japan, only about 100 types are known to be edible and about 40 types are known to be poisonous.

Poisoning by poisonous mushrooms accounts for 70% of natural poisoning and 60% of deaths. According to the statistics of the Ministry of Health, Labor and Welfare, about 250 poisoning by poisonous mushrooms occurred in the five years from 2002 to 2006, and the total number of patients exceeded 800, and several people died. As for the cause of food poisoning, it is known that there are many poisoning cases of Omphalotus japonicus, Entoloma rhodopolius, and Clitocybe acromelalga. The cause of such poisoning by poisonous mushrooms is that considerable skill is required to accurately identify the mushrooms by their form, and the general person mistakenly eats poisonous mushrooms and eats them.

The identification of these species of mushrooms is based on the form of the fruiting bodies, but accurate identification requires considerable skill and is a highly skilled professional belonging to a research institution such as a university or a museum. I have to rely on. Therefore, it is extremely difficult for a general staff of a medical institution or an administrative organization such as a forensic research institute or a health center to identify a mushroom.

When a patient suspected of poisoning a poisonous mushroom occurs, it is necessary to promptly and appropriately treat it. To this end, it is important to accurately identify the causative mushroom. However, in order to accurately identify, it is necessary to retain some form of fruiting body, so that fruiting body fragments and finely cuts like the cause of food poisoning when an addicted patient or death occurs Even those that do not retain the original mushroom characteristics such as form and color, such as cooked mushrooms, cannot be easily identified by experts.

Regarding the identification of mushroom varieties based on the PCR method, a technique for amplifying and detecting a microsatellite region by the PCR method has been reported (Patent Document 1). In addition, it has been reported that detection of pathogenic bacteria can be detected by amplification of ribosomal RNA via PCR (Patent Document 2). In addition, according to Non-Patent Documents 1 and 2, a method for examining these varieties by sequencing the base sequence of the ITS region of ribosomal RNA of false black hearts and hallucinogenic mushrooms has been reported.

JP-A-11-318465 JP 2005-261228 A Yoshito Shimono, Susumu Takamatsu, Takeshi Ozaki: Abstracts of Annual Meeting of the Mycological Society of Japan, No. 44, 43 (2000) Takuro Maruyama et al .: Food Hygiene Magazine No. 44, paragraphs 44-48 (2003)

In order to accurately identify poisonous mushrooms by visual inspection, the shape of the fruit body needs to be retained to some extent, such as broken fruit bodies, mushrooms cut into pieces, cooked mushrooms, etc. There is a problem that even experts who cannot retain the original characteristics of mushrooms such as color cannot be identified.

In addition, a technique for identifying mushrooms by amplifying a gene encoding ribosomal RNA with a universal primer using a PCR method and determining the base sequence by direct sequencing is used (Non-patent Documents 1 and 2). Since it takes a lot of time and money to determine the base sequence, development of a further improvement method has been desired.

Furthermore, since some strains are heteronuclear coexistents, there may be mutations between different nuclei. In addition, in the case of wild mushrooms, they may adhere to or mix with other filamentous fungi or yeast fruit bodies. In addition, foreign substances are more prominently mixed in mushrooms after cooking or edible, and in these cases, it is very difficult to determine the base sequence by direct sequencing. In that case, an operation such as subcloning the PCR product into a recombinant plasmid using Escherichia coli as a host is required, and more time and cost are required.

In particular, for the moss agaricus, the mycelium cannot be separated and cultured, and since there is no means to acquire genetic resources only by collecting wild mushrooms, gene analysis examples themselves have not been performed.

Furthermore, so far, specific detection methods for microorganisms such as bacteria and fungi using the PCR method are known, but no detection method for poisonous mushrooms such as moss agaricus has been reported. It could not be detected by the specific detection method.

As a result of diligent research to solve the above-mentioned problems, the present inventors have isolated DNA containing a gene encoding 5.8S ribosomal RNA of Kusaura Benitatake and Doxasaco, and further quickly and highly sensitively uses Kusaura Benitatake or Dokusasaco. The present inventors have succeeded in producing a detectable primer and completed the present invention.

According to the present invention, there is provided an oligonucleotide that specifically hybridizes with at least a part of the polynucleotide in the spacer region of the ribosomal RNA gene of Kusaura Benitatake or Dokusaco. When this oligonucleotide is used, it is possible to detect and identify Kusaura Agaricus or doxasaco through hybridization such as Southern blot, microarray, PCR method, etc., and it is possible to detect these mushrooms regardless of the form of fruit bodies.

In addition, according to the present invention, there is provided an oligonucleotide that specifically hybridizes with at least a part of DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2. When this nucleotide is used, it becomes possible to more easily detect and identify Kusaura Agaricus or doxasaco through hybridization such as Southern blotting, microarray, and PCR method.

Moreover, according to the present invention, a DNA comprising the base sequence represented by SEQ ID NO: 1 or 2 is provided. By referring to this DNA, nucleotides for the detection of Kusaura Benitatake and Doxasaco can be designed more simply and quickly. Further, by using this DNA as a positive control, it is possible to more reliably determine the presence or absence of these poisons.

In addition, according to the present invention, a 5 ′ primer that hybridizes with at least a part of the polynucleotide of the spacer region of Kusaura Agaricus or Doxasaco, and a 3 ′ that hybridizes with at least a part of the polynucleotide of the spacer region of Kusaura Benitake or Doxasaco. A primer set consisting of a combination with a side primer is provided. This primer makes it possible to quickly and easily detect and identify Kusaura Amanita or Doxasaco using PCR, and to detect these mushrooms regardless of the form of fruiting bodies.

Further, according to the present invention, a 5 ′ primer that hybridizes with at least a part of the DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2, and a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 or 2 A primer set consisting of a combination with a 3 ′ primer that hybridizes with at least a part is provided. This primer makes it possible to more easily detect and identify Kusaura Benitatake or Doxasaco using the PCR method.

Further, according to the present invention, a DNA comprising an oligonucleotide that specifically hybridizes with at least a part of a spacer region of a ribosomal RNA gene of Kusaura Benitatake or Doxasaco, or a polynucleotide having a base sequence represented by SEQ ID NO: 1 or 2. A microarray is provided. This microarray enables comprehensive simultaneous analysis of many types of mushrooms and foods.

In addition, according to the present invention, there is provided a detection kit for Kusaura Benitatake or Doxasaco comprising the above primer set. This detection kit makes it possible to quickly and easily detect and identify mulberry fly sardines or doxasaco using the PCR method, and to detect these poisons regardless of the form of fruiting bodies.

In addition, according to the present invention, there is provided a diagnostic kit for Kusaura Agaric poisoning or Dokusasaco poisoning comprising the above primer set. This diagnostic kit targets the contents of digestive organs, vomiting, urine, etc. of patients suspected of being addicted, and enables rapid and simple detection / identification of Kusaura Benitatake or Dokusasaco by using the PCR method. Allows diagnosis of addiction.

Moreover, according to the present invention, there is provided a method for detecting Kusaura Amanita or Dokusasaco, which comprises amplifying at least a part of the Ribosomal RNA gene of Kusaura Benitatake or Dokusasaco using the above primer set. This detection method makes it possible to easily and specifically detect these poisons by selecting regions having different sequences among mushroom species as targets for the PCR method.

According to the present invention, there is also provided a detection kit and a detection method for a poisonous mushroom present in a cooked food, which hybridizes with at least a part of the polynucleotide in the spacer region of the ribosomal RNA gene 5 There are provided a detection kit and a detection method comprising a primer set comprising a combination of a 'side primer and a 3' primer that hybridizes with at least a part of the polynucleotide of the spacer region of the venom ribosomal RNA gene. Since this detection kit and detection method is cooked, it is possible to detect poisonous mushrooms in foods whose fruit body form is not clear by using the PCR method targeting the spacer region of the ribosomal RNA gene. enable.

In addition, according to the present invention, there is provided a diagnostic kit for poisonous mushroom poisoning, a 5 ′ primer that hybridizes with at least a part of the polynucleotide in the spacer region of the poisonous ribosomal RNA gene, and the poisonous ribosome. There is provided a diagnostic kit comprising a primer set consisting of a combination of a 3 ′ primer that hybridizes with at least a part of the polynucleotide of the spacer region of the RNA gene. This diagnostic kit makes it possible to detect poisonous mushrooms whose form of fruiting body has become unclear after consumption by using the PCR method targeting the spacer region of the ribosomal RNA gene. It has an advantageous effect on the diagnosis of poisoning.

According to the present invention, there are provided nucleotides, primer sets, detection methods and kits for specific and rapid detection and identification of poisonous mushrooms (Chrysalis communis, Doxasaco, etc.).

[Explanation of terms]
The term “poisonous mushroom” as used herein includes a component that has a harmful effect on mammals, particularly humans, and cannot be edible due to that component (provides a significant harmful effect when consumed). I mean. Further, in the present specification, an edible mushroom under a specific condition may be regarded as a poison other than an edible condition. For example, mushrooms that cannot be eaten raw (including harmful components that are lost by heating) may be considered poisonous under raw eating conditions.

In addition, the term “Casaura Benitatake” in this specification refers to the Astragalus genus Ipponshimida (scientific name: Entololoma rhodopolius). Because the shape of the fruit body is similar to the edible mushrooms Enoloma sarcopum, Honyo shimeji, and Hatake shimeji (Lyophyllum decastes), they are confused and prone to addiction. Hazardous components are hemolytic proteins, muscarin, etc., which cause digestive and nervous system symptoms, particularly abdominal pain, vomiting, diarrhea, etc., and if the symptoms are severe, death may occur. Furthermore, as of the filing date, it is not possible to separate and culture the mycelium of Kusaura Amanita.

In addition, “Dokusasako” in the present specification refers to Dokusasako (scientific name: Clitocybe acromelalga) belonging to the genus Kaytake of the genus Amanita. It is confused with other edible mushrooms and other edible mushrooms. Particularly edible mushrooms, Clitocybe gibba, Armillaria mellea, Lactarius akahatu, Lactarius hatutake, Lactarius chrysorrheus, easy to mix with Clitocybe clavipe. Known harmful components include clitidine and achromeric acid. Eating doxasako causes mild nausea, and after a few days, the end of the body, such as the limbs, nose, and penis, swells as if burned red, causing severe pain in that part and lasting for more than a month.

The above two types of mushrooms are poisonous mushrooms that cause a lot of poisoning in the country after Tsukiyotake, especially because of the ability to identify the mushrooms, and the diagnosis and status after the poisoning. Development of technology for grasping is strongly desired.

Further, the “ribosomal RNA gene” in the present specification refers to the entire gene including a region encoding each subunit of ribosomal RNA (rRNA) and a non-coding region therebetween. As shown in FIG. 1a, the ribosomal RNA gene includes ribosomal RNAs having different sedimentation coefficients (S), that is, portions encoding 18S (small subunit), 5.8S, 28S (large subunit), and 5S ribosomal RNA. Exists. Among these ribosomal RNA genes, in particular, the portion consisting of 18S, 5.8S, ITS1 and 2 is transcribed as a single RNA, which is later spliced into each portion. It is also referred to as “ribosomal RNA precursor (region)” and substantially corresponds to the region of the base sequence represented by SEQ ID NO: 1 or 2.

In addition, the “spacer region” in the present specification refers to a region that exists between the coding region and the coding region and does not encode amino acids (non-coding region). In the present specification, on the ribosomal RNA gene. The spacer region in The spacer region can be suitably used in the present invention because the difference in base sequence between species is larger than that of the coding region. As shown in FIGS. 1a and b, on the chromosome, one ribosomal RNA gene has a structure connected in tandem, and the spacer region includes ITS1 and 2 and 5S sandwiching the coding region of the 5.8S subunit. There are known IGS1 and IGS2 that sandwich the code region of a subunit. ITS1 and ITS2 are referred to as ITS (Internal Transcribed Spacer) regions because they are spliced to RNA after transcription. In the present invention, an ITS region that is relatively conserved within the species is preferably used, but an IGS region can also be used.

In addition, both the ITS region and the IGS region may have different sequences in the repetition of the tandem structure, but the variation width is smaller than the difference between species (the homology may be 50% or less) It does not affect the results of the present invention. However, when designing primers or nucleotides, it is not the base sequence itself shown in Sequence Listing 1 or 2, but 90%, more preferably 95% identity with this, or substitution / deletion of several bases -The inserted base sequence may be used. The identity at this time can be calculated using standard initial parameters in blast2seq provided by NCBI (National Center for Biotechnology Information).

In addition, the “nucleotide” in the present specification refers to a nucleotide or nucleoside consisting of a naturally occurring base, sugar and intersugar linkage, and in particular, an oligomer (for example, about 2 to 100 bp) and a polymer (for example, 100 bp). -) Are referred to as "oligonucleotide" and "polynucleotide", respectively. The oligonucleotide according to the present embodiment includes a non-naturally occurring monomer that functions in the same manner, an oligomer or a polymer including a fluorescent molecule or the like and a monomer labeled with a radioisotope.

In the present specification, the “primer” refers to an oligonucleotide that is required to hybridize with a template and start the PCR reaction in the PCR reaction. Based on the template desired to be amplified in the PCR reaction, it is more preferable to hybridize with the template and to generate a PCR product, preferably a specific PCR product (in chain length or sequence). Is designed so that the primer itself contains its template-specific sequence. Such a primer can be used by those skilled in the art by using well-known common knowledge and programs such as BLAST and databases such as the ratio of primer GC (guanine / cytosine) and melting temperature. It can be easily designed. In this case, the 3 ′ region of the primer is preferably complementary to the template strand, but a restriction enzyme recognition sequence, a tag, or the like can be added to the 5 ′ side. Moreover, although a general primer is not restricted to this, Usually, it is designed so that it may have a chain length of 10 bp-100 bp, preferably 15 bp-35 bp. In particular, primers suitable for the present invention will be described in detail in the description of the embodiments.

In the present specification, “specifically hybridizes” means that another polynucleotide (or oligonucleotide) binds complementarily to a certain polynucleotide through hydrogen bonding or the like and serves as a comparative control. It means a state in which it does not bind to a polynucleotide under the same conditions. It does not necessarily have to be specific for all other polynucleotides, and may have specificity according to the purpose of use. For example, in the detection of a poisonous mushroom, it may be said that “specifically hybridizes” if it does not bind to an edible mushroom polynucleotide.

Hybridization conditions can be selected according to the intended use of the nucleotide. For example, if it is a nucleotide as a primer used for PCR, it is selected so that it may hybridize on the conditions at the time of annealing in PCR. In addition, when used in Southern blots, microarrays, etc., it is selected so as to hybridize under the hybridization conditions. Preferably, those that hybridize under stringent hybridization conditions are preferred, but not limited thereto as long as they satisfy the purpose. (For details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology (Wiley Interscience Publishers, 1995).) “Stringent conditions” defined here are: Conditions that include denaturing agents such as formamide during hybridization, eg, 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM at 42 ° C. Examples include, but are not limited to, those using sodium phosphate buffer at pH 6.5, 750 mM sodium chloride, and 75 mM sodium citrate, and those appropriately modified.

In this specification, “cooking” refers to a commonly used method for processing food, such as processing by various types of heating, shape processing (cutting), or mixing with other seasonings or foods. That means. The processing by heating is not limited to this, but for example, boiled (mainly about 100 ° C.), fried (mainly about 180 ° C.), or baked (mainly 200 ° C. or more) is often used.

Embodiment
Hereinafter, embodiments of the present invention will be described.

Certain embodiments are oligonucleotides that specifically hybridize with at least a portion of the polynucleotide of the spacer region of the ribosomal RNA gene of Kusaura Amanita or Doxasaco. By using this oligonucleotide, it becomes possible to detect and identify Kusaura Agaricus or doxasaco by gene-related technology through hybridization such as Southern blot, microarray, PCR method, etc., and detection of these poisons regardless of the form of fruiting bodies. It becomes possible.

Yet another embodiment is an oligonucleotide that specifically hybridizes with at least a portion of DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2. When this oligonucleotide is used, it is possible to detect and identify Kusaura Agaricus or doxasaco through hybridization such as Southern blot, microarray, PCR method, etc., and it is possible to detect these mushrooms regardless of the form of fruit bodies. By designing oligonucleotides that hybridize based on this base sequence, the above nucleotides can be obtained simply and reliably and used to detect these poisons without newly sequencing the moss agaricus or doxasaco. it can.

That is, by using the oligonucleotide of the above embodiment, by using the method of the present invention, compared to the conventional identification method using the form of the fruiting body, which could only be performed by experts with advanced technical skills, Anyone can identify poisonous mushrooms easily, quickly and reliably. In addition, these methods using the nucleotides enable detection with higher objectivity and reproducibility than the conventional method of identifying in the form of fruit bodies. In addition, this nucleotide is synthesized using a technique such as PCR or nucleic acid chemical synthesis, and is provided after confirming that it does not hybridize with a comparative control (including confirmation on the sequence).

The oligonucleotide of the above embodiment is selected in length and sequence depending on the purpose of use, but when used as a probe or primer, the hybridizing portion is 10-100 bases, preferably 15-50 bases, more preferably 15-30 bases. In addition, it is desirable that this nucleotide has 80%, more preferably 90%, and still more preferably 95% identity with the sequence to be hybridized. This is the case for oligonucleotides).

Yet another embodiment is a DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2. By referring to this DNA, nucleotides for the detection of Kusaura Benitatake and Dokusaco can be designed more easily and rapidly. Further, by using this DNA as a positive control, it is possible to more reliably determine the presence or absence of these poisons.

In general, mushrooms are heteronuclear in some strains, and there may be mutations between different nuclei. In addition, there are many cases where it adheres to or mixes with other filamentous fungi (such as molds) or the fruit bodies of yeast, and sequencing in a situation without clues may be difficult. In particular, it is very difficult to perform analysis using a gene-related technique for moss agaricus, since it is impossible to isolate and culture. The present inventor succeeded in sequencing the vicinity of the ribosomal RNA precursor region (SEQ ID NO: 1) of Kusaura Agaricus for the first time by collecting and analyzing a large amount of fresh Kusaura Amanita after extensive efforts. This makes it possible to perform detection quickly and easily without going through a very difficult sequencing work, especially in the mulberry fly.

The DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 in the above embodiment includes DNA containing a gene encoding 5.8S ribosomal RNA of Kusaura Benitatake or Doxasaco and its adjacent region (ITS region). For example, it can be obtained as follows.

(Isolation of 5.8S ribosomal RNA gene and its adjacent region)
(1) Preparation of genomic DNA Examples of the supply source of genomic DNA include fruit bodies of Kusaura Amanita and Doxasaco collected from nature, or mycelium of Doxasaco that has been separated and stored.

For example, fruit bodies collected from nature are thoroughly washed with water, water is removed with a paper towel or the like, cut into several millimeters, and pulverized in liquid nitrogen. Subsequently, genomic DNA can be extracted from the crushed fruit bodies by a method using a surfactant, a common method known in the art such as protease treatment, a commercially available genomic DNA extraction kit, or the like.

(2) Amplification of 5.8S ribosomal RNA gene by PCR and its adjacent region In the genome of fungi such as filamentous fungi and yeast generally containing mushrooms, the region encoding 5.8S ribosomal RNA and its adjacent region are shown in FIG. It is known that it is such a structure. White et al. Universal Oligonucleotide Primers ITS1 (SEQ ID NO: 7) and ITS4 (SEQ ID NO: 8) capable of amplifying the 5.8S ribosomal RNA gene of the fungus and its flanking region [White et al .: PCR protocols, Academic Press, San Diego, pp. 315 (1990)], the target DNA can be obtained by performing a PCR reaction using the genomic DNA obtained in (1) above as a template.

(3) Determination of base sequence After subcloning the obtained PCR fragment into an appropriate vector such as pT7-blue T-vector (Novagen), the base sequence is determined. Alternatively, direct sequencing is performed using PCR fragments. The base sequence can be determined by a known method such as a chemical modification method of Maxa Mugilbert or a dideomisinucleotide chain termination method using M13 phage. Usually, an automatic base sequencer (for example, 3130 manufactured by Applied Biosystems) is used. Sequencing is performed using a genetic analyzer or the like.

As described above, the target DNA can be obtained. By changing the primers ITS1 and ITS4 used at this time to other appropriate primers, the DNA of the spacer region of the ribosomal RNA gene and its sequence are obtained. In addition, it is also possible to obtain the DNA of the corresponding region of other mushrooms and the sequence thereof.

Further, regarding the DNA consisting of the base sequence shown in Sequence Listing 1 or 2, since the base sequence is disclosed in this specification, other simpler acquisition methods are possible. For example, these DNAs may be obtained by chemical synthesis, obtained by PCR using cDNA or genomic DNA of this DNA as a template, or obtained by hybridization using a DNA fragment having this base sequence as a probe. May be.

Still another embodiment includes a 5 ′ primer that hybridizes with at least a part of the spacer region polynucleotide of the ribosomal RNA gene of Kusaura Benitatake or Doxasaco, and the polynucleotide of the spacer region of the ribosomal RNA gene of Kusaura Benitatake or Doxasaco. It is a primer set which consists of a combination with the 3 'side primer which hybridizes with at least one part. This primer set enables specific detection / identification of moss agaricus or doxasaco using the PCR method targeting the spacer region of a ribosomal RNA gene that is specific depending on the species.

In still another embodiment, a 5 ′ primer that hybridizes with at least a part of DNA consisting of the base sequence shown in SEQ ID NO: 1 or 2, and a DNA consisting of the base sequence shown in SEQ ID NO: 1 or 2 It is a primer set which consists of a combination with the 3 'side primer which hybridizes with at least one part. By designing a primer based on the base sequence shown in SEQ ID NO: 1 or 2, the number of experiments and conditions required such as new sequencing is reduced, and a detection system using the PCR method can be made more easily. It becomes possible to construct.

In the detection / identification method using the PCR method of the above embodiment, unlike the conventional PCR method and the method using the direct sequence, it does not take time and effort for the sequence and cannot be identified even by an expert. Also, identification can be made from cooked kusaura sagittarius and dokusasako fruit body pieces that do not retain the original form of the fruit body. In addition, detection and identification of mushrooms using an edible mushroom after cooking as a sample is also included in the embodiment of the detection and identification method of the present invention.

The creation of the primer (set) according to the above embodiment will be exemplified and described below.
(Synthesis of primer for detection of Kusaura Benitatake and Dokusasaco)
SEQ ID NO: 1 exemplifies the base of the moss agaricus, and SEQ ID NO: 2 exemplifies the base sequence used for the detection of Doxusasako. By comparing these base sequences, a sequence characteristic to Kusaura Benitatake or Dokusaco can be found, and a synthetic oligonucleotide having the sequence can be used as a primer (set) according to this embodiment.

For example, the 5′-side primer and 3′-side primer that can be used for the detection of Kusaura Benitatake or Dokusaco can be selected from any region of the base sequence represented by SEQ ID NO: 1 or 2. However, a region in which a difference is recognized between strains of different species, a region having a high GC content is not included, and a region that does not hybridize with primers is preferable.

For example, as a 5′-side primer, in SEQ ID NO: 1, there is a difference between mushrooms [Entoloma sarcopum, Lyophyllum shimeji, Lyophyllum decastes] that are morphologically similar to those of Kusaura Benitatake. The region to be obtained is selected to be 10-50 bases, more preferably 15-25 bases, and used as a primer. These primers can be used in any arbitrary combination.

In addition, in SEQ ID NO: 2, mushrooms [Clitocybe gibba], Armillaria mellea, Lactarius akahatu, Lactarius hatutake, Lactarius chrysorrheus, Lactarius chrysorrheus, Clitocybe clavipe)] can be selected as a region by selecting 10-50 bases, more preferably 15-25 bases. At least these primers can be used in any arbitrary combination.

Next, the detection method by PCR according to the present invention can be performed, for example, as follows.
(Detection of Kusaura Benitatake and Dokusasaco using the primer of the present invention)
(1) Preparation of analyte sample solution Preparation of an analyte sample solution suspected of containing poisonous mushrooms is a method of using a surfactant from raw fruit bodies, boiled, fried, or baked fruit body pieces. DNA may be extracted by the usual method of (1), and this is purified using phenol, chloroform, ethanol or the like to obtain a sample solution for the specimen. A commercially available DNA extraction kit can also be used.

(2) Detection of Pseudomonas aeruginosa and Doxasaco by PCR The detection of the poisonous venom by using the primer specific for Pleurotus edulis and Doxasaco according to the present invention, coding region of ribosomal RNA, spacer region, 5.8S ribosomal RNA gene or its adjacent region. After amplification, the amplified fragment is fractionated by agarose gel electrophoresis, polyacrimido gel electrophoresis, etc., and the gel is stained with ethidium bromide, etc., and the presence or absence of the amplified fragment is confirmed under a UV lamp. be able to.

Amplification can be performed by PCR according to standard protocols [see, eg, Sambrook, J et al .: Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)]. That is, the DNA sample is denatured into a single strand. Next, a 5 ′ primer complementary to the 5 ′ side of one strand of the target DNA sequence, a 3 ′ primer complementary to the 3 ′ side of the other strand, DNA used as a template, and four deoxynucleotides Using a reaction system containing triphosphate (dATP, dCTP, dGTP, dTTP or dUTP) and a DNA polymerase, the template polynuclease and the above two primers are annealed. Next, a complementary strand is synthesized on each template from four types of deoxynucleotide triphosphates by DNA polymerase. Then, after the resulting double strand is made into a single strand by heat denaturation, the same annealing as described above is repeated using each strand as a template, and the complementary strand synthesis procedure is repeated to exponentially extract only the portion sandwiched between the primers. Can be increased.

Thus, the DNA sequence of the portion narrowed by the primer is subjected to the above-described series of operations for N cycles (usually N = 20-40), whereby a DNA fragment amplified theoretically 2N times is obtained. Since DNA polymerase can synthesize hundreds to thousands of bases per minute, one cycle can be performed in 5 to 7 minutes. Therefore, for example, in the case of amplification of 25 cycles, the process can be completed in about 3 hours. In this case, assuming that the efficiency of each cycle is 90%, (1 + 0.9) 25, that is, about 1 million times can be amplified. it can. Furthermore, recent progress in PCR technology is remarkable, and the time for one cycle can be shortened to within one minute. From these facts, when PCR is used, detection can be performed very quickly and with extremely high sensitivity compared to other methods.

As the DNA polymerase used in the PCR method, it is desirable to use a polymerase that is heat resistant at the DNA cleavage temperature, such as Taq or Tth polymerase. By using these, it is not necessary to replenish the enzyme every time heat denaturation of each cycle, so that it can be carried out more easily and quickly.

Furthermore, in the above embodiment, by using real-time PCR, detection can be performed easily and very quickly in one step or with a short number of steps. As the real-time PCR, for example, various fluorescent PCR-based techniques can be used. Examples of fluorescent PCR-based techniques include intercalator methods using various fluorescent nucleic acid labeling agents such as SYBR GREEN I (for example, LightCycler (registered trademark: Roche), ABI Prizm 7700 Sequence Detection System (registered trademark)). (Perkin Elmer, Applied Biosystems)), TaqMan probe method for monitoring amplification in real time using the 5 ′ exonuclease activity of Taq DNA polymerase enzyme, but is not limited thereto.

In one embodiment, the DNA fragment to be amplified is a DNA fragment containing a part of the adjacent region of the 5.8S ribosomal RNA gene possessed by Kusaura Benitatake or Doxasaco. In the PCR method or the like, the DNA sequence of the portion sandwiched between the primers is amplified, so the size of the DNA fragment depends on the type of primer used. Finally, the amplified DNA fragment is stained using an agarose gel or acrylamide gel, and the presence or absence of the amplified fragment can be confirmed under a UV lamp.

In the above description of the embodiment, DNA has been taken up. However, since the ITS region is also present in mRNA before splicing, it is possible to perform PCR on RNA, and the embodiment of the present invention. include. In this case, RNA extraction and conversion to DNA by reverse transcriptase are performed before the above PCR reaction, but these reactions can be performed within the scope of ordinary technical knowledge in this technical field. .

Moreover, further embodiment is a primer set in which said 5 'side primer contains the base sequence shown by sequence number 3 or 5. By using this primer set, it is possible to more reliably discriminate and detect other sasabeniko or dokusasaco.

Moreover, further embodiment is a primer set in which said 3 'side primer contains the base sequence shown by sequence number 4 or 6. By using this primer set, it is possible to more reliably discriminate and detect other sasabeniko or dokusasaco.

For example, when the above primer is used, 109 bp (when using a primer set containing SEQ ID NOs: 3 and 4 for Kusaura Benitake) or 108 bp (a primer set containing SEQ ID NOs: 5 and 6 for Doxasaco) DNA fragment) is amplified.

In still another embodiment, an oligonucleotide that specifically hybridizes with at least a part of the spacer region of the ribosomal RNA gene of Kusaura Benitatake or Doxasaco, or the polynucleotide of the base sequence represented by SEQ ID NO: 1 or 2. A DNA microarray provided. This microarray enables comprehensive simultaneous analysis of many types of mushrooms and foods, and is very effective in investigating the cause of food poisoning. The microarray according to the present embodiment can be provided by a method including a step of fixing a probe oligonucleotide to a chip.

Oligonucleotides for microarray probes are preferably designed to be oligonucleotides with a chain length of at least 15 bp. The microarray includes one or more arbitrary probes per spot, and one or two or more oligonucleotides according to the present invention are included in any spot. This oligonucleotide may be fixed to the microarray chip by physical bonding or chemical bonding.

The material of the microarray chip may be all known types of substances, preferably those used in ordinary microarrays, reactive groups capable of immobilizing oligonucleotides on the chip surface, nitrocellulose or 3 Subsequent structure formation may be further included. For example, silicon wafers, glass, polycarbonate, membranes, polymer films such as polystyrene or polyurethane, and porous materials are used. Oligonucleotides can be immobilized by a method used in a normal microarray production method, for example, a photolithography method, a piezoelectric printing method, a micro pipetting method, or a spotting method.

The above oligonucleotide can be immobilized at 2 pg-100 pg per spot on the microarray. Further, the number of spots on the microarray may be 1 or more, preferably 2 to 3 times. At this time, the spots may be, for example, 50 μm to 500 μm in diameter and 10 μm to 500 μm between the spots, but it is preferable to appropriately adjust the size and density of the spots according to the resolution of the microarray analysis system. The microarray according to the present embodiment is installed in a well of a microwell plate, for example, a 96-well plate, and sample molecules, labels, and hybridization are performed using automatic equipment (Biomek, Genetix robo, etc.) using an existing 96-well plate It is also possible to process the cleaning process mechanically.

Still another embodiment is a detection kit and a detection method for Kusaura Agaricus or Doxasaco, which include the above primer set. This detection method is characterized by amplifying at least a part of the ribosomal RNA gene of Kusaura Benitatake or Dokusaco. The detection kit and the detection method enable rapid and simple detection and identification of Kusaura Benitatake or Dokusasaco using the PCR method, and detection regardless of the form of the fruiting body is possible. It is also possible to combine a plurality of poisonous mushroom detection kits into a single detection kit. In this case, as in the case of using a microarray, multiple types of mushrooms can be detected and identified at the same time. It is very effective in investigating the cause of

A further embodiment is a detection method according to the above embodiment, wherein at least part of the ribosomal RNA gene amplified by the PCR method is at least part of 5.8S ribosomal RNA. Targeting the 5.8S ribosomal RNA of Kusaura Benitatake or Doxasaco enables more certain differentiation of other similar mushrooms.

Yet another embodiment is a diagnostic kit for Kusaura Agaric poisoning or Dokusako poisoning comprising the above primer set. This diagnostic kit makes it possible to quickly and easily detect and identify Kusaura Agaric or Doxusasako by using the PCR method. In addition, this diagnostic kit makes it possible to detect even Kusaura Benitatake or Dokusako, whose form of fruiting body has become unclear after consumption, by using the PCR method targeting the spacer region of the ribosomal RNA gene. Effective in diagnosing poisoning by these mushrooms.

Yet another embodiment is a diagnostic kit for poisonous mushroom poisoning, comprising a 5 ′ primer that hybridizes with at least a part of the polynucleotide of the spacer region of the poisonous ribosomal RNA gene, and the poisonous mushroom. A diagnostic kit comprising a primer set comprising a combination of 3′-primers that hybridize with at least part of the polynucleotide in the spacer region of the ribosomal RNA gene. This diagnostic kit makes it possible to detect poisonous mushrooms whose form of fruiting body has become unclear after consumption by using the PCR method targeting the spacer region of the ribosomal RNA gene. Effective in diagnosing poisoning.

In the above embodiment, specifically, a sample is prepared from the contents of the digestive organs of the patient, vomit, feces and urine, and amplification of DNA fragments derived from these poisonous mushrooms is attempted using the PCR method. Mushrooms can be detected from a sample that has been partially digested with protease, nuclease, or the like because of its large amount of fiber and poor digestion. In addition, since the target DNA fragment contained in these samples is often cleaved, the primer is designed to amplify a fragment of 30-500 bp, more preferably 30-300 bp, still more preferably 50-150 bp. However, this is not limited to this because it depends on the state of the target sample. Under severe conditions, since the abundance of fragments of 500 bp or more is small (see Examples described later), DNA fragments to be amplified can be detected for most samples at 500 bp or less. If it is 300 bp or less, more reliable detection is possible, and if it is 150 bp or less, the certainty of detection can be further increased, and it is advantageous for amplification by PCR. On the other hand, if the DNA fragment to be amplified is 30 bp or more, it can be distinguished from the primer dimer, and if it is 50 bp or more, the primer dimer can be more reliably distinguished.

Yet another embodiment is a detection kit or detection method for a venom present in cooked food, which hybridizes with at least a portion of the polynucleotide in the spacer region of the ribosomal RNA gene of the venom. A detection kit or a detection method comprising a primer set comprising a combination of a 5′-side primer and a 3′-side primer that hybridizes with at least a part of the polynucleotide in the spacer region of the venom ribosomal RNA gene. This detection kit or detection method can detect poisonous mushrooms in foods whose form of fruiting body is not clear because they are cooked by using the PCR method targeting the spacer region of the ribosomal RNA gene. enable. In other words, it is possible to quickly and easily identify a causal toxin in a meal, which has been difficult in the past, by examining the meal when suspicious symptoms are observed as food poisoning using the PCR method. . Moreover, even if the form does not change significantly after cooking, the color and size of the fruiting body changes depending on the breeding environment, which is effective in identifying poisonous mushrooms that are difficult to identify by form.

The present inventors have revealed for the first time that poisonous mushrooms can be detected by a PCR reaction even after cooking that has been applied in the direction of degrading nucleic acids such as by applying heat. It became. Furthermore, it has been clarified that detection can be performed more reliably by using short fragments as amplification targets for mushroom DNA that has undergone such physical or chemical changes. That is, since it depends on the condition of the sample, it is not limited to this. However, since the target DNA fragment in these samples is often cleaved, the primer is 30-500 bp, more preferably 30-300 bp. More preferably, it is designed to amplify a 50-150 bp fragment. Under severe conditions, since the abundance of fragments of 500 bp or more is small (see Examples described later), DNA fragments to be amplified can be detected for most samples at 500 bp or less. If it is 300 bp or less, more reliable detection is possible, and if it is 150 bp or less, the certainty of detection can be further increased, and it is advantageous for amplification by PCR. On the other hand, if the DNA fragment to be amplified is 30 bp or more, it can be distinguished from the primer dimer, and if it is 50 bp or more, the primer dimer can be more reliably distinguished.

In summary, the mushroom identification method using the gene in the above embodiment is less dependent on the mushroom form, faster work, objectivity / reproduction than the conventional visual identification method by experts. It has many advantages such as improvement of reliability and certainty, cost reduction, and ease of implementation by anyone who has acquired a general gene-related technique. In addition to the benefits described above, the benefits include the identification of mushrooms that cause poisoning in emergency medical settings, the cause of food poisoning due to the inability to identify mushrooms in institutions that require food poisoning data collection such as health centers, etc. It is also effective from the viewpoint of the decrease in "" or the progress of forensic appraisal at forensic research institutes.

The oligonucleotides, primer sets, diagnostic kits, detection kits, and detection methods described by the above embodiments are not intended to limit the present invention, but are disclosed for the purpose of illustration. The technical scope of the present invention is defined by the description of the scope of claims, and those skilled in the art can make various design changes within the technical scope of the invention described in the scope of claims. For example, in the above embodiment, an ITS region, or an ITS1 region or an ITS2 region may be used instead of the spacer region of the ribosomal RNA gene, and the diagnostic kit, diagnostic marker, and detection method of this aspect are of the present invention. Needless to say, it is included in the technical scope.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this is an example and this invention is not limited to these. The commercially available reagents mentioned in the examples were used according to the manufacturer's instructions unless otherwise indicated.

[Example 1]
<Preparation of DNA from fruit body>
The mushrooms collected from the field were cut into 3 cm squares for each “bulk”. Furthermore, what was used for "stir-fried" was sliced to 3 mm thickness. When cooking them, “baking” was baked on a hot plate at 240 ° C. and baked on each side for 2 minutes. “Fry” was fried for 2 minutes on a hot plate of oiled 240 ° C. In “Fried”, each mushroom was sprinkled with commercially available tempura flour and fried in 180 ° C. oil for 2 minutes. In "boiled", mushrooms were placed in boiling water and boiled for 30 minutes, 60 minutes, 120 minutes and 180 minutes.

Raw mushrooms and cooked mushrooms were lightly dampened with a paper towel, frozen with liquid nitrogen, and then crushed until the mushrooms shattered.

300 mg of mushrooms pulverized in liquid nitrogen was placed in a 1.5 ml microtube, and 500 μl of Extraction Buffer having the composition shown in Table 1 was added. 33 μl of 20% SDS was added, mixed thoroughly, and incubated in a 65 ° C. water bath for 1 hour. Thereafter, 167 μl of 5M potassium acetate was added, mixed thoroughly, and left on ice for 20 minutes. Thereafter, the mixture was centrifuged at 13,000 rpm for 30 minutes at 4 ° C., and the supernatant was transferred to a new microcentrifuge tube containing 333 μl of isopropanol and mixed. This was centrifuged at 13,000 rpm for 5 minutes at 4 ° C. to precipitate the DNA, and the supernatant was completely removed, followed by drying.

To the dried DNA pellet, 100 μl of TE2 buffer having the composition shown in Table 2 was added and dissolved, 5 μl of RNase solution (10 mg / ml) was added, and incubated in a 37 ° C. water bath for 1 hour. Then, 500 μl of an equal mixture of Tris saturated phenol (phenol containing 0.1% 8-hydroxyquinoline saturated with Tris-HCl (pH 8.0)) and chloroform-isoamyl alcohol (24: 1) was added and mixed by inversion. Thereafter, the mixture was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected in a new microcentrifuge tube.

1/10 of the recovered amount of 3M sodium acetate (pH 5.2) and twice the amount of 99.5% ethanol were added, and the mixture was allowed to stand at −80 ° C. for 30 minutes. And it centrifuged at 13,000 rpm for 20 minutes at 4 degreeC, and removed the supernatant liquid. To this, 500 μl of 70% ethanol was added and centrifuged at 4 ° C. and 13,000 rpm for 5 minutes, and the supernatant was removed and dried. After drying, 20 μl of TE buffer shown in Table 3 was added to dissolve the DNA to obtain a DNA extract.

[Example 2]
<Detection of DNA from mushroom fruit body after cooking>
(1) Confirmation of mushroom fruit body DNA after cooking It was examined whether sufficient DNA was extracted from the cooked mushroom for PCR reaction. The prepared poisonous mushrooms (Tsukiyotake, Kusaura Benitatake, Kakimeji) were cooked as described in [Example 1] to obtain a DNA extract. FIG. 2 shows a part of the result of examining the size of the obtained DNA by agarose electrophoresis.

As shown in FIG. 2, in the case of cooking “baked”, “fried” or “fried”, the DNA was slightly decomposed compared to uncooked, but a large DNA of about 20 kbp was confirmed. In addition, even in the case of a small size, DNA having a size of about 5 kbp could be confirmed. On the other hand, DNA cooked “boiled” was more fragmented than “baked”, “stir-fried” and “fried” cooked foods, especially boiled for more than 120 minutes. Even so, DNA of 500 bp or more could be confirmed by electrophoresis, and some DNA of about 2 kbp could be confirmed at maximum. This was thought to be due to the fact that "boiled", "stir-fried" and "fried" cooking were hot but only exposed to heat for a short time, whereas "boiled" was exposed to heat for a long time. . However, it became clear that in any cooking method, a fragment having a length detectable by PCR can be extracted.

(2) Amplification of the DNA obtained from the fruit body after cooking by PCR reaction Using the DNA obtained in (1), PCR was carried out using universal primers to determine whether the DNA was amplified (conditions For details of these, see [Example 3]). As a result, as shown in FIG. 3, a part of the ribosome gene could be amplified by the PCR method, even when DNA extracted from any cooked mushroom was used as a template.

From the above results, it has been clarified that ribosomal DNA can be amplified and detected by PCR from mushroom fruit bodies that have been cooked such as “baked”, “fried”, and “fried”.

Example 3
<Amplification and sequencing of ITS region including 5.8S ribosomal RNA coding region of Kusaura Benitatake or Doxasaco>
(1) Preparation of Primer (Universal Primer) for Obtaining ITS Region Containing 5.8S Ribosomal RNA Coding Region Primers are prepared according to the method of White et al. [White et al .: PCR protocols, Academic Press, San Diego, pp. 315 (1990)], a primer for amplifying the ITS region containing the fungal 5.8S ribosomal RNA gene was synthesized. That is, 5′-GCATTGAATTCTGAACACA-3 ′ (ITS1 primer, SEQ ID NO: 7) was synthesized as a 5 ′ sense primer sequence, and 5′-GCATTGAATTCTGAACACA-3 ′ ((ITS4 primer, SEQ ID NO: 8) was synthesized as a 3 ′ antisense primer. The synthetic oligonucleotide was chemically synthesized.

(2) Amplification and sequencing by DNA of a DNA containing the 5.8S ribosomal RNA coding region of Kusaura Benitatake or Doxasaco as a template from the fruiting body of Kusaura Benitatake or Dokusasaco as described in [Example 1] PCR was performed using the prepared DNA and using the primers ITS1 and ITS4 prepared in (2) above as primers. The composition of the PCR reaction solution is as shown in Table 4 below.

PCR is performed using a thermal cycler (Takara, TP-650 type), with heat denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 1 minute as one cycle. 30 cycles were performed. The obtained PCR product was sequenced by a fluorescence automatic DNA sequencer (Applied Biosystems 3130xl type) using a big dye terminator cycle sequencing kit (Applied Biosystems).

As a result, as shown in FIG. 4, in SEQ ID NO: 1 obtained from Kusaura Benitatake, Kusaura Benitatake is a morphologically similar mushroom, Enoloma sarcopum, Lyophyllum shimeji, Hatake shimeji (Lyophyllum) It became clear that there were many differences with decastes). In this sequence, the ITS1 region is 12-248 and the ITS2 region is 407-903 (each further includes the sequences of primer ITS1 and primer 4).

Further, as shown in FIG. 5, in SEQ ID NO: 2 obtained from Dokusasako, Dokusasako is a morphologically similar mushroom, Clitocybe gibba, Ararataria mellea, Lactarius akahatu, It became clear that there were many differences among the bamboo shoots (Lactarius hatutake), Kitachichitake (Lactarius chrysorrheus), and Hotei shimeji (Clitocybe clavipes). In this sequence, the ITS1 region is 12-252 and the ITS2 region is 411-631 (each further including the sequences of primer ITS1 and primer 4).

Example 4
<Specific Detection of DNA Fragments Derived from Kusaura Benitatake and Dokusaco>
(1) Preparation of DNA from mushroom fruiting body 19 kinds of mushrooms (see Table 5) including moss agaricus and doxasaco were prepared in the same manner as in [Example 1].

(2) Selection and synthesis of oligonucleotide primers Comparison of other mushrooms with similar morphologically similar base sequences derived from Kusaura Benitatake or Doksasaco decoded in [Practical example 2]. A base sequence region that does not include a region with a high content and does not hybridize with the primers was selected (FIGS. 4 and 5, encircled portion). That is, 5′-TTTGAGAACTGCTGTGAAAATC-3 ′ (SEQ ID NO: 3) was selected as a sense primer for detecting mulberry fly shoots, and 5′-GGCACAAAGTCCCTATATGTTTA-3 ′ (SEQ ID NO: 4) was selected as an antisense primer. 5′-GGTGCACACCTGATAACCA-3 ′ (SEQ ID NO: 5) was selected as a sense primer for detection of scab, and 5′-AGCTTAAGCTTTCGCACCAG-3 ′ (SEQ ID NO: 6) was selected as an antisense primer. Chemical synthesis of the above primers was performed according to a conventional method.

(3) Specific Detection of DNA Fragments Derived from Kusaura Benitatake and Dokusaco The DNA prepared in (1) above was amplified using the PCR method by the following method using the primer synthesized in (2) above. That is, SYBRR Green Realtime PCR Master Mix (TOYOBO) was used and PCR was performed by adding each primer to a concentration of 50 μM. The reaction conditions were 20 cycles of denaturation at 95 ° C. for 5 seconds, annealing at 65 ° C. for 5 seconds, and extension at 72 ° C. for 15 seconds. The composition of the PCR reaction solution is as shown in Table 6 below.

(4) Detection of amplified DNA fragment The reaction solution containing the amplified DNA fragment obtained by PCR according to (3) above was subjected to electrophoresis on a 2% agarose gel together with a DNA size marker, and then stained with ethidium bromide. Some of the results are shown in FIGS. 6 and 7, respectively. In PCR (lanes 1, 2 and 3 in FIG. 6) carried out using this primer against Kusaura Agaricus, a specific DNA fragment of 109 base pairs was obtained, and PCR carried out using this primer against Doxusaco ( In lane 1) of FIG. 7, a specific DNA fragment of 108 base pairs was obtained. No band could be detected from mushrooms other than Kusaura Benitake (lane 4-21 in FIG. 6) and mushrooms other than doxasaco (lane 2-19 in FIG. 7).

From the above results, while the conventional identification method using the form of the fruiting body could only be performed by experts with advanced technical skills, anyone using the method of the present invention can easily, quickly and accurately. It has been clarified that Kusaura Benitatake or Dokusasaco can be identified.

In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

As described above, the oligonucleotide, primer, and diagnostic kit, detection kit and method using the same for detection of poisonous mushrooms according to the present invention can be obtained by using gene-related technology. ) Specific and rapid detection and identification.

It is a figure which shows the area | region which codes ribosomal RNA on genomic DNA. It is a figure which shows the area | region which codes 5.8S ribosomal RNA on genomic DNA, and its adjacent region. The arrow indicates the position of the universal primer. It is an electrophoresis photograph which shows the result of the agarose electrophoresis of DNA extracted from the cooked poisonous mushroom. It is an electrophoresis photograph which shows the result of having performed PCR amplification reaction using a universal primer with respect to DNA extracted from the cooked poisonous mushroom. It is a figure which shows the base sequence of the ITS area | region (However, the primer site | part of ITS1 and ITS4 is excluded) and its alignment which contain the 5.8S ribosomal RNA gene of a mushroom which resembles the shape of Kusaura Benitake. The boxed part is the part used as a primer for specific detection this time. It is a figure which shows the base sequence of the ITS area | region (However, the primer site | part of ITS1 and ITS4 is excluded) and its alignment which contain the 5.8S ribosomal RNA gene of a mushroom which resembles the shape of Kusaura Benitake. It is a figure which shows the base sequence (except for the primer part of ITS1 and ITS4) of the ITS area | region containing the 5.8S ribosomal RNA gene of the mushroom which resembles the form of Doxasako, and the alignment. The boxed part is the part used as a primer for specific detection this time. It is a figure which shows the base sequence (except for the primer part of ITS1 and ITS4) of the ITS area | region containing the 5.8S ribosomal RNA gene of the mushroom which resembles the form of Doxasako, and the alignment. It is an electrophoresis photograph which shows the amplification result by PCR using the Kusaura Agaricus specific primer set containing the base sequence of sequence number 3 and 4. It is an electrophoresis photograph which shows the amplification result by PCR using the docasaco specific primer containing the base sequence of sequence number 5 and 6.

Claims (15)

  An oligonucleotide that specifically hybridizes with at least a portion of the polynucleotide in the spacer region of the ribosomal RNA gene of Doxasaco.   An oligonucleotide that specifically hybridizes with at least a portion of DNA consisting of the base sequence represented by SEQ ID NO: 2.   A DNA microarray comprising the oligonucleotide according to claim 1 or 2, comprising a chain length in the range of 15 to 100 bases.   DNA consisting of the base sequence represented by SEQ ID NO: 2. A 5 ′ primer that hybridizes with at least a portion of the polynucleotide in the spacer region of the ribosomal RNA gene of Doxasaco, and a 3 ′ primer that hybridizes with at least a portion of the polynucleotide in the spacer region of the ribosomal RNA gene of Doxasako A combination of
The primer set, wherein the 5′-side primer has a chain length in the range of 15 to 100 bases, and the 3′-side primer has a chain length in the range of 15 to 100 bases. 5′-side primer that hybridizes with at least a part of the polynucleotide consisting of the base sequence represented by SEQ ID NO: 2 and 3′-side primer that hybridizes with at least a part of the polynucleotide composed of the base sequence represented by SEQ ID NO: 2 In combination with
The primer set, wherein the 5′-side primer has a chain length in the range of 15 to 100 bases, and the 3′-side primer has a chain length in the range of 15 to 100 bases.   The primer set according to claim 5 or 6, wherein the 5 'primer includes a base sequence represented by SEQ ID NO: 5.   The primer set according to claim 5 or 6, wherein the 3 'primer includes a base sequence represented by SEQ ID NO: 6.   A detection kit for Doxasasako, comprising the primer set according to any one of claims 5 to 8.   9. A diagnostic kit for doxasaco poisoning comprising the primer set according to any one of claims 5 to 8.   A method for detecting the doxasaco, comprising amplifying at least a part of the ribosomal RNA gene of doxasaco using the primer set according to any one of claims 5 to 8. A detection kit for poisonous mushrooms present in cooked foods,
A 5 ′ primer that hybridizes with at least a portion of the polynucleotide in the spacer region of the venom ribosomal RNA gene, and a 3 ′ that hybridizes with at least a portion of the polynucleotide in the spacer region of the venom ribosomal RNA gene. It consists of a combination with the side primer,
The 5 ′ primer comprises a primer set consisting of a chain length in the range of 15 to 100 bases, and the 3 ′ primer comprises a chain length in the range of 15 to 100 bases,
Detection kit. A diagnostic kit for poisonous mushroom poisoning,
A 5 ′ primer that hybridizes with at least a portion of the polynucleotide of the poisonous spacer region, and a 3 ′ primer that hybridizes with at least a portion of the polynucleotide of the spacer region of the venomous ribosomal RNA gene. A combination of
The 5 ′ primer comprises a primer set consisting of a chain length in the range of 15 to 100 bases, and the 3 ′ primer comprises a chain length in the range of 15 to 100 bases,
Diagnostic kit.   The diagnostic kit according to claim 13, wherein at least a part of the ribosomal RNA gene is at least a part of 5.8S ribosomal RNA. A method for detecting poisonous mushrooms present in cooked foods,
A 5 ′ primer that hybridizes with at least a portion of the polynucleotide in the spacer region of the venom ribosomal RNA gene, and a 3 ′ that hybridizes with at least a portion of the polynucleotide in the spacer region of the venom ribosomal RNA gene. Amplifying at least a part of the polynucleotide encoding the spacer region of the ribosomal RNA gene of the poisonous mushroom using a primer set consisting of a combination with a side primer,
The detection method, wherein the 5′-side primer has a chain length in the range of 15 to 100 bases, and the 3′-side primer has a chain length in the range of 15 to 100 bases.