Method for detecting target plant genus Number:7,144,702 from the United States Patent and Trademark Office (PTO) owispatent

Title: Method for detecting target plant genus

Abstract: A method for detecting species in a target plant genus comprises the steps of conducting PCR using at least one member selected from the group consisting of primers (A) and (B), which can hybridize under stringent conditions to a nucleic acid molecule having a common nucleotide sequence for all species in the target plant genus in 45S rRNA precursor gene sequence thereof, wherein 3' end of primer (A) can complementarily bind to a base in ITS-1 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule while 3' end of primer (B) can complementarily bind to a base in ITS-2 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule, and identifying the presence of the resulting amplification product from PCR containing at least a part of ITS-1 or ITS-2 sequence of the target plant genus.The method for detecting species in a target plant genus, particularly an allergenic plant genus such as the genus Fagopyrum, can make it possible to detect with high sensitivity, for example, about 1 ppm of the plant(s) in cases where the plant(s) is contained in a food ingredient or food product.

Patent Number: 7,144,702 Issued on 12/05/2006 to Hirao,   et al.

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Inventors:	Hirao; Takashi (Higashi-Osaka, JP), Hiramoto; Masayuki (Higashi-Osaka, JP)
Assignee:	House Foods Corporation (Osaka-fu, JP)
Appl. No.:	10/285,061
Filed:	October 31, 2002

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Foreign Application Priority Data

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Nov 01, 2001 [JP]			2001-336571
Sep 27, 2002 [JP]			2002-284222

Current U.S. Class:	435/6 ; 435/91.1; 435/91.2
Current International Class:	C12Q 1/68 (20060101); C12P 19/34 (20060101)

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References Cited [Referenced By]

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U.S. Patent Documents

	5876977		March 1999		Wang et al.
	5962665		October 1999		Kroeger et al.

Other References

Lott et al., Nucleotide Sequence Analysis of the 5.8S rDNA and Adjacent ITS2 Region of Candida albicans and Related Species. Yeast (1993) 9:1199. cited by examiner . Holzhauser et al., Polymerase chain reaction (PCR) for detection of potentially allergenic hazelnut residues in complex food matrixes. Eur. Food Res. Technol. (2000) 211: 360-365. cited by examiner . Hartmann et al., Extensive Ribosomal DNA Genic Variation in the Columnar Cactus Lophocereus. Mol. Evol. (2001) 53: 124-134. cited by examiner . Lavin et al., The Dalbergioid Legumes (Fabeceae): Delimitation of a pantropical monophyletic clade. Amer. J. Bot. (2001) 88: 503-533. cited by examiner . Buck et al., Design Strategies and Performance of Custom DNA Sequencing Primers. BioTechniques (1999) 27: 528-536. cited by examiner . Jong Hee Shin et al., Rapid Identification of up to Three Candida Species in a Single Reaction Tube by a 5' Exonuclease Assay Using Fluorescent DNA Probes, Journal of Clinical Microbiology, Jan. 1999, p. 165-170, vol. 37, No. 1 (Abstract). cited by other . Jana Proft et al., Identification of six sibling species of the Anopheles macilipennis complex (Diptera: Culicidae) by a polymerase chain reaction assay, Parasitol Res (1999), 85: 837-843. cited by other . Michael Allmann et al., Polymerase chain reaction (PCR): a possible alternative to immunochemical methods assuring safety and quality of food; Z Lebensm Unters Forsch (1993) 196:248-251. cited by other.

Primary Examiner: Fredman; Jeffrey
Attorney, Agent or Firm: Hoffmann & Baron, LLP

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Claims

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What is claimed is:

1. A method for detecting species in the genus Arachis in cases where even one species of the genus Arachis is contained in a subject to which the method is to be applied such as a food ingredient or product, which comprises the steps of: a) collecting 45S rRNA precursor gene sequences containing a ITS sequence of the genus Arachis and plants thought to be related species thereof and then selecting the region common to the genus Arachis in the 45 rRNA precursor gene sequences; b) selecting the base specific to the genus Arachis from the region; c) designing primers (A) and/or (B) having the specific base at 3' end thereof, which primers can hybridize under stringent conditions to a nucleic acid molecule having a common nucleotide base sequence for all species in the genus Arachis in 45S rRNA precursor gene sequence thereof, wherein 3' end of primer (A) can complementarily bind to a base in ITS-1 sequence of the genus Arachis when the primer hybridizes to the nucleic acid molecule while 3' end of primer (B) can complementarily bind to a base in ITS-2 sequence of the genus Arachis when the primer hybridizes to the nucleic acid molecule; d) isolating DNA from the subject to which the method is to be applied; e) conducting PCR amplification for the isolated DNA using at least one member selected from the group consisting of the primers (A) and (B); and f) identifying the presence of the resulting amplification product from PCR containing at least a part of ITS-1 or ITS-2 sequence of the genus Arachis.

2. The method of claim 1, wherein the nucleic acid molecule having a common nucleotide sequence for all species in the genus Arachis in 45S rRNA precursor gene sequence thereof is a nucleic acid molecule having a common specific nucleotide sequence for all species in the genus Arachis in ITS-1 sequence thereof.

3. The method of claim 1, wherein the nucleic acid molecule having a common nucleotide sequence for all species in the genus Arachis in 45S rRNA precursor gene sequence thereof is a nucleic acid molecule having a common specific nucleotide sequence for all species in the genus Arachis in ITS-2 sequence thereof.

4. The method of claim 2, wherein the step of conducting PCR comprises using the primers (A) and (C), which primer (C) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in which ITS-1, 5.8S rRNA gene, ITS-2 and LSU rRNA gene sequences of the genus Arachis are continuously bonded.

5. The method of claim 3, wherein the step of conducting PCR comprises using the primers (B) and (D), which primer (D) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in which SSU rRNA gene, ITS-1, 5.8S rRNA gene and ITS-2 sequences of the genus Arachis are continuously bonded.

6. The method of claim 2, wherein the step of conducting PCR comprises using the primers (A) and (E), which primer (E) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in which SSU rRNA gene and ITS-1 sequences of the genus Arachis are continuously bonded.

7. The method of claim 3, wherein the step of conducting PCR comprises using the primers (B) and (F), which primer (F) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in which ITS-2 and LSU rRNA sequences of the genus Arachis are continuously bonded.

8. The method of claim 2, wherein the common specific nucleotide sequence for all species in the genus Arachis in ITS-1 sequence thereof is a nucleotide sequence selected from the group consisting of SEQ NO:17 and a complementary nucleotide sequence thereof.

9. The method of claim 1, wherein the primer (A) is selected from the group consisting of SEQ NOs:18, 19 and 20.

10. The method of claim 3, wherein the common specific nucleotide sequence for all species in the genus Arachis in ITS-2 sequence thereof is selected from the group consisting of SEQ NO:23 and a complementary nucleotide sequence thereof.

11. The method of claim 1, wherein the primer (B) is SEQ NO:24.

12. The method of claim 9, wherein the step of conducting PCR comprises the steps of conducting PCR amplification at an annealing temperature of the primer and template DNA used in PCR amplification higher than Tm value of the primer, and subsequently continuing the PCR amplification at an annealing temperature near the Tm value.

13. The method of claim 4, wherein the primer (C) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in 5.8S rRNA gene sequence of the genus Arachis, and 3' end of primer (C) can complementanly bind to a base in 5.8S rRNA gene sequence of the genus Arachis when the primer hybridizes to the nucleic acid molecule.

14. The method of claim 5, wherein the primer (D) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in 5.8S rRNA gene sequence of the genus Arachis, and 3' end of primer (D) can complementarily bind to a base in 5.8S rRNA gene sequence of the genus Arachis when the primer hybridizes to the nucleic acid molecule.

15. The method of claim 13, wherein the part of nucleotide sequence in 5.8S rRNA gene sequence of the genus Arachis is selected from the group consisting of SEQ NO:1 and a complementary nucleotide sequence thereof.

16. The method of claim 14, wherein the part of nucleotide sequence in 5.8S rRNA gene sequence of the genus Arachis is selected from the group consisting of SEQ NO:1 and a complementary nucleotide sequence thereof.

17. The method of claim 4, wherein the primer (C) is selected from the group consisting of SEQ NOs:2, 3 and 4.

18. The method of claim 5, wherein the primer (D) is selected from the group consisting of SEQ NOs:5, 6 and 7.

19. The method of claim 4, wherein the step of conducting PCR comprises using the primer (A) selected from the group consisting of SEQ NOs:18, 19 and 20 and the primer (C) selected from the group consisting of SEQ NOs:2, 3 and 4.

20. The method of claim 1, wherein the step of conducting PCR comprises using the primer (A) selected from the group consisting of SEQ NOs:18, 19 and 20 and the primer (B) of SEQ NO:24.

21. The method of claim 5, wherein the step of conducting PCR comprises using the primer (B) of SEQ NO:24 and the primer (D) selected from the group consisting of SEQ NOs:5, 6 and 7.

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Description

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CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Japanese Patent Application Nos. 2002-284222 filed on Sep. 27, 2002 and Japanese Patent Application No. 2001-336571 filed on Nov. 1, 2001.

BACKGROUND OF THE INVENTION

The 45S rRNA precursor gene sequence (Small Subunit ribosomal RNA (SSU rRNA) gene.about.Internal Transcribed Spacer-1 (ITS-1).about.5.8S ribosomal RNA (5.8S rRNA) gene.about.Internal Transcribed Spacer-2 (ITS-2).about.Large Subunit ribosomal RNA (LSU rRNA) gene) has been used for the classification of species. For example, according to the method developed by Shin J H, et al. (J. Clin. Microbiol., 37: 165 170(1999)), 5 candida species (fungi) of the genus Candida can be detected and identified using two primers hybridized to the 5.8S rRNA and 28S rRNA (LSU rRNA) gene sequences common to fungi and 5 separate probes each of which can specifically hybridize to the ITS-2 sequence of its corresponding species. The method is different from the present invention as described below. Firstly, the method is aimed at fungi, specifically candida (fungi). Secondly, the method does not use the primers, which hybridize to ITS-1 or ITS-2 sequence. Consequently, these primer pairs do not assure the specificity to the genus Candida, whereas each of five probes can independently recognize its corresponding candida species (fungi) of the genus Candida. In other words, only one species of the genus Candida can be detected and identified when a single set of the primer pair and a probe is used. Thirdly, the above publication does not describe about the sensitivity of the detection, which is very important for the detection methods of allergenic plants in food. Lastly, the method needs expensive reagents and instruments due to the use of probes.

According to the method developed by Proft J, et al. (Parasitol. Res., 85: 837 843(1999)), a certain anopheles mosquito can be classified into one of 6 species of the genus Anopheles using 6 primer pairs. The method uses a primer that can hybridize to the 5.8 rRNA gene sequence common to the six anopheles mosquito species of the genus Anopheles in combination with 6 primer pairs each of which can specifically hybridize to the ITS-2 sequence of its corresponding anopheles mosquito species of the genus Anopheles. Based on the size of the amplification product obtained by PCR method, the anopheles mosquito of interest can be classified into one of the 6 species of the genus Anopheles. The method is different from the present invention as described below. Firstly, the method is aimed at mosquitoes, specifically the anopheles mosquitoes. Secondly, due to the properties of designed primer pairs, only one species of the genus Anopheles can be detectable when a single primer pair is used. Thirdly, an object of the method is to identify a specimen exclusively derived from a single species of mosquitoes. Consequently, the object of the method is not to analyze anopheles mosquitoes in a mixture. Lastly, the above publication does not describe about the sensitivity of the detection, which is very important for the detection methods of allergenic plants in food.

Thus, the conventional methods mentioned above are to detect one specific species in a mixture and to identify a bio specimen exclusively derived from a single species of the genus, and therefore, the methods do not relate to a method for detecting the target genus broadly in cases where even one kind of the target genus is contained in a mixture. In addition, the primer sequences common to several species are located on SSU rRNA, 5.8S rRNA and LSU rRNA gene sequence, and therefore, primer sequences common to several species are not found in ITS-1 or ITS-2 sequence.

On the other hand, regarding detection of allergenic plants in food, a method for detecting whether some wheat is contained in a food sample of interest is disclosed by Allmann M, et al. (Z Lebensm Unters Forsch, 196: 248 251(1993)). The method uses primers which specifically hybridize to a IGS sequence between 25S rRNA (LSU rRNA) and 18S rRNA (SSU rRNA) gene sequences of wheat. However, it is hard to evaluate the specificity of the primers by simulation and the like because the primers have to be designed based on little information about the IGS sequence in the method. Therefore, it would be difficult to judge the reliability of the analysis.

SUMMARY OF THE INVENTION

An object of the present inventions is to provide a method for detecting species (a plant or plants) in a target plant genus, particularly an allergenic plant genus such as the genus Fagopyrum, which makes it possible to detect with high sensitivity, for example, about 1 ppm of the plant(s) in cases where the plant(s) is contained in a food ingredient or food product.

Since a trace of allergenic food ingredients, particularly plants in an allergenic plant genus may be unintentionally contaminated in the food ingredient or product at the stages of production, distribution and fabrication, it is important that providers of the food ingredient or product conduct quality control to detect whether these plants have contaminated the food ingredient or product.

For example, regarding buckwheat, though it is reported that patients are affected with anaphylaxis by pillows made of buckwheat chaff and die due to anaphylactic shock and traces of buckwheat may effect a severe symptom in allergic patients for buckwheat, there is no method for detecting buckwheat in the food ingredient or product in the world. For example, it is considered that contamination of buckwheat into the food ingredient or product occurs in a case where buckwheat grown near a field cultivated with plants other than buckwheat is contaminated in the food ingredient harvest time. Therefore, in order to find the contamination of trace of unintended buckwheat, it is desirable that a method for detecting buckwheat be built up, wherein the method can detect as sensitive as possible, for example, even 1 ppm of the buckwheat in a food ingredient and product. Furthermore, as for grain allergies, it is said that some cross-reaction occurs among taxonomically related plants, and therefore, it is desirable that the method be able to detect a wide range of any plants in the genus Fagopyrum without limiting the detectable plants to eatable buckwheat.

Regarding a method for detecting peanuts, an ELISA kit, which can detect about 2.5 ppm of peanuts using specific antibodies for proteins inherent to peanuts, have been sold and used in the world. When positive finding in ELISA, whether it is false positive or truly positive can be confirmed in detail by Western Blot etc., but it confirms only the size of protein involving antigen-antibody reaction. A method for detecting a DNA inherent to peanuts has not been reported. In order to detect peanuts in a food ingredient and product through a variety of processing steps, it is desirable that there is built up a method for detecting target DNA sequences, which will have a relatively high resistivity against the processing rather than proteins. Furthermore, as it is the same as in buckwheat, it is desirable that the method be able to detect a wide range of plants in the genus Arachis.

Thus, it is important to detect a plant(s) in the allergenic plant genus with high sensitivity in cases where even only one kind of the plants is contained in the food ingredient, product and the like.

In cases of genetically modified products and the like, DNA sequences to be detected are limited to recombinant DNA sequences. On the other hand, 6 in cases of plants which originally exist in nature, there has not been clear knowledge how to choose a target DNA sequence from a large number of DNA sequences, and whether the thus chosen DNA sequence is useful and universal for a variety of plants. It has been conducted to choose a specific protein to a target plant, and to detect a DNA sequence coding for the protein, but it is necessary to choose a separate specific protein to each plant. Furthermore, even if such a specific protein can be chosen, when the copy number of a DNA sequence coding for the protein is small, there are some cases where the method may not have a sufficient sensitivity and therefore it will be disadvantage for the detection of traces of a contaminating plant.

Under such circumstances, in order to develop a method for detecting a plant(s) in an allergenic plant genus and the like in cases where even only one kind of the plants is contained in a food ingredient and product, the present inventors have focused their attention on the gene sequences of a target plant genus to vigorously conduct the research. In order to detect whether one specific plant has contaminated a food ingredient or product, it may be conducted to detect a specific gene sequence of the plant in the food ingredient and product. However, in order to detect a case where even only one kind of the plants is contained in a genus in a food ingredient and product, such method is very complicated and inefficient because it is necessary to repeat the same operation for respective plants in a specific genus.

In order to solve this problem, the inventors have conducted further research, collected some information on gene sequences of plants in the genus Fagopyrumn (21 sequences registered in GenBank) and in other genus and studied on a variety of viewpoint, and thereby, the inventors have found that a specific common sequence for plants in the genus Fagopyrum, which differs from a sequence of plants in other genus, is present in gene sequences of the plant in the genus Fagopyrum (21 sequences registered in GenBank). As the result of an investigation conducted based on this knowledge for other plant genus such as the genus Arachis, the inventors have also found that there is similar tendency among them.

Based on this knowledge, it has been found that a method for detecting each allergenic plant genus using a sequence of 45S rRNA precursor gene, as a sequence which exists as a sequence having a large copy number in plant DNA and is specific to each allergenic plant genus, can be useful in attaining the object. When positive indication appears in PCR, differently from ELISA, as an amplification product can be analyzed not only in the size thereof but also in detail sequence thereof by sequencing the amplification product, it can be confirmed more precisely whether it is false positive or truly positive. Furthermore, it has been found that, by choosing a region including ITS-1 or ITS-2 sequence as a target sequence, the method is useful in detecting trace of plants in the target plant genus in a mixture because the specific sequence can be obtained and common region of sequences for plants in the genus can be chosen. Moreover, as the sequence of 45S rRNA precursor gene is present in most plants, it can be advantageously applied on a variety of plants.

Based on this knowledge, the present inventions have been completed. In this connection, the following method for detecting plants can be applied not only to the allergenic plant genus but also to other plant genus.

Accordingly, the present invention provides a method for detecting species (a plant(s)) in a target plant genus, which comprises the steps of conducting PCR using at least one member selected from the group consisting of primers (A) and (B), which can hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence common to all species in the target plant genus in 45S rRNA precursor gene sequence thereof, wherein 3' end of primer (A) can complementarily bind to a base in a ITS-1 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule while 3' end of primer (B) can complementarily bind to a base in a ITS-2 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule, and identifying the presence of the resulting amplification product from PCR containing at least a part of the ITS-1 or ITS-2 sequence of the target plant genus.

Herein, the phrase "hybridize under stringent conditions" means that two DNA fragments hybridize to each other under the standard hybridization condition described by Sambrook J. et al. (Expression of Cloned Genes in E. coli (Molecular Cloning : A laboratory Manual (1989)) Cold Spring Harbor Laboratory Press, New York, USA, 9.47 9.62 and 11.45 11.61). More specifically, for example, it means that a hybridization and washing (for example, about 2.0.times.SSC, 50.degree. C.) are conducted on the basis of Tm value obtained by the following equation. Tm=81.5+16.6(log.sub.10[Na.sup.+])+0.41(fraction G+C)-(600/N)

In addition, the term genus as used in the present specification means a group including all species in the genus or some species chosen from the species in the genus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 1.

FIG. 2 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 1.

FIG. 3 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 1.

FIG. 4 is nucleotide sequences obtained by a sequence analysis of the target amplification product from buckwheat chaff.

FIG. 5 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 3.

FIG. 6 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 3.

FIG. 7 is nucleotide sequences obtained by a sequence analysis of the target amplification product from Shirahana soba.

FIG. 8 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 5.

FIG. 9 is nucleotide sequences obtained by a sequence analysis of the target amplification product from peanut.

FIG. 10 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 6.

FIG. 11 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 6.

FIG. 12 is nucleotide sequences obtained by a sequence analysis of the target amplification product from peanut.

FIG. 13 is 45S rRNA precursor gene structure.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Although a target plant genus to be detected by the method of the present invention may be any plant genus, because the method can detect a presence of trace of a plant(s) in the target plant genus in a food ingredient or product, the method is particularly useful in detecting whether plants in the allergenic plant genus such as the genus Fagopyrum, genus Arachis, genus Triticum and genus Glycine are contaminated in the food ingredient or product.

The method of the present inventions uses at least one member selected from the group consisting of primers (A) and (B), which can hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence common to all species in the target plant genus in a 45S rRNA precursor gene sequence thereof, wherein 3' end of primer (A) can complementarily bind to a base in a ITS-1 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule, while 3' end of primer (B) can complementarily bind to a base in a ITS-2 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule to conduct PCR amplification for DNA isolated from a subject to which the method is to be applied. In the PCR amplification, based on a conventional procedure described in publications, for example, Saiki RK, et al., Science, 230: 1350 1354(1985) and Shyokubutsu no PCR Zikken Protocol--Idenshi no Tanri.cndot.Hatsugen Kara Genome Kaiseki Made--(Saiboukougaku Bessatsu Saiboukougaku Series 2), General Editors Shimamoto, K. and Sasaki, T., Shujunsha Co., Ltd., Tokyo, 1995 and the like, optimal conditions are chosen from appropriate modification of temperature and time of each step of denaturation, annealing and extension, a kind and concentration of enzyme (DNA polymerase), concentrations of DNTP, primer and magnesium chloride, an amount of template DNA and the like.

In addition, PCR amplification may be conducted at an annealing temperature of the primer and the template DNA higher than Tm value of the primer, preferably the Tm value plus 10 to 3.degree. C., and subsequently at an annealing temperature near the Tm value, preferably the Tm value plus 7 to 0.degree. C. , wherein the Tm value is determined by commercially available software such as HYB Simulator.TM. version 4.0 (Advanced Gene Computing Technologies, Inc.) and Primer Express.TM. version 1.5 (PE Applied Biosystems).

After the PCR amplification of DNA isolated from a subject to be studied such as a food ingredient or product, the resulting reaction solution is analyzed by for example, electrophoresis to determine whether the target plant genus is present in the subject. The determination is based on whether any PCR amplification products having target size are present in the reaction solution after the PCR amplification, and if the PCR amplification products are present in the reaction solution, whether at least a part of the ITS-1 or ITS-2 sequence of the target plant genus is present in the sequence of the PCR amplification products. That is, if the PCR amplification products, which have the target size and contain at least a part of the ITS-1 or ITS-2 sequence of the target plant genus, are present in the reaction solution, the studied subject is contaminated by a plant(s) in the target plant genus. On the other hand, if the PCR amplification products are not present in the reaction solution or even though it exists, unless it contains at least a part of ITS-1 or ITS-2 sequence of the target plant genus, the studied subject is not contaminated by a plant(s) in the target plant genus. Furthermore, the method of the present invention can detect with high sensitivity, for example, about 1 ppm level of a contamination.

For example, at least 2 primers may be used in the method of the present invention. In cases where at least 2 kinds of the target plant genus are detected at the same time, at least 3 primers may be used provided that it is important to use at least one member selected from the group consisting of primers (A) and (B), which can hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence common to all species in the target plant genus in 45S rRNA precursor gene sequence thereof, wherein 3' end primer (A) can complementarily bind to a base in ITS-1 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule while 3' end of primer (B) can complementarily bind to a base in ITS-2 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule. In this connection, examples of the primer (A) include primers, which can hybridize to a nucleic acid molecule having a boundary between a ITS-1 sequence and a 5.8S rRNA gene sequence or which can hybridize to a nucleic acid molecule having a boundary between a ITS-1 sequence and a SSU rRNA gene sequence. Likewise, examples of the primer (B) include primers, which can hybridize to a nucleic acid molecule having a boundary between a ITS-2 sequence and a 5.8S rRNA gene sequence or which can hybridize to a nucleic acid molecule having a boundary between a ITS-2 sequence and a LSU rRNA gene sequence. Preferably the primers (A) and (B) have at least 15 bases, more preferably 15 to 30 bases. Since the ITS-1 sequence and the ITS-2 sequence contain many specific sequences for species, the primer (A) or (B), which has a specificity common to the target plant genus, can be obtained by choosing a suitable nucleic acid molecule having a specific nucleotide sequence common to the target plant genus in the ITS-1 and ITS-2 sequences, as a nucleic acid molecule having a nucleotide sequence common to the target plant genus in the 45S rRNA precursor gene sequence. One or two or more member(s) selected from the group consisting of the primer (A) and the primer (B) may also be used, but if at least two members are used, the method of the present invention can become more highly sensitive to the target plant genus, particularly genus Fagopyrum.

In another embodiment of the method for detection of the present invention, primer (A) is used together with a primer (C) which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence continuously bonded ITS-1, 5.8S rRNA gene, ITS-2 and LSU rRNA gene of the target plant genus. Alternatively, primer (A) is used together with a primer (E) which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence continuously bonded SSU rRNA gene and ITS-1 of the target plant genus. In a further embodiment of the method for detection of the present invention, the primer (B) is used together with a primer (D) which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence continuously bonded SSU rRNA gene, ITS-1, 5.8S rRNA gene and ITS-2 of the target plant genus. Alternatively, primer (B) is used together with a primer (F) which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence continuously bonded ITS-2 and LSU rRNA gene of the target plant genus. In this connection, 5.8S rRNA gene is highly preservative and contains many sequences common to a great majority of plants. Therefore, as a primer (C), by appropriately choosing a primer, which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence of 5.8S RRNA gene, wherein 3' end thereof can complementarily bond to a nucleotide sequence in 5.8S rRNA gene sequence when the primer hybridizes to the nucleic acid molecule, or as s primer (D), by appropriately choosing a primer, which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence of 5.8S rRNA gene, wherein 3' end thereof can complementarily bond to a nucleotide sequence in 5.8S rRNA gene sequence when the primer hybridizes to the nucleic acid molecule, the resulting primer can be commonly used for a variety of plants. If said primer is fixed and a common specific primer is chosen for the species in the target plant genus from the ITS-1 or ITS-2 region thereof, then the primers can be easily designed to detect with high sensitivity the contaminated plants in the target plant genus. Preferably, the primers (C) to (F) have at least 15 bases, more preferably 15 to 30 bases.

When these primers are designed, it will be sufficient to design them based on, for example, PCR Hou Saizensen--Kisogizyutsu Kara Ouyou Made (Tanpakushitsu.cndot.Kakusan.cndot.Kouso Rinzizoukan), ed. Sekiya, T. and Fujinaga, K., Kyoritsu Shuppan Co. Ltd., Tokyo, 1997, Baio Zikken Illustrated 3 Hontouni Hueru PCR (Saiboukougaku Besshi Me de Miru Zikken Note Series), Nakayama, H., Shujunsha Co., Ltd., Tokyo, 1996 or PCR Technology: Principles and Applications of DNA Amplification, ed. Erlich, H. A., Stockton Press, Inc., NY, 1989. However, since there is a low possibility that the target DNA is decomposed when the DNA is detected in un-processed materials, the primers may be those which can induce an amplification product within 700 bases, and since there is a possibility that the target DNA is decomposed to become short when the DNA is detected in processed foods, the primers, which can induce an amplification product within 200 bases, are preferable in view of that the primers provide high sensitivity.

In view of the above, it is preferable that the primer (C) or (D) be able to hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence indicated by SEQ NO:1 or a complementary nucleotide sequence thereof. Said primer is preferable because the region indicated by SEQ NO:1 has an especially high homology, a primer which hybridize to any region of 5.8S rRNA gene sequence may be used because the sequences of species in the allergenic plant genus have a high homology over almost the whole region of 5.8S rRNA gene sequence. More preferably, it is a primer, which can hybridize under stringent conditions to a nucleic acid molecule having positions 11 to 63 of the nucleotide sequence of SEQ NO:1 or a complementary nucleotide sequence thereof. Preferably, primer (C) is an oligonucleotide indicated by any of SEQ NO:2, 3 or 4, which hybridizes to the nucleic acid molecule of SEQ NO:1. Preferably, primer (D) is also an oligonucleotide indicated by any of SEQ NO:5, 6 or 7, which hybridizes to a complementary strand of SEQ NO:1. Said primers have to hybridize under stringent conditions specific to the target nucleic acid molecule and 3' end thereof have to be a complementary base to the target part of DNA sequence so that the hybridized primers can function as one primer and an extension reaction occurs. Therefore, as long as the primers meet the above requirement, the primers may be an oligonucleotide indicated by any nucleotide sequence of SEQ NOs:2 to 7, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto.

The specific nucleotide sequence common to the target plant genus in ITS-1 or ITS-2 sequence can be identified by obtaining the ITS-1.about.5.8S rRNA gene.about.ITS-2 sequence of a plant(s) in the target plant genus to be detected and other plant genus from GenBank, conducting an alignment and searching a region having a high specificity common to the target plant genus. In addition, among the regions thus identified, a base, which can assure that the base is specific to the target plant genus and not to plants thought to be related species thereof, can be determined as 3' end of the primers to select a primer sequence.

When the target plant genus is the genus Fagopyrum, examples of a commonly specific nucleotide sequences in the ITS-1 sequence thereof include a nucleotide sequence indicated by any of SEQ NO:8, 9 or 10, or a complementary nucleotide sequence thereof Preferably, they include a nucleotide sequence of positions 11 to 61 of the nucleotide sequence of SEQ NO:8 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 11 to 67 of the nucleotide sequence of SEQ NO:9 or a complementary nucleotide sequence thereof In addition, SEQ NO:10 is particularly useful as a region for selecting primers for detecting specifically F. esculentum (common buckwheat), F. tataricum (Tartarian buckwheat), F. homotropicuam and/or F. cyniosum, which are members of the genus Fagopyrum.

Preferably, the primer (A) is an oligonucleotide indicated by any of SEQ NOs:11 to 16 wherein the oligonucleotide indicated by any of SEQ NOs:11 to 14 hybridizes to a complementary strand of SEQ NO:8 and the oligonucleotide indicated by any of SEQ NOs:15 and 16 hybridizes to a nucleic acid molecule of SEQ NO:9. The primer (A) may also be an oligonucleotide indicated by any nucleotide sequence of SEQ NOs:11 to 16, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto. Furthermore, examples of the common specific nucleotide sequence in ITS-2 include a nucleotide sequence indicated by any of SEQ NO:21 or 22, or a complementary nucleotide sequence thereof These nucleotide sequences are particularly useful as a region for selecting primers for detecting specifically F. esculentum (common buckwheat), F. tataricum (Tartarian buckwheat), F. homotropicum and/or F. cymsosum, which are members of the genus Fagopyrum. In addition, it is preferable to use a combination of the primer of any of SEQ NOs:11 to 14 and the primer of any of SEQ NOs:15, 16 or 2 to 4.

When the target plant genus is the genus Arachis, examples of a common specific nucleotide sequences in the ITS-1 sequence thereof include a nucleotide sequence indicated by SEQ NO:17, or a complementary nucleotide sequence thereof. Preferably, they include a nucleotide sequence of positions 1 to 60 of the nucleotide sequence of SEQ NO:17 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 43 to 99 of the nucleotide sequence of SEQ NO:17 or a complementary nucleotide sequence thereof. More preferably, they include a nucleotide sequence of positions 11 to 50 of the nucleotide sequence of SEQ NO:17 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 53 to 89 of the nucleotide sequence of SEQ NO:17 or a complementary nucleotide sequence thereof

Preferably, the primer (A) is an oligonucleotide indicated by any of SEQ NOs:18 to 20, which hybridizes to a complementary strand of SEQ NO:17. The primer (A) may also be an oligonucleotide indicated by any nucleotide sequence of SEQ NOs:18 to 20, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto. Furthermore, examples of the common specific nucleotide sequence in ITS-2 sequence of the genus Arachis include a nucleotide sequence of SEQ NO:23 or a complementary nucleotide sequence thereof Preferably, it is a nucleotide sequence of positions 11 to 47 of the nucleotide sequence of SEQ NO:23 or a complementary nucleotide sequence thereof. Moreover, it is preferable that the primer (B) be an oligonucleotide indicated by SEQ NO:24, which hybridizes to a nucleic acid molecule of SEQ NO:23. The primer (B) may also be an oligonucleotide indicated by any nucleotide sequence of SEQ NO:24, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto. In addition, it is preferable to use a combination of the primer of any of SEQ NOs:18 to 20 and the primer of any of SEQ NOs: 2 to 4, a combination of the primer of any of SEQ NOs:18 to 20 and the primer of SEQ NO:24 or a combination of the primer of SEQ NO:24 and the primer of any of SEQ NOs:5 to 7, and more preferably, a combination of the primer of any of SEQ NOs:18 to 20 and the primer of any of SEQ NOs:2 to 4.

When the target plant genus is genus Tritictim, examples of common specific nucleotide sequences in ITS-2 sequence thereof include a nucleotide sequence indicated by any of SEQ NO:25, 26 or 27, or a complementary nucleotide sequence thereof. Preferably, it is a nucleotide sequence of positions 11 to 50 of the nucleotide sequence of SEQ NO:25 or a complementary nucleotide sequence thereof, a nucleotide sequence of positions 11 to 47 of the nucleotide sequence of SEQ NO:26 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 11 to 47 of the nucleotide sequence of SEQ NO:27 or a complementary nucleotide sequence thereof.

Preferably, the primer (B) is an oligonucleotide indicated by any of SEQ NOs:28 to 30 wherein the oligonucleotide of SEQ NO:28 hybridizes to the complementary strand of SEQ NO:25, the oligonucleotide of SEQ NO:29 hybridizes to the nucleic acid molecule of SEQ NO:26 and the oligonucleotide of SEQ NO:30 hybridizes to the nucleic acid molecule of SEQ NO:27. The primer (B) may also be an oligonucleotide indicated by any nucleotide sequence of SEQ NOs:28 to 30, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto. In addition, it is preferable to use a combination of the primer of SEQ NO:28 and at least one primer selected from the group consisting of SEQ NOs:29 and 30.

When the target plant genus is genus Glycine, examples of commonly specific nucleotide sequences in ITS-2 sequence thereof include a nucleotide sequence indicated by any of SEQ NO:31, 32 or 33, or a complementary nucleotide sequence thereof Preferably, it is a nucleotide sequence of positions 11 to 48 of the nucleotide sequence of SEQ NO:31 or a complementary nucleotide sequence thereof, a nucleotide sequence of positions 11 to 55 of the nucleotide sequence of SEQ NO:32 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 11 to 52 of the nucleotide sequence of SEQ NO:33 or a complementary nucleotide sequence thereof

Preferably, the primer (B) is an oligonucleotide indicated by any of SEQ NOs:34 to 41 wherein the oligonucleotide of SEQ NO:34 hybridizes to a complementary strand of SEQ NO:31, the oligonucleotide of any of SEQ NOs:35 to 40 hybridizes to a nucleic acid molecule of SEQ NO:32 and the oligonucleotide of SEQ NO:41 hybridizes to a nucleic acid molecule of SEQ NO:33. The primer (B) may also be an oligonucleotide indicated by any nucleotide sequence of SEQ NOs:34 to 41, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto. It is preferable to use a combination of the primer of SEQ NO:34 and at least one primer selected from the group consisting of SEQ NOs:35 to 41.

In order to design these primers and to evaluate the designed primers, a PCR simulation may be used.

For example, in order to design the primer for detecting the genus Fagopyrum, a common region having a high specificity for all of the 21 DNA sequences of plants in genus Fagopyrum including eatable buckwheat (common buckwheat and Tartarian buckwheat) is selected from the region of ITS-1.about.5.8S rRNA gene.about.ITS-2 sequence, and further, a base, which can assure the specificity to other plants, is selected as 3' end of the primer to determine the primer sequence. However, the species in the genus Fagopyrum have the ITS-1.about.5.8S rRNA gene.about.ITS-2 sequence from which a part thereof is deleted and from which a number of bases are deleted, which differ from each other, and therefore, it is necessary to conduct further selection in order to obtain a same size of amplification product for the 21 plants in the genus Fagopyrum. If the same size of amplification product can be obtained for the 21 plants in the genus Fagopyrum, the presence of the genus Fagopyrum can be easily detected. In the genus Fagopyrum, particularly by selecting the primer (A) and the primer (C) or two primers (A), the simulation has confirmed that the same size of amplification product would be obtained for all of 21 plants in the genus Fagopyrum. There can be designed primers by which nonspecific products can be easily identified in light of the size of the products.

As mentioned above, regarding the designed primer, it was confirmed by PCR simulation whether or not the target amplification product could be obtained and the results were almost the same as the results of actual PCR, and therefore, the simulation results possess high reliability. In this connection, the above described PCR simulation software, Amplify 1.0 (Bill Engels) and the like can be used in the PCR simulation.

An amplification of DNA sequence using the primers described above can be conducted by PCR methods (Polymerase Chain Reaction: for example, Saiki R K, et al., Science, 230: 1350 1354(1985)), as well as LAMP (Loop-Mediated Isothermal Amplification: Notomi T, et al., Nucleic Acids Res., 28: e 63(2000)) or by other appropriate methods. In addition, though the amplification product is generally detected by electrophoresis, other methods can be used.

EXAMPLES

The present invention will be described more specifically with reference to the following Examples.

Example 1

A. Design of Oligonucleotide Primers for Detection of DNA from Buckwheat

1) DNA Sequences of the Genus Fagopyrum

Regarding the genus Fagopyrum, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 21 DNA sequences registered in GenBank were examined to select suitable regions for the primers. 1: Fagopyrum urophyllum (AB000342) 2: Fagopyrum urophyllu (AB000341) 3: Tartarian buckwheat: Fagopyrum tataricuim (sub_species: potanini) (AB000340) 4: Tartarian buckwheat: Fagopyrum tataricum (AB000339) 5: Fagopyrum statice (AB000338) 6: Fagopyrum statice (AB000337) 7: Fagopyrum pleioramosum (AB000336) 8: Fagopyrum lineare (AB000335) 9: Fagopyrum leptopodum (AB000334) 10: Fagopyrum homotropicum (AB00333) 11: Fagopyrum gracilipes (AB000332) 12: Common buckwheat: Fagopyrum esclilentum ancestralis (AB000331) 13: Common buckwheat: Fagopyrum esculentum (AB000330) 14: Fagopyrum cymosum (AB000329) 15: Fagopyrum cymosum (AB000328) 16: Fagopyrum cymosum (AB000327) 17: Fagopyrum cymosum (AB000326) 18: Fagopyrum cymosum (AB000325) 19: Fagopyrum cymosum (AB00324) 20: Fagopyrum capillatum (AB000323) 21: Fagopyrum callianthum (AB000322) (2) DNA Sequences of other Common Allergenic Plants

As sequences of peanut, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected. 1: peanut: Arachis hypogaea (AF156675) 2: wheat: Triticum aestivum (AJ301799) 3: soybean: Glycine max (U60551) 4: walnut: Juglans regia (AF303809) 5: matsutake mushroom: Thicholoma matsutake (U62964) 6: peach: Prunus persica (AF185621) 7: apple: Malus x domestica (AF186484) 8: Valencia orange: Citrus sp. (E08821) (3) DNA Sequences of Plants Widely Used for a Food Ingredient

As sequences of corn, brown rice, pepper and mustard, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected. 1: corn: Zea mays (U46648) 2: brown rice: Oryza sativa (AF169230) 3: pepper: Piper nigrum (AF275197) 4: mustard: Sinapis alba (X15915) (4) Oligonucleotide Primer Synthesis and Evaluation

Among ITS-1 sequences of the aforementioned 21 DNA sequences of the genus Fagopyrum, there was determined a nucleotide sequence which would specifically hybridize to all of the 21 DNA sequences of the genus Fagopyrum through the study of the ITS-1 sequences. The thus determined nucleotide sequence is indicated as SEQ NO:11. Subsequently, the oligonucleotide primer with SEQ NO:11 was synthesized.

TABLE-US-00001 Sense primer 5'-GGA CCA CGA ACA GAA GCG CGT CCC G-3' (SEQ NO:11)

From among 5.8S rRNA gene sequences of the aforementioned 21 DNA sequences of the genus Fagopyrum and 8 DNA sequences of other common allergenic plants, there was determined a nucleotide sequence which would hybridize to all of these sequences through the study of the sequences. The thus determined nucleotide sequence is indicated as SEQ NO:3. Subsequently, the oligonucleotide primer with the SEQ NO:3 was synthesized.

TABLE-US-00002 Antisense primer (SEQ NO:3) 5'-ATC GCA TTT CGC TAC GTT CTT CAT CG-3'

Regarding the sense and antisense primer pair, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels). As a result, it was predicted that target 140 bp amplification products would be obtained from the aforementioned 21 DNA sequences of the genus Fagopyrum. In contrast, no 140 bp amplification product was predicted from the aforementioned 8 DNA sequences of common allergenic plants other than genus Fagopyrum (peanut, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange) and the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard). However, the results of the simulation indicated some possibility that nonspecific amplification products, which were different from the target one in size, would be obtained from soybean, apple and orange in light of weak amplified signals. On the other hand, no amplification product was predicted from the 5 DNA sequences of other common allergenic plants (peanut, wheat, walnut, matsutake mushroom and peach) and the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard). The simulation results are shown in Tables 1A and 1B. The meanings of symbols and numerical values in Tables 1A and 1B are explained below. .star-solid.: An obtained amplification product whose size almost matched to the target product size 140 bp (.+-.10 bp), which would be obtained. W 2 6: Probability of obtaining amplification products

High Probability - - - W6>W5>W4>W3>W2 - - - Low Probability Numerical values followed by bp:

Each value was obtained by subtracting 2 from the value obtained in the simulation. (-): No amplification product was predicted.

TABLE-US-00003 TABLE 1A SEQ NO:11 & SEQ NO:3 primer: Amplification products Scientific Name GenBank (Common Name) Accession Genus Fagopyrum No. W6 W5 W4 W3 W2 .star-solid.Fagopyrum AB000342 140 -- -- -- -- urophyllum bp .star-solid.Fagopyrum AB000341 140 -- -- -- -- urophyllum bp .star-solid.Fagopyrum tataricum AB000340 140 -- 64 -- -- (Tartarian buckwheat) bp bp .star-solid.Fagopyrum tataricum AB000339 140 -- 64 -- -- (Tartarian buckwheat) bp bp .star-solid.Fagopyrum statice AB000338 140 -- -- -- -- bp .star-solid.Fagopyrum statice AB000337 140 -- -- -- -- bp .star-solid.Fagopyrum AB000336 140 -- -- -- -- pleioramosum bp .star-solid.Fagopyrum lineare AB000335 140 -- -- -- -- bp .star-solid.Fagopyrum AB000334 140 -- -- -- -- leptopodum bp .star-solid.Fagopyrum AB000333 140 -- 326 -- -- homotropicum bp bp .star-solid.Fagopyrum gracilipes AB000332 140 -- -- -- -- bp .star-solid.Fagopyrum esculentum AB000331 140 -- 326 -- -- (Common buckwheat) bp bp .star-solid.Fagopyrum esculentum AB000330 140 -- 325 -- -- (Common buckwheat) bp bp .star-solid.Fagopyrum cymosum AB000329 140 -- -- 333 -- bp bp .star-solid.Fagopyrum cymosum AB000328 140 -- -- 321 -- bp bp .star-solid.Fagopyrum cymosum AB000327 140 -- -- 321 -- bp bp .star-solid.Fagopyrum cymosum AB000326 140 -- -- -- -- bp .star-solid.Fagopyrum cymosum AB000325 140 -- -- 321 -- bp bp .star-solid.Fagopyrum cymosum AB000324 140 -- -- 333 -- bp bp .star-solid.Fagopyrum capillatum AB000323 140 -- -- -- -- bp .star-solid.Fagopyrum AB000322 140 -- -- -- -- callianthum bp

TABLE-US-00004 TABLE 1B SEQ NO:11 & SEQ NO:3 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Other Common Allergenic Plants Arachis hypogaea AF156675 -- -- -- -- -- (Peanut) Triticum aestivum AJ301799 -- -- -- -- -- (Wheat) Glycine max (Soybean) U60551 -- -- -- 227 -- bp Juglans regia (Walnut) AF303809 -- -- -- -- -- Tricholoma matsutake U62964 -- -- -- -- -- (Matsutake mushroom) Prunus persica (Peach) AF185621 -- -- -- -- -- Malus x domestica AF186484 -- -- 275 -- -- (Apple) bp Citrus sp. (Valencia E08821 -- -- 312 -- -- orange) bp 215 bp Plants Widely Used for a Food Ingredient Zea mays (Corn) U46648 -- -- -- -- -- Oryza sativa AF169230 -- -- -- -- -- (Brown rice) Piper nigrum (Pepper) AF275197 -- -- -- -- -- Sinapis alba (Mustard) X15915 -- -- -- -- --

B. Preparation of Template DNA for PCR (1) Samples Used for DNA Isolation Buckwheat:

Commercially available seeds of Shirahana soba (common buckwheat) and Dattan soba (Tartarian buckwheat) were used.

Buckwheat Chaff:

Commercially available buckwheat chaff used for pillows was used.

Pepper:

Commercially available seeds of black pepper and white pepper were used.

Soybean, Wheat, Corn and Mustard:

Leaves that germinated from commercially available seeds of soybean, wheat, corn and mustard were used.

Preparation of Black Pepper Powder Containing Buckwheat Chaff:

0.1 g of ground buckwheat chaff was added to 0.9 g of ground black pepper to obtain black pepper powder containing 10% buckwheat chaff.

(2) Isolation of DNA from Buckwheat, Buckwheat Chaff, Black Pepper, White Pepper and Black Pepper Powder Containing Buckwheat Chaff

DNA isolation was conducted by using the QIAGEN Genomic-tip according to the procedures described in the Genomic DNA Handbook with a few modifications thereto as stated below.

Into a 15 ml-tube was transferred 1 g of a ground sample, added 4 ml of Carlson Lysis Buffer (0.1 M Tris-HCl (pH 9.5), 2% CTAB, 1.4 M Polyethylene Glycol# 6000, 20 mM EDTA), 8 .mu.l of RNase A (100 mg/ml), 10 .mu.l of 2-mercaptoethanol and 80 .mu.l of proteinase K (20 mg/ml) thereto and mixed, and the resulting mixture was incubated for 20 min. at 74.degree. C.

After cooling down to room temperature, to the mixture was added 5 ml of phenol/chloroform/isoamyl alcohol (25/24/1) at room temperature and mixed well by inverting the tube. After centrifuging them, a resulting upper water layer, was collected. The water layer was mixed well with the same volume of chloroform/isoamyl alcohol (24/1) and after centrifuging, a resulting upper water layer was collected. The water layer was mixed well with chloroform/isoamyl alcohol (24/1), and after centrifuging, a resulting water layer was collected again and used in the next step.

Half of the volume of the water layer obtained above was subjected to isopropanol precipitation to collect crude DNA. The collected crude DNA was dissolved in 500 .mu.l of Buffer QBT and the resulting solution was applied to the Genomic-tip 20/G column equilibrated with 1 ml of Buffer QBT to adsorb DNA. Subsequently, the column was washed with 5 ml of Buffer QBT and then with 2 ml of Buffer QC. Finally, DNA was eluted with 1.7 ml of Buffer QF, and the resulting eluate was subjected to isopropanol precipitation to collect DNA, which was then dissolved in 40 .mu.l of sterilized ultrapure water. After the concentration of the resulting DNA preparation was determined, the DNA preparation was used for a PCR template.

(3) Isolation of DNA from Leaves of Wheat, Soybean, Corn and Mustard

DNA isolation was conducted by using the QIAGEN DNeasy Plant Mini Kit according to the procedures described in the DNeasy Plant Mini Kit Handbook mentioned below.

0.5 g of a ground sample was transferred to a 15 ml-tube, added 3 ml of Buffer AP1 and 30 .mu.l of RNase A (100 mg/ml), and mixed well with them. Then the resulting mixture was incubated for 15 min. at 65.degree. C. 975 .mu.l of Buffer AP2 was added to the mixture. The resulting mixture was incubated for 10 min. on ice and then centrifuged to obtain a supernatant. The supernatant was applied to a QIAshredder Spin Column and a flow-through fraction was obtained by centrifuging the column. To the flow-through fraction was added 0.5 volume of Buffer AP3 and 1 volume of ethanol, and mixed. The resulting mixture was divided into halves to be applied to two separate DNeasy Spin Columns. 650 .mu.l of the mixture was applied to a DNeasy Spin Column and the column was centrifuged for 1 min. at 6,000.times.g to adsorb DNA. This step was repeated with the remaining mixture. In order to wash the column, to the column was added 500 .mu.l of Buffer AW and was centrifuged for 1 min. at 6,000.times.g. To the column was added 500 .mu.l of Buffer AW again and was centrifuged for 1 min. at a maximum speed to flush out the remaining Buffer AW. Finally, to the column was added 120 .mu.l of preheated (65.degree. C.) Buffer AE and was centrifuged for 1 min. at 6,000.times.g to obtain a DNA eluate. After the concentration thereof was determined, the DNA eluate was used for a PCR template.

(4) Preparation of DNA Solutions for Evaluation of Sensitivity (Buckwheat in Black Pepper Powder)

A DNA preparation derived from black pepper powder containing 10% buckwheat chaff was diluted stepwise with a DNA preparation from black pepper to obtain black pepper DNA solutions containing 1%, 0.1%, 100 ppm, 10 ppm, 1 ppm, 100 ppb, and 10 ppb of buckwheat chaff DNA. Both DNA preparations used above were obtained according to the procedures described in (2).

(5) Preparation of DNA Solutions for Evaluation of Sensitivity (Buckwheat in Wheat)

A DNA preparation derived from buckwheat seeds was diluted stepwise with a DNA preparation from wheat leaves to obtain wheat leaf DNA solutions containing 1 ppm, 100 ppb, 10 ppb, and 1 ppb of buckwheat seed DNA. The DNA preparation from buckwheat seeds was obtained according to the procedures described in (2). The DNA preparation from wheat leaves was obtained according to the procedures described in (3).

C. PCR

PCR was conducted using the QIAGEN HotStarTaq Master Mix Kit according to the procedures described in the HotStarTaq PCR Handbook as stated below.

PCR was carried out using final volumes of 25 .mu.l of a solution containing 12.5 .mu.l of 2.times. HotStarTaq Master Mix (HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl.sub.2, 400 .mu.M each dNTP), 0.2 .mu.M of each primer (SEQ NO:11 and SEQ NO:3), the template DNA and sterilized ultrapure water in 0.2-ml microcentrifuge tubes. Amplification was performed using a GeneAmp PCR System 9600 (Applied Biosystems) according to the following PCR program: pre-incubation at 95.degree. C. for 15 min.; 45 cycles consisting of denaturation at 95.degree. C. for 1 min., annealing at 68.degree. C. for 2 min. and extension at 72.degree. C. for 1 min.; followed by a final extension at 72.degree. C. for 4 min. The PCR reaction mixture was electrophoresed on a 2% agarose gel containing ethidium bromide. After the electrophoresis, the gel was analyzed using a FluorImager 595 (Amersham Pharmacia Biotech). The results are shown in FIGS. 1 3. The meanings of numerical values, abbreviations and symbols used in FIGS. 1 3 are as follows. M: 100 bp DNA Ladder Marker (-): Negative control (no DNA) Numerical values above sample names: the amounts of the template DNA Arrow: indicates the target amplification product (140 bp)

The quality of each of the template DNA used here was sufficient enough to be used for PCR based on the result of a separate PCR, in which target products were obtained using a primer pair to amplify a part of a plant chloroplast DNA.

D. PCR Results

PCR described above was conducted using primers designed in the present invention. The results are shown in FIGS. 1 3. As shown in FIG. 1, target 140 bp amplification products, predicted from the simulation results of the 21 DNA sequences of the genus Fagopyrum, were obtained from Shirahana soba (common buckwheat) and Dattan soba (Tartarian buckwheat). On the other hand, no 140 bp amplification product was obtained from wheat, mustard, soybean, corn, and white pepper. However, nonspecific amplification products whose sizes were different from the target product were obtained from soybean (approximately 230 bp) and wheat (approximately 2,300 bp) Because these results almost matched the simulation results shown in Table 1, the simulation results were thought to be reliable. Consequently, it was confirmed that a wide range of plants in the genus Fagopyrum including common buckwheat and Tartarian buckwheat were detectable using the present invention.

As shown in FIG. 2, target 140 bp amplification products, predicted from the simulation results of the 21 DNA sequences of the genus Fagopyrum, were obtained from black pepper powder samples containing 10 to 1 ppm of buckwheat chaff. This result showed that buckwheat chaff present in an amount of more than 1 ppm in black pepper is detectable.

As shown in FIG. 3, the target 140 bp amplification products, predicted from the simulation results of the 21 DNA sequences of the genus Fagopyrum, were obtained from the wheat DNA sample containing 10 to 1 ppm of buckwheat DNA.

The nonspecific amplification product that was obviously different from the target product in size did not interfere in the detection of 1 ppm of buckwheat DNA. This result showed that buckwheat DNA present in an amount of more than 1 ppm in wheat DNA is detectable.

E. Preparation of a Sequencing Sample

(1) Purification of the Amplification Product from Buckwheat Chaff

Purification of the amplification product from buckwheat chaff obtained in section D was conducted by using the QIAGEN QIAquick PCR Purification Kit according to the procedures described in the QIAquick Spin Handbook as stated below.

To 1 volume of PCR reaction mixture was added 5 volumes of Buffer PB and mixed well. After being spun down by centrifugation, to a QIAquick Spin Column was applied the mixture and centrifuged for 1 min. at 10,000.times.g to adsorb DNA. Then, to the column was added 750 .mu.l of buffer PE to wash and centrifuged for 1 min. at 10,000.times.g. In addition, the column was centrifuged for 1 min. at 10,000.times.g to