Patent Application
20080280304
The 45S rRNA precursor gene sequence (Small Subunit ribosomal RNA (SSU rRNA) gene-Internal Transcribed Spacer-1 (ITS-1)˜5.8S ribosomal RNA (5.8S rRNA) gene˜Internal Transcribed Spacer-2 (ITS-2)˜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.
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, 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 Fagopyrum (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×SSC, 50° C.) are conducted on the basis of Tm value obtained by the following equation.
Tm=81.5+16.6(log10[Na+])+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.
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 (S.), 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 R K, et al., Science, 230: 1350-1354 (1985) and Shyokubutsu no PCR Zikken Protocol—Idenshi no Tanri—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° C., and subsequently at an annealing temperature near the Tm value, preferably the Tm value plus 7 to 0° C., wherein the Tm value is determined by commercially available software such as HYB Simulator™ version 4.0 (Advanced Gene Computing Technologies, Inc.) and Primer Express™ 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 (13) 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 (1B) 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 (13), 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∩Kakusan∩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˜5.8S rRNA gene˜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. homotropicum and/or F. cymosum, 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. cymosum, 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 (13) 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 Triticum, 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˜5.8S rRNA gene˜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˜5.8S rRNA gene˜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.
The present invention will be described more specifically with reference to the following Examples.
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 urophyllum (AB000341)
3: Tartarian buckwheat: Fagopyrum tataricum (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 (AB000333)
11: Fagopyrum gracilipes (AB000332)
12: Common buckwheat: Fagopyrum esculentum 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 (AB000324)
20: Fagopyrum capillatum (AB000323)
21: Fagopyrum callianthum (AB000322)
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: 7 Ticholoma matsutake (U62964)
6: peach: Prunus persica (AF185621)
7: apple: Malus×domestica (AF186484)
8: Valencia orange: Citrus sp. (E08821)
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)
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.
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.
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.
★: 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.
Arachis hypogaea (Peanut)
Triticum aestivum (Wheat)
Glycine max (Soybean)
Juglans regia (Walnut)
Tricholoma matsutake
Prunus persica (Peach)
Malus x domestica (Apple)
Citrus sp. (Valencia orange)
Zea mays (Corn)
Oryza sativa (Brown rice)
Piper nigrum (Pepper)
Sinapis alba (Mustard)
Commercially available seeds of Shirahana soba (common buckwheat) and Dattan soba (Tartarian buckwheat) were used.
Commercially available buckwheat chaff used for pillows was used.
Commercially available seeds of black pepper and white pepper were used.
Leaves that germinated from commercially available seeds of soybean, wheat, corn and mustard were used.
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 μl of RNase A (100 mg/ml), 10 μl of 2-mercaptoethanol and 80 μl of proteinase K (20 mg/ml) thereto and mixed, and the resulting mixture was incubated for 20 min. at 74° 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 μl 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 μl 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 μl of RNase A (100 mg/ml), and mixed well with them. Then the resulting mixture was incubated for 15 min. at 65° C. 975 μl 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 μl of the mixture was applied to a DNeasy Spin Column and the column was centrifuged for 1 min. at 6,000×g to adsorb DNA. This step was repeated with the remaining mixture. In order to wash the column, to the column was added 500 μl of Buffer AW and was centrifuged for 1 min. at 6,000×g. To the column was added 500 μL 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 μl of preheated (65° C.) Buffer AE and was centrifuged for 1 min. at 6,000×g to obtain a DNA eluate. After the concentration thereof was determined, the DNA eluate was used for a PCR template.
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).
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).
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 μl of a solution containing 12.5 μl of 2× HotStarTaq Master Mix (HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, 400 μM each dNTP), 0.2 μM 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° C. for 15 min.; 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 68° C. for 2 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° 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
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.
PCR described above was conducted using primers designed in the present invention. The results are shown in
As shown in
As shown in
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.
(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×g to adsorb DNA. Then, to the column was added 750 μl of buffer PE to wash and centrifuged for 1 min. at 10,000×g. In addition, the column was centrifuged for 1 min. at 10,000×g to remove Buffer PE completely. Finally, to the column was added 50 μl of Buffer EB, let stand for 1 min., and then centrifuged for 1 min. at 10,000×g. The resulting eluted DNA was used for a sequencing sample.
PCR for sequencing of the purified amplification product from buckwheat chaff obtained in (1) was conducted by using the Applied Biosystems BigDye Terminator Cycle Sequencing FS Ready Reaction Kit according to the procedures described in the manufacturer's manual as stated below.
The PCR for sequencing was carried out using final volumes of 20 μl of a solution containing 8 μl of BigDye Terminator RR Mix, 3.2 μmol of primer (SEQ NO:3), 2 ng 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 96° C. for 1 min.; 25 cycles consisting of denaturation at 96° C. for 10 secs. and annealing and extension at 60° C. for 1 min.
Subsequently, removal of the excess dye-labeled dideoxynucleotides from the sequencing reaction mixture was conducted by using the Amersham Pharmacia Biotech AutoSeq G-50 according to the procedures described in the manufacturer's manual as stated below.
The AutoSeq G-50 column was uncapped and 100 μl of 10 mM EDTA was added to the resin in the column. The column was then capped and the resin inside was suspended thoroughly by vortexing. The cap was then loosened and the bottom closure of the column was snapped off. The column was then uncapped and placed in a 2-ml microcentrifuge tube for support and centrifuged for 1 min. at 2,000×g. The column was then placed in a new 2-ml microcentrifuge tube and the sample was applied to the resin in the column. After being capped, the column was centrifuged for 1 min. at 2,000×g, and the flow-through fraction obtained was used in the next step.
The sample for sequencing from buckwheat chaff obtained in E (2) was analyzed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The obtained nucleotide sequence of the amplification product was compared with the sequence of common buckwheat, Fagopyrum esculentum (AB000330), registered in GenBank. The result is shown in
Number Symbols Nucleotide numbers of amplification products
Asterisks (*): Identical nucleotides between two nucleotide sequences
Dash (-): Unidentifiable nucleotide
S: Mixed nucleotide with C and G
Single underline: The sense primer region
Double underline: The antisense primer region
As shown in
The DNA sequences described in Example 1 (1) “DNA Sequences of the Genus Fagopyrum”, (2) “DNA Sequences of Other Common Allergenic Plants” and (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” were examined to select suitable regions for the primers.
As representatives of the DNA sequences of related species of the genus Fagopyrum, 5.8S rRNA gene, 1TS-1 and ITS-2 sequences in the following 27 DNA sequences registered in GenBank were selected. In this connection, the 27 DNA sequences were selected as representatives of the DNA sequences of related species of the genus Fagopyrum, each of which had the highest score in the corresponding genus other than genus Fagopyrum and a score of 60 bits or more among sequences of species belonging to the corresponding genus selected from sequences registrated in GenBank through a BLAST homology search using the ITS-1 sequence of buckwheat (Fagopyrum esculentum AB000330).
1: Aconogonum sp. Won 152 (AF189731)
2: Fallopia scandens (AF040069)
3: Polygonum virginianum (U51274)
4: Rumex acetosella (AF189730)
5: Talinum paraguayense (L78056)
6: Bruinsmia styracoides (AF396438)
7: Talinella pachypoda (L78054)
8: Rehderodendron kwangtungense (AF396448)
9: Pterostyrax corymbosus (AF396445)
10: Anredera cordifolia (L78086)
11: Cistanthe quadripetala (L78062)
12: Xenia vulcanensis (L78060)
13: Talinopsis frutescens (L78058)
14: Talinaria palmeri (L78052)
16: Phemeranthus confertiflorus (L78039)
17: Montiopsis umbellata (L78033)
18: Grahamia bracteata (L78028)
19: Herniaria glabra (AJ310965)
20: Alluaudia duwosa (L78011)
21: Sinojackia xylocarpa (AF396451)
22: Halesia macgregori (AF396442)
23: Changiostyrax dolichocarpa (AF396439)
24: Alectryon subdentatus (AF314765)
25: Anacampseros recurvata (L78014)
26: Weinmannia racemosa (AF485597)
27: Bursera tecomaca (AF080029)
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 and would not induce nonspecific amplification products from soybean through the study of the ITS-1 sequences. The thus determined nucleotide sequence is indicated as SEQ NO: 14. Subsequently, the oligonucleotide primer with SEQ NO:14 was synthesized.
The oligonucleotide primer with SEQ NO:3 was also used as an antisense primer, the same as in Example 1.
Regarding the sense and antisense primer pair, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1. As a result, it was predicted that target 146 bp amplification products would be obtained from the aforementioned 21 DNA sequences of the genus Fagopyrum. In contrast, obtaining of any 146 bp amplification product was not predicted to be obtained from the aforementioned 8 DNA sequences of other common allergenic plants (peanut, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used as a food ingredient (corn, brown Lice, pepper and mustard) and the 2 DNA sequences among related species of the genus Fagopyrum belonging to Polygonaceae and the 23 DNA sequences of related species of the genus Fagopyrum not belonging to Polygonaceae. In this connection, the results of simulation indicated some possibility that an amplification product, whose size almost matched the target product size of 146 bp, would be obtained from the sequences of Aconogonum sp. Won 152 and Fallopia scandens in the related species of the genus Fagopyrum belonging to Polygonaceae. However, by sequence analysis of the amplification products, it is possible to identify either the genus Fagopyrum or the related species thereof. The simulation results are shown in Tables 2A to 2C. The meanings of symbols and numerical values in Tables 2A to 2C are explained below.
★: An obtained amplification product whose size almost matched the target product size 146 bp (±10 bp).
W 2-6: Probability of obtaining amplification products
High Probability—W6>W5>W4>W3>W—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.
Related Species of the genus Fagopyrum:
Sequences similar to the ITS-1 sequence of Fagopyrum esculentum (AB000330) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected from among them. Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Table 2C as the representative of the DNA sequences of related species of the genus Fagopyrum.
Arachis hypogaea (Peanut)
Triticum aestivum (Wheat)
Glycine max (Soybean)
Juglans regia (Walnut)
Tricholoma matsutake
Prunus persica (Peach)
Malus x domestica (Apple)
Citrus sp. (Valencia orange)
Zea mays (Corn)
Oryza sativa (Brown rice)
Piper nigrum (Pepper)
Sinapis alba (Mustard)
Fagopyrum Belonging to
Polygonum virginianum
Rumex acetosella
Talinum paraguayense
Bruinsmia styracoides
Talinella pachypoda
Rehderodendron kwangtungense
Pterostyrax corymbosus
Anredera cordifolia
Cistanthe quadripetala
Xenia vulcanensis
Talinopsis frutescens
Talinaria palmeri
Portulaca sp.
Phemeranthus confertiflorus
Montiopsis umbellata
Grahamia bracteata
Herniaria glabra
Alluaudia dumosa
Sinojackia xylocarpa
Halesia macgregori
Changiostyrax dolichocarpa
Alectryon subdentatus
Anacampseros recurvata
Weinmannia racemosa
Bursera tecomaca
The DNA samples isolated from buckwheat, pepper, wheat, soybean, corn and mustard in Examples 1B (2) and (3) and the DNA solutions for evaluation of sensitivity prepared in Example 1B (5) were used.
PCR was conducted in the same way as in Example 1C, except for the use of the following primer and PCR program.
Each primer of SEQ NO: 14 and SEQ NO:3 was used at 0.2 μM of final concentration.
PCR was conducted according to the following PCR program.
Pre-incubation at 95° C. for 15 min.; thereafter 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 66° C. for 2 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 mm.
The results are shown in
(−): Negative control (no DNA)
Numerical values above sample names: the amounts of the template DNA
Arrow: indicating the target amplification product (146 bp)
The quality of each of the template DNAs 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 plant chloroplast DNA.
PCR described above was conducted using primers designed in the present invention. The results are shown in
As shown in
The nonspecific amplification product that was obviously different from the target 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.
(1) Purification of the Amplification Product from Shirahana Soba (Common Buckwheat)
Purification of the amplification product from Shirahana soba (common buckwheat) obtained in D was conducted in the same way as in Example 1E
(1) “Purification of the Amplification Product from Buckwheat Chaff”.
PCR for sequencing of the purified amplification product from Shirahana soba (common buckwheat) obtained in (1) was conducted in the same way as in Example 1E (2) “Sequencing PCR Reaction and Removal of Excess Dye-Labeled Dideoxynucleotides”, except for the use of primers of SEQ NO:14 and SEQ NO:3).
The sample for sequencing from Shirahana soba (common buckwheat) obtained in E (2) was analyzed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The obtained nucleotide sequence of the amplification product was compared with the sequence of common buckwheat, Fagopyrum esculentum (AB000331) and F. homotropicum (AB000340) in GenBank. The result is shown in
Number Symbols: Nucleotide numbers of amplification products
Asterisks (*): Nucleotides of Shirahana soba (common buckwheat), which are identical with those of both nucleotide sequences of F. esculentum (AB000331) and F. homotropicum (AB000340)
*: Nucleotides of Shirahana soba (common buckwheat), which are identical with those of only one of the nucleotide sequences of F. esculentum (AB000331) and F. homotropicum (AB000340)
Single underline The sense primer region
Double underline The antisense primer region
As shown in
Regarding the genus Arachis, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 11 DNA sequences registered in GenBank were examined to select suitable regions for the primers.
1: Arachis batizocoi (AF203553)
2: Arachis correntina (AF203554)
3: Arachis hermannii (AF203556)
4: Arachis hoehnei (AJ320395)
5: Arachis hypogaea (AF156675)
6: Arachis magna (AF203555)
7: Arachis major (AF203552)
8: Arachis palustris (AF203557)
9: Arachis pintoi (AF203551)
10: Arachis triseminata (AF204233)
11: Arachis villosa (AF203558)
The DNA sequences described in Example 1A (2) “DNA Sequences of Other Common Allergenic Plants” were selected. Regarding buckwheat, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequence registered in GenBank were also selected.
1: buckwheat: Fagopyrum esculentum (AB000330)
The DNA sequences described in Example 1A (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” were selected.
Regarding French bean, lima bean, lentil, chickpea, mung bean and adzuki bean, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected. In the case of adzuki bean, only the ITS-1 sequence of Vigna angularis var. nipponensis (AB059747) was selected because the 5.8S rRNA gene sequence was not registered in GenBank.
1: French bean: Phaseolus vulgaris (AF115169)
2: lima bean: Phaseolus lunatus (AF115175)
3: lentil: Lens culinaris subsp. culinaris (AF228066)
4: chickpea: Cicer arietinum (AJ237698)
5: mung bean: Vigna radiata (X14337)
6: adzuki bean: Vigna angularis var. nipponensis (AB059747)
As representatives of the DNA sequences of related species of the genus Arachis, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 69 DNA sequences registered in GenBank were selected. In this connection, the 69 DNA sequences were selected as representatives of the DNA sequence of related species of the genus Arachis, each of which had the highest score in the corresponding genus other than genus Arachis and a score of 60 bits or more among sequences of species belonging to the corresponding genus selected from ITS-1 sequence of buckwheat through BLAST homology search. (Arachis hypogaea AF 156675)
1: Stylosanthes acuminata (AJ320282)
2: Stylosanthes angustifolia (AJ320284)
3: Stylosanthes aurea (AJ320285)
4: Stylosanthes biflora (AJ320289)
5: Stylosanthes bracteata (AJ320346)
6: Stylosanthes calcicola (AJ320348)
7: Stylosanthes campestris (AJ320291)
8: Stylosanthes capitata (AJ320350)
9: Stylosanthes cayennensis (AJ320292)
10: Stylosanthes erects (AJ320352)
11: Stylosanthes fruticosa (AJ320356)
12: Stylosanthes gracilis (AJ320296)
13: Stylosanthes grandifolia (AJ320299)
14: Stylosanthes guianensis subsp. dissitiflora (AJ320301)
15: Stylosanthes hamata (AJ320365)
16: Stylosanthes hippocampoides (AJ320317)
17: Stylosanthes hispida (AJ320328)
18: Stylosanthes humilis (AJ320323)
19: Stylosanthes ingrata (AJ320329)
20: Stylosanthes leiocarpa (AJ320332)
21: Stylosanthes linearifolia (AJ320367)
22: Stylosanthes macrocarpa (AJ320369)
23: Stylosanthes macrocephala (AJ320371)
24: Stylosanthes macrosoma (AJ320333)
25: Stylosanthes mexicana (AJ320374)
26: Stylosanthes montevidensis (AJ320336)
27: Stylosanthes pilosa (AJ320377)
28: Stylosanthes scabra (AJ320382)
29: Stylosanthes seabrana (AJ320384)
30: Stylosanthes sericeiceps (AJ320386)
31: Stylosanthes subsericea (AJ320387)
32: Stylosanthes sundaica (AJ320389)
33: Stylosanthes sympodialis (AJ320391)
34: Stylosanthes tomentosa (AJ320337)
35: Stylosanthes tuberculata (AJ320392)
36: Stylosanthes viscosa (AJ320340)
37: Ormocarpum bernierianum (AF189036)
38: Ormocarpum coeruleum (AF189037)
39: Ormocarpum drakei (AF189039)
40: Ormocarpum flavum (AF189041)
41: Ormocarpum keniense (AF068155)
42: Ormocarpum kirkii (AF068152)
43: Ormocarpum klainei (AF189044)
44: Ormocarpum megalophyllum (AF068154)
45: Ormocarpum muricatum (AF068156)
46: Ormocarpum orientale (AF068159)
47: Ormocarpum pubescens (AF189045)
48: Ormocarpum rectangulare (AF189046)
49: Ormocarpum schliebenii (AfF189047)
50: Ormocarpum sennoides (AF068153)
51: Ormocarpum somalense (AF 189048)
52: Ormocarpum trachycarpum (AF189049)
53: Ormocarpum trichocarpum (AF068158)
54: Ormocarpum verrucosum (AF189050)
55: Chapmannia floridana (AF203543)
56: Chapmannia prismatica (AJ320400)
57: Chapmannia somalensis (AF203544)
58: Ormocarpopsis aspera (AF068148)
59: Ormocarpopsis calcicola (AF068145)
60: Ormocarpopsis itremoensis (AF068149)
61: Ormocarpopsis mandrarensis (AF068147)
62: Ormocarpopsis parvifolia (AF068144)
63: Ormocarpopsis tulearensis (AF068146)
64: Diphysa humilis (AF068162)
65: Diphysa macrophylla (AF189029)
66: Diphysa suberosa (AF189034)
67: Spigelia coelostylioides (AF177992)
68: Spigelia hedyotidea (AF 178005)
69: Spigelia marilandica (AF177991)
Among ITS-1 sequences of the aforementioned 11 DNA sequences of the genus Arachis, there was determined three nucleotide sequences which would specifically hybridize to all of the 11 DNA sequences of the genus Arachis through the study of the ITS-1 sequences. The thus determined nucleotide sequences are indicated as SEQ NOs:18, 19 and 20. Subsequently, the oligonucleotide primers with SEQ NOs:18, 19 and 20 were synthesized.
As an antisense primer, the oligonucleotide primer with SEQ NO:3 was also used, the same as in Example 1.
Regarding the sense and antisense primer pairs, a simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), in the same manner as in Example 1. As a result, it was predicted that 156 to 157 bp (a combination of the primers with SEQ NOs:18 and 3), 114 to 116 bp (a combination of the primers with SEQ NOs:19 and 3) and 113 to 115 bp (a combination of the primers with SEQ NOs:20 and 3) of target amplification products would be obtained from the aforementioned 11 DNA sequences of the genus Arachis.
Furthermore, regarding the sense and antisense primer pairs, it was predicted whether amplification products would be obtained from the aforementioned 8 DNA sequences of common allergenic plants other than peanut (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used as a food ingredient (corn, rise, pepper and mustard) and the 6 DNA sequences of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean).
Regarding the combination of the primers with SEQ NOs:18 and 3, the result of a simulation indicated that desired amplification products having almost 156 bp would not be obtained from 7 DNA sequences of common allergenic plants other than peanut (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach and orange), the 4 DNA sequences of plants widely used as a food ingredient (corn, brown rice, pepper and mustard) and the 6 DNA sequences of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean). In this connection, the results of the simulation indicated some possibility that amplification products having almost 156 bp would be obtained from apple from among the other common allergenic plants. However, by a sequence analysis of the amplification products, it is possible to identify either peanut or apple. The simulation results are shown in Tables 3A and 3B. The meanings of symbols and numerical values in Tables 3A and 3B are explained below.
★: An obtained amplification product whose size almost matched the target product size 156 bp (±10 bp).
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.
(−*): No annealing site of the primer (SEQ NO: 18) was predicted within the ITS-1 sequence of Vigna angularis var. nipponensis (adzuki bean).
In the case of adzuki bean, only the ITS-1 sequence was selected because the 5.8S rRNA gene sequence of Vigna angularis var. nipponensis (AB059747) was not registered in GenBank.
Fagopyrum esculentum
Triticum aestivum
Glycine max
Juglans regia
Tricholoma matsutake
Prunus persica
Citrus sp.
Zea mays
Oryza sativa
Piper nigrum
Sinapis alba
Phaseolus vulgaris
Phaseolus lunatus
Lens culinaris subsp.
culinaris
Cicer arietinum
Vigna radiata
Vigna angularis var.
nipponensis
Regarding the combination of the primers with SEQ NOs:20 and 3, no amplification product having almost 114 bp was predicted from 6 DNA 5 sequences of common allergenic plants other than peanut (wheat, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used as a food ingredient (corn, brown lice, pepper and mustard) and the 5 out of 6 DNA sequences of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea and mung bean). In this connection, the results of the simulation indicated some possibility that nonspecific amplification products having almost 114 bp would be obtained from buckwheat and from soybean from among the other common allergenic plants and from adzuki bean in the leguminous plants widely used for a food ingredient in even weak amplified signals. Here, regarding the DNA sequence of adzuki bean (Vigna angulans var. nipponensis AB059747), as the ITS-1 sequence was registered in GenBank, but the 5.8S rRNA gene sequence was not registered therein, the amplification product having almost 100 bp was estimated based on the predicted annealing site of SEQ NO: 20 to the ITS-1 sequence and the assumption that Vigna angulans var. nipponensis (adzuki bean) had the same 5.8S rRNA gene sequence as Arachis hypogaea (peanut) and the primer with SEQ NO: 3 had an annealing site within the 5.8S rRNA gene sequence. However, despite the prediction of amplification, the probability of obtaining these amplification products compared with obtaining the target amplification product of the genus Arachis were lower than the probability of obtaining amplification for peanut, and by a sequence analysis of the amplification products, it is also possible to identify whether they are peanut or not.
In addition, regarding the combination of the primers with SEQ NOs:20 and 3, no amplification products having almost 100 bp were predicted from the 69 DNA sequences of related species of both the genus Arachis belonging to leguminous plants and those not belonging to leguminous plants. The simulation results are shown in Tables 4A to 4E. The meanings of symbols and numerical values in Tables 4A to 4E are explained below.
As to the simulation in which SEQ NO: 19 and SEQ NO: 3 were used, the result is not shown here because it was later found that this primer pair was not suitable for PCR analysis to detect the genus Arachis.
★: An obtained amplification product whose size almost matched the target 114 bp (±10 bp).
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.
Related Species of the genus Arachis:
Sequences similar to the ITS-1 sequence of Arachis hypogaea (AF156675) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected among them. Each sequence having highest score in each genus and having a score of 60 bits or more is shown in the following Tables 4D-4E as the representatives of the DNA sequences of related species of the genus Arachis.
(+*): An annealing site of the primer (SEQ NO: 20) was predicted within the ITS-1 of Vigna angularis var. nipponensis (adzuki bean).
In the case of adzuki bean, only the ITS-1 sequence registered in GenBank was used because the 5.8S rRNA gene sequence of Vigna angularis var. nipponensis (AB059747) was not registered in GenBank. Furthermore, the size of the amplification product (approximately 100 bp) was estimated based on the predicted annealing site of the SEQ NO: 20 within the ITS-1 sequence and the assumption described below. It was assumed that Vigna angularis var. nipponensis (adzuki bean) had the same 5.8S rRNA gene sequence as Arachis hypogaea (peanut) and the primer with the SEQ NO: 3 had an annealing site within the 5.8S rRNA gene sequence.
Triticum aestivum
Glycine max
Juglans regia
Tricholoma matsutake
Prunus persica
Malus x domestica
Citrus sp.
Zea mays
Oryza sativa
Piper nigrum
Sinapis alba
Phaseolus vulgaris
Phaseolus lunatus
Lens culinaris subsp.
Culinaris
Cicer arietinum
Vigna radiata
nipponensis
Stylosanthes acuminata
Stylosanthes angustifolia
Stylosanthes aurea
Stylosanthes biflora
Stylosanthes bracteata
Stylosanthes calcicola
Stylosanthes campestris
Stylosanthes capitata
Stylosanthes cayennensis
Stylosanthes erecta
Stylosanthes fruticosa
Stylosanthes gracilis
Stylosanthes grandifolia
Stylosanthes guianensis subsp.
dissitiflora
Stylosanthes hamata
Stylosanthes hippocampoides
Stylosanthes hispida
Stylosanthes humilis
Stylosanthes ingrata
Stylosanthes leiocarpa
Stylosanthes linearifolia
Stylosanthes macrocarpa
Stylosanthes macrocephala
Stylosanthes macrosoma
Stylosanthes mexicana
Stylosanthes montevidensis
Stylosanthes pilosa
Stylosanthes scabra
Stylosanthes seabrana
Stylosanthes sericeiceps
Stylosanthes subsericea
Stylosanthes sundaica
Stylosanthes sympodialis
Stylosanthes tomentosa
Stylosanthes tuberculata
Stylosanthes viscosa
Ormocarpum bernierianum
Ormocarpum coeruleum
Ormocarpum drakei
Ormocarpum flavum
Ormocarpum keniense
Ormocarpum kirkii
Ormocarpum klainei
Ormocarpum megalophyllum
Ormocarpum muricatum
Ormocarpum orientale
Ormocarpum pubescens
Ormocarpum rectangulare
Ormocarpum schliebenii
Ormocarpum sennoides
Ormocarpum somalense
Ormocarpum trachycarpum
Ormocarpum trichocarpum
Ormocarpum verrucosum
Chapmannia floridana
Chapmannia prismatica
Chapmannia somalensis
Ormocarpopsis aspera
Ormocarpopsis calcicola
Ormocarpopsis itremoensis
Ormocarpopsis mandrarensis
Ormocarpopsis parvifolia
Ormocarpopsis tulearensis
Diphysa humilis
Diphysa macrophylla
Diphysa suberosa
Spigelia coelostylioides
Arachis Not Belonging to
Spigelia hedyotidea
Spigelia marilandica
6 commercially available peanuts were used.
Leaves that germinated from commercially available seeds of Shirahana soba (common buckwheat), wheat, 2 soybeans, 2 adzuki beans and corn were used.
(2) Isolation of DNA from Peanut
DNA isolation was conducted by using the QIAGEN Genomic-tip and the resulting isolate was purified by MACHEREY-NAGEL NucleoSpin as stated below.
Into a 15 ml-tube 1 g of a ground sample was transferred, added 10 ml of Buffer G2, 100 μl of Proteinase K (20 mg/ml) and 10 μl of RNase A (100 mg/ml), and they were mixed. The resulting mixture was incubated for 1 hour at 50° C. Then the resulting mixture was centrifuged for 10 min. at 3,000×g to obtain a supernatant. The resulting supernatant was applied to the Genomic-tip 20/G column equilibrated with 1 ml of Buffer QBT to adsorb DNA to the column. Subsequently, the column was washed with 4 ml of Buffer QC and DNA was eluted with 1 ml of preheated (50° C.) Buffer QF. To the eluate was added 4 volume of Buffer NT2, mixed with it, and then the resulting mixture was divided into two halves to be applied to two separate NucleoSpin Extract Columns. 650 μl of the mixture was applied to a NucleoSpin Extract Column and then the column was centrifuged for 1 min. at 6,000×g to adsorb DNA to the column. This step was repeated with the remaining mixture. In order to wash the column, to the column was added 600 μl of Buffer NT3 and was centrifuged for 1 min. at 6,000×g. To the column was added 600 μl of Buffer NT3 again and was centrifuged for 1 min. at a maximum speed to flush out the Buffer NT3 remaining in the column. Finally, to the column was added 100 μl of Buffer NE and was centrifuged for 1 min. at a maximum speed to obtain a DNA eluate from the column and the resulting eluate was subjected to isopropanol precipitation to collect DNA, which were then dissolved in 40 μl 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 Shirahana Soba (Common Buckwheat), Wheat, Soybean, Adzuki Bean and Corn
DNA isolation was conducted by using the QIAGEN DNeasy Plant Mini Kit according to the procedures described in the DNeasy Plant Mini Kit Handbook as shown below.
Into a 1.5 ml-tube 50 mg of a ground sample was transferred, added 600 μl of Buffer AP1 and 6 μl of RNase A (100 mg/ml), and they were mixed well. Then the resulting mixture was incubated for 1 hour at 65° C. 2001cl of Buffer AP2 was then added to the mixture. The resulting mixture was incubated for 10 min. on ice and then centrifuged to obtain a supernatant. The resulting 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 with them. The resulting mixture was divided into two halves to be applied to two DNeasy Spin Columns. 650 μl of the mixture was applied to a DNeasy Spin Column and the column was centrifuged for 1 min. at 6,000×g to adsorb DNA to the column. This step was repeated with the remaining mixture. In order to wash the column, to the column was added 500 μl of Buffer AW and was centrifuged for 1 min. at 6,000×g. To the column was added 500 μl of Buffer AW again and was centrifuged for 1 min. at a maximum speed to flush out the Buffer AW remaining in the column. Finally, to the column was added 100 μl of preheated (65° C.) Buffer AE and was centrifuged for 1 min. at a maximum speed, and added another 100 μl of preheated (65° C.) Buffer AE and was centrifuged for 1 min. at a maximum speed to obtain a DNA eluate from the column, and the resulting eluate was subjected to isopropanol precipitation to collect DNA, which were then dissolved in 50 μl of sterilized ultrapure water. After the concentration of the resulting DNA preparation was determined, the DNA eluate was used for a PCR template.
A DNA preparation derived from peanut seeds was diluted stepwise with a DNA preparation from wheat leaves to obtain wheat leaf DNA solutions containing 10 ppm and 1 ppm of peanut seed DNA. The DNA preparation from peanut 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).
PCR was conducted using the Applied Biosystems AmpliTaq Gold(R) & 10×PCR Buffer II & MgCl2 Solution with dNTP as stated below.
PCR was carried out using final volumes of 25 μl of a solution containing 2.5 μl of 10×PCR Buffer II, 0.125 μl of AmpliTaq Gold (5 U/μl), 2.5 μl of dNTPs Mix (2 mM each), 1.5 μl of MgCl2 Solution (25 mM), 0.5 μM of each primer (SEQ NO:18 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 2400 (Applied Biosystems) according to the following PCR program: pre-incubation at 95° C. for 15 min.; 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 66° C. for 2 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° 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
(−): Negative control (no DNA)
Numerical values above sample names: the amounts of the template DNA
Arrow: indicates the target amplification product (156 bp)
PCR described above was conducted using primers designed in the present invention. The results are shown in
(1) Purification of the Amplification Product from Peanut
A purification of the amplification product from peanut obtained in D was conducted in the same way as in Example 1E (1) “Purification of the Amplification Product from Buckwheat Chaff”.
PCR for sequencing of the purified amplification product from peanut obtained in (1) was conducted in the same way as in Example 1E (2) “Sequencing PCR Reaction and Removal of Excess Dye-Labeled Dideoxynucleotides”, except for use of primers with SEQ NOs: 18 and 3.
The sample for sequencing from peanut obtained in E (2) was analyzed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The obtained nucleotide sequence of the amplification product was compared with the sequence of the genus Arachis, Arachis hypogaea (AF156675), A. correntina (AF203554) and A. villosa (AF203558) in GenBank. The result is shown in
Number Symbols: Nucleotide numbers of amplification products
Asterisks (*): Nucleotides of the peanuts, which are identical with those of all of the nucleotide sequences of Arachis hypogaea (AF156675), A. correntina (AF203554) and A. villosa (AF203558) in GenBank
Single underline: The sense primer region
Double underline: The antisense primer region
As shown in
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 μl of a solution containing 12.5 μl of 2× HotStarTaq Master Mix (HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, 400 μM each dNTP), 0.5 μM of each primer (SEQ NO:20 and SEQ NO:3), the template DNA and sterilized ultrapure water in 0.2-ml microcentrifuge tubes. Amplification was performed using a Sequence Detection System ABI PRISM 7700 (Applied Biosystems) according to the following PCR program: pre-incubation at 95° C. for 15 min.; 25 cycles consisting of denaturation at 95° C. for 30 secs., annealing and extension at 75° C. for 30 secs. respectively and 30 cycles consisting of denaturation at 95° C. for 30 secs., annealing and extension at 72° C. for 30 secs. respectively; followed by a final extension at 72° C. for 5 min. The resulting 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
(−): Negative control (no DNA)
Numerical values above sample names: the amounts of the template DNA
Arrow: indicates the target amplification product (114 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 plant chloroplast DNA.
I. PCR Results (Part 2: a combination of primers with SEQ NOs:20 and 3)
PCR described above was conducted using primers designed in the present invention. The results are shown in
As shown in
Furthermore, the nonspecific amplification product from wheat that was obviously different from the target product in size did not interfere in the detection of 1 ppm of peanut DNA.
In this connection, as shown in Examples 1, 2 and 3, where a W value, which shows a possibility of obtaining amplification products in a simulation by Amplify, is not more than W4, it has been found that the amplification products, which differ from the target one, are obtained in some cases and not obtained in other cases in actual PCR. Please note that Tables 1 to 4 show the data of the simulation results of Amplify which are of W2 value or higher but do not show those of a lower W value such as W1 and W0 wherein the possibility of obtaining the amplification products is considered to be low.
(1) Purification of the Amplification Product from Peanut
The purification of the amplification product from peanut obtained in I was conducted in the same way as in Example 1E (1) “Purification of the Amplification Product from Buckwheat Chaff”.
PCR for sequencing of the purified amplification product from peanut obtained in (1) was conducted in the same way as in Example 1E (2) “Sequencing PCR Reaction and Removal of Excess Dye-Labeled Dideoxynucleotides”, except for use of primers with SEQ NOs:20 and 3.
The sample for sequencing from peanut obtained in J (2) was analyzed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The obtained nucleotide sequence of the amplification product was compared with the sequence of Arachis hypogaea (AF156675), A. correntina (AF203554), A. villosa (AF203558), A. major (AF203552) and A. hermannii (AF203556) in the genus Arachis and the nucleotide sequence of the commercially available peanut obtained in G. The result is shown in
Number Symbols: Nucleotide numbers of amplification products
Commercially available peanuts: A part of the nucleotide sequence of the commercially available peanuts obtained in G
Asterisks (*): Nucleotides of the peanuts, which are identical with those of all of the sequences of Arachis hypogaea (AF156675), A. correntina (AF203554), A. villosa (AF203558), A. major (AF203552) and A. hermannii (AF203556) in the genus Arachis and the sequence of the commercially available peanut obtained in G.
Single underline: The sense primer region
Double underline: The antisense primer region
As shown in
The DNA sequences described in Example 1A (1) “DNA sequences of the Genus Fagopyrum”, (2) “DNA Sequences of other common allergenic plants” and (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” were examined to select suitable regions for the primers.
The DNA sequences described in Example 2A (2) “DNA Sequence of Plants in Related Species of the Genus Fagopyrum” were selected.
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: 15. Subsequently, the oligonucleotide primer with SEQ NO:15 was synthesized.
The oligonucleotide primer with SEQ NO:14 was also used as an antisense primer, the same as in Example 2.
Regarding the sense and antisense primer pair, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1, to examine whether a target size of amplification products would be obtained from the 21 DNA sequences of the genus Fagopyrum, the 8 DNA sequences of common allergenic plants other than buckwheat (peanut, wheat, soybean, walnut, matsutake mushroom, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard) and the 27 sequences of related species of the genus Fagopyrum.
The simulation results are shown in Tables 5A and 5C. The meanings of symbols and numerical values in Tables 5A and 5C are explained below.
★: An obtained amplification product whose size almost matched the target 140 bp (±10 bp).
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.
Related Species of the genus Fagopyrum:
Sequences similar to the ITS-1 sequence of Fagopyrum esculentum (AB000330) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected from among them. Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Tables 5B-5C as the representative of the DNA sequences of related species of the genus Fagopyrum.
The DNA samples isolated from Shirahana soba (common buckwheat) and Dattan soba (Tartalian buckwheat) in Example 1B (2) were diluted stepwise with sterilized ultrapure water to use them. The DNA samples isolated from white pepper in Example 1B (2), mustard in Example 1B (3), peanut in Example 3B (2) and wheat, soybean, and corn in Example 3B (3) were also used. In addition, the DNA samples isolated from brown lice seeds and Fallopia convolvulus in the same way as in Example 1B (3) were used. The DNA samples from Fallopia convolvulus were diluted stepwise with sterilized ultrapure water to use.
PCR was conducted in essentially the same way as in Example 1C, except for use of the following primers and PCR program.
Each primer of SEQ NO:14 and SEQ NO:15 was used at 0.5 μM of a final concentration.
PCR was conducted according to the following PCR program.
Pre-incubation at 95° C. for 15 min.; thereafter 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 66° C. for 2 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 min.
Regarding the primers of the present invention, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels) to examine the specificity to the ITS-1-5.8S rRNA gene sequence of each plant. As a result, as shown in Tables 5A to 5C, it was predicted that target 101 bp amplification products would be obtained from the aforementioned 21 DNA sequences of the genus Fagopyrum. On the other hand, it was predicted that no target 101 bp amplification product would be obtained from the 8 DNA sequences of other common allergenic plants (peanut, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard) and the 27 DNA sequences of related species of the genus Fagopyrum both belonging to Polygonaceae and not belonging to Polygonaceae. Also, no nonspecific amplification product could be predicted. As a result, it was confirmed that a wide range of plants in the genus Fagopyrum would be specifically detectable using the present invention.
Fagopyrum lineare
Fagopyrum gracilipes
Arachis hypogaea
Triticum aestivum
Glycine max
Juglans regia
Tricholoma matsutake
Prunus persica
Malus x domestica
Citrus sp.
Zea mays
Oryza sativa
Piper nigrum
Sinapis alba
Aconogonum sp. Won 152
Fagopyrum Belonging to
Fallopia scandens
Polygonum virginianum
Rumex acetosella
Talinum paraguayense
Bruinsmia styracoides
Talinella pachypoda
Rehderodendron
kwangtungense
Pterostyrax corymbosus
Anredera cordifolia
Cistanthe quadripetala
Xenia vulcanensis
Talinopsis frutescens
Talinaria palmeri
Portulaca sp.
Phemeranthus
confertiflorus
Montiopsis umbellata
Grahamia bracteata
Herniaria glabra
Alluaudia dumosa
Sinojackia xylocarpa
Halesia macgregori
Changiostyrax dolichocarpa
Alectryon subdentatus
Anacampseros recurvata
Weinmannia racemosa
Bursera tecomaca
PCR described above was conducted using primers designed in the present invention. In this case, target 101 bp amplification products, predicted from the simulation results of the ITS-1˜5.8S rRNA gene sequences of the genus Fagopyrum, were obtained from 500 to 50 fg of Shirahana soba (common buckwheat) DNA and Dattan soba (Tartarian buckwheat) DNA. As a result, it was found that even where 500 to 50 fg of buckwheat DNA is present, the buckwheat can be detected. In this connection, such sensitivity corresponds to a sensitivity wherein there can be detected 10 to 1 ppm of buckwheat DNA contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples. On the other hand, no amplification product having 101 bp or nonspecific amplification products was obtained from wheat, peanut, soybean, corn, mustard, pepper and brown rice. Furthermore, regarding Fallopia convolvulus, when an amount of template DNA is 50 to 5 ng, a target size of an amplification product was obtained with a very weak signal, but when an amount of template DNA is 500 pg or less, no amplification product in a target size or nonspecific amplification product was obtained at all. In this connection, regarding Fallopia convolvulus, when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples, even if 1% of Fallopia convolvulus was present in sample DNA, the level of Fallopia convolvulus DNA corresponds to a non-detected level as false positive. By modification of PCR program, there is a possibility that amplification products in a target size will not be obtained from 50 to 5 ng of DNA from Fallopia convolvulus.
Consequently, in conjunction with the results of specificity studied by PCR simulation and of sensitivity and specificity studied by PCR, it was confirmed that a wide range of the genus Fagopyrum including common buckwheat and Tartarian buckwheat were detectable using the present invention.
The DNA sequences described in Example 3A (1) “DNA Sequences of the Genus Arachis”, Example 3A (2) “DNA Sequences of Other Common Allergenic Plants”, Example 3A (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” and Example 3A (4) “DNA Sequences of Leguminous Plants Widely Used for a Food Ingredient” were examined to select suitable regions for the primers. In addition, as a DNA sequence of adzuki bean, 5.8S rRNA gene, ITS-2 sequences in the following DNA sequences registered in GenBank were selected.
1: adzuki bean: Vigna angularis vars nipponensis (AB060088)
As representatives of the DNA sequences of related species of the genus Arachis, respective 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 45 DNA sequences registered in GenBank were examined to select suitable regions for the primers. In this connection, the 45 DNA sequences were selected as representations, each of which was of highest Score in the species which were other than peanut (Arachis hypogaea AF156675) and were of Score of 60 bits or more among sequences of said species hit to ITS-2 sequence of peanut through BLAST homology search.
1: Chapmannia floridana (AF203543)
2: Chapmannia gracilis (AF203546)
3: Chapmannia prismatica (AJ320400)
4: Chapmannia reghidensis (AF204232)
5: Chapmannia sericea (AF203548)
6: Chapmannia somalensis (AF203544)
7: Chapmannia tinireana (AF203547)
8: hebrigiella gracilis (AF203561)
9: Fissicalyx fendleri (AF189061)
10: Stylosanthes acuminata (AJ320282)
11: Stylosanthes angustifolia (AJ320284)
12: Stylosanthes aurea (AJ320285)
13: Stylosanthes biflora (AJ320289)
14: Stylosanthes bracteata (AJ320346)
15: Stylosanthes calcicola (AJ320348)
16: Stylosanthes campestris (AJ320291)
17: Stylosanthes capitata (AJ320350)
18: Stylosanthes cayennensis (AJ320292)
19: Stylosanthes erecta (AJ320352)
20: Stylosanthes fruticosa (AJ320356)
21: Stylosanthes gracilis (AJ320296)
22: Stylosanthes grandifolia (AJ320299)
23: Stylosanthes guianensis subsp. dissitiflora (AJ320301)
24: Stylosanthes hamata (AJ320365)
25: Stylosanthes hippocampoides (AJ320316)
26: Stylosanthes hispida (AJ320328)
27: Stylosanthes humilis (AJ320327)
28: Stylosanthes ingrata (AJ320329)
29: Stylosanthes leiocarpa (AJ320332)
30: Stylosanthes linearifola (AJ320367)
31: Stylosanthes macrocarpa (AJ320369)
32: Stylosanthes macrocephala (AJ320371)
33: Stylosanthes macrosoma (AJ320333)
34: Stylosanthes mexicana (AJ320373)
35: Stylosanthes montevidensis (AJ320336)
36: Stylosanthes pilosa (AJ320377)
37: Stylosanthes scabra (AJ320382)
38: Stylosanthes seabrana (AJ320384)
39: Stylosanthes sericeiceps (AJ320386)
40: Stylosanthes subsericea (AJ320387)
41: Stylosanthes sundaica (AJ320389)
42: Stylosanthes sympodialis (AJ320391)
43: Stylosanthes tomentosa (AJ320337)
44: Stylosanthes tuberculata (AJ320392)
45: Stylosanthes viscosa (AJ320340)
In addition, if PCR simulation is conducted by selecting a primer hybridized to ITS-1 sequence, the DNA sequences described in Example 3A
(5) “DNA Sequences of Plants in Related Species to the Genus Arachis” were also selected.
(a) Among 5.8S rRNA gene sequences of the aforementioned 11 DNA sequences of the genus Arachis and the aforementioned 8 DNA sequences of other common allergenic plants, there was determined nucleotide sequences which would hybridize to all DNA sequences of these plants through the study of the sequences. The thus determined nucleotide sequence is indicated as SEQ NO:7. Subsequently, the oligonucleotide primer with SEQ NO:7 was synthesized.
Among ITS-2 sequences of the aforementioned 11 DNA sequences of the genus Arachis, there was determined nucleotide sequences which would specifically hybridize to all of the 11 DNA sequences of the genus Arachis through the study of the sequences. The thus determined nucleotide sequence is indicated as SEQ NO:24. Subsequently, the oligonucleotide primer with SEQ NO:24 was synthesized.
(b) Furthermore, in another combination of primers, the primer of SEQ NO:18 described in Example 3 was selected as sense primer on the ITS-1 sequence and the primer of SEQ NO:24 was selected as antisense primer on the ITS-2 sequence.
Regarding the sense and antisense primer pairs, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1 to examine whether target size of amplification products are obtained from the 11 DNA sequences of the genus Arachis, the 8 DNA sequences of common allergenic plants other than peanut (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the 6 DNA sequence of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean) and the DNA sequences of plants in related species of the genus Arachis.
(a) The simulation results using a combination of primers of SEQ NOs:7 and 24 are shown in Tables 6A to 6D. The meanings of symbols and numerical values in Tables 6A to 6D are explained below.
★: An obtained amplification product whose size almost matched the target 140 bp (±10 bp).
W 2-6: Probability to obtain 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.
Related Species of the genus Arachis:
Sequences similar to the ITS-2 sequence of Arachis hypogaea (AF156675) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or one were selected among them. Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Tables GB-6D as the representative of the DNA sequences of related species of the genus Arachis.
(−*): No annealing site of the primer (SEQ NO: 24) was predicted within the ITS-2 sequence of Vigna angularis var. nipponensis (adzuki bean).
In the case of adzuki bean, only the ITS-2 sequence was selected because the 5.8S rRNA gene sequence of Vigna angularis var. nipponensis (AB059747) was not registered in GenBank.
(b) The simulation results using a combination of primers of SEQ NOs:18 and 24 are shown in Tables 7A to 7E. The meanings of symbols and numerical values in Tables 7A to 7E are explained below.
★: An obtained amplification product whose size almost matched the target 140 bp (±10 bp).
W 2-6: Probability to obtain 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.
Related Species of the genus Arachis:
Sequences Similar to the ITS-1 or ITS-2 sequence of Arachis hypogaea (AF156675) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected among them. Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Tables 7B-7E as the representative of the DNA sequences of related species of the genus Arachis.
(−*): No annealing site of the primer (SEQ NO: 18) was predicted within the ITS-1 sequence of Vigna angularis var. nipponensis (adzuki bean) and no annealing site of the primer (SEQ NO:24) was predicted within the ITS-2 sequence of Vigna angularis var. nipponensis (adzuki bean).
In the case of adzuki bean, only either the ITS-1 or ITS-2 sequence was selected respectively because full length of the ITS-1˜5.8S rRNA gene˜ITS-2 sequence of Vigna angularis var. nipponensis (AB059747) was not registered in GenBank.
A DNA preparation from peanut isolated in Example 3B (2) was diluted stepwise with sterilized ultrapure water to use.
PCR was conducted in substantially the same way as in Example 1C, except for use of following primers and PCR program.
(a) Each primer of SEQ NO:7 and SEQ NO:24 was used at 0.5 μM of final concentration.
(b) Each primer of SEQ NO:18 and SEQ NO:24 was used at 0.5, M of final concentration.
PCR was conducted according to the following PCR program.
Pre-incubation at 95° C. for 15 min.; thereafter 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 68° C. for 1 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 min.
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 plant chloroplast DNA.
Regarding the primers of the present invention (the combination of the primers of SEQ NOs:7 and 24), the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels) to examine the specificity to 5.8S rRNA gene˜ITS-2 sequence of each plants. As a result, as shown in Tables GA to GD, it was predicted that target 253 to 259 bp amplification products would be obtained from the aforementioned 11 DNA sequences of the genus Arachis. On the other hand, it was predicted that no target 253 to 259 bp amplification products would be obtained from the 8 DNA sequences of other common allergenic plants (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the 6 DNA sequence of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean) and the 41 DNA sequences of plants in related species of the genus Arachis belonging to leguminous plants. In this connection, among the plants in related species of the genus Arachis belonging to leguminous plants, it was predicted by simulation that amplification products having almost 253 to 259 bp would be obtained from the DNA sequences of Stylosanthes cayennensis, Stylosanthes hispida, Stylosanthes viscosa and Fissicalyx fendleri, but these amplification products can be identified by sequence analysis. Optionally, whether these amplification products are peanut may be also identified by PCR showing in Example 3 and the like. As a result, it was confirmed that wide ranges of plants in the genus Arachis would be specifically detectable using the present invention.
Fagopyrum esculentum
Triticum aestivum (Wheat)
Glycine max (Soybean)
Juglans regia (Walnut)
Tricholoma matsutake
Prunus persica (Peach)
Malus x domestica
Citrus sp.
Zea mays
Oryza sativa
Piper nigrum
Sinapis alba
Phaseolus vulgaris
Phaseolus lunatus
Lens culinaris subsp.
culinaris (Lentil)
Cicer arietinum
Vigna radiata
Vigna angularis var.
nipponensis
Chapmannia floridana
Arachis Belonging to leguminous
Chapmannia gracilis
Chapmannia prismatica
Chapmannia reghidensis
Chapmannia sericea
Chapmannia somalensis
Chapmannia tinireana
Fiebrigiella gracilis
Stylosanthes acuminata
Stylosanthes angustifolia
Stylosanthes aurea
Stylosanthes biflora
Stylosanthes bracteata
Stylosanthes calcicola
Stylosanthes campestris
Stylosanthes capitata
Stylosanthes erecta
Stylosanthes fruticosa
Stylosanthes gracilis
Stylosanthes grandifolia
Stylosanthes guianensis
Stylosanthes hamata
Stylosanthes
hippocampoides
Stylosanthes humilis
Stylosanthes ingrata
Stylosanthes leiocarpa
Stylosanthes linearifolia
Stylosanthes macrocarpa
Stylosanthes macrocephala
Stylosanthes macrosoma
Stylosanthes mexicana
Stylosanthes
montevidensis
Stylosanthes pilosa
Stylosanthes scabra
Stylosanthes seabrana
Stylosanthes sericeiceps
Stylosanthes subsericea
Stylosanthes sundaica
Stylosanthes sympodialis
Stylosanthes tomentosa
Stylosanthes tuberculata
PCR described above was conducted using primers designed in the present invention. In this case, target 253 to 259 bp amplification products, expected from the simulation results of the 5.8S rRNA gene˜ITS-2 sequences of the genus Arachis, were obtained from 500 to 50 fg of peanut DNA. It is found from the results that even where 500 to 50 fg of peanut DNA is present, the peanut can be detected. In this connection, this sensitivity correspond to a sensitivity wherein there can be detected 10 to 1 ppm of peanut DNA contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples.
Consequently, in conjunction with the results of specificity studied by PCR simulation, and of sensitivity and specificity studied by PCR, it was confirmed that a wide range of the genus Arachis including peanut were detectable using the present invention.
Regarding the primers of the present invention (the combination of the primers of SEQ NOs:18 and 24), the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels) to examine the specificity to ITS-1˜5.8S rRNA gene˜ITS-2 sequence of each plant. As a result, as shown in Tables 7A to 7E, it was predicted that target 384 to 390 bp amplification products would be obtained from the aforementioned 11 DNA sequences of the genus Arachis. On the other hand, it was predicted that no target 384 to 390 bp amplification products would be obtained from the 8 DNA sequences of other common allergenic plants (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown lice, pepper and mustard), the 7 DNA sequence of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean), the 71 DNA sequences of plants in related species of the genus Arachis belonging to leguminous plants and the 3 DNA sequences of plants in related species of the genus Arachis not belonging to leguminous plants. Among the plants in related species of the genus Arachis belonging to leguminous plants, it was predicted by simulation that amplification products, which were almost matched 384 to 390 bp of the target one in size, would be obtained from the DNA sequences of Stylosanthes cayennensis, Stylosanthes hispida, Stylosanthes viscosa and Fissicalyx fendleri, but these amplification products can be identified by sequence analysis. Optionally, whether these amplification products are peanut may be also identified by PCR showing in Example 3 and the like. As a result, it was confirmed that wide ranges of plants in the genus Arachis would be specifically detectable using the present invention.
Fagopyrum esculentum
Triticum aestivum
Glycine max (Soybean)
Juglans regia (Walnut)
Tricholoma matsutake
Prunus persica (Peach)
Citrus sp.
Zea mays
Oryza sativa
Piper nigrum
Sinapis alba
Phaseolus vulgaris
Phaseolus lunatus
Lens culinaris subsp.
culinaris (Lentil)
Cicer arietinum
Vigna radiata (Mung bean)
Vigna angularis var.
nipponensis
Vigna angularis var.
nipponensis
Stylosanthes acuminata
Stylosanthes angustifolia
Stylosanthes aurea
Stylosanthes biflora
Stylosanthes bracteata
Stylosanthes calcicola
Stylosanthes campestris
Stylosanthes capitata
Stylosanthes erecta
Stylosanthes fruticosa
Stylosanthes gracilis
Stylosanthes grandifolia
Stylosanthes guianensis
Stylosanthes hamata
Stylosanthes
hippocampoides
Stylosanthes humilis
Stylosanthes ingrata
Stylosanthes leiocarpa
Stylosanthes linearifolia
Stylosanthes macrocarpa
Stylosanthes macrocephala
Stylosanthes macrosoma
Stylosanthes mexicana
Stylosanthes
montevidensis
Stylosanthes pilosa
Stylosanthes scabra
Stylosanthes seabrana
Stylosanthes sericeiceps
Stylosanthes subsericea
Stylosanthes sundaica
Stylosanthes sympodialis
Stylosanthes tomentosa
Stylosanthes tuberculata
Stylosanthes
hippocampoides
Stylosanthes humilis
Stylosanthes mexicana
Ormocarpum bernierianum
Ormocarpum coeruleum
Ormocarpum drakei
Ormocarpum flavum
Ormocarpum keniense
Ormocarpum kirkii
Ormocarpum klainei
Ormocarpum
megalophyllum
Ormocarpum muricatum
Ormocarpum orientale
Ormocarpum pubescens
Ormocarpum rectangulare
Ormocarpum schliebenii
Ormocarpum sennoides
Ormocarpum somalense
Ormocarpum trachycarpum
Ormocarpum trichocarpum
Ormocarpum verrucosum
Chapmannia floridana
Chapmannia prismatica
Chapmannia somalensis
Chapmannia gracilis
Chapmannia reghidensis
Chapmannia sericea
Chapmannia tinireana
Ormocarpopsis aspera
Ormocarpopsis calcicola
Ormocarpopsis
itremoensis
Ormocarpopsis
mandrarensis
Ormocarpopsis parvifolia
Ormocarpopsis
tulearensis
Diphysa humilis
Diphysa macrophylla
Diphysa suberosa
Fiebrigiella gracilis
Spigelia coelostylioides
Spigelia hedyotidea
Spigelia marilandica
PCR described above was conducted using primers designed in the present invention. In this case, target 253 to 259 bp amplification products, 5 expected from the simulation results of the ITS-1˜5.8S rRNA gene˜ITS-2 sequences of the genus Arachis, were obtained from 500 to 50 fg of peanut DNA. It is found from the results that even where 500 to 50 fg of peanut DNA is present, the peanut can be detected. In this connection, this sensitivity corresponds to a sensitivity wherein there can be detected 10 to 1 ppm of peanut DNA contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples.
Consequently, in conjunction with the results of specificity studied by PCR simulation, and the results of sensitivity and specificity studied by PCR, it was confirmed that a wide range of plants in the genus Arachis including peanut were detectable using the present invention.
Regarding the genus Triticum, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 29 DNA sequences registered in GenBank were examined to select suitable regions for the primer.
1: Triticum aestivum (AF440679)
2: Triticum aestivum (AF440676)
3: Triticum aestivum (AF438191)
4: Triticum aestivum (AF438188)
5: Triticum aestivum (AF438187)
6: Triticum aestivum (AF438186)
7: Triticum baeoticum (AJ238901)
8: Triticum urartu (AJ301803)
9: Triticum turgidum subsp. dicoccum (AJ301801)
10: Triticum monococcum (AJ301800)
11: Triticum aestivum (AJ301799)
12: Triticum monococcum (AJ245404)
13: Triticum turgidum (AJ238919)
14: Triticum turgidum (AJ238918)
15: Triticum turgidum (AJ238917)
16: Triticum turgidum (AJ238915)
17: Triticum turgidum (AJ238913)
18: Triticum turgidum (AJ238912)
19: Triticum turgidum (AJ238911)
20: Triticum timopheevii (AJ238924)
21: Triticum timopheevii (AJ238923)
22: Triticum timopheevii (AJ238922)
23: Triticum timopheevii (AJ238921)
24: Triticum timopheevii (AJ238920)
25: Triticum turgidum (AJ238916)
26: Triticum turgidum (AJ238914)
27: Triticum urartu (AJ238902)
28: Triticum aestivum (Z11761)
29: Triticum monococcum (L11581)
(2) DNA Sequences of Other Common Allergenic Plants and Plants Widely Used for a Food Ingredient
The DNA sequences described in Example 1A (2) “DNA Sequences of Other Common Allergenic Plants” and (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” were selected. Regarding buckwheat, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected.
1: Buckwheat: Fagopyrum esculentum (AB000330)
Regarding rye, barley and oats, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected.
1: Rye: Secale cereale (L36504)
2: Barley: Hordeum vulgare (AF440678)
3: Oat: Avena sativa (Z96893)
(4) DNA Sequences of Related Species of the genus Triticum
As representatives of the DNA sequences of related species of the genus Triticum, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 70 DNA sequences registered in GenBank were selected. In this connection, the 70 DNA sequences were selected as representatives of the DNA sequences of related species of the genus Triticum, each of which had the highest score in the corresponding genus other than genus Fagopyrum and a score of 60 bits or more among sequences of species belonging to the corresponding genus selected from sequences registrated in GenBank through a BLAST homology search using the ITS-2 sequence of wheat (Triticum aestivum Z11761).
1: Ancestral species of wheat: Aegilops sharonensis (AF149195)
2: Taeniatherum caput-medusae (L36505)
3: Agropyron puberulum (L36482)
4: Thinopyrum intermedium (AF507809)
5: Lophopyrum elongatum (L36495)
6: Pseudoroegneria spicata (L36502)
7: Peridictyon sanctum (L36497)
8: Australopyrum pectinatum (L36484)
9: Amblyopyrum muticum (AF149202)
10: Henrardia persica (L36491)
11: Eremopyrum bonaepartis (L36490)
12: Crithopsis delileana (L36487)
13: Psathyrostachys fragilis (L36498)
14: Heteranthelium piliferum (L36492)
15: Critesion violaceum (L36488)
16: Secale sylvestre (AJ409210)
17: Haynaldia villosa (L36489)
18: Bromus tectorum (L36485)
19: Helictotrichon gervaisii (AJ389134)
20: Festuca lasto (AF303418)
21: Lagurus ovatus (AJ389166)
22: Poa pratensis (AF171183)
23: Pseudarrhenatherum longifolium (AJ389162)
24: Alopecurus vaginatus (Z96921)
25: Calamagrostis epigejos (AJ306448)
26: Thisetum spicatum (AJ389168)
27: Koeleria pyramidata (Z96911)
28: Beckmannia eruciformis (AJ389164)
29: Lolium persicum (AF171157)
30: Diarrhena americana (AF019798)
31: Arrhenatherum elatius (AF019795)
32: Deschampsia christophersenii (AF486267)
33: Piptochaetium fimbriatum (L36523)
34: Vulpia fasciculata (AF303402)
35: Phalaris truncata (L36522)
36: Holcus lanatus (Z96919)
37: Merxmuellera stricta (AF019871)
38: Brachypodium mexicanum (AF019805)
39: Austrostipa nodosa (AF019804)
40: Ampelodesmos mauritanica (AF019799)
41: Nassella viridula (L36521)
42: Melica imperfecta (L36519)
43: Achnatherum hymenoides (L36507)
44: Austrodanthonia auriculata (AF367604)
45: Notodanthonia laevis (AF019875)
46: Oryzopsis exigua (AF019801)
47: Chionochloa rigida (AF367597)
48: Thysanolaena maxima (AF019854)
49: Monachather paradoxus (A-F019852)
50: Stipagrostis zeyheri (A-F019845)
51: Arundo donax (AF019809)
52: Zingeria biebersteiniana (AJ428836)
54: Briza minor (L36510)
55: Thibolium hispidum (AF367602)
56: Rytidosperma pumilum (AF019878)
57: Karroochloa purpurea (AF019874)
58: Centropodia glauca (AF019861)
59: Cortaderia archboldii (AF367620)
60: Lamprothyrsus peruvianus (AF367605)
61: Imperata cylindrica (A1F345653)
62: Zizania latifolia (A-F169234)
63: Prionanthium ecklonii (AF019866)
64: Pentaschistis aspera (AFO 19865)
65: Pentameris macrocalycina (A-FO19864)
66: Molinia caerulea (AF019857)
67: Dregeochloa pumilla (AFO 19853)
68: Diplopogon setaceus (AF019851)
69: Amphipogon amphopogonoides (AFO 19850)
70: Aristida purpurea (AF019807)
Among ITS-2 sequences of the aforementioned 29 DNA sequences of the genus Triticum, there was determined nucleotide sequences which would specifically hybridize to all of the 29 DNA sequences of the genus Triticum through the study of the sequences. The thus determined nucleotide sequences are indicated as SEQ NOs:28, 29 and 30. Subsequently, the oligonucleotide primers with SEQ NOs:28, 29 and 30 were synthesized.
Regarding the sense and antisense primer pairs, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1 to examine whether target size of amplification products are obtained from the 29 DNA sequences of the genus Triticum, the 8 DNA sequences of common allergenic plants other than wheat (buckwheat, peanut, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the DNA sequences of rye, barley and oat and the DNA sequences of Aegilops termed ancestral species of wheat and plants in related species of the genus Triticum used for breed improvement of wheat belonging to the tribe Triticeae. The simulation results are shown in Tables 8A to 8F. The meanings of symbols and numerical values in Tables 8A to 8F are explained below.
★: An obtained amplification product whose size almost matched the target 140 bp (±10 bp).
W 2-6: Probability to obtain 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.
Related Species of the genus Triticum:
Sequences similar to the ITS-2 sequence of Triticum aestivum (Z11761) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected from among them. Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Tables 8C-8F as the representative of the DNA sequences of related species of the genus Triticum.
Commercially available seeds of wheat were used.
(2) DNA Isolation from Wheat
A DNA was isolated from wheat in the same way as in Example 1B (3). The isolated DNA preparation of wheat was diluted stepwise with sterilized ultrapure water to use as template DNA for PCR.
PCR was conducted in the substantially same way as Example 1C, except for use of the following primers and PCR program.
The primer of SEQ NO:28 was used at 0.5 μM of final concentration and each primer of SEQ NOs:29 and 30 was used at 0.25 μM of final concentration.
PCR was conducted according to the following PCR program.
Pre-incubation at 95° C. for 15 min.; thereafter 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 66° C. for 1 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 min.
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 plant chloroplast DNA.
Regarding the primers of the present invention, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels) to examine the specificity to ITS-2 sequence of each plant. As a result, as shown in Tables 8A to 8F, it was predicted that target 93 to 95 bp amplification products would be obtained from the aforementioned 29 DNA sequences of the genus Triticum. On the other hand, it was predicted that no target 93 to 95 bp amplification product would be obtained from the 8 DNA sequences of common allergenic plants other than wheat (buckwheat, peanut, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown lice, pepper and mustard), the DNA sequences of rye, barley and oat, the 2 DNA sequences of related species of the genus Triticum belonging to the tribe Triticeae and the 51 DNA sequences of related species of the genus Triticum not belonging to the tribe Triticeae. In this connection, it was predicted by simulation that amplification products having almost 93 to 95 bp would be obtained from the DNA sequences of Aegilops termed ancestral species of wheat, plants in related species of the genus Triticum used for breed improvement of wheat belonging to the tribe Triticeae and some plants in related species of the genus Triticum not belonging to tribe Triticeae.
dicoccum
Arachis hypogaea
Fagopyrum esculentum
Glycine max (Soybean)
Juglans regia (Walnut)
Tricholoma matsutake
Prunus persica (Peach)
Malus x
domestica
Citrus sp.
Zea mays
Oryza sativa (Brown rice)
Piper nigrum (Pepper)
Sinapis alba (Mustard)
Secale cereale (rye)
Hordeum vulgare (Barley)
Avena sativa (Oat)
caput-medusae
Triticum Belonging to Tribe
fragilis
piliferum
Critesion violaceum
Secale sylvestre
Helictotrichon
gervaisii
Festuca lasto
Poa pratensis
Pseudarrhenatherum
longifolium
Alopecurus vaginatus
Calamagrostis epigejos
Trisetum spicatum
Koeleria pyramidata
Beckmannia eruciformis
Lolium persicum
Diarrhena americana
Arrhenatherum elatius
Deschampsia
christophersenii
Piptochaetium
fimbriatum
Vulpia fasciculata
Phalaris truncata
Holcus lanatus
Merxmuellera stricta
Brachypodium mexicanum
Austrostipa nodosa
Ampelodesmos
mauritanica
Nassella viridula
Melica imperfecta
Achnatherum hymenoides
Austrodanthonia auriculata
Notodanthonia laevis
Oryzopsis exigua
Chionochloa rigida
Thysanolaena maxima
Monachather paradoxus
Stipagrostis zeyheri
Arundo donax
Zingeria biebersteiniana
Centotheca lappacea
Briza minor
Tribolium hispidum
Rytidosperma pumilum
Karroochloa purpurea
Centropodia glauca
Cortaderia archboldii
Lamprothyrsus
peruvianus
Imperata cylindrica
Zizania latifolia
Prionanthium ecklonii
Pentaschistis aspera
Pentameris macrocalycina
Molinia caerulea
Dregeochloa pumilla
Diplopogon setaceus
Amphipogon amphopogonoides
Aristida purpurea
PCR described above was conducted using primers designed in the present invention. In this case, target 93 to 95 bp amplification products, expected from the simulation results of the ITS-2 sequences of the genus Triticum, were obtained from 500 to 50 fg of wheat DNA. It is found from the results that even where 500 to 50 fg of wheat DNA is present, the wheat can be detected. In this connection, this sensitivity correspond to a sensitivity wherein there can be detected 10 to 1 ppm of peanut DNA contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples.
Consequently, in conjunction with the results of specificity studied by PCR simulation, and the results of sensitivity studied by PCR, it was confirmed that a wide range of the genus Triticum including wheat, ancestral species of wheat, and the majority of the tribe Triticeae were specifically detectable at a high sensitivity using the present invention.
Regarding the genus Glycine, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 50 DNA sequences registered in GenBank were examined to select suitable regions for the primer.
6: Glycine soja (U60550)
7: Glycine soja (AF144653)
8: Glycine soja (AJ009790)
9: Glycine soja (AJ009791)
10: Glycine soja (AJ224109)
14: Glycine cyrtoloba (U60548)
15: Glycine tomentella (AF023447)
16: Glycine tomentella (U60544)
17: Glycine microphylla (U60537)
18: Glycine tomentella (U60542)
19: Glycine arenaria (U60543)
20: Glycine tabacina (U60539)
21: Glycine curvata (U60547)
22: Glycine tomentella (AJ011345)
23: Glycine pindanica (U60546)
24: Glycine lactovirens (U60540)
25: Glycine albicans (U60541)
26: Glycine argyrea (U60535)
27: Glycine tomentella (AF023446)
28: Glycine latifolia (U60538)
29: Glycine clandestina (U60534)
30: Glycine tomentella (AF023445)
31: Glycine dolichocarpa (AJ011340)
32: Glycine dolichocarpa (AJ224110)
33: Glycine canescens (AF023444)
34: Glycine hirticaulis (U60545)
35: Glycine tomentella (AJ011342)
36: Glycine dolichocarpa (AJ011341)
37: Glycine canescens (U60533)
38: Glycine canescens (AJ011348)
39: Glycine tabacina (AJ009788)
40: Glycine tabacina (AJ009789)
41: Glycine latrobeana (U60536)
42: Glycine tomentella (AJ011344)
43: Glycine tomentella (AJ011343)
44: Glycine tomentella (AJ011338)
45: Glycine tabacina (AJ011346)
46: Glycine dolichocarpa (AJ011339)
47: Glycine tabacina (AJ224111)
48: Glycine falcata (U60549)
49: Glycine latifolia (AJ009786)
50: Glycine tabacina (AJ011347)
The DNA sequences described in Example 1A (2) “DNA Sequences of Other Common Allergenic Plants” and (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” were selected. Regarding buckwheat, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected.
1: buckwheat: Fagopyrum esculentum (AB000330)
The DNA sequences described in Example 3A (4) “DNA Sequences of Leguminous Plants Widely Used for a Food Ingredient” were selected. Regarding adzuki bean, ITS-2 sequences in the following DNA sequences registered in GenBank were selected.
1: adzuki bean: Vigna angularis var. nipponensis (AB060088)
As representatives of the DNA sequences of related species of the genus Glycine, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 5 DNA sequences registered in GenBank were selected. In this connection, the 5 DNA sequences were selected as representatives of the DNA sequences of related species of the genus Glycine, each of which had the highest score in the corresponding genus other than genus Glycine and a score of 60 bits or more among sequences of species belonging to the corresponding genus selected from sequences registrated in GenBank through a BLAST homology search using the ITS-2 sequence of soybean (Glycine max U60551).
1: Ophrestia radicosa (AF467-484)
2: Myrospermum sousanum (AF187086)
3: Amphicarpaea bracteata (AF417019)
4: Amphicarpaea edgeworthii (AF417013)
5: Strophostyles umbellata (AFO69115)
Among ITS-2 sequences of the aforementioned 50 DNA sequences of the genus Glycine, there was determined nucleotide sequences which would specifically hybridize to all of the 50 DNA sequences of the genus Glycine through the study of the sequences. The thus determined nucleotide sequences are indicated as SEQ NOs:34 to 41. Subsequently, the oligonucleotide primers with SEQ NOs:34 to 41 were synthesized.
Regarding the sense and antisense primer pairs, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1 to examine whether target size of amplification products are obtained from the 50 DNA sequences of the genus Glycine, the 8 DNA sequences of common allergenic plants other than soybean (buckwheat, peanut, wheat, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the 6 DNA sequence of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean) and the 5 DNA sequences of related species of the genus Glycine. The simulation results are shown in Tables 9A to 9C. The meanings of symbols and numerical values in Tables 9A to 9C are explained below.
★: An obtained amplification product whose size almost matched the target 87 to 89 bp (±10 bp).
W 2-6: Probability to obtain 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.
Related Species of the genus Arachis:
Sequences similar to the ITS-2 sequence of Glycine max (U60551) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected from among them Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Table 9C as the representative of the DNA sequences of related species of the genus Glycine.
Commercially available seeds of soybean were used.
(2) DNA Isolation from Soybean
A DNA was isolated from seeds of soybean in the same way as in Example 1B (3). The isolated DNA preparation of soybean was diluted stepwise with sterilized ultrapure water to use as template DNA for PCR.
PCR was conducted in the substantially same way as Example 1C, except for use of the following primers and PCR program.
The primer of SEQ NO:34 was used at 0.5,LM of final concentration and each primer of SEQ NOs:36 and 37 was used at 0.25 μM of final concentration.
PCR was conducted according to the following PCR program.
Pre-incubation at 95° C. for 15 min.; thereafter 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 68° C. for 1 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 mm.
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 plant chloroplast DNA.
Regarding the primers of the present invention, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels) to examine the specificity to ITS-2 sequence of each plant. As a result, as shown in Tables 9A to 9C, it was predicted that target 87 to 89 bp amplification products would be obtained from the aforementioned 50 DNA sequences of the genus Glycine. On the other hand, it was predicted that no target 87 to 89 bp amplification product would be obtained from the 8 DNA sequences of other common allergenic plants (buckwheat, peanut, wheat, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the 6 DNA sequence of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean) and the 3 DNA sequences of related species of the genus Glycine belonging to leguminous plants. In this connection, it was predicted by simulation that amplification products having almost 87 to 89 bp would be obtained from the DNA sequences of Amphicarpaea edgeworthii and Ophrestia radicosa, but the former can be identified by sequence analysis whether the genus Glycine or not and the latter may be able to be identified by appropriately using a commercially available ELISA kit of soybeans and various types of PCR and the like reported.
Glycine dolichocarpa
Arachis hypogaea (Peanut)
Fagopyrum esculentum
Triticum aestivum (Wheat)
Juglans regia (Walnut)
Tricholoma matsutake
Prunus persica (Peach)
Malus x domestica
Citrus sp.
Zea mays (Corn)
Oryza sativa (Brown rice)
Piper nigrum (Pepper)
Sinapis alba
Phaseolus vulgaris
Phaseolus lunatus
Lens culinaris subsp.
culinaris (Lentil)
Cicer arietinum (Chickpea)
Vigna radiata (Mung bean)
Vigna angularis var.
nipponensis (Adzuki bean)
Myrospermum sousanum
Amphicarpaea bracteata
Strophostyles umbellata
PCR described above was conducted using primers designed in the present invention. In this case, target 87 to 89 bp amplification products, expected from the simulation results of the ITS-2 sequences of the genus Glycine, were obtained from 500 to 50 fg of soybean DNA. It is found from the results that even where 500 to 50 fg of soybean DNA is present, the soybean can be detected. In this connection, this sensitivity corresponds to a sensitivity wherein there can be detected 10 to 1 ppm of soybean DNA contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples.
Consequently, in conjunction with the results of specificity studied by PCR simulation, and the results of sensitivity studied by PCR, it was confirmed that a wide range of the genus Glycine including soybean were specifically detectable at a high sensitivity using the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2001-336571 | Nov 2001 | JP | national |
| 2002-284222 | Sep 2002 | JP | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11581872 | Oct 2006 | US |
| Child | 12139701 | US | |
| Parent | 10285061 | Oct 2002 | US |
| Child | 11581872 | US |
