Patent Application
20080064028
Hereinafter, the present invention will be described more specifically with reference to the Examples.
Shirahana buckwheat (common buckwheat; Fagopyrum esculentum, diploid) and Dattan buckwheat (tatary buckwheat; Fagopyrum tataricum, diploid) seeds from Takano were used.
Commercially-available products were used.
Leaves germinated from commercially-available seeds were used.
DNA extraction was conducted using Genomic-tip manufactured by QIAGEN with reference to QIAGEN Genomic DNA Handbook and User-Developed Protocol: Isolation of genomic DNA from plants using the QIAGEN Genomic-tip according to procedures below.
In a 15-ml tube, 1 g of a pulverized sample was introduced, 4 ml of Carlson Lysis Buffer (0.1 M Tris-HCl (pH 9.5), 2% CTAB, 1.4 M Polyethylene Glycol #6000, and 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) were added and mixed, followed by incubation at 74° C. for 20 minutes. After being returned to room temperature, 5 ml of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the resulting mixture and well mixed. An aqueous layer was then collected therefrom by centrifugation. This aqueous layer was supplemented and well mixed with the same amount of chloroform:isoamyl alcohol (24:1). An aqueous layer was then collected therefrom by centrifugation. After the same amount of chloroform:isoamyl alcohol (24:1) was again added to the aqueous layer and mixed, an aqueous layer was collected therefrom by centrifugation.
A ½ aliquot was taken from the obtained aqueous layer and subjected to isopropanol precipitation to collect the resulting precipitate. The precipitate was dissolved in 500 μl of Buffer QBT and applied to Genomic-tip 20/G Column equilibrated with 1 ml of Buffer QBT, to which DNA was then adsorbed. Then, the Column was washed with 5 ml of Buffer QBT and subsequently with 2 ml of Buffer QC. Finally, a precipitate collected by elution with 1.7 ml of Buffer QF and isopropanol precipitation was dissolved in 40 μl of sterilized ultrapure water. A DNA concentration in the resulting solution was measured, and the DNA solution appropriately diluted with sterilized ultrapure water was used as a template DNA sample for PCR.
DNA extraction was conducted using DNeasy Plant Maxi Kit manufactured by QIAGEN with reference to DNeasy Plant Maxi Kit Handbook according to procedures below.
In a 50-ml tube, 2 g of a pulverized sample was introduced, 10 ml of Buffer AP1 and 20 μl of RNase A (100 mg/ml) were added and mixed. The resulting mixture was incubated at 65° C. for 15 minutes and then centrifuged at approximately 3,000×g for 10 minutes. A 4-ml aliquot of the resulting supernatant was collected into a 15-ml tube, to which 1.8 ml of Buffer AP2 was in turn added. The resulting mixture was left in ice for 10 minutes and centrifuged at approximately 3,000×g for 10 minutes. The resulting supernatant was applied to QIAshredder Spin Column and centrifuged at approximately 3,000×g for 5 minutes. A 5-ml aliquot of the resulting flow-through solution was collected into a 50-ml tube, to which 7.5 ml of Buffer AP3/E was in turn added and mixed. The resulting mixture was applied to DNeasy Spin Column and centrifuged at approximately 3,000×g for 5 minutes to have DNA adsorbed to the Column. Then, 12 ml of Buffer AW was added to the Column and centrifuged at approximately 3,000×g for 5 minutes, followed by the washing of the Column. Again, 12 ml of Buffer AW was added thereto and centrifuged at approximately 3,000×g for 10 minutes, followed by the washing of the Column. Finally, 1 ml of Buffer AE preincubated at 65° C. was added to the Column and left for 10 minutes. The Column was then centrifuged at approximately 3,000×g for 5 minutes to elute DNA from the Column. A DNA concentration in the resulting solution was measured, and the DNA solution appropriately diluted with sterilized ultrapure water was used as a template DNA sample for PCR.
DNA extraction was conducted using DNeasy Plant Maxi Kit manufactured by QIAGEN in combination with NucleoSpin Extract 2 in 1 manufactured by MACHEREY-NAGEL with reference to QIAGEN Genomic DNA Handbook and NucleoSpin Extract 2 in 1 For Direct Purification of PCR Products according to procedures below.
In a 15-ml tube, 1 g of a pulverized sample was introduced, 10 ml of Buffer G2, 100 μl of proteinase K (20 mg/ml), and 10 μl of RNase A (100 mg/ml) were added and mixed, followed by incubation at 50° C. for 1 hour. The resulting mixture was centrifuged at approximately 3,000×g for 10 minutes to obtain its supernatant. The obtained supernatant was applied to Genomic-tip 20/G Column equilibrated with 1 ml of Buffer QBT, to which DNA was then adsorbed. Then, the Column was washed with 4 ml of Buffer QC. DNA was then eluted with 1 ml of Buffer QF preheated to 50° C. To the resulting eluate, 4 volumes of Buffer NT2 was added and mixed. Then, 650-μl/run of the resulting mixture solution was applied to two NucleoSpin Extract Columns and centrifuged at approximately 6,000×g for 1 minute to have DNA adsorbed to the Columns. This was repeated until the whole amount of the mixture solution was treated. Then, 600 μl of Buffer NT3 was added to the Column and centrifuged at approximately 6,000×g for 1 minute, followed by the washing of the Column. Again, 600 μl of Buffer NT3 was added thereto and centrifuged at the maximum speed for 1 minute to completely remove the Buffer NT3 remaining in the Column. Finally, 100 μl of Buffer NE was added to the Column and centrifuged at the maximum speed for 1 minute to elute DNA from the Column. A precipitate collected by isopropanol precipitation was dissolved in 50 μl of sterilized ultrapure water. A DNA concentration in the resulting solution was measured, and the DNA solution appropriately diluted with sterilized ultrapure water was used as a template DNA sample for PCR.
DNA extraction was conducted using DNeasy Plant Mini Kit manufactured by QIAGEN with reference to DNeasy Plant Mini Kit Handbook according to procedures below.
In a 15-ml tube, 0.5 g of a pulverized sample was introduced, 3 ml of Buffer AP1 and 30 μl of RNase A (100 mg/ml) were added and mixed, followed by incubation at 65° C. for 15 minutes. To this mixture, 975 μl of Buffer AP2 was added and left on ice for 10 minutes. The mixture was centrifuged to obtain its supernatant. The obtained supernatant was applied to QIAshredder Spin Column, which was in turn centrifuged to obtain a flow-through solution from the Column. To this flow-through solution, 0.5 volumes of Buffer AP3 and 1 volume of ethanol were added and mixed. Then, 650-μl/run of the resulting mixture solution was applied to two DNeasy Spin Columns and centrifuged at approximately 6,000×g for 1 minute to have DNA adsorbed to the Columns. This was repeated until the whole amount of the mixture solution was treated. Then, 500 μl of Buffer AW was added to the Column and centrifuged at approximately 6,000×g for 1 minute, followed by the washing of the Column. Again, 500 μl of Buffer AW was added thereto and centrifuged at the maximum speed for 1 minute to completely remove the Buffer AW remaining in the Column. Finally, 120 μl of Buffer AE preincubated at 65° C. was added to the Column and centrifuged at approximately 6,000×g for 1 minute to elute DNA from the Column. A DNA concentration in the resulting solution was measured, and the DNA solution appropriately diluted with sterilized ultrapure water was used as a template DNA sample for PCR.
Sequences universal to the ITS-1-5.8S rRNA gene sequences of the following 21 sequences registered in GenBank of plants belonging to the genus Fagopyrum were used as primer sequences:
Then, oligo DNA primers (manufacture by QIAGEN, OPC-purified oligonucleotides) having the following sequences were synthesized and used as primers for PCR that detect a portion of the ITS-1-5.8S rRNA gene sequence of buckwheat (hereinafter, referred to as buckwheat PCR):
A PCR simulation software Amplify 1.0 (Bill Engels) was used to confirm whether a result of the simulation showed that a PCR amplification product was obtained with the primers for detecting buckwheat, based on 21 sequences of plants belonging to the genus Fagopyrum, 8 sequences of likely-to-be-allergenic plants other than buckwheat (peanut, wheat, soybean, walnut, matsutake mushroom, peach, apple, and orange), 4 sequences of plants frequently used as food ingredients (maize, rice, pepper, and mustard), and 27 sequences of related plant species of buckwheat. The related plant species of buckwheat used herein refer to plants other than the genus Fagopyrum, which attained Score 60 bits or more when the ITS-1 sequence portion in the nucleotide sequence (AB000330) of common buckwheat, Fagopyrum esculentum, registered in GenBank was subjected to BLAST homology search. This time, the sequence of a species attaining the highest score in a genus to which each of the plants belonged was selected as a representative sequence of the genus. The PCR simulation was conducted for the ITS-1-5.8S rRNA gene-ITS-2 sequence region of that sequence. The GenBank Accession Number of the sequence used in the simulation and a result of the simulation are shown in Tables 1A to 1C. Abbreviated letters and symbols in Tables 1A to 1C are as shown below:
Filled-in asterisk: those expected to yield a PCR amplification product having a size around a target size (±10 bp)
W value: Possibility of yielding a PCR amplification product
Numeric (bp): the size (bp) of a PCR amplification product
−: those expected to yield no PCR amplification product
Arachis hypogaea (Peanut)
Triticum aestivum (Wheat)
Glycine max (Soybean)
Juglans regia (Walnut)
Tricholoma matsutake
Prunus persica (Peach)
Malus x domestica
Citrus sp.
Zea mays
Oryza sativa (Rice)
Piper nigrum (Pepper)
Sinapis alba (Mustard)
Aconogonum sp. Won 152
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
As shown in Tables 1A to 1C, it was expected from the result of the simulation that a PCR amplification product having a target size of 101 bp was obtained from the 21 sequences of plants belonging to the genus Fagopyrum. In addition, it was expected that a PCR amplification product having the target size and a non-specific PCR amplification product were not obtained from the 8 sequences of likely-to-be-allergenic plants other than buckwheat (peanut, wheat, soybean, walnut, matsutake mushroom, peach, apple, and orange), the 4 sequences of plants frequently used as food ingredients (maize, rice, pepper, and mustard), and the 27 sequences of related plant species of buckwheat.
Buckwheat PCR was conducted using HotStarTaq Master Mix Kit manufactured by QIAGEN according to procedures below.
Primers of SEQ ID NOs: 14 and 15 (0.5 μM each at a final concentration) and template DNA were added to 12.5 μl of 2×HotStartTaq Master Mix (HotStar Taq DNA Polymerase, PCR buffer with 3 mM MgCl2, and 400 μM each dNTP), whose final volume was adjusted with sterilized ultrapure water to 25 μl to make a reaction solution, which was in turn placed in a 0.2-ml microtube and reacted using a thermal cycler GeneAmp PCR System 9600 manufactured by Applied Biosystems according to the following PCR steps: enzyme activation at 95° C. for 15 minutes; 45 cycles of denaturation at 95° C. for 1 minute, annealing at 66° C. for 2 minutes, and extension 72° C. for 1 minute; and final extension at 72° C. for 4 minutes. The resulting PCR reaction solution was subjected to ethidium bromide-containing 2% agarose gel electrophoresis and analyzed with a fluorescent image analyzer FluorImager 595 manufactured by Amersham Biosciences. The results are shown in
M: 100-bp DNA Ladder Marker
(−): No addition of template DNA
Numeric: Amount of template DNA added
Arrow: Target band (approximately 101 bp) of PCR amplification product
The extracted plant DNA was confirmed to have a purity level capable of PCR amplification by obtaining a PCR amplification product with primers for amplifying a portion of plant chloroplast DNA (data not shown).
As a result of buckwheat PCR, a PCR amplification product having a size of approximately 101 bp expected from the target ITS-1-5.8S rRNA gene sequence of buckwheat was obtained from 500 to 50 fg of Shirahana buckwheat (common buckwheat) and Dattan buckwheat DNAs, as shown in
As a result of buckwheat PCR, a PCR amplification product having the target size and a non-specific PCR amplification product were not obtained from 50 ng each of the DNAs of the wheat leaf, peanut seed, soybean leaf, maize leaf, mustard leaf, and white pepper, and rice, as shown in
The nucleotide sequence of the Shirahana buckwheat DNA-derived PCR amplification product thus obtained was analyzed by double-strand direct sequencing using primers of SEQ ID NOs: 14 and 15. The obtained nucleotide sequence was compared with the nucleotide sequence (AB000330) of common buckwheat, Fagopyrum esculentum, registered in GenBank to confirm that the nucleotide sequence of the Shirahana buckwheat DNA-derived PCR amplification product matched 100% to the target site of the nucleotide sequence (AB000330) of common buckwheat (Fagopyrum esculentum) registered in GenBank: This demonstrated that PCR using the primers amplified and detected a portion of the ITS-1-5.8S rRNA gene sequence of buckwheat.
These results showed that buckwheat PCR using the primers could detect, with high sensitivity and specificity, the ITS-1-5.8S rRNA gene sequences of the general plants belonging to the genus Fagopyrum. We decided to use the present primers in PCR that quantified the copy number of the ITS-1-5.8S rRNA gene sequence of buckwheat (hereinafter, referred to as a quantitative PCR method for a buckwheat sequence).
Next, the detection of, by PCR, a standard plant sample used in correction was investigated.
In the present Example, statice, a spermatophyte not described in an upland weed list by The Weed Science Society of Japan, whose seed was easily available was used as the standard plant sample.
Based on the DNA sequence (AJ222860) of statice registered in GenBank, primers having the following sequences for PCR that detected a portion of the ITS-1 sequence of statice (hereinafter, referred to as statice PCR) were designed to synthesize oligo DNA primers (manufactured by QIAGEN, OPC-purified oligonucleotides):
Statice PCR was conducted basically in the same way as the above Example 1.C.(3) except that the above-described primers were used at a final concentration of 0.2 μM each. The results are shown in
The extracted plant DNA was confirmed to have a purity level capable of PCR amplification by obtaining a PCR amplification product with primers for amplifying a portion of plant chloroplast DNA (data not shown).
As a result of statice PCR, a PCR amplification product having a size of approximately 101 bp expected from the target ITS-1 sequence of statice was obtained from 50 ng of the DNA of the statice seed, as shown in
Thus, the primers for detecting statice DNA are presumed to have specificity to statice DNA.
Next, confirmation of whether statice was suitable as the standard plant sample was conducted. Namely, statice PCR was conducted to confirm that statice did not contaminate a food or a food ingredient.
As a result of statice PCR, a PCR amplification product having a target size and a non-specific PCR amplification product were not obtained from 50 ng each of the DNAs of the seeds of 5 types of wheat, 5 types of corn grits, and 3 types of mustard, as shown in
A quantitative PCR method for a buckwheat sequence established as described below was conducted to confirm whether or not buckwheat contaminated the sample of the statice seed. As a result of the quantitative PCR method for the buckwheat sequence, it was confirmed that the fluorescent signal indicating amplification was not found from the DNA of the statice seed, and that contamination was not observed (data not shown).
The nucleotide sequence of the statice DNA-derived PCR amplification product thus obtained was analyzed by double-strand direct sequencing using primers of SEQ ID NOs: 57 and 58. The obtained nucleotide sequence was compared with the nucleotide sequence (AJ222860) of statice, Limonium sinuatum, registered in GenBank to confirm that the nucleotide sequence of the statice DNA-derived PCR amplification product matched 100% to the target site of the nucleotide sequence (AJ222860) of statice (Limonium sinuatum) registered in GenBank. It could be confirmed that the statice PCR amplified and detected a portion of the target ITS-1 sequence of statice.
These results suggested that mutual contamination did not take place between statice and food ingredients, and that the statice was suitable as the standard plant sample for correction. We thus decided to use the primers of SEQ ID NOs: 57 and 58 in PCR that quantified the copy number of the ITS-1 sequence of statice (hereinafter, referred to as a quantitative PCR method for a statice sequence).
The target amplification product of buckwheat and the target amplification product of statice were ligated by a PCR method and introduced into a TA cloning vector. The TA cloning vector was introduced into E. coli and amplified, thereby constructing a plasmid for standard curves for quantitatively analyzing the copy numbers of buckwheat and statice.
At first, oligo DNA primers (manufactured by QIAGEN, OPC-purified oligonucleotides) having sequences below were synthesized and used as primers. These primers contain the primer sites for buckwheat and statice used in the above-described buckwheat PCR and statice PCR.
A ligation plasmid was constructed using HotStarTaq Master Mix Kit manufactured by QIAGEN with reference to the method by Jayaraman K. et al. (1992. A PCR-Mediated Gene Synthesis Strategy Involving the Assembly of Oligonucleotides Representing Only One of the Strands, BioTechniques 12: 392-398) according to procedures below.
To 25 μl of 2×HotStartTaq Master Mix (HotStar Taq DNA Polymerase, PCR buffer containing 3 mM MgCl2, and 400 μM each dNTP), dNTP (500 μM at a final concentration) was added, primers of SEQ ID NOs: 60 and 63 (1.0 μM each at a final concentration) as outer primers, primers of SEQ ID NOs: 61 and 62 (25 nM each at a final concentration) as bridging primers were added. As template DNAs, the PCR amplification product with the target DNA sequence of buckwheat PCR obtained in Example 1.C.(4) and the PCR amplification product with the target DNA sequence of statice PCR obtained in Example 1.D.(3) were added. The final volume was adjusted with sterilized ultrapure water to 50 μl to make a reaction solution, which was in turn placed in a 0.2-ml microtube and reacted using a thermal cycler PTC-200 DNA Engine manufactured by MJ Research according to the following PCR steps: enzyme activation at 95° C. for 15 minutes; 15 cycles of denaturation at 95° C. for 1 minute, annealing at 40° C. for 1 minute, and extension 72° C. for 1 minute; and 30 cycles of denaturation at 95° C. for 1 minute, annealing at 66° C. for 1 minute, and extension 72° C. for 1 minute. The resulting PCR reaction solution was subjected to ethidium bromide-containing 2% agarose gel electrophoresis and analyzed with a fluorescent image analyzer FluorImager 595 manufactured by Amersham Biosciences. The nucleotide sequence of the resulting PCR amplification product was analyzed by double-strand direct sequencing using primers of SEQ ID NOs: 60 and 63.
As a result of ligation PCR, a PCR amplification product having an expected size of approximately 200 bp was obtained (data not shown). As a result of nucleotide sequence analysis, it was confirmed that this PCR amplification product contained the target DNA sequences of buckwheat PCR and statice PCR (data not shown).
Using pGEM-T Easy Vector System (manufactured by Promega), the PCR amplification product thus obtained was TA-cloned into pGEM-T Easy Vector, with which E. coli (E. coli JM109 (DH5α)) was then transformed. A transformant, having the approximately 220-bp inserted fragment that could be confirmed to contain the target DNA sequences of buckwheat PCR and statice PCR by colony PCR and nucleotide sequence analysis, was subjected to liquid culture in a LB medium. QIAGEN Hi Speed Plasmid Midi Kit manufactured by QIAGEN was used to extract and purify the plasmid from the resulting culture. The nucleotide sequence of the DNA fragment inserted into the purified plasmid was analyzed by double-strand sequencing using primers for the sequence on the plasmid. As a result, it was confirmed that the nucleotide sequence of the DNA fragment inserted into the plasmid of the transformant contained the target DNA sequences of buckwheat PCR and statice PCR, as intended (data not shown).
The number (copy number) of the plasmid molecules was calculated based on the plasmid length and the absorbance (Abs. 260 nm) of the above-described plasmid extracted and purified. The plasmid was diluted with 5 ng/μl salmon sperm DNA (manufactured by Wako Pure Chemical Industries, fibrous sodium deoxyribonucleate from salmon testis dissolved in sterilized ultrapure water) to prepare a dilution series of the plasmid for standard curves at 109 to 101 copies/2.5 μl. We decided to use this dilution series in the generation of standard curves for the quantitative PCR methods for buckwheat and statice sequences.
A TaqMan MGB probe (manufactured by Applied Biosystems Japan, reporter dye FAM) having a sequence below was synthesized and used as a probe for detecting a buckwheat sequence. A sequence universal to 21 sequences registered in GenBank as the ITS-1-5.8S rRNA gene sequences of plants belonging to the genus Fagopyrum was employed as the probe sequence.
A Quantitative PCR method for a buckwheat sequence was conducted using QuantiTect Probe PCR Kit manufactured by QIAGEN according to procedures below.
Primers of SEQ ID NOs: 14 and 15 (0.2 μM each at a final concentration), the TaqMan MGB probe of SEQ ID NO: 64 (0.2 μM at a final concentration), and template DNA were added to 12.5 μl of 2×QuantiTect Probe PCR Master Mix. The final volume was adjusted with sterilized ultrapure water to 25 μl to make a solution, which was in turn dispensed into a 96-well PCR plate. For standard curves, a solution supplemented with the dilution series of the plasmid DNA for standard curves instead of the template DNA was dispensed. The 96-well PCR plate into which each of the solutions was dispensed was loaded in a real-time PCR device Sequence Detection System 7700 manufactured by Applied Biosystems, in which the solution was reacted according to the following PCR steps: at 50° C. for 2 minutes; 95° C. for 15 minutes; and 45 cycles of denaturation at 95° C. for 1 minute, annealing at 66° C. for 2 minutes, and extension at 72° C. for 1 minute. Every reaction was conducted with the same samples in duplicate (in 2 wells). After the completion of reaction, fluorescence data taken during the extension step was analyzed. A baseline was first set to cycles 0 to 1 and then appropriately set to within a range before a cycle where the increase of fluorescence was confirmed to begin. A threshold line was set according to the method described in Kuribara H et al., 2002, Novel Reference Molecules for Quantitation of Genetically Modified Maize and Soybean, Journal of AOAC International 85: 1077-1089. The results are shown in
The extracted plant DNA was confirmed to have a purity level capable of PCR amplification by success of obtaining a PCR amplification product with primers for amplifying a portion of plant chloroplast DNA (data not shown).
As a result of the quantitative PCR method for the buckwheat sequence, a fluorescent signal indicating amplification was found from the DNA from the Shirahana buckwheat seed, as shown in
This specificity corresponds to a specificity level at which, when PCR is conducted with 50 ng of DNA extracted from a certain sample supplemented with statice as a template, the sample is not quantified as a false positive even if the sample was black bindweed (related species of buckwheat), one species of weeds that are 100% inedible.
As a result of the quantitative PCR method for the buckwheat sequence, a quantitative property and sensitivity where a standard curve having a correlation coefficient of 0.999 and a slope of −3.504 could be drawn with 108 to 101 copies of the plasmid for standard curves could be confirmed, as shown in
These results demonstrated that the quantitative PCR method for the buckwheat sequence using the primers of SEQ ID NOs: 14 and 15 together with the probe of SEQ ID NO: 64 could detect, with high sensitivity and specificity, the ITS-1-5.8S rRNA gene sequences of the general plants belonging to the genus Fagopyrum and quantify their copy numbers. We decided to use the present quantitative PCR method for the buckwheat sequence in combination with a quantitative PCR method for a statice sequence for correction shown below in the measurement of the amount of contaminating buckwheat.
A TaqMan MGB probe (manufactured by Applied Biosystems Japan, reporter dye FAM) having a sequence below was synthesized and used as a probe for detecting a statice sequence.
A quantitative PCR method for a statice sequence was conducted basically in the same way as Example 1.F.(2) except that primers of SEQ ID NOs: 57 and 58 were used at a final concentration of 0.2 μM each and the TaqMan MGB probe of SEQ ID NO: 59 was used at a final concentration of 0.2 μM. The results are shown in
As a result of the quantitative PCR method for the statice sequence, a fluorescent signal indicating amplification was found from the DNA from the statice seed, as shown in
As a result of the quantitative PCR method for the statice sequence, a quantitative property that could draw a standard curve having a correlation coefficient of 0.999 and a slope of −3.386 with 108 to 101 copies of the plasmid for standard curves could be confirmed, as shown in
These results demonstrated that the quantitative PCR method for the statice sequence using the primers of SEQ ID NOs: 57 and 58 together with the probe of SEQ ID NO: 59 could specifically detect the ITS-1 sequence of statice and quantify its copy number. We decided to use the present quantitative PCR method for the statice sequence for correction in combination with the quantitative PCR method for the buckwheat sequence shown in Example 1.F. in the measurement of the amount of contaminating buckwheat.
Excellent Light Blue for a cut flower (single lot) sold by Sakata Seed Corporation was used.
The buckwheat flour of Shirahana buckwheat (common buckwheat; Fagopyrum esculentum, diploid), the buckwheat flour of Dattan buckwheat (F. tataricum, diploid), the buckwheat flour of Takane Ruby (F. esculentum, diploid), and the buckwheat flour of Great Ruby (F. esculentum, tetraploid) sold by Takano Co., Ltd. were used. Shirahana buckwheat flour was used in the preparation of an artificially contaminated sample.
Commercially-available Norin 61 was used.
Commercially-available chemical-free Akita Komachi brown rice was used.
Pulverization was performed with Ultra Centrifugal Mill ZM1 (manufactured by Retsch) equipped with a rotor (made of stainless steel, 24-edged) and a screen (made of stainless steel, 0.20 mm).
The parts of the mill such as a sample holder, a sample lid, a rotor, a screen, fasteners, and a jig were washed with water, immersed in 10% bleaching solution, washed with water, and dried, before and after use for the pulverization of the sample. The main body of the mill was washed with an air gun and wiped, and then used.
Before the pulverization of the sample in large amounts, a portion thereof or commercially-available freeze-dried maize with cornhusk not contaminated with buckwheat and statice was pulverized. DNA was then extracted therefrom to confirm the present or absence of a fluorescent signal indicating amplification from 50 ng of the template DNA by the quantitative PCR methods for the buckwheat sequence and the statice sequence shown in Example 1.F and Example 1.G. When no fluorescent signal was observed, the mill was assessed as being not contaminated, and the work proceeded to do the pulverization of the sample in large amounts illustrated below. When a fluorescent signal was observed, the mill was assessed as being contaminated. In this case, the mill was washed again, and brown rice (1 kg) already confirmed to have no contamination with buckwheat and statice was pulverized in this mill. After the washing of the mill and the replacement of its screen with a new one, the commercially-available freeze-dried maize with cornhusk not contaminated with buckwheat and statice was pulverized again, and the presence or absence of a fluorescent signal was confirmed in the same way as above. After the mill could be assessed as being not contaminated with buckwheat and statice, the work proceeded to do the pulverization of the sample in large amounts illustrated below.
In the mill that was confirmed to have no contamination with buckwheat, approximately 1 kg of statice was pulverized. Ten 2-g aliquots were sampled from the pulverized powder, and DNA was extracted therefrom with DNeasy Plant Maxi Kit by the method described in Example 1.B.(2) to confirm the absence of a fluorescent signal indicating amplification from 50 ng of the template DNA by the quantitative PCR method for the buckwheat sequence (data not shown). The powder of statice not contaminated with buckwheat was secured by these procedures.
In the mill that was confirmed to have no contamination with buckwheat and statice, approximately 500 g of rice was pulverized. Five 2-g aliquots were sampled from the pulverized powder, and DNA was extracted therefrom with DNeasy Plant Maxi Kit by the method described in Example 1.B.(2) to confirm the absence of a fluorescent signal indicating amplification from 50 ng of the template DNA by the quantitative PCR methods for the buckwheat sequence and the statice sequence (data not shown). The same procedures were conducted for wheat. The pulverized powders of rice and wheat not contaminated with buckwheat and statice were obtained by these procedures.
Six anti-static OP bags (manufactured by Fukusuke Kogyo, PZ type No. 6 (special anti-statice treatment) reclosable with a zipper and three sides sealed), in which 45.00 g of the rice pulverized powder was weighed and placed, were prepared and numbered 1 through 6. In the bag No. 1, 5.00 g of buckwheat flour was weighed and placed. The contents of the bag were manually mixed for 15 minutes with the top of the bag closed, to obtain the rice pulverized powder containing 10% buckwheat flour. Subsequently, 5.00 g of this powder of rice containing 10% (100, 000 ppm) buckwheat flour was weighed and placed in the bag No. 2. The contents of the bag were manually mixed for 15 minutes with its mouth closed, to obtain the powder of rice containing 1% (10,000 ppm) buckwheat flour. These dilution and mixing procedures were repeated to prepare the rice pulverized powders containing 100,000 to 1 ppm of buckwheat flour.
The wheat pulverized powders containing 100,000 to 1 ppm of buckwheat flour were prepared in the same way as above.
In an anti-static OP bag (manufactured by Fukusuke Kogyo, PZ type No. 5 (special anti-statice treatment) reclosable with a zipper and three sides sealed), 12.5 g of the rice pulverized powder containing 10 ppm of buckwheat flour and 12.5 g of the wheat pulverized powder containing 10 ppm of buckwheat flour were weighed and placed. The contents of the bag were manually mixed for 15 minutes with top of the bag closed, to obtain the pulverized powder of rice and wheat containing 10 ppm of buckwheat flour
For determining the particle size of buckwheat flour with the assumption that the buckwheat flour was a globular, the particle size distribution measurement (laser diffraction/scattering method, dry process, under the condition of a pressure of 0.5 kg/cm2) of Shirahana buckwheat flour was conducted. The measurement was outsourced to Seishin Enterprise Co., Ltd., Powder Technology Centre. As a result, the particle size of the Shirahana buckwheat flour in terms of a particle size (median size) (×50) was 80.941 μm.
For determining the density (density including inter- and intra-particle voids and pores) of buckwheat flour, the bulk density measurement (Mercury (Hg) method: a method where buckwheat flour is placed in a cell having a fixed volume, which is then filled with mercury) of Shirahana buckwheat flour was conducted. The measurement was outsourced to Seishin Enterprise Co., Ltd., Powder Technology Centre. As a result, the bulk density of the Shirahana buckwheat flour (by the Hg method) was 1.181 g/cm3.
Volume occupied by buckwheat flour=(volume of cell)−(volume of mercury added)
Bulk density of buckwheat flour(Hg method)=(volume of buckwheat flour added)/(volume occupied by buckwheat flour)
Weight per particle of buckwheat flour was calculated from the measured values (the particle size of 80.941 μm and the density of 1.181 g/cm3) of the Shirahana buckwheat flour to make a trial calculation of the particle number of the buckwheat flour in the artificially contaminated samples of varying buckwheat flour concentrations. The results are shown in Table 2. This result revealed that, when a sample for DNA extraction was sampled from the artificially contaminated sample containing 10 ppm of contaminating buckwheat of interest in quantification, 4 g or more of the sample for DNA extraction was required for placing at least approximately 100 particles of buckwheat flour in the sample that had been sampled. We decided to sample a 5-g aliquot for DNA extraction.
Six samples were sampled from Shirahana buckwheat flour and three samples were sampled from each of Takane Ruby buckwheat flour, Great Ruby buckwheat flour, and Dattan buckwheat flour. These samples were used in DNA extraction.
Three samples were sampled from each of the wheat pulverized powder containing 100 ppm of Shirahana buckwheat flour, the wheat pulverized powder containing 10 ppm of Shirahana buckwheat flour, the rice pulverized powder containing 10 ppm of Shirahana buckwheat flour, and the pulverized powder of wheat and rice containing 10 ppm of Shirahana buckwheat flour, and used in DNA extraction.
DNA extraction was conducted using Genomic-tip manufactured by QIAGEN with reference to QIAGEN Genomic DNA Handbook and User-Developed Protocol: Isolation of genomic DNA from plants using the QIAGEN Genomic-tip according to procedures below.
In a 50-ml tube, 5 g of the sample and 1 g of the statice pulverized powder were placed and to which 30 ml of Carlson Lysis Buffer (0.1 M Tris-HCl (pH 9.5), 2% CTAB, 1.4 M Polyethylene Glycol #6000, and 20 mM EDTA), 60 μl of RNase A (100 mg/ml), 75 μl of 2-mercaptoethanol, and 600 μl of proteinase K (20 mg/ml) were added. For further enhancing the dispersibility of the sample, three zirconia balls (manufactured by Nikkato, YTZ ball, φ7 mm) were added to the mixture and mixed for 10 minutes or more with a shaker (manufactured by Iwaki Sangyo, KM Shaker V-DX) at Speed 100 until lumps were eliminated, followed by incubation at 74° C. for 20 minutes. During the incubation, the tube was manually shaken and mixed every five minutes.
Following centrifugation at 3,000×g for 10 minutes, 4 ml of the resulting supernatant was collected into a 15-ml tube and 5 ml of phenol:chloroform:isoamyl alcohol (25:24:1) was added and well mixed. After this mixture was centrifuged at 3,000×g for 10 minutes, the resulting supernatant (aqueous layer) was collected into a 15-ml tube and 3.5 ml of chloroform:isoamyl alcohol (24:1) was added and well mixed. After this mixture was centrifuged at 3,000×g for 10 minutes, the resulting supernatant (aqueous layer) was collected into a 15-ml tube and subjected again to extraction with chloroform:isoamyl alcohol (24:1) and centrifugation to collect a supernatant (aqueous layer). A precipitate collected from a 150-μl aliquot of the supernatant (aqueous layer) by isopropanol precipitation was dissolved in 100 μl of sterilized ultrapure water and 900 μl of Buffer QBT was added. The resulting solution was applied to Genomic-tip 20/G Column equilibrated with 1 ml of Buffer QBT, to which DNA was then adsorbed. Then, the Column was washed with 4 ml of Buffer QC. Finally, a precipitate collected by DNA elution with 1 ml of Buffer QF and isopropanol precipitation was dissolved in 40 μl of sterilized ultrapure water. A DNA concentration in the resulting solution was measured, and the DNA solution appropriately diluted with sterilized ultrapure water was used as a template DNA sample for PCR.
The quantitative PCR methods for the buckwheat sequence and the statice sequence were conducted by the method described in Example 1.F. and Example 1.G. Based on the standard curves, the copy number of the buckwheat sequence and the copy number of the statice sequence of 50 ng of DNA extracted from 100% buckwheat flour supplemented with the statice standard were quantified. Based on the quantitative values, “the copy number of the statice sequence/the copy number of the buckwheat sequence=Lo/Fo ratio” was calculated. The Lo/Fo ratio of each buckwheat flour sample was calculated by simultaneously measuring the same samples in 2 wells and obtaining the average of ratios from two measurements.
As a result of Lo/Fo ratio measurement, the Lo/Fo ratio was 2.36 for the Shirahana buckwheat flour (6 extracted samples each measured in duplicate in two wells), 3.25 for the Takane Ruby buckwheat flour, 2.70 for the Great Ruby buckwheat flour, and 4.75 for the Dattan buckwheat flour (3 extracted samples each measured in duplicate in two wells), as shown in Table 3. We decided that the amount of contaminating buckwheat was determined using the Lo/Fo ratio of the Shirahana buckwheat flour obtained here and “the copy number of the buckwheat sequence/the copy number of the statice sequence=Fs/Ls ratio” of the artificially contaminated sample calculated in Example 2.G. The raw data of a variety of buckwheat flour samples in Lo/Fo ratio measurement is shown in Tables 4A and 4B.
Fagopyrum:
Limonium:
Fagopyrum:
Limonium:
The measurement value of the Dattan buckwheat flour deviated most from the measurement value of the Shirahana buckwheat flour and however, was only about twice the measurement value of the Shirahana buckwheat flour. Thus, the present method is considered to have sufficient precision as a quantifying method by PCR.
G. Calculation of “Copy Number of Buckwheat Sequence/Copy Number of Statice Sequence Ratio” in DNA Extracted from Artificially Contaminated Sample Supplemented with Statice Standard and Calculation of Amount of Buckwheat Contaminating Artificially Contaminated Sample
The quantitative PCR methods for the buckwheat sequence and the statice sequence were conducted by the method described in Example 1.F. and Example 1.G. Based on the standard curves, the copy number of the buckwheat sequence and the copy number of the statice sequence of 50 ng of DNA extracted from the artificially contaminated sample supplemented with the statice standard were quantified. Based on the quantitative values, “the copy number of the buckwheat sequence/the copy number of the statice sequence=Fs/Ls ratio” was calculated. The Fs/Ls ratio of the artificially contaminated sample was calculated by extracting 3 samples from the same sample, each of which was measured in 2 wells. The amount (μg) of buckwheat contaminating the artificially contaminated sample (1 g) was determined using the Fs/Ls ratio calculated here and the Lo/Fo ratio calculated in Example 2.F according to an equation below.
Amount of contaminating buckwheat(ppm(μg/g))=Fs/Ls×Lo/Fo×1,000,000
As a result of Fs/Ls ratio measurement and the calculation of the amount of contaminating buckwheat, a reasonable value could be obtained in both of two measurements for the wheat pulverized powder containing 100 ppm of Shirahana buckwheat flour, the wheat pulverized powder containing 10 ppm of Shirahana buckwheat flour, the rice pulverized powder containing 10 ppm of Shirahana buckwheat flour, and the pulverized powder of wheat and rice containing 10 ppm of Shirahana buckwheat flour, as shown in Table 5. The raw data of a variety of artificially contaminated sample in Fs/Ls ratio measurement is shown in Tables 6A and 6B.
Fagopyrum:
Limonium:
Fagopyrum:
Limonium:
The same seeds as Example 1.A.(1) and Example 1.A.(2) were used.
The same leaves as Example 1.A.(3) were used.
Commercially-available products were used.
Leaves germinated from commercially-available seeds were used.
DNA extraction was conducted in the same way as Example 1.B.(2).
DNA extraction was conducted in the same way as Example 1.B.(3).
DNA extraction was conducted in the same way as Example 1.B.(4).
DNA extraction was conducted using Genomic-tip manufactured by QIAGEN with reference to QIAGEN Genomic DNA Handbook according to procedures below.
In a 50-ml tube, 1 g of a pulverized sample was introduced and 10 ml of Buffer G2, 200 μl of proteinase K (20 mg/ml), and 20 μl of RNase A (100 mg/ml) were added and mixed, followed by incubation at 50° C. for 1 hour. The resulting mixture was then centrifuged at approximately 3,000×g for 10 minutes to obtain its supernatant. The supernatant from which oil contents and powders were removed was further centrifuged at approximately 3,000×g for 10 minutes to obtain its supernatant. The obtained supernatant was applied to Genomic-tip 20/G Column equilibrated with 1 ml of Buffer QBT, to which DNA was then adsorbed. Then, the Column was washed with 4 ml of Buffer QC. A precipitate collected by elution with 1 ml of Buffer QF preheated to 50° C. and isopropanol precipitation was dissolved in 100 μl of sterilized ultrapure water. A DNA concentration in the resulting solution was measured, and the DNA solution appropriately diluted with sterilized ultrapure water was used as a template DNA sample for PCR.
DNA extraction was conducted using DNeasy Plant Maxi Kit manufactured by QIAGEN with reference to DNeasy Plant Maxi Kit Handbook according to procedures below.
In a 15-ml tube, 1 g of a pulverized sample was introduced and 10 ml of Buffer AP1 and 10 μl of RNase A (100 mg/ml) were added and mixed, followed by incubation at 65° C. for 60 minutes. The resulting solution was then centrifuged at approximately 3,000×g for 10 minutes to obtain its supernatant. To this supernatant, 1.5 ml of Buffer AP2 was added. The resulting mixture was left on ice for 10 minutes and centrifuged to obtain its supernatant. The obtained supernatant was applied to QIAshredder Spin Column to obtain a flow-through solution from the Column by centrifugation. To this flow-through solution, 1.5 volumes of Buffer AP3 and 1 volume of ethanol were added and mixed. The resulting mixture was applied to DNeasy Spin Column and centrifuged at approximately 1,500×g for 1 minute to have DNA adsorbed to the Column. Then, 10 ml of Buffer AW was added to the Column and centrifuged at approximately 1,500×g for 1 minute, followed by the washing of the Column. Again, 10 ml of Buffer AW was added to the Column and centrifuged at approximately 1,500×g for 1 minute. Subsequently, the Buffer AW that remained in the Column was completely eliminated by centrifugation at approximately 3,000×g for 10 minutes. Finally, 1 ml of sterilized ultrapure water preincubated at 65° C. was added to the Column and left for 5 minutes. The Column was then centrifuged at approximately 3,000×g for 5 minutes to elute DNA from the Column. A precipitate collected by isopropanol precipitation was dissolved in 100 μl of sterilized ultrapure water. A DNA concentration in the resulting solution was measured, and the DNA solution appropriately diluted with sterilized ultrapure water was used as a template DNA sample for PCR.
Sequences universal to the ITS-1 sequences of the following 11 sequences registered in GenBank of plants belonging to the genus Arachis were used as primer sequences. Concerning Arachis hypogaea among these plants, a sequence obtained from the analysis of a commercially-available peanut was also used, in place of Arachis hypogaea (AF156675) registered in GenBank.
Then, oligo DNA primers (manufacture by QIAGEN, OPC-purified oligonucleotides) having sequences below were synthesized and used as primers for PCR that detected a portion of the ITS-1 sequence of a peanut (hereinafter, referred to as peanut PCR).
A PCR simulation software Amplify 1.0 (Bill Engels) was used to confirm whether a result of the simulation showed that a PCR amplification product was obtained with the primers for detecting a peanut, based on 11 sequences of plants belonging to the genus Arachis, 8 sequences of likely-to-be-allergenic plants other than a peanut (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach, apple, and orange), 8 sequences of plants frequently used as food ingredients (maize, rice, pepper, mustard, carrot, shiitake mushroom, Chinese cabbage, and turnip), 6 sequences of plants of the family Leguminosae (kidney bean, lima bean, lentil, chickpea, mung bean, and adzuki bean), 69 sequences of related plant species of a peanut, and statice. The related plant species of a peanut used herein refer to plants other than the genus Arachis, which attained Score 60 bits or more when the ITS-1 sequence portion in the nucleotide sequence (AF156675) of a peanut, Arachis hypogaea, registered in GenBank was subjected to BLAST homology search. This time, the sequence of a species attaining the highest score in a genus to which each of the plants belonged was selected as a representative sequence of the genus. The PCR simulation was conducted for the ITS-1-5.8S rRNA gene-ITS-2 sequence region of that sequence. The GenBank Accession Number of the sequence used in the simulation and a result of the simulation in the case of using the combination of the primers of SEQ ID NOs: 21 and 65 are shown as a representative in Tables 7A to 7E. Abbreviated letters and symbols in Tables 7A to 7E are as shown below:
Filled-in asterisk: those expected to yield a PCR amplification product having a size around a target size (±10 bp)
W value: Possibility of yielding a PCR amplification product
Numeric (bp): the size (bp) of a PCR amplification product
−: those expected to yield no PCR amplification product
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
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 viscose
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
Spigelia hedyotidea
Spigelia marilandica
Phaseolus vulgaris
Cicer arietinum (Chickpea)
Lens culinaris subsp.
culinaris (Lentil)
Phaseolus lunatus (Lima bean)
Vigna angularis var.
nipponensis (Adzuki bean)
Vigna radiate (Mung bean)
Fagopyrum_esculentum
Triticum aestivum (Wheat)
Glycine max (Soybean)
Juglans regia (Walnut)
Tricholoma matsutake
Prunus persica (Peach)
Malus x domestica (Apple)
Malus x domestica (Apple)
Malus x domestica (Apple)
Citrus sp.
Zea mays (Maize)
Oryza sativa (Rice)
Piper nigrum (Pepper)
Sinapis alba (White mustard)
Brassica nigra (Black mustard)
Brassica juncea
Brassica rapa subsp. rapa
Brassica chinensis
Lentinula edodes
Daucus carota (Carrot)
Limonium sinuatum
As shown in Tables 7A to 7C, it was expected from the result of the simulation that a PCR amplification product having a target size of 76 bp was obtained from the 11 sequences of plants belonging to the genus Arachis when the combination of the primers of SEQ ID NOs: 21 and 65 was used. In addition, it was expected that a PCR amplification product having the target size and a non-specific PCR amplification product were not obtained from the 7 sequences of likely-to-be-allergenic plants other than a peanut (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach, and orange), the 8 sequences of plants frequently used as food ingredients (maize, rice, pepper, mustard, carrot, shiitake mushroom, Chinese cabbage, and turnip), the 6 sequences of plants of the family Leguminosae (kidney bean, lima bean, lentil, chickpea, mung bean, and adzuki bean), the 69 sequences of related plant species of a peanut, and the statice. Because there was expected the possibility that a non-specific PCR amplification product having a different size from the target size but having a weak signal was obtained from the apple, we decided to subject the apple to additional confirmation by actual PCR. The PCR simulation gave results from which a PCR amplification product having the target size was also expected to be obtained from the sequences of plants belonging to the genus Arachis in both cases where the combination of the primers having the sequences shown in SEQ ID NOs: 21 and 66 was used and where the combination of the primers of SEQ ID NOs: 21 and 26 was used.
Peanut PCR was conducted using HotStarTaq Master Mix Kit manufactured by QIAGEN according to procedures below.
Primers (0.2 μM each at a final concentration) and template DNA were added to 12.5 μl of 2×HotStartTaq Master Mix (HotStar Taq DNA Polymerase, PCR buffer with 3 mM MgCl2, and 400 μM each dNTP), whose final volume was adjusted with sterilized ultrapure water to 25 μto make a reaction solution. This was in turn introduced in a 0.2-ml microtube and reacted using a thermal cycler GeneAmp PCR System 9600 manufactured by Applied Biosystems according to the following PCR steps: enzyme activation at 95° C. for 15 minutes; 45 cycles of denaturation at 95° C. for 30 seconds, and annealing and extension at 68° C. for 30 seconds; and final extension at 72° C. for 4 minutes. The resulting PCR reaction solution was subjected to ethidium bromide-containing 2% agarose gel electrophoresis and analyzed with a fluorescent image analyzer FluorImager 595 manufactured by Amersham Biosciences. The results in the case of using the combination of the primers SEQ ID NOs: 21 and 65 are shown as a representative in
M: 100-bp DNA Ladder Marker
(−): No addition of template DNA
Numeric: Amount of template DNA added
Arrow: Target band (approximately 76 bp) of PCR amplification product
The extracted plant DNA was confirmed to have a purity level capable of PCR amplification by obtaining a PCR amplification product with primers for amplifying a portion of plant chloroplast DNA or a Rubisco gene sequence (data not shown).
As a result of peanut PCR, a PCR amplification product having a size of approximately 76 bp expected from the target ITS-1 sequence of a peanut was obtained from 500 fg of the peanut DNA, as shown in
As a result of peanut PCR, a PCR amplification product having the target size and a non-specific PCR amplification product were not obtained from 50 ng each of the DNAs of the apple seed, wheat leaf, buckwheat leaf, adzuki bean leaf, soybean leaf, maize leaf, and statice seed, as shown in
The nucleotide sequence of the peanut DNA-derived PCR amplification product obtained using the combination of the primers of SEQ ID NOs: 21 and 65 was analyzed by double-strand direct sequencing using primers of SEQ ID NOs: 21 and 65. The obtained nucleotide sequence was compared with the nucleotide sequence of a commercially-available peanut, Arachis hypogaea, to confirm that the nucleotide sequence of the peanut DNA-derived PCR amplification product matched 100% to the target site of the nucleotide sequence of the commercially-available peanut (Arachis hypogaea) (data not shown). This demonstrated that PCR using the primers amplified and detected a portion of the ITS-1 sequence of a peanut. In addition, from the obtained result, the PCR was found to amplify and detect a portion of the ITS-1 sequence of a peanut in both cases where the combination of the primers of SEQ ID NOs: 21 and 66 was used and where the combination of the primers of SEQ ID NOs: 21 and 26 was used (data not shown).
These results showed that peanut PCR using the primers can detect, with high sensitivity and specificity, the ITS-1 sequences of the general plants belonging to the genus Arachis. We decided to use these primers in PCR that quantified the copy number of the ITS-1 sequence of a peanut (hereinafter, referred to as a quantitative PCR method for a peanut sequence).
A TaqMan MGB probe (manufactured by Applied Biosystems Japan, reporter dye FAM) having a sequence below was synthesized and used as a probe for detecting a peanut sequence. A sequence universal to 11 sequences registered in GenBank as the ITS-1 sequences of plants belonging to the genus Arachis and the sequence obtained from the analysis of the commercially-available peanut was employed as the probe sequence.
A Quantitative PCR method for a peanut sequence was conducted using QuantiTect Probe PCR Kit manufactured by QIAGEN according to procedures below.
Primers (0.2 μM each at a final concentration), the TaqMan MGB probe of SEQ ID NO: 34 (0.1 μM at a final concentration), and template DNA were added to 12.5 μl of 2×QuantiTect Probe PCR Master Mix. The final volume was adjusted with sterilized ultrapure water to 25 μl to make a solution, which was in turn dispensed into a 96-well PCR plate. The 96-well PCR plate into which the solution was dispensed was loaded in a real-time PCR device Sequence Detection System 7700 manufactured by Applied Biosystems, in which the solution was reacted according to the following PCR steps: at 95° C. for 15 minutes; 45 cycles of denaturation at 95° C. for 30 seconds, and annealing and extension at 68° C. for 30 seconds; and final extension at 72° C. for 4 minutes. Every reaction was conducted with the same samples in duplicate (in 2 wells). After the completion of reaction, fluorescence data taken during the extension step was analyzed. A baseline was first set to cycles 0 to 1 and then appropriately set to within a range before a cycle where the increase of fluorescence was confirmed to begin. A threshold line was set according to the method described in Kuribara H et al., 2002, Novel Reference Molecules for Quantitation of Genetically Modified Maize and Soybean, Journal of AOAC International 85: 1077-1089. The results in the case of using the combination of the primers of SEQ ID NOs: 21 and 65 are shown as a representative in
The extracted plant DNA was confirmed to have a purity level capable of PCR amplification by obtaining a PCR amplification product with primers for amplifying a portion of plant chloroplast DNA or Rubisco gene sequence (data not shown).
As a result of the quantitative PCR method for the peanut sequence, a fluorescent signal indicating amplification was found from the DNA of the peanut seed, as shown in
As a result of the quantitative PCR method for the peanut sequence, a quantitative property and sensitivity where a standard curve having a correlation coefficient of 0.996 and a slope of −3.911 could be drawn with the peanut DNA in an amount ranging from 50 ng to 500 fg could be confirmed, as shown in
These results demonstrated that the quantitative PCR methods for the peanut sequence using the primers of SEQ ID NOs: 21 and 65 together with the probe of SEQ ID NO: 34, the quantitative PCR methods for the peanut sequence using the primers of SEQ ID NOs: 21 and 66 together with the probe of SEQ ID NO: 34, and the quantitative PCR methods for the peanut sequence using the primers of SEQ ID NOs: 21 and 26 together with the probe of SEQ ID NO: 34 could detect, with high sensitivity and specificity, the ITS-1 sequences of the general plants belonging to the genus Arachis and quantify the copy number of the peanut sequence as long as the plasmid for standard curves containing the target sequence of a peanut and the standard curves were generated. The present quantitative PCR method for the peanut sequence can be used in combination with the quantitative PCR method for the statice sequence for correction in the measurement of the amount of a contaminating peanut.
Dough (having a diameter of 6 cm and a thickness of 1 mm) prepared by adding 35 g of water and 0.8 g of a salt to 80 g of wheat containing 100 ppm (hereinafter, W/W) of buckwheat was subjected to any of the following four heat treatments: (1) baking (160° C., 10 min), (2) frying (185° C., 5 sec), (3) steaming (100° C., 10 min), and boiling (100° C., 10 min), and used as a processed product model that was cooked. They were then mixed with a statice standard sample, followed by DNA extraction in the same way as above. Buckwheat contained in the heated sample was quantified using a primer set consisting of oligonucleotide having a sequence shown in SEQ ID NO: 14 and oligonucleotide having a sequence shown in SEQ ID NO: 15, in combination with a probe having a sequence shown in SEQ ID NO: 64. Based on the measured quantitative value of buckwheat in the processed product, a buckwheat concentration in the wheat used was determined, when water contents were taken into consideration. As a result, the buckwheat concentration was 145 ppm for (1) the baked product, 56 ppm for (2) the fried product, 198 ppm for (3) the steamed product, and 143 ppm for (4) the boiled product, and a sufficient quantitative property was shown. Thus, it was suggested that quantification by the method of the present invention could be performed in all of the most general heat treatments, baking, frying, steaming, and boiling, in food processing. Therefore, it is considered that the method of the present invention can maintain this quantitative property for the processed food by processing other than the above-described processing. Thus, the method of the present invention is applicable to a wide range of processed foods.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
A PCR method of the present invention that quantifies a plant belonging to a specific plant genus that contaminates a food or a food ingredient can detect and quantify the presence of a very small amount of the plant belonging to the specific plant genus in the food or the food ingredient and as such, is especially effective in the detection of the presence or absence of a plant belonging to an allergenic plant genus such as the genus Fagopyrum, the genus Arachis, the genus Triticum, and the genus Glycine, and in the quantification of the plant. The PCR method of the present invention is a method in which correction for influences such as the DNA extraction efficiency of each sample to be examined and the inhibition of PCR reaction is conducted not by externally adding purified DNA as a standard to conduct correction for influences such as the inhibition of PCR reaction in a reaction solution but by simultaneously extracting DNA derived form a specific plant genus to be detected and DNA derived from a standard plant from a sample externally supplemented with a standard plant sample other than purified DNA to conduct a quantitative PCR method. This method allows highly reliable quantification because of being capable of measurement under a condition where influences such as DNA extraction efficiency and the inhibition of PCR reaction are uniform between the standard plant sample and the sample derived from the specific plant genus to be detected. The method of the present invention has an advantage that the method is capable of correction for influences such as DNA extraction efficiency and the inhibition of PCR reaction and even for difference in DNA content among samples to be examined. This method also allows the proper quantitative detection of a plant belonging to a specific plant genus in a DNA-free food ingredient such as salts or a food containing the ingredient.
Thus, the present invention is useful for quantitatively detecting a plant belonging to an allergenic specific plant genus that contaminates a food or a food ingredient. In addition, quantitative analysis by the PCR method can reliably exclude a false positive, if any, by subjecting its PCR amplification product to DNA sequence analysis, and as such, can be said to have excellent industrial applicability.
The quantitative PCR method of the present invention can have a dynamic range wider than those of ELISA methods and can achieve sufficiently high specificity and sensitivity for quantitatively detecting a specific ingredient contaminating a food or a food ingredient. Moreover, the method used in combination with synthesized materials (primer and probe) can attain the high reproducibility and reliability of a measurement result.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-139513 | May 2003 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP04/06913 | 5/14/2004 | WO | 00 | 11/15/2005 |
