JP5366900B2 - Leek group detection primer and detection method - Google Patents

Description

The present invention is intended to detect the main edible species of the genus Allium, leeks, onions and wakegi, and when the plant is contained in food ingredients, products, etc., it is highly sensitive even if the amount is small. The present invention relates to a method for detecting a leek group that can be detected by the PCR method, PCR primers used in the method, and the like.

  “Leek” is one of the plants often used in processed foods. On the other hand, “green onions” are similar in appearance to the waggi, so there are some that are treated differently from the original species in the market, and differ depending on regional characteristics and dietary habits. It is known that there is.

  In other words, white leeks are the mainstream in eastern Japan, but in the eastern Japan “Wakegi” refers to a variety of leeks called leeks, or a young leaf onion of the Hakata Universal Allium type. In the genetic classification, it is leek (Allium fistulosum).

  In western Japan, green leek (leaf onion) is the mainstream, but in Western Japan “Wakegi” is Wakegi (Allium wakegi). It is an interspecific hybrid of divide and leek onion, and the scientific name of Allium Wakegi is officially recognized. Unlike leeks, it does not produce seeds, but increases with underground bulbs (bulbs).

  As described above, there are a plurality of types of so-called “green onions” in the market, and the names of tools used vary depending on the appearance and regional characteristics. In other words, the name and the actual variety may be confused. On the other hand, it is necessary for food manufacturers to promptly check these correct varieties for materials to be used and to display them appropriately.

  In addition, when using onions for processed foods, dried products are often used. In this case, it is often cut into small pieces, and may be misidentified as other plant species or vinyl pieces due to its form. In addition, even when trying to confirm these, it may be difficult to distinguish them from “green onions” only by microscopic observation or appearance.

  From this point of view, it would be convenient if a test method using a gene that can be tested quickly even with a small amount of sample could be used. That is, if the method is to extract DNA from an object and examine the sequence of the gene, it is possible to quickly test using a small sample amount.

  On the other hand, for example, Patent Document 1 is disclosed as a method for examining the sequence of such a gene. Patent Document 1 targets two internal transcription spacer regions 1 (ITS1) or internal transcription spacer region 2 (ITS2) and is designed based on the base sequences of the regions present on both sides of ITS1 or ITS2. A set is constructed, and the primer set is used to carry out an amplification reaction using DNA prepared from foreign substances as a template. In this method, the sample is identified by determining the base sequence of the amplified nucleic acid fragment and specifying the species from the base sequence.

JP2004-337069

  By the method of the said patent document 1, a leek and a scallion can be distinguished. However, this method is complicated because it requires a step of determining the base sequence.

  In addition, when the above-mentioned purpose is considered, if there is a primer specific to each leek and scallion, it can be rapidly detected by PCR. However, until now, there has been no PCR primer targeting leek and scallion.

Therefore, the present inventors can detect a specific gene with high sensitivity for each genus Allium so that each leek and scallion can be distinguished quickly and easily using DNA extracted from the target. An object was to develop a primer and a detection method thereof.

  In order to solve the above-mentioned problems, the present inventors have attempted to create specific primers for each of leek and scallion. However, of these three types, the forage is an interspecific hybrid and has both the onion and onion genomes. In such hybrid plants, it was difficult to find a primer specific to wakegi. That is, if an attempt is made to prepare a primer for detecting an onion, it becomes a primer that also reacts with the onion, and it has been difficult to specifically detect the onion with only a pair of primer sets.

  Therefore, the present inventors have adopted a method of creating a primer that specifically detects onion from different viewpoints. And, it was made possible to judge the onion based on the results of both the onion and the primer that specifically detects the onion.

  That is, the present inventors prepared an onion (Allium fistulosum) specific primer and an onion (Allium cepa) specific primer. And the detection method which can detect a leek, an onion, and a scallion was able to be constructed | assembled by utilizing these primers.

Specifically, the present invention provides the following PCR primers and PCR primer sets.
Item 1. 1. PCR primer consisting of DNA of a maximum of 30 bases containing the base sequence of base numbers 10 to 21 in SEQ ID NO: 1 on the 3 ′ end side 2. PCR primer consisting of a DNA of 30 nucleotides at the maximum containing the nucleotide sequence of nucleotide numbers 10 to 21 in SEQ ID NO: 2 on the 3 ′ end side. 3. PCR primer consisting of a DNA of a maximum of 30 bases containing the base sequence of base numbers 13-24 in SEQ ID NO: 3 in the sequence listing on the 3 ′ end side 4. PCR primer consisting of a DNA of up to 30 bases containing the base sequence of base numbers 10 to 21 in the sequence listing in the sequence listing at the 3 ′ end. A PCR primer set comprising the primer according to claim 1 and the primer according to claim 2.
Item 6. A PCR primer set comprising the primer according to claim 3 and the primer according to claim 4.
Item 7. A step of extracting DNA from the sample, a step of performing PCR using the primer set according to claim 5 using this DNA as a template, and whether or not leeks are present in the sample by detecting the amplified DNA And a method for detecting leeks.
Item 8. A step of extracting DNA from the sample, a step of performing PCR using the primer set according to claim 6 using this DNA as a template, and whether or not onion is present in the sample by detecting the amplified DNA A method for detecting onions.
Item 9. A step of extracting DNA from a sample; a step of performing PCR using the primer set of claim 5 using the DNA as a template; a step of performing PCR using the primer set of claim 6; And a step of detecting whether or not the sample is present in the sample by detecting the DNA amplified by the primer set.

  By conducting a PCR test using the primer of the present invention, it is possible to quickly determine onions, onions, and onions that are difficult to distinguish by visual observation. In the foreign matter analysis, it can be quickly determined whether or not the foreign matter is derived from the leek group.

Hereinafter, the present invention will be described in detail.
Primers for detecting leeks The PCR primer set of the present invention includes those having a characteristic base sequence for leeks among those belonging to the genus Allium and those having a characteristic base sequence for onions. In addition, the leek group referred to in the present invention refers to leek (Allium fistulosum), onion (Allium cepa), and scallion (Allium wakegi).

  Each primer set was designed as follows. Candidate genes were selected from data in the Allium ITS region by NCBI search. The ITS area will be described below. Nuclear ribosomal DNA (nrDNA) has a repetitive sequence structure with 5'-18S-ITS1-5.8S-ITS2-26S-3 'as a unit.

  The number of copies is said to be 6400 copies in wheat from 1 copy of protozoa, but in many animals it is said to be less than 1000 copies. The internal transcription spacer region 1 (ITS1) and the internal transcription spacer region 2 (ITS2) are non-conserved regions specific to each plant species, and are composed of a base sequence having a relatively short base length and adjacent bases on both sides thereof. The sequences, ie, 18S rRNA and 5.8S rRNA in the case of ITS1, and 5.8S rRNA and 25S 26S rRNA in the case of ITS2) are widely conserved in plants in general and have a region of the base sequence that defines the plant and other species. ing.

  Although this ITS region shows differences between species in the genus Allium, differences between varieties are rare. Therefore, the ITS region is suitable as a target sequence for a leeks genus-specific primer in that it can specifically detect leeks and can cover a large variety of leeks.

  Specifically, the ITS region (internal Transcribed Spacer region) of nuclear ribosomal DNA was extracted, compared, and a base sequence portion specific to each onion and onion was selected. Furthermore, after determining a specific DNA region that can obtain an amplified DNA fragment of an appropriate size that can be detected by PCR from the selected site, and selecting a PCR detection primer set that can achieve the purpose from that region, Performance was evaluated. In consideration of the range of onions to be detected and the similarity of the base sequences, a primer set capable of detecting onions and a primer set capable of detecting onions close to the onions were developed.

  Using this primer set, specific regions of onions and onions are amplified by PCR, and the amplified DNA fragments are subjected to agarose gel electrophoresis, and DNA fragments of a specific size are detected from the electrophoresis pattern. By doing so, the presence of onion and onion-derived DNA can be determined. In addition, the presence of the onion DNA can be determined from the results of the onion and onion. In addition to gel electrophoresis, the real-time PCR apparatus can determine the presence of allium-derived DNA by melting curve analysis of the amplified DNA fragment.

  Furthermore, primers were designed so as to cross with other species and not to form primer dimers. In consideration of the case of using primers together, the primer usage conditions were designed to be set uniformly.

The base sequences of the primers provided by the present invention are as follows. S represents a sense primer and AS represents an antisense primer.
Sequence number 1 (leek S): TTG CGT TGT TTG GAT GGG TTC
Sequence number 2 (leek AS): CAC TCA CAG TGT AAG TTT ACC
Sequence number 3 (onion S): TTG AAG TAA GAT GTA GAG TAG AAC
Sequence number 4 (onion AS): ATC ACT CAC AGT ATG TTT ACA
In addition, each of the above primers may be a primer having a maximum of 30 bases as a whole by adding an arbitrary sequence to the 5 ′ end side. Furthermore, for each primer, detection is possible if 12 bases from the 3 ′ end match the template DNA.

  Therefore, the present invention proposes, as a first primer, a PCR primer consisting of DNA of a maximum of 30 bases containing the base sequence of base numbers 10 to 21 in SEQ ID NO: 1 in the sequence listing on the 3 ′ end side. Next, as a second primer, a PCR primer composed of DNA of a maximum of 30 bases containing the base sequence of base numbers 10 to 21 in SEQ ID NO: 2 in the sequence listing on the 3 ′ end side is proposed. Moreover, it is preferable to use these primers as a set. By using these primers as a set, allium fistulosum can be detected.

Next, the present invention proposes, as a third primer, a PCR primer consisting of DNA of a maximum of 30 bases containing the base sequence of base numbers 13 to 24 in SEQ ID NO: 3 in the sequence listing on the 3 ′ end side. Furthermore, as a fourth primer, a PCR primer consisting of a DNA of 30 nucleotides at the maximum containing the nucleotide sequence of nucleotide numbers 10 to 21 in SEQ ID NO: 4 in the sequence listing on the 3 ′ end side is proposed. Moreover, it is preferable to use these primers as a set. By using these primers as a set, onion (Allium cepa) can be detected.

How to detect leek groups
-Inspection object Although the inspection object in this detection method is not specifically limited, a foodstuff, a foreign material, etc. are mentioned specifically ,. Moreover, a drink etc. are also possible. Specifically, raw materials and processed foods that are raw or heated as foods can be mentioned. Further, since the present invention uses a PCR method, it can be detected if a trace amount of DNA is present. Therefore, if there is a trace amount of food, it can be detected.

-DNA extraction process There is no particular limitation on the form of the sample for extracting DNA from the test object. General well-known DNA extraction methods (Notice of Food Health Department, Ministry of Health, Labor and Welfare, Ministry of Health, Labor and Welfare, and inspection methods for foods containing allergens, November 06, 2002, No. 1106001) and various DNA extraction kits on the market [ For example, Nucleon PhytoPure, plant and fungal DNA extraction kits (Amersham Biosciences Corp., USA), DNA Extraction Isoplant II kit (Nippon Gene Co. Ltd., Japan), DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany), etc.] Any sample capable of recovering DNA can be applied to the above detection method.

-PCR process using this primer set The method of detecting leek of the present invention comprises a step of extracting DNA from a sample, a step of performing PCR using the primer set of the present invention described above using this DNA as a template, and amplification. By detecting the thus-prepared DNA, it can be confirmed whether or not onions and onions were present in the sample. In addition, since the onion is an interspecific hybrid and has both the onion and onion genomes, it can be judged from the results of the onions and the results of the onions.

  In the PCR reaction step, PCR is performed using the extracted DNA as a template and a primer set for detecting the genus Allium. As the PCR apparatus, for example, a commercially available apparatus of a block type or a capillary type can be used. In order to rapidly amplify DNA, a capillary type device is preferable. In addition, if a capillary-type real-time PCR apparatus is used, the increase in DNA fragments as amplification products can be monitored in real time, so that the presence or absence of leek group DNA can be quickly grasped. The correspondence between the types of primers used and the detectable leek groups is shown in Table 1 below.

・ Detection of amplified DNA fragments
The presence or absence of DNA fragments, which are PCR amplification products, can be confirmed by gel electrophoresis of the amplification product with a normal PCR device, or with a melting curve analysis of the amplification product with a real-time PCR device. By deriving the melting temperature (Tm) value of the PCR amplification product from the analysis, it can be confirmed whether the PCR amplification product is the target amplification product. Specifically, the onion and onion DNA can be detected by each primer set. The size and Tm of theoretical DNA fragments obtained by PCR amplification for each species are generally as shown in Table 2 below.

In addition, Table 3 shows how to detect with each primer when the test subjects are leek, onion and scallion. In addition, as shown in Table 3, it is determined that the onion is detected when both the onion and the onion are positive.

By performing the PCR process using the above three kinds of leek genus-specific primers, it is possible to specifically identify and detect not only leeks but also onions close to the leeks, or onions used as leeks.

・ Simultaneous identification using multiplex primer For the onion detection primer (SEQ ID NO: 1 and SEQ ID NO: 2) and onion detection primer (SEQ ID NO: 3 and SEQ ID NO: 4) of the present invention, these primers should be mixed and used. You can also. That is, by performing PCR using a type in which these primers are mixed, it can be determined whether any of the onion, onion, or onion is present in the test sample.

  In the PCR reaction system, the number of moles added to the reaction tube of the primer sets of SEQ ID NO: 3 and SEQ ID NO: 4 for detecting onion is different from the difference in easiness of annealing of each primer to the template DNA. The number of moles added to the reaction tube of the primer set of SEQ ID NO: 1 and SEQ ID NO: 2 for detecting s is preferably about 3 times.

  When PCR is performed using this multiplex, detection of target size amplification products is considered positive for onion DNA if a band is confirmed at 378 bp, and onion DNA is positive if a band is confirmed at 330 bp. If a band is confirmed at both positions of 330 bp and 378 bp, it is regarded as positive for DNA. Moreover, when a band is not confirmed in any position or a band is confirmed in other positions, it can be regarded as negative.

・ Appraisal is rare when the object to be inspected is a mixture of onion and onion, but even in the case where the object to be inspected is a mixture of onion and onion, it can be distinguished from the onion in the present invention. is there. Regarding the method of distinguishing between a green onion and an onion, or from an onion, the amount of each DNA is estimated using a real-time PCR method, and can be discriminated by the ratio. That is, the amount of template DNA originally present is compared with the number of cycles in which an exponential amplification period is recognized using real-time PCR.

  The ratio of onion-derived DNA amount and onion-derived DNA amount is constant when the onion is the test object, but when the onion is a mixture, the ratio of onion is not as long as the ratio of onion to onion is not 1: 1. Since the ratio is not the same as in the case, it can be distinguished.

As a specific method, PCR amplification is performed using a real-time PCR method, and the number of cycles in the exponential amplification phase is measured. This cycle number is known as a C _T (Threshold Cycle) value in a PCR apparatus manufactured by Applied Biosystems. Also, it is known as a CP (Crossing Point) value in a PCR device manufactured by Roche Diagnostics.

The number of cycles in the exponential amplification period (C _T value, CP value) is calculated by drawing an auxiliary line with a certain fluorescence signal: F (Fluorescence) value in the exponential amplification region. The number of cycles calculated from the intersection.

For example, when using the light cycler (Roche Diagnostics) and calculating the CP value (F = 1), when using the primer of the present invention, the CP value ratio (F = 1) (Onion / Onion): It will be a constant value around 1.2, but if it deviates from it (CP value ratio (F = 1) if the ratio of onion is large, 1.3 or more, CP (if the ratio of onion is large) F = 1) value ratio: around 1.1) is found to be a mixture of leeks and onions.

─Confirmation of detection sensitivity of PCR analysis using primer set for detecting leek DNA─
The following tests were conducted to confirm the detection sensitivity of PCR primers for various onions and onions. The onion (Allium fistulosum) and the onion (Allium cepa) used for the sample used the material for in-house use. For these purified DNAs, a part of each sample (freeze-dried) was washed with 70% ethanol, then quickly washed with sterilized water, and then crushed with a multi-bead shocker (Yasui Kikai, Osaka). Prepared using DNeasy plant Mini kits (Qiagen GmbH, Germany). After measuring the amount of these Allium plant DNAs, the concentration of DNA was adjusted to 1 ng / μL using sterilized water, and then serial dilution was performed from 1000 pg to 0.001 pg to prepare DNA samples of each concentration.

A 20 μL reaction solution containing 2 μL of DNA sample solution to be donated to PCR was prepared using SYBR Premix Ex Taq ^™ (Perfect Real Time) manufactured by Takara Bio Inc. The composition of the PCR reaction solution was as shown in Table 4 below.

  PCR reaction was performed under the following conditions. The equipment used was LightCycler (Roche Diagnostics GmbH, Germany), and the amplification reaction conditions were as follows. That is, in the case of the leek specific primer, the primer set of SEQ ID NO: 1 and SEQ ID NO: 2 was used. In the case of onion-specific primers, the primer sets of SEQ ID NO: 3 and SEQ ID NO: 4 were used. As temperature conditions, after a cycle of 95 ° C for 1 minute was performed, the temperature was raised to 95 ° C at a rate of 20 ° C / second, kept at the same temperature for 5 seconds, and then 60 ° C at a rate of 20 ° C / second. The temperature was lowered to (annealing temperature), then kept at the same temperature for 10 seconds, further raised to 72 ° C. at a rate of 10 ° C./second, and then subjected to 40 amplification cycles comprising three steps of keeping at the same temperature for 20 seconds.

  The PCR amplification product was recorded as the SYBR Green I-dependent fluorescence amount at the final step of each cycle. At the end of the amplification cycle, the melting curve was raised to 97 ° C at a rate of 20 ° C / s, then lowered to 60 ° C at a rate of 20 ° C / s, kept at the same temperature for 10 seconds, and then 0.2 ° C It was obtained by recording the fluorescence intensity every 0.2 seconds during a slow temperature increase to 97 ° C. at a rate of / sec. The Tm value of the PCR amplification product obtained from the melting curve by the PCR device software was used to estimate the presence or absence of a specific amplification product by PCR. Furthermore, by separating the PCR amplification product (2 μL of the reaction solution) by 2.0% (wt / vol) agarose gel electrophoresis, staining the separated gel with ethidium bromide, and then visualizing the amplification product under UV irradiation The presence or absence of the amplification product was confirmed. A 100 bp DNA Ladder (New England BioLabs Inc., USA) was used as a molecular weight marker. The results of these PCRs are listed in Table 5. + (Plus) in the table indicates that an amplification product of the target size was detected by electrophoresis, and-(minus) indicates that the amplification product was not detected.

  In addition, when using a leek specific primer, detection of target size amplification products is considered positive if a band is confirmed at a position of 378 bp, and no band is confirmed at any position, or a band is present at other positions. If confirmed, it is considered negative. In addition, when an onion-specific primer is used, if a band is confirmed at 330 bp, it is regarded as positive, and if no band is confirmed at any position, or a band is confirmed at other positions, it is regarded as negative. When both a leek-specific primer and an onion-specific primer are used, if a band is confirmed at the position of 378 bp, it is considered positive for the onion DNA, and if a band is confirmed at the position of 330 bp, it is regarded as positive for the onion DNA, both 330 bp and 378 bp If a band is confirmed at the position of, it is considered positive for DNA. In addition, if a band is not confirmed at any position, or a band is confirmed at other positions, it is considered negative. Judgment was made by the method as described above. The results are shown in Table 5 below.

As described in Table 5, the detection limit of each primer was as follows: leek specific primer: 0.05 pg, onion specific primer: 1 pg. Each electropherogram is shown in FIG.

─Confirmation of detection specificity of primer set for detecting leek genus DNA─
In order to examine the detection specificity of the present invention, the detection specificity was confirmed using various genes. Purified human DNA used was purchased from Semain (CeMines, LLC, USA).
Purified DNA of cattle, pigs, chickens, sea cucumbers, skipjack, sockeye salmon, yellowtail, scallops, king crab, cuttlefish, lobster, and octopus were frozen after freezing the (muscle) meat portion of each sample purchased from the store, Instrument, Osaka) using a sample prepared using Nucleon PhytoPure, plant and fungal DNA extraction kits (Amersham Biosciences Corp., USA) or DNA Extraction Isoplant II kit (Nippon Gene Co. Ltd., Japan) .

  Wheat and corn DNA were purchased from BioChain Institute, Inc., USA. The purified buckwheat DNA used was a buckwheat flour purchased from a store using a multi-bead shocker, and a pulverized sample prepared using Nucleon PhytoPure, plant and fungal DNA extraction kits.

  The green onions (former rice cake, chicken leg bowl, black thousand, Kotaro, Koka Kogane, Kosuge), onion (white seeds), Asatsuki are obtained from the company's own materials department, green onions (Nobunaga, Asahi Kujo, Shimonita Kaoru, All-purpose rice, onions (pecoros, red onion), bamboo shoots, soybeans, sesame, spinach, carrots, burdock, asparagus, cabbage, and leek were purchased from a store, and rice was obtained from a domestic farmer. For these purified DNAs, DNeasy plant Mini kits (Qiagen GmbH) were prepared from samples that had been partially washed with 70% ethanol, then quickly washed with sterilized water, and then crushed with a multi-bead shocker (Yasui Kikai, Osaka). , Germany) was used. Purified DNA of Aspergillus niger IFO 9642, Saccharomyces cerevisiae IFO 0282, Escherichia coli JCM 1649T, and Bacillus cereus DSM 4312 is suitable for each strain. After culturing in a medium, a solution prepared from the culture using a Puregene Yeast and Gram-Positive DNA Isolation kit (Gentra Systems Inc., USA) was used. All purified DNAs have been subjected to RNA removal by RNase.

The equipment used in the PCR reaction was LightCycler (Roche Diagnostics GmbH, Germany), and the amplification reaction conditions were the same as in Example 1 except for the amplification cycle. Amplification cycles were performed 35 times. The results of these PCRs are shown in Table 6. In the table, + (plus) indicates that the amplification product was detected, and-(minus) indicates that the amplification product was not detected.

As a result of the specificity test with other species DNA using two types of leeks, nonspecific amplification was not observed. A specificity test was conducted on leek and onion-specific primers using 10 varieties of onion and 3 onions, which were generally considered to be edible, and it was detected in all onion and onion varieties tested. In addition, the onion and onion-specific primers were both detectable in the onion.

─Detection of leek-derived DNA in commercial processed foods by PCR analysis using a primer set for detecting leek genus DNA─
For commercial products that clearly stated the use of Allium plants as a raw material, three types of Allium primers were used, and an implementation test was conducted to confirm whether they could actually be detected.

  As a sample, a leek genus plant in a commercially processed product was used. That is, prepare commercially available foods that clearly specify the use of Allium plants as raw materials, take 1 to 10 mg (dry weight) of Allium plants contained in those commercially available products, 70% of each sample After washing with ethanol and further with sterilized water, it was crushed with a multi-bead shocker (Yasui Kikai, Osaka). Each DNA was extracted from these disruptions using DNeasy plant Mini kits (Qiagen GmbH, Germany). At the time of extraction, RNA was also removed by RNase.

  After measuring the amount of DNA, adjust the DNA concentration to 1 ng / μL. Concentration adjustment was not performed for those with a concentration of 1 ng / μL or less due to low concentration. The equipment used in the PCR reaction was LightCycler (Roche Diagnostics GmbH, Germany), and the amplification reaction conditions were the same as in Example 1. The results of these PCRs are listed in Table 7. In the table, + (plus) indicates that the amplification product was detected, and-(minus) indicates that the amplification product was not detected.

In addition, PCR amplification products amplified from food sample DNA are converted into BigDye Terminator v3.1 Cycle.
Using Sequencing kit (Applied Biosystems), direct sequencing was performed in both directions, and then the base sequence was analyzed with Applied Biosystems 310 Genetic Analyzer (Applied Biosystems). Analyze the analyzed base sequence data with GenBank nucleotide sequence database (BLAST search) or the DNA database owned by the inventors, and assign the species of DNA derived from PCR amplification products based on the similarity of the base sequence. Identified. The above results are summarized as follows.

  That is, when PCR primers for detecting leek (PCR primer set consisting of SEQ ID NOS: 1 and 2) are used, amplification products are confirmed only in DNA derived from foods that use leek and scallion as ingredients. The Tm value of the product was about 85.8 ° C. No amplification product was seen when DNA from other food samples was used. As a result of analyzing the base sequence of the PCR amplification product, the base sequence of the PCR amplification product amplified from the green onion-derived DNA in the commercial food was identical to the ITS region sequence of the green onion. That is, PCR using the PCR primers does not detect leek-derived DNA or other DNA from leek-free food samples in multiple commercial foods, but can detect leek-derived DNA specifically from leek-containing food samples. confirmed.

When PCR primers for onion detection (PCR primer set consisting of SEQ ID NOs: 3 and 4) are used, amplification products are confirmed only in food-derived DNA using onions and bamboo shoots as ingredients. The Tm value was about 85.4 ° C. No amplification product was seen when DNA from other food samples was used. As a result of analyzing the base sequence of the PCR amplification product, the base sequence of the PCR amplification product amplified from the onion-derived DNA in the commercial food was identical to the ITS region sequence of the onion.

─ Simultaneous appraisal using multiplex primer (onion + onion) ─
Using the primer group of the present invention, it was verified that the primers of SEQ ID NOs: 1 to 4 were put and tested by one PCR.

  SEQ ID NOs: 1 and 2 (onion-specific primer) and SEQ ID NOs: 3 and 4 (onion-specific primer) were mixed with onion primer 1 at a ratio of onion primer 3 to perform a detection test. As samples, PCR was carried out using the onion DNA (1 ng / μL), onion DNA (1 ng / μL), and onion DNA (1 ng / μL) with the reaction composition shown in Table 8 below.

  The instrument used in the PCR reaction was a LightCycler (Roche Diagnostics GmbH, Germany), and the amplification reaction conditions were the same as in Example 1 except that the number of amplification cycles was 35. An electropherogram is shown in FIG.

All of the leek and scallion primer groups (SEQ ID NOs: 1 to 4) were subjected to the PCR reaction at a time, but for the leek, the amplified product could be detected at a position of 378 bp as prescribed. In addition, as for the onion, an amplification product at a position of 330 bp could be confirmed as prescribed. Furthermore, amplification products could be detected at both the 378 bp position and 330 bp position, as was also the case with wakegi. In addition, amplification products other than the target were not observed.

─Appraisal in case of mixed onion and onion─
If a leek and an onion are mixed in the test object, two bands will be detected in the same manner as the wakegi, and only whether there is an amplified product or not It cannot be judged. On the other hand, an implementation test was performed to determine whether each DNA amount was estimated using a real-time PCR method and could be discriminated by the ratio.

  Real-time PCR was carried out by comparing the amount of template DNA originally present, based on the number of cycles in which an exponential amplification period was observed.

  As samples, using onion DNA (1 ng / μL), onion DNA (1 ng / μL), and onion DNA (1 ng / μL), make samples diluted 2, 4, and 8 times, The mixing ratio of onion DNA and onion DNA was changed. The PCR reaction was performed with the composition shown in Table 9.

  The equipment used in the PCR reaction was LightCycler (Roche Diagnostics GmbH, Germany), and the amplification reaction conditions were the same as in Example 1 except that the number of amplification cycles was 35.

  For real-time PCR analysis, PCR amplification was performed with a Light Cycler (Roche Diagnostics), and the CP value (F = 1) was measured.

  The number of cycles in the exponential amplification period (CP value) is calculated from the intersection of the auxiliary line and the amplification curve by drawing an auxiliary line with a certain fluorescence signal: F (Fluorescence) value in the exponential amplification region. This is the calculated number of cycles. In this example, the CP value (F = 1) was calculated. The results are shown in Table 10.

CP value ratio (F = 1) for onion (onion / onion): constant value around 1.2, but if it deviates from it (CP value ratio (F = 1) if there is a large percentage of onion): When the onion ratio is 1.3 or more, the CP (F = 1) value ratio: around 1.1) is a mixture of onion and onion. Thus, in most cases, it is possible to distinguish between a mixture of a leek and an onion and a case of a leek.

It is the electrophoretic diagram which showed the detection limit of each primer. It is the electrophoretic diagram which showed the result of simultaneous identification using a multiplex primer.

Claims (5)

  A PCR primer consisting of a DNA with a maximum of 30 bases containing the base sequence of base numbers 10 to 21 in SEQ ID NO: 1 on the 3 ′ end side and 3 base sequences of base numbers 10 to 21 in SEQ ID NO: 2 of the sequence list A PCR primer set consisting of a PCR primer consisting of DNA consisting of a maximum of 30 bases at the end.   A PCR primer consisting of a DNA with a maximum of 30 bases containing the base sequence of base numbers 13 to 24 in SEQ ID NO: 3 on the 3 ′ end side and 3 base sequences of base numbers 10 to 21 in SEQ ID NO: 4 of the sequence list A PCR primer set consisting of a PCR primer consisting of DNA consisting of a maximum of 30 bases at the end.   A step of extracting DNA from the sample, a step of performing PCR using the primer set according to claim 1 using the DNA as a template, and whether or not leeks are present in the sample by detecting the amplified DNA And a method for detecting leeks.   A step of extracting DNA from the sample, a step of performing PCR using the primer set according to claim 2 using the DNA as a template, and whether or not onion is present in the sample by detecting the amplified DNA A method for detecting onions.   A step of extracting DNA from a sample; a step of performing PCR using the primer set of claim 1 using the DNA as a template; a step of performing PCR using the primer set of claim 2; And a step of detecting whether or not the sample is present in the sample by detecting the DNA amplified by the primer set.