JP5271556B2 - Peach detection primer set and peach detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple means for specifically detecting Prunus persica in high sensitivity by differentiating the Prunus persica from other fruit of close species even from a sample containing a plurality of plant species. <P>SOLUTION: The primer set for detecting the Prunus persica by specifically amplifying a DNA fragment originated from trnS-trnG intergenic spacer gene of the Prunus persica is constituted of an oligonucleotide having a specific base sequence, and an oligonucleotide having another specific base sequence (with the proviso that a primer set constituted of an oligonucleotide having a combination of specified base sequences is excluded). <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a primer set and a peach detection method that can detect peach causing food allergy specifically and with high sensitivity.

  Peach (Prunus persica) is a plant of the genus Prunus, and it is known that ingestion of peach causes oral allergy (OAS), gastrointestinal and systemic symptoms, and anaphylactic shock. Some peach allergic patients cross-react with cherry peaches, apricots, plums, apples, pears and pollen. Peach is currently included in 20 foods that contain allergens and are based on specific raw materials, and it is necessary to establish a peach detection method to ensure food safety and security.

  As a conventional peach detection method, an ELISA method has been reported in which detection is performed using an antibody that specifically reacts with a peach allergen protein (Non-patent Document 1). Here, in an antigen-antibody reaction such as ELISA, there is a risk of cross-reacting with a protein other than the peach targeted by the antibody. Therefore, if a positive result is obtained by ELISA, it is necessary to confirm whether it is a false positive by a method such as PCR. However, no PCR method for detecting peaches has been reported so far, and the development of a peach detection PCR method using the presence or absence of a DNA sequence peculiar to peaches as an index is required.

  As a method of distinguishing peaches and related species using DNA, for the purpose of creating gene linkage maps of peaches and related species, a method using RAPD markers and SSR markers (non-patent document 2), peaches and related species There is a report of a method for analyzing the difference in the base sequence of PCR amplification products (Non-patent Document 3) for the purpose of performing a systematic analysis of species. However, these methods are not intended to detect peaches present in foods composed of various raw materials. The PCR primers used in these methods are assumed to be used when the test sample is composed of a single plant species (eg, peach). In the case of a sample containing multiple plant species, The possibility of reacting with DNA derived from plant species other than peach is very high. Therefore, when a sample containing a plurality of plant species such as food is used as a test sample, it is difficult to specifically detect only the peach in the sample and determine the presence or absence of the peach. Therefore, these methods cannot be used as a method for detecting peach in food.

Duffort OA, Polo F., Lombardero M., Diaz-Perales A., Sanchez-Monge R., Garcia-Casado G., Salcedo G., Barber D. Immunoassay to quantify the major peach allergen Pru p 3 in foodstuffs. Differential allergen release and stability under physiological conditions.J Agric Food Chem. 2002, 50, 7738-7741 Bliss FA, Arulsekar S., Foolad MR, Becerra V., Gillen AM, Warburton ML, Dandekar AM, Kocsisne GM, Mydin KK An expanded genetic linkage map of Prunus based on an interspecific cross between almond and peach.Genome 2002, 45, 520-529 Lee S., Wen J. A phylogenetic analysis of Prunus and the Amygdaloideae (Rosaceae) using ITS sequences of nuclear ribosomal DNA. American Journal of Botany 2001, 88, 150-160

  Accordingly, an object of the present invention is to provide a simple means for detecting peaches specifically and with high sensitivity by distinguishing them from other closely related fruits even in a sample containing a plurality of plant species. .

  As a result of intensive studies to solve the above problems, the present inventors designed based on a peach-specific base sequence in the trnS-trnG intergenic spacer region on the peach chloroplast genome. When PCR was performed using primers, a DNA fragment of a specific size was amplified in a sample containing peach, and it was found that peach can be detected specifically and with high sensitivity, and the present invention has been completed.

That is, the present invention includes the following inventions.
(1) A peach detection primer set that specifically amplifies a DNA fragment derived from the peach trnS-trnG intergenic spacer gene, the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1, 2 or 4; A peach detection primer set comprising an oligonucleotide having the base sequence shown in 3 or 5 (however, an oligonucleotide having the base sequence shown in SEQ ID NO: 4 and an oligonucleotide having the base sequence shown in SEQ ID NO: 5) Excluding primer sets that are configured).
5'- aattggtcgtaataaaaagtcaaaa-3 '(SEQ ID NO: 1)
5'-tggtcgtaataaaaagtcaaaa-3 '(SEQ ID NO: 2)
5'-cgtaaacgctctaattttaatgg-3 '(SEQ ID NO: 3)
5'- aattggtcgtaataaaaagtcaata-3 '(SEQ ID NO: 4)
5'-cgtaaacgctctaattttaatag-3 '(SEQ ID NO: 5)
(2) Peach detection using a DNA extracted from a sample as a template, performing PCR using any of the primer sets described in (1), and detecting the presence or absence of an amplification product obtained by the PCR Method.
(3) The amplification product is determined using whether or not the base sequence obtained by removing the primer sequence from the base sequence of the amplification product includes the peach characteristic base portion “g” at the 829th position of the base sequence shown in SEQ ID NO: 6 The method according to (2), comprising a step of confirming whether the DNA is amplified from peach-derived DNA.
(4) A peach detection kit comprising the primer set according to (1).

  ADVANTAGE OF THE INVENTION According to this invention, the peach detection primer set which can detect the peach which may cause food allergy specifically and with high sensitivity is provided. According to the peach detection primer set of the present invention, the presence or absence of a small amount of peach in food can be specifically and highly sensitively detected by a single PCR. In addition, since the amplification product obtained by PCR using the primer set of the present invention is relatively short, about 74 to 77 bp, even when the DNA is fragmented during the food processing process, the peach DNA is longer than when the amplification product is long. Can be detected with high sensitivity. Furthermore, in a sample from which the target amplification product was obtained by PCR using the primer set of the present invention, the amplification product was obtained due to contamination of peach DNA by confirming the base sequence of the amplification product. Therefore, the reliability of the inspection result can be ensured.

  The peach detection primer set of the present invention is a primer set that specifically amplifies a DNA fragment derived from the trnS-trnG intergenic spacer gene of peach, and the amplified DNA fragment contains a peach characteristic base portion. .

  Here, “a DNA fragment derived from the peach trnS-trnG intergenic spacer gene” refers to a DNA fragment that is amplified when PCR is performed using the peach trnS-trnG intergenic spacer gene as a template. -Amplify DNA fragment derived from trnG intergenic spacer gene "only when PCR is performed using peach trnS-trnG intergenic spacer gene as a template. In the case of the primer set of 2 and 5, a 74 bp amplification product) is obtained, and when the other is used as a template, the amplification product cannot be obtained. The “peach characteristic base portion” refers to the 829th “g” in the base sequence (SEQ ID NO: 6) of the peach registered in GenBank with Accession Number AY500733.

  In the present invention, “peach detection” means that the presence or absence of a peach that may be contained in a food material or food due to contamination is determined by the presence or absence of a target amplification product by PCR. Moreover, the "peach" here refers to the Rosaceae cherry genus (Prunus persica) and nectarine (Prunus persica var. Nucipersica). Rosaceae (Prunus persica), which includes white peach, yellow peach and banana, is equivalent to “peach” that is usually cultivated and edible in Japan, “white peach”, “white birch”, “river” Examples include, but are not limited to, Nakajima white peach, Shimizu white peach, Golden peach, Okubo, Akatsuki, Yuzora, and hybrids of these. Nectarine (Prunus persica var. Nucipersica), also known as Tsubaki peach or oil peach, is equivalent to “Nectarine” that is usually grown and edible in Japan. ”,“ Fantasia ”,“ flavor top ”, and hybrids thereof, but are not limited thereto.

  The oligonucleotide constituting the peach detection primer set of the present invention is designed based on a peach-specific nucleotide sequence in the trnS-trnG intergenic spacer region on the peach chloroplast genome.

  In designing a peach detection primer, first, a “peach-specific base sequence” is specified. This “peach-specific base sequence” can be identified by aligning and comparing the base sequences of a plurality of trnS-trnG intergenic spacer regions collected from peaches and other fruits. Specifically, the base sequences of the trnS-trnG intergenic spacer region of peach and other fruits are compared, and the portion contains a plurality of bases common to peach cultivars. A base sequence containing a base that can be distinguished from the base sequence is specified as a base sequence specific to peach.

  The plurality of base sequences used for the alignment can be obtained by searching using a DNA database such as GenBank. In addition, the base sequences of peach cultivars that were not in the database can be obtained by newly analyzing the base sequences independently. For alignment of base sequences, alignment software (for example, CLUSTALW, URL: http://www.ddbj.nig.ac.jp/) published on the Internet can be used.

  Next, a primer is designed based on the specified base sequence. First, the above-mentioned “base common to peaches and distinguishable from other fruits” is arranged at the 3 ′ end of the primer. For primer design, for example, “Forefront of PCR-From Basic Technology to Application” (Protein / Nucleic Acid / Enzyme Special Issue 1996 Kyoritsu Publishing Co., Ltd.) or “Bio-Experiment Illustrated 3 Really Renewable PCR: Cell Engineering” Attached sheet "Experimental note series to see" (by Hiroki Nakayama, Shujunsha Co., Ltd.), "PCR technology-Principle and application of DNA amplification" (Henry A Erlich, supervised by Kuniyuki Kato, Takara Shuzo Co., Ltd.) do it. In the case of detection with processed foods, it is possible that DNA is degraded and shortened, so the target amplification product obtained from the primer set is as short as possible. It is preferable for the purpose of obtaining high sensitivity.

  On the other hand, even theoretically designed primers may not have the required sensitivity and specificity. Therefore, the primer set of the present invention is determined by actually performing PCR using the designed primer and confirming that necessary sensitivity and specificity can be obtained.

  Furthermore, reproducibility may not be obtained due to differences in the type of PCR apparatus. The reason why different results can be obtained between different types of PCR instruments is thought to be due to subtle differences in sample temperature during the PCR cycle. In particular, a factor that greatly affects the sensitivity and specificity of PCR is considered to be a subtle temperature difference during the annealing reaction in which the primer and the template DNA bind. Therefore, in the present invention, in addition to the above primer design, the 3'-side base can be modified in addition to the above-described primer design with the goal of obtaining similar test results with a plurality of types of PCR devices.

Modification of the 3 ′ base of the primer can be performed by referring to, for example, the method described in Pettersson M., Bylund M., Alderborn A. Molecular haplotype determination using allele-specific PCR and pyrosequencing technology.Genomics 2003, 82, 390-396. Can be done.
As described above, the “base common to peaches and distinguishable from other fruits” is set at the 3 ′ end of the primer. If the base is the mismatched base that is different from the sequence of the peach or peach-related species that targets the second base from the 3 'end of the primer, the second 1 from the 3' end of the primer will be used for the peach DNA. Only the base is mismatched, but at least the first and second two bases from the 3 ′ end are successively mismatched to the peach related species DNA. By doing so, it was considered that even if the annealing temperature is somewhat different due to differences in PCR devices, PCR temperature conditions can be obtained that detect only peach DNA and not erroneously detect peach related species DNA. Based on this concept, the second base from the 3 ′ end of the designed primer oligonucleotide was replaced with a mismatch base.

  The oligonucleotide constituting the peach detection primer set of the present invention was synthesized based on the primer design and modification method as described above, and specifically, the nucleotide sequences shown in SEQ ID NOs: 1 to 3 were used. An oligonucleotide having the base sequence shown in SEQ ID NO: 4 in which the second base “a” from the 3 ′ end of SEQ ID NO: 1 is replaced with a mismatch base “t”, the second from the 3 ′ end of SEQ ID NO: 3 These are five types of oligonucleotides having the base sequence shown in SEQ ID NO: 5 in which the base “g” is replaced with the mismatch base “a”.

The base sequences shown in the above SEQ ID NOs are as follows.
Sequence number 1: 5'- aattggtcgtaataaaaagtcaaaa-3 '
Sequence number 2: 5'-tggtcgtaataaaaagtcaaaa-3 '
Sequence number 3: 5'-cgtaaacgctctaattttaatgg-3 '
Sequence number 4: 5'- aattggtcgtaataaaaagtcaata-3 '
Sequence number 5: 5'-cgtaaacgctctaattttaatag-3 '

  Oligonucleotides that serve as primers can be synthesized using methods commonly known in the art as methods for synthesizing oligonucleotides, such as the phosphotriethyl method, phosphodiester method, etc., using a commonly used automatic DNA synthesizer. is there.

  The primer set for peach detection of the present invention is a combination of oligonucleotides designed and modified as described above, specifically, an oligonucleotide having the base sequence shown in SEQ ID NO: 1 or 2 or 4, and SEQ ID NO: 3 Or an oligonucleotide having the base sequence shown in 5. However, a primer set composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 4 and an oligonucleotide having the base sequence shown in SEQ ID NO: 5 is excluded.

  The oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1 or 2 or 4 can be annealed to a nucleotide sequence complementary to a nucleotide sequence specific to peach in the peach trnS-trnG intergenic spacer gene. Used as a forward primer in the detection primer set. The oligonucleotide having the base sequence shown in SEQ ID NO: 3 or 5 can be annealed to the specific base sequence described above and used as a reverse primer for the peach detection primer set.

  As shown in the Examples below, the peach DNA detection sensitivity is 5 fg for the primer set consisting of SEQ ID NOs: 1 and 3 and the primer set consisting of SEQ ID NOs: 2 and 5, and 50 fg for the primer set consisting of SEQ ID NOs: 1 and 5. The primer set consisting of Nos. 2 and 3 and the primer set consisting of SEQ ID Nos. 4 and 3 were 500 fg, and it was confirmed that none of the primer sets erroneously detected Sakura peach, which is a peach-related species. On the other hand, in the primer set consisting of SEQ ID NOs: 4 and 5, despite the fact that mismatch bases are introduced into both the forward primer and the reverse primer, cherry peach is erroneously detected even under the condition that peach DNA 500 fg is detected in actual PCR. It was detected and judged to be inappropriate as a peach detection primer.

  Therefore, a primer set consisting of SEQ ID NO: 2 and 5, a primer set consisting of SEQ ID NO: 1 and 3, a primer set consisting of SEQ ID NO: 1 and 5, a primer set consisting of SEQ ID NO: 2 and 3, a primer consisting of SEQ ID NO: 4 and 3 The set can be suitably used as a primer set for peach detection. Among them, a primer set consisting of SEQ ID NOs: 2 and 5, a primer set consisting of SEQ ID NOs: 1 and 3, and a primer set consisting of SEQ ID NOs: 1 and 5 From the viewpoint of robustness that can maintain sensitivity and specificity even when the PCR apparatus is changed, the primer set consisting of SEQ ID NOs: 2 and 5 is particularly preferable.

  In addition, in the sample from which the target amplification product was obtained, the base sequence of the amplification product, in particular the base sequence excluding the primer sequence of the amplification product, was the peach base sequence (Accession Number AY500733) registered in GenBank. : Verify whether the amplified product was obtained by mixing with peach DNA by confirming whether it contains the peach-characteristic base part “g” at position 829 of the trnS-trnG intergenic spacer gene of SEQ ID NO: 6) Therefore, higher reliability of the inspection result can be ensured.

  The primer set can also be made into a kit. The kit of the present invention only needs to contain at least the above primer set, and if necessary, PCR extraction reagents (excluding primer sets) such as DNA extraction reagents, PCR buffers and DNA polymerases, and positive reactions. A DNA solution containing a PCR amplification region as a control, a detection reagent such as a staining agent or electrophoresis gel, and instructions may be included.

  The present invention also provides a method for detecting peaches using the above primer set. The “peach” that can be detected by the method of the present invention is as described above. This method includes a step of performing PCR using the above-described primer set using DNA extracted from a sample as a template, and detecting the presence or absence of a target amplification product obtained by the PCR.

  The sample is not particularly limited as long as it is a food material or food that may contain peach. The detection result obtained by the method of the present invention can be used for allergy labeling of foods, and for confirmation of the presence or absence of unintentional mixing by the producer on the production line.

  For extraction of DNA from a sample, any method known to those skilled in the art as a nucleic acid extraction method may be used. For example, phenol / chloroform method, cell lysis with a surfactant, cell lysis with a protease enzyme, physical lysis with glass beads It can be performed by a destruction method, a treatment method that repeats freeze-thaw, or the like. As the reagent, various DNA extraction kits sold by manufacturers may be used. Depending on the sample extraction, filtration with a membrane filter or homogenization is performed.

PCR amplification is not particularly limited except that the above primer set is used, and may be performed according to a conventional method. Specifically, a cycle including denaturation of template DNA, annealing of the primer to the template, and primer extension reaction using a thermostable enzyme (DNA polymerase such as Taq polymerase or Tth DNA polymerase from Thermus themophilis) is repeated. Thus, a fragment containing a specific base sequence of the trnS-trnG intergenic spacer region is amplified. The composition of the PCR reaction solution and the PCR reaction conditions (temperature cycle, number of cycles, etc.) should be determined by a person skilled in the art based on preliminary experiments, etc. under conditions such that a PCR amplification product can be obtained with high sensitivity in PCR using the above primer set. Can be selected and set appropriately. An example of the annealing conditions described above is to perform in a PCR reaction solution containing about 1.5 mM MgCl ₂ at 58 ° C. for 1 minute, but this is only an example, and the annealing temperature, PCR reaction solution The composition, annealing time, and the like can be appropriately set according to the length of the oligonucleotide sequence serving as a primer, the base composition, and the like. A series of these PCR operations can be performed using a commercially available PCR kit or PCR apparatus according to the operating instructions. As the PCR apparatus, GeneAmp PCR System 9700 (Applied Biosystems) is usually used, but GeneAmp PCR System 9600 (Applied Biosystems) can also be used.

  Whether or not a target amplification product has been obtained by PCR can be confirmed using methods such as agarose gel electrophoresis, DNA hybridization, and real-time PCR. The fragment length of the target amplification product is the number of bases in the region sandwiched between both primers in the base sequence of the trnS-trnG intergenic spacer region to be detected. For example, when a primer set consisting of SEQ ID NO: 2 and SEQ ID NO: 5 is used, the fragment length of the target amplification product is about 74 bp. Such a relatively short target amplification product is effective for the purpose of obtaining high sensitivity even in processed foods.

  In addition, in this method, regarding the sample (sample in which the target amplification product is detected) that is determined to be mixed with peach from the above PCR results, the obtained target amplification product is truly derived from peach DNA. It can be confirmed that the peach characteristic base portion in the base sequence of the amplification product is used as an index. Specifically, the target amplification product is purified by agarose electrophoresis, etc., the band is cut out and the DNA is extracted, and the obtained DNA fragment is determined by the direct sequence method or inserted into an appropriate vector. Then, after cloning and culturing in E. coli etc., the base sequence of the obtained DNA fragment is confirmed. The sequence can be confirmed by a general method such as the Sanger method or the Maxam-Gilbert method. Among the confirmed nucleotide sequences, the nucleotide sequence excluding the primer sequence is the peach characteristic in the 829th of the trnS-trnG intergenic spacer gene of the peach base sequence (Accession Number AY500733: SEQ ID NO: 6) registered in GenBank. When the base portion “g” is included, it can be determined that the amplification product is amplified from peach-derived DNA.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
(Example 1) Design and synthesis of peach detection primer In designing a peach detection primer, peach (Prunus persica) was set as a detection target, and plants other than peach were set not to be detected.

  When designing primers for peach detection, peach trnS-trnG intergenic spacer region 1 sequence obtained from GenBank, trnS-trnG intergenic spacer region 101 sequence from non-peach rose plants, and 6 commercially available peach varieties analyzed by direct sequencing By aligning and comparing the 6 sequences of the trnS-trnG intergenic spacer region of (Shirakaba, Kawanakajima Hakuto, Golden Peach, Flavor Top, Hidemine, Tomo), we searched for a peach-specific base sequence.

Peach base sequence (GenBank):
Peach: Prunus persica (AY500733)

Among the base sequences of rosaceae plants other than peaches that have been aligned (GenBank):
Sakura Peach: Prunus avium (AY871252)
Plum: Prunus salicina (AY500722)
Apricot: Prunus armeniaca (AY500725)
Ume: Prunus mume (AY500726)
Almond: Prunus dulcis (AY500730)
Spino peach: Prunus spinosa (AY500720)
Prune: Prunus domestica (AY500719)
A type of prune: Prunus insititia (AY500718)
A kind of cherry genus: Prunus davidiana (AY500731)
A kind of cherry genus: Prunus mira (AY500732)
Related species of Juneberry: Amelanchier arborea (EF127115)
Related species of hawthorn: Crataegus laevigata (EF127093)

  The alignment results are shown in FIG. First, by alignment, a base sequence containing a plurality of bases common to peaches and including bases that can be distinguished from other fruits in the common bases was specified as a base sequence specific to peaches.

  Among the specified base sequences specific to the above peaches, a region that is common to peaches and that is different from other fruits is a base corresponding to the 3 'end of the primer, and contains about 30 bp in the 5' direction. Was studied as a primer design region. A base sequence common to peaches and different from other fruits was arranged between the forward primer and reverse primer design regions. As a result, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 (5′-aattggtcgtaataaaaagtcaaaa-3 ′) as a forward primer and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2 with 3 bases deleted from the 5 ′ end of SEQ ID NO: 1 (5 ′ -tggtcgtaataaaaagtcaaaa-3 ') and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 (5'-cgtaaacgctctaattttaatgg-3') was selected as a reverse primer.

  The 3 ′ ends of the nucleotide sequences of SEQ ID NOs: 1 and 2 selected as the forward primer both correspond to the 814th base of the peach base sequence (Accession Number AY500733) reported to GenBank. It is a base that can be distinguished from non-peach fruits (Prunus genus is the same as peach and cannot be distinguished). The 3 ′ end of the nucleotide sequence of SEQ ID NO: 3 selected as the reverse primer is also equivalent to the 844th base of the GenBank peach base sequence (Accession Number AY500733), and is a base that can distinguish peach and non-peach fruits ( In the genus Prunus, P. mira and P. davidiana are the same as peach and cannot be distinguished). The base sequence between SEQ ID NO: 1 or 2 as the forward primer and SEQ ID NO: 3 as the reverse primer also contains the 829th base of the GenBank peach base sequence (Accession Number AY500733). It is a base that can distinguish fruits other than (in Prunus, only P. mira is the same as peach and cannot be distinguished). Both P. mira and P. davidiana are not cultivars intended for edible use, and are very unlikely to be mixed into food. There was no problem.

  Among the designed primers, a primer set consisting of SEQ ID NOs: 2 and 3 is used, and the sequence is highly homologous to both the detection target peach (1 sequence) and the sequence of the forward primer and the reverse primer in the Blast search. Hits, non-peach roses (top 99 sequences: Spiraea, Aronia, Malus, Amelanchier, Mespilus, Oemleria, Maddenia, Crataegus, Prunus) and Blast search did not hit Aligned Rosaceae (2 sequences: Prunus spp.), Major fruits, grains, vegetables (14 sequences: orange, grapes, blueberries, rice, wheat, corn, soybeans, potatoes, carrots, spinach, lettuce, cucumbers PCR simulation using Amplify 1.0 (Bill Engels) was performed on the trnS-trnG intergenic spacer region of eggplant and tomato. As a result, it was expected that the target amplification product of the primer set consisting of SEQ ID NOs: 2 and 3 was obtained only for the peach and P. mira sequences, and no target amplification product was obtained from any other sequence.

  Since SEQ ID NO: 1 has the same 3 ′ end as SEQ ID NO: 2, even when a PCR simulation is performed with a primer set consisting of SEQ ID NO: 1 and 3, the target is the same as the primer set consisting of SEQ ID NO: 2 and 3. It was expected that amplification products were obtained only for the peach and Prunus mira sequences, and no target amplification product was obtained from any other sequence.

(Example 2) Specificity and sensitivity evaluation of primer set using SEQ ID NO: 1 for forward primer and SEQ ID NO: 3 for reverse primer
(1) Preparation of DNA sample As a peach sample, white varieties, white peach, Kawanakajima white peach, golden peach, flavor top, Hidemine, peach varieties (seed), as a plant sample other than peach, primer among peach related species Sakura peach (seed), which had the highest homology with the primer in the nucleotide sequence expected to bind to was used. For the seeds, the seed coat was peeled off with a razor without touching it directly, and the embryos were taken out and used for DNA extraction. About 0.1 g of seeds was put in a 2 ml tube (manufactured by Eppendorf), one zirconia bead having a diameter of 7 mm (manufactured by Nikkato Co., Ltd.), and finely pulverized with Retsch MM 300 (manufactured by QIAGEN). About 0.1 g of pulverized seeds was charged with 15 ml of buffer G2 (QIAGEN), 100 μl Proteinase K (20 mg / ml) (QIAGEN), 20 μl RNase A (100 mg / ml) (QIAGEN) After adding to a 50 ml tube and mixing, it was kept warm at 50 ° C. for 2 hours. The mixed solution was transferred to a 2 ml tube and centrifuged at about 20,000 × g for 5 minutes to obtain the supernatant. The obtained supernatant was subjected to Genomic-tip 20 / G (QIAGEN) equilibrated with 1 ml of buffer QBT (QIAGEN) in advance to adsorb the DNA to the tip. Thereafter, the tip was washed with 6 ml of buffer QC (QIAGEN), and the DNA was eluted with 1 ml of buffer QF (QIAGEN) preheated to 50 ° C. The precipitate recovered by isopropanol precipitation was dissolved in 50 μl of TE buffer (pH 8.0). The spectrum of 220-350 nm of the DNA solution was measured with a spectrophotometer, and the DNA concentration in the solution was calculated from the absorbance at 260 nm of the sample having the maximum value at 260 nm. For peaches, the DNA solution is diluted to 20 ng / μl with TE buffer (pH 8.0), and then serially diluted with TE buffer (pH 8.0) containing 20 ng / μl salmon sperm DNA. It was. For cherry tree, a DNA template diluted with TE buffer (pH 8.0) to 20 ng / μl was used as a template DNA sample for PCR. In the PCR 1 reaction solution, 500 fg, 50 fg, and 5 fg were used as peach DNA samples, and 50 ng was used as cherry peach DNA samples.

  All template DNA samples diluted to 20 ng / μl were prepared from the literature (Watanabe T., Akiyama H., Maleki S., Yamakawa H., Iijima K., Yamazaki F., Matsumoto T., Futo S., Arakawa F ., Watai M., and Maitani T., 2007. A specific qualitative detection method for peanut (Arachis hypogaea) in foods using polymerase chain reaction; Journal of Food Biochemistry (2006) 30: 215-233) It was confirmed by PCR that the purity was such that a PCR amplification product was obtained.

(2) PCR reaction PCR was performed using each template DNA sample prepared as described above.
The PCR reaction was carried out using the PCR reaction solution composition and the PCR reaction conditions shown in Table 1 below, using SEQ ID NO: 1 as the forward primer and SEQ ID NO: 3 as the reverse primer, and placing them in a 0.2 ml tube, and using the ABI PRISM 7700 (Applied Biosystems) (Manufactured by company: Equipment equivalent to GeneAmp PCR System 9600).

(3) Detection
8 μl of the solution after the PCR reaction was subjected to 3% agarose gel electrophoresis containing ethidium bromide. At the same time, 20bp DNA Ladder and 100bp DNA Ladder (Takara Bio Inc.) were used as molecular weight markers so that the amount of DNA in the 100bp band would be 5ng, and visualized by UV irradiation. Further, it was determined whether or not the fragment of the PCR amplification product could be confirmed more clearly than 5 ng of the 100 bp band.

  As a result of the determination, a target amplification product having a fragment length of about 77 bp was confirmed in 5 fg, 50 fg, and 500 fg of peach DNA, while no target amplification product was observed in 50 ng of cherry pink DNA.

  From these results, PCR using the primer sets of SEQ ID NOs: 1 and 3 can specifically detect peaches, and can detect 5 fg (0.1 ppm weight / weight) of peach DNA in 50 ng of salmon sperm DNA, That is, it was shown that peach can be detected with a sensitivity of 0.1 ppm peach DNA / sample DNA level.

(Example 3) Specificity and sensitivity evaluation of primer set using SEQ ID NO: 2 for forward primer and SEQ ID NO: 3 for reverse primer Samples were peach DNA 500fg, and homology with primer sequences as representative among peach related species Cherry peach DNA 50 ng, which is highly probable and could be amplified, was used.

  The PCR reaction was performed according to the PCR reaction solution composition and PCR reaction conditions shown in Table 2 below, using SEQ ID NO: 2 as the forward primer and SEQ ID NO: 3 as the reverse primer, and placed in a 0.2 ml tube, and the GeneAmp PCR System 9600 (Applied Bio) System).

  The detection was confirmed by agarose electrophoresis in the same manner as in Example 2 (3). As a result, when the primer sets of SEQ ID NOs: 2 and 3 were used, a target amplification product having a fragment length of about 74 bp was confirmed with 500 fg of peach DNA, and no target amplification product was found with 50 ng of cherry pink DNA.

  From this result, PCR using the primer sets of SEQ ID NOs: 2 and 3 can specifically detect peach, and can detect 500 fg (10 ppm weight / weight) of peach DNA in 50 ng of salmon sperm DNA, that is, 10 ppm. It was shown that peach can be detected with sensitivity of peach DNA / sample DNA level.

Example 4 Design and Synthesis of Peach Detection Primer Introduced with Mismatch Base Second from the 3 ′ end of SEQ ID NO: 1, originally “a” is mismatched with the base sequences of peach and peach related species The oligonucleotide (5′-aattggtcgtaataaaaagtcaata-3 ′) having the base sequence (SEQ ID NO: 4) substituted for “t”, the second from the 3 ′ end of SEQ ID NO: 3, originally “g”, And oligonucleotides (5′-cgtaaacgctctaattttaatag-3 ′) having a base sequence (SEQ ID NO: 5) substituted with “a” which mismatches with the base sequence of peach-related species were synthesized according to the usual oligonucleotide synthesis method. .

  A combination of the primers of SEQ ID NOs: 2 and 5 is selected from the primers of SEQ ID NOs: 1, 2 and 3 designed in Example 1 and the primers of SEQ ID NOs: 4 and 5 described above, and a primer set comprising this combination is used. In the same manner as in Example 1, PCR simulation using Amplify 1.0 (Bill Engels) was performed on the base sequences of trnS-trnG intergenic spacer regions of peach and other plants. As a result, in PCR using the primer set consisting of SEQ ID NOs: 2 and 5, target amplification products can be obtained only for peach and Prunus mira sequences, and no target amplification products can be obtained from any other sequences. It was done.

  Combined with the results of the PCR simulation in Example 1, the same results were obtained regardless of which of SEQ ID NO: 3 or SEQ ID NO: 5 in which the second base from the 3 ′ end of SEQ ID NO: 3 was replaced with a mismatched base was used as a reverse primer. Therefore, it was considered that the mismatch of the second base from the 3 ′ end did not affect the PCR simulation results. Therefore, in the primer set in which SEQ ID NO: 1 or 2 or 4 is the forward primer and SEQ ID NO: 3 or 5 is the reverse primer, the target amplification product is converted into the peach and Prunus mira sequences in any combination of PCR simulations. It was expected that no target amplification product could be obtained from any other sequence.

(Example 5) Specificity and sensitivity evaluation of primer sets in which the combination of forward primer and reverse primer is SEQ ID NO: 2 and 5, SEQ ID NO: 1 and 5, SEQ ID NO: 4 and 3, and SEQ ID NO: 4 and 5 The template DNA sample prepared in Example 2, ie, peach DNA 5fg, 50fg, 500fg, and 50ng of cherry peach DNA having high homology with the primer sequence and being likely to be amplified as representative of peach related species.

  The PCR reaction was carried out using the PCR reaction solution composition and PCR reaction conditions shown in Table 2 above (however, the temperature in the annealing reaction step was 48 ° C or 52 ° C instead of 60 ° C), and the combination of the forward primer and the reverse primer was SEQ ID NO: 2. 5 and 5, SEQ ID NOS: 1 and 5, SEQ ID NOS: 4 and 3, and SEQ ID NOS: 4 and 5, respectively, are put into 0.2 ml tubes, and GeneAmp PCR System 9700 or GeneAmp PCR System 9600 is used as a PCR device. I went.

  The detection was confirmed by agarose electrophoresis in the same manner as in Example 2 (3). The results are shown in Table 3 below.

  When the primer set consisting of SEQ ID NOs: 2 and 5 was used, a target amplification product having a fragment length of about 74 bp was confirmed at 5 fg, 50 fg, and 500 fg of peach DNA at both annealing temperatures of 48 ° C. and 52 ° C., and No target amplification product was observed with 50 ng of cherry DNA.

  When the primer set consisting of SEQ ID NOs: 1 and 5 was used, a target amplification product having a fragment length of about 77 bp was confirmed at 50 fg and 500 fg of peach DNA at both annealing temperatures of 48 ° C and 52 ° C. No target amplification product was seen at 50 ng.

  When a primer set consisting of SEQ ID NOs: 4 and 3 was used, a target amplification product of about 77 bp fragment length was confirmed with peach DNA 500 fg at both annealing temperatures of 48 ° C. and 52 ° C., and target with 50 ng of cherry peach DNA No amplification product was seen.

  When a primer set consisting of SEQ ID NOs: 4 and 5 was used, a target amplification product having a fragment length of about 77 bp in peach DNA 50 fg and 500 fg was confirmed. However, a target amplification product was also observed with 50 ng of cherry peach DNA, and an annealing temperature at which no target amplification product was observed could not be found.

  From these results, the best form is a primer set consisting of SEQ ID NOs: 2 and 5, and under the tested PCR conditions, peach DNA can be detected with a sensitivity of 5 fg in the annealing temperature range of at least 4 ° C. It was shown not to detect. The primer sets consisting of SEQ ID NOs: 1 and 5 and SEQ ID NOs: 4 and 3 were both able to detect peach DNA 50-500fg and confirmed that they were not detected by mistake, but the primers consisting of SEQ ID NOs: 2 and 5 The set was more sensitive. Although PCR using the above three primer sets has different sensitivities, it can specifically detect peaches and can detect at least 500 fg (10 ppm weight / weight) of peach DNA in 50 ng of salmon sperm DNA. Since peach can be detected with a sensitivity of at least 10 ppm peach DNA / sample DNA level, both can be used for peach detection. Furthermore, with these primer sets, the same PCR results can be obtained at different annealing temperatures by 4 ° C. Therefore, even if a different type of PCR device is used, the same detection results can be obtained with good reproducibility under the same PCR conditions, so any laboratory with a general PCR device can be used. In addition, anyone who has received general training in the field can easily use it. On the other hand, the primer set consisting of SEQ ID NOs: 4 and 5 has mistakenly detected cherry peach even under the condition of detecting peach DNA 500 fg in actual PCR, even though mismatch bases are introduced into both forward primer and reverse primer. Since it was detected, it was considered inappropriate as a primer for peach detection.

(Example 6) Specificity and sensitivity evaluation of primer set using SEQ ID NO: 2 for forward primer and SEQ ID NO: 5 for reverse primer
(1) Preparation of DNA sample Samples are peach, white birch, Kawanakajima white peach, golden peach, flavor top, Hidemine, peach peach (seed), non-peach plant samples such as plum (seed), apricot (seed) ), Cherry peach (seed), ume (seed), almond (leaf), prune (seed), apple (seed), pear (seed), pear (seed), strawberry (fruit), raspberry (fruit), aloe vera ( Leaf), pineapple (fruit), papaya (fruit), orange (fruit), mandarin (fruit), melon (fruit), persimmon (seed), fig (fruit), mango (fruit), banana (fruit), avocado ( Fruit), blueberry (fruit), grape (fruit), kiwi (fruit), rice (seed), soybean (seed), corn (seed), wheat (seed), potato (tuber), carrot (root), onion Bulbs), Chinese cabbage (leaves), spinach (leaf), it was used a total of 36 kinds of cucumber (fruit), tomato (fruit).

  For the seeds, the seed coat was peeled off with a razor without touching it directly, and the embryos were taken out and used for DNA extraction. The fruits, tubers, roots and bulbs were peeled off with a razor so as not to touch them directly, and the inside was taken out and used for DNA extraction. About 0.1 g of seeds or leaves and about 2 g of fruits, tubers, roots or bulbs divided into about 1 g each are put into 2 ml tubes (manufactured by eppendorf), and zirconia beads with a diameter of 7 mm (Nikkato Corporation) 1 product) was put and finely pulverized with Retsch M 300 (QIAGEN). About 0.1 g of ground seeds or leaves, about 2 g of fruits, tubers, roots or bulbs, 15 ml of buffer G2 (QIAGEN), 100 μl Proteinase K (20 mg / ml) (QIAGEN), The mixture was added to a 50 ml tube containing 20 μl of RNase A (100 mg / ml) (manufactured by QIAGEN), mixed, and then incubated at 50 ° C. for 2 hours.

  Thereafter, template DNA samples diluted to 20 ng / μl were prepared in the same manner as in the operation after transferring the mixed solution to the 2 ml tube in Example 2 (1). In addition, each template DNA sample diluted to 20 ng / μl was confirmed to have a level of purification at which a PCR amplification product was obtained by plant DNA detection PCR, as in (1) of Example 2.

(2) PCR reaction PCR was performed using each template DNA sample prepared as described above.
The PCR reaction was performed according to the PCR reaction solution composition and PCR reaction conditions shown in Table 4 below, using SEQ ID NO: 2 as the forward primer and SEQ ID NO: 5 as the reverse primer, and placed in a 0.2 ml tube, and the GeneAmp PCR System 9700 or GeneAmp PCR in the PCR device. Performed using System 9600.

  The detection was confirmed by agarose electrophoresis in the same manner as in Example 2 (3). 2 to 5 show the results of detection by electrophoresis when PCR was performed with GeneAmp PCR System 9700 using the primer set consisting of SEQ ID NOs: 2 and 5 under the conditions shown in Table 4. As shown in FIG. 2, in peaches of 6 varieties tested, a target amplification product having a fragment length of about 74 bp was clearly confirmed when peach DNA 50fg and 500fg were used as templates. On the other hand, as shown in FIGS. 3 to 5, in 36 types of plants other than peaches, a target amplification product having a fragment length of about 74 bp was not observed even when 50 ng of DNA was used as a template. The same results as in FIGS. 2 to 5 were also obtained with GeneAmp PCR System 9600. From these results, PCR using the primer set consisting of SEQ ID NOs: 2 and 5 can specifically detect peach, and can detect 50 fg (1 ppm weight / weight) of peach DNA in 50 ng of salmon sperm DNA, that is, 1 ppm. It has been shown that peaches can be detected with a level of sensitivity, and that robustness can be obtained with reproducibility even with different PCR instruments.

(Example 7) Verification by confirmation of base sequence of target amplification product When the base sequence of the target amplification product obtained from peach (Shirakaba) was confirmed by the direct sequence method, the base sequence excluding the primer sequence of the target amplification product was It had the following base sequence.
5'- ttaaaagaaaagat g ggatcataaaacaa -3 '(SEQ ID NO: 7)

  When the base sequence of SEQ ID NO: 7 was aligned with the peach base sequence registered in GenBank (Accession Number AY500733: SEQ ID NO: 6), the 15th base (double underlined portion) counted from the 5 ′ end of SEQ ID NO: 7 ) And the 829th base which is the peach characteristic base part of SEQ ID NO: 6 were overlapped and both were “g”, so that it was confirmed that the target amplification product was amplified from peach-derived DNA.

(Example 8) Confirmation of detection sensitivity by model processed food
(1) Preparation of peach standard sample Peach skin was peeled off and the pulp part from which the seed was removed was freeze-dried and pulverized, and immediately mixed with an equal amount of calcium carbonate to homogenize. This was used as a peach standard sample.

(2) Protein quantification of peach standard sample 2 g of peach standard sample is collected in a 50 mL tube, and buffer for protein extraction (0.5% SDS, 2% 2-mercaptoethanol, 0.5M NaCl, 0.1M Tris-HCl (pH8.6) )) 20 mL was added and mixed well to disperse the solid and extracted with shaking at room temperature for 16 hours. The extract was centrifuged at 10,000 × g for 30 minutes, and then the supernatant was filtered through a microfilter having a pore size of 0.8 μm to obtain a protein extract. With respect to 16 μl of the obtained protein extract, the protein concentration was quantified using 2-D Quant kit (GE Healthcare Bioscience) according to the description of the kit.

(3) Selection of model processed foods Foods commonly found in the market that may contain peaches were listed as candidates, and among them, processed foods that were thought to be difficult to detect peach DNA were selected. Among them, the first model processed food was selected as a jam that is considered to be the most severely degraded DNA in the processing process and likely to be undetectable. Second, we selected cookies that are considered likely to be undetectable due to the relatively low DNA concentration of the added peach due to the large amount of DNA in the model processed food.

(4) Production of model processed food Jam was produced as shown in FIG. 6 and cookies were produced as shown in FIG. For both jams and cookies, peach standard samples with / without additives were prepared. As for the additive, a peach standard sample was added so that the peach protein per final weight of the model processed food was 10 μg / g. As for the additive products, both jams and cookies were prepared twice, and the additive-free products were prepared once each.

(5) Preparation of DNA sample In the same manner as in (1) of Example 2, DNA was extracted once from 2 g of the pulverized and homogenized model processed food, and the DNA concentration was measured. Those having a concentration higher than 20 ng / μl were diluted with TE buffer (pH 8.0) to 20 ng / μl, and those thinner than 20 ng / μl were directly used as DNA samples. The DNA sample extracted from the additive corresponds to 10 μg / g peach protein. A DNA sample extracted from a jam to which a peach standard sample was added was diluted 10-fold with a DNA sample extracted from a jam to which a peach standard sample was not added. A cookie prepared in the same manner was used as a cookie 1/10 diluted DNA sample. A 1/10 diluted DNA sample corresponds to 1 μg / g peach protein. 2.5 μl of these DNA samples were used for PCR per reaction solution.

(6) PCR reaction Each prepared DNA sample was repeated twice, and a PCR reaction was performed in the same manner as in Example 6, (2).

(7) Detection It confirmed by agarose electrophoresis similarly to (2) of Example 2.
(8) Confirmation of base sequence of target amplification product The base sequence was confirmed in the same manner as in Example 7.
(9) Detection Result and Confirmation Result of Base Sequence Table 5 shows the amount of peach protein added to the prepared model processed food and the detection result of the target amplification product by the peach detection PCR method. An electropherogram is shown in FIG.

  As is clear from the results in Table 5 and FIG. 8, a target amplified product with a fragment length of about 74 bp was clearly confirmed in the DNA sample of the model processed food to which the peach standard sample corresponding to 10 μg / g of peach protein was added. On the other hand, the target amplification product was not observed in the DNA sample of the model processed food to which the peach standard sample was not added. Furthermore, the target amplification product was clearly confirmed from a DNA sample corresponding to 1 μg / g of peach protein.

  When the base sequence of the target amplification product obtained from the jam and cookies added with the peach standard sample was aligned with the peach base sequence (Accession Number AY500733) registered in GenBank, both base sequences were the same as SEQ ID NO: 7 In addition, the 15th base counted from the 5 ′ end (corresponding to the double underline in SEQ ID NO: 7) and the 829th base which is the peach characteristic base part of the peach base sequence (Accession Number AY500733) overlap, Since it was “g”, it was confirmed that the target amplification product was amplified from peach-derived DNA. From this, it was shown that PCR using this primer set detects peach mixed at a peach protein level of 10 μg / g and possibly even at a level of 1 μg / g level in multiple foods.

The alignment of the nucleotide sequence of trnS-trnG intergenic spacer gene of peach and peach related species is shown. Primer set of the present invention (primer consisting of the primer of SEQ ID NO: 2 and the primer of SEQ ID NO: 5) for DNA samples (500 fg, 50 fg, 5 fg) derived from peach (white birch, Kawanakajima white peach, golden peach, flavor top, Hidemine, Tomo) The sensitivity evaluation test result (agarose gel electrophoresis photograph of PCR amplification product) is shown. Arrow: Band of amplification product of approximately 74 bp. Primer set of the present invention (50 ng) derived from peach (birch) DNA sample (500 fg), plum, apricot, cherry peach, ume, almond, prune, apple, pear, pear, strawberry, raspberry 2 shows a specificity evaluation test result (agarose gel electrophoresis photograph of a PCR amplification product) of a primer set comprising a primer of No. 2 and a primer of SEQ ID NO: 5. Arrow: Band of amplification product of approximately 74 bp. The present invention for DNA samples (50 ng) derived from peach (birch), aloe vera, pineapple, papaya, orange, tangerine, melon, strawberry, fig, mango, banana, avocado, blueberry, grape, kiwi The specificity evaluation test result (agarose gel electrophoresis photograph of the PCR amplification product) of the primer set (primer set comprising the primer of SEQ ID NO: 2 and the primer of SEQ ID NO: 5) is shown. Arrow: Band of amplification product of approximately 74 bp. Primer set (sequence) of the present invention for DNA sample (500 fg) derived from peach (Kawanakajima Hakuto), rice, soybean, corn, wheat, potato, carrot, onion, Chinese cabbage, spinach, cucumber, tomato The result of a specificity evaluation test (agarose gel electrophoresis photograph of a PCR amplification product) of a primer set comprising a primer of No. 2 and a primer of SEQ ID No. 5 is shown. Arrow: Band of amplification product of approximately 74 bp. A flow diagram of a method for producing a jam is shown. A flow diagram of a method for making cookies is shown. Primer of the present invention for DNA sample (50 fg) derived from peach (birch), jam added, jam added 1/10 diluted, jam not added, cookie added, cookie added 1/10 diluted, cookie not added DNA sample (50 ng) The sensitivity evaluation test result (agarose gel electrophoresis photograph of the PCR amplification product) of the set (primer set comprising the primer of SEQ ID NO: 2 and the primer of SEQ ID NO: 5) is shown. Arrow: Band of amplification product of approximately 74 bp.

Claims (4)

A peach detection primer set that specifically amplifies a DNA fragment derived from the trnS-trnG intergenic spacer gene of peach, comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1, 2 or 4, and SEQ ID NO: 3 or 5 composed of an oligonucleotide comprising the nucleotide sequence shown in, peach detection primer set (provided that consists of an oligonucleotide comprising the nucleotide sequence shown in SEQ ID NO: 5 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4 Excluding primer set).
5'- aattggtcgtaataaaaagtcaaaa-3 '(SEQ ID NO: 1)
5'-tggtcgtaataaaaagtcaaaa-3 '(SEQ ID NO: 2)
5'-cgtaaacgctctaattttaatgg-3 '(SEQ ID NO: 3)
5'- aattggtcgtaataaaaagtcaata-3 '(SEQ ID NO: 4)
5'-cgtaaacgctctaattttaatag-3 '(SEQ ID NO: 5) A method for detecting a peach, comprising a step of performing PCR using a DNA extracted from a sample as a template and using the primer set according to claim 1 and detecting the presence or absence of an amplification product obtained by the PCR. The amplification product is a peach-derived DNA using as an index whether or not the base sequence obtained by removing the primer sequence from the base sequence of the amplification product contains the peach characteristic base portion “g” at the 829th position of the base sequence shown in SEQ ID NO: 6 The method according to claim 2, further comprising the step of confirming whether or not the signal has been amplified.   A peach detection kit comprising the primer set according to claim 1.

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