KR102011741B1 - Meltiplex pcr primers for detection of fruits and vegetables causing allergy, and use thereof - Google Patents

Abstract

The present invention provides a primer set that binds to the Pru p 2.01A gene; Primer sets that bind to the Cyclophilin gene; A set of primers that bind to the Pectin methylesterase inhibitor gene; And a primer set that binds to the Mdtl1 gene. Provided is a multiplex PCR primer set for allergen-induced fruit vegetable detection comprising at least two primer sets selected from the group consisting of:

Description

Multiplex PCR Primer for Detection of Allergen-Induced Fruits and Its Uses {MELTIPLEX PCR PRIMERS FOR DETECTION OF FRUITS AND VEGETABLES CAUSING ALLERGY, AND USE THEREOF}

The present invention relates to a multiplex PCR primer capable of quickly detecting allergen-producing fruit vegetables, and an allergen-inducing food detection method and detection kit using the same.

Allergy refers to a rapid immune response in vivo caused by antigen-antibody reactions in contact with a specific foreign substance.

The living body produces antibodies and lymphocytes that specifically react to antigens, and when they come into contact with antigens, they produce various immune responses.

The immune response is one of the important defense mechanisms for the self-preservation of the living body, and usually acts protectively against the living body, but sometimes it is called an allergy when the mechanism adversely affects the living body and causes a disorder.

Antigens that cause allergic reactions are called allergens. Typical allergens include pollen, drugs, vegetable fibers, bacteria, food, dyes, and chemicals. When allergens are foods, they are called food allergies.

Food allergy is when a person ingests a food that is harmless to a normal person. An allergen in the food causes an excessive immune response in the human body.

150 million people worldwide (6-7 million of the US, 2.5% of the developing world's population, 5-8% of the country) suffer from food allergies. The form of food allergy is manifested in the form of hives, itching or anaphylaxis in the respiratory, digestive and skin areas and occurs within 1 to 2 hours of food ingestion. Few people have food allergies, but they can be life threatening in severe cases by allergens. Therefore, in terms of food safety, analyzing foods that cause allergies has emerged as a very important issue.

There is no specific method for preventing and treating food allergies, and the only way to prevent them is to ensure that sensitive consumers do not consume the foods through accurate labeling of foods that cause food allergies. This applies equally to the food itself, as well as to processed foods manufactured and processed using it as raw material.

KFDA includes eggs (poultry), milk, buckwheat, peanuts, beans, wheat, mackerel, crab, shrimp, pork, peaches, tomatoes, sulfurous acid, walnuts, chicken, beef, squid and shellfish (oysters, abalone and mussels). Food allergy labeling system for 21 kinds is implemented. The food allergy labeling system extends to processed and fast food as well as processed foods.

However, it is not uncommon for allergic reactions to one or more foods, and respiratory allergies such as allergic rhinitis and food allergies are commonly reported. If the raw foods are taxonomically adjacent to each other, or if the allergens have similar molecular structures, they can cause the same allergic reaction, which is called cross-reaction.

An example of a cross reaction is pollen-related food allergies. Pollen allergy sufferers are allergic to at least one food.

Typical examples include Birch pollen- related allergic reactions, which include allergic reactions in one or more foods, including fruits such as pears, apples, melons, and kiwis, nuts such as peanuts and hazelnuts, and vegetables such as corn and lettuce. Can be seen. Such cross-reaction occurs because the specific allergens causing allergy are the same, and in order to identify allergens, it is necessary to detect allergens of specific food raw materials that can predict the cross-reaction.

In order to detect allergens of certain food ingredients in foods, a method of testing for the presence of allergens is required, but ELISA or EIA methods for detecting allergens are most commonly used.

However, since allergen, a protein to be tested, may be easily lost in various stages of processing in which various raw materials are mixed, there is a problem in that the test is low in accuracy and takes a long time to detect. Since allergens can cause fatal reactions in the presence of very small amounts, there is a need for highly sensitive allergen detection methods.

On the other hand, since DNA is more stable to heat than protein, analysis of DNA in foods can effectively detect microorganisms or raw materials present in small amounts.

In particular, using polymerase chain reaction (PCR), a primer set amplifies a portion of the DNA sequence, so that a small amount of the target substance present in a small amount can be amplified by one or more times. Therefore, in the case of processed foods that go through several processes, DNA analysis using PCR rather than protein can effectively detect allergens.

Therefore, in the situation where the development of an allergen-induced food detection method capable of accurately analyzing various allergens is steadily emerging, the inventors have developed an allergen-induced food detection method having improved sensitivity and accuracy by using a specific primer set.

It is an object of the present invention to provide multiplex PCR primers capable of detecting tomatoes, apples, peaches, and kiwis that cause certain allergies at once.

It is also an object of the present invention to provide a method capable of detecting the presence or absence of allergen-inducing vegetables in foods and processed foods by a real-time PCR method using a primer for detecting allergens.

According to an aspect of the present invention, a primer set that binds to the Pru p 2.01A gene; Primer sets that bind to the Cyclophilin gene; A set of primers that bind to the Pectin methylesterase inhibitor gene; And a primer set that binds to the Mdtl1 gene. Provided is a multiplex PCR primer set for allergen-induced fruit vegetable detection comprising at least two primer sets selected from the group consisting of:

In one embodiment, the primer set for detecting the Pru p 2.01A gene may be composed of a nucleotide sequence represented by SEQ ID NO: 1 and a nucleotide sequence represented by SEQ ID NO: 2.

In one embodiment, the primer set for detecting the Cyclophilin gene may be composed of a nucleotide sequence represented by SEQ ID NO: 3 and a nucleotide sequence represented by SEQ ID NO: 4.

In one embodiment, the primer set for detecting the Pectin methylesterase inhibitor gene may be composed of a nucleotide sequence represented by SEQ ID NO: 5 and a nucleotide sequence represented by SEQ ID NO: 6.

In one embodiment, the primer set for detecting the Mdtl1 gene may be composed of the nucleotide sequence represented by SEQ ID NO: 7 and the nucleotide sequence represented by SEQ ID NO: 8.

In one embodiment, the allergen may be tomato, apple, peach and kiwi.

According to another aspect of the invention, there is provided a method for detecting allergen-induced food comprising the step of performing a polymerase chain reaction (PCR) using the primer.

According to another aspect of the present invention, there is provided an allergen-inducing food detection kit comprising the primer set, and a PCR reaction mixture.

In one embodiment, the PCR reaction mixture may comprise a reaction buffer, dNTP and DNA polymerase.

Primer set for multiplex PCR of the present invention can determine the presence or absence of allergen-causing vegetables, such as tomatoes, apples, peaches, kiwis at once, and easily and simply the presence of allergen-inducing vegetables in food and processed foods Can be determined.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a primer set according to an embodiment of the present invention wheat (1), soybean (2), buckwheat (3), tomato (4), apple (5), peach (6), kiwi (7), pine nuts (8), walnuts (9), peanuts (10), sesame seeds (11), corn (12), rice (13), barley (14), potatoes (15), peppers (16), cherries (17), plums (18), pistachios (19), almonds (20), cashew nuts (21), hazelnuts (22) and perilla (23) is the result of performing a Simplex PCR.
2 is a result of performing multiplex PCR on the positive control (1), tomato (2), apple (3), peach (4) and kiwi (5) with a primer set according to an embodiment of the present invention.
Figure 3 is a set of primers according to an embodiment of the present invention as a set of wheat (2), soybean (3), buckwheat (4), tomato (5), apple (6), peach (7), kiwi (8) , Pine nuts (9), walnuts (10), peanuts (11), sesame seeds (12), corn (13), rice (14), barley (15), potato (16), pepper (17), cherry (18) The results of performing multiplex PCR on, plums 19, pistachios 20, almonds 21, cashew nuts 22, hazelnuts 23, and perilla 24. Positive control is 1 and negative control is N.
4 is a result of confirming the sensitivity of the flex PCR with a primer set according to an embodiment of the present invention. The gDNA of the positive control group was 50 ng 1 time, 10 ng 2 times, 2 ng 3 times, 0.4 ng 4 times, 0.08 ng 5 times, 0.016 ng 6 times, 0.0032 ng 7 times, and negative control. Is N.

Hereinafter, with reference to the accompanying drawings will be described the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

When a part is said to "include" a certain component, this means that it may further include other components, without excluding other components, unless specifically stated otherwise.

Unless defined otherwise, molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing can be performed by conventional techniques commonly used in the field of recombinant DNA within the capabilities of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and reference books.

Unless defined otherwise herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art.

Various scientific dictionaries that include the terms included herein are well known and available in the art. Any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing herein and some methods and materials are described. Depending on the context used by those skilled in the art, the present invention is not limited to specific methodologies, protocols, and reagents, because it can be used in a variety of ways.

As used herein, the singular encompasses the plural objects unless the context clearly dictates otherwise. Also, unless otherwise indicated, nucleic acids are written from left to right, 5 'to 3', respectively, and amino acid sequences are written from left to right, amino to carboxyl directions. Hereinafter, the present invention will be described in more detail.

According to an aspect of the present invention, a primer set that binds to the Pru p 2.01A gene; Primer sets that bind to the Cyclophilin gene; A set of primers that bind to the Pectin methylesterase inhibitor gene; And a primer set that binds to the Mdtl1 gene. Provided is a multiplex PCR primer set for allergen-induced fruit vegetable detection comprising at least two primer sets selected from the group consisting of:

The "multiplex PCR" refers to a method of amplifying a plurality of target genes in the same reaction solution by using a plurality of primer pairs simultaneously.

Unlike the conventional PCR, the “primer set” can simultaneously amplify Pru p 2.01A, Cyclophilin, Pectin methylesterase inhibitor, and Mdtl1 through a single PCR reaction.

By analyzing the Pru p 2.01A, Cyclophilin, Pectin methylesterase inhibitor and Mdtl1 at once, it is possible to simultaneously determine the presence of allergen-causing fruits, tomatoes, apples, peaches and kiwi.

The design of the primer set is subject to various constraints such as A, G, C, T content ratio, preventing the formation of primer conjugate (dimer), prohibition of repeating three or more times of the same base sequence. In addition, conditions such as template DNA, primer concentration, dNTP concentration, Mg ²⁺ concentration, reaction temperature and reaction time should be appropriate in a single PCR reaction condition.

In the multiplex PCR, two or more genes in a target sample may be identified at a time by performing PCR simultaneously by mixing two or more primer pairs.

In the primer design, the same conditions as in the case of single PCR are more strictly required. In addition, in order to distinguish the gene products that are amplified on the gel after completion of the reaction, the size of the gene product must be distinguished, respectively, the size constraints are followed. In setting the reaction conditions, since common reaction conditions for all primers to be PCR must be set at one time, a very difficult condition setting process is required compared to a single PCR.

The primer is a short nucleic acid sequence comprising a free 3 'hydroxyl group, which may form a base pair and a base pair of complementary nucleic acids, and perform strand copying of the nucleic acid template. Can serve as a starting point for

The primers can initiate DNA synthesis in the presence of reagents for polymerization and four different nucleoside triphosphates at appropriate buffers and temperatures, and hybridize with nucleic acids present in the target material to synthesize specific genes of the target material. Can be used to amplify.

The primer may preferably be a single chain in consideration of the efficiency of amplification, may be a deoxyribonucleotide (deoxyribonucleotide).

The primers can include naturally occurring dNMPs (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer may also include ribonucleotides.

Hybridization means that two single stranded nucleic acids form a duplex structure by pairing complementary base sequences.

Such hybridization may occur when perfect complementarity between single stranded nucleic acid sequences occurs or when some mismatch base is present. The degree of complementarity required for the hybridization may vary depending on the hybridization reaction conditions, and in particular, may be controlled by temperature.

In general, if the hybridization temperature is high, the hybridization is likely to occur in a perfect match, and if the hybridization temperature is low, hybridization may occur even if some mismatches exist.

The primer set may be hybridized to a specific gene present in the target raw material, and if the desired raw material is present in the sample, the amplified product is amplified because the nucleotide sequence of the characteristic gene is heavy by a polymerase chain reaction. The presence or absence of the raw material can be determined through.

The primers may incorporate additional features that do not change the basic properties. That is, the nucleic acid sequence can be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution of one or more homologs of nucleotides and uncharged linkages, such as phosphonates, phosphoresteres, phosphoramidates or carbamates, or phosphorothioates or phosphorodithioates. Modification of the nucleotides to charged linkers, etc. is possible. Nucleic acids may also include one or more of nucleases, toxins, antibodies, signal peptides, proteins such as poly L-lysine, insertion agents such as acridine or psoralen, chelating agents such as metals, radioactive metals, iron oxidizing metals, and alkylating agents. It may have additional covalently linked residues.

The primer sequence can also be modified using a label that can provide a detectable signal directly or indirectly. The primer can include a label that can be detected using spectroscopy, photochemistry, biochemistry, immunochemistry or chemical means. Useful labels include ³² P, fluorescent dyes, electron dense reagents, enzymes (generally used in ELISAs), biotin or hapten and proteins for which antiserum or monoclonal antibodies are available.

The primers were subjected to cloning and restriction enzyme digestion of appropriate sequences and to phosphoester ester methods such as Narang (1979, Meth, Enzymol. 68: 90-99), and diethylphosphoramidate methods such as Beaucage. (1981, Tetrahedron Lett. 22: 1859-1862), and any other well-known methods, including direct chemical synthesis, such as the solid support method of US Pat. No. 44,580,66.

The polymerase chain reaction may be performed according to general reaction conditions using a conventional PCR device known in the art, or may be performed by some modification.

The primer set for detecting the Pru p 2.01A gene may be composed of a nucleotide sequence represented by SEQ ID NO: 1 and a nucleotide sequence represented by SEQ ID NO: 2.

The primer set for detecting the Cyclophilin gene may be composed of a nucleotide sequence represented by SEQ ID NO: 3 and a nucleotide sequence represented by SEQ ID NO: 4.

The primer set for detecting the Pectin methylesterase inhibitor gene may be composed of a nucleotide sequence represented by SEQ ID NO: 5 and a nucleotide sequence represented by SEQ ID NO: 6.

The primer set for detecting the Mdtl1 gene may be composed of a nucleotide sequence represented by SEQ ID NO: 7 and a nucleotide sequence represented by SEQ ID NO: 8.

The multiplex PCR primer set may detect tomatoes, apples, peaches, and kiwi which are allergens.

According to another aspect of the invention, there is provided a method for detecting allergen-induced food comprising a; performing a polymerase chain reaction (PCR) using the primer set.

The primer set can effectively detect the target material through the polymerase chain reaction.

The "Polymerase Chain Reaction (PCR)" is a method well known in the art as a method of amplifying exponentially the starting nucleic acid material by synthesizing a nucleic acid using a polymerase in a chain, a specific nucleic acid of a specific nucleic acid This concept includes both qualitative analysis PCR to confirm the presence, quantitative PCR to measure the amount of specific nucleic acid, and real-time PCR that enables qualitative and quantitative analysis by tracking the PCR process in real time. The polymerase chain reaction may be performed according to general PCR reaction conditions using conventional PCR equipment known in the art, or may be carried out with some modifications.

The detection method may be performed by amplifying a specific DNA using the primer set, and then checking the amplification reaction of the specific DNA. The checking of the amplification of the specific DNA is performed by electrophoresis on the PCR reaction product. It may be carried out by confirming whether or not match the size of the expected DNA product.

The "amplification reaction" refers to a reaction for amplifying a nucleic acid molecule. Various amplification reactions are widely reported in the art, for example, polymerase chain reaction (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed) Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction (LCR), Gap-LCR (WO 90). / 01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (WO 88/10315), self-sustained sequence replication (WO 90 / 06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CPPCR) (US Pat. No. 4,437,975) ), Random priming Arbitrarily primed polymerase chain reaction (APPCR) (US Pat. Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA) (US Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification and loop-mediated isothermal amplification; LAMP), a real-time polymerase chain reaction is known, and an amplification reaction can be performed through a multiplex polymerase chain reaction.

The multiple polymerase chain reaction can be economical and time can be saved because a variety of samples can be confirmed by one amplification reaction.

The sample of the template DNA used in the detection method can be obtained from all foods in which allergenic foods can be found, preferably processed foods mixed with various food ingredients.

The template DNA may be obtained through a DNA extraction method commonly used in the art, such as alkaline extraction, hot water extraction, column extraction, and phenol / chloroform extraction, but is not limited thereto.

The detection method can detect a target DNA of 0.4 ng / μL or more concentration.

The inventors have optimized primer sequences for target gene detection, and the primer sets are highly sensitive and can effectively amplify even very low levels of target DNA, such as 0.4 ng / μL of target DNA.

According to another aspect of the present invention, there is provided an allergen-inducing food detection kit comprising the primer set, and a PCR reaction mixture.

The PCR reaction mixture may include reaction buffer, dNTP and DNA polymerase, and may include all reagents available for PCR reaction by those skilled in the art.

Hereinafter, with reference to the accompanying drawings will be described in detail with respect to embodiments of the present invention, it is apparent that the present invention is not limited by the following examples.

Example 1 Primer Design

To detect allergen-induced foods, primers specific to the DNA sequences of peach, tomato, kiwi and apple allergens were designed using the Primer Designer Program (Version 3.0; Scientific and Educational Software, USA).

For DNA sequences of peach, tomato, kiwi and apple allergens, refer to the Genebank sequence registered with the Gene Bank (www.ncbi.nlm.nih.gov) operated by NCBI under the National Institutes of Health.

Similar sequences were collected by BLAST (www.ncbi.nlm.nih.gov/BLAST/) analysis and specific primers were designed in consideration of Tm values and G + C content (Table 1).

SEQ ID NO: Primer Classification Gene name Size (bp) Genbank No. detection One Pru p 2.201-F Pru p 2.01A 209 EU424257.1 peach 2 Pru p 2.201-R 3 Sola 5-F Cyclophilin 146 M55019.1 tomato 4 Sola 5-R 5 Act 6-F Pectin methylesterase inhibitor 127 AB091088.1 Kiwi 6 Act 6-R 7 Mal 2-F Mdtl1 105 AF090143 Apple 8 Mal 2-R 9 18S-F 18S rRNA 172 AJ272181.1 plant 10 18S-R

Pru p 2.01A (Genbank accession No. EU424257.1) encodes peach allergen (Pru p 2.01A). The presence of Pru p 2.01A can detect peach allergens added to foods and the size of amplified products amplified by Pru p 2.201-F / R is 209 bp.

Cyclophilin (Genbank accession No. M55019.1) encodes tomato allergen (Cyclophilin). The presence of cyclophilin can detect tomato allergens added to foods and the size of the amplified product amplified by Sola 5-F / R is 146 bp.

Pectin methylesterase inhibitor (Genbank accession No. AB091088.1) encodes a kiwi allergen (Pectin methylesterase inhibitor). The presence of a pectin methylesterase inhibitor can detect kiwi allergens added to foods and the size of the amplified product amplified by Act 6-F / R is 127 bp.

Mdtl1 (Genbank accession No. AF090143) encodes apple allergen (Mdtl1). The presence of Mdtl1 can detect apple allergens added to foods and the size of amplified products amplified by Mal 2-F / R is 105 bp.

18S rRNA (Genbank accession No. AJ272181.1) encodes 18S rRNA, and the size of the amplification product amplified by 18S-F / R is 172 bp.

The primers were designed up to about 200 bp for effective application to processed foods. 18S rRNA primer sets were designed as internal controls to confirm failure of the PCR reaction.

Example 2 Sample Preparation

To evaluate the performance of the primer set, wheat, soy, buckwheat, tomato, apple, peach, kiwi, pine nut, walnut, peanut, sesame, corn, rice, barley, potato, pepper, cherry, plum, pistachio, almond Twenty-three fruits and vegetables from all over the country were selected, including cashew nuts, hazelnuts and perilla. Each sample was obtained from the domestic market, homogenized with liquid nitrogen, and stored at -20 ° C.

A total of nine target foods, including juice, dried fruit and fruit powder of each allergenic fruit vegetable, were obtained from the domestic market.

1 g of each sample was extracted with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Tomato, peach, kiwi, apple, cherry, and plum samples were collected by the CTAB method.

The remaining samples used a Qiagen kit. The Qiagen Kit method was modified to add 500 ml of chloroform and centrifuged for 15 minutes at 13,000 rpm. Centrifuged supernatants were loaded on a DNeasy Mini Spin Column.

All processed food samples were extracted by CTAB method, and DNA concentration and purity were measured by UV / VIS Spectrophotometer (Mecasys, Korea).

Example 3 Simplex and Multiplex PCR Conditions

Simplex PCR Condition

Simplex and multiplex PCR were performed with the primer set of Example 1.

PCR composition was mixed with 2.5 μl of 10X Ex Taq buffer, 200 μM of dNTP mix, 0.5 U of Ex Taq polymerase (TaKaRa, Japan), 400 nM forward and reverse primer, and 50 ng template DNA.

Using GeneAtlas (ASTEC, Japan) the reaction mixture at 94 ° C. for 5 minutes; (94 degreeC 30 second, 60 degreeC 30 second, 72 degreeC 30 second) It amplified by 40 times, 72 degreeC 10 minutes. Those without template DNA were set as negative controls.

PCR products were stained with EtBr (ethidium bromide) and electrophoresed in 0.5X Tris-acetate-EDTA buffer 2% agarose gel.

Multiplex PCR Condition

Multiplex PCR conditions were optimized for primer concentration and annealing temperature. Multiplex PCR reaction composition is shown in Table 2.

reagent Final concentration volume Sample DNA 50 ng 1 μL Distilled water - Up to 25 μL 10X PCR buffer (MgCl ₂ added) 1X 2.5 μL dNTP 200 μM 2 μL Tap DNA polymerase 1 U 1 μL Pru p 2.201-F / R
(Peach primer set) 800 nM 1 μL Sola 5-F / R
(Tomato primer set) 400 nM 1 μL Act 6-F / R
(Kiwi primer set) 1200 nM 1 μL Mal 2-F / R
(Apple primer set) 200 nM 1 μL 18S rRNA-F / R 400 nM 1 μL

Using GeneAtlas (ASTEC, Japan) the reaction mixture at 94 ° C. for 5 minutes; (94 degreeC 30 second, 62 degreeC 30 second, 72 degreeC 30 second) It amplified by 40 times, 72 degreeC 10 minutes (Table 3). Those without template DNA were set as negative controls.

Temperature time Cycle number 94 ℃ 5 minutes One 94 ℃ 30 seconds 40 62 ℃ 20 seconds 72 ℃ 30 seconds 72 ℃ 7 minutes One

PCR products were stained with EtBr (ethidium bromide) and electrophoresed in 0.5X Tris-acetate-EDTA buffer 3% agarose gel.

Example 4 PCR Product Sequencing

The PCR product amplified with the primer set of Example 1 was purified from 1% agarose gel using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA), and pGEM-T easy vector (Promega) according to the manufacturer's protocol. , Madison, WI, USA). After ligation, each plasmid was inserted into E. coli .

DNA-spinTM Plasmid DNA Purification Kit (Intron Biotechnology, Seongnam, Korea) was extracted and sequenced.

Sequence analysis was entrusted to Genotech (Genotech, Korea), and the results were analyzed by BLSAT search of NCBI database.

Experimental Example 1: Confirmation of specificity through Simplex PCR

Before optimizing Multiplex PCR, Simplex PCR was performed to confirm the specificity of the designed primers.

Wheat (1), soybeans (2), buckwheat (3), tomatoes (4), apples (5), peaches (6), kiwi (7), pine nuts (8) under Simplex PCR conditions set in Example 3 , Walnut (9), Peanut (10), Sesame (11), Corn (12), Rice (13), Barley (14), Potato (15), Pepper (16), Cherry (17), Plum (18) 23 kinds of foods including pistachios (19), almonds (20), cashew nuts (21), hazelnuts (22), and perilla (23) were analyzed.

As a result, sequencing revealed bands of 209, 146, 127 and 105 bp sizes, which are expected products of each primer set (FIG. 1).

Experimental Example 2 Multiplex PCR Optimization

In consideration of the results of the Simplex PCR of Experimental Example 1, the concentration, amplification temperature and time of each primer set were optimized.

Primer concentration and amplification temperature were set to control the intensity of the band and the presence of unintentional bands due to cross reaction and competition between the primers. Generally, in order to optimize Multiplex PCR, the amplification temperature should be lowered by 4 to 6 ° C, but the optimization temperature can be considered when there is a possibility of finding an independent band.

Among the amplification temperature range of 60 ~ 63 ℃ 62 ℃ was the most effective amplification time, 20 seconds was the most effective.

The concentration of each primer set was set such that each band had a similar intensity.

Tomato (2), apple (3), peach (4) and kiwi (5) were analyzed under the Multiplex PCR conditions set in Example 3 (FIG. 2). The gDNA mixed with the DNA of tomato, apple, peach and kiwi was set to the positive control group (1), and to the negative control group (N) containing no template DNA.

Referring to FIG. 2, specific bands of 146, 105, 209, and 127 bp sizes of tomatoes, apples, peaches, and kiwis, respectively, were found, thereby distinguishing four allergenic fruits.

Experimental Example 3: Confirmation of specificity and sensitivity of Multiplex PCR

In order to confirm the specificity of the Multiplex PCR set in Example 3, wheat (2), soybean (3), buckwheat (4), tomato (5), apple (6), Peach (7), Kiwi (8), Pine Nut (9), Walnut (10), Peanut (11), Sesame (12), Corn (13), Rice (14), Barley (15), Potato (16) , Pepper (17), cherry (18), plum (19), pistachio (20), almond (21), cashew nut (22), hazelnut (23) and perilla (24) were analyzed (FIG. 3).

The gDNA mixed with the DNA of tomato, apple, peach and kiwi was set to the positive control group (1), and to the negative control group (N) containing no template DNA.

Referring to FIG. 3, 18S rRNA bands were found in all samples and specific bands of 146, 105, 209 and 127 bp sizes were found in tomatoes, apples, peaches and kiwis, respectively, but not in the remaining samples.

The results suggest that it is possible to specifically detect allergens of tomatoes, apples, peaches and kiwis under the Multiplex PCR condition.

Sensitivity test of Multiplex PCR was used by diluting gDNA mixed with DNA of tomato, apple, peach and kiwi by 50, 10, 2, 0.4, 0.08, 0.016, 0.0032 ng concentration (Fig. 4).

Referring to Figure 4, tomatoes, apples, peaches and kiwi were detected at 0.4 ng concentration. However, some bands were not detected at concentrations below 0.08 ng. Based on FIG. 5, the detection limit DNA concentration of Multiplex PCR is 0.0032 ng for 18S rRNA, 0.016 ng for peach and apple, and 0.4 ng for tomato and kiwi.

Experimental Example 4: Application to Commercial Foods

Four juices, four dried fruits and one fruit powder were analyzed by the Multiplex PCR conditions (Table 4).

division Allergens contained Food form Test result Food 1 tomato juice tomato Food 2 Dry fruit tomato Food 3 Apple juice Apple Food 4 Dry fruit Apple Food 5 Dry fruit Apple Food 6 Fruit powder Apple Food 7 peach juice peach Food 8 Dry fruit peach Food 9 Kiwi juice Kiwi

Referring to Table 4, allergens contained in foods 1 to 9 were distinguishable, and 18S rRNA was amplified in all samples.

The results suggest that the multiplex PCR primer set for detecting allergen-induced fruit and vegetable of the present invention can be a useful tool for detecting allergens of various foods by PCR technique.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the invention is indicated by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the invention.

<110> Kyung Hee University <120> MELTIPLEX PCR PRIMERS FOR DETECTION OF FRUITS AND VEGETABLES          CAUSING ALLERGY, AND USE THEREOF <130> GKH17P-0222-KR <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> Pru p 2.201-F <400> 1 gcaaccggaa ttagcaac 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pru p 2.201-R <400> 2 aaatcttgac ccccgttctc 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Sola 5-F <400> 3 ggagccaaat tcaacgatg 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Sola 5-R <400> 4 acgacgtgct ttccgttga 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Act 6-F <400> 5 aaatctgtcc caaaactcgc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Act 6-R <400> 6 ttagcactgg cctgagctat 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mal 2-F <400> 7 cttgccttgc gtttggtgat 20 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mal 2-R <400> 8 ggcactgctt ctcaaagatc tca 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18S-F <400> 9 cgaaagcatt tgccaaggat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18S-R <400> 10 ccggaaccca aagactttga 20

Claims (9)

A primer set for multiplex PCR consisting of a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 2 that bind to the Pru p 2.01A gene;
A primer set for multiplex PCR consisting of a primer of SEQ ID NO: 3 and a primer of SEQ ID NO: 4 that binds to the Cyclophilin gene;
A primer set for multiplex PCR consisting of a primer of SEQ ID NO: 5 and a primer of SEQ ID NO: 6 that binds to a Pectin methylesterase inhibitor gene; And
A primer set for multiplex PCR consisting of a primer of SEQ ID NO: 7 and a primer of SEQ ID NO: 8 that binds to the Mdtl1 gene,
Multiplex PCR primer set for simultaneous detection of one or more allergenic fruit vegetables selected from the group consisting of tomato, apple, peach and kiwi. delete delete delete delete delete A method for detecting allergenic foods comprising the step of performing a polymerase chain reaction (PCR) using the primer set of claim 1. An allergen-inducing food detection kit comprising the primer set of claim 1 and a PCR reaction mixture. The method of claim 8,
The PCR reaction mixture is an allergy-inducing food detection kit comprising a reaction buffer, dNTP and DNA polymerase.

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