CN109295250B - Detection kit and method for food-borne plant allergen components - Google Patents
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Abstract
The invention discloses a detection kit and a detection method for food-borne plant allergen components, and relates to the technical field of transgenic detection. The detection kit comprises one or more combinations of the following primer pairs: 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16 and 17-18 SEQ ID NO; the kit can be used for detecting food-borne plant allergen components in a sample, and has high specificity and sensitivity and accurate and reliable detection results.
Description
Technical Field
The invention relates to the technical field of transgenic detection, in particular to a detection kit and a method for food-borne plant allergen components.
Background
Food allergy is an adverse reaction of people to food, and belongs to an allergic reaction of organisms to foreign substances. The mild food allergy can cause allergic symptoms of skin and gastrointestinal tract, and the severe food allergy can cause asthma attack, anaphylactic shock, life threatening and even death. Data from the world health organization indicate that 22% -25% of people worldwide currently suffer from allergic disease and increase at a rate of 23 times per 10 years, with food allergy accounting for the vast majority of allergic disease and becoming a global concern. There are 100-125 cases of food allergy death in the united states each year, the incidence of allergic reactions has increased manyfold in the united kingdom over the last decade, and food allergy occurs in up to 150 million people each year. Food allergy has been regarded as a serious public health problem in recent 20 years. Since there is no specific method for treating food allergy, strict avoidance of foods containing allergic ingredients is the most effective therapy. Some developed countries have legislated the problem of allergens in food. Developed countries of the European Union (directive 2003/89/EC), the United states (food allergen labeling and consumer protection laws, 2004), and Japan (food sanitation laws, 2002) have developed food labels for food use that require import and export by various regulations to clarify food components, so that the harm of allergens to patients can be effectively avoided. The recommended marking requirements when the food possibly contains the sensitizing substances are also specified in the food safety law of the people's republic of China and the general rules of prepackaged food labels in China. Food safety is the most critical component in food quality, and food allergy has become an important food safety issue among them. Meanwhile, as developed countries have made strict requirements on the identification of allergens in import and export foods, food allergens even become a barrier to import and export trade. Therefore, how to rapidly and accurately detect the allergen in food is one of the problems that needs to be solved at present.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a detection kit for food-borne plant allergen components, which can be used for simultaneously detecting common food-borne plant allergen components such as peanut, walnut, pistachio nut, sesame, hazelnut, soybean, almond, cashew nut, carrot and the like, and has the characteristics of high detection speed, high sensitivity, good specificity, high throughput, convenience in operation and the like.
The invention also aims to provide a method for detecting food-borne plant allergen components, which can simultaneously detect common food-borne plant allergen components such as peanut, walnut, pistachio nut, sesame, hazelnut, soybean, almond, cashew nut, carrot and the like, and has the characteristics of high detection speed, high sensitivity, good specificity, high throughput and the like.
The invention is realized by the following steps:
in one aspect, the invention provides a food-borne plant allergen component detection kit, which comprises a nucleic acid combination, wherein the nucleic acid combination comprises one or more of the following primer pairs for detecting food-borne plant allergen components:
a first primer pair for detecting peanut components, which comprises an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2,
a second primer pair for detecting walnut components, which comprises an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4,
a third primer pair for detecting pistachio nut components, which comprises an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6,
a fourth primer pair for detecting sesame components, which comprises an upstream primer shown in SEQ ID NO.7 and a downstream primer shown in SEQ ID NO.8,
a fifth primer pair for detecting the hazelnut component, comprising an upstream primer shown in SEQ ID NO.9 and a downstream primer shown in SEQ ID NO.10,
a sixth primer pair for soybean component comprising an upstream primer represented by SEQ ID NO.11 and a downstream primer represented by SEQ ID NO.12,
a seventh primer pair for detecting almond components, which comprises an upstream primer shown in SEQ ID NO.13 and a downstream primer shown in SEQ ID NO.14,
an eighth primer pair for detecting the cashew component, comprising an upstream primer shown by SEQ ID NO.15 and a downstream primer shown by SEQ ID NO.16,
a ninth primer pair for detecting carrot components, comprising an upstream primer shown by SEQ ID NO.17 and a downstream primer shown by SEQ ID NO. 18.
The primer pair in the detection kit provided by the invention can carry out multiple PCR on the 9 primer pairs in the same PCR reaction system through reasonable design, so that non-specific amplification is avoided, the detection sensitivity and specificity are improved, and the detection kit can simultaneously detect common food-borne plant allergen components such as peanut, walnut, pistachio nut, sesame, hazelnut, soybean, almond, cashew nut, carrot and the like.
For example, the detection method using the above-mentioned kit may be: taking total DNA of a food sample to be detected as a template, carrying out PCR amplification on the 9 pairs of specific primer combinations, respectively using target sequences of each pair of primers as specific target sequences of food-borne plant allergen components, simultaneously designing a multiple PCR amplification product to obtain probes, fixing each probe on a membrane chip, hybridizing the amplification product with the probes, developing color by using a developing solution, and observing a detection result by naked eyes or an imaging instrument. If the sample has corresponding allergen components, the amplified PCR product is hybridized with the corresponding probe and is observed by color development, so that the detection effect is realized.
Further, in some embodiments of the present invention, the above-mentioned nucleic acid combination further comprises an internal reference primer pair comprising an upstream primer shown by SEQ ID NO.19 and a downstream primer shown by SEQ ID NO. 20.
The internal reference primer pair can detect chloroplast tRNA-Leu (trnL) genes of plants, and comprises an upstream primer shown in SEQ ID NO.19 and a downstream primer shown in SEQ ID NO. 20.
Further, in some embodiments of the present invention, the kit further comprises a membrane chip on which one or more of the probes having the base sequences shown in SEQ ID nos. 21 to 29 are immobilized.
The amplification products corresponding to each probe are as follows:
SEQ ID NO.21 is a peanut probe hybridizable to the PCR product of the first primer pair;
SEQ ID No.22 is a walnut probe hybridizable to the PCR product of the second primer pair;
SEQ ID No.23 is a pistachio probe hybridizable to a PCR product of the third primer pair;
SEQ ID NO.24 is a sesame probe, which can hybridize with the PCR product of the fourth primer pair;
SEQ ID NO.25 is a hazelnut probe, which can hybridize with the PCR product of the fifth primer pair;
SEQ ID NO.26 is a soybean probe hybridizable with the PCR product of the sixth primer pair;
SEQ ID NO.27 is an almond probe hybridizable to the PCR product of the seventh primer pair;
SEQ ID NO.28 is a cashew probe, which can hybridize with the PCR product of the eighth primer pair;
SEQ ID NO.29 is a carrot probe that hybridizes to the PCR product of the ninth primer pair.
Further, in some embodiments of the present invention, an internal reference probe (trnL probe) is further immobilized on the membrane chip, and the base sequence thereof is shown in SEQ ID NO. 30. The internal reference probe can be hybridized and combined with a PCR product of the internal reference primer pair.
Further, in some embodiments of the present invention, one or both of the following control probes are further immobilized on the membrane chip: a positive control probe and a negative control probe;
the base sequence of the positive control probe is shown as SEQ ID NO. 31;
the base sequence of the negative control probe is shown as SEQ ID NO. 32.
Further, in some embodiments of the invention, the nucleic acid combination further comprises: a positive oligonucleotide single-stranded DNA molecule for hybridization and combination with the positive probe, wherein the base sequence of the positive oligonucleotide single-stranded DNA molecule is shown as SEQ ID NO. 33.
The positive oligonucleotide single-stranded DNA can bind to the positive probe, but not to the negative probe. Therefore, the position of the positive probe on the membrane chip is spotted, and the position of the negative probe is not spotted, so that the hybridization result is reliable, and the detection result is more reliable.
Further, in some embodiments of the present invention, the 5 'end of the upstream primer or the 5' end of the downstream primer in each primer pair is labeled with any one of digoxin, fluorescein isothiocyanate, and biotin.
The marker existing at the 5 'end of the upstream primer or the 5' end of the downstream primer can enable the amplification product to be provided with a corresponding marker, after the PCR product is combined with the probe, the amplification product is easily combined with catalytic enzyme provided with the corresponding marker through the marker, and then whether the corresponding amplification product exists at the position of the corresponding probe or not can be judged according to the color development condition of the catalytic enzyme catalysis substrate, so that the food-borne plant allergen in the sample can be judged to be classified.
When the label at the 5' -end of the primer is biotin, a color reaction can be performed using a catalytic enzyme (Streptavidin, SA) labeled with alkaline phosphatase. The biotin and streptavidin have extremely high affinity, and the reaction is highly specific. Each streptavidin molecule has 4 binding sites for biotin molecules, and thus streptavidin can bind biotin in a multivalent form at the same time. The biotin-labeled primer is used in combination with alkaline phosphatase-labeled streptavidin to cascade-amplify the detection signal, and finally the detection signal can be detected through color reaction or chemiluminescence.
Accordingly, when the label at the 5' -end of the primer is fluorescein isothiocyanate, a color reaction can be performed using a catalytic enzyme labeled with a fluorescein isothiocyanate antibody. When the 5' -end of the primer is digoxin, the color reaction can be performed by using a catalytic enzyme labeled with a digoxin antibody.
Further, in some embodiments of the present invention, the detection kit further comprises PCR reaction components and a compounding solution;
wherein the PCR reaction components comprise UNG enzyme, dNTPs, Taq DNA polymerase and Mg2+And a PCR buffer.
Uracil in DNA is removed by using uracil-N-glycosylase (UNG enzyme), dUTP and dTTP are mixed according to a certain proportion when PCR reaction liquid is prepared, so that an amplification product contains deoxyuracil, and the product is sensitive to the UNG enzyme, so that a newly prepared reaction system can be treated by the UNG enzyme before PCR, and aerosol pollution is avoided to a certain extent. dNTPs are used as raw materials of PCR reaction and applied to synthesizing a new DNA sequence by taking original DNA as a template; through the action of TaqDNA polymerase, a DNA chain is easier to open, so that the reaction is easier to perform; mg (magnesium)2+The activator of TaqDNA polymerase can activate the function of TaqDNA polymerase; the PCR reaction buffer solution enables the reaction to react in a stable environment, and avoids influencing the activity of TaqDNA polymerase and further influencing the reaction. The PCR reaction buffer solution enables the reaction to proceed smoothly.
In addition, the preparation solution comprises a deactivating solution, a deactivating cleaning solution, a hybridization cleaning solution, a catalytic enzyme solution, an enzyme labeling cleaning solution 1, an enzyme labeling cleaning solution 2, a developing solution and a developing cleaning solution.
Wherein the deactivating solution comprises 100mmol/L NaOH, and the deactivating cleaning solution comprises 2 XSSPE and 0.1% SDS; the hybridization solution comprised 2 XSSPE and 0.1% SDS; hybridization washes included 2 × SSPE and 0.5% SDS; the enzyme labeling solution comprises 2 xSSPE and 0.5% SDS; the enzyme-labeled cleaning solution 1 comprises 2 xSSPE and 0.5% SDS; the enzyme-labeled cleaning solution 2 comprises 1M Tris-HCl, pH9.5, 5M NaCl, 1M MgCl2。
The catalytic enzyme solution contains alkaline phosphatase, glucose oxidase or horseradish peroxidase, and is labeled with streptavidin, fluorescein isothiocyanate antibody or digoxin antibody. When in use, the catalytic enzyme solution is added into the enzyme labeling solution in a proper proportion and mixed.
SSPE (salt sodium phosphate EDTA, SSPE) buffer is a common nucleic acid hybridization buffer; the SSPE buffer solution comprises NaCl and NaH2PO4·H2O (or NaH)2PO4·2H2O) and EDTA-Na2。
The procedure for preparing 1L of SSPE buffer was as follows: dissolve 17.53g NaCl, 27.6g NaH with 800mL distilled water2PO4·H2O (or 31.2g of NaH)2PO4·2H2O) and 7.4g of EDTA-Na2Then, the pH was adjusted to 7.4 with 10M NaOH (about 6 mL), the volume was adjusted to 1L, and the mixture was filtered and autoclaved.
By using the various preparation solutions, impurities are removed, interference is eliminated, and results are more accurate, clear and reliable.
The color developing solution contains a substrate which is catalyzed by a catalytic enzyme to develop color.
When the catalytic enzyme is alkaline phosphatase, the chromogenic solution is a chemical chromogenic substrate NBT/BCIP of the alkaline phosphatase, containing 0.15mg/mL BCIP, 0.30mg/mL NBT, 100mmol/L Tris-HCl, 5mmol/L MgCl2, and the pH is 9.5); the color developing cleaning solution is double distilled water.
It should be noted that in other embodiments, alkaline phosphatase may be replaced by horseradish peroxidase.
When the catalytic enzyme is horseradish peroxidase, the substrate contained in the color development solution is any one of TMB (Tetramethylbenzidine ), ABTS (2,2' -Azinobis- (3-ethylbenzidine-6-sulfonate, 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) and OPD (o-Phenylenediamine ), wherein TMB, ABTS and OPD are substrates of horseradish peroxidase, and color development reaction is carried out under the catalytic action of horseradish peroxidase, so that the detection result is visualized.
Alternatively, glucose oxidase is used instead of alkaline phosphatase and the substrate is glucose. Glucose is oxidized by Glucose Oxidase (GOD) to produce gluconic acid and hydrogen peroxide, which can be detected by a horseradish peroxidase-TMB chromogenic system.
In a word, the detection kit can simultaneously detect common food-borne plant allergen components such as peanuts, walnuts, pistachios, sesames, hazelnuts, soybeans, almonds, cashews, carrots and the like, and has the characteristics of high detection speed, high sensitivity, good specificity, high throughput, convenience in operation and the like.
In another aspect, the present invention provides a method for detecting allergen components of food-borne plants, comprising: carrying out PCR amplification reaction on DNA of a sample to be detected;
the PCR reaction system of the PCR amplification reaction contains a reference primer pair and one or more of the following primer pairs:
a first primer pair for detecting peanut components, which comprises an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2,
a second primer pair for detecting walnut components, which comprises an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4,
a third primer pair for detecting pistachio nut components, which comprises an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6,
a fourth primer pair for detecting sesame components, which comprises an upstream primer shown in SEQ ID NO.7 and a downstream primer shown in SEQ ID NO.8,
a fifth primer pair for detecting the hazelnut component, comprising an upstream primer shown in SEQ ID NO.9 and a downstream primer shown in SEQ ID NO.10,
a sixth primer pair for soybean component comprising an upstream primer represented by SEQ ID NO.11 and a downstream primer represented by SEQ ID NO.12,
a seventh primer pair for detecting almond components, which comprises an upstream primer shown in SEQ ID NO.13 and a downstream primer shown in SEQ ID NO.14,
an eighth primer pair for detecting the cashew component, comprising an upstream primer shown by SEQ ID NO.15 and a downstream primer shown by SEQ ID NO.16,
a ninth primer pair for detecting carrot components, comprising an upstream primer shown by SEQ ID NO.17 and a downstream primer shown by SEQ ID NO. 18;
the 5 'end of the upstream primer or the 5' end of the downstream primer in each primer pair is labeled with any one of digoxin, fluorescein isothiocyanate and biotin.
Further, in some embodiments of the present invention, the molar ratio of the primer carrying the marker to the primer not carrying the marker in each primer pair is greater than 1 in the PCR reaction system.
The asymmetric PCR amplification technology is characterized in that unequal amounts of upstream primers and downstream primers (or the upstream primers and the downstream primers with different amplification extension conditions) are adopted in the PCR amplification process, a large amount of single-stranded DNA is generated after PCR amplification, and the single-stranded DNA can be effectively hybridized with corresponding probes fixed on a support membrane, so that the detection sensitivity is improved.
Further, in some embodiments of the present invention, the molar ratio of the primer carrying the label to the primer not carrying the label in each primer pair is 1.2 to 1.7 in the PCR reaction system.
Preferably, in some embodiments of the present invention, the molar ratio of the primer carrying the marker to the primer not carrying the marker in each primer pair in the PCR reaction system is 1.5.
Further, in some embodiments of the present invention, the PCR reaction system further comprises an internal reference primer pair comprising an upstream primer shown in SEQ ID NO.19 and a downstream primer shown in SEQ ID NO. 20. The 5 'end of the upstream primer or the 5' end of the downstream primer in the internal reference primer pair is marked with any one marker of digoxin, fluorescein isothiocyanate and biotin, and the type of the marker is the same as that of other detection primers.
Further, in some embodiments of the present invention, the PCR reaction system further comprises a positive oligonucleotide single-stranded DNA molecule for hybridization binding with the positive probe, the base sequence of the positive oligonucleotide single-stranded DNA molecule is shown as SEQ ID NO. 33. The 5' end of the positive oligonucleotide single-stranded DNA molecule is marked with any one of digoxin, fluorescein isothiocyanate and biotin, and the type of the marker is the same as that of other detection primers.
Further, in some embodiments of the invention, after the PCR amplification reaction, the method further comprises: a hybridization step;
the hybridization step comprises: reacting a PCR reaction product obtained by the PCR amplification reaction with a membrane chip, and carrying out hybridization treatment;
wherein one or more probes with base sequences shown as SEQ ID NO.21-29 are fixed on the membrane chip.
Loading a probe on a membrane chip, hybridizing the probe and a PCR product, and then developing color through the probe and a marker marked on a primer; the experimental result can be directly judged by naked eyes through the color reaction; the method is simple and easy to implement.
Further, in some embodiments of the present invention, the membrane chip is selected from any one of a nitrocellulose membrane, a nylon membrane, and a thin film having a three-dimensional pore size structure.
One of a nitrocellulose membrane, a nylon membrane or a film with a three-dimensional pore structure is selected as a supporting membrane, so that a probe is conveniently loaded on the membrane to prepare a membrane chip; for example, the membrane with the three-dimensional pore structure can conveniently adsorb and fix the probe molecules on the membrane, thereby facilitating the subsequent reaction.
Further, in some embodiments of the present invention, an internal reference probe having a base sequence shown in SEQ ID NO.30 is immobilized on the membrane chip.
Further, in some embodiments of the present invention, a positive control probe having a base sequence represented by SEQ ID NO.31 and a negative control probe having a base sequence represented by SEQ ID NO.32 are immobilized on the membrane chip.
In a word, the detection method can simultaneously detect common food-borne plant allergen components such as peanut, walnut, pistachio nut, sesame, hazelnut, soybean, almond, cashew nut, carrot and the like, and has the characteristics of high detection speed, high sensitivity, good specificity, high throughput, easy observation of results and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic view showing the positions of probes immobilized on a membrane chip in example 1 of the present invention.
FIG. 2 shows the results of the test in example 3 of the present invention.
FIG. 3 shows the results of the test in example 4 of the present invention.
FIG. 4 shows the results of the test in example 5 of the present invention.
FIG. 5 shows the results of the test in example 6 of the present invention.
FIG. 6 shows the results of the detection in example 7 of the present invention.
FIG. 7 shows the results of the test in example 8 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The detection kit for food-borne plant allergen components provided by the embodiment comprises a nucleic acid combination and a membrane chip, wherein the nucleic acid combination comprises 10 primer pairs and positive oligonucleotide single-stranded DNA, and each primer pair is as follows:
a first primer pair for detecting peanut components, which can amplify a peanut Arah3 gene:
it comprises an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO. 2;
a second primer pair for detecting walnut components, which can amplify walnut Jug r2 gene:
it comprises an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO. 4;
a third primer pair for detecting pistachio nut components, which can amplify the intergenic region sequence of 18S rRNA and 5.8S rRNA of pistachio nuts:
it comprises an upstream primer shown as SEQ ID NO.5 and a downstream primer shown as SEQ ID NO. 6;
a fourth primer pair for detecting sesame components, which amplifies a sesame 2S albumin mRNA gene sequence:
it comprises an upstream primer shown as SEQ ID NO.7 and a downstream primer shown as SEQ ID NO. 8;
a fifth primer pair for detecting the components of hazelnuts, which amplifies the hazelnut oleosin gene:
it comprises an upstream primer shown as SEQ ID NO.9 and a downstream primer shown as SEQ ID NO. 10;
and a sixth primer pair for detecting soybean components, which is used for amplifying soybean Lectin genes:
it comprises an upstream primer shown as SEQ ID NO.11 and a downstream primer shown as SEQ ID NO. 12;
and a seventh primer pair for detecting almond components, which is used for amplifying the almond Pru dul gene:
it comprises an upstream primer shown as SEQ ID NO.13 and a downstream primer shown as SEQ ID NO. 14;
the eighth primer pair for detecting the cashew component is used for amplifying the cashew ana o 3 gene:
it comprises an upstream primer shown as SEQ ID NO.15 and a downstream primer shown as SEQ ID NO. 16;
a ninth primer pair for detecting carrot components, which is used for amplifying the carrot afp gene;
it comprises an upstream primer shown as SEQ ID NO.17 and a downstream primer shown as SEQ ID NO. 18.
In addition, the internal reference primer pair aiming at the trnL gene of the plant chloroplast comprises an upstream primer shown in SEQ ID NO.19 and a downstream primer shown in SEQ ID NO. 20.
The base sequence of the positive oligonucleotide single-stranded DNA is shown in SEQ ID NO. 33.
The 5 'end of the downstream primer and the 5' end of the positive oligonucleotide single-stranded DNA in each primer pair carry biotin labels.
It should be noted that, in other embodiments, the nucleic acid combination may include one or two or three or four or five or six or seven or eight of the first to ninth primer pairs, which may be selected according to the detection requirement, and all fall within the protection scope of the present invention.
Probes were immobilized on the membrane chip, and the probes included 12 types, and the base sequences (5 '-3') of the probes were as follows:
the 12 kinds of probes were immobilized at the corresponding positions on the support film in the order of the positions shown in FIG. 1. In other embodiments, the order of immobilization of the probes may be different from that of the present embodiment.
The film chip manufacturing method can refer to the following method:
printing a table on the membrane by using a printer, soaking the nylon membrane in 50mL of PBS (phosphate buffer solution) containing 0.5-5% (volume: volume) of glutaraldehyde, and incubating for 2 hours by slowly swinging a shaking table; the membrane was then washed 4 times in PBS, 50mL each for 5 minutes; finally, the film is put in the air for drying and sealed for storage.
With simultaneous 0.5M NaHCO3Adjusting the concentration of the probe (the 5' end of which is modified by amino) to 10 mu M at pH8.4, and respectively diluting the positive control probe and the negative control probe to 5 mu M; and spotting the detection probes, the positive control probes and the negative control probes on a nylon membrane in sequence. And (3) placing the film chip with the dotted probe into an oven, and baking for 2 hours at 80 ℃ for later use.
FIG. 1 shows a schematic diagram of the probe position of the membrane chip, in which PC represents a Positive Control (Positive Control); NC denotes a Negative Control (Negative Control).
It should be noted that, in other embodiments, the probes immobilized on the membrane chip may be one or more of the above 12 probes, which may be selected according to the detection requirement, and all fall within the protection scope of the present invention.
Example 2
The method for detecting the food-borne plant allergen components by adopting the detection kit in the embodiment 1 comprises the following specific steps:
2.1 extracting genome DNA of a sample to be detected, and determining the concentration of the sample nucleic acid by using a trace nucleic acid determinator to obtain a DNA template of the sample to be detected.
2.2 multiplex PCR System and conditions
Reaction system of PCR amplification: 10 XPCR Buffer (containing Mg)2+5 μ L of UNG enzyme;
dNTP(2.5mM each)：5μL；
biotin-labeled downstream primer (20. mu.M): 1.5 mu L; upstream primer (20 μ M): 1 mu L of the solution; (Note: 13 primer pairs (provided in example 1) were added, 1.5. mu.L of the downstream primer and 1. mu.L of the upstream primer for each primer pair;
DNA template of the sample to be tested: 100 ng;
positive oligonucleotide DNA ((20. mu.M): 0.01. mu.L;
add ddH2O to a total volume of 50. mu.L.
And carrying out PCR (polymerase chain reaction) cyclic amplification on the reaction system, wherein the PCR cyclic conditions are as follows, the reaction process consists of pre-denaturation and multiple PCR cycles, and the conditions are as follows:
UNG enzyme digestion reaction: at 37 ℃ for 5 minutes
Multiplex PCR:
pre-denaturation: the temperature is 95 ℃, and the time is 3 minutes; and (3) PCR circulation: 35 cycles consisting of: denaturation: the temperature is 95 ℃, and the time is 15 seconds; annealing: the temperature is 55 ℃, and the time is 30 seconds; extension: the temperature is 72 ℃, and the time is 15 seconds; and finally, extension: the temperature is 72 ℃, and the time is 3 minutes; and (3) storage: the temperature was 4 ℃.
2.3 Membrane chip hybridization:
after the reaction is finished, the PCR product is thermally denatured at 95 ℃ for 5 minutes and is ice-cooled for more than 5 minutes. And adding 20 mu L of the cooled PCR product into the hybridization solution, performing membrane chip spot hybridization detection, and sequentially and automatically completing the steps of deactivation, deactivation cleaning, hybridization cleaning, enzyme labeling cleaning, color development cleaning and the like, thereby performing membrane chip hybridization detection. The hybridization process was as follows:
(1) the membrane chip (provided in example 1) with the immobilized probe was placed in a hybridization cassette with the chip label facing up, 1mL of a deactivating solution (100mmol/L NaOH) was added, and incubation was carried out at 37 ℃ for 8 min;
(2) adding 1mL of deactivating detergent (2 XSSPE, 0.1% SDS), and incubating at 60 deg.C for 5 min;
(3) removing the deactivation cleaning solution (2 XSSPE, 0.1% SDS) by suction, adding 1mL of hybridization system solution (the product after PCR is denatured and then added into the hybridization solution (2 XSSPE, 0.1% SDS) for mixing), and incubating for 45min at 45 ℃ by a horizontal shaking table at 90 rpm;
(4) absorbing hybridization system liquid, adding 1mL of preheated hybridization cleaning liquid (2 xSSPE, 0.5% SDS), shaking and cleaning at 52 ℃ for 5min at 90rpm of a horizontal shaking table, and washing for 2 times;
(5) removing the hybridization cleaning solution by suction, adding preheated enzyme labeling solution (streptavidin-labeled alkaline phosphatase (1mg/mL) and adding the enzyme labeling solution into 999.5 mu L of enzyme labeling solution (2 xSSPE, 0.5% SDS) according to the proportion of 1: 2000), horizontally shaking at 42 ℃ for 90rpm, and oscillating and incubating for 30 min;
(6) absorbing enzyme-labeled solution, adding 1mL of preheated enzyme-labeled cleaning solution 1(2 XSSPE, 0.5% SDS), shaking and cleaning at 42 ℃ for 5min at 90rpm in a horizontal shaking table, and washing for 2 times;
(7) adsorbing enzyme-labeled cleaning solution 1, adding preheated enzyme-labeled cleaning solution 2 (containing 0.3mol/L NaCl and 20mmol/L NaH)2PO4，20mmol/L C10H14N2Na2O8·2H2O, pH is 7.4), 1mL, shaking and cleaning for 5min at 37 ℃ by a horizontal shaking table at 90rpm, and washing for 2 times;
(8) removing the enzyme-labeled cleaning solution 2 by suction, adding 1mL of developing solution (a mixture containing BCIP/NBT, namely 5-bromo-4-chloro-3-indolylphosphoric acid and nitrotetrazolium chloride), and standing at room temperature for developing for 15 min.
(9) Absorbing the color development liquid, adding 1mL of deionized water, washing for 2 times at room temperature, and judging the result after the film chip is dried. The specific hybridization procedure is shown in Table 1.
TABLE 1 automated hybridization Instrument assay procedure
| Procedure | Reagent | Temperature (. degree.C.) | Time (min) |
| Deactivation | Deactivating liquid | 37 | 8 |
| Deactivating cleaning | Deactivating cleaning fluid | 60 | 5 |
| Hybridization of | Hybrid liquid | 45 | 45 |
| Hybrid cleaning | Hybridization cleaning solution | 52 | 5 |
| Hybrid cleaning | Hybridization cleaning solution | 52 | 5 |
| Enzyme label | Enzyme labeling liquid | 42 | 30 |
| Enzyme-labeled cleaning 1 | Enzyme-labeled cleaning solution 1 | 42 | 5 |
| Enzyme-labeled cleaning 1 | Enzyme-labeled cleaning solution 1 | 42 | 5 |
| Enzyme-labeled cleaning 2 | Enzyme-labeled cleaning solution 2 | 37 | 5 |
| Enzyme-labeled cleaning 2 | Enzyme-labeled cleaning solution 2 | 37 | 5 |
| Color development | Color developing liquid | 37 | 15 |
| Color development cleaning | Deionized water | 37 | 5 |
| Color development cleaning | Deionized water | 37 | 5 |
Wherein: the deactivating solution comprises 100mmol/L NaOH and the deactivating wash comprises 2 XSSPE and 0.1% SDS; the hybridization solution comprised 2 XSSPE and 0.1% SDS; hybridization washes included 2 × SSPE and 0.5% SDS; the enzyme labeling solution comprises 2 xSSPE and 0.5% SDS; the enzyme-labeled cleaning solution 1 comprises 2 xSSPE and 0.5% SDS; the enzyme-labeled cleaning solution 2 comprises 1M Tris-HCl, pH9.5, 5M NaCl, 1M MgCl2。
SSPE (salt sodium phosphate EDTA, SSPE) buffer is a common nucleic acid hybridization buffer; the SSPE buffer solution comprises NaCl and NaH2PO4·H2O (or NaH)2PO4·2H2O) and EDTA-Na2。
The procedure for preparing 1L of SSPE buffer was as follows: dissolve 17.53g NaCl, 27.6g NaH with 800mL distilled water2PO4·H2O and 7.4g of EDTA-Na2Adjusting pH to 7.4 with 10M NaOH solution (about 6 mL), diluting to 1L, filtering, and concentratingAnd (5) steam pressing sterilization.
The color development liquid is an alkaline phosphatase chemical color development substrate NBT/BCIP, and contains 0.15mg/mL BCIP, 0.30mg/mL NBT, 100mmol/L Tris-HCl, 5mmol/L MgCl2 and pH 9.5); the color developing cleaning solution is double distilled water.
(10) And (4) analyzing results:
in the actual sample detection process, blank control, negative control and positive control should be set, so as to improve the accuracy of the detection result.
a) Blank control: using water as a blank control sample as a template, and according to the result of the detection method, developing PC (positive quality control points), not developing NC (negative quality control points), and not developing other targets;
b) negative control: using negative control samples (plant DNA except peanut, walnut, pistachio nut, sesame, hazelnut, soybean, almond, cashew nut and carrot) as templates, and performing the detection method, wherein PC (positive quality control point) is developed, NC (negative quality control point) is not developed, and internal reference is developed;
c) positive control: using a positive control sample (DNA containing one or a mixture of a plurality of food-borne plant allergen components in peanut, walnut, pistachio nut, sesame, hazelnut, soybean, almond, cashew nut and carrot) as a template, and performing the detection method to obtain a result, wherein PC (positive quality control point) is developed, NC (negative quality control point) is not developed, internal reference is developed, and a corresponding target point is developed (for example, if the positive control sample contains peanut, the film chip peanut target point area is developed);
it is an effective experiment to satisfy the above three conditions at the same time, and then it can be judged whether the sample to be tested is a common food-borne plant allergen component (peanut, walnut, pistachio nut, sesame, hazelnut, soybean, almond, cashew nut, carrot, or a mixture thereof) according to the color rendering result.
It should be noted that: the result can be judged and read by naked eyes or by a scanner, so that errors caused by artificial subjective judgment are reduced. And counting the signal value of each target point by scanner interpretation software according to a large amount of data, setting the threshold value of each target point, and judging the target point to be positive if the signal value is greater than the threshold value and judging the target point to be negative if the signal value is less than the threshold value.
Example 3
The method of the embodiment 2 is adopted to detect the DNA of peanut, walnut, pistachio nut, sesame, hazelnut, soybean, almond, cashew nut and carrot seed or tuber purchased in the market as a template by the kit of the embodiment 1, and the result is shown in figure 2, and the result of figure 2 shows that each food-borne plant allergen component is detected, and the samples have no cross reaction with each other, which indicates that the kit of the embodiment 1 has better specificity.
Example 4
The same amount of genomic DNA of a mixed sample of peanut, walnut, pistachio nut, sesame, hazelnut, soybean, almond, cashew nut and carrot was extracted according to the instructions of the plant genomic DNA extraction kit (cat # DP305) of Tiangen Biochemical technology (Beijing) Ltd. Using this as a template, the method of example 2 was performed using the kit of example 1, and the results are shown in FIG. 3. from the results of FIG. 3, the sample was a plant and contained the plant allergens peanut, walnut, pistachio nut, sesame, hazelnut, soybean, almond, cashew nut and carrot. The kit of example 1 has good specificity and sensitivity, and can detect each component in a mixed sample.
Example 5
The pig genome DNA of the sample containing no food-borne plant allergen component was extracted according to the instructions of the plant genome DNA extraction kit (cat # DP305) of Tiangen Biochemical technology (Beijing) Ltd, and the detection was carried out by the method of example 2 using the kit of example 1 using this as a template, and the result is shown in FIG. 4, and it can be seen from the result of FIG. 4 that the sample contains no plant component.
Example 6
The genomic DNA of the biscuit sample was extracted according to the instructions of the plant genomic DNA extraction kit (cat # DP305) of Tiangen Biochemical technology (Beijing) Ltd. The results of the tests performed by the method of example 2 using the kit of example 1 as a template are shown in fig. 5, and it can be seen from the results of fig. 5 that the sample contains plant components, specifically, plant allergens peanut, walnut, pistachio, hazelnut, and soybean.
Example 7
Oat genomic DNA was extracted according to the instructions of the plant genomic DNA extraction kit (cat # DP305) of Tiangen Biochemical technology (Beijing) Ltd. Using this as a template, the test was carried out by the method of example 2 using the kit of example 1, and the results are shown in FIG. 6, from which it can be seen that the sample contained the plant components, but did not contain the plant allergen components listed in this chip, and the results were in accordance with the expected results.
Example 8
Extracting pistachio nut genome DNA according to the instruction of plant genome DNA extraction kit (cargo number: DP305) of Tiangen Biochemical technology (Beijing) Ltd. After the concentration measurement using a microanalyzer, the nucleic acid gradient was diluted to a concentration of 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, or 0.1 ng/. mu.L, and the detection was carried out by the method of example 2 using the kit of example 1 using this as a template, and the results are shown in FIG. 7. As can be seen from the results of the figure, the kit can detect the pistachio nucifera nucleic acid at the lowest level of 0.1 ng/. mu.L, namely the absolute sensitivity of the kit for detecting the pistachio nucifera nucleic acid can reach 0.1 ng/. mu.L.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Sichuan HuaHan, Sanchuang Biotech Co., Ltd
<120> detection kit and method for food-borne plant allergen components
<160> 33
<170> PatentIn version 3.5
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Claims (8)
1. A detection kit for food-borne plant allergen components is characterized by comprising a nucleic acid combination, wherein the nucleic acid combination is as follows:
a first primer pair for detecting peanut components, which comprises an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2,
a second primer pair for detecting walnut components, which comprises an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4,
a third primer pair for detecting pistachio nut components, which comprises an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6,
a fourth primer pair for detecting sesame components, which comprises an upstream primer shown in SEQ ID NO.7 and a downstream primer shown in SEQ ID NO.8,
a fifth primer pair for detecting the hazelnut component, comprising an upstream primer shown in SEQ ID NO.9 and a downstream primer shown in SEQ ID NO.10,
a sixth primer pair for soybean component comprising an upstream primer represented by SEQ ID NO.11 and a downstream primer represented by SEQ ID NO.12,
a seventh primer pair for detecting almond components, which comprises an upstream primer shown in SEQ ID NO.13 and a downstream primer shown in SEQ ID NO.14,
an eighth primer pair for detecting the cashew component, comprising an upstream primer shown by SEQ ID NO.15 and a downstream primer shown by SEQ ID NO.16,
a ninth primer pair for detecting carrot components, comprising an upstream primer shown by SEQ ID NO.17 and a downstream primer shown by SEQ ID NO. 18;
the kit also comprises a membrane chip, wherein a probe with a base sequence shown in SEQ ID NO.21-29 is fixed on the membrane chip.
2. The food-borne plant allergen component detection kit according to claim 1, wherein one or two of the following control probes are further immobilized on said membrane chip: a positive control probe and a negative control probe;
the base sequence of the positive control probe is shown as SEQ ID NO. 31;
the base sequence of the negative control probe is shown as SEQ ID NO. 32.
3. The food-borne plant allergen component detection kit according to claim 2, wherein said nucleic acid combination further comprises: a positive oligonucleotide single-stranded DNA molecule for hybridization and combination with the positive probe, wherein the base sequence of the positive oligonucleotide single-stranded DNA molecule is shown as SEQ ID NO. 33.
4. The food-borne plant allergen component detection kit according to any one of claims 1 to 3, wherein the 5 'end of the upstream primer or the 5' end of the downstream primer in each primer pair is labeled with any one of digoxin, fluorescein isothiocyanate and biotin.
5. A method for detecting allergen components of food-borne plants is characterized by comprising the following steps: carrying out PCR amplification reaction on DNA of a sample to be detected;
the PCR reaction system of the PCR amplification reaction contains the following primer pair combinations:
a first primer pair for detecting peanut components, which comprises an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2,
a second primer pair for detecting walnut components, which comprises an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4,
a third primer pair for detecting pistachio nut components, which comprises an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6,
a fourth primer pair for detecting sesame components, which comprises an upstream primer shown in SEQ ID NO.7 and a downstream primer shown in SEQ ID NO.8,
a fifth primer pair for detecting the hazelnut component, comprising an upstream primer shown in SEQ ID NO.9 and a downstream primer shown in SEQ ID NO.10,
a sixth primer pair for soybean component comprising an upstream primer represented by SEQ ID NO.11 and a downstream primer represented by SEQ ID NO.12,
a seventh primer pair for detecting almond components, which comprises an upstream primer shown in SEQ ID NO.13 and a downstream primer shown in SEQ ID NO.14,
an eighth primer pair for detecting the cashew component, comprising an upstream primer shown by SEQ ID NO.15 and a downstream primer shown by SEQ ID NO.16,
a ninth primer pair for detecting carrot components, comprising an upstream primer shown by SEQ ID NO.17 and a downstream primer shown by SEQ ID NO. 18;
the 5 'end of the upstream primer or the 5' end of the downstream primer in each primer pair is marked with any one marker of digoxin, fluorescein isothiocyanate and biotin;
after the PCR amplification reaction, the method further comprises: a hybridization step;
the hybridization step comprises: reacting a PCR reaction product obtained by the PCR amplification reaction with a membrane chip, and carrying out hybridization treatment;
wherein, a probe with a base sequence shown in SEQ ID NO.21-29 is fixed on the membrane chip.
6. The method for detecting allergen components of food-borne plants according to claim 5, wherein the molar ratio of the primer carrying said marker to the primer not carrying said marker in each primer pair is greater than 1 in the PCR reaction system.
7. The method of claim 6, wherein the molar ratio of the primer with the marker to the primer without the marker in each primer pair is 1.2-1.7 in the PCR system.
8. The method for detecting allergen components of food-borne plants according to claim 5, wherein said membrane chip is selected from the group consisting of nitrocellulose membrane, nylon membrane, and a membrane having a three-dimensional pore size structure.
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