CN114214452B - Chip kit for detecting plant-derived allergen components - Google Patents
Chip kit for detecting plant-derived allergen components Download PDFInfo
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- CN114214452B CN114214452B CN202111651658.5A CN202111651658A CN114214452B CN 114214452 B CN114214452 B CN 114214452B CN 202111651658 A CN202111651658 A CN 202111651658A CN 114214452 B CN114214452 B CN 114214452B
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention provides a chip kit for detecting plant-derived allergen components, which comprises a microfluidic chip, wherein the microfluidic chip is provided with a reaction tank, and is characterized in that the reaction tank comprises a reaction tank for detecting at least one of wheat allergen components, cashew nut allergen components, almond allergen components, carrot allergen components, peanut allergen components, sesame allergen components and celery allergen components, and LAMP amplification reaction is independently carried out in the reaction tank. The invention develops a microfluidic chip for simultaneously detecting various plant-derived components of food, realizes the rapid detection of various plant-derived allergen components, and provides a guarantee for the selection of allergen detection methods.
Description
Technical Field
The invention belongs to the technical field of food allergen component detection, and particularly relates to a chip kit for detecting plant-derived allergen components.
Background
The incidence of food allergy is continuously rising in the global scope, and the food allergy is one of the food safety problems which are increasingly concerned, so that the research on the food allergen detection method is of great significance.
The methods for detecting food allergen components commonly used at home and abroad at present are divided into two main types: immunological detection methods and nucleic acid detection methods. The nucleic acid detection technology has the characteristics of sensitivity, rapidness and accuracy, and the nucleic acid component of the food after deep processing still maintains certain stability, so the nucleic acid detection technology becomes a main detection technology.
Loop-mediated isothermal amplification (LAMP) is a novel technique for specifically, efficiently and rapidly amplifying nucleic acids at a constant temperature by using 4 specially designed primers capable of recognizing 6 sites on a target sequence and a DNA polymerase having strand displacement activity. The gene chip technology is to fix a large number of probe molecules on a support and then hybridize with the marked sample molecules, and the quantity and sequence information of the sample molecules are obtained by detecting the hybridization signal intensity of each probe molecule.
DNA has higher stability than protein, but the protein in food may be denatured by complicated food processing, such as heat treatment or baking, and the original sensitization is lost, so that the situation that the gene chip detects allergen gene but the food is not necessarily sensitized. For complex foods, the detection result of the gene chip is more information as a risk early warning, and other detection methods are needed to be combined to determine whether the sensitization exists.
Digital microfluidic technology, also known as chip technology, has numerous advantages in the field of life sciences research. Including their high potential in portability, can provide high throughput, high-automation detection platforms and significant reductions in rare or expensive reagent or sample consumption. Digital microfluidic technology has been applied to create immunoassay devices that greatly simplifies and expands complex experimental procedures by automatically delivering, mixing, incubating, and washing analytes on a chip.
The micro-fluidic chip technology is a novel gene chip technology, and provides a wide application prospect for detecting food allergens by the characteristics of multi-index parallel detection, uniform reaction of each detection hole, controllable result and easy automation. In microfluidic technology, how to achieve a more uniform distribution of samples and physical isolation between different reaction wells after distribution is two key technical points. In the prior art, a microfluidic chip, especially a disc-type microfluidic chip, has uneven distribution of samples to different reaction cavities in the high-flux detection process, and the samples can react immediately after being added, so that the control of the reaction process of the samples cannot be realized.
Food allergens are proteins and other related substances that are capable of generating an immune or allergic response to a specific population. Food allergy is an allergic reaction of the body to foreign substances. Food allergy has become an important food safety problem at present, and the incidence rate of allergic diseases tends to rise year by year. More than 20% of developed countries suffer from allergic diseases, about 1% of adults and 2.5% of children worldwide suffer from food allergies, causing more than 150 deaths per year and more than 2 tens of thousands of first aid treatments. There are currently about 160 foods containing food allergens that can cause allergic reactions, so it is imperative to develop a high throughput, high sensitivity, and rapid food allergen detection disc chip.
Disclosure of Invention
In view of the above, the invention provides a chip kit for detecting plant-derived allergen components, which applies a loop-mediated isothermal amplification technology and a microfluidic chip technology to the field of food allergen detection, realizes rapid, convenient, accurate and high-flux detection, develops a microfluidic chip for simultaneously detecting multiple food plant-derived components, realizes rapid detection of multiple plant-derived allergen components, and provides a guarantee for selection of allergen detection methods.
The technical scheme of the invention is as follows:
the chip kit for detecting the plant-derived allergen component comprises a microfluidic chip, wherein the microfluidic chip is provided with a reaction tank; preferably, the microfluidic chip of the present invention is a butterfly microfluidic chip in the prior art.
The reaction tank comprises at least one of a wheat allergen component, a cashew nut allergen component, a almond allergen component, a carrot allergen component, a peanut allergen component, a sesame allergen component and a celery allergen component, and the LAMP amplification reaction is independently carried out in the reaction tank.
Further, the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the wheat allergen component is SEQ ID NO. 1 to SEQ ID NO. 6;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the cashew nut allergen components is SEQ ID NO 7 to SEQ ID NO 12;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the almond allergen component is SEQ ID NO. 13-18;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the carrot allergen components is SEQ ID NO. 19-24;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the peanut allergen components is SEQ ID NO. 25-SEQ ID NO. 30;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the sesame allergen component is SEQ ID NO. 31-36;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the celery allergen component is SEQ ID NO. 37-SEQ ID NO. 42.
According to the nucleic acid sequences of genes of wheat, cashew, peach kernel, carrot, peanut, sesame and celery published on NCBI, the conserved sequences in the gene sequences are found out through BLAST comparison in a database. Then, logging in a primer online design webpage of Japanese Rong Yan chemical company to design a plurality of specific lamp primers, wherein each set of primers comprises two outer primers F3 and B3, two inner primers FIP and BIP and two loop primers LoopF and LoopB. And screening each set of primers, wherein the primers comprise specificity, reaction time and reaction temperature, and finally, a set of primers with good specificity is obtained.
Further, the microfluidic chip also comprises a reaction tank of LAMP primer groups for blank control, negative control and amplification positive control.
Further, the blank control reaction well is not spotted;
the reaction tank of the negative control is replaced by double distilled water for the DNA template;
the reaction pool for amplifying the positive control is embedded with a DNA fragment containing shrimp mitochondrion 12SrRNA genes and an LAMP primer group thereof, and the primer group sequence is SEQ ID NO. 43-SEQ ID NO. 46. In particular, the DNA fragment containing shrimp mitochondria 12SrRNA gene is prepared according to SN/T4419.22-2016 (part 22 of the detection method of common allergen LAMP System for export food): the primers and corresponding DNA templates of shrimp in shrimp.
The invention also provides a detection method of the chip kit for detecting the plant-derived allergen component, which is characterized by comprising the following specific steps of:
1) Preparing a microfluidic gene chip:
the microfluidic gene chip is the microfluidic gene chip described in the claims;
2) Extracting nucleic acid of a sample to be detected;
3) Preparing a isothermal amplification reaction system:
in a system area, absorbing a isothermal amplification reagent, adding the isothermal amplification reagent into a centrifuge tube, covering a tube cover, and then moving the centrifuge tube to a template sample adding area;
adding the nucleic acid of the step 2) in the template sample adding area;
4) Sample adding of the microfluidic gene chip:
sucking the nucleic acid amplification reaction system prepared in the step 3) in a template area, and adding the prepared nucleic acid amplification reaction system into the main channel of the microfluidic gene chip from a sample inlet and outlet;
taking a sealing film, and covering the sealing film on the sample inlet and outlet;
centrifuging the microfluidic gene chip subjected to sample adding on a low-speed centrifuge for standby;
5) And (3) isothermal amplification:
preheating a constant-temperature amplification microfluidic chip nucleic acid analyzer;
feeding the microfluidic gene chip into a nucleic acid analyzer;
amplifying at a constant temperature of 65 ℃ for 60 minutes;
6) Judging results;
7) After detection, the fluorescence data are processed and analyzed by software.
In the invention, the reaction system of LAMP amplification is shown in the following table:
the method for extracting the DNA of the plant-derived ingredients of the food comprises the following steps:
(1) Fresh plant tissue 100 mg or dry weight tissue 20 mg is taken and fully ground by adding liquid nitrogen. 400. Mu.l of buffer FGA and 6. Mu.l of RNase A (10 mg/ml) were added, vortexed for 1min and left at room temperature for 10 min.
(2) 130 μl of buffer LP2 was added, mixed well and vortexed for 1 min.
(3) Centrifuge at 12,000 rpm (13,400 Xg) for 5 min and transfer the supernatant to a new centrifuge tube.
(4) 1.5 volumes of buffer LP3 (e.g., 500. Mu.l of filtrate plus 750. Mu.l of buffer LP 3) were added (before use, it was checked whether absolute ethanol had been added) and immediately mixed by shaking thoroughly for 15 sec, at which time flocculent precipitate may occur.
(5) The solution obtained in the last step and flocculent precipitate are added into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), the solution is centrifuged for 30 sec at 12,000 rpm (13,400 Xg), waste liquid is poured out, and the adsorption column CB3 is placed into the collecting pipe.
(6) 600 μl of rinse solution PW (before use, check if absolute ethanol has been added) was added to the column CB3, centrifuged at 12,000 rpm (13,400Xg) for 30 sec, the waste liquid was decanted, and the column CB3 was placed in a collection tube.
Note that: if the adsorption column membrane is green, 500. Mu.l of absolute ethanol is added to the adsorption column CB3, and the mixture is centrifuged at 12,000 rpm (13,400 Xg) for 30 sec, and the waste liquid is poured off and the adsorption column CB3 is placed in a collection tube.
(7) Repeating the operation step (6).
(8) The adsorption column CB3 is put back into a collecting pipe, and is centrifuged at 12,000 rpm (13,400 Xg) for 2 min, and the waste liquid is poured out. The adsorption column CB3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material.
(9) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 μl of elution buffer solution TB into the middle part of the adsorption film, standing at room temperature for 2-5 min, centrifuging at 12,000 rpm (13,400×g) for 2 min, and collecting the solution into the centrifuge tube.
The invention applies the loop-mediated isothermal amplification technology and the microfluidic chip technology to the field of food allergen detection, realizes rapid, convenient, accurate and high-flux detection, develops the microfluidic chip for simultaneously detecting various food plant source components, realizes rapid detection of various animal source components, has better convenience and higher rapidness, specificity and sensitivity, and provides guarantee for the selection of an allergen detection method.
Drawings
FIG. 1 is a graph showing the results of chip detection of the allergen components of brown sugar honey fried dough twist (positive wheat and sesame);
FIG. 2 is a graph showing the results of chip detection of allergen components of nut flaky pastries (cashew, peanut positive);
FIG. 3 is a graph showing the results of chip detection of dry steamed allergen components (carrot, wheat positive);
fig. 4 is a graph showing the detection result of a chip of allergen components of crispy cashew cake (positive for wheat and cashew);
FIG. 5 is a graph showing the results of chip detection of nut chocolate allergen ingredients (almond positive);
FIG. 6 is a graph showing the results of chip detection of allergen components of vegetable dumplings (positive wheat and celery).
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The chip kit for detecting the plant-derived allergen components comprises a microfluidic chip, wherein the microfluidic chip is provided with a reaction tank, and the reaction tank comprises at least one of a wheat allergen component, a cashew nut allergen component, a almond allergen component, a carrot allergen component, a peanut allergen component, a sesame allergen component and a celery allergen component, and the LAMP amplification reaction is independently carried out in the reaction tank.
Further, the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the wheat allergen component is SEQ ID NO. 1 to SEQ ID NO. 6;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the cashew nut allergen components is SEQ ID NO 7 to SEQ ID NO 12;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the almond allergen component is SEQ ID NO. 13-18;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the carrot allergen components is SEQ ID NO. 19-24;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the peanut allergen components is SEQ ID NO. 25-SEQ ID NO. 30;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the sesame allergen component is SEQ ID NO. 31-36;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the celery allergen component is SEQ ID NO. 37-SEQ ID NO. 42.
Further, the microfluidic chip also comprises a reaction tank of LAMP primer groups for blank control, negative control and amplification positive control.
Further, the blank control reaction well is not spotted;
the reaction tank of the negative control is replaced by double distilled water for the DNA template;
the reaction pool for amplifying the positive control is embedded with a DNA fragment containing shrimp mitochondrion 12SrRNA genes and an LAMP primer group thereof, and the primer group sequence is SEQ ID NO. 43-SEQ ID NO. 46.
Example 2
The detection method of the chip kit for detecting the plant-derived allergen component of the embodiment 1 is characterized by comprising the following specific steps:
1) Preparing a microfluidic chip:
2) Extracting nucleic acid of a sample to be detected;
3) Preparing a isothermal amplification reaction system:
in a system area, absorbing a isothermal amplification reagent, adding the isothermal amplification reagent into a centrifuge tube, covering a tube cover, and then moving the centrifuge tube to a template sample adding area;
adding the nucleic acid of the step 2) in the template sample adding area;
4) Sample adding of the microfluidic gene chip:
sucking the nucleic acid amplification reaction system prepared in the step 3) in a template area, and adding the prepared nucleic acid amplification reaction system into the main channel of the microfluidic gene chip from a sample inlet and outlet;
taking a sealing film, and covering the sealing film on the sample inlet and outlet;
centrifuging the microfluidic gene chip subjected to sample adding on a low-speed centrifuge for standby;
5) And (3) isothermal amplification:
preheating a constant-temperature amplification microfluidic chip nucleic acid analyzer;
feeding the microfluidic gene chip into a nucleic acid analyzer;
amplifying at a constant temperature of 65 ℃ for 60 minutes;
6) Judging results;
7) After detection, the fluorescence data are processed and analyzed by software.
In the invention, the reaction system of LAMP amplification is shown in the following table:
the method for extracting the DNA of the plant-derived ingredients of the food comprises the following steps:
(1) Fresh plant tissue 100 mg or dry weight tissue 20 mg is taken and fully ground by adding liquid nitrogen. 400. Mu.l of buffer FGA and 6. Mu.l of RNase A (10 mg/ml) were added, vortexed for 1min and left at room temperature for 10 min.
(2) 130 μl of buffer LP2 was added, mixed well and vortexed for 1 min.
(3) The supernatant was centrifuged at 12,000 rpm (13,400 Xg) for 5 min and transferred to a new centrifuge tube.
(4) 1.5 volumes of buffer LP3 (e.g., 500. Mu.l of filtrate plus 750. Mu.l of buffer LP 3) were added (before use, it was checked whether absolute ethanol had been added) and immediately mixed by shaking thoroughly for 15 sec, at which time flocculent precipitate may occur.
(5) The solution obtained in the last step and flocculent precipitate are added into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), the solution is centrifuged for 30 sec at 12,000 rpm (13,400 Xg), waste liquid is poured out, and the adsorption column CB3 is placed into the collecting pipe.
(6) 600 μl of rinse solution PW (before use, check if absolute ethanol has been added) was added to the column CB3, centrifuged at 12,000 rpm (13,400Xg) for 30 sec, the waste liquid was decanted, and the column CB3 was placed in a collection tube.
Note that: if the adsorption column membrane is green, 500. Mu.l of absolute ethanol is added to the adsorption column CB3, and the mixture is centrifuged at 12,000 rpm (13,400 Xg) for 30 sec, and the waste liquid is poured off and the adsorption column CB3 is placed in a collection tube.
(7) Repeating the operation step (6).
(8) The adsorption column CB3 is put back into a collecting pipe, and is centrifuged at 12,000 rpm (13,400 Xg) for 2 min, and the waste liquid is poured out. The adsorption column CB3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material.
(9) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 μl of elution buffer solution TB into the middle part of the adsorption film, standing at room temperature for 2-5 min, centrifuging at 12,000 rpm (13,400×g) for 2 min, and collecting the solution into the centrifuge tube.
The invention applies the loop-mediated isothermal amplification technology and the microfluidic chip technology to the field of food allergen detection, realizes rapid, convenient, accurate and high-flux detection, develops the microfluidic chip for simultaneously detecting various food plant source components, realizes rapid detection of various animal source components, has better convenience and higher rapidness, specificity and sensitivity, and provides guarantee for the selection of an allergen detection method.
And (3) chip detection:
1. constructing a chip reaction system;
the composition of the chip reaction system is shown in the following table:
；
2. sensitivity:
mixing the crushed wheat flour and rice flour to obtain the wheat with the mass fraction of 1%,0.5%,0.1% and 0.05%, and performing chip detection to determine the sensitivity of the method for detecting allergen wheat components. The results showed that 1% wheat flour, 0.5% wheat flour, 0.1% wheat flour, and the reaction results were positive; the reaction result of 0.05% wheat flour is negative, namely the detection sensitivity of the chip for detecting allergen wheat components is 0.1%. The results are shown in the following table:
。
the detection sensitivity of the chip of the other 7 allergen components is detected, and the result shows that the detection sensitivity of the allergen wheat component and the allergen peanut component in the 8 allergens is 0.1 percent; the detection sensitivity of the allergen sesame component, the cashew component, the almond component, the celery component and the carrot component is 0.5 percent.
3. Specificity (specificity)
50 parts of materials purchased in the market, including products containing wheat, peanut, sesame, cashew, peach kernel, celery and carrot components and products without the allergen components, are subjected to chip detection after DNA extraction, and the detection results are shown in figures 1-6, and are consistent with the labels.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art. It should be noted that technical features not described in detail in the present invention may be implemented by any prior art.
Sequence listing
<110> Guangdong product quality supervision and inspection institute (national quality technology administration Guangzhou electric safety inspection institute, guangdong province laboratory, hua' an laboratory)
<120> a chip kit for detecting plant-derived allergen components
<160> 46
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<213> Artificial sequence (Artificial Sequence)
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caacaacaat tttctcagcc 20
<210> 2
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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cagatcattg accagagg 18
<210> 3
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ctgaatgaac ggtggttgtt caacaacaat tcccgcag 38
<210> 4
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ctctacaaca acaggtgaac cgatgacacc agtgacacag 40
<210> 5
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<213> Artificial sequence (Artificial Sequence)
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tgattgttgc ggttgttg 18
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<213> Artificial sequence (Artificial Sequence)
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tttcctcttg cagcaatg 18
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ttcgcagtcc tcctcctg 18
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gcagcattcc ctcaagct 18
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gccgttggca actctgctca atttaccgag ccattgtgga 40
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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agttcgaaga gcagcagcga tttatagcgt cctcccctct g 41
<210> 11
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ggcccgagtc ttcttcaacc 20
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<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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tccggaactg tcaaaggtac g 21
<210> 13
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<212> DNA
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caaccacagg gttcgagtt 19
<210> 14
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
aagttgctgg cggaatgg 18
<210> 15
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
catccagttc gggcccagaa ggcatcccaa gctagcttca g 41
<210> 16
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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tccacggacg cctctggaaa ttgcacgtga cctgacca 38
<210> 17
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
tggcaccgga gtgtctagg 19
<210> 18
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gttcgtctgt gccactgc 18
<210> 19
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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<210> 20
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<212> DNA
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aatttttgct gtgcccaagg 20
<210> 21
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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<210> 22
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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atcgcgacat cggtggttgt ccctcgagtt ccctactgc 39
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ggcacgggag gccaatt 17
<210> 24
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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aagaagacct tcttgtgtcg ttg 23
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
atccaacgtg acgaggattc 20
<210> 26
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
tcgctctggt tctccatga 19
<210> 27
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
tccagcgcct ctccgatcat acccgtacag ccctagtcag 40
<210> 28
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
tcctctcagc accaagagag gttgcaatgc ctcgcacatg 40
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
ggactagggc tgtacggatc 20
<210> 30
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gttgcaatga gctgaacgag t 21
<210> 31
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
atggtggtga agaggatga 19
<210> 32
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
gataactcgg aattggcatt g 21
<210> 33
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
ctcgtccacg ttcctcagcc tggaaatgag cactggg 37
<210> 34
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
ttaggcaagc agtgaggcag ctagccctct ggtaaacct 39
<210> 35
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
ctgctcggac tgctgatt 18
<210> 36
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
taccaggagg gccaatca 18
<210> 37
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
caccctcact atatataccc tg 22
<210> 38
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
gatcaaagat attttacctt ctgga 25
<210> 39
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
gccattgcca aataaagatt cgagatcaaa cccatctctt taacacat 48
<210> 40
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
gaaatcatca gaaattgagc acccacttaa aaggagaaag gagacc 46
<210> 41
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
ggaaaaggag agagattggt ttttg 25
<210> 42
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
tgtcaaggct tttggctgg 19
<210> 43
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
tagaaaccga cctggctc 18
<210> 44
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
tcataagatt aagttacttt aggga 25
<210> 45
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
gcagcagtta taaaggaagg tctcggtctg aactcaaatc atgt 44
<210> 46
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
ccttaattca acatcgaggt cgacagcgta atcttctttg agag 44
Claims (1)
1. The chip kit for detecting the plant-derived allergen components comprises a microfluidic chip, wherein the microfluidic chip is provided with a reaction tank, and is characterized in that the reaction tank comprises a detection device for detecting at least one of a wheat allergen component, a cashew nut allergen component, a almond allergen component, a carrot allergen component, a peanut allergen component, a sesame allergen component and a celery allergen component, and LAMP amplification reaction is independently carried out in the reaction tank;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the wheat allergen component is SEQ ID NO. 1 to SEQ ID NO. 6;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the cashew nut allergen components is SEQ ID NO 7 to SEQ ID NO 12;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the almond allergen component is SEQ ID NO. 13-18;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the carrot allergen components is SEQ ID NO. 19-24;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the peanut allergen components is SEQ ID NO. 25-SEQ ID NO. 30;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the sesame allergen component is SEQ ID NO. 31-36;
the sequence of the primer group for nucleotide amplification in the reaction tank for detecting the celery allergen component is SEQ ID NO. 373-SEQ ID NO. 42;
the microfluidic chip also comprises a reaction tank of LAMP primer groups for blank control, negative control and amplification positive control;
the reaction tank of the blank control is not spotted;
the reaction tank of the negative control is replaced by double distilled water for the DNA template;
the reaction pool for amplifying the positive control is embedded with a DNA fragment containing shrimp mitochondrion 12SrRNA genes and an LAMP primer group thereof, and the primer group sequence is SEQ ID NO. 43-SEQ ID NO. 46.
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| CN202111651658.5A CN114214452B (en) | 2021-12-31 | 2021-12-31 | Chip kit for detecting plant-derived allergen components |
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| CN114214452B true CN114214452B (en) | 2024-04-05 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060462A (en) * | 2013-01-18 | 2013-04-24 | 天津生物芯片技术有限责任公司 | Gene chip and detection kit for detecting common food allergens |
| CN106676166A (en) * | 2016-10-11 | 2017-05-17 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Detection reagent for accurately identifying oat ingredient and detection method |
| CN109295250A (en) * | 2018-10-31 | 2019-02-01 | 四川华汉三创生物科技有限公司 | A kind of detection kit and method of food-borne plant hypersensitive ultimate constituent |
| CN113234570A (en) * | 2021-03-31 | 2021-08-10 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | High-flux disc type chip for detecting animal-derived ingredients of food and detection method |
-
2021
- 2021-12-31 CN CN202111651658.5A patent/CN114214452B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060462A (en) * | 2013-01-18 | 2013-04-24 | 天津生物芯片技术有限责任公司 | Gene chip and detection kit for detecting common food allergens |
| CN106676166A (en) * | 2016-10-11 | 2017-05-17 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Detection reagent for accurately identifying oat ingredient and detection method |
| CN109295250A (en) * | 2018-10-31 | 2019-02-01 | 四川华汉三创生物科技有限公司 | A kind of detection kit and method of food-borne plant hypersensitive ultimate constituent |
| CN113234570A (en) * | 2021-03-31 | 2021-08-10 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | High-flux disc type chip for detecting animal-derived ingredients of food and detection method |
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| CN114214452A (en) | 2022-03-22 |
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