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    • 专利摘要:
    The invention relates to a crustacean allergen test card and application thereof. The test card includes: a back card, which is provided with a sample loading zone and a test zone, wherein the sample loading zone is connected to the test zone so that a sample in the sample loading zone moves to the test zone; and the test zone is provided with a nucleic acid aptamer-hydrogel membrane tube; and a face card, which is provided with a sample loading well that is adapted for loading the sample into the sample loading zone. The test card of the invention with high sensitivity and simple operate can quantitatively and qualitatively determine the content of crustacean allergens in foods.
    公开号:NL2025926A
    申请号:NL2025926
    申请日:2020-06-26
    公开日:2021-02-01
    发明作者:Fu Linglin；Wang Yanbo；Zhou Jinru；Li Huan；Wang Chong；Zhang Qiaozhi；Chen Jian
    申请人:Univ Zhejiang Gongshang；
    IPC主号:
    专利说明:

    [0001] [0001] 1. Technical Field
    [0002] [0002] The invention relates to the field of allergen tests, and in particular to a crustacean allergen test card and application thereof.
    [0003] [0003] 2. Description of Related Art
    [0004] [0004] In recent years, there are many reports on food-borne allergies such as shrimps and crabs. Among the eight major categories of allergenic foods proposed by the IO Food and Agriculture Organization of the United Nations, crustaceans such as shrimps and crabs and products thereof are an important category. According to statistics, about 40% of adults and children in Asian countries are allergic to crustaceans.
    [0005] [0005] At present, a large number of countries across the globe, including the United States, the European Union, and New Zealand, have issued legislation mandating I5 labelling of allergenic ingredients contained in food products to alert consumers and prevent them from eating these foods by mistake. Therefore, it is a basic and primary task to test the allergen content for the production, evaluation and labeling of the food products containing allergenic ingredients. Arginine kinase (AK) and tropomyosin (TM), as two main allergens in the crustaceans, are widely present in the crustaceans such as shrimps and crabs.
    [0006] [0006] In the related art, the methods for detecting the arginine kinase in the crustaceans mainly include ELISA, RT-PCR, LC-MS and other techniques. These methods all require high-end instruments and equipment and professional operators, with long test time and complicated operation.
    [0007] [0007] Hence, there is a need to improve the crustacean allergen detection method.BRIEF SUMMARY OF THE INVENTION
    [0008] [0008] The invention is intended to solve one of the technical problems in the related art at least to a certain extent. To this end, an object of the invention is to provide a crustacean allergen test card. The test card with high sensitivity and simple operation can quantitatively and qualitatively determine the content of crustacean allergens in food.
    [0009] [0009] Therefore, according to one aspect of the invention, the invention provides a crustacean allergen test card. According to an embodiment of the invention, the test card includes: a back card, which 1s provided with a sample loading zone and a test zone, 1 wherein the sample loading zone is connected to the test zone so that a sample in the sample loading zone moves to the test zone; and the test zone is provided with a nucleic acid aptamer-hydrogel membrane tube; and a face card, which is provided with a sample loading well that is adapted for loading the sample into the sample loading zone.
    [0010] [0010] With the test card according to the embodiments of the invention, the nucleic acid aptamer-hydrogel in the nucleic acid aptamer-hydrogel membrane tube specifically binds to the tropomyosin and the arginine kinase in the sample to affect the behavior pattern of the hydrogel in the capillary; the changes in migration time at different concentrations are recorded; and then the concentrations of the crustacean allergen tropomyosin and arginine kinase in the sample under test are converted according to the change curve to achieve the level of quantitative testing. With the advantages of simple structure, easy operation, high sensitivity, easy storage, and good reliability, the test card of the invention can quantitatively and qualitatively determine the content of crustacean allergens in food, and has extremely high promotion and the practical value. Laboratory staff can operate it with little training.
    [0011] [0011] In addition, the crustacean allergen test card provided according to the above embodiments of the invention may also have the following additional technical features.
    [0012] [0012] According to an embodiment of the invention, the nucleic acid aptamer-hydrogel membrane tube comprises a silica capillary and a nucleic acid aptamer-hydrogel; nucleic acid aptamers in the nucleic acid aptamer-hydrogel are adapted for specifically binding tropomyosin and arginine kinase.
    [0013] [0013] According to an embodiment of the invention, the nucleic acid aptamers in the nucleic acid aptamer-hydrogel comprise a tropomyosin nucleic acid aptamer and an arginine kinase nucleic acid aptamer, the tropomyosin nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID No: 1 and SEQ ID No: 2; and the arginine kinase nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID No: 3 and SEQ ID No: 4.
    [0014] [0014] According to an embodiment of the invention, the silica capillary has a length of 8-10 cm and an inner diameter of 295-305 um; and the nucleic acid aptamer-hydrogel has a length of 1-1.5 mm.
    [0015] [0015] According to an embodiment of the invention, the test zone is provided with scales at both sides.
    [0016] [0016] According to an embodiment of the invention, the sample loading well is 2 funnel-shaped.
    [0017] [0017] According to an embodiment of the invention, the sample loading zone is covered with a piece of filter paper.
    [0018] [0018] According to an embodiment of the invention, the filter paper is a piece of circular filter paper; and optionally, the filter paper has a diameter of 1-1.5 cm and a pore size of 2-3 um.
    [0019] [0019] According to an embodiment of the invention, the crustacean allergen test card is prepared by the following steps:
    [0020] [0020] (1) preparing a nucleic acid aptamer-hydrogel, wherein nucleic acid IO aptamers comprise a tropomyosin nucleic acid aptamer and arginine kinase nucleic acid aptamer;
    [0021] [0021] (2) inserting a heated silica capillary into the nucleic acid aptamer-hydrogel to obtain a nucleic acid aptamer-hydrogel membrane tube;
    [0022] [0022] (3) fixing the nucleic acid aptamer-hydrogel membrane tube in the test zone;
    [0023] [0023] (4) covering the sample loading zone with a piece of filter paper;
    [0024] [0024] (5) providing the test zone with scales at both sides; and
    [0025] [0025] (6) combining the face card and the back card together to obtain the crustacean allergen test card.
    [0026] [0026] According to an embodiment of the invention, in step (2), the silica capillary is inserted into the nucleic acid aptamer-hydrogel to a depth of 1-1.5 mm over a time of 3-5 s; and
    [0027] [0027] According to an embodiment of the invention, in step (2), the silica capillary is heated at 90-95°C over a heating time of 10-15 min.
    [0028] [0028] According to another aspect of the invention, the invention provides a crustacean allergen test method. According to an embodiment of the invention, the method includes: plotting a standard curve of tropomyosin and a standard curve of arginine kinase; loading a sample to be tested into the test zone through the sample loading well in the foregoing crustacean allergen test card, and recording a time when the nucleic acid aptamer-hydrogel in the nucleic acid aptamer-hydrogel membrane tube reaches an end point, to obtain a test value of the time when the nucleic acid aptamer-hydrogel reaches the end point; and substituting the test value of the time when the nucleic acid aptamer-hydrogel reaches the end point into the standard curve of tropomyosin and the standard curve of arginine kinase, to obtain a tropomyosin concentration and an arginine 3 kinase concentration in the sample to be tested. Hence, when used to test the crustacean allergens, the method with high sensitivity, simple operation and short detecting time can quantitatively and qualitatively determine the content of crustacean allergens in food, and has extremely high promotion and the practical value.
    [0029] [0029] Additional aspects and advantages of the invention will be partially given in the description below, will become apparent partially from the description below, or will be learned through the practice of the invention.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
    [0030] [0030] FIG 1 is a schematic structural front view of a crustacean allergen test card according to an embodiment of the invention;
    [0031] [0031] FIG 2 is a graph showing fitting results of an arginine kinase standard curve according to an embodiment of the invention;
    [0032] [0032] FIG 3 is a graph showing fitting results of a tropomyosin standard curve according to an embodiment of the invention;
    [0033] [0033] FIG 4 is a graph showing cross-reactivity results of arginine kinase according to an embodiment of the invention; and
    [0034] [0034] FIG 5 is a graph showing cross-reactivity results of tropomyosin according to an embodiment of the invention.DETAILED DESCRIPTION OF THE INVENTION
    [0035] [0035] The technical solution of the invention will be described below by means of particular embodiments in detail. It should be understood that one or more method steps mentioned in the invention does not exclude the presence of other method steps before or after the combination steps, or the insertion of other method steps among these explicitly mentioned steps. It should also be understood that these embodiments are merely for illustrating the invention but not to limit the scope of the invention. Moreover, unless otherwise stated, the number of each method step only serves as a convenient tool for identifying each method step, but not to limit the order of each method step or to limit the scope for implementing the invention. The change or adjustment of a relative relationship among these numbers without any substantial change to the technical content should be construed as falling within the scope for implementing the invention.
    [0036] [0036] To better understand the above technical solutions, the exemplary embodiments of the invention will be described in more detail. Although the exemplary 4 embodiments of the invention are shown, it should be understood that the invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.
    [0037] [0037] The test materials used in the invention are all common commercial products, which are all commercially available.
    [0038] [0038] According to an aspect of the invention, the invention provides a crustacean allergen test card. The inventor screened and modified a large number of crustacean arginine kinase and tropomyosin from a DNA library to obtain an arginine kinase nucleic acid aptamer and a tropomyosin aptamer, both capable of specifically binding to the crustacean arginine kinase and tropomyosin, thereby preparing a nucleic acid aptamer, then, the nucleic acid aptamer-hydrogel in the nucleic acid aptamer-hydrogel membrane tube specifically bound to the tropomyosin and the arginine kinase in the sample to affect the behavior pattern of the hydrogel in the capillary; the change in migration time at different concentrations was recorded; and then the concentrations of the crustacean allergen tropomyosin and arginine kinase in the sample under test were converted according to the change curve to achieve the level of quantitative testing. As such, the presence and amount of the crustacean allergen in the sample were determined. With the advantages of simple structure, easy operation, high sensitivity, easy storage, and good reliability, the test card of the invention can quantitatively and qualitatively determine the content of crustacean allergens in food, and has extremely high promotion and the practical value.
    [0039] [0039] To facilitate understanding of the test card according to the embodiment of the invention, the test card is explained and illustrated as follows with reference to FIG. 1 according to the embodiment of the invention.
    [0040] [0040] Back card 10: According to an embodiment of the invention, the back card 10 is not particularly limited in type as long as it does not react with the sample to be tested. Those skilled in the art may make their own choices as needed. According to some specific embodiments of the invention, PET plastic may be selected as the back card. The back card 10 is provided with a sample loading zone 11 and a test zone 12; the sample loading zone 10 is connected to the test zone 12 so that the sample in the sample loading zone 11 moves to the test zone 12; and the test zone 12 is provided with a nucleic acid aptamer-hydrogel membrane tube 121. According to an embodiment of the invention, the 5 nucleic acid aptamer-hydrogel membrane tube 121 includes a silica capillary and a nucleic acid aptamer-hydrogel; and the nucleic acid aptamers in the nucleic acid aptamer-hydrogel are adapted for specifically binding tropomyosin and arginine kinase. Hence, the combination of the sample to be tested with the nucleic acid aptamers may affect the movement of the hydrogel.
    [0041] [0041] According to an embodiment of the invention, the nucleic acid aptamers in the nucleic acid aptamer-hydrogel comprise a tropomyosin nucleic acid aptamer and an arginine kinase nucleic acid aptamer; the tropomyosin nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID No: 1 and SEQ ID No: 2; and the arginine kinase nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID No: 3 and SEQ ID No: 4. Hence, the sample to be tested may specifically bind to the nucleic acid aptamers with high affinity, thereby enabling the test with high sensitivity, strong specificity and simple operation.
    [0042] [0042] According to an embodiment of the invention, the silica capillary has a length of 8-10 cm and an inner diameter of 295-305 um; and the nucleic acid aptamer-hydrogel has a length of 1-1.5 mm. Hence, the behavior pattern of the hydrogel in the capillary may be better observed, thereby enabling the test with simple operation.
    [0043] [0043] According to an embodiment of the invention, the test zone is provided with scales at both sides. Hence, the start point and the end point for the hydrogel to move the capillary may be marked to better facilitate recording the migration time of the hydrogel, thereby enabling the test with simple operation.
    [0044] [0044] Face card 20: The face card 20 is engaged with the back card 10 to form the test card. According to the embodiment of the invention, the face card 20 is not particularly limited in type. Those skilled in the art may make their own choices as needed.
    [0045] [0045] According to an embodiment of the invention, the sample loading zone 11 is covered with a piece of filter paper 211. The filter paper 211 may prevent impurities in the sample from entering the sample loading zone 11 and affecting the movement of the hydrogel. According to an embodiment of the invention, the filter paper 211 may be a piece of circular filter paper; and the circular filter paper has a diameter of 1-1.5 cm and a pore size of 2-3 um. Hence, impurities in the sample may be well filtered out.
    [0046] [0046] Crustacean Allergen Test Method
    [0047] [0047] According to another aspect of the invention, the invention provides a crustacean allergen test card. The inventor found that when used to test the crustacean allergens, the method exhibits high sensitivity, short detecting time, low production cost and simple operation, and can quantitatively and qualitatively determine the content of crustacean allergens in food, and has extremely high promotion and the practical value.
    [0048] [0048] According to an embodiment of the invention, the method includes the following steps.
    [0049] [0049] A standard curve of tropomyosin and a standard curve of arginine kinase are plotted. According to an embodiment of the invention, standard solutions of the tropomyosin and the arginine kinase are prepared at a gradient of a series of concentrations, and are tested with several test cards from the same batch; and the standard curves are plotted with the logarithmic concentration values of the standard solutions as the abscissa and the time when the hydrogel reaches the end point as the ordinate.
    [0050] [0050] A sample to be tested is loaded into the test zone through the sample loading well in the foregoing crustacean allergen test card, and a time when the hydrogel in the nucleic acid aptamer-hydrogel membrane tube reaches an end point is recorded to obtain a test value of the time when the nucleic acid aptamer-hydrogel reaches the end point.
    [0051] [0051] The test value of the time when the hydrogel reaches the end point is substituted into the standard curve of tropomyosin and the standard curve of arginine kinase, to obtain a tropomyosin concentration and an arginine kinase concentration in the sample to be tested.
    [0052] [0052] Hence, when used to test the crustacean allergens, the method exhibits high sensitivity, short detecting time, and simple operation, and can quantitatively and qualitatively determine the content of crustacean allergens in food, and has extremely high promotion and the practical value.
    [0053] [0053] The invention will be described with reference to the specific embodiments below. It should be noted that these embodiments are merely for a descriptive purpose, but not to limit the invention in any manner.
    [0054] [0054] Embodiment 1
    [0055] [0055] Preparation of Arginine Kinase:
    [0056] [0056] (1) 50 g of Pacific white shrimp muscle were weighed, with heads, tails, shells and veins removed.
    [0057] [0057] (2) The shrimp muscle was chopped into paste with a knife, dissolved in a buffer A (50 mM NaCl, 2 mM NaHCO; 10 mM EDTA), homogenized with a homogenizer and allowed to stand at 4°C for 2 h.
    [0058] [0058] (3) A resulting solution after standing in step (2) was centrifuged at 8000 r/min at 4°C for 20 min, to obtain a supernatant, which was added into 70% ammonium sulfate and then allowed to stand at 4°C for 8 h.
    [0059] [0059] (4) The resulting supernatant after standing in step (3) was centrifuged at 8000 r/min at 4°C for 20 min, to obtain a supernatant, which was added into 90% ammonium sulfate and then allowed to stand at 4°C for 6 h.
    [0060] [0060] (5) The resulting supernatant after standing in step (4) was centrifuged at 8000 r/min at 4°C for 20 min, to obtain a precipitate, which was dissolved in a Buffer B (20 mM Tris-HCL, 1 mM NaCl, pH 8.0) to obtain an arginine kinase solution.
    [0061] [0061] (6) The arginine kinase solution was eluted at a gradient with a 0.5 M NaCl solution through a Source 15Q anion exchange column to collect an eluted product, thereby obtaining the arginine kinase for later use.
    [0062] [0062] Preparation of Tropomyosin:
    [0063] [0063] (1) 50 g of Pacific white shrimp muscle were weighed, with heads, tails, shells and veins removed.
    [0064] [0064] (2) The shrimp muscle was chopped into paste with a knife, dissolved in a buffer A (50 mmol/L KCI and 2 mmol/L NaHCO), fully homogenized and then extracted at 4°C for 20 min.
    [0065] [0065] (3) A resulting solution after extraction in step (2) was centrifuged at 10000 r/min at 4°C for 20 min, to obtain a precipitate, which was resuspend in the Buffer A 10 times the volume of the precipitate and then centrifuged at 10000 r/min at 4°C for 20 min to obtain a precipitate. This process was repeated 5 times.
    [0066] [0066] (4) The resulting precipitate from step (3) was fully washed with pre-cooled acetone till colorlessness, filtered with six layers of gauze, and then dried at 8 room temperature to remove impurities such as fat and fat-soluble pigment, to obtain shrimp acetone powder.
    [0067] [0067] (5) The shrimp acetone powder was dissolved in the Buffer B (0.02 mol/L Tris-HCI, 1 mol/L KCl and 0.1 mmol/L DTT, pH 7.5), and then extracted for 72 hours.
    [0068] [0068] (6) A resulting extracted solution from step (5) was filtered with six layers of gauze to obtain a supernatant, which was heated in a water bath for 20 min.
    [0069] [0069] (7) The supernatant obtained after heating in step (6) was centrifuged at 10000 r/min at 4°C for 20 min, to obtain a supernatant, which was slowly added into 30% ammonium sulfate and then allowed to stand at 4°C for 1 h.
    [0070] [0070] (8) A resulting liquid after standing was centrifuged at 10000 r/min at 4°C for 20 min to obtain a precipitate, which was reconstituted with 1 M PBS to obtain a tropomyosin solution.
    [0071] [0071] (9) The tropomyosin solution was eluted at a gradient with a 0.5 M NaCl solution through a DEAE Sepharose F.F. anion exchange column to collect an eluted product, thereby obtaining the tropomyosin for later use.
    [0072] [0072] Screening of Nucleic Acid Aptamers:
    [0073] [0073] A random ssDNA library of crustacean arginine kinase was built. (Library Capacity: 1014; Fragment Length: 40 bp; the synthesis of the library was commissioned to Sangon Biotech (Shanghai) Co., Ltd.)
    [0074] [0074] According to this embodiment, three screening modes were alternatively used.
    [0075] [0075] In the first mode of screening, the ssDNA library was incubated with the tropomyosin and the arginine kinase (both prepared in Embodiment 1) at 37°C for 1 h, and then added to a graphene solution (graphene powder, 5 mg/ml, purchased from Aladdin Reagent (Shanghai) Co., Ltd.). A resulting mixture was cultured at 37°C to adsorb free and loose ssDNA. Next, the mixture was centrifuged at 12,000 r/min for 15 min to separate and remove unbound ssDNA and graphene from the precipitate, and the remaining supernatant containing the ssDNA bound to the arginine kinase was subjected to asymmetric PCR amplification (the ratio of a restrictive primer to a non-restrictive primer was 1:50; the sequences were: 5’-CAG GGG AGC GAG CG-3’and 5’-ATG AGG CAG GGG CCT CG-3°; Amplification Condition: transition at 95°C for 1 min; Cycling Condition: 95°C, 30 s; 50°C, 30 s; 72°C, 1 min; extended, 72°C, 3 min). Subsequently, a PCR product was purified and the ssDNA was prepared through exonuclease digestion. The screened nucleic acid aptamer binding buffer (BB: 50 mM Tris, 150 mM NaCl, 2 mM 9
    [0076] [0076] In the second mode of screening, a new ssDNA library was first incubated with a graphene solution at 37°C for 1 h; then a resulting mixture was centrifuged at 12,000 rpm for 15 min; and a resulting precipitate was washed and then centrifuged, which was repeated for three times. The resulting precipitate was reconstituted and then added with the arginine kinase for mixed culture to obtain an ssDNA bound to the arginine kinase, which was then subjected to the asymmetric PCR amplification (in the same way as in the first mode of screening). Subsequently, a PCR product was purified and the ssDNA was prepared through exonuclease digestion. Finally, the purified single-stranded DNA was taken as a subpool for the next round.
    [0077] [0077] In the third mode of screening, the ssDNA was incubated with ovalbumin, papain, and tropomyosin at 37°C for 1 h; graphene was added into a resulting mixture to continue the incubation; the mixture was centrifuged at 12,000 rpm for 15 min; a supernatant was removed from the centrifuged mixture; and the ssDNA in the precipitate were separated from the graphene for asymmetric PCR amplification. The resulting ssDNA after the amplification was the finally obtained nucleic acid aptamers.
    [0078] [0078] After multiple screenings, 8 nucleotide sequences were obtained. Data analysis was performed to obtain a dissociation constant (Kd), where a small dissociation constant indicated a strong affinity. Among others, the nucleotide sequence with the strongest affinity was the desired sequence. There are 4 sequences in total, named as SEQ ID No: 1-4.
    [0079] [0079] Among others, the sequence of SEQ ID No: 1 is:
    [0080] [0080] 5’-CTACCCAGAACTCTCGCGTTAGCTCAGTCGCTGTTTCGGGGCC AA-3’;
    [0081] [0081] the sequence of SEQ ID No: 2 is:
    [0082] [0082] 5’-CTACGAACGATGATGAGTCCAGAGACATATTACTTCGCGCATA AA-3’;
    [0083] [0083] the sequence of SEQ ID No: 3 is:
    [0084] [0084] 5 -CCAGGCCGCCAACGTTGACCTAGAAGCACTGCCAGACCCG-3; and
    [0085] [0085] the sequence of SEQ ID No: 4 is:
    [0086] [0086] S’-CTACCAGGCCGCCAACGTTGACCTAGAAGCACTGCCGACC-3’. 10
    [0087] [0087] Embodiment 2
    [0088] [0088] With the method according to the embodiment of the invention, the crustacean allergen test card was prepared by the following steps.
    [0089] [0089] Preparation of Nucleic acid aptamer-hydrogel Membrane Tube:
    [0090] [0090] Sangon Biotech (Shanghai) Co., Ltd was commissioned to synthesize two strands of DNA, and DNA, that are complementary to the nucleic acid aptamers obtained in Embodiment 1. The prepared stock solution of the DNA; and DNA, strands were placed in a centrifuge tube containing 4% acrylamide respectively, and then vacuum-dried at 25°C for 10 min to remove air.
    [0091] [0091] Then, a 1.4% (v/v) initiator (0.05 g of APS dissolved in 0.5 mL of ultrapure water) and a catalyst (25 uL TEMED dissolved in 0.5 mL of ultrapure water) were added to the stock solutions of the DNA, and DNA: strands, respectively. Vacuum drying was performed again at 25°C for 15 min to produce linear-stranded PS-DNA; and PS-DNA:. A 110 uM PS-DNA; solution and a 110 uM PS-DNA: solution were mixed with 65 pM I5 nucleic acid aptamer (obtained from Embodiment 1) in a Buffer (77 mM NaHPO,, 23 mM NaH:PO,, 50 mM NaCl, 5 mM MgCl,), incubated in a dry bath at 65°C for 5 min, and then slowly cooled to room temperature (repeated three times) to obtain a nucleic acid aptamer-hydrogel.
    [0092] [0092] Among others, a DNA; sequence corresponding to SEQ ID No: 1 is: 5°-AGT TCT GGG TAG-3’, which is named as SEQ ID No: 5; and a DNA; sequence corresponding to SEQ ID No: 1 is: 5°-TGG CCC CGA AAC-3’, which is named as SEQ ID No: 6.
    [0093] [0093] A DNA; sequence corresponding to SEQ ID No: 2 is: 5’-CAT CGT TCG TAG-3’, which is named as SEQ ID No: 7; and a DNA; sequence corresponding to SEQ ID No: 2is: 5°-TTT GTA CGC GAA-3’, which is named as SEQ ID No: 8.
    [0094] [0094] A DNA; sequence corresponding to SEQ ID No: 3 is: 5’-TTG GCG GCC TGG-3’, which is named as SEQ ID No: 9; and a DNA: sequence corresponding to SEQ ID No: 3 is: 5’-CGG GTC TGG CAG-3’, which is named as SEQ ID No: 10.
    [0095] [0095] A DNA; sequence corresponding to SEQ ID No: 4 is: 5°-GCG GCC TGG TAG-3’, which is named as SEQ ID No: 11; and a DNA: sequence corresponding to SEQ ID No: 41s: 5’-GGT CGG CAG TGC-3’, which is named as SEQ ID No: 12.
    [0096] [0096] Preparation of the nucleic acid aptamer-hydrogel membrane tube:
    [0097] [0097] A silica capillary (300 + 5 um) was immersed in a piraha solution (H,SO4:H,0,=7:3) at 100°C for 1 h, then immersed in acetone and ethanol respectively, 11 ultrasonically treated for 10 min, fully washed with ultrapure water, and then blown dry with nitrogen gas. Then, the silica capillary was heated at 90°C for 10 min, and the hot silica capillary was inserted into the nucleic acid aptamer-hydrogel prepared above for 3 s (to a depth of 1 mm), and then kept in cold storage for later use.
    [0098] [0098] Preparation of Back Card:
    [0099] [0099] White PET (Polyethylene Terephthalate) plastic with a thickness of 1 mm was cut into strips of 10 cm x 3 cm. Then, the nucleic acid aptamer-hydrogel membrane tube with a length of 8 cm was fixed to the back card at the test zone, with the side containing the nucleic acid aptamer-hydrogel close to the sample loading zone. Then, scale values of 0-8 cm were engraved on both sides of the membrane tube in a direction from the sample loading zone to the test zone. Then, the sample loading zone was covered with a piece of filter paper having a diameter of 1 cm.
    [00100] [00100] Preparation of Face Card:
    [00101] [00101] The face card was a transparent PDMS (Polydimethylsiloxane) plastic face card with a reserved sample loading well facing the test zone in the back card.
    [00102] [00102] Finally, the prepared face and back cards were combined together to obtain the crustacean allergen test card.
    [00103] [00103] Embodiment 3
    [00104] [00104] Testing of Crustacean Allergens:
    [00105] [00105] Designation of Standard Curve of Tropomyosin and Standard Curve of Arginine Kinase:
    [00106] [00106] The arginine kinase solutions at different concentrations (1, 5, 10, 50, 100, 500, 1000 pg/mL) obtained in Embodiment 1 were tested with several crustacean allergen test cards from the same batch obtained in Embodiment 2. With the test card placed horizontally, 100 pL of standard solution was loaded to the sample loading well, and then a timer with a resolution of tens of milliseconds was used to record a time required for the nucleic acid aptamer-hydrogel to pass through the 8 cm long capillary. A standard curve was plotted with the logarithmic value of the concentration as the abscissa and the time when the nucleic acid aptamer-hydrogel reaches the end point as the ordinate. The results are shown in FIG 2, with a regression equation as follows: y=-7.573x+25.13, R°=0.9984.
    [00107] [00107] The tropomyosin solutions at different concentrations (1, 5, 10, 50, 100, 500, 1000 pg/mL) obtained in Embodiment 1 were tested with several crustacean allergen test cards from the same batch obtained in Embodiment 2. With the test card placed horizontally, 100 pL of standard solution was loaded to the sample loading well, and then 12 a timer with a resolution of tens of milliseconds was used to record a time required for the sample to pass through the 8 cm long capillary. A standard curve was plotted with the logarithmic value of the concentration as the abscissa and the time when the nucleic acid aptamer-hydrogel reaches the end point as the ordinate. The results are shown in FIG 3, with a regression equation as follows: y=-8.222x+28.06, R’=0.9958.
    [00108] [00108] Preparation of Samples:
    [00109] [00109] 2 g of Pacific white shrimp muscle were weighed and then ground into powder with liquid nitrogen. Then, a protein infusion was extracted from the powder using a KeyGEN total protein extraction kit (KeyGEN, Nanjing, China). The specific steps are as follows.
    [00110] [00110] (1) 1 pL of protease inhibitor, 10 pL of phosphatase inhibitor and 5 pL of 100 mM PMSF were added to a pre-cooled 1 mL Lysis Buffer respectively to prepare a mixed solution, which was evenly mixed and then stored on ice for several minutes for later use.
    [00111] [00111] (2) A fresh Pacific white shrimp was provided with the head, tail, shell and vein removed. The shrimp muscle was chopped into paste with a knife, placed into a mortar, added with liquid nitrogen and ground till powder.
    [00112] [00112] (3) 0.1 g of shrimp powder was quickly weighed and placed into a 1.5 mL pre-cooled centrifuge tube, added with 1 mL of the above mixed solution, mixed evenly and allowed to stand at 4°C for 2 h.
    [00113] [00113] (4) A resulting solution from the standing in step (3) was centrifuged at 10,000 r/min at 4°C for 5 min, to obtain a supernatant, which was stored at -80°C in aliquots to obtain a shrimp total protein infusion, which should be prevented from repeated freezing and thawing.
    [00114] [00114] (5) The extracted shrimp total protein infusion was eluted with a 0.2-0.4 M NaCl solution through an ANX Sepharose Fast Flow anion exchange chromatographic column to collect an eluted product, which was subjected to PBS dialysis to obtain the impure protein mixture solution of the Pacific white shrimp.
    [00115] [00115] (6) 1 mL of the crude protein mixture solution of the Pacific white shrimp was obtained and added with different amounts of AK (arginine kinase), with the AK concentrations being 5, 10, 20, 50 ng/mL. Among others, 6 parallel samples were provided for each concentration for sample testing and recovery rate determination. During sample testing, the impure protein mixtures of AK and Pacific white shrimp at different concentrations (5, 10, 20, 50 ug/mL) were tested with several crustacean allergen test 13 cards from the same batch obtained in Embodiment 2. With the test card placed horizontally, 100 uL of sample was loaded to the sample loading well, and then a timer with a resolution of tens of milliseconds was used to record a time required for the sample to pass through the 8 cm long capillary. The test results are as shown in Table 1, where the recovery rate is 93-103% with respect to the concentration range of 5-50 pg/mL, indicating that this method can be applied to the testing of the arginine kinase in actual samples.
    [00116] [00116] Table 1 Test results of manually loaded samples (n = 6) Actual Concentration Detected Sample Value | Recovery
    [00117] [00117] (7) 1 mL of the impure protein mixture solution of the Pacific white shrimp was obtained and added with different amounts of TM (tropomyosin), with the TM concentrations being 5, 10, 20, 50 ug/mL. Among others, 6 parallel samples were provided for each concentration for sample testing and recovery rate determination. During sample testing, the impure protein mixtures of TM and Pacific white shrimp at different concentrations (5, 10, 20, 50 pg/mL) were tested with several crustacean allergen test cards from the same batch obtained in Embodiment 2. With the test card placed horizontally, 100 uL of sample was loaded to the sample loading well, and then a timer with a resolution of tens of milliseconds was used to record a time required for the sample to pass through the 8 cm long capillary. The test results are as shown in Table 2, where the recovery rate is 94-102% with respect to the concentration range of 5-50 pg/mL, indicating that this method can be applied to the testing of the tropomyosin in actual samples.
    [00118] [00118] Table 2 Test results of manually loaded samples (n = 6) Actual Concentration | Detected Sample Value | Recovery Rate [en 14
    [00119] [00119] Embodiment 3
    [00120] [00120] The crustacean allergen test card obtained in Embodiment 2 was evaluated, with the specific method as follows.
    [00121] [00121] Precision Test:
    [00122] [00122] With the method in Embodiment 3, the tropomyosin and arginine kinase standard solutions at the concentrations of 10, 20, and 50 ug/mL were continuously tested 6 times respectively, with the test results shown in Table 3. The results show that the relative standard deviation of the arginine kinase content is 1.6-3.6%, and the relative standard deviation of the tropomyosin content is 2.7-3.6%, indicating that the test method was stable.
    [00123] [00123] Table 3 Test results on method precision (n = 6) Concentration / Sample | Test Time RSD% (ng/mL) 17°53", 17 "45" 1830 1737", 10 2.6 1822" ,17 25" we 15°25", 14°45" 14/20" 1455", Arginine . 20 3.6 Kinase 15°30" 1550" 12°25", 11 "53". 11 44° 1230", 50 3.4 12°15" ,11 "50° 20°30",19°45",19'36", 19" 16", 10 3.5 1855", 18 45" 17°28", 17 "35", 1655 1645", Tropomyosin 20 2.7 17 45.17 53" 14 10°, 1355 1345 1320", 50 3.6 1430" .14 37"
    [00124] [00124] Cross Reaction Test:
    [00125] [00125] The method in Embodiment 3 was used to test the shrimp impure protein mixture solution, namely a mixture solution containing other shrimp proteins besides the arginine kinase. Shrimp impure protein mixture solutions with concentrations of 10, 20, 30, 15 and 40 pg/mL were prepared for a specific test. The results are shown in FIG 4, indicating that the method 1s highly specific and shows no cross reaction to other shrimp proteins.
    [00126] [00126] In summary, the nucleic acid aptamer-hydrogel in the nucleic acid aptamer-hydrogel membrane tube in the invention specifically binds to the tropomyosin and the arginine kinase in the sample to affect the behavior of the hydrogel in the capillary; the change in migration time at different concentrations are recorded; and then the concentrations of the crustacean allergen tropomyosin and arginine kinase in the sample under test according to the change curve to achieve the level of quantitative testing. With the advantages of simple structure, easy operation, high sensitivity, easy storage, and good reliability, the test card of the invention can quantitatively and qualitatively determine the content of crustacean allergens in food, and has extremely high promotion and the practical value. Laboratory staff can operate it with little training.
    [00127] [00127] In the description of the present specification, the description of reference terms such as "an embodiment”, "some embodiments”, “example”, "specific example", or "some examples” is intended to indicate the inclusion of a specific feature, structure, material or characteristic as described by the embodiment or example into at least one embodiment or example of the present embodiment. In the present description, the schematic statement of the above terms should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific feature, structure, material or characteristic described can be incorporated in any one or more embodiments or examples in an appropriate manner. In addition, various embodiments or examples described in the present specification can be integrated and combined by those skilled in the art.
    [00128] [00128] Although the embodiments of the present invention have been illustrated and described above, it should be understood that the embodiments above are to be exemplary but not to limit the present invention. Those skilled in the art may make alternations, modifications, substitutions and variations to the embodiments above within the scope of the present invention. 16
    SEQUENCE LISTING <110> Zhejiang Gongshang University <120> CRUSTACEAN ALLERGEN TEST CARD AND APPLICATION THEREOF <130> 2019 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 45 <212> DNA <213> Artificial sequence <400> 1 ctacccagaa ctctcgcgtt agctcagtcg ctgtttcggg gccaa 45 <210> 2 <211> 45 <212> DNA <213> Artificial sequence <400> 2 ctacgaacga tgatgagtcc agagacatat tacttcgcgc ataaa 45 <210> 3 <211> 40 <212> DNA <213> Artificial sequence <400> 3 ccaggccgcc aacgttgacc tagaagcact gccagacccg 40 <210> 4 <211> 40 <212> DNA <213> Artificial sequence <400> 4 ctaccaggcc gccaacgttg acctagaagc actgccgacc 40 <210> 5 <211> 12 <212> DNA <213> Artificial sequence <400> 5 agttctgggt ag 12
    <210> 6
    <211> 12
    <212> DNA
    <213> Artificial sequence
    <400> 6 tggccccgaa ac 12 <210> 7
    <211> 12
    <212> DNA
    <213> Artificial sequence
    <400> 7 catcgttcgt ag 12 <2105 8
    <211> 12
    <212> DNA
    <213> Artificial sequence
    <400> 8 catcgttcgt ag 12 <2105 9
    <211> 12
    <212> DNA
    <213> Artificial sequence
    <400> 9 ttggcggcct gg 12 <210> 10
    <211> 12
    <212> DNA
    <213> Artificial sequence
    <400> 10
    Cgggtctggc ag 12 <21e> 11
    <211> 12
    <212> DNA
    <213> Artificial sequence
    <400> 11 gcggcctggt ag 12 <2105 12
    <211> 12
    <212> DNA
    <213> Artificial sequence
    <400> 12 ggtcggcagt gc 12
    权利要求:
    Claims (10)
    [1]
    A crustacean allergy test card, comprising: a back card (10) having a sample loading zone (11) and a test zone (12), the sample loading zone (11) connected 1s to the test zone (12) so that a sample is in the sample loading zone (11) moves to the test zone (12) and the test zone has a nucleic acid aptamer hydrogel membrane tube (121); and a front card (20) having a sample loading source (21), the sample loading source (21) adapted to load the sample to the sample loading zone (11).
    [2]
    The shellfish allergy test card of claim 1, wherein the nucleic acid aptamer hydrogel membrane tube (121) contains a silica capillary and a nucleic acid aptamer hydrogel; the nucleic acid aptamers in the nucleic acid aptamer hydrogel are adapted to bind specifically to tropomyosin and arginine kinase.
    [3]
    The shellfish allergy test card of claim 2, wherein the nucleic acid aptamers in the nucleic acid aptamer hydrogel contain a tropomyosin nucleic acid aptamer and an arginine kinase nucleic acid aptamer, the tropomyosin nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID No: 1 and SEQ ID No: 2 and the arginine kinase nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID No: 3 and SEQ ID No: 4.
    [4]
    The shellfish allergy test card of claim 2, wherein the silica capillary has a length of 8-10 cm and an inner diameter of 295-305 µm; and the nucleic acid aptamer hydrogel is 1-1.5 mm in length.
    [5]
    The crustacean allergy test card according to any one of the preceding claims, wherein the test zone (12) is scaled on both sides.
    [6]
    The shellfish allergy test card according to any one of the preceding claims, wherein the sample loading source (21) is funnel-shaped.
    [7]
    The shellfish allergy test card according to any one of the preceding claims, wherein the sample loading source (21) is covered with a piece of filter paper (211), the piece of filter paper being a circular piece of filter paper with a diameter of 1-1.5 cm and a pore size. of 2-3 µm.
    [8]
    The crustacean allergy test card according to any one of the preceding claims, wherein the crustacean allergy test card is prepared according to the following steps: (1) preparing a nucleic acid aptamer hydrogel, wherein the nucleic acid 17 aptamers in the nucleic acid aptamer hydrogel is a tropomyosin nucleic acid. aptamer and an arginine kinase nucleic acid contain aptamer; (2) inserting a heated silica capillary into the nucleic acid aptamer hydrogel to obtain the nucleic acid aptamer hydrogel membrane tube (121); (3) fixing the nucleic acid aptamer hydrogel membrane tube in the test zone (12), (4) covering the sample loading source (21) with a piece of filter paper (211), (5) scaling the test zone (12) on both sides ; and (6) joining the front card (20) and the back card (10) to obtain the crustacean allergy test card.
    [9]
    The shellfish allergy test card according to claim 8, wherein in step (2) the silica capillary is inserted 1 to 1.5 mm into the nucleic acid aptamer hydrogel for a time of 3-5 sec; and in step (2) the silica is capillary heated to 90-95 ° C over a heating time of 10-15 minutes.
    [10]
    A method for crustacean allergy testing, comprising plotting a standard curve for tropomyosin and a standard curve for arginine kinase; loading a sample to be tested into the test zone via the sample loading source in the crustacean allergy test card according to any one of claims 1 to 9, and recording a time period in which nucleic acid aptamer hydrogel in nucleic acid aptamer hydrogel membrane tube (121) reaches an end point to obtain a test value for the time period in which the nucleic acid aptamer hydrogel in the nucleic acid aptamer hydrogel membrane tube (121) reaches the end point; and substituting the test value of the time period when the nucleic acid aptamer hydrogel in the nucleic acid aptamer hydrogel membrane tube (121) reaches the endpoint in the tropomyosin standard curve and the arginine kinase standard curve to obtain a tropomyosin concentration and an arginine kinase concentration. in the sample to be tested.
    18
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    同族专利:
    公开号 | 公开日
    CN110456059A|2019-11-15|
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    CN201910583426.7A|CN110456059A|2019-07-01|2019-07-01|A kind of detection card of shellfish allergens and its application|
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