Food-borne pathogen detection test paper as well as preparation method and application thereof

Abstract

The invention provides a food-borne pathogen detection test paper based on isothermal amplification and near-infrared fluorescence chromatography technology and a preparation method and application thereof, immunomagnetic beads are adopted to carry out pathogen rapid enrichment and purification on a food specimen, an isothermal amplification method is adopted to carry out rapid amplification and marking on food-borne pathogens, and an RPA method is adopted to carry out rapid detection on the food-borne pathogens. Amplification and marking of a target gene are completed within 15 minutes, near-infrared fluorescence chromatography is adopted, an amplification product is rapidly detected, and 10 cps / mL of the target gene can be detected through RPA amplification; the sensitivity of the near-infrared fluorescence immunochromatography is 100 times higher than that of the colloidal gold immunochromatography, the detection sensitivity can be remarkably improved by combining the near-infrared fluorescence immunochromatography and the colloidal gold immunochromatography, large and expensive instruments, thermostats and handheld fluorescence chromatography equipment are not used, and the method can be conveniently applied to the field of food-borne pathogen detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of pathogen detection, and particularly relates to a foodborne pathogen detection test strip based on isothermal amplification and near-infrared fluorescence chromatography technology, a preparation method thereof, and an application thereof. Background Art

[0002] Foodborne diseases refer to diseases caused by pathogenic factors such as toxic and harmful substances (including biological pathogens) that enter the human body through ingestion. Such diseases usually have infectivity or toxicity, and can generally be divided into infectious and toxic types, including common food poisoning, intestinal infectious diseases, zoonotic infectious diseases, parasitic diseases, and diseases caused by chemical toxic and harmful substances. The diseases caused by foodborne pathogens are extremely harmful, and these harms are mainly reflected in the following aspects: physical discomfort and pain, organ damage, impaired immune system, nervous system and systemic symptoms, etc.

[0003] Since import and export commodities involve cross-border transactions, their quality and safety are directly related to the national image and the smooth progress of international trade. Therefore, the detection work not only requires high sensitivity to accurately identify potential risk factors, but also requires high time efficiency so as to take timely measures to avoid delaying transactions and causing economic losses. In the market circulation link, there are a wide variety of commodities with a fast circulation speed. Once problems occur, their influence range is extensive and rapid. Therefore, when detecting commodities in the market circulation, high-sensitivity detection technology and short detection time are also required. The freshly made and sold commodities have more stringent requirements for detection sensitivity and time. Freshly made and sold commodities usually refer to commodities made on-site and directly sold to consumers, such as street snacks, freshly baked bread, etc. The freshness and safety of such commodities are crucial to consumers. Since the production and sales process of freshly made and sold commodities is relatively short and often not subjected to strict pretreatment and packaging, the detection work needs to be completed within an extremely short time and requires accurate detection results. This requires testing institutions and personnel to have a high level of professional quality and advanced testing equipment to ensure the sensitivity and accuracy of the detection work.

[0004] Vibrio parahaemolyticus belongs to the genus Vibrio of the family Vibrionaceae and is a food- and water-borne pathogen. Vibrio parahaemolyticus has similar biological characteristics and the same pathogenicity, both of which can cause diarrhea and food poisoning. Vibrio parahaemolyticus was first isolated from a food poisoning patient in Japan by Fujino et al. in 1953 and is also known as halophilic bacteria. This bacterium can grow in a medium with a relatively high salt concentration (above 3% - 4%) and cannot reproduce in a salt-free medium, hence its name. Vibrio parahaemolyticus is distributed all over the world and is usually present in bays, coastal areas, and seafood. People often get food poisoning by eating contaminated seafood such as crabs, oysters, and shrimp that have not been properly processed. Food poisoning caused by Vibrio parahaemolyticus accounts for a high proportion among bacterial food poisonings, and its harm is only second to Salmonella, Escherichia coli, Staphylococcus, and Clostridium botulinum. There are reports of food poisoning caused by Vibrio parahaemolyticus every year in coastal areas of our country. The basic symptoms after people are poisoned include: diarrhea (watery or bloody stools), abdominal cramps, nausea, vomiting, and headache. There have also been reports of fever and chills, but they are less common.

[0005] Currently, the detection technologies for foodborne pathogens include: identification methods based on cultivation techniques (national standard methods); nucleic acid detection methods (including PCR, qPCR, isothermal amplification techniques, and other nucleic acid detection methods); immunological detection methods (colloidal gold immunochromatography; upconversion, etc.). However, the classic cultivation method takes a long time, and the immunological method has low sensitivity, which cannot meet the requirements of rapid detection. In addition, nucleic acid detection methods generally require fluorescence quantitative PCR instruments or other equipment and are not suitable for on-site rapid detection. None of the existing detection methods can meet the requirements of rapid detection. Summary of the Invention

[0006] The present invention provides a foodborne pathogen detection test strip based on isothermal amplification and near-infrared fluorescence chromatography techniques, as well as its preparation method and application. Immunomagnetic beads are used to rapidly enrich and purify pathogens from food specimens. The isothermal amplification method is used to rapidly amplify and label foodborne pathogens. The recombinase polymerase amplification (RPA) method is used to complete the amplification and labeling of target genes within 15 minutes. Near-infrared fluorescence chromatography is used to rapidly detect the amplification products, making it conveniently applicable to the field of foodborne pathogen detection.

[0007] In the first aspect, the present invention provides a preparation method for a foodborne pathogen detection test strip, including the following steps:

[0008] Step 1: Prepare the FAM monoclonal antibody fluorescent microsphere marker, including: taking activated carboxyl latex microspheres, using lysine as an intermediate bridging molecule, and coupling with a fluorescent dye to obtain carboxyl latex fluorescent microspheres; activating the carboxyl latex fluorescent microspheres and coupling with the FAM monoclonal antibody to obtain a FAM monoclonal antibody-carboxyl latex microsphere immunofluorescent complex; performing blocking and suspension to obtain the FAM monoclonal antibody fluorescent microsphere marker;

[0009] Step 2: Dilute the FAM monoclonal antibody fluorescent microsphere marker with a suspension and coat it on a fluorescent pad, and dry it at 55 °C for 2 h;

[0010] Step 3: Coat goat anti-mouse IgG and BIOTIN monoclonal antibody on a nitrocellulose membrane to obtain a coated membrane;

[0011] Step 4: Take the fluorescent pad prepared in Step 2 and the coated membrane prepared in Step 3, and combine them with a sample pad and a water absorption pad to form the foodborne pathogen detection test strip.

[0012] In some embodiments, in Step 1, activate the carboxyl latex microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC); activate the carboxyl latex fluorescent microspheres with EDC.

[0013] In some embodiments, in Step 1, the fluorescent dye is selected from Cy series dyes (Cy7.5), AlexaFluor series dyes (Alexa Fluor 750), IRDye series dyes (IRDye 800), Dylight series dyes (Dylight 800), etc., preferably Dylight800 NHS Ester. The addition amount of the fluorescent dye is 7 g of fluorescent dye per 100 g of antigen or antibody, and the antigen or antibody refers to the antigen antibody bound to the surface of the microspheres during the preparation of the fluorescent immunomicrospheres.

[0014] In some embodiments, in Step 1, the blocking includes: adding 100 uL of blocking agent per 100 ug of labeled antigen or antibody, and reacting at room temperature for 1 h. Preferably, the blocking agent is 1% casein aqueous solution or 1% bovine serum albumin aqueous solution.

[0015] In some embodiments, in Step 1, the suspension includes: resuspending the blocked microspheres with a suspension, adding 100 uL of suspension per 100 ug of labeled antigen or antibody, and standing overnight at 2-8 °C for reaction.

[0016] Preferably, the suspension includes: 20 mM boric acid buffer, 10% sucrose, 5% trehalose, 1% bovine serum albumin, 2% Tween20, pH 8.0.

[0017] In some embodiments, in step 2, the FAM monoclonal antibody fluorescent microsphere label is diluted 3000-fold.

[0018] In some embodiments, in step 2, the fluorescence pad is made of glass fiber.

[0019] In some embodiments, in step 3, the coating film is dried at 65 °C for 2 h.

[0020] In some embodiments, in step 3, a C line and a T line are formed on the coating film. Among them, C line: coat goat anti-mouse IgG polyclonal antibody on the nitrocellulose membrane, and add trehalose as a protective agent to the coating buffer; T line: coat BIOTIN monoclonal antibody on the nitrocellulose membrane, and add trehalose as a protective agent to the coating buffer. Preferably, the formulation of the C line coating buffer is: 10 mM PBS, 5% trehalose, pH 7.5; the formulation of the T line coating buffer is: 10 mM PBS, 5% trehalose, pH 7.5.

[0021] In a second aspect, the present invention provides a foodborne pathogen detection kit, which includes a foodborne pathogen detection test strip and a diluent obtained by the above preparation method. The diluent contains: 10 mM PBS buffer, 1% bovine serum albumin, 1% casein, 0.1% sucrose, 0.4% Tween-20, pH 7.0.

[0022] In a third aspect, the present invention provides a method for using the foodborne pathogen detection kit, including the following steps:

[0023] (1) Sample treatment: Add 1 μL of the isothermal amplification product to be tested to 99 μL of the diluent, and vortex and mix evenly to obtain a sample treatment solution;

[0024] (2) Sampling: Take 60 μL of the sample treatment solution and add it to the sample well of the test strip, place it horizontally, and chromatograph for 10 minutes;

[0025] (3) Detection: Insert the test strip into a fluorescence detector to detect the test paper card;

[0026] (4) Result interpretation: When the fluorescence detector shows "positive", it means that the sample contains Vibrio parahaemolyticus; when it shows "negative", it means that the sample does not contain Vibrio parahaemolyticus or the bacterial level is lower than the detection limit.

[0027] In some embodiments, the method of use further includes rapid enrichment and purification of pathogens in the sample using immunomagnetic beads, specifically including the following steps:

[0028] a. Rinse or resuspend the sample with PBS;

[0029] b. Add 10 μL of M1 magnetic beads to 1 mL of the sample;

[0030] c. Add calcium chloride to a final concentration of 6 mM, place on a mixer, and mix in a 37 °C incubator for 30 min;

[0031] d. Adsorb the magnetic beads with a magnetic stand, discard the supernatant, wash three times with PBS, and discard the supernatant;

[0032] e. Dissociate the sample from the magnetic beads with PBS containing 30 mM EDTA;

[0033] f. Boil the dissociated product for 5 min, centrifuge at 8000 rpm for 3 min, centrifuge to obtain the supernatant, and the supernatant can be directly used for RPA amplification.

[0034] In some embodiments, the usage method further includes rapidly amplifying and labeling foodborne pathogens by an isothermal amplification method. For Vibrio parahaemolyticus, the following primers and probes are used to obtain an isothermal amplification product:

[0035] Forward primer: 5′-ATTTCTGAGCTTATTGGCGGTTTCTGTCGG-3′

[0036] Reverse primer: 5′-biotin-TAACAGGCTCGCAAACGAATGAAAAGGTGG-3′

[0037] Probe: 5′-FAM-CAAGAGTCAACGTCGCCTGAAACTGTTCAC-(THF)-ACTACACCGTC GGCA-spacer C3-3′.

[0038] The present invention completes the amplification and labeling of the target gene within 15 minutes by an isothermal amplification technique (RPA method) to obtain an isothermal amplification product, and then uses the foodborne pathogen detection kit of the present invention to detect the isothermal amplification product. If the sample contains Vibrio parahaemolyticus, a product with both BIOTIN and FAM will appear in the amplification product, which can be detected by a test strip that recognizes Biotin and FAM and shows a positive result. The antibodies involved are Biotin mouse monoclonal antibody and FAM mouse monoclonal antibody, and both antibodies can be purchased externally. For example: Biotin monoclonal antibody 5D4 from Zhuhai Bomei Biotechnology Co., Ltd., and FITC monoclonal antibody 9B7 from Zhuhai Bomei Biotechnology Co., Ltd.

[0039] The present invention provides a simple and easy method for detecting Vibrio parahaemolyticus in a sample. The operator only needs to add the pre-diluted specimen to be tested to the test strip card, and read the fluorescence value after 10 minutes of reaction. The detector directly gives a negative or positive result, and the result interpretation is objective.

[0040] The foodborne pathogen detection test strip of the present invention, its preparation method and application have the following beneficial effects:

[0041] 1. High sensitivity. The RPA amplification can detect the target gene at 10 cps / mL; and the sensitivity of near-infrared fluorescence immunochromatography is 100 times higher than that of colloidal gold immunochromatography itself. The combination of the two can further greatly improve the detection sensitivity.

[0042] 2. Do not use large and expensive instruments. A thermostat plus a handheld fluorescence chromatographic device is sufficient; it is suitable for portable on-site detection.

[0043] 3. Low cost. The cost of a single detection is less than 10 yuan. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0045] Figure 1 It is a detection legend for the 1000-fold dilution of the positive amplification product in Example 2 of the present invention;

[0046] Figure 2 It is a detection legend for the 10,000-fold dilution of the positive amplification product in Example 2 of the present invention;

[0047] Figure 3 It is a detection legend for the 100,000-fold dilution of the positive amplification product in Example 2 of the present invention;

[0048] Figure 4 It is a detection legend for the 1,000,000-fold dilution of the positive amplification product in Example 2 of the present invention;

[0049] Figure 5 It is a detection legend for the 100-fold dilution of the negative amplification product in Example 2 of the present invention;

[0050] Figure 6 It is a detection legend for the colloidal gold control reagent in Comparative Example 1 of the present invention.

[0051] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0052] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0053] Example 1: Preparation of a nucleic acid isothermal amplification product detection test strip card for Vibrio parahaemolyticus

[0054] Preparation of a FAM monoclonal antibody fluorescent microsphere label

[0055] The carboxyl latex microspheres were fully activated with EDC under the condition of 50 mM Hepes buffer. Under the condition of using lysine as an intermediate bridging molecule, a fluorescent dye (product name: Dylight800 NHS Ester, manufacturer: Thermo Scientific, catalog number: 46421, addition amount: 7 μg of dye was added per 100 μg of antigen or antibody) was added, and 7 μL of Dylight800 was added, and it was allowed to stand in the dark for 1 h; chemical coupling occurred at room temperature to form fluorescent microspheres; the carboxyl latex fluorescent microspheres were fully activated with EDC under the condition of 50 mM Hepes buffer, and FAM monoclonal antibody was added. At room temperature, the FAM monoclonal antibody chemically coupled with the lysine carboxyl group on the carboxyl latex fluorescent microspheres to form a FAM monoclonal antibody-carboxyl latex microsphere immunofluorescent complex; then the preparation of the FAM monoclonal antibody fluorescent microsphere label could be completed through the blocking and suspension procedures.

[0056] Specifically included:

[0057] Blocking: 100 μL of a blocking agent (1% casein aqueous solution or 1% bovine serum albumin aqueous solution) was added per 100 μg of labeled antigen or antibody, and it was allowed to act at room temperature for 1 h.

[0058] Suspension: The blocked microspheres were resuspended with a suspension. 100 μL of the suspension was added per 100 μg of labeled antigen or antibody, and it was allowed to stand overnight at 2 - 8 °C for reaction. (Suspension: 20 mM boric acid buffer, 10% sucrose, 5% trehalose, 1% bovine serum albumin, 2% Tween 20, pH 8.0).

[0059] Among them:

[0060] The coupling of the FAM monoclonal antibody and the fluorescent microspheres is specifically as follows:

[0061] Labeling step: (Taking the labeling of 100 μL of microsphere solution as an example)

[0062] 1. Add 10 μL of P0112 carboxyl latex microspheres to 90 μL of 50 mM hepes to dilute the microspheres to w% = 0.5%.

[0063] 2. Dilute the EDC stock solution 10-fold with 50 mM hepes to 5 mg / mL, take 20 μL and add it to the diluted microsphere solution, and shake and activate at room temperature for 30 min.

[0064] 3. Centrifuge at 14680 rpm for 10 min to discard the supernatant, and resuspend with 100 μL of 50 mM hepes solution.

[0065] 4. Take 1 μL of lysine stock solution and add it to 684 μL of 1*PBS solution to prepare a lysine solution.

[0066] 5. Add 10 μL of lysine solution to the microsphere solution, and shake and bind at room temperature for 30 min.

[0067] 6. Centrifuge at 14680 rpm for 10 min to discard the supernatant, and resuspend with 100 μL of 50 mM hepes solution.

[0068] 7. Add 7 μL of Dylight800, and let it stand in the dark for 1 h.

[0069] 8. Centrifuge at 14680 rpm for 10 min to discard the supernatant, and resuspend with 100 μL of 50 mM hepes solution.

[0070] 9. Dilute the EDC stock solution 10-fold with 50 mM hepes to 5 mg / mL, take 20 μL of EDC and add it to the solution in step 8, and shake and activate at room temperature for 30 min.

[0071] 10. Centrifuge at 14680 rpm for 10 min to discard the supernatant, and resuspend with 100 μL of 50 mM hepes solution.

[0072] 11. Add 100 ug of FAM monoclonal antibody, and shake and bind at room temperature for 1 h.

[0073] 12. Centrifuge at 14680 rpm for (5 - 10 min) to discard the supernatant, resuspend with 100 uL of blocking agent (1% casein aqueous solution or 1% bovine serum albumin aqueous solution), and place it at 4 °C overnight for static blocking.

[0074] 13. Centrifuge at 14680 rpm for (10 - 15 min) to discard the supernatant, and resuspend with 100 μL of suspension.

[0075] Preparation of FAM Monoclonal Antibody Fluorescent Microsphere Labeled Fluorescent Pad

[0076] The prepared FAM monoclonal antibody fluorescent microsphere label was diluted with a suspension and evenly coated on a fluorescent pad made of glass fiber, and dried at 55 °C for 2 h.

[0077] Among them, the suspension: 20 mM boric acid buffer, 10% sucrose, 5% trehalose, 1% bovine serum albumin, 2% Tween 20, pH 8.0, and the dilution factor was 3000-fold dilution.

[0078] Coating:

[0079] C line: Coated with goat anti-mouse IgG polyclonal antibody on the nitrocellulose membrane. The formula of the C line coating buffer is: 10 mM PBS, 5% trehalose, pH 7.5;

[0080] T line: Coated with BIOTIN monoclonal antibody on the nitrocellulose membrane. The formula of the T line coating buffer is: 10 mM PBS, 5% trehalose, pH 7.5.

[0081] Dried at 65 °C for 2 h.

[0082] Preparation of the lateral flow test strip for detecting the nucleic acid isothermal amplification product of Vibrio parahaemolyticus

[0083] (1) Prepare the FAM monoclonal antibody fluorescent microsphere label according to the recorded fluorescent microsphere labeling method for FAM monoclonal antibody.

[0084] (2) Coat goat anti-mouse IgG and BIOTIN monoclonal antibody on the nitrocellulose membrane to obtain a coated membrane.

[0085] (3) Among them, (1) the FAM monoclonal antibody fluorescent microsphere label and (2) the coated membrane form the lateral flow test strip for detecting the nucleic acid isothermal amplification product of Vibrio parahaemolyticus.

[0086] (4) Among them, (3) together with the diluent (10 mM PBS buffer, 1% bovine serum albumin, 1% casein, 0.1% sucrose, 0.4% Tween-20, pH 7.0) and the instruction manual of the detection kit form the test kit for detecting the nucleic acid isothermal amplification product of Vibrio parahaemolyticus.

[0087] Example 2: Detection of the nucleic acid isothermal amplification product of Vibrio parahaemolyticus

[0088] Use immunomagnetic beads to rapidly enrich and purify the pathogen in the sample, which specifically includes the following steps:

[0089] a. Rinse or resuspend the sample with PBS;

[0090] b. Take 10 μL of M1 magnetic beads and add them to 1 mL of the sample, and the volume can be enlarged proportionally;

[0091] c. Add calcium chloride to a final concentration of 6 mM, place it on a mixer, and mix in a 37 °C incubator for 30 min;

[0092] d. Use a magnetic stand to adsorb the magnetic beads, discard the supernatant, wash three times with PBS, and discard the supernatant;

[0093] e. Dissociate the sample from the magnetic beads with PBS containing 30 mM EDTA;

[0094] f. Boil the dissociated product for 5 min, centrifuge at 8000 rpm for 3 min, centrifuge to collect the supernatant, and it can be directly used for RPA amplification.

[0095] Rapid amplification and labeling of foodborne pathogens using isothermal amplification method

[0096] Forward primer: 5′-ATTTCTGAGCTTATTGGCGGTTTCTGTCGG-3′

[0097] Reverse primer: 5′-biotin-TAACAGGCTCGCAAACGAATGAAAAGGTGG-3′

[0098] Probe: 5′-FAM-CAAGAGTCAACGTCGCCTGAAACTGTTCAC-(THF)-ACTACACC

[0099] GTCGGCA-spacer C3-3′

[0100] Perform amplification using an RPA kit (T00001A-48 Jiangsu Qitian Gene Biotechnology Co., Ltd.).

[0101] (1) Reagent preparation: Take out the reaction tubes in aluminum foil packaging from the kit, tear open the packaging, take out the reaction tubes required for the experiment on the same day, place them on a 96-well plate, and take out buffer V for standby. Immediately seal the remaining reaction tubes after unpacking with an aluminum foil tape and store them at -20 °C.

[0102] (2) Preparation of the reaction system:

[0103]

[0104]

[0105] (3) Manually flick and mix the above reaction system, centrifuge briefly, add it to the reaction tube, and gently flick by hand to fully dissolve and mix the lyophilized powder (this step cannot be mixed with a vortex shaker), and centrifuge briefly to collect the liquid at the bottom of the tube.

[0106] (4) Sampling: Open the reaction unit, add 5 μL of magnesium acetate to the inner side of the tube in each reaction unit, and then add 10 μL of negative control, the template DNA to be tested, and positive control to each reaction unit. Mix well and centrifuge again.

[0107] (5) Place the reaction tube in a constant temperature water bath at 37 °C and let it stand for 4 min.

[0108] (6) After thoroughly mixing the reaction tube by flicking it with fingers, place it in a hand-held centrifuge for a short centrifugation.

[0109] (7) Place the reaction tube in the water bath and react at 37 °C to obtain the amplification product.

[0110] Rapid detection of the amplification product is carried out by near-infrared fluorescence chromatography

[0111] 1. Sample treatment: Add 1 μL of the isothermal amplification reaction product to be tested to 99 μL of the diluent, and vortex to mix well to obtain the sample treatment solution;

[0112] 2. Sampling: Take 60 μL of the sample treatment solution and add it to the sample well of the test strip, place it horizontally, and perform chromatography for 10 minutes;

[0113] 3. Detection: Insert the test strip into the fluorescence detector to detect the test strip card;

[0114] 4. Result interpretation: If the fluorescence detector shows "positive", it means that the sample contains Vibrio parahaemolyticus; if it shows "negative", it means that the sample does not contain Vibrio parahaemolyticus or the bacterial level is lower than the detection limit.

[0115] Specific calculation process:

[0116] The fluorescence detector scans the fluorescence intensity at the control line (C line) of the test strip card, and through signal conversion and peak area integration, obtains the C area;

[0117] The fluorescence detector scans the fluorescence intensity at the test line (T line) of the test strip card, and through signal conversion and peak area integration, obtains the T area;

[0118] By calculating the ratio of the T area to the C area, the T / C is obtained;

[0119] By detecting the T / C values of a large number of samples without Vibrio parahaemolyticus and calculating the average value of their T / C values, the negative T / C average value is obtained; calculating the SD of their T / C values, the negative T / C SD is obtained;

[0120] By the formula negative T / C average value + 2 * negative T / C SD, the cut-off value of this detection reagent is obtained;

[0121] The positivity or negativity of the sample to be tested is determined by comparing the T / C value of the sample to be tested with the cut-off value;

[0122] If the T / C value of the sample to be tested is greater than the cut-off value, it is determined as positive;

[0123] If the T / C value of the sample to be tested is less than the cut-off value, it is determined as negative.

[0124] The larger the T / C, the more target products with both Biotin and FAM in the isothermal amplification products, indicating a higher content of Vibrio parahaemolyticus in the sample to be tested, that is, the more initial template numbers.

[0125] Positive sample: Vibrio parahaemolyticus is added as a template in the isothermal amplification, and the amplification products are serially diluted with a diluent to become the positive samples to be tested.

[0126] Negative sample: Vibrio parahaemolyticus is not added as a template in the isothermal amplification, and the amplification products are serially diluted with a diluent to become the positive samples to be tested.

[0127] The results are shown in Table 1 and Table 2.

[0128] Table 1

[0129] Amplification product Dilution factor of positive amplification product Area of C Area of T T / C 1000 658451 1295424 1.96738 1000 691215 1319687 1.90923 1000 620304 1229795 1.98257 10000 1230717 640734 0.52062 10000 1360227 696321 0.51192 10000 1211124 642824 0.53077 100000 1664848 169216 0.10164 100000 1631866 171074 0.10483 100000 1683207 180874 0.10746 1000000 1796818 19382 0.01079 1000000 2056191 18298 0.0089 1000000 2002991 18442 0.00921

[0130] Table 2

[0131]

[0132] Figures 1-4 Detection legends of the positive amplification products diluted 1000-fold, 10000-fold, 100000-fold, and 1000000-fold are shown; Figure 5 Detection legend of the negative amplification products diluted 100-fold is shown.

[0133] Comparative Example 1: The isothermal amplification products of positive samples were serially diluted and detected by the colloidal gold method

[0134] The preparation methods of the colloidal gold test strip and the near-infrared immunofluorescence test strip are generally the same. The difference is that the immunogold, that is, the colloidal gold-FAM monoclonal antibody conjugate, is laid on the colloidal gold conjugate pad of the colloidal gold test strip, while the fluorescent immunomicrospheres, that is, the near-infrared fluorescent microspheres-FAM monoclonal antibody conjugate, are laid on the fluorescent conjugate pad of the near-infrared immunofluorescence test strip. The preparation process of the colloidal gold-FAM monoclonal antibody conjugate is described in detail below.

[0135] Add 20uL 0.1M K2CO3 solution to 1mL of 40000 parts per million 60nm colloidal gold solution, mix well, add 15ug FAM mouse monoclonal antibody and mix well, label at room temperature for 20min, add 100uL 100uL blocking agent (1% casein aqueous solution or 1% bovine serum albumin aqueous solution) to block at room temperature for 15min, centrifuge at 12000rpm* (5min) to discard the supernatant, and use 2mL suspension to reconstitute. Suspension: 20mM boric acid buffer, 10% sucrose, 5% trehalose, 1% bovine serum albumin, 2% Tween20, pH 8.0. Take 600uL of the reconstituted immune gold solution and evenly apply it on the glass fiber fluorescent pad, and dry it at 55℃ for 2h to obtain the colloidal gold binding pad.

[0136] Colloidal gold control reagent test example Figure 6 It can be seen that when the isothermal amplification products of the same positive sample were subjected to gradient dilution detection, the detection limit of the colloidal gold method was 10,000 times dilution of the amplification product, while the detection limit of the near-infrared fluorescence immunochromatography method could reach 1,000,000 times, which is 100 times the sensitivity of the colloidal gold method. This shows that the near-infrared fluorescence immunochromatography method can significantly enhance the detection of positive samples when detecting the isothermal amplification products of Vibrio parahaemolyticus, and avoid the problem of false negatives to a great extent.

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a foodborne pathogen detection test strip, comprising the following steps: Step 1: Prepare the FAM monoclonal antibody fluorescent microsphere label, including: Taking activated carboxyl latex microspheres, using lysine as an intermediate bridging molecule, and coupling with a fluorescent dye to obtain carboxyl latex fluorescent microspheres; Activating the carboxyl latex fluorescent microspheres, coupling with FAM monoclonal antibody to obtain a FAM monoclonal antibody-carboxyl latex microsphere immunofluorescent complex; performing blocking and suspension to obtain the FAM monoclonal antibody fluorescent microsphere label; Step 2: Diluting the FAM monoclonal antibody fluorescent microsphere label with a suspension, coating it on a fluorescent pad, and drying it at 55 °C for 2 h; Step 3: Coating goat anti-mouse IgG and BIOTIN monoclonal antibody on a nitrocellulose membrane to obtain a coated membrane; Step 4: Taking the fluorescent pad prepared in Step 2 and the coated membrane prepared in Step 3, and combining them with a sample pad and an absorbent pad to form the foodborne pathogen detection test strip.

2. The preparation method according to claim 1, wherein In Step 1, the fluorescent dye is Dylight800NHSEster.

3. The preparation method according to claim 1, wherein, In Step 1, the blocking includes: adding 100 uL of a blocking agent to every 100 ug of labeled antigen or antibody, and reacting at room temperature for 1 h; the blocking agent is a 1% casein aqueous solution or a 1% bovine serum albumin aqueous solution.

4. The preparation method according to claim 1, wherein, In Step 1, the suspension includes: resuspending the blocked microspheres with a suspension, adding 100 uL of the suspension to every 100 ug of labeled antigen or antibody, and standing overnight at 2-8 °C; the suspension includes: 20 mM boric acid buffer, 10% sucrose, 5% trehalose, 1% bovine serum albumin, 2% Tween20, pH 8.

0.

5. The preparation method according to claim 1, wherein, In Step 3, a C line and a T line are formed on the coated membrane, wherein, C line: coating goat anti-mouse IgG polyclonal antibody on the nitrocellulose membrane, and adding trehalose as a protective agent to the coating buffer; T line: coating BIOTIN monoclonal antibody on the nitrocellulose membrane, and adding trehalose as a protective agent to the coating buffer.

6. The preparation method according to claim 5, wherein, The formulation of the C line coating buffer is: 10 mM PBS, 5% trehalose, pH 7.5; the formulation of the T line coating buffer is: 10 mM PBS, 5% trehalose, pH 7.

5.

7. A foodborne pathogen detection kit, the kit contains a foodborne pathogen detection test strip obtained by the preparation method according to any one of claims 1-6, and a diluent, the diluent contains: 10 mM PBS buffer, 1% bovine serum albumin, 1% casein, 0.1% sucrose, 0.4% Tween-20, pH 7.

0.

8. A method for using the foodborne pathogen detection kit according to claim 7, comprising the following steps: (1) Sample treatment: Adding 1 uL of the isothermal amplification product to be tested into 99 uL of the diluent, vortexing and mixing evenly to obtain a sample treatment solution; (2) Sampling: Taking 60 uL of the sample treatment solution and adding it into the sample well of the test strip, placing it horizontally, and performing chromatography for 10 minutes; (3) Detection: Inserting the test strip into a fluorescence detector to detect the test paper card; (4) Result interpretation: If the fluorescence detector shows "positive", it indicates that Vibrio parahaemolyticus is present in the sample; if it shows "negative", it indicates that Vibrio parahaemolyticus is not present in the sample or the bacterial level is below the detection limit.

9. The usage method according to claim 7, wherein, It also includes using immunomagnetic beads to rapidly enrich and purify pathogens in the sample, specifically including the following steps: a. Rinse or resuspend the sample with PBS; b. Add 10 μL of M1 magnetic beads to 1 mL of the sample; c. Add calcium chloride to a final concentration of 6 mM, place it on a mixer, and mix in a 37°C incubator for 30 min; d. Adsorb the magnetic beads with a magnetic stand, discard the supernatant, wash three times with PBS, and discard the supernatant; e. Dissociate the sample from the magnetic beads with PBS containing 30 mM EDTA; f. Boil the dissociated product for 5 min, centrifuge at 8000 rpm for 3 min, take the supernatant after centrifugation, and it can be directly used for RPA amplification.

10. The usage method according to claim 7, wherein, It also includes using an isothermal amplification method to rapidly amplify and label foodborne pathogens. For Vibrio parahaemolyticus, the following primers and probes are used to obtain an isothermal amplification product: Forward primer: 5′-ATTTCTGAGCTTATTGGCGGTTTCTGTCGG-3′ Reverse primer: 5′-biotin-TAACAGGCTCGCAAACGAATGAAAAGGTGG-3′ Probe: 5′-FAM-CAAGAGTCAACGTCGCCTGAAACTGTTCAC-(THF)-ACTACACCGTC GGCA-spacerC3-3′.

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