Immunochromatography kit and immunochromatography test strip detection method for ricin

Abstract

The invention provides an immunochromatography kit and an immunochromatography test strip detection method for ricin, and belongs to the technical field of toxin detection. The method comprises the following steps: mixing and incubating an oligonucleotide chain substrate labeled with a fluorophore and biotin, a buffer solution, a BSA solution and a sample to be detected, and adding ammonia water to terminate the reaction to obtain a reaction solution; adding the reaction solution to an immunochromatography test strip, incubating in a dark place, detecting a fluorescence signal value, and determining whether ricin exists according to a difference value between a final sample fluorescence signal value and a final fluorescence signal value of negative control; the sample to be detected is enriched by using antibody-coated magnetic beads; if the fluorescence signal value of the sample is lower than the negative control fluorescence signal value-3 times of the standard deviation, it is judged that ricin exists. No cross reaction exists between the detection method and an abrus precatorius toxin sample, and the detection sensitivity can reach 0.17 ng / mL. When the kit is applied to detection of a milk simulation sample, the sensitivity can reach 1.53 ng / mL.

Description

Technical Field

[0001] The invention belongs to the technical field of toxin detection, and in particular relates to an immunochromatographic kit and an immunochromatographic test strip detection method for ricin. Background Art

[0002] Ricin toxin (RT) is derived from Ricinus communis, a plant of the Euphorbiaceae family. It is a plant toxin protein. It is highly toxic and easy to prepare. Therefore, being able to quickly identify RT on site is of great significance to maintaining national security and long-term social stability.

[0003] RT is composed of two polypeptide chains, A and B, connected by multiple disulfide bonds. The B chain is non-toxic but can mediate the entry of the A chain into cells, and the A chain mainly plays a toxic role. RT is very similar to abrin toxin (AT) in structure and biological characteristics. They are both type II ribosome-inactivating proteins (RIPs) with N-glycosidase activity, which can specifically and irreversibly hydrolyze adenine (A) on the 28S rRNA of the ribosome of organisms. 4324 ), resulting in the inability of the ribosome to bind to the elongation factor 2, thereby inhibiting the translation function of the ribosome, inhibiting protein synthesis, and then exerting a toxic effect. N-glycosidase activity causes a base deletion site (apurinic / apyrimidinic site, AP Site) to be formed at the corresponding position after adenine is detached. The inventors discovered for the first time that AT has AP lyase activity, which can continue to act on the formed AP site and break the nucleic acid chain after exerting N-glycosidase activity. In addition, a rapid and sensitive fluorescence signal detection method was established based on this enzyme activity (patent number: ZL 202311802616.6).

[0004] The enzyme activity of toxins not only plays an important role in organisms, but is also often used as a means of detection. Since inactivated toxins do not pose a hazard, detection methods based on enzyme activity are favored by many scholars. At present, the detection methods based on RT enzyme activity mainly focus on N-glycosidase activity, and detection is achieved by detecting the content of adenine through mass spectrometry technology. The advantages of this method are mainly strong specificity and high sensitivity, and it can detect whether RT and AT are toxic. However, this method often takes about 2 to 5 hours from incubation of toxins and substrates to on-machine detection (the length of incubation time depends on the content of toxins in the sample), which is time-consuming and not conducive to rapid detection. In addition, the instrument is expensive and costly.

[0005] If the AP lyase activity of RT can be combined with immunochromatographic test strip technology to establish a portable and highly sensitive detection method, it will be of great significance for the detection, identification and characterization of RT in various sample matrices. Summary of the invention

[0006] In view of this, the object of the present invention is to provide an immunochromatographic kit and an immunochromatographic test strip detection method for ricin toxin, so as to solve the problems that the existing RT detection method is time-consuming and not convenient for rapid detection.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an immunochromatographic kit for detecting ricin, comprising an immunochromatographic test strip, an oligonucleotide chain substrate labeled with a fluorescent group and biotin;

[0009] The oligonucleotide chain is a single-stranded polyadenine nucleotide, and the number of adenines in the single-stranded polyadenine nucleotide is 6 to 15;

[0010] The immunochromatographic test strip comprises a sample pad, a cellulose membrane and a water-absorbing pad which are overlapped in sequence, and a detection line and a quality control line are arranged in sequence on the cellulose membrane starting from one end close to the sample pad.

[0011] The detection line is coated with streptavidin that can specifically bind to biotin, and the quality control line is coated with green fluorescent microspheres.

[0012] Preferably, it also includes a buffer solution, a BSA solution and ammonia water.

[0013] Preferably, the cellulose membrane is Hi-Flow TM Plus Membrane 180.

[0014] The present invention also provides a method for detecting ricin by an immunochromatographic test strip, comprising the following steps:

[0015] 1) After mixing and incubating the oligonucleotide chain substrate labeled with fluorescent group and biotin, buffer, BSA solution, and the sample to be tested, ammonia water is added to terminate the reaction to obtain a reaction solution;

[0016] 2) adding the reaction solution to an immunochromatographic test strip, incubating in the dark, detecting the fluorescence signal value, and determining whether ricin is present based on the difference between the final fluorescence signal value of the sample and the final fluorescence signal value of the negative control;

[0017] The sample to be tested is enriched using antibody-coated magnetic beads;

[0018] The presence of ricin can be determined when the fluorescence signal value of the sample is lower than the fluorescence signal value of the negative control minus 3 times the standard deviation.

[0019] Preferably, in step 1), the concentration of the oligonucleotide chain substrate labeled with fluorescent group and biotin is 0.01 μM to 25 μM.

[0020] Preferably, the buffer in step 1) is ammonium citrate buffer, and the concentration of the ammonium citrate buffer is 2-3 mM.

[0021] Preferably, the concentration of the ammonia water in step 1) is 50-400 mM.

[0022] Preferably, the antibody-coated magnetic beads are Dynabeads TM .

[0023] Preferably, the temperature of the light-proof incubation in step 2) is 22 to 27° C., and the time of the light-proof incubation is 10 to 20 minutes.

[0024] Preferably, in step 1), the concentration of BSA is 80-120 μg / mL; the volume ratio of the oligonucleotide chain substrate labeled with fluorescent group and biotin, buffer, BSA solution, and the sample to be tested is 7-9:7-9:7-9:7-9;

[0025] The incubation temperature is 55-62° C., and the incubation time is 40-80 min.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention independently designs the single-stranded DNA reaction substrate sequence, and performs FAM fluorescent group and biotin modification on both ends. And for the first time, the AP lyase activity of AT is combined with the immunochromatography technology, utilizing the AP lyase activity of AT and using the nucleic acid substrate as the "antigen", and capturing the reaction substrate by fixing streptavidin to the T line of the test strip, and detecting AT by the line elimination method. The reaction principle is that AT first exerts its N-glycosidase activity to act on the adenine on the oligonucleotide chain, and then further exerts the AP lyase activity to break the oligonucleotide substrate. When the negative sample is chromatographed, the biotin on the substrate will be retained by the streptavidin fixed on the T line, and the T line has a higher fluorescence signal value. When the positive sample is chromatographed, the broken substrate containing biotin will be retained by the streptavidin fixed on the T line, and the broken substrate containing the fluorescent group will migrate, thereby reducing the fluorescence signal value of the T line (such as Figure 1 shown).

[0028] The reaction system of the method of the present invention has simple components, including only bovine serum albumin, ammonium citrate buffer and oligonucleotide substrates modified with fluorescent groups and biotin. The present invention constructs a complete test strip detection system by optimizing the components and concentration of the reaction system, the length of the reaction substrate, the type of NC membrane of the test strip, the type and concentration of the T-line fixed antibody, etc. The detection method only needs to be incubated at 59°C for 1h in the early stage to complete the detection. When the sample toxin reacts directly and is not enriched by antibody-coated magnetic beads, the detection sensitivity of the method can reach 1.65ng / mL; after enrichment with antibody-coated magnetic beads, no cross reaction with the abrin toxin sample is found, and the detection sensitivity can reach 0.17ng / mL. In addition, it is applied to the detection of milk simulation samples, and the sensitivity can reach 1.53ng / mL. The reaction system components and reaction conditions of the present invention are simple, the reaction time is short, and no expensive detection instruments and complex professional operations are required. It breaks through the technical barriers of the current RT detection method and can be used for on-site rapid screening and rapid detection of in vitro RT poisoning samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the principle of ricin immunochromatographic test strip detection of the present invention;

[0030] Figure 2 The influence of different nucleic acid substrates on reaction efficiency and detection effect;

[0031] Figure 3 It is the effect of different buffer solutions on the detection effect;

[0032] Figure 4 The effect of different ammonia concentrations on the detection effect;

[0033] Figure 5 It is the effect of different streptavidin markers on the T line on the test strip on the detection sensitivity;

[0034] Figure 6 The influence of different NC membranes of the test strips on the detection effect;

[0035] Figure 7 The effect of different types of magnetic beads on the enrichment of ricin;

[0036] Figure 8 is a linear gradient of different concentrations of reaction substrate DNA10A;

[0037] Fig. 9 This is the test result of the sensitivity of water samples when magnetic beads coated with antibodies are not used for sample enrichment;

[0038] Fig.10 It is the test result of the sensitivity of water samples when using antibody-coated magnetic beads to enrich samples;

[0039] Fig.11 It is the test result of the sensitivity of milk samples when using antibody-coated magnetic beads for sample enrichment;

[0040] Fig.12 This is the specific verification result of ricin. DETAILED DESCRIPTION

[0041] The present invention provides an immunochromatographic kit for detecting ricin, comprising an immunochromatographic test strip, an oligonucleotide chain substrate labeled with a fluorescent group and biotin;

[0042] The oligonucleotide chain is a single-stranded polyadenine nucleotide, and the number of adenines in the single-stranded polyadenine nucleotide is 6 to 15;

[0043] The immunochromatographic test strip comprises a sample pad, a cellulose membrane and a water-absorbing pad which are overlapped in sequence, and a detection line and a quality control line are arranged in sequence on the cellulose membrane starting from one end close to the sample pad.

[0044] The detection line is coated with streptavidin that can specifically bind to biotin, and the quality control line is coated with green fluorescent microspheres.

[0045] In the present invention, the immunochromatography kit further comprises a buffer solution, a BSA solution and ammonia water, and the cellulose membrane is a Hi-Flow TM Plus Membrane 180.

[0046] The present invention also provides a method for detecting ricin by an immunochromatographic test strip, comprising the following steps:

[0047] 1) After mixing and incubating the oligonucleotide chain substrate labeled with fluorescent group and biotin, buffer, BSA solution, and the sample to be tested, ammonia water is added to terminate the reaction to obtain a reaction solution;

[0048] 2) adding the reaction solution to an immunochromatographic test strip, incubating in the dark, detecting the fluorescence signal value, and determining whether ricin is present based on the difference between the final fluorescence signal value of the sample and the final fluorescence signal value of the negative control;

[0049] The sample to be tested is enriched using antibody-coated magnetic beads;

[0050] The presence of ricin can be determined when the fluorescence signal value of the sample is lower than the fluorescence signal value of the negative control minus 3 times the standard deviation.

[0051] In the present invention, the sample to be tested is enriched using antibody-coated magnetic beads, and the oligonucleotide chain substrate labeled with fluorescent group and biotin, buffer, BSA solution, and the sample to be tested are mixed and incubated, and then ammonia water is added to terminate the reaction to obtain a reaction solution; the antibody-coated magnetic beads are preferably Dynabeads TM ; The concentration of the oligonucleotide chain substrate labeled with fluorescent groups and biotin is preferably 0.01μM to 25μM, and more preferably 0.25μM to 12.5μM; the buffer is preferably ammonium citrate buffer, and the concentration of the ammonium citrate buffer is preferably 2 to 3mM, and more preferably 2.2 to 2.8mM; the concentration of BSA is preferably 80 to 120μg / mL, and more preferably 90 to 110μg / mL; the volume ratio of the oligonucleotide chain substrate labeled with fluorescent groups and biotin, the buffer, the BSA solution, and the sample to be tested is preferably 7 ~9:7~9:7~9:7~9, more preferably 7.5~8.5:7.5~8.5:7.5~8.5:7.5~8.5; the incubation temperature is preferably 55~62°C, more preferably 56~61°C; the incubation time is preferably 40~80min, more preferably 50~70min; the concentration of the ammonia water is preferably 50~400mM, more preferably 80~300mM; the volume ratio of the ammonia water to the buffer solution is preferably 7~9:7~9, more preferably 7.5~8.5:7.5~8.5.

[0052] In the present invention, the reaction solution is added to the immunochromatographic test strip, incubated in the dark, and the fluorescence signal value is detected. The difference between the final sample fluorescence signal value and the final fluorescence signal value of the negative control is used to determine whether ricin is present. The temperature of the light-proof incubation is preferably 22 to 27°C, more preferably 23 to 26°C; the time of the light-proof incubation is preferably 10 to 20 minutes, more preferably 12 to 18 minutes; the sample fluorescence signal value is lower than the negative control fluorescence signal value minus 3 times the standard deviation to determine the presence of ricin.

[0053] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] Main reagents and instruments used in the present invention

[0055] (1) Main reagents

[0056] Dynabeads TM Antibody coupling kit (14311D), Dynabeads TM M-270 streptavidin (65305): Thermo Fisher Scientific;

[0057] Bovine serum albumin (B2064), ammonium citrate (25102): Sigma-Aldrich (Shanghai) Trading Co., Ltd.;

[0058] Streptavidin (T line) (2022042602): Bovo Biotechnology;

[0059] Streptavidin (189737): Sigma-Aldrich (Shanghai) Trading Co., Ltd.;

[0060] Hi-Flow TM Plus Membrane 130 and Hi-Flow TM Plus Membrane 180: Millipore, USA;

[0061] Ammonia (1336-21-6): Sigma-Aldrich (Shanghai) Trading Co., Ltd.;

[0062] All oligonucleotide substrates were synthesized by GenScript Biotech Co., Ltd.

[0063] (2) Main instruments

[0064] Biological safety cabinet: NUAIRE, USA;

[0065] Vortex mixer: Jiangsu Haimen Qilin Bell Instrument Co., Ltd.

[0066] HμLaMixer TM Sample mixer (15920D): Thermo Fisher Scientific;

[0067] Constant temperature incubator: Tianjin Sateris Laboratory Analysis Instrument Manufacturing Plant;

[0068] FAM nucleic acid analyzer (JW-1901): Chengdu Weirui Biotechnology Co., Ltd.

[0069] The detection method of the present invention mainly consists of two parts: a reaction system and a chromatography system. The experiment is divided into two parts. The first part is the reaction stage. RT and a single-stranded DNA substrate are incubated at a constant temperature of 59°C for 1 hour in a 2.5mM ammonium citrate buffer (pH 4) for reaction. In this stage, AT mainly exerts AP lyase activity to cut the single-stranded DNA substrate. The second part is the chromatography stage. After the reaction is completed, a certain concentration of ammonia water is added to adjust the pH of the system, and then the system is added to the sample loading hole of the test strip for chromatography, and the fluorescence signal value is detected to achieve the detection of the sample.

[0070] Example 1

[0071] Optimization of oligonucleotide strand substrate

[0072] Three oligonucleotide chain substrates of different lengths were designed, namely DNA6A, DNA10A and DNA15A. The 5' ends of all nucleotide chains were labeled with FAM fluorescent group and the 3' ends were labeled with biotin. They were then synthesized by GenScript Biotech Co., Ltd. (as shown in Table 1).

[0073] Table 1 Sequence information of single-stranded DNA substrates of different lengths

[0074]

[0075] This experiment involves the reaction stage of RT and substrate in the early stage. In order to compare the effects of oligonucleotide chain substrates of different lengths on the reaction efficiency and detection effect, three oligonucleotide chain substrates of different lengths, DNA6A, DNA10A and DNA15A, were selected as the research objects for exploration (see Table 1). Different substrates were diluted to 12.5μM with sterile water. The total volume of the early reaction system was 64μL, with 16μL of each component, including oligonucleotide chain substrate, 100μg / mL BSA, 2.5mM ammonium citrate buffer (pH 4) and 10μg / mL RT. After the reaction system was mixed and incubated at 59℃ for 1h, 16μL of 100mM ammonia water was added to the reaction system to terminate the reaction as a chromatography system. After mixing evenly, 75μL was added to the spotting area of ​​the test strip, and the fluorescence signal value was detected using a FAM nucleic acid analyzer after incubation at room temperature in the dark for 15min.

[0076] Experimental results: Figure 2 As shown by Figure 2 It can be seen that when the substrate DNA10A was used for the reaction, compared with the negative control, the fluorescence signal value decreased most significantly and the reaction effect was the best. Therefore, DNA10A was selected as the final reaction substrate for subsequent experiments.

[0077] Example 2

[0078] Screening of buffers for chromatography

[0079] This experiment does not involve the reaction stage of RT and substrate in the early stage, and directly optimizes the subsequent chromatography system. PBS, PBST, ammonium citrate buffer (pH 4), and ammonium citrate buffer (pH 4) + ammonia water were used as buffers respectively. Sterile water was used to dilute the reaction substrate DNA10A to a concentration of 12.5 and 25 μM, respectively. The concentration of ammonium citrate buffer was 2.5 mM, and the concentration of ammonia water was 100 mM. Each reaction system contained 16 μL of buffer, 16 μL of reaction substrate, 16 μL of 100 μg / mL BSA solution, and the remaining components were filled to 80 μL with sterile water. After fully blowing and mixing, 75 μL of the mixture was drawn and added to the sample area of ​​the test strip, incubated at room temperature in the dark for 15 minutes, and then the test strip was inserted into the FAM nucleic acid analyzer to detect the fluorescence signal value.

[0080] Experimental results: Figure 3 As shown. Figure 3 It can be seen that under the four buffer systems, the fluorescence signal value increased after the substrate concentration increased. The pH value has a great influence on the chromatography process and fluorescence signal detection. The neutral buffer is more conducive to chromatography and detection. The ammonium citrate buffer (pH 4) buffer system seriously inhibits the detection of the fluorescence signal value, but the situation is greatly improved after using ammonium citrate buffer (pH 4) + ammonia water as a buffer to adjust the pH of the system. Although the effect is still inferior to the detection effect of PBS and PBST buffer systems, since ammonium citrate buffer (pH 4) is the best reaction system for RT to exert AP lyase activity, the subsequent experiments selected the mode of ammonium citrate buffer + ammonia water to adjust pH as the chromatography system for experiments.

[0081] Example 3

[0082] Optimizing the concentration of ammonia in the chromatography system

[0083] This experiment does not involve the reaction stage of RT and substrate in the early stage, and directly optimizes the subsequent chromatography system. It is determined that the final chromatography system on the test strip is an ammonium citrate buffer + ammonia water to adjust the pH system. The concentration of ammonia water in the system is selected as the research object, and the concentration of ammonium citrate buffer is 2.5mM unchanged. The concentrations of ammonia water are set to 50mM, 100mM, 200mM and 400mM respectively. Use sterile water to dilute the reaction substrate DNA10A to a concentration of 12.5μM. The chromatography system contains 16μL of reaction substrate, 16μL of ammonia water of different concentrations + 16μL of ammonium citrate buffer 16μL of 100μg / mL BSA solution, and the remaining components are filled with sterile water to 80μL. After fully blowing and mixing, 75μL of the mixture is drawn and added to the test strip spot area, incubated at room temperature in the dark for 15min, and then the test strip is inserted into the FAM nucleic acid analyzer to detect the fluorescence signal value.

[0084] Experimental results: Figure 4 As shown. Figure 4 It can be seen that the best reaction effect is achieved when the concentration of ammonia water is 100 mM. Therefore, the subsequent chromatography system is 2.5 mM ammonium citrate buffer (pH 4) + 100 mM ammonia water.

[0085] Example 4

[0086] Optimizing the Streptavidin T-Line Marker on the Test Strip

[0087] In order to screen for streptavidin (SA) with higher detection sensitivity, two different brands of SA were selected to mark the T line of the NC membrane as the detection line. The SA of Sigma-Aldrich (Shanghai) Trading Co., Ltd. is referred to as JK SA, and the SA of Bowo Bio is referred to as WR SA. The concentration of SA is 5 mg / mL. Prepare 80 μL of chromatography system, 16 μL of each component, including substrate DNA10A with concentrations of 2.5 μM, 5 μM, and 12.5 μM, 100 μg / mL BSA, 2.5 mM ammonium citrate buffer (pH 4), 100 mM ammonia water and sterile water. After mixing the system, take 75 μL and add it to the sample area of ​​the test strip. After incubation at room temperature in the dark for 15 minutes, use FAM nucleic acid analyzer for detection.

[0088] Experimental results: Figure 5 As shown by Figure 5 It can be seen that with the increase of substrate concentration, the fluorescence signal value of WRSA is significantly higher and can show a good gradient change, while the fluorescence signal value of JKSA is low and does not show a gradient change with the increase of substrate concentration, and has reached saturation. Therefore, WRSA was selected to be fixed on the T line of the test strip in subsequent experiments.

[0089] Example 5

[0090] Optimizing the NC membrane of the test strip

[0091] In order to select the most suitable NC membrane for AT detection, two different NC membranes were used, Hi-Flow TM PlusMembrane 130 (M130 for short) and Hi-Flow TMPlus Membrane 180 (abbreviated as M180) was made into a test strip for comparison of detection effects. 80μL of the chromatography system was prepared, with 16μL of each component, including 12.5μM substrate DNA10A, 100μg / mL BSA, 2.5mM ammonium citrate buffer (pH 4), 100mM ammonia water and sterile water. After the system was mixed, 75μL was added to the sample addition area of ​​the test strip, incubated at room temperature in the dark for 15min, and then detected using a FAM nucleic acid analyzer.

[0092] Experimental results: Figure 6 As shown. Figure 6 It can be seen that the chromatography effect of using the M180 NC membrane is better and the fluorescence signal intensity is higher, and the M180 NC membrane will be used for subsequent experiments.

[0093] Example 6

[0094] Screening for the best magnetic beads to use during the reaction

[0095] In the face of the detection of complex matrix samples, monoclonal antibody-coated magnetic beads are needed to enrich ricin in complex matrix samples. Therefore, the enrichment effect of different types of magnetic beads was explored. DynabeadsTM Antibody Coupling Kit (14311D) is referred to as Dead, and DynabeadsTM M-270 Streptavidin (65305) is referred to as M270. The experiment was carried out using two types of antibody-coated Dead and M270 magnetic beads. 25μg of antibody was coated with 1mg of magnetic beads, and 50μg of magnetic beads with a concentration of 1mg / mL were used for each sample. 500μL of RT samples diluted with sterile water at a concentration of 20ng / mL were incubated with the two magnetic beads at room temperature for 1h, and then adsorbed with a magnetic stand for 1min, and the supernatant was removed. Sterile water was used as a negative control. Add the treated magnetic beads to the reaction system, a total of 64 μL, 16 μL of each component, including 12.5 μM substrate, 100 μg / mL BSA, 2.5 mM ammonium citrate buffer (pH 4) and sterile water. After incubation at 59°C for 1 hour, add 16 μL of 100 mM ammonia water. Gently blow and mix the system and adsorb it on a magnetic stand for 1 minute. Take 75 μL of supernatant and add it to the sample well of the test strip. Incubate at room temperature in the dark for 15 minutes and then use a FAM nucleic acid analyzer for detection.

[0096] Experimental results: Figure 7 As shown. Figure 7 It can be seen that the detection effect using Dead magnetic beads is better than that using M270 magnetic beads, so Dead magnetic beads were used for subsequent detection.

[0097] Example 7

[0098] Linear gradient of reaction substrate

[0099] The linear gradient range of the reaction substrate DNA10A was explored using the optimized reaction system. The substrate was diluted to 0.05μM, 0.25μM, 0.5μM, 2.5μM, 5μM, 12.5μM, 25μM, and 50μM with sterile water, and added to the optimized chromatography system, including 16μL of 2.5mM ammonium citrate buffer (pH 4), 16μL of reaction substrate, 16μL of 100μg / mL BSA, 16μL of 100mM ammonia water, and 16μL of sterile water. After thorough mixing, 75μL was drawn and added to the sample well of the test strip, and the FAM nucleic acid analyzer was used for detection after incubation at room temperature for 15min.

[0100] Experimental results: Figure 8 As shown. Figure 8 It can be seen that when the substrate concentration is 0.01 μM-25 μM, it has a good linear relationship with the detected fluorescence signal value.

[0101] Example 8

[0102] Optimization of detection sensitivity by adding samples directly into the reaction system

[0103] When the antibody-coated magnetic beads were not used for sample enrichment, the sensitivity of the sample was tested using the optimized reaction system (16 μL per component, including 12.5 μM substrate DNA10A, 100 μg / mL BSA, 2.5 mM ammonium citrate buffer (pH 4), the sample to be tested, and 100 mM ammonia water). Sterile water was used as a negative control, and the reaction was carried out at 59°C. The detection concentrations of RT were 0.3125 ng / mL, 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL. The size of the reaction system was 64 μL, including 16 μL of substrate with a final concentration of 5 μM, 16 μL of BSA with a final concentration of 20 μg / mL, 16 μL of ammonium citrate buffer (pH 4) with a final concentration of 0.5 mM, and 16 μL of different concentrations of RT. After gently blowing and mixing the system, incubate at 59℃ for 1h, and finally add 16μL of 100mM ammonia water. After mixing, take 75μL of the system and add it to the sample well of the test strip. After incubating at room temperature in the dark for 15min, use FAM nucleic acid analyzer for detection. The sample is judged as positive if the fluorescence signal value of the sample is lower than the fluorescence signal value of the negative control -3 times the standard deviation. The test results are shown in the figure.

[0104] Experimental results: Fig. 9 As shown. Fig. 9It can be seen that when the sample is not enriched using antibody-coated magnetic beads, the detection limit of this detection method is 1.65 ng / mL.

[0105] Example 9

[0106] Sensitivity test of samples added to the reaction system after enrichment with antibody-coated magnetic beads

[0107] Antibody-coated Dead magnetic beads were used to enrich water and milk samples, 25 μg monoclonal antibody was coated with 1 mg magnetic beads, and 50 μg magnetic beads were used for each sample. The volume of water and milk samples was 500 μL. After flipping and incubating with monoclonal antibody-coated magnetic beads for 1 hour at room temperature, they were placed on a magnetic stand for adsorption for 1 minute, and the supernatant was discarded. The treated magnetic beads were added to the optimized system (16 μL of each component, including 12.5 μM substrate DNA10A, 100 μg / mL BSA, 2.5 mM ammonium citrate buffer (pH 4), the sample to be tested, and 100 mM ammonia water), gently blow and mix, and flip and incubate at 59 ° C for 1 hour. The concentration of RT in the simulated water sample was set to 0.15625ng / mL, 0.3125ng / mL, 0.625ng / mL, 1.25ng / mL, 2.5ng / mL, 5ng / mL, 10ng / mL, and 20ng / mL, and the concentration of AT in the simulated milk sample was set to 0.15625ng / mL, 0.3125ng / mL, 0.625ng / mL, 1.25ng / mL, 2.5ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, and 40ng / mL, all with sterile water as a negative control to explore the sensitivity of the detection method. After the incubation, 16μL of 100mM ammonia water was added to the system, and then blown and sucked evenly, placed on a magnetic stand for adsorption for 1min, 75μL of the system was drawn and added to the sample well of the test strip, and incubated at room temperature in the dark for 15min and then detected using a FAM nucleic acid analyzer. A sample whose fluorescence signal value is lower than the negative control fluorescence signal value - 3 times the standard deviation can be judged as a positive sample.

[0108] Experimental results: Fig.10 and Fig.11 As shown. Fig.10 It can be seen that after enrichment with antibody-coated magnetic beads, the detection limit of RT in water samples by this detection method is 0.17 ng / mL. Fig.11 It can be seen that the detection limit of the milk simulation sample is 1.53ng / mL.

[0109] Example 10

[0110] Specificity verification

[0111] At the same time, RT and AT were used to verify the specificity of the detection method, and all toxin concentrations were diluted to 20 ng / mL with sterile water. Except for the different toxins, the other reactions and steps were consistent with the RT simulation sample detection method. The specific detection method is: the water samples were enriched using antibody-coated Dead magnetic beads, 25 μg monoclonal antibody was coated with 1 mg magnetic beads, and 50 μg magnetic beads were used for each sample. The volume of the water sample was 500 μL, and it was incubated with monoclonal antibody-coated magnetic beads at room temperature for 1 hour, then placed on a magnetic stand for adsorption for 1 minute, and the supernatant was discarded. The treated magnetic beads were added to the optimized system (16 μL of each component, including 12.5 μM substrate DNA10A, 100 μg / mL BSA, 2.5 mM ammonium citrate buffer (pH4), sterile water and 100 mM ammonia water), gently blown to mix, and then incubated at 59 ° C for 1 hour. All toxin concentrations in the simulated water samples were diluted to 20 ng / mL using sterile water, and sterile water was used as a negative control to explore the specificity of the detection method.

[0112] Experimental results: Fig.12 As shown. Fig.12 It can be seen that AT has no cross reaction with RT during the detection process, and the detection method has good specificity.

[0113] It can be seen from the above examples that the detection method of the present invention has simple reaction system components and reaction conditions, short reaction time, strong specificity, high sensitivity, and is convenient and fast.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An immunochromatographic kit for detecting ricin, characterized in that: It includes an immunochromatographic test strip, an oligonucleotide chain substrate labeled with a fluorescent group and biotin; The oligonucleotide chain is a single-stranded polyadenine nucleotide, and the number of adenines in the single-stranded polyadenine nucleotide is 6 to 15; The immunochromatographic test strip comprises a sample pad, a cellulose membrane and a water-absorbing pad which are overlapped in sequence, and a detection line and a quality control line are arranged in sequence on the cellulose membrane starting from one end close to the sample pad. The detection line is coated with streptavidin that can specifically bind to biotin, and the quality control line is coated with green fluorescent microspheres.

2. The immunochromatography kit according to claim 1, characterized in that Also included are buffer, BSA solution and ammonia.

3. The immunochromatography kit according to claim 1, characterized in that The cellulose membrane is Hi-Flow TM PlusMembrane 180.

4. A method for detecting ricin by immunochromatographic test strips, characterized in that: The following steps are involved: 1) After mixing and incubating the oligonucleotide chain substrate labeled with fluorescent group and biotin, buffer, BSA solution, and the sample to be tested, ammonia water is added to terminate the reaction to obtain a reaction solution; 2) adding the reaction solution to an immunochromatographic test strip, incubating in the dark, detecting the fluorescence signal value, and determining whether ricin is present based on the difference between the final fluorescence signal value of the sample and the final fluorescence signal value of the negative control; The sample to be tested is enriched using antibody-coated magnetic beads; The presence of ricin can be determined when the fluorescence signal value of the sample is lower than the fluorescence signal value of the negative control minus 3 times the standard deviation.

5. The immunochromatographic test strip detection method according to claim 4, characterized in that: In step 1), the concentration of the oligonucleotide chain substrate labeled with fluorescent group and biotin is 0.01 μM to 25 μM.

6. The immunochromatographic test strip detection method according to claim 4, characterized in that: Step 1) The buffer is an ammonium citrate buffer, and the concentration of the ammonium citrate buffer is 2-3 mM.

7. The immunochromatographic test strip detection method according to claim 4, characterized in that: Step 1) The concentration of the ammonia water is 50-400 mM.

8. The immunochromatographic test strip detection method according to claim 4, characterized in that: The antibody-coated magnetic beads are Dynabeads TM .

9. The immunochromatographic test strip detection method according to claim 4, characterized in that: Step 2) The temperature of the dark-proof incubation is 22-27° C., and the time of the dark-proof incubation is 10-20 min.

10. The immunochromatographic test strip detection method according to claim 4, characterized in that: Step 1) The concentration of BSA is 80-120 μg / mL; the volume ratio of the oligonucleotide chain substrate labeled with fluorescent group and biotin, buffer, BSA solution, and the sample to be tested is 7-9:7-9:7-9:7-9; The incubation temperature is 55-62° C., and the incubation time is 40-80 min.

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