WO2017197914A1 - 一种基于抗原-适配体的竞争法检测试纸条及其应用以及利用适配体检测黄曲霉毒素b1或m1的试纸条 - Google Patents

一种基于抗原-适配体的竞争法检测试纸条及其应用以及利用适配体检测黄曲霉毒素b1或m1的试纸条 Download PDF

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WO2017197914A1
WO2017197914A1 PCT/CN2017/070786 CN2017070786W WO2017197914A1 WO 2017197914 A1 WO2017197914 A1 WO 2017197914A1 CN 2017070786 W CN2017070786 W CN 2017070786W WO 2017197914 A1 WO2017197914 A1 WO 2017197914A1
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aptamer
aflatoxin
test strip
detection
sample
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PCT/CN2017/070786
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English (en)
French (fr)
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陈爱亮
朱超
张桂兰
杨曙明
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中国农业科学院农业质量标准与检测技术研究所
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Priority claimed from CN201610325660.6A external-priority patent/CN105784990B/zh
Priority claimed from CN201610625665.0A external-priority patent/CN106290831A/zh
Application filed by 中国农业科学院农业质量标准与检测技术研究所 filed Critical 中国农业科学院农业质量标准与检测技术研究所
Publication of WO2017197914A1 publication Critical patent/WO2017197914A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to the field of biomedical detection, and in particular to an antigen-aptamer-based competition method for detecting test strips and applications thereof, and a test strip for detecting aflatoxin B1 or M1 using an aptamer .
  • a nucleic acid aptamer is a single-stranded oligonucleotide, mostly single-stranded DNA, between 10 and 100 bp in length, usually obtained by in vitro screening from a random sequence of oligonucleotide libraries using specific targets.
  • As a novel bio-recognition molecule it can bind specifically to the target substance with high affinity. Compared with antibodies, it has the advantages of easy synthesis, stable performance, easy modification, and has been used for the detection of various substances.
  • targets that have been screened for aptamers in the literature.
  • the established aptamer-based detection methods mainly include hundreds of methods such as colorimetry, fluorescence, and electrochemistry, but based on adaptation so far.
  • Colloidal gold immunochromatographic strip technology is one of the most common and most popular forms of rapid testing products. It is widely used in clinical diagnosis, food safety and environmental analysis, such as the famous early pregnancy test strips and thin skin. Meat test strips, etc. As a specific recognition molecule, aptamers can also be used to develop rapid detection products based on test strips. However, there are few reports on aptamer strips, and there are only a few articles. The general principle of these documents is to fix the complementary strand of the aptamer at the test strip detection line. When the sample contains the target, the aptamer labeled with fluorescent or colloidal gold and the target in the sample can not be combined with the test strip.
  • the complementary strands at the detection line are combined, and the fluorescent signal at the detection line is reduced or no signal; conversely, when there is no target in the sample, the aptamer labeled with fluorescent or colloidal gold flows through the test strip and complements the detection line. Chain binding, a strong signal appears at the detection line, and the target content in the sample is judged based on the fluorescence or colloidal gold signal size.
  • the principle of the detection method is mainly based on the competition between the aptamer-target and the aptamer-complementary chain, it is difficult to design a relatively reliable detection method. Since the lengths of the different aptamers are different, the length of the complementary strands is also different, so that the affinity of the aptamer and the complementary strand is also different.
  • the affinity of the aptamer to the complementary strand is often greater than the affinity of the aptamer to the target, resulting in undetectable; the current literature solution is to truncate the complementary strand, only part of it The complementary strand is immobilized to the detection line, thereby reducing the affinity of the complementary strand and the aptamer, but this method requires a cumbersome exploration, and when the complementary strand length is too short, the aptamer cannot hybridize with the complementary strand, thereby causing undetectability.
  • Aflatoxins mainly include aflatoxins B1, B2, M1, M2, G1, G2, wherein aflatoxin toxin B1 (AFB1) is one of the most toxic and carcinogenic in the aflatoxin family.
  • Aflatoxin M1 (AFM1) is a metabolite of aflatoxin B1 and its basic structure is a difuran ring of oxaphthalene.
  • the detection methods of aflatoxin B1 or M1 mainly include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA).
  • the TLC method has the advantage of simple equipment, its sensitivity is low.
  • High performance liquid chromatography has the characteristics of high sensitivity, strong separation ability, good specificity and reliable measurement results.
  • Enzyme-linked immunosorbent assay is a commonly used one.
  • the detection method is fast, sensitive, quantitative, and has low requirements on sample purity. It is especially suitable for the detection of large quantities of samples, but it is not suitable for the site because it requires a microplate reader and skilled personnel and a relatively long detection time. Quick check. Therefore, there is an urgent need to develop a rapid and simple method for rapid detection of aflatoxin B1 or M1.
  • An antigen-aptamer-based competition method for detecting a test strip comprising a sample absorption pad, a marker pad, a reaction film, an absorbent pad and a bottom plate;
  • the marker pad is coated with a detection probe, which is an aptamer of the detection target labeled with an indicator showing the signal intensity;
  • a detection zone and a quality inspection zone are disposed on the reaction film
  • the detection zone is fixedly coated with a target antigen
  • the quality inspection zone fixing package is provided with a quality control probe.
  • the quality inspection area is also called the quality control area.
  • the invention proposes to use the target antigen instead of the complementary strand to fix the target antigen to the test strip detection line, so that the aptamer has the same affinity with the target in the sample and the target on the detection line, thereby ensuring the feasibility of the detection method and accuracy. And the experimental conditions are simpler and easier to explore.
  • the antigen-aptamer-based competition method as described above detects a test strip, and when the target is an organic macromolecular target, the target antigen is the target itself;
  • the target antigen is a conjugate of the inorganic small molecule target and a carrier protein.
  • the macromolecule referred to herein refers to a substance which can be directly immobilized on a membrane and exposes an antigenic epitope
  • a small molecule refers to a substance which needs to be immobilized on a membrane by coupling a carrier protein and exposes an antigenic epitope
  • the test strip is detected by an antigen-aptamer-based competition method as described above, including but not limited to casein, bovine serum albumin or chicken ovalbumin.
  • Ovalbumin consists of 386 amino acids with a molecular weight of approximately 43 Kd.
  • OVA can assist the immobilization of small molecules onto the nitrocellulose membrane and expose its antigenic epitope, thereby facilitating the binding of the antigen to the aptamer.
  • casein or casein albumin can be used as the carrier protein.
  • the test strip is detected by an antigen-aptamer-based competition method as described above, and the indicator for indicating signal intensity includes any of fluorescent substance, biotin, radioisotope, electron dense substance, colloidal gold or enzyme.
  • the indicator for indicating signal intensity includes any of fluorescent substance, biotin, radioisotope, electron dense substance, colloidal gold or enzyme.
  • the test strip is detected by an antigen-aptamer-based competition method as described above, the fluorescent label comprising Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, amino acridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescence , 5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, CY7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE,
  • the test strip is detected by an antigen-aptamer-based competition method as described above, and the fluorescent substance includes any one of PE, CY5, CY7, per-CP, TRITC, Alexa Fluor 647, and quantum dots.
  • the test strip is detected by an antigen-aptamer-based competition method as described above, the nucleic acid sequence of the quality control probe being complementary to the aptamer or complementary to the sequence ligated to the aptamer.
  • the type of the ligated base is selected based on the nucleotide sequence of the aptamer so as not to exhibit a complementary pairing with the aptamer, and the nucleotide sequence which does not affect the efficiency of binding of the aptamer to the target is correct.
  • the test strip is detected by an antigen-aptamer-based competition method as described above, and the sequence linked to the aptamer is one of PolyA, PolyT, PolyG or PolyC.
  • the number of bases is from 6 to 24.
  • the sequence ligated to the aptamer is at the 5' end of the aptamer nucleotide sequence.
  • the test strip is detected by an antigen-aptamer-based competition method as described above, and the sequence linked to the aptamer is PolyT18.
  • nucleic acid sequence of the QC probe complementary to the sequence ligated to the above aptamer is:
  • the antigen-aptamer assay as described above detects the use of test strips in the detection of food contaminants, clinical disease markers, and the like, particularly in the detection of aflatoxin B1 and thrombin.
  • the application as described above comprises:
  • step 2) detecting the sample to be tested with a test strip, obtaining a signal intensity ratio of the sample to be tested in the detection zone and the quality control zone of the test strip, and obtaining a target in the sample to be tested according to the regression equation obtained in step 1) The content.
  • the ratio of the fluorescent or color-developing signal of the standard in the detection area and the control area of the test paper includes the following steps: taking the target standard or the sample to be tested On the sample absorption pad of the test paper, after 10 minutes, the test strip is placed in the test strip detection instrument ESEQuant-LR3 for detection, respectively, to determine the fluorescence or color signal intensity of the detection zone and the quality control zone;
  • the signal intensity (T) is higher than the fluorescence signal intensity (C) in the upper control region, and the T/C value is obtained, which is the ratio of the fluorescence signal of the detection zone and the quality control zone.
  • the antigen-aptamer-based competition method for detecting test strips has the advantages of simple design, reliable results, high sensitivity, accurate quantification, strong specificity, simple and convenient, and short detection time. Compared with the prior art, the beneficial effects of the present invention are:
  • the invention avoids the complicated complementary chain truncation design of the current aptamer test strip, and the use of the antigen instead of the complementary strand makes the design and development of the aptamer-based test strip easier and simpler;
  • test strip of the invention sets the detection area/quality control area signal ratio as the data normalization method, reduces the experimental error of different operators and even different instruments scanning between different strips, and samples the different strips. Substituting the value into the standard curve, the exact quantitative effect can be achieved;
  • the test strip of the invention since the aptamer is used as the recognition molecule, based on the high affinity and high specificity of the aptamer to the target, the test strip of the invention has the advantages of high sensitivity and high specificity;
  • the nucleic acid aptamer has very good thermal stability, so the test strip of the invention can also be stored and transported at normal temperature, and has the advantages of long shelf life and reliable results.
  • Another object of the present invention is to provide a test paper for detecting aflatoxin B1 or M1.
  • the test paper for detecting aflatoxin B1 or M1 comprises a sample absorption pad, a marker pad, a reaction film and an absorbent pad;
  • the marker pad comprises a detection probe which is a fluorescein-labeled aflatoxin B1 aptamer or an aflatoxin aptamer comprising a linker sequence, the aflatoxin B1 aptamer
  • the nucleotide sequence is sequence 1;
  • the reaction film includes a detection zone and a quality control zone
  • the detection zone is coated with a conjugate of an aflatoxin B1 hapten and a carrier protein;
  • the carrier protein is, but not limited to, ovalbumin or bovine serum albumin.
  • the aflatoxin B1 hapten is capable of binding to the aflatoxin B1 aptamer
  • the QC region is coated with a QC probe, and the nucleotide sequence of the QC probe is the complement of the aflatoxin B1 aptamer or the complement of the aptamer ligation sequence.
  • the 5' end of the quality control probe is labeled with biotin and is covalently bonded to the reaction membrane by coupling of streptavidin with biotin thereon.
  • the fluorescein is, but not limited to, Cy5, Alexa Fluor 647, , quantum dots, colloidal gold, or the like.
  • the test strip further includes a bottom plate, and the sample absorbent pad, the marker pad, the reaction film, and the absorbent pad are fixed on the bottom plate.
  • Another object of the present invention is to provide a method for detecting the content of aflatoxin B1 or M1 in a sample to be tested.
  • the method for detecting the content of aflatoxin B1 or M1 in a sample to be tested comprises the following steps:
  • the ratio of the fluorescent signal of the standard in the detection zone and the control zone of the test strip includes the following steps: taking the aflatoxin B1 or M1 standard The product or the sample to be tested is added dropwise to the sample absorption pad of the test paper. After 5 minutes, the test strip is placed in the test strip detection instrument. In ESEQuant-LR3, the detection is performed to determine the fluorescence signal intensity of the detection zone and the quality control zone respectively; the fluorescence signal intensity (A) of the detection zone is compared with the fluorescence signal intensity of the upper control zone (B) to obtain the A/B value. The ratio of the fluorescence signal of the detection zone and the quality control zone.
  • the method further comprises: pre-processing the sample to be tested; and when the sample to be tested is peanut meal, the method for pre-processing the sample to be tested is: weighing 2g sample in a 50mL centrifuge tube, add 8mL of n-hexane and 10mL of 70% aqueous methanol solution, shake for 5min, room temperature 4000 rpm / separation of the heart for 10min; remove the upper layer of liquid, take 0.5mL of the lower layer of liquid to add 0.5mL of deionized water, mix, 0.5 mL of the mixed liquid was added to 0.5 mL of a 35% aqueous methanol solution, and shaken for 30 s; 100 ⁇ L was taken for analysis (sample dilution factor was 20 times).
  • the detection principle of the test paper of the invention is: dropping a sample on the sample absorption pad, and if the sample contains aflatoxin B1 or M1, the aflatoxin B1 or M1 moves to the marker pad by chromatography and fluorescein
  • the labeled aflatoxin B1 aptamer binds to form a complex and drives the fluorescein-labeled aptamer together under chromatography.
  • the free fluorescein-labeled aptamer not bound by the aflatoxin B1 or M1 in the sample will bind to the aflatoxin B1 coupled to the carrier protein on the detection zone. Fluorescent signal.
  • the test paper for detecting aflatoxin B1 or M1 of the invention has the advantages of high sensitivity, precise quantification, strong specificity, simplicity and convenience, and short detection time. Since fluorescein is used as a marker instead of colloidal gold, very few fluorescent signals can be detected by the instrument, thus greatly improving the sensitivity of detection, and the sensitivity of detecting aflatoxin B1 can reach 0.1 ng/ml.
  • the quality control tape (dot) as the data normalization method, the experimental error of different operators and even different instrument scanning between different paper strips is reduced, and the sample values measured by different paper strips are substituted into the standard curve, that is, accurate quantification can be achieved. Effect.
  • the test strip can be inserted into the sample, and the experimental result can be obtained within 20 minutes, which can greatly improve the detection efficiency.
  • FIG. 1 is a schematic structural view of a test strip provided by the present invention.
  • Figure 2 is a structural formula of BSA-conjugated AFB1 hapten.
  • Figure 3 is a schematic diagram showing the principle of a fluorescent test strip for detecting AFB1.
  • the first peak represents the detection area signal value (indicated by the arrow).
  • Fig. 5 is a standard curve showing the linear relationship between AFB1 concentration and T/C value in Example 1 of the present application.
  • Figure 6 is a standard curve showing the linear relationship between AFM1 concentration and T/C value in Example 1 of the present application.
  • Fig. 7 is a graph showing the detection results specific to AFB1/AFM1 test paper in Example 1 of the present application; the first peak represents the detection region signal value (indicated by an arrow).
  • the first peak represents the detection area signal value
  • the second peak represents the quality inspection area signal value (arrow Show).
  • Figure 9 is a standard curve showing the linear relationship between AFB1 concentration and T/C value in Example 2 of the present application.
  • Example 10 is a signal control value of a quality control zone and a detection zone when different concentrations of thrombin react with a test strip in Example 2 of the present application;
  • Figure 11 is a standard curve showing the linear relationship between thrombin concentration and T/C value in Example 2 of the present application.
  • Synthetic fluorescein-labeled detection probes and biotin-labeled quality control probes were designed separately.
  • the detection probe is a fluorescein-labeled aflatoxin B1 aptamer, and the fluorescein is, but not limited to, Cy5.
  • the aflatoxin B1 aptamer can bind to the aflatoxin B1 hapten in the detection zone.
  • the nucleotide sequence of the aflatoxin B1 aptamer is as follows:
  • the control sequence is a complementary single-stranded DNA molecule of the biotin-labeled aflatoxin B1 aptamer, which can be bound to the detection probe by base complementation.
  • the nucleotide sequence of the QC sequence is:
  • the structural formula of the conjugate AFB1-BSA is shown in Figure 2.
  • the carboxyl group of the aflatoxin B1 hapten forms the peptide bond with the amino group of the carrier protein BSA.
  • the preparation method of the above conjugate firstly preparing AFB1 ⁇ (AFB1-O) by deuteration reaction; then preparing AFB1 artificial antigen by carbodiimide method; finally, according to AFB1 and modified bovine serum albumin (C-BSA) React To the coupled product, the conjugate AFB1-BSA.
  • AFB1 ⁇ (AFB1-O) by deuteration reaction
  • AFB1 artificial antigen by carbodiimide method
  • C-BSA modified bovine serum albumin
  • the detection probe was coated on a marker pad (Shanghai Jieyi Biotechnology Co., Ltd., JY-BX101) (coating concentration: 0.1 ⁇ M) to obtain a marker pad coated with the detection probe;
  • the reaction membrane includes a detection zone and a quality control zone.
  • the conjugate AFB1-BSA was coated in the reaction membrane formation detection zone at a coating concentration of 1 ⁇ M;
  • the quality control probe is an aptamer complementary sequence of labeled biotin, which forms a control region on the reaction membrane by a covalent bond of streptavidin coupled with biotin thereon, and the coating concentration is 1 ⁇ M.
  • the test strip consists of the sample absorption pad (Shanghai Jieyi Biotechnology Co., Ltd., JY-BX111), the marker pad of the coated detection probe prepared in the above 1), the reaction film prepared in the above 2), and the absorbent pad are sequentially adhered. It is attached to the bottom plate, and the detection area of the reaction film is adjacent to the marker pad, and the quality control area of the reaction film is adjacent to the water absorption pad, as shown in the upper diagrams of FIGS. 1 and 3.
  • the sample flows under capillary action to the marker pad and continues to flow forward with the fluorescently labeled probe mixture.
  • the detection probe When flowing to the detection zone, the detection probe will bind to the fixed hapten at the detection zone, and the high fluorescence intensity is detected by the test strip detection instrument ESEQuant-LR3; when flowing to the quality control zone, the remaining detection probe will In combination with the quality control probe, high fluorescence intensity also appears in the quality control zone.
  • the aflatoxin B1 in the sample is less and less, and the remaining free detection probes will be more and more, and the more the hapten is bound at the detection zone, the stronger the fluorescence intensity, and thus the fluorescence intensity can be
  • the change determines the concentration of aflatoxin B1 in the sample.
  • Aflatoxin B1 or M1 standard is added dropwise to the sample absorption pad of the test paper. After 5 minutes, the test strip is placed in the test strip detection instrument ESEQuant-LR3 for detection, and the detection area and quality control are respectively determined. The fluorescence signal intensity of the region; the fluorescence signal intensity (A) of the detection zone is compared with the fluorescence signal intensity (B) of the upper control zone to obtain the A/B value, and the obtained A/B value is used to prepare the aflatoxin B1 or M1 standard.
  • the concentration of the product corresponds to a linear regression curve of the fluorescence signal ratio, and the regression equation is calculated;
  • aqueous solutions of different concentrations of AFB1 (Fermentek Ltd, AF028) shown in Fig. 4 were prepared and measured by the test strips prepared in Example 1, respectively.
  • the fluorescence intensity at the detection zone and the QC zone was determined using a test strip tester ESEQuant-LR3. As a result, as shown in Fig. 4, it can be seen that as the AFB1 concentration increases, the fluorescence intensity at the detection zone gradually becomes smaller.
  • Detection zone The fluorescence intensity and the fluorescence intensity ratio of the quality control area (Table 1), the standard curve for the detection of aflatoxin B1 is shown in Figure 5. The results show that the sensitivity of the test strip is 0.5 ng/mL; the linear range is 0.5 ng/mL- 50 ⁇ g/mL.
  • aqueous solutions of different concentrations of AFM1 (Belling Technology Co., Ltd., 6795-23-9) were prepared and tested with the test strips prepared in Example 1.
  • the test strip detection instrument ESEQuant-LR3 was used to measure the fluorescence intensity at the detection area and the quality control area respectively. Using the ratio of the fluorescence intensity of the detection area to the fluorescence intensity of the quality control area (Table 2), the standard curve for detecting aflatoxin M1 was plotted.
  • the results show that the sensitivity of the test strip is 10 ng/mL; the linear range is 10-1000 ng/mL.
  • OTA Fermentek Ltd, OC030a
  • ZEN Balinger Technology Co., Ltd., 17924
  • the fluorescence intensity of the detection zone and the quality control zone was measured by the test strip detection instrument ESEQuant-LR3. The results are shown in Fig. 7. It is shown that AFG1, AFB2, OTA, ZEN, DON and blank samples can not cause fluorescence at the detection zone. The change also indicates that the method has good specificity for aflatoxin B1 or M1.
  • a sample of peanut aflatoxin B1 (content 143.47 ng/mL), which was determined by the National Standard Method (LC-MSMS), was selected and measured using this test strip.
  • test strip detection instrument ESEQuant-LR3 100 ⁇ L of the prepared sample to be tested was added dropwise to the sample absorption pad of the test paper, and the sample chromatography was observed to flow along the nitrocellulose membrane until it was adsorbed by the above absorbent pad; after 5 minutes, the test strip was placed.
  • the detection is performed to determine the fluorescence signal intensity of the detection zone and the quality control zone respectively; the fluorescence signal intensity (A) of the detection zone is compared with the fluorescence signal intensity of the upper control zone (B), The obtained calculated value is substituted into the regression equation of the above step 1, and the concentration of aflatoxin B1 in the sample to be tested is calculated.
  • the fluorescence intensity (A) of the detection zone was 962.06, the fluorescence intensity (B) of the control zone was 1210.75, A/B was 0.7946, and the calculated concentration of aflatoxin B1 in the sample to be tested was 171.05 ng/mL.
  • the error with the measured value of the national standard method is less than 10%, indicating that the method has good accuracy.
  • a sample of peanut aflatoxin M1 (content 261.54 ng/mL), which was determined by the National Standard Method (LC-MSMS), was selected and measured using this test strip.
  • test strip detection instrument ESEQuant-LR3 100 ⁇ L of the prepared sample to be tested was added dropwise to the sample absorption pad of the test paper, and the sample chromatography was observed to flow along the nitrocellulose membrane until it was adsorbed by the above absorbent pad; after 5 minutes, the test strip was placed.
  • the detection is performed to determine the fluorescence signal intensity of the detection zone and the quality control zone respectively; the fluorescence signal intensity (A) of the detection zone is compared with the fluorescence signal intensity of the upper control zone (B), The obtained calculated value is substituted into the regression equation of the above step 1, and the concentration of aflatoxin M1 in the sample to be tested is calculated.
  • the fluorescence intensity (A) of the detection zone was 1006.77
  • the fluorescence intensity (B) of the control zone was 1234.65
  • the A/B was 0.815432.
  • the calculated concentration of aflatoxin M1 in the sample to be tested was 312.61 ng/mL.
  • the error with the measured value of the national standard method is less than 20%, indicating that the method has good accuracy.
  • Example 2 The same procedures as in Example 1 were carried out, and the same materials, reagents and the like as in Example 1 were used unless otherwise specified. the difference lies in:
  • the detection probe is a fluorescein Cy5-labeled aflatoxin B1 aptamer with the ligated sequence PolyT18; the aflatoxin B1 aptamer can be used in the detection zone of Aspergillus flavus Toxin
  • the B1 hapten binds, and the nucleotide sequence of the aflatoxin B1 aptamer is as follows:
  • the QC probe is a biotinylated PolyA18 that binds to the aflatoxin B1 aptamer linker sequence and can be complementary to the base of the test probe linker PolyT18.
  • the nucleic acid sequence of the quality control probe is:
  • the aflatoxin B1 standard is added dropwise to the sample absorption pad of the test paper. After 10 minutes, the test strip is placed in the test strip detection instrument ESEQuant-LR3 for detection, and the detection area and the quality control area are respectively determined. Fluorescence signal intensity; the fluorescence signal intensity (T) of the detection zone is compared with the fluorescence signal intensity (C) of the upper control zone to obtain the T/C value, and the concentration of the aflatoxin B1 standard is corresponding to the fluorescence signal T/C ratio. Linear regression curve to calculate the regression equation;
  • Aqueous solutions of different concentrations of AFB1 (Fermentek Ltd, AF028) shown in Fig. 8 were prepared and measured using the test strips prepared in Example 1, respectively.
  • the fluorescence intensity at the detection zone and the QC zone was determined using a test strip tester ESEQuant-LR3. The results are shown in Fig. 8. It can be seen that as the concentration of AFB1 increases, the fluorescence intensity at the detection zone gradually decreases, and the fluorescence intensity of the quality control region gradually increases. Using the ratio of the fluorescence intensity of the detection zone to the fluorescence intensity of the quality control zone (Table 3), the standard curve for the detection of aflatoxin B1 is shown in Figure 9. The results show that the sensitivity of the test strip is 0.1 ng/mL; the linear range is 0.1 ng. /mL ⁇ 1000ng / mL.
  • the standard curve method in the sensitivity and linear range of aflatoxin B1 was examined in the first part of this example. The results are shown in Fig. 9.
  • the concentration of aflatoxin B1 in the sample to be tested is calculated.
  • the standard curve method in the sensitivity and linear range of aflatoxin B1 was examined in the first part of this example. The results are shown in Fig. 9.
  • test strip 100 ⁇ L of the prepared sample to be tested was added dropwise to the sample absorption pad of the test paper, and the sample chromatography was observed to flow along the nitrocellulose membrane until it was adsorbed by the above absorbent pad; after 10 minutes, the test strip was placed.
  • Test strip The detection instrument ESEQuant-LR3 is tested to determine the fluorescence signal intensity of the detection zone and the quality control zone respectively; the fluorescence signal intensity (T) of the detection zone is compared with the fluorescence signal intensity (C) of the upper control zone, and the calculation will be obtained. The value is substituted into the regression equation of the above step 1, and the concentration of aflatoxin B1 in the sample to be tested is calculated.
  • Synthetic colloidal gold labeled detection probes and biotinylated quality control probes were designed separately.
  • the detection probe is a colloidal gold-labeled thrombin aptamer with a linker sequence PolyT20; the thrombin aptamer can bind to a thrombin antigen in the detection zone, and the nucleotide sequence of the thrombin aptamer is as follows:
  • the colloidal gold synthesis method is: heating and boiling 100 mL of a 1 mM HAuCl 4 solution, adding 10 mL of a 38.8 mM trisodium citrate solution with vigorous stirring, continuing to boil for 20 minutes, and then cooling at room temperature, and the obtained colloidal gold particle size is 13 nm.
  • the excess aptamer was then removed by centrifugation at 16,000 rpm for 15 min and then resuspended in buffer (100 mL PB solution, 0.5 g PEG 20000, 1 g sucrose, 0.1 mL Tween-20, 0.02 g MgSO 4 , and 0.05 g (NH 4 ) 2 SO 4 )) .
  • the QC probe is a biotin-labeled PolyT20 that binds to the thrombin aptamer linker sequence and can be complementary to the base of the test probe linker PolyA20.
  • the nucleotide sequence of the QC sequence is:
  • the detection probe was coated on a marker pad (Shanghai Jieyi Biotechnology Co., Ltd., JY-BX101) (coating concentration: 0.1 ⁇ M) to obtain a marker pad coated with the detection probe, and dried at 37 degrees overnight.
  • a marker pad Shanghai Jieyi Biotechnology Co., Ltd., JY-BX101
  • the reaction membrane includes a detection zone and a quality control zone.
  • the thrombin antigen is coated in the reaction film formation detection zone at a coating concentration of 1 mg/mL;
  • the quality control probe is a complementary sequence of the aptamer ligation sequence of the labeled biotin, which forms a covalent bond on the reaction membrane by coupling a streptavidin to form a covalent bond with the biotin thereon, and the coating concentration It is 5 ⁇ M.
  • the test strip consists of the sample absorption pad (Shanghai Jieyi Biotechnology Co., Ltd., JY-BX111), the marker pad of the coated detection probe prepared in the above 1), the reaction film prepared in the above 2), and the absorbent pad are sequentially adhered. It is attached to the bottom plate, and the detection area of the reaction film is adjacent to the marker pad, and the quality control area of the reaction film is adjacent to the water absorption pad, as shown in FIG.
  • the sample flows under capillary action to the marker pad 2 and continues to flow forward with the colloidal gold-labeled probe mixture.
  • the aptamer 9 labeled with the detection target for displaying the signal intensity will bind to the thrombin antigen immobilized at the detection zone 6, and a colloidal gold-red band will appear, passing through the test strip.
  • the detection instrument ESEQuant-LR3 detects a high color rendering signal intensity; when flowing to the quality control zone 7, the remaining aptamer 9 labeled with the indicator for displaying the signal intensity is fixed and quality controlled.
  • the quality control probe 11 of the area also has a red strip in the quality control area 7, and the color signal intensity is detected by the test strip detection instrument ESEQuant-LR3.
  • the strip detection instrument ESEQuant - LR3 detects changes in the intensity of the color signal in the detection zone and the quality control zone to determine the concentration of thrombin in the sample.
  • aqueous solutions of different concentrations of thrombin (Sigma, Cat# T6884) shown in Fig. 10 were prepared and measured with the test strips prepared in Example 2, respectively.
  • the color development intensity at the detection zone and the quality control zone was measured by the test strip detection instrument ESEQuant-LR3. As a result, as shown in Fig. 10, it can be seen that as the thrombin concentration increases, the color development intensity at the detection zone gradually becomes smaller.
  • the standard curve for detecting thrombin was plotted as shown in Fig. 11. The results showed that the sensitivity of the test strip was 1 nM; the linear range was 1 nM-100 nM.
  • the standard curve method for detecting thrombin sensitivity and linear range is obtained.
  • Mouse serum was taken and human thrombin was added at a final concentration of 2 nM, 10 nM, and 50 nM, respectively.

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Abstract

涉及生物医学检测领域，具体而言，涉及一种基于抗原-适配体的竞争法检测试纸条及其应用以及利用适配体检测黄曲霉毒素B1或M1的试纸条，所述基于抗原-适配体的竞争法检测试纸条包括样品吸收垫、标记物垫、反应膜、吸水垫和底板；所述标记物垫包被有检测探针，所述检测探针为标有用于显示信号强度的指示剂的检测靶标物的适配体；所述反应膜上设置有检测区和质检区；所述检测区固定包被有靶标物抗原；所述质检区固定包被有质控探针。所述检测黄曲霉毒素B1或M1的试纸，包括样品吸收垫、标记物垫、反应膜和吸水垫；所述标记物垫包被检测探针，所述检测探针为荧光素标记的黄曲霉毒素B1适配体；所述反应膜包括检测区和质控区；所述检测区包被黄曲霉毒素B1半抗原与载体蛋白形成的偶联物；所述质控区包被质控探针，所述质控探针由亲和素偶联生物素标记的黄曲霉毒素B1适配体或其连接序列的互补单链DNA分子形成的偶联物。检测黄曲霉毒素B1或M1的试纸具有灵敏度高、精确定量、特异性强、简单方便和检测时间短的优点。

Description

一种基于抗原-适配体的竞争法检测试纸条及其应用以及利用适配体检测黄曲霉毒素B1或M1的试纸条 技术领域
本发明涉及生物医学检测领域，具体而言，涉及一种基于抗原-适配体的竞争法检测试纸条及其应用，以及一种利用适配体检测黄曲霉毒素B1或M1的试纸条。
背景技术
核酸适配体是一种单链的寡核苷酸，多为单链DNA，长度在10～100bp之间，通常是利用特异的靶标从随机序列的寡核苷酸文库中通过体外筛选获得，作为一种新型生物识别分子，可以与靶标物质高亲和力特异性结合。与抗体相比，具有易于合成制备，性能稳定，便于修饰等优点，已被用于多种物质的检测。目前文献报道的已经筛选到适配体的靶标有几百种，建立的基于适配体的检测方法主要包括比色法、荧光法、电化学等上百种方法，但到目前为止基于适配体的商品化的检测产品还几乎没有。例如目前利用筛选到的AFB1的适配体(也可以检测AFM1)建立了多种检测方法，如纳米金比色法、荧光染料PicoGreen法等，但这些方法容易受到多种基质干扰，灵敏度较低，不适合现场应用。
胶体金免疫层析试纸条技术是目前最常见最成熟也最受欢迎的快速检测产品形式之一，被广泛应用与临床诊断、食品安全以及环境分析等，比如著名的早孕试纸条、瘦肉精试纸条等。适配体作为一种特异性的识别分子，也可以被用来开发基于试纸条形式的快速检测产品，但是目前有关适配体试纸条的文献报道却很少，仅有几篇文献。这些文献一般采用的原理是在试纸条检测线处固定适配体的互补链，样品中含有靶标时，标记有荧光或胶体金的适配体和样品中的靶标结合，则不能和试纸条检测线处的互补链结合，检测线处荧光信号降低或不出信号；反之，样品中没有靶标时，标记有荧光或胶体金的适配体流经试纸条时和检测线处的互补链结合，检测线处出现较强的信号，从而根据荧光或胶体金信号大小判断样品中靶标含量。
然而，在实际的应用中，由于该检测方法的原理主要是基于适配体-靶标和适配体-互补链的竞争来实现的，因此很难设计比较可靠的检测方法。由于不同适配体长度不同，因此其互补链的长度也不同，从而适配体与互补链的的亲和力也不同。由于适配体链一般比较长，所以适配体与互补链的亲和力经常大于适配体与靶标的亲和力，从而导致无法检测；目前文献的解决方法都是通过将互补链截短，仅将部分互补链固定到检测线上，从而降低互补链和适配体的亲和力，但是这种方法需要繁杂的摸索，且互补链长度过短时适配体无法与互补链杂交，从而也导致无法检测。
近年来，具有急慢性毒性、致突变性、致癌性和致畸性的黄曲霉毒素引起社会各界的高度关注。黄曲霉毒素主要包括黄曲霉毒素B1、B2、M1、M2、G1、G2，其中黄曲霉素毒素B1(AFB1)是黄曲霉毒素家族中毒性和致癌性最强的一种。黄曲霉毒素M1(AFM1)是黄曲霉毒素B1的代谢物，其基本结构为一个二呋喃环的氧杂萘邻酮。目前，黄曲霉毒素B1或M1的检测方法主要包括薄层色谱法(TLC)、高效液相色谱法(HPLC)、酶联免疫吸附法(ELISA)。TLC法虽然具有设备简单的优点，但其灵敏度低。高效液相色谱法具有灵敏度高、分离能力强、特异性好和测定结果可靠等特点，但如果样品成分复杂，在进行液相色谱分离前，需对样品作彻底有效的净化处理，不适合于大批量样品的检测，并且设备仪器昂贵，不易普及。酶联免疫吸附法是目前常使用的一种 检测方法，它具有快速、灵敏、可定量、对样品纯度要求不高，特别适用于大批量样品的检测，但由于需要酶标仪和操作熟练的人员以及检测时间相对较长，所以不适合现场快速检测。因此急需开发新的快速简单灵敏的黄曲霉毒素B1或M1的快速检测方法。
有鉴于此，特提出本发明。
发明内容
本发明的目的在于提供一种基于抗原-适配体的竞争法检测试纸条以及一种利用适配体检测黄曲霉毒素B1或M1的试纸条，以解决上述问题。
为了实现本发明的上述目的，特采用以下技术方案：
一种基于抗原-适配体的竞争法检测试纸条，所述试纸条包括样品吸收垫、标记物垫、反应膜、吸水垫和底板；
所述标记物垫包被有检测探针，所述检测探针为标有显示信号强度的指示剂的检测靶标物的适配体；
所述反应膜上设置有检测区和质检区；
所述检测区固定包被有靶标物抗原；
所述质检区固定包被有质控探针。
质检区也称为质控区。
本发明提出了利用靶标抗原代替互补链的方法，将靶标抗原固定到试纸条检测线，这样适配体与样品中的靶标和检测线上的靶标亲和力一致，保证了检测方法的可行性和准确性。且实验条件也更简单，摸索起来更容易。
优选的，如上所述的基于抗原-适配体的竞争法检测试纸条，当所述靶标物为有机物大分子靶标时，所述靶标物抗原即为靶标物本身；
当所述靶标物为无机小分子靶标时，所述靶标物抗原为所述无机小分子靶标与载体蛋白的偶联物。
这里所指的大分子是指可以直接固定到膜上并暴露抗原表位的物质，小分子是指需要通过偶联载体蛋白才能固定到膜上并暴露抗原表位的物质。
优选的，如上所述的基于抗原-适配体的竞争法检测试纸条，所述载体蛋白包括但不限于酪蛋白、牛血清白蛋白或鸡卵清白蛋白。
鸡卵清白蛋白(Ovalbumin，OVA)由386个氨基酸组成，分子量约43Kd。OVA作为载体蛋白可以协助小分子物质固定到硝酸纤维素膜上并暴露其抗原表位，从而利于抗原与适配体的结合。
同样的，载体蛋白也可选用酪蛋白(Casein)或牛血清白蛋白(BSA)等。
优选的，如上所述的基于抗原-适配体的竞争法检测试纸条，所述显示信号强度的指示剂包括荧光物质、生物素、放射性同位素、电子致密物、胶体金或酶中的任一种。
优选的，如上所述的基于抗原-适配体的竞争法检测试纸条，所述荧光标记包括Alexa 350、Alexa 405、Alexa 430、Alexa 488、Alexa 555、Alexa 647、AMCA、氨基吖啶、BODIPY 630/650、BODIPY 650/665、BODIPY-FL、BODIPY-R6G、BODIPY-TMR、BODIPY-TRX、5-羧基-4′，5′-二氯-2′，7′-二甲氧基荧光素、5-羧基-2′，4′，5′，7′-四氯荧光素、5-羧基荧光素、5-羧基罗丹明、6-羧基罗丹明、6-羧基四甲基罗丹明、Cascade Blue、Cy2、Cy3、Cy5、CY7、6-FAM、丹磺酰氯、荧光素、HEX、6-JOE、NBD(7-硝基苯并-2-氧杂-1，3-二唑)、Oregon Green 488、Oregon Green 500、Oregon Green514、Pacific Blue、邻苯二甲酸、对苯二甲酸、间苯二甲酸、甲酚固紫、甲酚蓝紫、亮甲酚蓝、对氨基苯甲酸、赤藓红、酞菁、偶氮甲碱、花青、黄嘌呤、琥珀酰荧光素、稀土金属穴状化合物、三双吡啶基二胺铕、铕穴状化合物或螯合物、二胺、双花青苷、La Jolla蓝染料、别藻蓝蛋白、allococyanin B、藻蓝蛋白C、藻蓝蛋白R、硫胺、藻红青蛋白、藻红蛋白R、REG、罗丹明绿、罗丹明异硫氰酸酯、罗丹明红、ROX、TAMRA、TET、TRIT(四甲基罗丹明异硫醇)、四甲基罗丹明和德克萨斯红中的一种或多种。
优选的，如上所述的基于抗原-适配体的竞争法检测试纸条，所述荧光物质包括PE、CY5、CY7、per-CP、TRITC、Alexa Fluor647以及量子点中的任一种。
优选的，如上所述的基于抗原-适配体的竞争法检测试纸条，所述质控探针的核酸序列与所述适配体互补或与所述适配体上连接的序列互补。
连接序列碱基的种类根据适配体的核苷酸序列进行选择，以不出现与适配体发生内部的互补配对，且不影响适配体与靶标结合的效率的核苷酸序列为准。
优选的，如上所述的基于抗原-适配体的竞争法检测试纸条，所述适配体上连接的序列为PolyA、PolyT、PolyG或PolyC中的一种。
优选的，碱基的数量为6～24个。
优选的，适配体上连接的序列在适配体核苷酸序列的5'端。
优选的，如上所述的基于抗原-适配体的竞争法检测试纸条，所述适配体上连接的序列为PolyT18。
与上述适配体上连接的序列互补的质控探针的核酸序列为：
5'-biotin-AAA-AAA-AAA-AAA-AAA-AAA-3'。
如上所述的抗原-适配体的竞争法检测试纸条在检测食品污染物、临床疾病标志物等中的应用，特别是在检测黄曲霉毒素B1和凝血酶中的应用。
优选的，如上所述的应用，包括：
1)、用试纸条检测靶标标准品，得到标准品在试纸条的检测区和质控区的信号强度比值，制作靶标标准品的浓度对应信号强度比值的线性回归曲线，计算回归方程；
2)、用试纸条检测待测样品，得到待测样品在试纸条的检测区和质控区的信号强度比值，根据步骤1)得到的回归方程，得到所述待测样品中靶标物的含量。
用上述的试纸条检测靶标标准品或待测样品，得到标准品在所述试纸的检测区和质控区的荧光或显色信号比值包括如下步骤：取靶标标准品或待测样品滴加到试纸的样品吸收垫上，10分钟后，将试纸条放入试纸条检测仪器ESEQuant-LR3中进行检测，分别测定检测区和质控区的荧光或显色信号强度；将检测区的荧光信号强度(T)比上质控区的荧光信号强度(C)，得到T/C值，为检测区和质控区的荧光信号比值。
本发明基于抗原-适配体的竞争法检测试纸条具有设计简单、结果可靠、灵敏度高、精确定量、特异性强、简单方便和检测时间短的优点。与现有技术相比，本发明的有益效果为：
1)、由于以抗原代替了目前试纸条检测区中的适配体的互补链，彻底避免了由于适配体-靶标和适配体-互补链亲和力差异使得过于杂交或杂交不上带来的检测误差，提高了检测方法的稳定性；
2)、本发明避免了目前适配体试纸条繁杂的互补链截短设计，利用抗原代替互补链使得基于适配体的试纸条设计开发更为容易简单；
3)、本发明试纸条通过设置检测区/质控区信号比值作为数据归一化方法，降低了不同纸条间不同操作人员乃至不同仪器扫描的实验误差，将不同纸条测得的样品值代入标准曲线，即可以达到精确定量的效果；
4)、由于采用了适配体作为识别分子，基于适配体对靶标的高亲和力和高特异性，因此本发明试纸条具有高灵敏高特异性的优点；
5)、核酸适配体具有非常好的热稳定性，因此本发明的试纸条还可以常温储运，具有货架期长、结果可靠等优点。
本发明的另一个目的是提供一种检测黄曲霉毒素B1或M1的试纸。
本发明所提供的检测黄曲霉毒素B1或M1的试纸，包括样品吸收垫、标记物垫、反应膜和吸水垫；
所述标记物垫包被检测探针，所述检测探针为荧光素标记的黄曲霉毒素B1适配体或含有连接序列的黄曲霉毒素适配体，所述黄曲霉毒素B1适配体的核苷酸序列为序列1；
所述反应膜包括检测区和质控区；
所述检测区包被黄曲霉毒素B1半抗原与载体蛋白形成的偶联物；所述载体蛋白为但不限于卵清白蛋白或牛血清白蛋白。
所述黄曲霉毒素B1半抗原能与所述黄曲霉毒素B1适配体结合；
所述质控区包被质控探针，所述质控探针的核苷酸序列为所述黄曲霉毒素B1适配体的互补序列或适配体连接序列的互补序列。
所述质控探针的5’末端标记生物素，且通过链酶亲和素与其上的生物素偶联形成共价键包被在反应膜上。
上述试纸条中，所述荧光素为但不限于Cy5、Alexa Fluor647、量子点、胶体金等。
上述试纸条还包括底板，且所述样品吸收垫、标记物垫、反应膜和吸水垫固定在所述底板上。
上述的试纸条在检测黄曲霉毒素B1或M1中的应用也是本发明保护的范围。
上述的试纸条在制备检测黄曲霉毒素B1或M1产品中的应用也是本发明保护的范围。
本发明的另一个目的是提供一种检测待测样品中黄曲霉毒素B1或M1含量的方法。
本发明所提供的检测待测样品中黄曲霉毒素B1或M1含量的方法，包括如下步骤：
(1)用上述的试纸条检测黄曲霉毒素B1或M1标准品，得到标准品在所述试纸的检测区和质控区的荧光信号比值，制作黄曲霉毒素B1或M1标准品的浓度对应荧光信号比值的一元线性回归曲线，计算回归方程；
(2)将所述黄曲霉毒素标准品替换为待测样品，用所述试纸检测待测样品，得到待测样品在所述试纸的检测区和质控区的荧光信号比值，根据步骤(1)的回归方程，得到所述待测样品中黄曲霉毒素的含量。
用上述的试纸条检测黄曲霉毒素B1或M1标准品或待测样品，得到标准品在所述试纸的检测区和质控区的荧光信号比值包括如下步骤：取黄曲霉毒素B1或M1标准品或待测样品滴加到试纸的样品吸收垫上，5分钟后，将试纸条放入试纸条检测仪器 ESEQuant-LR3中，进行检测，分别测定检测区和质控区的荧光信号强度；将检测区的荧光信号强度(A)比上质控区的荧光信号强度(B)，得到A/B值，为检测区和质控区的荧光信号比值。
所述步骤(2)中所述检测待测样品之前，还包括将待测样品进行前处理步骤；当所述待测样品为花生粕时，所述待测样品前处理的方法为：称取2g样品于50mL离心管中，加入8mL正己烷和10mL 70％的甲醇水溶液，振荡5min，室温4000转/分离心10min；去除上层液体，取0.5mL下层液体加入0.5mL去离子水，混匀，再取混匀液体0.5mL加入0.5mL 35％的甲醇水溶液，振荡30s；取100μL进行分析(样品稀释倍数为20倍)。
本发明试纸的检测原理为：在样品吸收垫上滴加样品，如果样品中有黄曲霉毒素B1或M1，则黄曲霉毒素B1或M1经层析作用移动到标记物垫的时候就会和荧光素标记的黄曲霉毒素B1适配体结合形成复合物，并带动荧光素标记的适配体一起在层析作用下上行。当移动到检测区时，未被样品中黄曲霉毒素B1或M1结合的游离的荧光素标记的适配体就会和检测区上的偶联在载体蛋白上的黄曲霉毒素B1结合，而有荧光信号。样品中黄曲霉毒素B1或M1越多，剩余游离的荧光素标记的适配体就会越少，检测区处的荧光信号越低，依此来检测样品中的黄曲霉毒素B1或M1的含量。
本发明的检测黄曲霉毒素B1或M1的试纸具有灵敏度高、精确定量、特异性强、简单方便和检测时间短的优点。由于以荧光素代替了胶体金作为标记物，很少的荧光信号即可以通过仪器检测出来，因此大大提高了检测的灵敏度，检测黄曲霉毒素B1灵敏度可达0.1ng/ml。通过设置质控带(点)作为数据归一化方法，降低了不同纸条间不同操作人员乃至不同仪器扫描的实验误差，将不同纸条测得的样品值代入标准曲线，即可以达到精确定量的效果。由于采用了适配体-样品-半抗原反应的原理，通过选择特异性强的适配体，与其他霉菌毒素基本没有交叉，因此避免了其他霉菌毒素的干扰。采用本方法，只需把试纸条插入样品，20分钟内即可获得实验结果，可以大大提高检测的效率。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明提供的试纸条的结构示意图；
图2为偶联BSA的AFB1半抗原的结构式。
图3为检测AFB1的荧光试纸条原理示意图。
图4为本申请实施例1中荧光试纸条检测不同AFB1浓度下检测区处荧光强度检测结果图；第一个波峰代表检测区信号值(箭头所示)。
图5为本申请实施例1中AFB1浓度与T/C值线性关系的标准曲线。
图6为本申请实施例1中AFM1浓度与T/C值线性关系的标准曲线。
图7为本申请实施例1中对AFB1/AFM1试纸特异性的检测结果图；第一个波峰代表检测区信号值(箭头所示)。
图8为本申请实施例2中荧光试纸条检测不同AFB1浓度下检测区处荧光强度检测结果图；第一个波峰代表检测区信号值，第二个波峰代表质检区信号值(箭头所示)。
图9为本申请实施例2中AFB1浓度与T/C值线性关系的标准曲线。
图10为本申请实施例2中不同浓度凝血酶与试纸条反应时的质控区和检测区信号值；
图11为本申请实施例2中凝血酶浓度与T/C值线性关系的标准曲线。
附图标记：
1样品吸收垫；
2标记物垫；
3反应膜；
4吸水垫；
5底板；
6检测区；
7质控区；
8待检靶标；
9标有用于显示信号强度的指示剂的检测靶标物的适配体；
10固定包被在检测区的靶标物抗原；
11固定包被在质控区的质控探针。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1检测黄曲霉毒素试纸条的制备及其在黄曲霉毒素B1和M1中的应用
一、检测黄曲霉毒素试纸条的制备
1、检测探针和质控探针的合成
分别设计合成荧光素标记的检测探针和生物素标记的质控探针。
其中检测探针为荧光素标记的黄曲霉毒素B1适配体，荧光素为但不限于Cy5。
黄曲霉毒素B1适配体可以和检测区的黄曲霉毒素B1半抗原结合，黄曲霉毒素B1适配体的核苷酸序列如下：
5'-Cy5-GTT-GGG-CAC-GTG-TTG-TCT-CTC-TGT-GTC-TCG-TGC-CCT-TCG-CTA-GGC-CCA-CA-3′(序列1)
质控序列为生物素标记的黄曲霉毒素B1适配体的互补单链DNA分子，可以与检测探针通过碱基互补结合。
质控序列的核苷酸序列为：
5'-biotin-TGT-GGG-CCT-AGC-GAA-GGG-CAC-GAG-ACA-CAG-AGA-GAC-AAC-ACG-TGC-CCA-AC-3'(序列2)
2、AFB1半抗原和载体蛋白BSA的偶联物AFB1-BSA制备
偶联物AFB1-BSA的结构式如图2，黄曲霉毒素B1半抗原的羧基和载体蛋白BSA的氨基形成肽键。
上述偶联物的制备方法：首先经过肟化反应制备AFB1肟(AFB1-O)；然后用碳二亚胺法制备AFB1人工抗原；最后根据AFB1与修饰过的牛血清蛋白(C-BSA)的反应得 到偶联产物，即偶联物AFB1-BSA。
3、制备试纸条
1)、标记物垫制备
将检测探针包被在标记物垫(上海杰一生物技术有限公司，JY-BX101)上(包被浓度为0.1μM)，得到包被检测探针的标记物垫；
2)、反应膜制备
反应膜包括检测区和质控区。
将偶联物AFB1-BSA按照包被浓度为1μM包被在反应膜形成检测区；
质控探针为标记生物素的适配体互补序列，其通过链酶亲和素与其上的生物素偶联形成共价键包被在反应膜上形成质控区，包被浓度为1μM。
3)、制备试纸条
试纸条由样品吸收垫(上海杰一生物技术有限公司，JY-BX111)、上述1)制备的包被检测探针的标记物垫、上述2)制备的反应膜、吸水垫依次按照顺序黏贴在底板上，且反应膜的检测区与标记物垫相邻，反应膜的检质控区与吸水垫相邻，如图1及图3上图所示。
二、试纸条检测原理
如图3中图所示，当不含黄曲霉毒素B1的样品加入样品吸收垫上时，样品在毛细作用下流动到标记物垫，并带着荧光标记的探针混合物一起继续向前流动。当流动到检测区时，检测探针会与检测区处固定的半抗原结合，经试纸条检测仪器ESEQuant-LR3检测出高荧光强度；当流动到质控区时，剩余的检测探针会与质控探针结合，质控区也出现高荧光强度。
如图3下图所示，当含有黄曲霉毒素B1的样品加入样品吸收垫上时，样品在毛细作用下流动到标记物垫，黄曲霉毒素B1与检测探针(适配体)结合，并在毛细作用下一起向前流动。当流动到检测区时，未与黄曲霉毒素B1结合的检测探针会与半抗原结合；当样品中的AFB1越多，结合的检测探针也就越来越多，剩余的游离检测探针就会越来越少，和检测区半抗原结合的检测探针也就越少，荧光轻度越低。反之，样品中的黄曲霉毒素B1越来越少，剩余的游离检测探针就会越来越多，和检测区处半抗原结合也就越多，荧光强度越强，从而可以根据荧光强度的变化判断样品中黄曲霉毒素B1的浓度。
三、试纸条检测方法
1、取黄曲霉毒素B1或M1标准品滴加到试纸的样品吸收垫上，5分钟后，将试纸条放入试纸条检测仪器ESEQuant-LR3中，进行检测，分别测定检测区和质控区的荧光信号强度；将检测区的荧光信号强度(A)比上质控区的荧光信号强度(B)，得到A/B值，将得到的A/B值制作黄曲霉毒素B1或M1标准品的浓度对应荧光信号比值的一元线性回归曲线，计算回归方程；
2、将黄曲霉毒素标准品B1或M1替换为待测样品，用上述试纸检测待测样品，记录在所述试纸的检测区和质控区的荧光信号比值，根据步骤(1)的回归方程，得到待测样品中黄曲霉毒素的浓度。
四、试纸条检测黄曲霉毒素B1或M1灵敏度、线性范围、特异性研究
1、检测黄曲霉毒素B1灵敏度与线性范围研究
配制图4所示的不同浓度AFB1(Fermentek Ltd,AF028)水溶液，分别用实施例1制备的试纸条进行测定。
用试纸条检测仪器ESEQuant-LR3分别测定检测区和质控区处的荧光强度。结果如图4所示，可以看出，随着AFB1浓度增加，检测区处荧光强度逐渐变小。利用检测区 荧光强度与质控区荧光强度比值(表1)，绘制检测黄曲霉毒素B1的标准曲线如图5所示，结果表明，试纸条的灵敏度0.5ng/mL；线性范围为0.5ng/mL-50μg/mL。
表1黄曲霉毒素B1标准品的检测结果
Figure PCTCN2017070786-appb-000001
2、检测黄曲霉毒素M1灵敏度与线性范围研究
按照上述方法，配制不同浓度AFM1(百灵威科技有限公司，6795-23-9)水溶液，分别用实施例1制备的试纸条进行测定。
用试纸条检测仪器ESEQuant-LR3分别测定检测区和质控区处的荧光强度，利用检测区荧光强度与质控区荧光强度比值(表2)，绘制检测黄曲霉毒素M1的标准曲线如图
6所示，结果表明，试纸条的灵敏度为10ng/mL；线性范围为10-1000ng/mL。
表2黄曲霉毒素M1标准品的检测结果
Figure PCTCN2017070786-appb-000002
3、特异性研究
配制浓度为100ng/mL的AFB1溶液、100ng/mL的AFM1溶液、浓度为100ng/mL的黄曲霉毒素G1(百灵威科技有限公司，1165-39-5)、浓度为100ng/mL的黄曲霉毒素B2(百灵威科技有限公司，7220-81-7)、浓度为100ng/mL的赭曲酶毒素Ochratoxin A(OTA，Fermentek Ltd,OC030a)溶液、浓度为100ng/mL的玉米赤酶烯酮Zearalenone(ZEN，百灵威科技有限公司，17924-92-4)溶液，浓度为100ng/mL的呕吐毒素Vomitoxin(DON，百灵威科技有限公司，51481-10-8)溶液和空白样品分别用试纸条进行测定。
用试纸条检测仪器ESEQuant-LR3分别测定检测区和质控区处的荧光强度，结果如图7所示，显示AFG1、AFB2、OTA、ZEN、DON以及空白样品等均不能引起检测区处荧光变化，这也说明本方法对黄曲霉毒素B1或M1具有很好的特异性。
五、试纸条在花生粕中黄曲霉毒素B1检测中的应用
1、标准曲线绘制
同实施例1中检测黄曲霉毒素B1灵敏度与线性范围研究中的标准曲线方法，得到的回归方程为：y＝-0.2076x+0.9881，R2＝0.99，为黄曲霉毒素B1标准曲线一元回归方程。
2、样品提取
选择一份经国标方法(LC-MSMS)测定过的花生粕黄曲霉毒素B1样品(含量143.47ng/mL)，用本试纸条进行测定。首先称取2g花生粕于50mL离心管中，加入8mL正己烷和10mL70％的甲醇水溶液，振荡5min，室温4000转/分离心10min；去除上层液体，取0.5mL下层液体加入0.5mL去离子水，混匀，再取混匀液体0.5mL加入0.5mL35％的甲醇水溶液，振荡30s；取100μL进行分析(样品稀释倍数为20倍)。
3、样品测定
取上述制备好的待测样品100μL滴加到所述试纸的样品吸收垫上，观察样品层析沿着硝酸纤维素膜流动，直到被上面的吸水垫吸附；5分钟后，将试纸条放入试纸条检测仪器ESEQuant-LR3中，进行检测，分别测定检测区和质控区的荧光信号强度；将检测区的荧光信号强度(A)比上质控区的荧光信号强度(B)，将得到的计算值代入上述步骤1的回归方程，计算得到待测样品中黄曲霉毒素B1的浓度。
3、检测结果：
检测区的荧光强度(A)为962.06，质控区的荧光强度(B)为1210.75，A/B为0.7946，计算得到的待测样品中黄曲霉毒素B1的浓度为171.05ng/mL。与国标方法测定值误差低于10％，说明本方法具有很好的准确度。
六、试纸条在花生粕中黄曲霉毒素M1检测中的应用
1、标准曲线绘制
同实施例1中检测黄曲霉毒素M1灵敏度与线性范围研究中的标准曲线方法，得到的回归方程为：y＝-0.321x+1.1987，R2＝0.9973。
2、样品提取
选择一份经国标方法(LC-MSMS)测定过的花生粕黄曲霉毒素M1样品(含量261.54ng/mL)，用本试纸条进行测定。首先称取2g花生粕于50mL离心管中，加入8mL正己烷和10mL70％的甲醇水溶液，振荡5min，室温4000转/分离心10min；去除上层液体，取0.5mL下层液体加入0.5mL去离子水，混匀，再取混匀液体0.5mL加入0.5mL35％的甲醇水溶液，振荡30s；取100μL进行分析(样品稀释倍数为20倍)。
3、样品测定
取上述制备好的待测样品100μL滴加到所述试纸的样品吸收垫上，观察样品层析沿着硝酸纤维素膜流动，直到被上面的吸水垫吸附；5分钟后，将试纸条放入试纸条检测仪器ESEQuant-LR3中，进行检测，分别测定检测区和质控区的荧光信号强度；将检测区的荧光信号强度(A)比上质控区的荧光信号强度(B)，将得到的计算值代入上述步骤1的回归方程，计算得到待测样品中黄曲霉毒素M1的浓度。
4、检测结果：
检测区的荧光强度(A)为1006.77，质控区的荧光强度(B)为1234.65，A/B为0.815432，计算得到的待测样品中黄曲霉毒素M1的浓度为312.61ng/mL。与国标方法测定值误差低于20％，说明本方法具有较好的准确度。
实施例2检测黄曲霉毒素B1试纸条的制备及在检测黄曲霉毒素B1(AFB1)中的应用
一、检测黄曲霉毒素B1试纸条的制备
执行与实施例1相同的步骤，如无特殊说明，使用与实施例1中相同的材料、试剂等。不同之处在于：
在检测探针和质控探针的合成中，检测探针为荧光素Cy5标记的带有连接序列PolyT18的黄曲霉毒素B1适配体；黄曲霉毒素B1适配体可以和检测区的黄曲霉毒素 B1半抗原结合，黄曲霉毒素B1适配体的核苷酸序列如下：
5'-Cy5-TTT-TTT-TTT-TTT-TTT-TTT-GTT-GGG-CAC-GTG-TTG-TCT-CTC-TGT-GTC-TCG-TGC-CCT-TCG-CTA-GGC-CCA-CA-3′(序列3)
质控探针为生物素标记的可以与黄曲霉毒素B1适配体连接序列结合的PolyA18，可以与检测探针连接序列PolyT18通过碱基互补结合。
质控探针的核酸序列为：
5'-biotin-AAA-AAA-AAA-AAA-AAA-AAA-3'(序列4)
二、试纸条检测原理
与实施例1中说明的试纸条检测原理相同。
三、试纸条检测方法
1)取黄曲霉毒素B1标准品滴加到试纸的样品吸收垫上，10分钟后，将试纸条放入试纸条检测仪器ESEQuant-LR3中，进行检测，分别测定检测区和质控区的荧光信号强度；将检测区的荧光信号强度(T)比上质控区的荧光信号强度(C)，得到T/C值，制作黄曲霉毒素B1标准品的浓度对应荧光信号T/C比值的线性回归曲线，计算回归方程；
2)将黄曲霉毒素标准品B1替换为待测样品，用上述试纸检测待测样品，记录在所述试纸的检测区和质控区的荧光信号比值，根据步骤(1)的回归方程，得到待测样品中黄曲霉毒素的浓度。
四、黄曲霉毒素B1试纸条在检测黄曲霉毒素B1中的应用
1、检测黄曲霉毒素B1灵敏度与线性范围研究
配制图8所示的不同浓度AFB1(Fermentek Ltd，AF028)水溶液，分别用实施例1制备的试纸条进行测定。
用试纸条检测仪器ESEQuant-LR3分别测定检测区和质控区处的荧光强度。结果如图8所示，可以看出，随着AFB1浓度增加，检测区处荧光强度逐渐变小，质控区荧光强度逐渐增加。利用检测区荧光强度与质控区荧光强度比值(表3)，绘制检测黄曲霉毒素B1的标准曲线如图9所示，结果表明，试纸条的灵敏度0.1ng/mL；线性范围为0.1ng/mL～1000ng/mL。
表3黄曲霉毒素B1标准品的检测结果
Figure PCTCN2017070786-appb-000003
2、黄曲霉毒素B1试纸条在花生粕中黄曲霉毒素B1检测中的应用
1)、标准曲线绘制
同本实施例第一部分中检测黄曲霉毒素B1灵敏度与线性范围研究中的标准曲线方 法，结果如图9所示，标准曲线为y＝-1.8999x+5.3963，R2＝0.9846。
2)、样品提取
选择5份经国标方法(LC-MSMS)测定过的花生粕黄曲霉毒素B1样品，用本试纸条进行测定。首先称取2g花生粕于50mL离心管中，加入8mL正己烷和10mL 70％的甲醇水溶液，振荡5min，室温4000转/分离心10min；去除上层液体，取0.5mL下层液体加入0.5mL去离子水，混匀，再取混匀液体0.5mL加入0.5mL 35％的甲醇水溶液，振荡30s；取100μL进行分析(样品稀释倍数为20倍)。
3)、样品测定
取上述制备好的待测样品100μL滴加到所述试纸的样品吸收垫上，观察样品层析沿着硝酸纤维素膜流动，直到被上面的吸水垫吸附；10分钟后，将试纸条放入试纸条检测仪器ESEQuant-LR3中，进行检测，分别测定检测区和质控区的荧光信号强度；将检测区的荧光信号强度(T)比上质控区的荧光信号强度(C)，将得到的计算值代入上述步骤1的回归方程，计算得到待测样品中黄曲霉毒素B1的浓度。
4)、检测结果：
5份花生粕实际样品检测结果如表4所示。从结果可以看出，本发明试纸条结果与LC-MS方法相比，回收率在110％～115％之间，回收率较好，说明本发明试纸条和LC-MS方法具有非常好一致性，同时可以看出本方法比LC-MS方法整体偏高，避免了可能的假阴性结果。另外本方法标准差小于10％，说明本方法具有很好的稳定性。因此可以用于实际样品中黄曲霉毒素B1的检测。
表4本发明试纸条检测花生粕中AFB1结果与LC-MS方法比较
Figure PCTCN2017070786-appb-000004
3、黄曲霉毒素B1试纸条在检测不同样品并与进口试剂盒比较
1)、标准曲线绘制
同本实施例第一部分中检测黄曲霉毒素B1灵敏度与线性范围研究中的标准曲线方法，结果如图9所示，标准曲线为y＝-1.8999x+5.3963，R2＝0.9846。
2)、样品提取
选择10份经进口黄曲霉毒素B1酶联免疫试剂盒(美国ROMER试剂盒)测定为阳性或阴性的不同粮油及饲料样品，用本试纸条进行测定。首先称取2g粮油或饲料样品于50mL离心管中，加入8mL正己烷和10mL 70％的甲醇水溶液，振荡5min，室温4000转/分离心10min；去除上层液体，取0.5mL下层液体加入0.5mL去离子水，混匀，再取混匀液体0.5mL加入0.5mL 35％的甲醇水溶液，振荡30s；取100μL进行分析(样品稀释倍数为20倍)。
3)、样品测定
取上述制备好的待测样品100μL滴加到所述试纸的样品吸收垫上，观察样品层析沿着硝酸纤维素膜流动，直到被上面的吸水垫吸附；10分钟后，将试纸条放入试纸条 检测仪器ESEQuant-LR3中，进行检测，分别测定检测区和质控区的荧光信号强度；将检测区的荧光信号强度(T)比上质控区的荧光信号强度(C)，将得到的计算值代入上述步骤1的回归方程，计算得到待测样品中黄曲霉毒素B1的浓度。
4)、检测结果：
结果见表5。检测10种不同的基质样品，回收率在85％-110％之间，说明本方法具有很好的适用性和准确度。
表5本发明试纸条检测不同基质AFB1结果与进口试剂盒比较
Figure PCTCN2017070786-appb-000005
实施例3检测凝血酶试纸条的制备及在检测凝血酶中的应用
一、检测凝血酶试纸条的制备
1、检测探针和质控探针的合成
分别设计合成胶体金标记的检测探针和生物素标记的质控探针。
其中检测探针为胶体金标记的带有连接序列PolyT20的凝血酶适配体；凝血酶适配体可以和检测区的凝血酶抗原结合，凝血酶适配体的核苷酸序列如下：
5′-SH-TT TTT TTT TTT TTT TTT TTT GGT TGG TGTGGT TGG-3′(序列5)
胶体金合成方法为：加热煮沸100mL浓度为1mM的HAuCl4溶液，剧烈搅拌下加入10mL浓度为38.8mM的柠檬酸三钠溶液，继续煮沸20分钟，然后室温冷却，得到的胶体金粒径为13nm。
胶体金与适配体偶联方法为：取10μL浓度为100μM巯基修饰的适配体、1μL醋酸钠(pH＝5.20)和0.5μL浓度为10mM三(2-羧乙基)膦盐酸盐混合避光孵育1小时，13000rpm离心20min后加入1mL合成好的胶体金室温孵育16小时以上。然后加入12μL Tris-Hac缓冲液(pH＝8.2)和100μL浓度1M的NaCl溶液进行盐化一天。然后16,000rpm离心15min去除多余的适配体，然后用缓冲液重悬备用(100mL PB溶液，0.5g PEG20000，1g sucrose，0.1mL Tween-20，0.02g MgSO4，and 0.05g(NH4)2SO4))。
质控探针为生物素标记的可以与凝血酶适配体连接序列结合的PolyT20，可以与检测探针连接序列PolyA20通过碱基互补结合。
质控序列的核苷酸序列为：
5′-AAA AAA AAA AAA AAA AAA AA-biotin-3′(序列6)
2、制备试纸条
1)、标记物垫制备
将检测探针包被在标记物垫(上海杰一生物技术有限公司，JY-BX101)上(包被浓度为0.1μM)，得到包被检测探针的标记物垫，37度过夜烘干。
2)、反应膜制备
反应膜包括检测区和质控区。
将凝血酶抗原按照包被浓度为1mg/mL包被在反应膜形成检测区；
质控探针为标记生物素的适配体连接序列的互补序列，其通过链酶亲和素与其上的生物素偶联形成共价键包被在反应膜上形成质控区，包被浓度为5μM。
3)、试纸条组装
试纸条由样品吸收垫(上海杰一生物技术有限公司，JY-BX111)、上述1)制备的包被检测探针的标记物垫、上述2)制备的反应膜、吸水垫依次按照顺序黏贴在底板上，且反应膜的检测区与标记物垫相邻，反应膜的检质控区与吸水垫相邻，如图1所示。
二、试纸条检测原理
如图1所示，当不含凝血酶的样品加入样品吸收垫1上时，样品在毛细作用下流动到标记物垫2，并带着胶体金标记的探针混合物一起继续向前流动。当流动到检测区6时，标有用于显示信号强度的指示剂的检测靶标物的适配体9会与检测区6处固定的凝血酶抗原结合，出现胶体金红色条带，经试纸条检测仪器ESEQuant-LR3检测出高显色信号强度；当流动到质控区7时，剩余的标有用于显示信号强度的指示剂的检测靶标物的适配体9会与固定包被在质控区的质控探针11，质控区7也出现红色条带，经试纸条检测仪器ESEQuant-LR3检测出显色信号强度。
如图1所示，当含有凝血酶的样品加入样品吸收垫上时，样品在毛细作用下流动到标记物垫，凝血酶与检测探针(适配体)结合，并在毛细作用下一起向前流动。当流动到检测区时，未与凝血酶结合的检测探针会与凝血酶抗原结合；当样品中的凝血酶越多，结合的检测探针也就越来越多，剩余的游离检测探针就会越来越少，和检测区凝血酶抗原结合的检测探针也就越少，胶体金显色强度越低。反之，样品中的凝血酶越少，剩余的游离检测探针就会越来越多，和检测区处凝血酶抗原结合也就越多，胶体金显色强度越强，通过纸条检测仪器ESEQuant-LR3检测检测区和质控区显色信号强度的变化即判断样品中凝血酶的浓度。
三、试纸条检测方法
1)取凝血酶标准品滴加到试纸的样品吸收垫上，10分钟后，将试纸条放入试纸条检测仪器ESEQuant-LR3中，进行检测，分别测定检测区和质控区的胶体金显色信号强度；将检测区的显色信号强度(T)比上质控区的显色信号强度(C)，得到T/C值，将得到的T/C值制作凝血酶标准品的浓度对应显色信号比值的线性回归曲线，计算回归方程；
2)将凝血酶标准品替换为待测样品，用上述试纸检测待测样品，记录在所述试纸的检测区和质控区的显色信号比值，根据步骤(1)的回归方程，得到待测样品中凝血酶的浓度。
四、凝血酶试纸条在检测凝血酶中的应用
1、检测凝血酶灵敏度与线性范围研究
配制图10所示的不同浓度凝血酶(Sigma，Cat#T6884)水溶液，分别用实施例2制备的试纸条进行测定。
用试纸条检测仪器ESEQuant-LR3分别测定检测区和质控区处的显色强度。结果如图10所示，可以看出，随着凝血酶浓度增加，检测区处显色强度逐渐变小。利用检测区显色强度与质控区显色强度比值(表6)，绘制检测凝血酶的标准曲线如图11所示，结果表明，试纸条的灵敏度1nM；线性范围为1nM-100nM。
表6凝血酶标准品的检测结果
Figure PCTCN2017070786-appb-000006
2、凝血酶试纸条在血清凝血酶检测中的应用
1)、标准曲线绘制
同本实施例第一部分中检测凝血酶灵敏度与线性范围研究中的标准曲线方法，得到的回归方程为：y＝-1.177x+2.4398，R2＝0.972，为凝血酶标准曲线一元回归方程。
2)、样品制备
取小鼠血清，分别加入终浓度分别为2nM、10nM、50nM的人凝血酶。
3)、样品测定
取上述制备好的待测样品100μL滴加到所述试纸的样品吸收垫上，观察样品层析沿着硝酸纤维素膜流动，直到被上面的吸水垫吸附；10分钟后，观察试纸条显色信号，并将试纸条放入试纸条检测仪器ESEQuant-LR3中，进行检测，分别测定检测区和质控区的显色信号强度；将检测区的显色信号强度(T)比上质控区的显色信号强度(C)，将得到的计算值代入上述步骤1的回归方程，计算得到待测样品中凝血酶的浓度。
4)、检测结果：
3份添加人凝血酶的小鼠血清样品测得结果如表7所示。从结果可以看出，本发明试纸条的回收率在105％～115％之间，标准差小于15％，具有很好的准确度和精密度，因此可以用于实际样品中凝血酶的检测。
表7小鼠血清凝血酶添加回收实验结果
添加浓度 实测浓度 回收率 标准差(％)
2nM 2.3 115％ 12.2
10nM 10.5 105％ 9.8.
50nM 55 110％ 8.7
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (18)

  1. 一种基于抗原-适配体的竞争法检测试纸条，其特征在于，所述试纸条包括样品吸收垫、标记物垫、反应膜、吸水垫和底板；
    所述标记物垫包被有检测探针，所述检测探针为标有用于显示信号强度的指示剂的检测靶标物的适配体；
    所述反应膜上设置有检测区和质检区；
    所述检测区固定包被有靶标物抗原；
    所述质检区固定包被有质控探针。
  2. 根据权利要求1所述的基于抗原-适配体的竞争法检测试纸条，其特征在于，当所述靶标物为有机物大分子靶标时，所述靶标物抗原即为靶标物本身；
    当所述靶标物为无机小分子靶标时，所述靶标物抗原为所述无机小分子靶标与载体蛋白的偶联物。
  3. 根据权利要求2所述的基于抗原-适配体的竞争法检测试纸条，其特征在于，所述载体蛋白包括但不限于酪蛋白、牛血清白蛋白或鸡卵清白蛋白。
  4. 根据权利要求1所述的基于抗原-适配体的竞争法检测试纸条，其特征在于，所述显示信号强度的指示剂包括荧光物质、生物素、放射性同位素、电子致密物、胶体金或酶中的任一种。
  5. 根据权利要求4所述的基于抗原-适配体的竞争法检测试纸条，其特征在于，所述荧光物质包括PE、CY5、CY7、per-CP、TRITC、Alexa Fluor647以及量子点中的任一种。
  6. 根据权利要求1所述的基于抗原-适配体的竞争法检测试纸条，其特征在于，所述质控探针的核酸序列与所述适配体互补或与所述适配体上连接的序列互补。
  7. 根据权利要求6所述的基于抗原-适配体的竞争法检测试纸条，其特征在于，所述适配体上连接的序列为PolyA、PolyT、PolyG或PolyC中的一种。
  8. 根据权利要求7所述的基于抗原-适配体的竞争法检测试纸条，其特征在于，所述适配体上连接的序列为PolyT18。
  9. 权利要求1～8任一项所述的抗原-适配体的竞争法检测试纸条在检测食品污染物、临床疾病标志物等中的应用，特别是其在检测黄曲霉毒素B1和M1和凝血酶中的应用。
  10. 如权利要求9所述的应用，其特征在于，包括：
    1)、用试纸条检测靶标标准品，得到标准品在试纸条的检测区和质控区的信号强度比值，制作靶标标准品的浓度对应信号强度比值的线性回归曲线，计算回归方程；
    2)、用试纸条检测待测样品，得到待测样品在试纸条的检测区和质控区的信号强度比值，根据步骤1)得到的回归方程，得到所述待测样品中靶标物的含量。
  11. 一种检测黄曲霉毒素B1或M1的试纸，包括样品吸收垫、标记物垫、反应膜和吸水垫；
    所述标记物垫包被检测探针，所述检测探针为荧光素标记的黄曲霉毒素B1适配体；所述黄曲霉毒素B1适配体的核苷酸序列为序列1；
    所述反应膜包括检测区和质控区；
    所述检测区包被黄曲霉毒素B1半抗原与载体蛋白形成的偶联物；
    所述质控区包被质控探针，所述质控探针的核苷酸序列为所述适配体的互补序列。
  12. 根据权利要求11所述的试纸条，其特征在于：所述黄曲霉毒素B1半抗原能与所述黄曲霉毒素B1适配体结合；
    所述质控探针的5’末端标记生物素。
  13. 根据权利要求11或12所述的试纸条，其特征在于：所述载体蛋白为卵清白蛋白或牛血清白蛋白；
    所述荧光素为Cy5、Alexa Fluor647、量子点或胶体金。
  14. 根据权利要求11-13中任一所述的试纸条，其特征在于：
    所述试纸中还包括底板，且所述样品吸收垫、所述标记物垫、所述反应膜和所述吸水垫顺次固定在所述底板上。
  15. 权利要求11-14任一所述的试纸条在检测黄曲霉毒素B1或M1中的应用。
  16. 权利要求11-14任一所述的试纸条在制备检测黄曲霉毒素B1或M1产品中的应用。
  17. 一种检测待测样品中黄曲霉毒素B1或M1含量的方法，包括如下步骤：
    (1)用权利要求11-14任一所述的试纸条检测黄曲霉毒素B1或M1标准品，得到标准品在所述试纸的检测区和质控区的荧光信号比值，制作黄曲霉毒素B1或M1标准品的浓度对应荧光信号比值的一元线性回归曲线，计算回归方程；
    (2)将所述黄曲霉毒素标准品替换为待测样品，用所述试纸检测待测样品，得到待测样品在所述试纸的检测区和质控区的荧光信号比值，根据步骤(1)的回归方程，得到所述待测样品中黄曲霉毒素的含量。
  18. 一种检测黄曲霉毒素B1或M1的试纸，包括样品吸收垫、标记物垫、反应膜和吸水垫；
    所述标记物垫包被检测探针，所述检测探针为含有连接序列的黄曲霉毒素适配体；
    所述反应膜包括检测区和质控区；
    所述检测区包被黄曲霉毒素B1半抗原与载体蛋白形成的偶联物；
    所述质控区包被质控探针，所述质控探针的核苷酸序列为所述适配体的连接序列的互补序列。
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