A Cas12a-specific immunochromatographic test strip and its preparation method and application

Abstract

The present invention provides a kind of Cas12a special immunochromatographic test strip and its preparation method and application, the method comprises steps: S1: sample pad and conjugate pad are soaked with different treatment solutions and then dried; S2: streaking of detection T line and quality control C line is performed on chromatographic membrane, and streaking conditions are respectively 1mg / mL sheep anti-rabbit secondary antibody and 2mg / mL r-SA containing 0.2% BSA; S3: chromatographic membrane, conjugate pad, sample pad, absorption pad are assembled on a sticky bottom plate successively, and sample pad, conjugate pad, chromatographic membrane and absorption pad are arranged successively; S4: shearing is made into the immunochromatographic test strip of prescribed width, sealed and preserved. The present invention prepares a kind of Cas12a special immunochromatographic test strip by reagent screening, material selection, condition optimization, etc., which is conducive to the application of the high-sensitive visual detection method based on Cas12a in the relevant fields such as rapid transgenic and on-site detection, and has practical application value.

Description

Technical Field

[0001] The present invention relates to the field of molecular detection technology, and more specifically to a Cas12a-specific immunochromatographic test strip, a preparation method thereof, and applications thereof. Background Art

[0002] Lateral flow chromatography (LFCA) is an effective technology for detecting target substances in samples, based on immune recognition or nucleic acid hybridization and antibody labeling technology, which enables the components (antigens, antibodies, proteins, nucleic acids, etc.) in the object to be detected to be separated on the reaction matrix through the difference in the movement speed of capillary force. This technology has the advantages of simple operation, low detection cost, and high detection efficiency. It is widely used in various on-site detection fields because it does not require equipment. By combining with isothermal amplification technology, a highly sensitive test strip method based on the CRISPR system has been developed, which completely breaks the limitations of the test strip method in terms of sensitivity.

[0003] Tsou et al. (2019) combined CRISPR / Cas12a with lateral flow test strips to directly and specifically detect HPV16 and HPV18 in liquid samples, with a detection limit of 0.24fM, the same as polymerase chain reaction, but in a shorter time. Based on the combination of loop-mediated isothermal amplification (LAMP), CRISPR / Cas12a "trans-cleavage" and lateral flow biosensor (LFB), an ultrasensitive HPV16 and HPV18 detection method (named CIALFB) was developed, which achieved highly sensitive detection of 3.1aM (about 1.8 copies) targets and had reliable specificity for clinical samples. Zhu et al. (2022) developed a PAM-free loop-mediated isothermal amplification (LAMP)-assisted CRISPR / Cas12a detection platform (named Cas-PfLAMP), which can be used to quickly (about 50 minutes) and accurately detect three rice pathogens with a sensitivity as low as 9 or 3 copies. These applications demonstrate the efficiency and high sensitivity of CRISPR test strips, and also provide new ideas for the rapid detection of GMOs.

[0004] However, most current CRISPR test strip-related studies use universal commercial nucleic acid-based test strips. Due to the poor compatibility between the universal test strips and the optimized CRISPR reaction system, the test strips have poor detection effects. Summary of the invention

[0005] The purpose of the present invention is to provide a Cas12a-specific immunochromatographic test strip and a preparation method and application thereof, so as to solve the problem of poor detection effect of CRISPR test strips in the prior art.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] According to a first aspect of the present invention, a method for preparing a Cas12a-specific immunochromatographic test strip is provided, comprising the following steps: S1: pretreatment of a sample pad and a conjugate pad: the sample pad and the conjugate pad are soaked in different treatment solutions and then dried for standby use, wherein the sample pad treatment solution is: 2% glucose, 1% Triton X-100, 1% BSA, 50mM boric acid, pH 8.0, and the composition of the conjugate pad treatment solution is: 1% sucrose, 3% BSA, 0.5% Tween-20, and the solvent is 0.01M Tris-base solution, pH 8.0; S2: streaking of the detection line T and the quality control line C: streaking of the detection line T and the quality control line C on the chromatographic membrane using a colloidal gold spotting system, the streaking condition for the detection line T is 1 mg / mL goat anti-rabbit secondary antibody containing 0.2% BSA, and the streaking condition for the quality control line C is 2 mg / mL r-SA; S3: assembly: assembling the streaked chromatographic membrane, conjugate pad, sample pad, and absorption pad on the adhesive bottom plate in sequence, with adjacent pads partially overlapping, wherein the sample pad, conjugate pad, chromatographic membrane, and absorption pad are arranged in sequence along the chromatographic direction, the quality control line C is close to the conjugate pad, and the detection line T is close to the absorption pad; S4: cutting: cutting the assembled composite plate into immunochromatographic test strips of a specified width, and sealing and storing them under dry conditions for later use.

[0008] Preferably, CN140 is selected as the chromatography membrane.

[0009] Preferably, 4 μL of colloidal gold-labeled FITC antibody, containing 0.8 μg of FITC antibody, is further applied to the pretreated conjugate pad.

[0010] Preferably, in step S4, the composite plate is cut into 3 mm wide immunochromatographic test strips.

[0011] According to a second aspect of the present invention, a Cas12a-specific immunochromatographic test strip prepared according to the above-mentioned preparation method is provided.

[0012] According to a third aspect of the present invention, there is provided an application of a Cas12a-specific immunochromatographic test strip in genetically modified crop detection, comprising the following steps: 1) extracting genomic DNA from a sample to be tested;

[0013] 2) RPA amplification: using the genomic DNA as a template, P-CaMV 35S and T-NOS as targets, and using RPA primers corresponding to P-CaMV 35S and T-NOS, respectively, to perform RPA amplification reaction; 3) Cas12a digestion reaction: performing Cas12a digestion on the RPA amplification product obtained in step 2), and the reaction conditions are 37°C for 15 min; 4) Immunochromatographic test strip detection: adding the Cas12a digestion product obtained in step 3) to the sample pad of the prepared immunochromatographic test strip, immersing one end of the sample pad in the chromatography buffer, and standing vertically for 3 to 7 minutes. After the buffer reaches the absorbent paper, the immunochromatographic test strip is taken out, and the color development of the C line and the T line is observed; when the immunochromatographic test strip simultaneously shows a clear detection line T and a quality control line C, it indicates that the target P-CaMV 35S and / or T-NOS are present; when the immunochromatographic test strip only shows a clear quality control line C, it means that both the target P-CaMV 35S and T-NOS are not present; when both the detection line T and the quality control line C on the immunochromatographic test strip are not visible, it means that the test has failed or the test strip is invalid.

[0014] In step 2), the RPA primers corresponding to the targets P-CaMV 35S and T-NOS are:

[0015] Primer pair for detection of P-CaMV 35S gene:

[0016] RPA-35S-F: 5'-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3',

[0017] RPA-35S-R: 5'-CCCTTACGTCAGTGGAGATATCACATCAATCC-3';

[0018] Primer pairs for detecting T-NOS gene:

[0019] RPA-NOS-F: 5'-TGAATCCTGTTGCCGGTCTTGCGATGATTATC-3',

[0020] RPA-NOS-R: 5'-TCTATCGCGTATTAAATGTATAATTGCGGGAC-3'.

[0021] In step 2), the amplification system of the RPA amplification reaction includes: 29.5 μL Primer free rehydration buffer, 2.4 μL of upstream and downstream primers (10 μM), 11.2 μL ddH2O, 2.5 μL MgOAC, and 2 μL DNA template.

[0022] In step 3), 15n-Biotin-ssDNA probe is used in the Cas12a enzyme digestion reaction system, and its final concentration is 250nM.

[0023] In step 4), the components of the chromatography buffer used are: 25 mM Tris, 150 mM NaCl, 0.05% Tween-20, 5% PEG 6000, and 0.1% BSA.

[0024] The Cas12a-specific immunochromatographic test strip provided by the present invention is prepared based on the Cas12a "trans-cleavage" mechanism. The detection process includes rapid genomic DNA extraction (5 min), RPA reaction (37 ° C, 12 min), Cas12a enzyme cleavage reaction (37 ° C, 15 min), and test strip detection (5 min). The whole process can be completed within 40 min (the detection principle is as follows Figure 1 As shown). The RPA reaction is used to amplify the target DNA fragment, and the Cas12a enzyme digestion reaction provides the analyte for the test strip. The digestion product is subjected to lateral flow immunochromatography, and the final result is displayed in the form of a test strip. According to a preferred embodiment of the present invention, the detection system targets the sample P-CaMV 35S or T-NOS, and the breakage of the 15nt-Biotin-ssDNA probe is used as the detection signal. The r-SA line position on the NC membrane of the test strip represents the quality control line (C line), and the secondary antibody line position represents the detection line (T line).

[0025] However, it should be understood that the detection of the target P-CaMV 35S or T-NOS is shown as an example of the main embodiment of the present invention, but the Cas12a dedicated immunochromatographic test strip of the present invention is not limited to the detection sample P-CaMV 35S or T-NOS, and can also be used for other target detection.

[0026] For test strip analysis, the analyte first binds to the gold-labeled FITC antibody on the binding pad and then flows to the absorption pad. The amounts of ssDNA probe, colloidal gold-labeled FITC antibody, r-SA, and goat anti-rabbit IgG antibody need to match each other, so the present invention optimizes the r-SA and secondary antibody fixed on the NC membrane during the test strip preparation process. The optimized r-SA is sufficient to capture all intact ssDNA probes bound to the gold-labeled complex. The optimized secondary antibody can ensure that the T line of the positive sample is clear. The optimized chromatography buffer helps to eliminate false positive signals.

[0027] According to the present invention, the detection of the presence of the target is positively correlated with the occurrence of the "trans-cutting" reaction of Cas12a, the breakage of the reporter probe, and the appearance of an obvious T line on the Cas12a special immunochromatographic test strip. Whether the ssDNA is broken or not determines the color development result of the test strip. After binding with crRNA, Cas12a has "trans-cutting" activity only when the target nucleic acid activator is present. When the target DNA is present, the complete ssDNA probe is cut off by the activated Cas12a, and the FITC end fragment of the broken ssDNA probe is captured by the T line, and the test strip shows an obvious T line; when the target is not present, the complete ssDNA is captured by the C line, and the test strip only shows a clear C line.

[0028] During the test process of the present invention, some phenomena that occurred can also be explained in combination with the detection principle. First, in terms of the selection of biotin capture objects, although biotin antibodies and ordinary streptavidin also have high affinity for biotin, they may not be suitable for test strip analysis. Special treatment methods such as thiolization combined with appropriate working buffer solutions enhance the hydrogen bonding, hydrophobic interaction, and electrostatic adsorption between recombinant streptavidin and the NC membrane, thereby enhancing the adsorption capacity of the NC membrane and being more conducive to immune binding on the solid phase carrier. In this experiment, compared with biotin antibodies and ordinary streptavidin, recombinant streptavidin (r-SA) is more easily immobilized on the NC membrane in the ruled area to form a stable C line.

[0029] Secondly, under negative conditions, when different concentrations of ssDNA probes are applied to Cas12a test strips, the color development of the C line is significantly different. At too low or too high concentrations, ssDNA cannot adapt to the test strip, resulting in overflow of the C line color and the appearance of unexpected T lines. When the ssDNA concentration is too low, there is a mismatch between the amount of FITC antigen carried by the double-labeled ssDNA and the amount of the gold-labeled antibody, resulting in a large amount of free gold-labeled antibodies unable to be bound by the C line and reach the T line, thereby causing the T line to develop color; when the ssDNA concentration is too high, due to the High-dose hook effect, a large amount of free ssDNA probes that are not bound to the gold-labeled antibody arrive at the C line before the ssDNA-gold-labeled antibody complex to occupy the r-SA binding site, resulting in the overflow of the gold-labeled complex to the T line. At the appropriate ssDNA concentration, the amount of ssDNA (double-end group labeling) matches the gold-labeled antibody and the streaking capture material, so that the negative sample has a stable C line and a very weak or no T line.

[0030] In addition, unlike the traditional test strips in which the C line is used as the secondary antibody, the Cas12a test strips of the present invention use the secondary antibody as the T line. Specifically, the cutting causes a large number of ssDNA reporter probes to break, and then the FITC group and the biotin group on the ssDNA are separated, and a large number of biotin-carrying probes that cannot bind to the gold-labeled complex occupy the binding site of streptavidin at the C line on the NC membrane and cannot develop color, and the gold-labeled complex migrates forward to the secondary antibody of the T line to make the T line develop color.

[0031] Another phenomenon is that compared with the obvious C line of the negative sample, the C line of the positive sample is significantly weaker. The same phenomenon also occurs in the application of other related methods. This is caused by the incomplete "trans" cutting of the ssDNA probe by Cas12a. The analysis product of the positive sample contains a large number of broken ssDNA probes and a small number of intact probes. The broken probes are related to the formation of the T line; the intact probe can bind to r-SA and the gold label complex at the same time, and its presence is the cause of the weak C line. Compared with the stable C line of the traditional band, the intensity of the C line of the positive sample is reduced to a certain extent, especially when the detection signal of the T line is very strong. More broken ssDNA signal reporters correspond to the enhancement of the T line and the weakening of the C line, which to a certain extent reflects the cutting efficiency of Cas12a.

[0032] According to the present invention, a Cas12a special immunochromatographic test strip is prepared, and immunological methods are successfully applied to nucleic acid detection. This study is conducive to giving full play to the rapid and on-site detection capabilities of RPA and Cas12a, overcoming the shortcomings of most nucleic acid detection methods that rely on complex equipment. This is the first time that Cas12a test strip technology has been applied to transgenic detection, providing a highly sensitive technical support for rapid detection of transgenics, which is of great significance to transgenic safety management.

[0033] In summary, the present invention has prepared a kind of Cas12a special immunochromatographic test strip with better detection effect through a large amount of screening and optimization work, and evaluated its detection effect. The final result shows that the whole detection process can be completed in 40min, and the detection limit of the prepared Cas12a test strip to P-CaMV 35S and T-NOS is as low as 1-10 copies (0.001ng genomic DNA) and the detection signal is stable under the detection limit concentration, and the specific detection result is consistent with the current standard, and the result of the actual sample application is consistent with the RPA agarose gel electrophoresis result. In summary, the present invention has successfully prepared a kind of Cas12a immunochromatographic test strip and applied to transgenic detection, which can further promote the application of ultrasensitive Cas12a test strips in transgenic field detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a detection principle diagram of a Cas12a-specific immunochromatographic test strip;

[0035] Figure 2 The results of NC membrane and biotin capture agent screening are shown in Figure 1. (A) NC membrane screening; (B) biotin capture agent screening;

[0036] Figure 3 This is the optimization result of the binding pad treatment solution;

[0037] Figure 4 Optimization of the preparation conditions of Cas12a-specific immunochromatographic test strips; N: negative control; P: positive control; (A) r-SA concentration optimization; (B) secondary antibody concentration optimization; (C) optimization of secondary antibody blocking conditions under negative / positive conditions; (D) comparison of the effects of the two schemes; (E) optimization of the amount of gold-labeled FITC antibody;

[0038] Figure 5 The conditions for using the Cas12a-specific immunochromatographic test strip were optimized; N: negative control; P: positive control; (A) ssDNA length optimization; (B) ssDNA concentration optimization; (C) chromatography buffer optimization;

[0039] Figure 6 It is the sensitivity analysis of the Cas12a-specific immunochromatographic test strip; wherein, the Ct value is the qPCR result; ****, p<0.0001; *, p<0.05; (A) Molecular sensitivity analysis results of gradient dilution plasmid; (A1 and A2) Test strip analysis results of gradient dilution plasmid; (A3 and A4) Fluorescence detection results of gradient dilution plasmid (measured by RPA-Cas12a-FS method); (B) Sensitivity analysis results of gradient dilution MON88017 genomic DNA; (B1 and B2) Test strip analysis results of gradient dilution genomic DNA; (B3 and B4) Fluorescence detection results of gradient dilution genomic DNA (measured by RPA-Cas12a-FS method); (C) Repeated detection results at LOD concentration in (A); (C1 and C2): Repeated detection results of test strip at LOD concentration in (A); (C3 and C4): Test strip band intensity ratio (T line / C line) in (C1) and (C2);

[0040] Figure 7 The agarose gel results of the RPA products of molecular sensitivity; (A) molecular sensitivity analysis results of gradient dilution plasmid; (B) sensitivity analysis results of gradient dilution MON88017 genomic DNA;

[0041] Figure 8It is a specific analysis of Cas12a-specific immunochromatographic test strips; among them, (A) 1–16: RRS, Bt11, KF6, KMD1, MON531, MON863, NK603, RF1, MS1, MIR604, MON88017, Bt176, MON88913, MON89788, MON87701, Sugar beet H7-1; (A1) agarose gel electrophoresis results of RPA products of samples; (A2) test strip analysis results of samples; (A3) test strip detection band quantification results; (B) 1-16: RRS, Bt11, KF6, KMD1, MON531, MON863, Bt176, NK603, MON88913, MON88017; (B1) agarose gel electrophoresis results of RPA products of samples; (B2) test strip analysis results of samples; (B3) test strip detection band quantification results;

[0042] Fig. 9 The figures are the test results of actual samples; wherein, N: blank control; 1-8: 8 random market samples; (A, B) agarose gel electrophoresis results of RPA products of T-NOS and P-CaMV 35S screening elements; (C, D) final test strip test results of T-NOS and P-CaMV 35S screening elements; (E, F) quantitative results of test strip test bands of positive standards. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0044] Materials and methods

[0045] 1 Reagents and Equipment

[0046] DNA-based ultra-fast nucleic acid release agent was purchased from Weifang Anpu Future Biotechnology Co., Ltd. Basic kits were purchased from Murray (Shanghai) Biotechnology Co., Ltd. LbCpf1 nuclease and RNase inhibitor were purchased from Shanghai Tolo Biotechnology Co., Ltd. and BBI Life Sciences Co., Ltd. Tecan Infinite 200PRO microplate reader was purchased from Tecan (Shanghai) Trading Co., Ltd. 96-well black / transparent flat-bottom polystyrene NBS microplates were purchased from Corning (Shanghai, China). Recombinant streptavidin (r-SA), colloidal gold-labeled rabbit anti-fluorescein FITC antibody (15 nm), goat anti-rabbit secondary antibody, polyethylene glycol PEG6000, and Sigma-Aldrich bovine serum albumin (BSA) were purchased from Beijing Borsi Technology Co., Ltd., Beijing Boosun Biotechnology Co., Ltd., Beijing Solebao Technology Co., Ltd., Beijing Solebao Technology Co., Ltd., and Beijing Borsi Technology Co., Ltd., respectively. Specific oligonucleotide probes were designed for the C-line and T-line marking materials of the test strip. The probes were labeled with FICT (fluorescein isothiocyanate) groups and Biotin groups at both ends, respectively. The required oligonucleotides were synthesized and purified by Sangon (Shanghai, China), and the relevant sequence information is listed in Table 1. All solvents and other chemicals were analytical grade reagents.

[0047] Table 1 Sequence information of RPA primers, crRNAs and ssDNA reporters

[0048]

[0049] Nitrocellulose membrane (NC membrane), sample pad, conjugate pad, absorption pad and PVC bottom plate were purchased from Merck Millipore. Bio-Dot CM4000 cutting machine and Bio-Dot XYZ3060 colloidal gold spotting system were purchased from Bio-Dot Trading (Shanghai) Co., Ltd.

[0050] The transgenic seed powder standard was obtained from AOCS (Urbana, IL, USA), and the sample information is listed in Table 2. Other seed samples were obtained from Shanghai Huacao Market.

[0051] Table 2 Test results of GM crop standards by current standards and this research method

[0052]

[0053]

[0054] Note: “√” means the test result is positive.

[0055] Example 1 Screening of test strip preparation materials and optimization of pretreatment methods

[0056] 1.1 Selection of NC membrane materials

[0057] In order to determine the materials suitable for preparing Cas12a test strips, this example screened two NC membranes: CN140 and CN95.

[0058] Results: Figure 2As shown in Figure A, different NC membranes have different porosities and adsorption capacities. There are significant differences between CN140 and CN95 in their adsorption capacities for r-SA, and CN140 is more suitable for this study.

[0059] 1.2 Screening of biotin capture reagents

[0060] On the other hand, although both biotin antibodies and streptavidin can specifically bind to biotin, their different affinities and immobilization capacities result in different application capabilities in the preparation of test strips. Each mole of streptavidin (SA) can bind to four moles of biotin, and often has a higher affinity than biotin. And because SA does not contain sugar groups and its isoelectric point is close to neutral, it has a lower nonspecific binding level than egg white avidin in detection. At the same time, the chemical properties, structure, steric hindrance, molecular weight and other factors of the capture agent have a great influence on its solid phase adsorption capacity, thereby affecting its application in test strip detection. This embodiment screened the Anti-Biotin (brand: Bioss), SA (brand: Sigma) and r-SA (brand: Bersee) that are widely used on the market.

[0061] Results: Figure 2 As shown in Figure B, it was found that the expected red bands did not appear at the line positions corresponding to Anti-Biotin and SA, indicating that the immobilization capacity of both on the NC membrane was poor and was not suitable for this study. Compared with Anti-Biotin and SA, the r-SA lined test strip showed obvious bands at the C line position, indicating that r-SA could be adsorbed on the NC membrane and successfully intercepted the complex containing colloidal gold and biotin (5'-Biotin-ssDNA probe-gold-labeled FITC antibody). Therefore, r-SA was finally selected for later research.

[0062] 1.3 Optimization of sample pad and conjugate pad pretreatment methods

[0063] In order to determine the pretreatment method of the material, this embodiment pretreated the sample pad and the conjugate pad differently before preparing the test strip. The material was soaked in the treatment solution and dried at 37°C for use. The sample pad treatment solution was: 2% glucose, 1% Triton X-100, 1% BSA, 50mM boric acid (pH 8.0). The four conjugate pad treatment solutions are as follows:

[0064] Treatment solution 1: 2% glucose, 1% Triton X-100, 1% BSA, 500 mM boric acid, pH 8.0;

[0065] Treatment solution 2: 2% glucose, 1% Triton X-100, 3% BSA, 500 mM boric acid, pH 8.0;

[0066] Treatment solution 3: 1% sucrose, 3% BSA, 0.5% Tween-20, the solvent is 0.01 M Tris-base solution (pH 8.0);

[0067] Treatment solution 4: 1% sucrose, 6% BSA, 0.5% Tween-20, the solvent is 0.01 M Tris-base solution (pH 8.0).

[0068] The conjugate pads soaked in treatment solutions 1, 2, 3, and 4 were dried and stored under the same conditions and subsequently applied to the same negative sample simultaneously.

[0069] Results: Figure 3 As shown in the figure, the conjugate pads treated with treatment solutions 1 and 2 are not conducive to the adsorption of r-SA by the NC membrane, while the conjugate pad corresponding to treatment solution 3 can make the test strip have an obvious C line. When the amount of BSA increases, the chromatographic resistance of the test strip corresponding to treatment solution 4 increases, and the sample solubility at the conjugate pad is extremely poor, which hinders the binding of the gold-labeled antibody and the antigen, and ultimately causes the C line and T line intensities to decrease at the same time. Finally, it was determined that the conjugate pad was pretreated with treatment solution 3, which had a relatively good effect.

[0070] Example 2 Determination of Cas12a test strip preparation conditions

[0071] 2.1 Optimization of marking conditions for C-line and T-line

[0072] In order to prepare the Cas12a test strip, this embodiment optimizes the streaking conditions of the C line and T line of the test strip. A colloidal gold spotting system was used at 1 μL / cm 2 The CN140 NC membrane was streaked at a streaking speed of 100 μg / mL. r-SA was used as the C line and goat anti-rabbit secondary antibody was used as the T line. At a secondary antibody concentration of 1 mg / mL, r-SA was streaked at concentrations of 0.5, 1, 1.5, 2, 2.5, and 3 mg / mL. At the same time, 2 mg / mL was selected as the streaking concentration of r-SA, and secondary antibodies with concentrations of 0.5, 1, and 2 mg / mL were used for streaking. After determining the concentration of the secondary antibody, 0.2%, 0.4%, 0.6%, 0.8%, and 1% BSA were used to block the T line to eliminate false positive signals.

[0073] Results: Figure 4As shown in A, with the increase of r-SA concentration, the C line band gradually becomes thinner and the T line band gradually becomes weaker. This is because in a short action time, when the r-SA concentration is low, r-SA cannot completely capture the ssDNA bound to the gold-labeled antibody, resulting in some antigens not being fixed by the C line and continuing to migrate to the T line, making the T line color; when the r-SA concentration increases, the gold-labeled complex is almost completely fixed at the C line, and the T line color becomes extremely weak. When r-SA far exceeds the number of antigens that match it, only a very small amount of r-SA is needed to completely capture the ssDNA, so the color development position appears at the bottom of the C line, and the band becomes narrower than that of low-concentration r-SA. When the r-SA concentration reaches 2 mg / mL, the T line almost no longer weakens. While ensuring sufficient r-SA, the economic benefits and color development effects are comprehensively considered, and 2 mg / mL of r-SA is finally determined as the line drawing condition for the C line.

[0074] When the amount of ssDNA is the same, T-NOS is targeted, and the C line position is streaked with the optimized r-SA concentration, and the streaking conditions of the secondary antibody are optimized under negative and positive conditions. The streaking conditions of the secondary antibody are related to the intensity of the final detection line (T line). Under ideal conditions, the T line does not appear in negative samples, and the T line of positive samples is clearly colored.

[0075] Results: Figure 4 As can be seen in B, when the concentration of the secondary antibody is 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, the test strips all show very weak T lines under negative conditions; when positive, a strong T line that can be clearly distinguished from the negative cannot be formed under the 0.5 mg / mL condition, and the T line intensity at 1 mg / mL is not significantly different from that at 2 mg / mL. Therefore, the secondary antibody streaking concentration was determined to be 1 mg / mL. To further optimize the T line color development effect, different proportions of BSA were added to the 1 mg / mL secondary antibody for nonspecific blocking. Figure 4 As can be seen from the C in the figure, the T line of the negative sample disappears when the secondary antibody solution contains 0.2% BSA. Figure 4 D in the figure shows that compared with the case without T-line BSA blocking, the weak band of the negative sample T-line disappeared under the condition of streaking with 0.2% BSA secondary antibody, and the T-line intensity of the positive sample decreased, but the color development of the positive and negative samples was clearly distinguished. Finally, 1 mg / mL of secondary antibody containing 0.2% BSA was used as the T-line streaking condition.

[0076] 2.2 Optimization of the dosage of gold-labeled FITC antibody

[0077] The amount of gold-labeled antibody used has a direct relationship with the color intensity of the test strip. This embodiment also optimizes the amount of gold-labeled FITC antibody used (μL). The prepared immunochromatographic test strip uses colloidal gold as the color developer.

[0078] Results: Figure 4 As shown in Figure E, too much gold-labeled antibody may cause mismatch between it and the amount of probe and r-SA, thus causing false positives, while too little gold-labeled antibody may cause insufficient color development of the test strip. After multiple tests, it was finally determined that 4 μL gold-labeled FITC antibody (containing 0.8 μg FITC antibody) was evenly distributed on the conjugate pad.

[0079] Example 3 Assembly of Cas12a test strips

[0080] Before assembly, the colloidal gold spotting system must be used to streak the secondary antibody and r-SA on the NC membrane (the secondary antibody streaking position is on the top), and the specific streaking conditions are determined according to the optimization test. Lay an appropriate amount of gold-labeled antibody on the binding pad. Assemble the streaked NC membrane, binding pad, sample pad, and absorption pad on the sticky bottom plate in sequence, with an overlap of 2mm between adjacent pads. Among them, the binding pad and sample pad are located at the lower end of the NC membrane in turn, and the absorption pad is located at the upper end of the NC membrane. After assembly, the composite plate is cut into 3mm wide test strips with a cutting machine and sealed and stored under dry conditions until use.

[0081] Example 4 Sample preparation, RPA amplification and Cas12a enzyme digestion experiment

[0082] The extraction of sample genomic DNA is carried out using a DNA-type ultra-fast nucleic acid releaser. The specific steps are: simply grind the seeds, take about 2 mg of seed powder and add it to 20 μL of releaser, let it stand for five minutes and then flick it to mix. The required DNA is present in the lysis supernatant. 100-200 ng of genomic DNA is used as a template for the amplification reaction.

[0083] The RPA amplification products corresponding to P-CaMV 35S and T-NOS involved in this study were cloned into the pUC57 vector to construct two standard reference plasmids for absolute sensitivity test. The concentration of the recombinant plasmid at A260 / 280 was determined by Nanodrop, and its molecular copy number was calculated. The quantitative plasmid was diluted in series to obtain 10 6 , 10 5 , 10 4 , 10 3 , 500, 250, 100, 50, 25, 10 and 1 copies / μL of plasmid samples.

[0084] Using sample genomic DNA as template, recombinase polymerase isothermal amplification (RPA) reaction was performed using RPA primers corresponding to P-CaMV 35S and T-NOS, respectively. The specific amplification system included: 29.5 μL Primer free rehydration buffer, 2.4 μL each of upstream and downstream primers (10 μM), 11.2 μL ddH2O, 2.5 μL MgOAC, and 2 μL DNA template. In order to verify the amplification reaction, all RPA products were electrophoresed using 4% agarose gel.

[0085] A 20μL Cas12a enzyme digestion system was constructed by optimizing the ssDNA probe, which contained 2μL 10×Cpf1Reaction Buffer, 1pmol Cpf1 (Cas12a), 10pmol crRNA, 1U RNase inhibitor, 1μL ssDNA probe (probe concentration and probe type determined according to the optimization results), 2μL amplification product, and DEPC water. The ssDNA probe suitable for the test strip was synthesized by Sangon and purified by HPLC. Its two ends were labeled with biotin and fluorescein isothiocyanate (FITC), corresponding to the r-SA on the NC membrane of the test strip and the gold-labeled FITC antibody on the binding pad, respectively. Different concentrations of 8nt-Biotin-ssDNA probe and 15nt-Biotin-ssDNA probe were used in the Cas12a enzyme digestion test, and the best probe and the best probe dosage were screened by the color development effect of the test strip. In addition, another YG-ssDNA was designed, with 6-carboxyfluorescein (6-FAM) and a black hole quencher (BHQ1) labeled at both ends. Biotin-ssDNA and YG-ssDNA were used for enzyme cleavage reactions in each enzyme cleavage experiment to verify the Cas12a trans-cleavage reaction.

[0086] Example 5 Optimization of the use conditions of Cas12a test strips

[0087] 5.1 Optimization of ssDNA probe dosage in Cas12a enzyme digestion system

[0088] First, the ssDNA probe in the Cas12a enzyme digestion system was optimized. Only when the amounts of the biotin and FITC dual-labeled ssDNA probes, the gold-labeled FITC antibody, and the r-SA match each other, the T line of the test strip will disappear under negative conditions, thereby avoiding false positives. The ssDNA was initially screened at final concentrations of 750nM, 500nM, and 250nM in the Cas12a enzyme digestion system. At the same time, the amount of 15nt ssDNA was further screened.

[0089] Results: Figure 5As shown in A, 15nt ssDNA is more suitable for test strip detection than 8nt ssDNA. Figure 5 As shown in B, the C line of the negative sample was the strongest and the T line was the weakest at 250 nM. Finally, 15 nt ssDNA was selected for the Cas12a enzyme digestion system, and its final concentration was determined to be 250 nM.

[0090] 5.2 Screening of chromatography buffer components

[0091] Secondly, when the test strip is analyzed, a chromatography buffer is required to provide chromatography power. Suitable chromatography buffer can provide favorable immunological reaction conditions, help separate active substances in the mixture, etc., thereby improving the sensitivity and specificity of the test strip analysis. In order to perform immunochromatographic tests, three kinds of chromatography buffers were prepared in this embodiment, and the applicable chromatography buffer was selected according to the actual color development effect of negative and positive samples.

[0092] Chromatography buffer 1: 10% PEG 6000, 0.1% BSA;

[0093] Chromatography buffer 2: 25 mM Tris, 150 mM NaCl, 0.05% Tween-20;

[0094] Chromatography buffer 3: 25 mM Tris, 150 mM NaCl, 0.05% Tween-20, 5% PEG 6000, 0.1% BSA.

[0095] Results: Figure 5 As shown in C, among the three chromatography buffers used in this embodiment, the C line intensity under the action of chromatography buffer 2 is weaker than that of chromatography buffers 1 and 3, and false positives occur in negative samples, which is not suitable for this study; the use effects of chromatography buffers 1 and 3 are both good, but the color intensity of C line and T line band under the action of chromatography buffer 3 is better, so chromatography buffer 3 is determined as the final chromatography buffer.

[0096] Example 6 Molecular sensitivity and stability test of Cas12a test strip

[0097] 6.1 Sensitivity test

[0098] Take 10 6 , 10 5 , 10 4 , 10 3The molecular sensitivity test was carried out with the standard plasmid at 50 ng / μL, 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, 0.001 ng / μL, 0.0001 ng / μL, 0.00001 ng / μL, 0.000001 ng / μL, and 0.000001 ng / μL as the detection object.

[0099] Results: In the molecular sensitivity test, for T-NOS screening elements, such as Figure 6 As shown in A1 in the figure, the T line intensity of the test strip corresponding to 1 copy of the plasmid is clearly different from that of the negative, and the C line intensity is significantly stronger than that of the negative, indicating that the prepared test strip can be as sensitive to T-NOS as low as 1 copy. Figure 6 As shown in A2 in the figure, the T line intensity of the test strip corresponding to 10 copies of the plasmid can be distinguished from the negative, while the T line of 1 copy of the plasmid is no different from the negative, and the detection limit is 10 copies. When the same template is used, Figure 6 As shown in A3 in FIG, only bands corresponding to 50 and 100 copies of plasmids can be observed for T-NOS and P-CaMV 35S, respectively, in RPA agarose gel electrophoresis, and the detection sensitivity is much lower than the method constructed in this study.

[0100] 6.2 Stability test

[0101] Multiple repeated tests were performed at the detection limit concentration determined by the absolute sensitivity test to explore the detection stability of the test strip.

[0102] Results: During the genomic DNA detection of MON88017 transgenic standard, Figure 6 As shown in B1 in Figure 1, the constructed Cas12a test strip method can achieve 10 -4 ng / μL or above genomic DNA detection, such as Figure 6 As shown in B2 in Figure 1, the detection of 0.01 ng / μL genomic DNA can be achieved for the P-CaMV 35S screening element. Figure 7 The weak electrophoresis bands shown in B make the test strip results clearer and easier to distinguish.

[0103] For the two screening elements, T-NOS and P-CaMV 35S, multiple repeated tests were performed at the detection limit determined by the molecular sensitivity test. The color development results of the test strips all indicated positive, and the response signal was stable. The results are as follows Figure 6 As shown in C.

[0104] Example 7 Specificity test of Cas12a test strip

[0105] For the two detection targets, P-CaMV 35S and T-NOS, the specificity of this research method was verified using 16 transgenic standards with different contents and transgenic components available in the laboratory.

[0106] Results: Figure 8 The detection results of A2 targeting T-NOS showed that the test strips of 11 transgenic samples containing T-NOS, including RRS, Bt11, KF6, KMD1, MON531, MON863, NK603, RF1, MS1, MIR604, and MON88017, all had very weak C lines and obvious T lines, indicating positive; Bt176, MON88913, MON89788, MON87701, and H7-1 did not contain T-NOS, and their corresponding test strips all had only obvious C lines, indicating negative. In the test results of P-CaMV 35S, the test strips of RRS, Bt11, KF6, KMD1, MON531, MON863, Bt176, NK603, MON88913 and MON88017 containing P-CaMV35S had obvious T lines, indicating positive; while RF1, MS1, MIR604, Sugar beet H7-1, MON89788 and MON87701 without P-CaMV 35S had no obvious T lines, indicating negative, such as Figure 8 The quantitative results of the color bands showed that the detection bands of positive and negative samples were significantly different, as shown in Figure 8 The results of T-NOS and P-CaMV 35S test strips are shown in A3 and B3. Figure 8 A1 and Figure 8 The agarose gel electrophoresis results shown in B1 are consistent with those in the current standards, indicating that the Cas12a test strip detection method proposed in the present invention has good specificity.

[0107] Example 8 Application of Cas12a test strips in actual samples

[0108] In order to verify the applicability and accuracy of the Cas12a test strips of the present invention in actual samples, this embodiment randomly selected 3 kinds of corn, 3 kinds of soybeans and 2 kinds of cotton seeds on the market and used this research method for detection. The test used a transgenic positive product with known information as a positive control, and set a blank control. The experimental results were compared with the agarose gel electrophoresis results of the genomic DNA amplification product.

[0109] According to the described detection process, 8 kinds of plant seeds randomly purchased from the market were tested using Cas12a special test strips. The genetically modified soybean standard RRS was used as a positive control, and a blank control without template was set up.

[0110] Results: The final test strip results for T-NOS and P-CaMV 35S screening elements are shown in Fig. 9 C and D. All samples were negative. Fig. 9 Figures E and F show that the quantitative results of the positive standard test strip T line are significantly different from those of the negative sample (p<0.0001). The test strip results are consistent with the agarose gel electrophoresis results of the RPA product (e.g. Fig. 9 The results of this study show that the method is practical and accurate.

[0111] Example 9 Performance comparison of different genetically modified crop detection methods

[0112] In order to fully evaluate the Cas12a test strip of the present invention, other related rapid detection methods of genetically modified organisms are summarized in Table 3 below. By comparison, the present invention is superior to most nucleic acid / protein-based detection methods in terms of detection sensitivity, and has the same detection sensitivity as the reported Cas12a-based fluorescence detection method (RPA-Cas12a-FS). At the same time, compared with most nucleic acid detection methods, the Cas12a test strip method has shorter detection time and better portability. In the portable test strip method, the proposed strategy has a sensitivity of (10 copies / μL or 29.1×10 -6 ng / mL), which is several orders of magnitude higher than the analytical capability of the protein test strip method, one order of magnitude higher than the sensitivity of the RPA test strip method (100 copies), and has better detection effects.

[0113] The biotin capture agent in the present invention selects streptavidin with better affinity than biotin antibodies, which helps to improve the stability of the test strip and other detection performance and greatly reduces the production cost. The detection sensitivity of the test strip prepared according to the present invention is 1-10 copies / μL, which is comparable to the detection level of commercial test strips (170 copies / μL; 9 copies / μL).

[0114] Table 3 Performance comparison of different GM crop detection methods

[0115]

[0116] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for detecting genetically modified crops, characterized in that: The following steps are involved: 1) Extracting genomic DNA from the sample to be tested; 2) RPA amplification: using the genomic DNA as a template, P-CaMV 35S and T-NOS as targets, and using RPA primers corresponding to P-CaMV 35S and T-NOS, respectively, to perform RPA amplification reaction; the RPA primers corresponding to the targets P-CaMV 35S and T-NOS are: Primer pair for detection of P-CaMV 35S gene: RPA-35S-F: 5'-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3', RPA-35S-R: 5'-CCCTTACGTCAGTGGAGATATCACATCAATCC-3'; Primer pairs for detecting T-NOS gene: RPA-NOS-F: 5'-TGAATCCTGTTGCCGGTCTTGCGATGATTATC-3', RPA-NOS-R: 5'-TCTATCGCGTATTAAATGTATAATTGCGGGAC-3'; 3) Cas12a digestion reaction: The RPA amplification product obtained in step 2) was digested with Cas12a at 37°C for 15 min; 4) Immunochromatographic test strip detection: The Cas12a cleavage product obtained in step 3) is added dropwise to the sample pad of the prepared immunochromatographic test strip, one end of the sample pad is immersed in the chromatography buffer, and it is vertically stood for 3 to 7 minutes. After the buffer reaches the absorbent paper, the immunochromatographic test strip is taken out, and the color development of the C line and the T line is observed; When the immunochromatographic test strip simultaneously displays a clear detection line T and a quality control line C, it indicates that the target P-CaMV 35S and / or T-NOS exist; when the immunochromatographic test strip only displays a clear quality control line C, it indicates that neither the target P-CaMV 35S nor T-NOS exists; when neither the detection line T nor the quality control line C on the immunochromatographic test strip appears, it indicates that the test has failed or the test strip is invalid.

2. The detection method according to claim 1, characterized in that: In step 2), the amplification system of the RPA amplification reaction includes: 29.5 μL Primer free rehydration buffer, 2.4 μL of each of the upstream and downstream primers at 10 μM, 11.2 μL ddH2O, 2.5 μL MgOAC, and 2 μL DNA template.

3. The detection method according to claim 1, characterized in that: In step 3), 15nt-Biotin-ssDNA probe is used in the Cas12a enzyme digestion reaction system, and its final concentration is 250nM.

4. The detection method according to claim 1, characterized in that: In step 4), the chromatography buffer used is: 25 mM Tris, 150 mM NaCl, 0.05% Tween-20, 5% PEG 6000, 0.1% BSA.

5. The detection method according to claim 1, characterized in that: The preparation method of Cas12a immunochromatographic test strip comprises the following steps: S1: Pretreatment of sample pad and conjugate pad: soak the sample pad and conjugate pad with different treatment solutions and then dry them for use. The sample pad treatment solution is: 2% glucose, 1% Triton X-100, 1% BSA, 50mM boric acid, pH 8.0; the conjugate pad treatment solution is: 1% sucrose, 3% BSA, 0.5% Tween-20, and the solvent is 0.01M Tris-base solution, pH 8.0; S2: streaking of the test line T and the quality control line C: streaking of the test line T and the quality control line C on the chromatographic membrane using a colloidal gold spotting system. The streaking condition for the test line T is 1 mg / mL goat anti-rabbit secondary antibody containing 0.2% BSA, and the streaking condition for the quality control line C is 2 mg / mL r-SA; S3: Assembling: Assemble the scribed chromatographic membrane, conjugate pad, sample pad and absorbent pad on the adhesive base plate in sequence, with the adjacent two pads partially overlapping, wherein the sample pad, conjugate pad, chromatographic membrane and absorbent pad are arranged in sequence along the chromatographic direction, with the quality control line C close to the conjugate pad and the detection line T close to the absorbent pad; S4: Cutting: Cut the assembled composite plate into immunochromatographic test strips of a specified width, and seal and store them in a dry condition for later use.

6. The detection method according to claim 5, characterized in that: CN140 was selected as the chromatography membrane.

7. The detection method according to claim 5, characterized in that: The method further includes applying 4 μL of colloidal gold-labeled FITC antibody, containing 0.8 μg of FITC antibody, to the conjugate pad after pre-treatment with the treatment solution.

8. The detection method according to claim 5, characterized in that: In step S4, the composite plate is cut into 3 mm wide immunochromatographic test strips.

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