CN116536392A - A method for identification of transgenic crops with single base specificity - Google Patents

Abstract

The invention relates to the field of molecular biology detection, and provides a transgenic crop identification method with single base specificity. The method comprises the following steps: taking target DNA as a template, and reacting by using a PCR primer group and an RPA primer group with biotin marks to obtain PCR or RPA amplification products; the PCR primer group comprises a sequence shown in SEQ ID NO:1 and a first forward primer with a sequence shown as SEQ ID NO:2, a first reverse primer; the RPA primer group comprises a first RPA primer group, a second RPA primer group and a third RPA primer group, and forward primer sequences of the three RPA primer groups are respectively shown in SEQ ID NO: 3. SEQ ID NO:5 and SEQ ID NO:6, the sequence of the second reverse primer is shown as SEQ ID NO:4 is shown in the figure; and adding the PCR or RPA amplification product into a detection system with a single base specific probe for detection, and obtaining an identification result. The invention has single base specificity, can realize the identification of transgenic crops, and has the advantages of high accuracy, good sensitivity, high detection speed, simple and convenient operation, low cost and the like.

Description

A method for identifying genetically modified crops with single base specificity

Technical Field

The invention relates to the field of molecular biological detection, and in particular to a method for identifying genetically modified crops with single-base specificity.

Background Art

In fields such as novel coronavirus, genotyping, identification of food pathogens and genetically modified crops, molecular diagnosis is usually involved, and molecular diagnosis often involves nucleic acid testing. The specificity, sensitivity and economy of nucleic acid testing directly affect the accuracy and practicality of molecular diagnostic results. Nucleic acid testing relies on nucleic acid amplification and detection technology.

In the prior art, gel electrophoresis has been widely used for qualitative analysis of amplified products, but this detection method is very cumbersome and time-consuming. Although methods such as fluorescent dyes and pyrophosphate colorimetric assays can achieve simplified detection of amplified products, these methods cannot distinguish between target gene amplification products and non-specific amplification products, resulting in unreliable detection results. In addition, conventional PCR (polymerase chain reaction)-ELISA (enzyme-linked immunosorbent assay) usually involves expensive fluorescent labeling, complex sample denaturation and lengthy hybridization processes, which increases the complexity of the operation, and ELISA analysis is also affected by primer dimers, resulting in false positive results.

It can be seen that the existing nucleic acid detection methods still have the problems of long detection time, high detection cost, and inability to distinguish between target gene amplification products and non-specific amplification products, resulting in poor reliability of detection results and inability to identify genetically modified crops.

Summary of the invention

In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for identifying genetically modified crops with single base specificity, which has the advantages of high accuracy, good sensitivity, fast detection speed, simple operation and low detection cost, and can well distinguish between target gene amplification products and non-specific amplification products, and can specifically identify sequences with single base differences to achieve the identification of genetically modified crops.

In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

The present invention provides a method for identifying transgenic crops with single base specificity, comprising the following steps:

Extracting target DNA from the sample to be tested;

Using the target DNA as a template, a PCR reaction is performed using a biotin-labeled PCR primer set to obtain a PCR amplification product; or an RPA reaction is performed using a biotin-labeled RPA primer set to obtain an RPA amplification product; wherein the PCR primer set includes a first forward primer having a sequence as shown in SEQ ID NO: 1 and a first reverse primer having a sequence as shown in SEQ ID NO: 2; the RPA primer set includes a first RPA primer set, a second RPA primer set and a third RPA primer set; the first RPA primer set includes a second forward primer having a sequence as shown in SEQ ID NO: 3 and a second reverse primer having a sequence as shown in SEQ ID NO: 4; the second RPA primer set includes a third forward primer having a sequence as shown in SEQ ID NO: 5 and a second reverse primer having a sequence as shown in SEQ ID NO: 4; the third RPA primer set includes a fourth forward primer having a sequence as shown in SEQ ID NO: 6 and a second reverse primer having a sequence as shown in SEQ ID NO: 4;

The PCR amplification product or the RPA amplification product is added to a detection system containing a single base specific probe for detection to obtain an identification result.

The single-base specific genetically modified crop identification method provided by the embodiment of the present invention can complete the entire process from sample preparation to result collection within 1.5 hours, greatly shortening the detection time and having a fast detection speed. In addition, the entire detection process does not require the use of expensive reagents and well-trained technicians, greatly reducing the detection cost. At the same time, the detection method has good sensitivity and specificity, can well distinguish target gene amplification products from non-specific amplification products, and can specifically identify sequences with single-base differences to achieve the identification of genetically modified crops, providing a low-cost and reliable detection approach for on-site molecular diagnosis with limited resources.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a schematic diagram of the principle of a method for identifying transgenic crops with single base specificity provided in an embodiment of the present invention;

FIG2 is an agarose gel electrophoresis result of each group of PCR amplification products in Example 1 of the present invention;

FIG3 is a result of a screening test of dCas9 reaction components in the detection system in Example 1 of the present invention;

FIG4 is a result showing the effect of the amount of dCas9 added on the signal-to-noise ratio of the detection system in Example 2 of the present invention;

FIG5 is a result showing the effect of the amount of sgRNA added on the signal-to-noise ratio of the detection system in Example 2 of the present invention;

FIG6 is a result showing the effect of the coating amount of the dCas9 antibody on the signal-to-noise ratio of the detection system in Example 3 of the present invention;

7 is a result showing the effect of the concentration of SA-HRP enzyme on the signal-to-noise ratio of the detection system in Example 4 of the present invention;

FIG8 is a graph showing the effect of the incubation time of the ternary complex and the dCas9 antibody coated therewith on the signal-to-noise ratio of the detection system in Example 5 of the present invention;

FIG9 is a graph showing the absorbance detection results of PCR amplification products of different crops in Example 6 of the present invention;

FIG10 is the agarose gel electrophoresis result of PCR amplification products of different crops in Example 6 of the present invention;

FIG11 is a test result of the repeatability of the identification method of the present invention at different template concentrations in Example 6 of the present invention;

FIG12 is a stability test result of the identification method of the present invention at different template concentrations in Example 6 of the present invention;

FIG13 is a diagram showing the mismatch sequences of target sequences near each group of PAM sites in Example 7 of the present invention;

FIG14 is a graph showing the absorbance detection results of PCR amplification products of each group of mismatched sequences in Example 7 of the present invention;

15 is the absorbance detection results of PCR amplification products of transgenic rice genomic DNA standard substances with different template concentrations in Example 8 of the present invention;

16 is a standard curve of the logarithm of the absorbance value-CaMV35S promoter concentration in Example 8 of the present invention;

17 is the test result of identifying samples with different transgenic rice contents using real-time fluorescence quantitative PCR method in Example 9 of the present invention;

FIG18 is a test result of samples with different transgenic rice contents identified by the identification method provided by the present invention in Example 9 of the present invention;

FIG19 is sequence information of three pairs of RPA primer sets provided in Example 10 of the present invention;

FIG20 is the agarose gel electrophoresis result of RPA amplification products using different RPA primer sets in Example 10 of the present invention;

FIG. 21 is the detection result of identifying different crops using the identification method of the present invention in Example 10 of the present invention.

DETAILED DESCRIPTION

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but the embodiments of the present invention are not limited thereto.

Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.

The PCR primer set and RPA primer set used in the examples of the present invention were synthesized by Shanghai Bioengineering Co., Ltd.

The experimental reagents used in the embodiments of the present invention, unless otherwise specified, are all conventional biochemical reagents and can be obtained from commercial channels; the amounts of the experimental reagents, unless otherwise specified, are the amounts of reagents used in conventional experimental operations; the experimental methods, unless otherwise specified, are all conventional methods.

The reagents and their sources involved in the following embodiments are as follows: dCas9 (SNAP-tag) nuclease, NEBuffer, and Anti-SNAP-tag antibody used as dCas9 antibody were purchased from NEB Co., Ltd. (USA); ultrapure water (DEPC water) treated with diethyl pyrocarbonate and sterilized at high temperature and high pressure was purchased from biosharp Co., Ltd. (China); PCR-related reagents, TaKaRa taq hotstart enzyme was purchased from Bio-Riyi Biotechnology Co., Ltd. (China); RPA-related reagents, DNA isothermal rapid amplification kit (RPA kit, catalog number: WLB8201KIT), were purchased from Weifang Anpu Future Biotechnology Co., Ltd. (China); Tween-20 was purchased from neoFroxx Co., Ltd. (Germany); bovine serum albumin (BSA), streptavidin horseradish peroxidase (SA-HRP), and tris hydroxymethylaminomethane (Tris base) were purchased from Sigma-Aldrich Trading Co., Ltd. (USA); TMB colorimetric reagents including A solution and B solution were purchased from MacLean Biochemical Technology Co., Ltd. (China); disodium hydrogen phosphate ( _{Na2HPO4 4} ), sodium dihydrogen phosphate (NaH ₂ PO ₄ ), and sulfuric acid (H ₂ SO4) were purchased from Sinopharm Chemicals Co., Ltd. (China); polystyrene 96-well microplates were purchased from Corning Incorporated (USA). All aqueous solutions were prepared with ultrapure water purified by the Millipore-XQ system.

As mentioned above, existing gel electrophoresis, fluorescent dyes and pyrophosphate colorimetric assay methods or conventional PCR-ELISA methods are easily affected by non-specific amplified fragments and have the disadvantage of insufficient specificity in practical applications. Although probe-based fluorescence detection methods that use TaqMan fluorescent probes and molecular beacon probes to specifically identify non-primer sequences in amplified products can significantly improve analytical specificity, such methods require a high-standard operating environment, rely on large and expensive instruments and skilled operators, are not suitable for grassroots testing, and cannot efficiently distinguish single-base differences in specific sequences.

SNP is a nucleic acid sequence polymorphism caused by the change of a single nucleotide base. SNP (single nucleotide polymorphism) is widely present in the biological world and has a wide range of uses. In the prior art, direct sequencing methods represented by Sanger sequencing and pyrophosphate sequencing are the most accurate methods for detecting SNPs. The sequencing results can directly reflect the specific information of SNPs. However, these methods are costly and require special equipment, are time-consuming, and have complex data analysis. In addition, these methods are suitable for whole-genome SNP scanning and can be used as a SNP site identification study for samples with unknown sequences, but are not suitable for routine large-scale screening and identification of known SNP sites. Classical detection methods based on gel electrophoresis analysis are also widely used in SNP detection, such as mismatch cleavage method, single-stranded conformation polymorphism (SSCP), enzyme-amplified polymorphic sequence (CAPS), allele-specific PCR (AS-PCR), etc. Although this type of technology does not require high equipment and has low investment costs, it has a slow detection speed and is difficult to achieve high throughput and simplified analysis. Fluorescence PCR-based technologies, such as real-time fluorescence PCR, digital PCR, and high-resolution melting curve (HRM), can be applied to SNP site detection. They can simplify operations, increase analysis speed, and are suitable for high-throughput analysis, but their sensitivity to single-base mutations is poor.

With the continuous development of biotechnology, some high-throughput and highly automated detection methods, such as DNA chips, denaturing high-performance liquid chromatography (DHPLC), and mass spectrometry detection technology, have been successfully applied to the detection of SNPs. However, these PCR and instrumental analysis technologies have high instrument and reagent costs, require specific instruments, and have high requirements for equipment hardware. Therefore, it is urgent to develop a highly specific, simple and rapid SNP detection technology to meet the needs of future commercial product biosafety supervision.

The embodiment of the present invention provides a method for identifying genetically modified crops with single base specificity, comprising the following steps:

S1. Extract the target DNA of the sample to be tested.

S2. Using the target DNA as a template, a PCR reaction is performed using a biotin-labeled PCR primer set to obtain a PCR amplification product; or a RPA reaction is performed using a biotin-labeled RPA primer set to obtain an RPA amplification product; wherein the PCR primer set includes a first forward primer having a sequence as shown in SEQ ID NO: 1 and a first reverse primer having a sequence as shown in SEQ ID NO: 2; the RPA primer set includes a first RPA primer set, a second RPA primer set and a third RPA primer set; the first RPA primer set includes a second forward primer having a sequence as shown in SEQ ID NO: 3 and a second reverse primer having a sequence as shown in SEQ ID NO: 4; the second RPA primer set includes a third forward primer having a sequence as shown in SEQ ID NO: 5 and a second reverse primer having a sequence as shown in SEQ ID NO: 4; the third RPA primer set includes a fourth forward primer having a sequence as shown in SEQ ID NO: 6 and a second reverse primer having a sequence as shown in SEQ ID NO: 4.

S3. Add the PCR amplification product or the RPA amplification product to a detection system containing a single base-specific probe for detection to obtain an identification result.

In some embodiments, the samples to be tested in the above step S1 can be some genetically modified crops (such as genetically modified corn, genetically modified soybeans, genetically modified rice, etc.), or some non-genetically modified crops (such as non-genetically modified corn, non-genetically modified soybeans, non-genetically modified rice, etc.).

The cauliflower mosaic virus 35S (CaMV35S) promoter is widely used in transgenic organisms, such as transgenic corn, transgenic soybean, transgenic rice, etc. Based on this, the present invention selects the CaMV35S promoter sequence as the target DNA for on-site screening and identification of transgenic crops.

In some embodiments, in the above step S2, since the CaMV35S promoter sequences in different transgenic crops are not exactly the same, the inventors of the present invention compared the CaMV35S promoter sequences of multiple transformants and, after multiple analyses, finally selected the following PCR primer set as the amplification primer for the CaMV35S promoter of multiple transgenic crops. The first forward primer and/or the first reverse primer in the PCR primer set carries a biotin label. The PCR primer set includes a first forward primer with a sequence as shown in SEQ ID NO: 1 and a first reverse primer with a sequence as shown in SEQ ID NO: 2. The sequence information of the first forward primer and the first reverse primer is as follows (from the 5' end to the 3' end):

Sequence information of the first forward primer: p35s-F:ATTGATGTGATATCTCCACTGACGT.

Sequence information of the first reverse primer: p35s-R:CCTCTCCAAATGAAATGAACTTCCT.

Moreover, a biotin-labeled RPA primer set was designed based on the CaMV35S promoter sequence. Specifically, the RPA primer set includes a first RPA primer set, a second RPA primer set, and a third RPA primer set, wherein the forward primer and/or reverse primer in the first RPA primer set, the second RPA primer set, and the third RPA primer set carries a biotin label. The first RPA primer set includes a second forward primer with a sequence as shown in SEQ ID NO: 3 and a second reverse primer with a sequence as shown in SEQ ID NO: 4; the second RPA primer set includes a third forward primer with a sequence as shown in SEQ ID NO: 5 and a second reverse primer with a sequence as shown in SEQ ID NO: 4; the third RPA primer set includes a fourth forward primer with a sequence as shown in SEQ ID NO: 6 and a second reverse primer with a sequence as shown in SEQ ID NO: 4. The sequence information is as follows (from 5' to 3'):

The sequence information of the second forward primer is as follows:

RPA-F1:GTCTTCAAAGCAAGTGGATTGATGTGATAT.

The sequence information of the third forward primer is as follows:

RPA-F2: CAAGTGGATTGATGTGATATCTCCACTGACGT.

The sequence information of the fourth forward primer is as follows:

RPA-F3: GATTGATGTGATATCTCCACTGACGTAAG.

The sequence information of the second reverse primer is as follows:

RPA-R: CTCTCCAAATGAAATGAACTTCCTTAT.

The primer combinations and amplified fragment sizes are shown in Table 1 below.

Table 1 Primer combinations and amplified fragment lengths of CaMV35S promoter

serial number Primer combinations Fragment length 1 (First RPA Primer Set) RPA-F1/RPA-R 101 2 (Second RPA Primer Set) RPA-F2/RPA-R 117 3 (Third RPA Primer Set) RPA-F3/RPA-R 106

In one embodiment, assuming that the sample to be tested is transgenic rice, the specific operation process of using the target DNA as a template and a biotin-labeled PCR primer set to perform a PCR reaction to obtain a PCR amplification product is as follows: using the rice genomic DNA (i.e., target DNA) containing the CaMV35S promoter sequence as a template, PCR amplification is performed to obtain a PCR amplification product. The PCR reaction system is: 2 μL 10×PCR buffer, 2 μL 10mM dNTPs, 1 μL target DNA, 0.5 μL 10 μM first forward primer, 0.5 μL 10 μM first reverse primer. The reaction procedure is: 98°C pre-denaturation for 60s; 98°C denaturation for 10s; 58°C annealing extension for 30s; 72°C for 1min, 35 cycles, and 72°C extension for 7min.

In another embodiment, assuming that the sample to be tested is transgenic rice, the specific operation process of performing RPA reaction using the target DNA as a template and using a biotin-labeled RPA primer set to obtain an RPA amplification product is as follows:

According to the instructions of the RPA kit, the total RPA reaction system is 50 μL: 29.4 μL Abuffer is added to each dry powder reaction tube; 2 μL second forward primer, 2 μL second reverse primer (or, 2 μL third forward primer and 2 μL third reverse primer, or, 2 μL fourth forward primer and 2 μL fourth reverse primer); 13.1 μL ddH ₂ O and 1 μL plant genomic DNA (i.e., target DNA) template, flick to fully dissolve the powder; finally, add 2.5 μL B Buffer and mix thoroughly to trigger the reaction, and then incubate at 37°C for 15 minutes. After the reaction is completed, add 50 μL purification buffer, mix well, centrifuge at 12000 rpm for 5 minutes, and take the supernatant to obtain the RPA amplification product.

In some embodiments, in the above step S3, the single-base specific probe is a complex of dCas9 protein and specific sgRNA; the sequence of the specific sgRNA is shown in SEQ ID NO: 7.

The specific sgRNA used in the embodiment of the present invention is composed of a direct repeat sequence forming a stem-loop structure and a spacer sequence complementary to the target sequence (target sequence). Exemplarily, online sgRNA design software (such as CHOPCHOP) can be used to design sgRNA. Specifically, the CaMV35S promoter sequence can be used as the target sequence, and the target gene sequence (i.e., the target sequence) can be input. All sequences containing PAM (protospacer adjacent motif) sites can be found in the target gene sequence, and then the sites between the upstream and downstream primers of PCR and RPA are selected according to the experiment, and the sgRNA with good specificity is selected to obtain the specific sgRNA. Among them, the sequence information of the specific sgRNA is as follows (from 5' end to 3' end):

agggucuugcgaaggauaguGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUA AGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUU UU.

Among them, the lowercase sequence in the sequence information of the above-mentioned specific sgRNA is a spacer sequence complementary to the target sequence (target sequence), and the uppercase sequence is a direct repeat sequence.

It should be noted that the "t" in the sequence information of the sgRNA in the sequence table represents uracil in the RNA sequence and thymine in the DNA sequence.

The above-mentioned specific sgRNA was synthesized by Beijing Qingke Biotechnology Co., Ltd.

The upstream primers are different at both ends of the DNA molecule because the nucleotide molecule is not symmetrical. There is a phosphate at the 5' position and -OH at the 3' position, that is, a phosphate end and a hydroxyl end. DNA replication always occurs from the 5' end to the 3' end because DNA polymerase can only add nucleotides to the 3' end. So whoever replicates first is the upstream, and the one after that is the downstream. Upstream and downstream are relative concepts, which refer to a certain sequence/base relative to another sequence/base.

In some embodiments, the above step S3 may specifically include the following steps:

The dCas9 protein and the specific sgRNA are pre-incubated to form a single-base specific probe; the PCR amplification product or the RPA amplification product is added to a detection system containing the single-base specific probe, so that the PCR amplification product or the RPA amplification product is incubated with the single-base specific probe to form a ternary complex; the ternary complex is added to a microplate coated with a dCas9 antibody, incubated to complete the immune reaction, and then streptavidin horseradish peroxidase is added for incubation, and then a color developing reagent is added to perform absorbance detection to obtain an identification result.

In conjunction with FIG1 , the principle of the method for identifying genetically modified crops with single base specificity provided in an embodiment of the present invention is as follows:

First, the target DNA of the sample to be tested is extracted; then, the target DNA is amplified using a biotin-labeled PCR primer set or a biotin-labeled RPA primer set to obtain a PCR amplification product or an RPA amplification product; then, the PCR amplification product or the RPA amplification product is accurately recognized and combined with a single-base specific probe (dCas9/sgRNA complex) by dCas9 in a detection system in which a single-base specific probe (dCas9/sgRNA complex) is present, forming a dCas9/sgRNA/DNA-Bio ternary complex; thereafter, the dCas9/sgRNA/DNA-Bio ternary complex can be captured by a microplate coated with a dCas9 antibody and combined with a SA-HRP probe through a biotin-streptavidin interaction; finally, colorimetric detection (absorbance detection) can be achieved in the presence of a TMB substrate to determine whether the sample to be tested is a genetically modified crop.

The identification method proposed in the embodiment of the present invention can identify the presence of the target sequence by naked eyes or by simply measuring absorbance. It is simple to operate, highly practical, and has low detection cost. It can be applied to on-site genetically modified crop screening and detection with limited resources.

In an embodiment of the present invention, the amount of dCas9 protein and the specific sgRNA will directly affect the formation of the dCas9/sgRNA complex, thereby indirectly affecting the sensitivity of the detection. When the molar ratio of the dCas9 protein to the specific sgRNA is 1:0.6-10, the sensitivity of the detection system is good. Preferably, when the molar ratio of the dCas9 protein to the specific sgRNA is 1.2:1.05-1.35, the sensitivity of the detection system is good; more preferably, when the molar ratio of the dCas9 protein to the specific sgRNA is 1:1, the sensitivity of the detection system is optimal.

In an embodiment of the present invention, since the dCas9/sgRNA/DNA-Bio ternary complex is fixed on the microplate by the immune binding of the dCas9 protein and the dCas9 antibody, the amount of dCas9 antibody coated on the microplate will directly affect the amount of the captured dCas9/sgRNA/DNA-Bio ternary complex, thereby affecting the sensitivity of the detection. When the coating concentration of the dCas9 antibody is 0.75μg/mL to 2μg/mL, the sensitivity of the detection system is better. When the coating concentration of the dCas9 antibody is 1μg/mL, the sensitivity of the detection system is optimal.

SA-HRP binds to biotin on the dCas9/sgRNA/DNA-Bio ternary complex and catalyzes TMB color development. If too little SA-HRP is added to the reaction, the reaction is insufficient, and too much SA-HRP may cause nonspecific adsorption, resulting in weak color development of the blank control. When the concentration of the streptavidin horseradish peroxidase is 0.5-7.5 μg/mL, the signal-to-noise ratio is large; when the concentration of the streptavidin horseradish peroxidase is 5 μg/mL, the signal-to-noise ratio is the largest.

In the step, the ternary complex is added to a microplate coated with a dCas9 antibody and incubated to complete the immune reaction. The incubation time is another important factor affecting the nonspecific adsorption of the immunoassay. When the incubation time is 10 to 50 minutes, the signal-to-noise ratio is large; when the incubation time is 30 minutes, the signal-to-noise ratio is the largest.

In an exemplary embodiment, 4 μL of 0.3 μM dCas9 protein, 4 μL of 0.3 μM specific sgRNA, 10 μL of 10×NEBuffer, and 69.5 μL of DEPC water can be mixed and incubated at 25°C for 10 min to form a dCas9/sgRNA complex, i.e., a single base-specific probe. Next, the dCas9/sgRNA complex obtained above is mixed with 2.5 μL of biotin-labeled PCR amplification product or RPA amplification product and incubated at 37°C for 15 min to form a dCas9/sgRNA/DNA-Bio ternary complex. Afterwards, 100 μL of dCas9 antibody dissolved in 0.1M Tris-HCl buffer at a concentration of 1 μg/mL is added as a coating solution to a high-affinity polystyrene 96-well microplate and coated overnight at 4°C. After that, pour off the unbound coating solution in each well and wash three times with 300 μL of washing buffer (phosphate buffer containing 0.10M of 0.050% Tween 20 and pH 7.4). Then add 150 μL of blocking buffer (phosphate buffer containing 1.0% BSA, 0.050% Tween-20, pH 7.4) to each well and block for 30 minutes at 37°C. After washing three times with washing buffer, add 90 μL of dCas9/sgRNA/DNA-Bio ternary complex solution to each well and incubate at 37°C for 30 minutes to complete the immune reaction. Wash three times with washing buffer. Add 100 μL of 5 μg/mL SA-HRP enzyme (streptavidin horseradish peroxidase) to the microplate and incubate at 37°C for 20 minutes. Wash three times with washing buffer. After washing, TMB colorimetric detection was performed. 50 μL of TMB solution A and 50 μL of TMB solution B were added to the immune complex after the above reaction and incubated in the dark for 7 minutes. Finally, 50 μL of 2.0M H ₂ SO ₄ was added to terminate the reaction. The results can be visually detected by the naked eye and quantified by measuring the absorbance value at 450 nm. If the nucleic acid to be tested is positive, the color of the solution is yellow, and the sample to be tested can be determined to be a genetically modified crop; if the nucleic acid to be tested is negative, the color of the solution does not change, and the sample to be tested can be determined to be a non-genetically modified crop.

The embodiment of the present invention uses a combination of clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 and TMB colorimetric method to perform nucleic acid detection, which is simple to operate, has good sensitivity and specificity, can accurately distinguish target gene amplification products from non-specific amplification products, and effectively avoid the interference of non-specific amplification products, thereby achieving specific identification of genetically modified crops, and can specifically identify sequences with single base differences.

The present invention has been tested for many times, and some test results are now cited as references to further describe the invention in detail, and the following is a detailed description in conjunction with specific embodiments.

Example 1 Specific detection of genetically modified crops

1) Agarose gel electrophoresis detection

Experimental groups: ① Using transgenic rice (KMD) genomic DNA as target DNA, the target DNA was amplified using a biotin-labeled PCR primer set to obtain a PCR amplification product. ② Using non-transgenic rice genomic DNA as target DNA, the target DNA was amplified using a biotin-labeled PCR primer set to obtain a PCR amplification product. ③ Blank control group (ddH ₂ O).

The PCR amplification products prepared in the above test groups ①②③ were respectively detected by agarose gel electrophoresis. The test results are shown in Figure 2. As shown in Figure 2, the target band can be observed in the test group ① (as shown in "1" in Figure 2), and the target band is not observed in the test group ② (as shown in "2" in Figure 2) and the blank control group ③ (as shown in "3" in Figure 2). M in Figure 2 represents DL2000.

2) Screening test of dCas9 reaction components in the detection system

By setting eight test groups a to h containing different reaction components, a dCas9/DNA-Bio complex for ELISA detection was prepared. Among them, the reaction components of test group a include: dCas9, Target sgRNA (specific sgRNA provided by the present invention), Target DNA (PCR amplification product with biotin labeling). The reaction components of test group b include: dCas9, Target sgRNA (specific sgRNA provided by the present invention), non-Target DNA. The reaction components of test group c include: dCas9, non-Target sgRNA (specific sgRNA not provided by the present invention), Target DNA (PCR amplification product with biotin labeling). The reaction components of test group d include: dCas9, Target DNA (PCR amplification product with biotin labeling). The reaction components of test group e include: Target sgRNA (specific sgRNA provided by the present invention), Target DNA (PCR amplification product with biotin labeling). The reaction components of test group f include: Target DNA (PCR amplification product with biotin labeling). The reaction components of test group g include: dCas9, Target sgRNA (specific sgRNA provided by the present invention). The reaction components of test group h (blank group) do not include dCas9, Target sgRNA (specific sgRNA provided by the present invention) and Target DNA (PCR amplification product labeled with biotin).

The dCas9/DNA-Bio complexes prepared from the above test groups a to h were tested by ELISA, and the test results are shown in Figure 3. The horizontal axis in Figure 3 is the component composition corresponding to the test groups a to h, and the vertical axis is the absorbance value at 450nm. As shown in Figure 3, the absorbance will increase significantly only in the detection system where dCas9, the correct sgRNA and the biotinylated target DNA exist at the same time.

Therefore, from the results of the above experiments 1) and 2), it can be seen that the identification method proposed in the present invention is specific for the detection of target DNA (genetically modified crops) and can be applied to the rapid on-site screening of genetically modified crops.

Example 2 Effect of the dosage of dCas9 and sgRNA on the signal-to-noise ratio of the detection system

When preparing the dCas9/sgRNA/DNA-Bio ternary complex, the amount of dCas9 and sgRNA will directly affect the formation of the dCas9-sgRNA complex, thereby indirectly affecting the sensitivity of the detection. During the experiment, the amount of sgRNA added was fixed at 4 μL, and the amount of 0.3 μM dCas9 added was set to 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL, respectively. As shown in Figure 4, the horizontal axis is the amount of dCas9 added (volume), and the vertical axis is the signal-to-blank ratio. As the amount of dCas9 added gradually increases, the signal-to-noise ratio of the detection system gradually increases. When the amount of dCas9 added is 4 μL, the signal-to-noise ratio reaches the maximum value. Subsequently, the amount of dCas9 added continues to increase, and the signal-to-noise ratio shows a downward trend. Therefore, 4 μL of 0.3 μM dCas9 is selected for subsequent experiments.

Next, the amount of 0.3 μM dCas9 fixed in the detection system was added to 4 μL, and the amount of 0.3 μM sgRNA added was set to 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL, respectively. As shown in Figure 5, the horizontal axis is the amount of sgRNA added (volume), and the vertical axis is the signal-to-blank ratio. As the amount of sgRNA added gradually increases, the signal-to-noise ratio of the detection system gradually increases. When the amount of sgRNA added is 4 μL, the signal-to-noise ratio reaches the maximum value. Subsequently, the amount of sgRNA added continues to increase, and the signal-to-noise ratio shows a downward trend. Therefore, 4 μL of 0.3 μM sgRNA was selected for subsequent experiments.

Combined with the test results of Figures 4 and 5, when the dosage ratio (volume ratio) of 0.3 μM dCas9 to 0.3 μM sgRNA is 4:3.5-4.5, that is, the molar ratio of dCas9 to sgRNA is 1.2:1.05-1.35, the signal-to-noise ratio of the detection system is large, and the detection sensitivity of the detection system is good; when the dosage ratio (volume ratio) of 0.3 μM dCas9 to 0.3 μM sgRNA is 1:1, that is, the molar ratio of dCas9 to sgRNA is 1:1, the signal-to-noise ratio of the detection system is the largest, and the detection sensitivity of the detection system is the best.

Example 3: Effect of Cas9 antibody coating concentration on the detection system

Since the dCas9/sgRNA/DNA-Bio ternary complex is fixed on the microplate through the immune binding of dCas9 protein and dCas9 antibody, the amount of dCas9 antibody coated on the microplate will directly affect the amount of captured dCas9/sgRNA/DNA-Bio ternary complex, thereby affecting the sensitivity of detection.

In the embodiment of the present invention, the PCR amplification product of transgenic rice containing CaMV35S promoter at 1 ng/μL was used as the signal value for detection, and the detection value of the PCR amplification product of ddH ₂ O was used as the no-template control (NTC). The coating concentrations of dCas9 antibodies were set to 0.25μg/mL, 0.5μg/mL, 1μg/mL, 2μg/mL, and 4μg/mL, respectively, and the effect of the coating amount of dCas9 antibodies on the dCas9/sgRNA detection system was studied and tested. The test results are shown in FIG6 , where the abscissa is the coating concentration of dCas9 antibodies and the ordinate is the signal-to-blank ratio. As shown in FIG6 , when the coating concentration of dCas9 antibodies is within the concentration range of 0.25μg/mL to 1μg/mL, the signal-to-blank ratio increases with the increase of the coating concentration of dCas9 antibodies. When the concentration is 1μg/mL, the signal-to-blank ratio is the largest, indicating that the immune response is saturated at this time. When the coating concentration of dCas9 antibodies continues to increase, the blank signal value increases, resulting in a decrease in the signal-to-blank ratio. Therefore, the preferred coating concentration of the dCas9 antibody in the embodiment of the present invention may be 0.75 μg/mL to 2 μg/mL, and the most preferred coating concentration of the dCas9 antibody is 1 μg/mL.

Example 4 Effect of SA-HRP enzyme concentration on the detection system

SA-HRP binds to biotin on the dCas9/sgRNA/DNA-Bio complex and catalyzes TMB color development. If too little SA-HRP is added to the reaction, the reaction is insufficient, and too much may cause nonspecific adsorption, resulting in weak color development of the blank control. Based on this, the following screening test was designed in the embodiment of the present invention to determine the preferred concentration range of SA-HRP.

4 μL 0.3 μM dCas9 protein, 4 μL 0.3 μM specific sgRNA, 10 μL 10×NEBuffer, and 69.5 μL DEPC water were mixed and incubated at 25°C for 10 min to form a dCas9/sgRNA complex, i.e., a single-base specific probe. Next, the dCas9/sgRNA complex obtained above was mixed with 2.5 μL of biotin-labeled PCR amplification product or RPA amplification product and incubated at 37°C for 15 min to form a dCas9/sgRNA/DNA-Bio ternary complex. Afterwards, 100 μL of dCas9 antibody dissolved in 0.1M Tris-HCl buffer at a concentration of 1 μg/mL was added as a coating solution to a high-affinity polystyrene 96-well microplate and coated overnight at 4°C. After that, pour off the unbound coating solution in each well and wash three times with 300 μL of washing buffer (phosphate buffer containing 0.050% Tween 20 at a concentration of 0.10M and a pH of 7.4). Then add 150 μL of blocking buffer (phosphate buffer containing 1.0% BSA, 0.050% Tween-20 at a concentration of 0.10M and a pH of 7.4) to each well and block at 37°C for 30 minutes. After washing three times with washing buffer, add 90 μL of dCas9/sgRNA/DNA-Bio ternary complex solution to each well and incubate at 37°C for 30 minutes to complete the immune reaction. Wash three times with washing buffer.

On the basis of the above test steps, 5 test groups were set up. 100 μL of SA-HRP enzyme with a concentration of 0.5 μg/mL, 1 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL was added to the microplate in the 5 test groups, respectively, and incubated at 37°C for 20 min. Wash 3 times with washing buffer. After washing, TMB colorimetric detection was performed, and 50 μL of TMB solution A and 50 μL of TMB solution B were added to the immune complex after the above reaction, and incubated in the dark for 7 min. Finally, 50 μL of 2.0M H ₂ SO ₄ was added to terminate the reaction. The results can be visually detected by the naked eye and quantified by measuring the absorbance value at 450 nm. The test results of the signal-to-noise ratio of each test group are shown in Figure 7, where the horizontal axis is the concentration of SA-HRP enzyme and the vertical axis is the signal-to-blank ratio. As shown in Figure 7, when the SA-HRP enzyme concentration ranges from 0.5 to 5 μg/mL, the signal-to-noise ratio is large; when the concentration of the streptavidin horseradish peroxidase is 5 μg/mL, the signal-to-noise ratio is the largest. Therefore, the preferred concentration of the streptavidin horseradish peroxidase in the embodiment of the present invention is 0.5 to 5 μg/mL, and the most preferred concentration of the streptavidin horseradish peroxidase is 5 μg/mL.

Example 5 Effect of the incubation time of the ternary complex and the dCas9 antibody coated immune response on the detection system

4 μL 0.3 μM dCas9 protein, 4 μL 0.3 μM specific sgRNA, 10 μL 10×NEBuffer, and 69.5 μL DEPC water were mixed and incubated at 25°C for 10 min to form a dCas9/sgRNA complex, i.e., a single-base specific probe. Next, the dCas9/sgRNA complex obtained above was mixed with 2.5 μL of biotin-labeled PCR amplification product or RPA amplification product and incubated at 37°C for 15 min to form a dCas9/sgRNA/DNA-Bio ternary complex. Afterwards, 100 μL of dCas9 antibody dissolved in 0.1M Tris-HCl buffer at a concentration of 1 μg/mL was added as a coating solution to a high-affinity polystyrene 96-well microplate and coated overnight at 4°C. After that, pour off the unbound coating solution in each well and wash three times with 300 μL of washing buffer (phosphate buffer containing 0.050% Tween 20 at a concentration of 0.10M and pH 7.4). Then add 150 μL of blocking buffer (phosphate buffer containing 1.0% BSA, 0.050% Tween-20 at a concentration of 0.10M and pH 7.4) to each well and block at 37°C for 30 minutes. Wash three times with washing buffer.

On the basis of the above steps, 5 experimental groups were set up, 90 μL of dCas9/sgRNA/DNA-Bio ternary complex solution was added to each well, and incubated at 37 °C. The incubation time of each experimental group was 5 min, 10 min, 30 min, 50 min, and 70 min, respectively, to complete the immune response.

Afterwards, each test group was washed three times with washing buffer. 100 μL of 5 μg/mL SA-HRP enzyme (streptavidin horseradish peroxidase) was added to the microplate and incubated at 37°C for 20 min. Washed three times with washing buffer. After washing, TMB colorimetric detection was performed, and 50 μL of TMB solution A and 50 μL of TMB solution B were added to the immune complex after the above reaction and incubated in the dark for 7 min. Finally, 50 μL of 2.0 M H ₂ SO ₄ was added to terminate the reaction, and the absorbance value of each test group at 450 nm was tested. The test results are shown in Figure 8, with the horizontal axis representing the incubation time and the vertical axis representing the signal-to-blank ratio.

As shown in Figure 8, as the incubation time increases, the absorbance values of each test group (positive sample) at 450nm gradually increase, and non-specific binding increases, and the A450 value of the blank control increases, so the signal-to-noise ratio decreases. When the incubation time is 30 minutes, the signal-to-noise ratio is the largest.

Example 6 Specificity, repeatability and stability of dCas9-mediated ELISA

Test samples: transgenic rice containing CaMV35S promoter, non-transgenic rice, non-transgenic corn, non-transgenic rapeseed and non-transgenic soybean.

Test method: Extract the DNA of each test sample above, amplify the DNA of each test sample using a biotin-labeled PCR primer set to obtain a PCR amplification product. The initial PCR template is 1.0 ng/μL. Mix 4μL 0.3μM dCas9 protein, 4μL 0.3μM specific sgRNA, 10μL 10×NEBuffer, and 69.5μL DEPC water, and incubate at 25°C for 10 minutes to form a dCas9/sgRNA complex. Then, mix the above-obtained dCas9/sgRNA complex with 2.5μL of biotin-labeled PCR amplification product or RPA amplification product, and incubate at 37°C for 15 minutes to form a dCas9/sgRNA/DNA-Bio ternary complex. Afterwards, 100 μL of dCas9 antibody dissolved in 0.1M Tris-HCl buffer at pH 8.0 at a concentration of 1 μg/mL was added as a coating solution to a high-affinity polystyrene 96-well microplate and coated overnight at 4°C. Afterwards, the unbound coating solution in each well was poured off and washed three times with 300 μL of washing buffer (phosphate buffer containing 0.050% Tween 20 at a concentration of 0.10M and a pH of 7.4). Then, 150 μL of blocking buffer (phosphate buffer containing 1.0% BSA, 0.050% Tween-20 at a concentration of 0.10M and a pH of 7.4) was added to each well and blocked at 37°C for 30 minutes. After washing three times with washing buffer, 90 μL of dCas9/sgRNA/DNA-Bio ternary complex solution was added to each well and incubated at 37°C for 30 minutes to complete the immune reaction. Wash three times with washing buffer. Add 100 μL of 5 μg/mL SA-HRP enzyme (streptavidin horseradish peroxidase) to the microplate and incubate at 37°C for 20 min. Wash three times with washing buffer. After washing, perform TMB colorimetric detection, add 50 μL of TMB solution A and 50 μL of TMB solution B to the immune complex after the above reaction, and incubate for 7 min in the dark. Finally, add 50 μL of 2.0 M H ₂ SO ₄ to terminate the reaction. The results can be visually detected by the naked eye and quantified by measuring the absorbance value at 450 nm.

The test results are shown in Figure 9. The absorbance values of non-GM crop samples (non-GM rice, non-GM corn, non-GM rapeseed and non-GM soybean) are all close to the blank signal. Under the same template concentration, the absorbance of transgenic rice containing the CaMV35S promoter increased significantly. The test results are consistent with the agarose gel electrophoresis results shown in Figure 10. Therefore, the identification method provided in the embodiment of the present invention can reliably distinguish between transgenic crops and non-GM crops, showing good specificity.

Among them, a in Figure 9 represents transgenic rice containing CaMV35S promoter, b represents non-transgenic rice, c represents non-transgenic corn, d represents non-transgenic rapeseed, e represents non-transgenic soybean, and f represents NTC (blank control). M in Figure 10 represents DL2000, and 1 to 6 correspond to a to f in Figure 9 respectively.

In addition, the repeatability of the method was studied by performing five parallel measurements at template concentrations of 500 copies/μL and 50 copies/μL, respectively. The test results are shown in FIG11 , and the relative standard deviations (RSDs) were 2.2% and 7.2%, respectively.

In addition, the stability of the dCas9-mediated ELISA detection system was also tested. The specific test method is as follows: After the dCas9 antibody was coated on the microplate and stored at 4°C for 1 day, 2 days, 3 days, 1 week and 2 weeks, it was only reduced by 10.7% and 5.2% compared with the initial absorbance of 500 copies/μL and 50 copies/μL (as shown in Figure 12). It can be seen that the dCas9-ELISA detection system has good repeatability and stability. The horizontal axis in Figure 12 is the storage time, and the vertical axis is the absorbance value of the sample at 450nm.

Example 7dCas9-mediated ELISA single base recognition ability test

The precise recognition and binding of the CRISPR/dCas9 system to the target DNA is highly dependent on the sequence of the specific sgRNA. In order to evaluate the single-base recognition ability of the identification method (dCas9-mediated ELISA) provided in the embodiment of the present invention, under the condition that the dosage ratio (volume ratio) of 0.3 μM dCas9 and 0.3 μM sgRNA is 1:1, that is, the molar ratio of dCas9 to sgRNA is 1:1, the mutations near the PAM site in the targeted CaMV35S sequence were tested, including four types of single-base mismatch targets (1MT-1, 1MT-2, 1MT-3 and 1MT-4), two-base mismatch targets (2MT), three-base mismatch targets (3MT) and twenty-base mismatch targets (20MT). Among them, the mismatch sequences of the target sequences near the PAM sites of each group are shown in Figure 13. The base "GGG" near the 3' end in FIG13 is the PAM site, and the gray bases in the sequences of 1MT-1, 1MT-2, 1MT-3, 1MT-4, 2MT, 3MT, and 20MT are mismatched bases.

The mismatched sequences of the target sequences near the PAM sites of the above-mentioned groups are subjected to PCR reaction using a PCR primer set with biotin labeling to obtain PCR amplification products. Then, each group of PCR amplification products is subjected to absorbance test according to the test method as described in Example 6, and the test results are shown in Figure 14. As can be seen from Figure 14, the absorbance values of 2MT, 3MT and 20MT are consistent with the blank signal, and are much smaller than the absorbance value of CaMV35S, indicating that the proposed method can easily distinguish multiple base differences of target DNA. Compared with polybasic mutations, the absorbance values of 1MT-1, 1MT-2, 1MT-3, and 1MT-4 increase. However, there are statistically significant differences (p<0.0001) in the absorbance signals between CaMV35S and 1MT groups (1MT-1, 1MT-2, 1MT-3, and 1MT-4). Therefore, the detection method of the ELISA mediated by dCas9 proposed in the embodiment of the present invention can detect single nucleotide polymorphism (SNP) sites. Among them, "****" in Figure 14 represents p < 0.0001.

Example 8 Sensitivity test of dCas9-mediated ELISA detection system

The transgenic rice genomic DNA standard material was diluted with ddH ₂ O to 500, 250, 175, 100, 50, 25 and 12.5 copies to prepare CaMV35S promoter quantitative standard samples.

Referring to the test method of Example 6 above, PCR amplification was performed on transgenic rice genomic DNA standard materials using different template concentrations, and the absorbance value of the obtained PCR amplification products was detected. The test results are shown in Figure 15. As can be seen from Figure 15, the PCR amplification products of the NTC sample (blank sample) did not appear yellow on the microplate (see the microplate illustration above Figure 15), and no signal was observed; the PCR amplification products containing the CaMV35S promoter quantitative standard sample appeared yellow on the microplate (see the microplate illustration above Figure 15). Moreover, as the concentration of the CaMV35S promoter quantitative standard sample gradually increased, the yellow color on the microplate gradually deepened. These results show that it is feasible to use the identification method provided in the embodiment of the present invention to accurately and visually identify the DNA of transgenic crops.

A standard curve (as shown in FIG. 16 ) was drawn based on the absorbance test value of each sample and the concentration of the CaMV35S promoter quantitative standard sample. The linear regression equation of the standard curve was: Y (a.u.) = 0.9144lgX (copy/μL) - 0.573, wherein Y and X represent the logarithm of the absorbance value and the concentration of the CaMV35S promoter quantitative standard sample, respectively. The correlation coefficient was 0.9993, and the detection limit (LOD) was 12.5 copies/μL. As shown in FIG. 16 , as the concentration of the CaMV35S promoter quantitative standard sample gradually increased, the absorbance values of each group of samples at 450 nm increased linearly. In the range of template concentration of 12.5 to 500 copies/μL, the logarithm of the absorbance test value of the sample and the concentration of the CaMV35S promoter quantitative standard sample showed good linearity.

Example 9 Actual sample detection

Homozygous KMD (transgenic rice) seeds were mixed with non-transgenic rice seeds to prepare three groups of test samples with KMD contents of 10%, 1.0% and 0.10%, respectively. Genomic DNA was extracted from the three groups of test samples, and the real-time fluorescence quantitative PCR verification was performed using the SN/T 4853.2-2017 standard. The test results are shown in Figure 17. As shown in Figure 17, the Ct values of the test samples with transgenic rice sample contents of 10%, 1.0% and 0.10% were 29.04, 32.25, and 35.56, respectively, while no signal was found in non-transgenic rice. Among them, curves a to d in Figure 17 represent the real-time fluorescence quantitative PCR curves of the test samples with transgenic rice sample contents of 10%, 1.0%, 0.10% and 0, respectively. The abscissa of Figure 17 is the number of cycles, and the ordinate is the relative fluorescence unit RFU (10^ ³ ).

The identification method provided in Example 6 of the present invention was used to detect the PCR amplification products of the samples to be tested with the above transgenic rice sample content of 10%, 1.0%, 0.10% and 0, and the color and absorbance value of the microplate were observed. The test results are shown in Figure 18, where the horizontal axis is the mixing ratio of the transgenic rice and non-transgenic rice content, and the vertical axis is the absorbance value of each sample at 450nm. As shown in Figure 18, the microplate corresponding to the blank sample (i.e., the sample to be tested with a transgenic rice sample content of 0) did not show color change. The microplate corresponding to the sample to be tested containing the transgenic rice sample showed yellow, and as the KMD content increased, the color of the microplate became more and more yellow, and the corresponding absorbance value also increased as the KMD content increased.

According to the above results, samples with 0.10% GM content can be distinguished from blank samples, indicating that the identification method provided by the embodiment of the present invention has good sensitivity and reaches the 0.9% identification threshold set by the European Union. Therefore, the identification method provided by the embodiment of the present invention can be used for the effective detection of genetically modified crops.

Example 10 dCas9-mediated ELISA nucleic acid detection based on RPA

Three pairs of RPA primer sets were designed based on the CaMV35S promoter sequence, and the sequence information of these three pairs of RPA primer sets is shown in Figure 19. The three pairs of RPA primer sets were tested by agarose gel electrophoresis, and the test results are shown in Figure 20. M in Figure 20 represents DL2000; 1-2 represents RPA primer set RPA-F1/RPA-R; 3-4 represents RPA primer set RPA-F2/RPA-R; 5-6 represents RPA primer set RPA-F3/RPA-R. 1, 3, and 5 are positive samples; 2, 4, and 6 are negative samples. As shown in Figure 20, the amplification target band of the RPA primer set RPA-F2/RPA-R is single and has no primer dimers, so the RPA-F2/RPA-R primer set was selected for subsequent experiments.

S1. Extract the target DNA of the samples to be tested. The samples to be tested are: genetically modified rice (Rice KMD), non-genetically modified rice (Non-GM-rice), non-genetically modified corn (Non-GM-maize), non-genetically modified rapeseed (Non-GM-rape), non-genetically modified soybean (Non-GM-soybean), and blank control group (NTC).

The genomic DNA of each sample (crop seed) to be tested was rapidly extracted within 1 min using a lysis buffer, and the extracted supernatant was directly used as a DNA template (target DNA). The lysis buffer was 50 mM Tris-HCl (pH 8.0), which contained the following components: 250 mM NaOH, 0.5 mM Na ₂ EDTA (pH 8.0), 0.1% BSA, 0.050% Tween-20, and 20 mM MgCl ₂ in a concentration of 50 mM Tris-HCl (pH 8.0).

S2. Using the target DNA as a template, an RPA reaction was performed using a biotin-labeled RPA primer set to generate an RPA amplification product within 15 minutes.

S3. The RPA amplification product obtained in step S2 is added to a detection system containing a single base-specific probe for detection. Within 60 minutes, it can be visually identified by the naked eye or quantitatively tested by simply measuring the absorbance. The test results are shown in Figure 21. As shown in Figure 21, the absorbance corresponding to the transgenic rice is significantly increased, while the absorbance of other non-transgenic crops is similar to that of the blank control group, which indicates that the RPA-based dCas9-mediated ELISA detection system provided in the embodiment of the present invention has good specificity for transgenic crops and can be used for on-site screening and identification of transgenic crops.

In summary, the method for identifying genetically modified crops with single-base specificity provided by the embodiment of the present invention uses the CRISPR-dCas9 system to develop a dCas9-ELISA detection system, and a PCR primer set or RPA primer set containing a biotin label is used to amplify the target DNA to obtain a PCR amplification product or an RPA amplification product; thereafter, the PCR amplification product or the RPA amplification product is added to the detection system in which a single-base specific probe (dCas9/sgRNA complex) is present, and the double-stranded target nucleic acid containing the biotin label is recognized by the dCas9-sgRNA to form a ternary complex, and then captured by the dCas9 antibody coated on the microplate, and then streptavidin-HRP is added for incubation and TMB reaction is performed, and then the detection result can be judged by the naked eye to judge the color change of the solution, and can also be judged by combining the absorbance value at a wavelength of 450nm with the instrument to determine the absorbance value, thereby realizing the identification of genetically modified crops. The method of the present invention has the advantages of good specificity, high sensitivity, fast detection speed, simple operation, low cost, etc., and can be applied to molecular diagnosis in resource-limited environments.

The identification method provided in the embodiment of the present invention can only be successfully detected when the detection system contains dCas9, correct sgRNA and biotinylated target DNA. The specific sgRNA designed by the present invention can successfully detect the CaMV35S promoter. The identification method provided in the embodiment of the present invention has good repeatability and stability. Combined with the microplate reader, the method of the present invention can realize the quantitative detection of the genome of genetically modified crops with a sensitivity of 12.5 copies/μL. In addition, the identification method provided in the embodiment of the present invention can well distinguish samples with a genetically modified component content of 0.1% and negative samples by the naked eye, and can be applied to the screening and identification of genetically modified crops in field environments with limited resources. In addition, the identification method provided in the embodiment of the present invention can be combined with rapid extraction and RPA technology, using a microplate pre-coated with dCas9 antibodies, and the extraction and amplification results can be completed within 75 minutes, which greatly shortens the detection time, and the qualitative detection can be completed without the aid of large instruments throughout the process, which greatly reduces the detection cost; at the same time, the method of the present invention also has good detection specificity, can specifically identify sequences with single-base differences, and can be applied to nucleic acid detection with single-base resolution.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention.

Claims (10)

1. A transgenic crop identification method with single base specificity, characterized in that, comprising the steps: Extract the target DNA of the sample to be tested; Using the target DNA as a template, use a biotin-labeled PCR primer set to perform a PCR reaction to obtain a PCR amplification product; or use a biotin-labeled RPA primer set to perform an RPA reaction to obtain an RPA amplification product; wherein, the The PCR primer set includes a sequence such as the first forward primer shown in SEQ ID NO: 1 and a sequence such as the first reverse primer shown in SEQ ID NO: 2; the RPA primer set includes the first RPA primer set , the second RPA primer set and the third RPA primer set; the first RPA primer set includes a sequence such as the second forward primer shown in SEQ ID NO: 3 and a sequence such as the first forward primer shown in SEQ ID NO: 4 Two reverse primers; the second RPA primer set includes a sequence such as the third forward primer shown in SEQ ID NO: 5 and a sequence such as the second reverse primer shown in SEQ ID NO: 4; the third The RPA primer set includes a fourth forward primer whose sequence is shown in SEQ ID NO: 6 and a second reverse primer whose sequence is shown in SEQ ID NO: 4; The PCR amplification product or the RPA amplification product is added into a detection system with a single base specific probe for detection, and an identification result is obtained. 2. The method according to claim 1, wherein the single-base specific probe is a complex of dCas9 protein and specific sgRNA; The sequence of the specific sgRNA is shown in SEQ ID NO:7. 3. method according to claim 2, is characterized in that, described PCR amplified product or RPA amplified product are added in the detection system that exists single base specificity probe and detect, obtain appraisal result, comprise: Pre-incubating the dCas9 protein and the specific sgRNA to form a single-base specific probe; Adding the PCR amplification product or the RPA amplification product into the detection system in which the single-base specific probe exists, so that the PCR amplification product or the RPA amplification product is compatible with the single-base specific probe The needles were incubated to form a ternary complex; Add the ternary complex to a microwell plate coated with dCas9 antibody, incubate to complete the immune reaction, then add streptavidin-horseradish peroxidase to incubate, then add a chromogenic reagent for absorbance detection , get the identification result. 4. The method according to claim 3, wherein the molar ratio of the dCas9 protein to the specific sgRNA is 1:0.6-10. 5. The method according to claim 4, wherein the molar ratio of the dCas9 protein to the specific sgRNA is 1:1. 6. The method according to claim 3, wherein the coating concentration of the dCas9 antibody is 0.75 μg/mL˜2 μg/mL. 7. The method according to claim 6, wherein the coating concentration of the dCas9 antibody is 1 μg/mL. 8. The method according to claim 3, characterized in that the concentration of the streptavidin-horseradish peroxidase is 0.5-7.5 μg/mL. 9. The method according to claim 8, characterized in that the concentration of the streptavidin-horseradish peroxidase is 5 μg/mL. 10. The method according to claim 3, characterized in that, in the step of adding the ternary complex to a microwell plate coated with dCas9 antibody, and incubating to complete the immune reaction, the incubation time is 10-50 min.