A method for detecting corn transformant CC-2

Abstract

The application discloses a method for detecting corn transformant CC-2, and particularly discloses a primer composition for detecting the transgenic corn transformant CC-2, wherein the primer composition comprises a primer composed of sequence 1 and sequence 2, and a crRNA shown in sequence 3. The detection method comprises the following steps: using the primer composition to perform isothermal amplification based on a multi-enzyme system isothermal nucleic acid detection technology, and performing CRISPR / Cas delta detection on the isothermal amplification product. The application takes the transgenic corn as the object, aims to establish a transgenic corn RPA-CRISPR / Cas delta isothermal amplification detection method, and tests the sensitivity, specificity and other aspects of the method, so as to develop and establish a rapid, convenient and high-sensitivity field transgenic detection method. The detection and monitoring of the transgenic corn can provide important scientific and technological support.

Description

Technical Field

[0001] This invention relates to the field of gene detection, and more specifically, to a method for detecting the maize transformant CC-2. Background Technology

[0002] Corn is also a crucial raw material for the feed industry and animal husbandry. With the increasing consumption of meat, eggs, and dairy products, its proportion as feed in the total grain demand is gradually increasing. To improve the regulation and management of genetically modified (GM) crops, there is an urgent need to establish rapid on-site detection methods. Currently, there are two main types of rapid detection methods for GM crops: one is protein-based rapid detection methods, primarily using protein test strips. However, protein-based methods require antibody preparation, which is time-consuming and costly. Furthermore, the detection sensitivity of protein test strips needs improvement. On the other hand, GM crop protein test strips can only detect exogenous proteins expressed by GM crops, limiting the detection targets and making it impossible to distinguish between different GM crop lines and transformants expressing the same protein. The other type is nucleic acid-based rapid detection methods. Nucleic acids are often used as targets for GM crop detection due to their high stability, ease of operation, and the specificity of nucleic acid sequences, which can distinguish between different GM crop lines and transformants. Traditional nucleic acid detection methods mainly utilize PCR technology, but PCR detection methods rely on sophisticated instruments and are time-consuming, failing to meet the needs of rapid on-site detection of GM crops. In recent years, an increasing number of researchers have focused on developing new in vitro nucleic acid amplification technologies, aiming to revolutionize traditional PCR technology and overcome its shortcomings. Isothermal nucleic acid amplification technology is a novel nucleic acid analysis technique that achieves nucleic acid amplification under constant temperature conditions. Compared to traditional PCR, isothermal nucleic acid amplification technology eliminates the need for thermal cycling equipment and offers advantages such as shorter processing time, higher sensitivity, and higher specificity for amplifying target sequences.

[0003] RPA (Recombinant DNA Polymerase) is a multi-enzyme isothermal nucleic acid detection technique that completes the amplification reaction under a multi-enzyme system. The process can be divided into four parts: Step 1: Formation of DNA nucleoprotein microfilaments. Under ATP-powered conditions, recombinase proteins combine with specific oligonucleotide primers to form a complex structure that can be elongated at both ends, called a DNA nucleoprotein microfilament. Step 2: Primer-target DNA pairing. The formed DNA nucleoprotein microfilaments approach the double-stranded DNA by elongating at the front and shortening at the back, and then perform screening and matching. After successful matching, the double-stranded DNA unwinds under the action of single-strand binding proteins, and the oligonucleotide primer pairs with the target sequence. Step 3: Primer extension. Under the action of BsuDNA polymerase, double-stranded DNA is extended from the 3' end of the primer to form new double-stranded DNA. The newly formed double-stranded DNA can serve as a new template for amplification, and the amplification product grows exponentially. RPA technology has high sensitivity and specificity. When RPA technology is combined with fluorescent probes for nucleic acid detection, the reaction time is short, the amplified products are numerous, and the results are intuitive. The peak time can be directly determined from the fluorescence signal value, and the process can generally be completed in 20 minutes. The instruments used are simple and convenient, meeting the needs of on-site testing. Leveraging the low equipment requirements of RPA technology, rapid detection methods have been established with numerous applications in areas such as bacterial and viral detection, and cancer research.

[0004] CRISPR (clustered regularly interspaced short palindromic repeats) consists of clustered, regularly spaced short palindromic repeats, composed of multiple unidirectional repeats and non-repetitive spacer sequences. Casδ is a newly identified superfamily of two CRISPR / Cas effectors by the Lai Jinsheng team at China Agricultural University, through large-scale microbial genome and metagenomic data mining. It comprises three members (Casδ-1, Casδ-2, and Casδ-3), with protein lengths ranging from 867 to 936 amino acids. Phylogenetic analysis reveals that Casδ is evolutionarily located at a transitional position between Cas12n and typical type V systems (such as Cas12b), serving as an evolutionary intermediate connecting these two systems. Its C-terminal loop structure is crucial for its catalytic activity. After Casδ proteins bind to their specifically recognized target DNA under the guidance of crRNA, their RuvC nuclease domain is activated, leading to efficient cleavage of surrounding single-stranded DNA sequences. This dual mode of "cis-cleavage activation and trans-cleavage execution" constitutes a natural signal amplification mechanism—after a single Casδ protein binds to its target, it can cleave a large number of non-specific single-stranded DNA reporter molecules, significantly enhancing the detection signal. The compact structure of Casδ and its simplified design, which eliminates the need for tracrRNA, give it unique advantages in certain applications, providing a foundation for developing highly sensitive molecular diagnostic tools.

[0005] CRISPR / Casδ is a novel gene-editing protein independently developed by China Agricultural University. It possesses both efficient gene editing and nucleic acid detection functions, demonstrating broad application prospects. The nucleic acid detection technology developed based on this protein showcases my country's independent innovation capabilities in this field, achieving independent control over key core technologies and filling technological gaps in related research areas. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid detection method for the transgenic maize transformant CC-2.

[0007] This invention provides a primer composition comprising a primer pair consisting of CC-F and CC-R, and crRNA as shown in sequence 1; The sequences of the primer pairs are shown in any one of A1)-A5): A1) The CC-F is as shown in Sequence 2, and the CC-R is as shown in Sequence 3; A2) The CC-F is as shown in Sequence 2, and the CC-R is as shown in Sequence 5; A3) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 3; A4) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 5; A5) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 7.

[0008] The application of the above-mentioned primer composition in the detection of transgenic maize transformant CC-2 should also be within the scope of protection of this invention.

[0009] This invention provides a rapid detection method for transgenic maize transformants, comprising the following steps: 1) Using the genome of the maize to be tested as a template, primers targeting the maize transformant were used to perform isothermal amplification based on multi-enzyme isothermal nucleic acid detection technology to obtain isothermal amplification products; 2) The isothermal amplification product from step 1) is subjected to CRISPR / Casδ detection using crRNA targeting the maize transformant. The detection results are used to determine whether the maize to be tested is the target maize transformant.

[0010] Furthermore, the isothermal amplification time in step 1) is 20 min.

[0011] Furthermore, the reaction temperature in step 1) is 37°C. Furthermore, the primer concentration in step 1) is 1.0 μmol / L.

[0012] Furthermore, in step 2), the concentration of Casδ protein is 0.5 μg / μL, and the concentration of crRNA is 0.5 μM.

[0013] Furthermore, the concentration of the ssDNA probe in step 2) is 1.0 μM.

[0014] Furthermore, the magnesium ion concentration in step 2) is 10 mM.

[0015] Furthermore, the reaction temperature in step 2) is 37°C.

[0016] Furthermore, the CRISPR / Casδ reaction time in step 2) is 10 min.

[0017] Furthermore, the primer combination includes a primer pair consisting of CC-F and CC-R, and the crRNA shown in sequence 1; The sequences of the primer pairs are shown in any one of A1)-A5): A1) The CC-F is as shown in Sequence 2, and the CC-R is as shown in Sequence 3; A2) The CC-F is as shown in Sequence 2, and the CC-R is as shown in Sequence 5; A3) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 3; A4) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 5; A5) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 7.

[0018] This invention focuses on the transgenic maize transformant CC-2, aiming to establish a method for detecting transgenic maize using RPA-CRISPR / Casδ isothermal amplification. The method's sensitivity and specificity were tested in multiple aspects, resulting in a rapid, convenient, and highly sensitive field detection method for transgenic maize. This will provide important scientific and technological support for the detection and monitoring of transgenic maize. Attached Figure Description

[0019] Figure 1 For the screening of ND207 primers.

[0020] Figure 2 For the screening of CC-2 primers.

[0021] Figure 3 Sensitivity screening for RPA-ND207 primers.

[0022] Figure 4 Sensitivity screening for RPA-CC-2 primers.

[0023] Figure 5This was used to test the RPA reaction temperature and primer concentration.

[0024] Figure 6 The increasing trend of RPA fluorescence signal value.

[0025] Figure 7 This is a test of the reaction time for RPA isothermal amplification.

[0026] Figure 8 To identify the Casδ protein cleavage activity, (a) terminal fluorescence value, (b) real-time fluorescence signal value, and (c) visualization results of excitation light at wavelengths of 440-460 nm.

[0027] Figure 9 Screening of crRNAs for Casδ-ND207 transformants; (a) terminal fluorescence values ​​of three crRNAs, (b) real-time reaction values ​​of three crRNAs, and (c) visual detection of three crRNAs.

[0028] Figure 10 To optimize the Casδ cleavage system, (a) terminal signal values ​​of reactions with different combinations of crRNA and Casδ concentrations, (b) real-time fluorescence reaction signals of reactions with different combinations of crRNA and Casδ concentrations, and (c) visualization detection of reactions with different combinations of crRNA and Casδ concentrations.

[0029] Figure 11 To optimize the concentration of ssDNA fluorescent probes in the Casδ system; (a) terminal fluorescence values ​​of ssDNA reactions with different concentrations; (b) real-time reaction values ​​of ssDNA reactions with different concentrations; (c) visualization detection of ssDNA reactions with different concentrations.

[0030] Figure 12 The reaction effects of Casδ systems with different reaction times are shown; (a) the terminal fluorescence values ​​of the reaction at different times, (b) the real-time reaction values ​​of the reaction at different times, and (c) the visual detection of the reaction at different times.

[0031] Figure 13 Screening of magnesium ion concentration in Casδ system; where (a) terminal fluorescence values ​​of reactions with different magnesium ion concentrations, (b) real-time reaction values ​​of reactions with different magnesium ion concentrations, and (c) visual detection of reactions with different magnesium ion concentrations.

[0032] Figure 14 Sensitivity testing of the Casδ-ND207 transformant system; (a) fluorescence values ​​at the reaction terminal under different copy numbers; (b) (c) (d) (e) real-time reaction values ​​and visualization detection under different copy numbers.

[0033] Figure 15For the specificity test of Casδ-ND207 transformants; among which, (a) the terminal fluorescence value of different transformants, (b) the real-time reaction value of different transformants, and (c) the visual detection of different transformants.

[0034] Figure 16 Screening of crRNAs for Casδ-CC-2 transformants; (a) terminal fluorescence values ​​of three crRNAs, (b) real-time reaction values ​​of three crRNAs, and (c) visual detection of three crRNAs.

[0035] Figure 17 Sensitivity testing of the Casδ-CC-2 transformant system; (a) fluorescence values ​​at the reaction terminal under different copy numbers; (b) (c) (d) (e) real-time reaction values ​​and visualization detection under different copy numbers.

[0036] Figure 18 For the specificity test of Casδ-CC-2 transformants; among which, (a) the terminal fluorescence value of different transformants, (b) the real-time reaction value of different transformants, and (c) the visual detection of different transformants. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] The Casδ sequence information in the following examples is disclosed in *Nucleic Acids Research*, 2025; the qualitative PCR detection method and its standardization for the transformation event-specific transformation of maize ND207 is disclosed in *Acta Agronomica Sinica*, 2023; the establishment and application of a dual-droplet digital polymerase chain reaction detection method for maize CC-2 is disclosed in *Food Science*, 2025; the study on the insect resistance effect of maize transformant DBN9936 on Bt transgenic maize (Ruifeng 125, DBN9936, DBN9978) against the Asian corn borer is disclosed in *Plant Protection*, 2021; and the Toxic Effects of Bt-(Cry1Ab+Vip3Aa) Maize ("DBN3601T" Event) on the Asian Corn Borer Ostrinia furnacalis (Guenée) in Southwestern maize is disclosed. The study on the insect resistance effect of Ruifeng 125 Bt transgenic maize (Ruifeng 125, DBN9936, DBN9978) against the Asian corn borer was published in *Plant Protection* in 2021. The real-time fluorescence quantitative PCR detection method for Ruifeng 8 in transgenic insect-resistant maize (Zhejiang University Ruifeng 8) was published in *Journal of Biosafety (Chinese and English)* in 2025. The public can obtain these methods from the inventors to replicate this experiment.

[0040] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged. Invention Overview This invention provides a rapid detection method for transgenic maize transformants. The specific procedure is as follows: Take a small amount of transgenic maize leaves containing the transformant ND207 / CC-2 into a 1.5 ml centrifuge tube, add 100 μL of buffer B1, grind the leaves until the solution changes from colorless to turbid green, add 100 μL of buffer B2, vortex to mix, and aspirate 1 μL of the supernatant as a template for RPA amplification. The RPA system consists of 29.4 μL of Buffer A, 2.5 μL of Buffer B, 2 μL of primers, and 50 μL of buffer B2. The optimal RPA amplification parameters are: primer concentration 1.0 μmol / L, isothermal reaction at 37℃, and reaction time 20 min. Take 5 μL of the RPA amplification product, add Casδ protein, crRNA, ssDNA fluorescent probe, magnesium ions, and buffer. The Casδ protein concentration is 0.5 μg / μL, crRNA concentration is 0.5 μM, ssDNA fluorescent probe is 1 μM, magnesium ions are 10 mM, the reaction temperature is 37℃, and the reaction time is 10 min. Irradiate with a portable, small excitation light source with a wavelength of 440-460nm, and observe the fluorescence signal with the naked eye using 500nm filter glasses.

[0042] Example 1 Sequence Preparation Follow the instructions for rapid DNA extraction and detection (Tiangen, KG203). Place a small amount of corn leaves in a 1.5 ml centrifuge tube and add 100 μl of buffer B1, ensuring the buffer completely covers the sample. Use a pestle to crush the sample, grinding until the solution becomes cloudy and discolored (from colorless to green). After crushing, add 100 μl of buffer B2 and vortex to mix. Let stand for a few seconds, then carefully aspirate the supernatant into another clean 1.5 ml centrifuge tube as a template for RPA amplification. The total extraction time for a single sample is within 5 minutes. It eliminates the need for liquid nitrogen grinding, reducing the risk of cross-contamination. Compared to traditional CTAB (approximately 4 hours) and conventional kit methods (approximately 1 hour), this method significantly saves time and is simple to operate, making it suitable for outdoor extraction.

[0043] The full-length T-DNA sequence of ND207 integrated into the maize genome is 8060 bp, containing three exogenous genes and their regulatory elements. To obtain the flanking sequences of ND207, PCR detection primers were designed targeting the 5' end of ND207 (Table 1). The genomic DNA of the homozygous ND207 maize variety was amplified by PCR using the primers provided by the developer. Sequencing analysis of the amplification products yielded the flanking sequences of ND207.

[0044] Table 1 Primers used for PCR amplification of transgenic maize ND207

[0045] To obtain the flanking sequence of CC-2, the research unit designed PCR detection primers targeting the 3' end of CC-2 (Table 2). PCR amplification was performed using genomic DNA from a homozygous CC-2 maize variety using primers provided by the researchers. Sequencing analysis of the amplification products yielded the flanking sequence of CC-2.

[0046] Table 2 Primers used for PCR amplification of transgenic maize CC-2

[0047] Example 2 Primer Design This study aimed to establish a line-specific RPA fluorescence isothermal amplification method for approved transgenic maize events. Due to the different target sequences, primer and probe designs needed to be tailored to different samples. Primer designs are shown in Tables 3 and 4. In previous experiments on RPA technology for detecting maize lines, the quality of the test sample affected the RPA amplification efficiency, and primer selection was quite cumbersome. Therefore, forward and reverse primer combinations were used for PCR amplification, and the fragment sizes of the amplified products are shown in Tables 5 and 6.

[0048] Table 3 Primer sequences used for RPA of transgenic maize ND207

[0049] Table 4 Primer sequences used for CC-2 RPA in transgenic maize

[0050] Table 5 Primer combinations used for RPA of transgenic maize ND207

[0051] Table 6 Primer combinations used for CC-2 RPA in transgenic maize

[0052] Primers were designed based on the specific sequence of transgenic maize, and an RPA detection method was established using an RPA reaction kit (Guangzhou Aidi Gene Technology Co., Ltd., catalog number WLB8201T). The entire reaction temperature was 37℃, and the total system volume was 50μL. The RPA reaction system is shown in Table 7. The reaction system was thoroughly mixed and vortexed, and then added to reaction tubes containing 0.2mL of lyophilized enzyme powder. After capping and thoroughly mixing and centrifuging, the tubes were immediately placed in the RPA reaction instrument and reacted at a constant temperature of 37℃.

[0053] Table 7 RPA Reaction System

[0054] In the CRISPR / Casδ system, gRNA is responsible for specifically recognizing the target nucleic acid and then guiding the Casδ protein to cleave it. The gRNA sequence consists of two parts: a direct repeat sequence and a spacer sequence. The optimal direct repeat sequence for CRISPR / Casδ is 5'-GUGCUGACGACCAGCACUAGAUGGUCGUUCAGGCAC-3', and the PAM recognition sequence is 5'-ATG. As shown in Table 8, after mixing the CRISPR / Casδ cleavage system in an EP tube, the reaction was carried out at 37°C. During the reaction, the fluorescence values ​​were monitored and recorded using a fluorescence reader. After the reaction, the results were observed and photographed under excitation light at a wavelength of 440-460 nm.

[0055] Table 8 CRISPR / Casδ Cutting System

[0056] Obtaining the flanking sequence of the exogenous T-DNA insertion site in transgenic plants is a key technical point in establishing a transformation event-specific qualitative RPA detection method. After extracting genomic DNA from homozygous ND207, PCR amplification of the ND207 homozygous maize genomic DNA was performed using primers ND207-5F and ND207-5R provided by the research unit. Sequencing analysis of the amplification products yielded a 254bp flanking sequence at the 5' end. The primer combinations listed in Table 5 were used for RPA amplification of the ND207 transformants. Primer combinations with a target band length between 200 and 300 bp, bright amplified bands without tailing, few primer dimers, and no non-specific amplification were selected. Figure 1 It can be seen that primer combinations 5'-1, 5'-2, 5'-3, 5'-5, and 5'-7 showed amplification signals, and the target band appeared on electrophoresis. There were no other non-specific bands. The size of the target band was consistent with expectations, and the band was clear and bright, so it can be used for the next screening step. Primers 5'-8 and 5'-9 showed amplification signals, but the target band on electrophoresis was weak. The product of 5'-4 was not homogeneous enough and showed tailing. Primer 5'-6 showed no amplification signal.

[0057] Genomic DNA was extracted from homozygous CC-2 individuals. PCR amplification of the CC-2 homozygous maize genomic DNA was performed using primers CC-2-3F and CC-2-3R provided by the research unit. Sequencing analysis of the amplified products yielded a 251 bp flanking sequence at the 3' end. RPA amplification of the CC-2 transformant was performed using primer combinations listed in Table 6. Primer combinations with a target band length between 200 and 300 bp, bright amplified bands without tailing, low primer dimer count, and no non-specific amplification were selected. Figure 2It can be seen that: 3'-1, 3'-2, 3'-4, 3'-5, and 3'-6 have amplification signals, the target band appears in electrophoresis, there are no other non-specific bands, the size of the target band is consistent with the expectation, and the band is clear and bright, so the next screening can be carried out. 3'-3 has amplification signals, the target band in electrophoresis is weak. 3'-8 product is not homogeneous enough and shows tailing phenomenon. 3'-7 and 3'-9 have no amplification signals.

[0058] Primer-template binding efficiency is one of the factors affecting the sensitivity of the RPA reaction. Under the same conditions, the DNA copy number of the ND207 specific transformant was set to 2 × 10⁻⁶. 7 2×10 5 2×10³, 2×10¹ and 2×10 0 Samples in copies / μL and a blank control were subjected to RPA amplification for 30 min, and the products were analyzed by gel electrophoresis. Figure 3 It can be seen that the primer combination 5'-1, 5'-2, 5'-3, 5'-5, 5'-7 is suitable for DNA concentrations of 2×10⁻⁶. 5 At DNA concentrations of 2×10³ copies / μL and above, the amplification bands are bright. The bands for 5'-1, 5'-2, 5'-3, 5'-5, and 5'-7 are weaker but still clearly distinguishable at a DNA concentration of 2×10³ copies / μL. The band for 5'-7 is also weaker but still clearly distinguishable at a DNA concentration of 2×10¹ copies / μL, while the bands for 5'-1 and 5'-2 are faintly visible and indistinct. When the DNA concentration is reduced to 2×10³ copies / μL... 0 At copies / μL, no clear amplification bands were observed with any primer combination. Therefore, through primer screening, primer combination 5'-7, namely ND207-5'-F3 and ND207-5'-R1, was selected to establish a specific qualitative RPA detection method for insect-resistant maize ND207 transformants.

[0059] Under the same conditions, the DNA copy number of the CC-2 specific transformant was set to 2 × 10⁻⁶. 7 2×10 5 2×10³, 2×10¹ and 2×10 0 RPA amplification was performed on samples of copies / μL and a blank control. The RPA products were then analyzed by electrophoresis. Figure 4 It can be seen that the primer combination 3'-1, 3'-2, 3'-5, 3'-6 at a copy number concentration of 2×10⁻⁶ 5 Amplification bands of copies / μL and above are bright, and primer combination 3'-4 at a DNA concentration of 2×10⁻⁴ are effective. 7 At a DNA concentration of 2×10⁶ copies / μL or higher, the amplification bands are bright, and at a DNA concentration of 2×10⁶... 5At a DNA concentration of 2 × 10⁻⁶ copies / μL, the amplified bands were weak but still identifiable. Primer combination 3'-1 at a DNA concentration of 2 × 10⁻⁶ copies / μL resulted in weaker bands, but they were still distinguishable. 3 The amplification bands at copies / μL were weak but clearly distinguishable; no clear amplification bands were observed at 3'-2, 3'-4, 3'-5, and 3'-6. When the DNA concentration was reduced to 2×10⁻⁶... 0 At copies / μL, no amplification band was observed with any primer combination. Therefore, through primer sensitivity screening, primer combination 3'-1, namely CC-2-3'-F1 and CC-2-3'-R1, was selected to establish a specific qualitative RPA detection method for transgenic maize CC-2 transformants.

[0060] Example 3: Optimization of Key Factors in RPA Reaction: Primer Concentration and Temperature Taking primers ND207-5'-F3 / ND207-5'-R1 as an example, the key factors of the RPA reaction, primer concentration and temperature, were optimized (primer concentrations were 0.2 μmol / L, 0.4 μmol / L, 0.6 μmol / L, 0.8 μmol / L, and 1.0 μmol / L; reaction temperatures were 33℃, 35℃, 37℃, 39℃, and 41℃, respectively). Other reaction conditions were the same as in Example 2, specifically: 1 μL of transformant DNA supernatant was used as the template for RPA amplification, along with 29.4 μL of Buffer A, 2.5 μL of Buffer B, 2 μL of each primer, and 50 μL of the RPA system. The RPA amplification reaction time was 20 min.

[0061] The results are as follows Figure 5 As shown, within the primer concentration range of 0.6–1.0 μmol / L and the reaction temperature range of 35–39 °C, relatively clear bands were observed, and the RPA reaction yielded good amplification. The optimal primer concentration was ultimately determined to be 1.0 μmol / L, and the optimal reaction temperature was 37 °C.

[0062] Example 4 Optimization of the time gradient of recombinase polymerase amplification (RPA) Using the optimal primer concentration and reaction temperature from Example 3, experiments were conducted on the recombinase polymerase amplification (RPA) time gradient in a systematic progressive experimental design to test the effect of different amplification times (5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min and 40 min) on the detection results.

[0063] The results are as follows Figure 6As shown, dynamic tracking by the real-time fluorescence quantitative monitoring system revealed that under isothermal conditions of 37℃, the reaction kinetics analysis showed that the fluorescence signal value (ΔRn) increased rapidly in the early stage, and the growth rate slowed down significantly after 15 min. At 20 min, it reached a value close to the plateau phase, and then continued to increase at a very slow rate, reaching the plateau phase at 30 min. This indicates that the amplification efficiency reached the theoretical maximum value at this time, and the fluorescence signal value no longer increased thereafter.

[0064] Meanwhile, to verify the correlation between real-time fluorescence data and product accumulation, the experimental group used agarose gel electrophoresis (2% agarose, 100V, 40min) to characterize the time-series sampled products. Figure 7 Electrophoresis analysis showed that, under the optimal combination of primer concentration of 1.0 μmol / L and reaction temperature of 37℃, faint specific bands appeared in the 15-minute experimental group, and clear specific bands appeared in the 20-minute experimental group.

[0065] The purified Casδ protein, crRNA, fluorescent probe, RPA amplification product nucleic acid template, buffer, and appropriate amount of water were mixed in an EP tube and reacted at 37℃. Fluorescence values ​​were monitored and recorded using a real-time fluorescence quantitative PCR instrument. The results were observed and photographed under excitation light at a wavelength of 440-460 nm. The results showed that the purified CRISPR / Casδ protein exhibited good cleavage activity, such as… Figure 8 As shown, however, the fluorescence signal level never reached the standard for visual detection (ΔRn < 1.0 × 10⁻⁶). 6 ).

[0066] Example 5: The effect of different crRNAs on detection results During the detection, three different designed crRNAs were used for three repeated reactions. In this process, the binary complex formed by the crRNA sequence and the CRISPR / Casδ protein in the system specifically recognizes the target sequence in the RPA reaction product via the PAM sequence, thereby activating the trans-cleavage activity of the CRISPR / Casδ protein. This process continuously cleaves the single-stranded DNA probe in the reaction system. After the probe is cleaved, its fluorophore separates from the quencher group. The quencher group no longer inhibits fluorescence, and the fluorophore begins to emit light, thus generating a fluorescent signal. The crRNA (crRNA-ND207) sequence and the single-stranded DNA probe sequence in the reaction system are shown in Table 9.

[0067] Table 9. Casδ-gRNA sequences and single-stranded DNA probe sequences used for ND207 detection.

[0068] Three crRNAs were added to a CRISPR / Casδ trans-cleavage system, and the fluorescence signal generated during cleavage was monitored in real time. A blank control group without the target was also included. The reaction was performed at 37℃ for 30 min in a real-time quantitative PCR instrument, with the FAM channel fluorescence signal collected every 1 min. After the reaction, images were taken under excitation light at a wavelength of 440-460 nm. The results showed... Figure 9 All three crRNAs produced fluorescent signals, indicating that they could effectively bind to the Casδ protein and its corresponding target, forming a stable ternary complex and successfully activating the trans-cleavage activity of Casδ. crRNA3 showed the strongest fluorescent signal and was therefore selected as the optimal crRNA for subsequent experiments. However, the fluorescence signal value was still not high enough for visual detection (ΔRn < 1.0 × 10⁻⁶). 6 There is still room for further optimization of the reaction system and conditions.

[0069] Orthogonal experiments were used to optimize the concentrations of Casδ protein and crRNA. Specifically, three different concentration gradients of Casδ protein (0.25, 0.5, and 1 μg / μL) were set up, and these were combined with the optimal crRNA3 (concentrations of 0.25, 0.5, and 1 μM, respectively). The results were detected in a CRISPR / Casδ trans-cleavage system. The mixture was incubated at 37°C for 30 min. Finally, the optimal concentration combination of Casδ protein and crRNA was determined by the intensity of the fluorescence signal. The results are as follows: Figure 10 As shown, when the Casδ protein concentration was 0.5 μg / μL and the crRNA concentration was 0.5 μM, the observed green fluorescence and fluorescence signal were stronger than those of other reaction groups, and the level of visualization detection was achieved.

[0070] Example 6: Effect of different single-stranded DNA probe concentrations The above reaction indicates that more Casδ protein is activated to produce trans-cleavage activity, enabling it to cleave more single-stranded DNA probes in a shorter time. However, the fluorescence signal intensity of the reaction solution is still not high at this time. After ruling out the influence of other conditions, it is speculated that this may be due to the insufficient concentration of single-stranded DNA probes in the reaction solution.

[0071] In real-time fluorescence RPA detection systems, the initial signal value and peak height are crucial for qualitative analysis. However, changes in the fluorescence signal value are controlled by the probe concentration, which also affects whether the fluorescence can be observed visually. Therefore, the concentration of the single-stranded DNA probe was increased, and five control groups were set up with single-stranded DNA probe concentrations of 0.2 μM, 0.4 μM, 0.8 μM, 1.0 μM, and 1.2 μM. The results were then analyzed. Figure 11 It was found that when the DNA probe concentration was 0.2 μM and 0.4 μM, the fluorescence signal value remained low and almost invisible to the naked eye. However, when the concentration was increased to 0.8 μM, the fluorescence signal of the reaction solution was significantly enhanced, with ΔRn reaching 1.0 × 10⁻⁶. 6 To meet the requirements of visual detection, the reaction solution can be irradiated with a portable small excitation light source with a wavelength of 440-460nm, which can produce green fluorescence visible to the naked eye under a 500nm filter, thus meeting the requirements of visual detection. When the wavelength is further increased to 1.0μM, both the reaction rate and the terminal fluorescence signal value are improved, but the reaction rate decreases when the wavelength is increased to 1.2μM.

[0072] Therefore, the optimal concentration of Casδ protein was determined to be 0.5 μg / μL, the optimal concentration of crRNA was 0.5 μM, and the optimal concentration of ssDNA probe was 1.0 μM.

[0073] Example 7: Effect of different reaction parameters on detection results Subsequently, the fluorescence signal values ​​of the CRISPR / Casδ detection system were analyzed at different time points. The results are shown in Figure 12, which show that the visual detection requirement can be met in 12 minutes.

[0074] like Figure 13 As shown, when detecting 1 μL of amplified product, without the addition of magnesium ions, the fluorescence signal rise rate was normal, and the fluorescence signal curve reached the visual detection level within 12 minutes. Adding MgSO4 to the CRISPR / Casδ reaction system beforehand, maintaining the magnesium ion concentration at 6 mM during detection, significantly accelerated the fluorescence signal rise rate, reaching the visual detection level within 11 minutes. Further increasing the magnesium ion concentration to 10 mM resulted in an even faster reaction rate, with the fluorescence signal curve reaching the visual detection level within 10 minutes. However, when the magnesium ion concentration was increased to 12 mM, the fluorescence signal terminal fluorescence value decreased, indicating a slight decrease in reaction rate. This is presumably due to excessive MgSO4 affecting the Casδ protein cleavage efficiency in the reaction system. In subsequent studies, the magnesium ion concentration in the CRISPR / Casδ reaction system was maintained at 10 mM, and the reaction time was further optimized to 10 minutes.

[0075] Example 8: Sensitivity Detection of ND207 Using flanking DNA sequences of ND207 at different concentration gradients (containing 2 × 10⁻⁶) 7 copies, 2×10 5 copies, 2×10 3 copies and 2×10 2The detection sensitivity of the CRISPR / Casδ system was evaluated using copies of DNA. Figure 14 As shown, for target DNA copy numbers of 2 × 10⁻⁶ 3 For samples with more than 2 copies, the fluorescence signal value of the reaction solution increases rapidly, reaching a visual detection level within approximately 10 minutes. The target DNA copy number is 2 × 10⁻⁶. 2 The ΔRn signal value of the copies' reaction could not reach the level of visual detection.

[0076] After reacting the CRISPR / Casδ reagent with the RPA amplification product for 10 min, the reaction solution was irradiated with a portable 440-460 nm wavelength excitation light source, and observed with the naked eye using 500 nm filter glasses. Positive samples showed a green fluorescent signal, while negative controls showed no fluorescent signal. The target DNA copy number was 2 × 10⁻⁶. 3 Samples with more than 2 × 10⁻⁶ copies showed detectable fluorescence signals within 10 minutes. 2 Although the fluorescence signal value increased during the reaction of copies, ΔRn failed to reach a level suitable for visual detection. These results confirm that the detection method based on the CRISPR / Casδ system has a sensitivity of 2 × 10⁻⁶. 3 The system contains copies of the Casδ protein, which exhibits trans-cleavage activity. Once activated, the Casδ protein continuously cleaves single-stranded DNA probes in solution, generating a strong fluorescent signal in a short time, thus demonstrating high detection sensitivity even when detecting low-concentration positive samples.

[0077] Example 9 Specificity detection of ND207 like Figure 15 As shown, after introducing specific sequences of other transgenic maize varieties into the reaction system, no fluorescence signals or visible products were observed, thus confirming that the method has good specificity.

[0078] Example 10 Selection of CC-2 crRNA sequence The optimized CRISPR / Casδ detection system, combined with three designed crRNAs, was used to perform fluorescence visualization detection of the RPA amplification products of transgenic maize CC-2. The crRNA (crRNA-CC-2) sequence and single-stranded DNA probe sequence in the reaction system are shown in Table 10.

[0079] Table 10. Casδ-gRNA sequences and single-stranded DNA probe sequences used for CC-2 detection.

[0080] Three crRNAs were added to a CRISPR / Casδ trans-cleavage system, and the fluorescence signal generated during cleavage was monitored in real time. A blank control group without the target was also included. The reaction was performed at 37℃ for 30 min in a real-time quantitative PCR instrument, with the fluorescence signal of the FAM channel collected every 1 min. After the reaction, images were taken under excitation light at a wavelength of 440-460 nm. The results showed... Figure 16 All three crRNAs produced fluorescent signals, indicating that they could effectively bind to the Casδ protein and its corresponding target, forming a stable ternary complex and successfully activating the trans-cleavage activity of Casδ. Among them, crRNA3 had the strongest fluorescent signal (sequence 1) and was therefore selected as the best crRNA for subsequent experiments.

[0081] Example 11 Sensitivity detection of the CC-2 detection system Using CC-2 flanking DNA sequences at different concentration gradients (containing 2 × 10⁻⁶) 7 copies, 2×10 5 copies, 2×10 3 copies and 2×10 2 The detection sensitivity of the CRISPR / Casδ system was evaluated using copies of DNA. Figure 17 As shown, the reaction solution was irradiated with a small 440-460 nm wavelength excitation light source, and observed with the naked eye using 500 nm filter glasses. For a target DNA copy number of 2 × 10⁻⁶, 3 For samples with more than 2 copies, the fluorescence signal value of the reaction solution increases rapidly, reaching a visual detection level within approximately 10 minutes. The appearance of a visible green fluorescence signal indicates that the cleavage reaction was complete, confirming that the sample is a transgenic maize variety containing the target transformant CC-2. The target DNA copy number is 2 × 10⁻⁶. 2 The ΔRn signal value of the copies' reaction could not reach the level of visual detection.

[0082] Example 12 Specificity detection of the CC-2 detection system DNA samples from transgenic varieties containing maize transformants CC-2, ND207, DBN9936, DBN3601T, Ruifeng 125, and Ruifeng 8 were tested in the reaction system, and the results are as follows: Figure 18 (as shown) Figure 18 The results show that, except for the transgenic maize variety containing the CC-2 transformant, no other transgenic varieties amplified the band, indicating that the primers have good specificity. This confirms the good specificity of the method, and the CC-2-CRISPR / Casδ detection method has been successfully established.

[0083] Under established optimal conditions, a two-step method for detecting transgenic transformants using RPA-CRISPR / Casδ was developed. The two-step nucleic acid detection method includes a pre-amplification step of the target nucleic acid and subsequent CRISPR / Casδ detection. The specific reaction procedure is as follows: DNA is rapidly extracted from maize leaves to be tested. 1 μL of DNA is added as a template to the RPA isothermal amplification reaction system. The system is then amplified at 37℃ for 20 min. 5 μL of the RPA amplification product is then added to the CRISPR / Casδ detection reaction solution, and the reaction is continued at 37℃ for approximately 10 min, thus completing the entire detection process for maize transformants ND207 and CC-2.

[0084] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A primer composition, characterized by, The primer combination includes a primer pair consisting of CC-F and CC-R, and the crRNA shown in sequence 1; The sequences of the primer pairs are shown in any one of A1)-A5): A1) The CC-F is as shown in Sequence 2, and the CC-R is as shown in Sequence 3; A2) The CC-F is as shown in Sequence 2, and the CC-R is as shown in Sequence 5; A3) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 3; A4) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 5; A5) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 7.

2. The use of the primer composition according to claim 1 in the detection of maize transformant CC-2.

3. A rapid detection method for the transgenic maize transformant CC-2, characterized in that, Includes the following steps: 1) Using the genome of the maize to be tested as a template, primers for maize transformant CC-2 were used to perform isothermal amplification based on multi-enzyme isothermal nucleic acid detection technology to obtain isothermal amplification products; 2) The isothermal amplification product from step 1) is detected by CRISPR / Casδ using crRNA targeting maize transformant CC-2. Based on the detection results, it is determined whether the maize to be tested is a maize material, combination, variety, etc. containing the target maize transformant.

4. The detection method according to claim 3, characterized in that, The isothermal amplification time in step 1) is 20 min.

5. The detection method according to claim 4, characterized in that, The reaction temperature in step 1) is 37°C.

6. The detection method according to claim 5, characterized in that, The primer concentration in step 1) is 1.0 μmol / L.

7. The detection method according to any one of claims 3-6, characterized in that, In step 2), the concentration of Casδ protein is 0.5 μg / μL, and the concentration of crRNA is 0.5 μM.

8. The detection method according to claim 5, characterized in that, In step 2), the concentration of the ssDNA probe is 1.0 μM and the magnesium ion concentration is 10 mM.

9. The detection method according to claim 6, characterized in that, The reaction temperature in step 2) is 37°C; the CRISPR / Casδ reaction time is 10 min.

10. The detection method according to claim 9, characterized in that, The primer combination includes a primer pair consisting of CC-F and CC-R, and crRNA as shown in sequence 1; The sequences of the primer pairs are shown in any one of A1)-A5): A1) The CC-F is as shown in Sequence 2, and the CC-R is as shown in Sequence 3; A2) The CC-F is as shown in Sequence 2, and the CC-R is as shown in Sequence 5; A3) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 3; A4) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 5; A5) The CC-F is as shown in Sequence 4, and the CC-R is as shown in Sequence 7.

Images