Multiplexed SNP detection method and kit based on rnase h2-dependent lamp and application

Abstract

The application discloses a rapid, single-tube, on-site multiplex SNP detection method (Comp-rhLAMP) based on RNase H2-dependent loop-mediated isothermal amplification (rhLAMP). The method realizes high specificity and multiplex SNP detection through a competitive one-step reaction (single tube, on-site) combined with an RNA-modified oligonucleotide probe. The technology amplifies target DNA under isothermal conditions, and simultaneously utilizes RNase H2 to specifically recognize and cut mutant DNA sequences, so that the detection can be completed within 30 minutes, the sensitivity reaches 1.69 copies / reaction, and the SNP recognition frequency is 0.5%. The method can be applied to rapid identification of herbicide-resistant genotypes in field samples, screening of gene-edited crops and typing of germplasm resources, and significantly improves the molecular detection efficiency in agricultural production due to the single-tube design, compatibility with portable equipment and visual result reading, thereby providing a reliable tool for agricultural management.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and more specifically, to a rapid detection method, kit, and application of multiple SNPs based on RNase H2-dependent LAMP. Background Technology

[0002] Single nucleotide polymorphisms (SNPs), as the most common genetic variations in the genome, play a crucial role in genetic characteristics, disease susceptibility, and herbicide resistance. In global agricultural production, herbicide resistance control is a vital aspect of modern agriculture, significantly impacting crop health and yield. However, herbicide resistance in weeds due to target gene mutations presents a severe challenge. Current weed control strategies include researching weed resistance mechanisms, developing novel herbicides, enhancing crop resistance, and breeding highly resistant crop varieties. However, distinguishing target resistance caused by single-base variations within a complex genomic context, especially rapid, multi-target resistance analysis and integrated field analysis, remains a significant technological bottleneck.

[0003] Current methods for verifying herbicide resistance mainly include phenotypic detection and molecular detection. Phenotypic detection, such as laboratory bioassays, assesses resistance by observing the plant's response to increasing herbicide dosages; however, this method is time-consuming and cumbersome. While molecular detection techniques improve specificity, these techniques rely on sophisticated instruments and skilled personnel, making them difficult to meet the needs of rapid field testing.

[0004] Isothermal amplification (IAT) technology offers advantages such as ease of operation, rapid detection speed, and high sensitivity, providing a new approach for rapid, on-site SNP detection. However, isothermal amplification technology lacks specificity for SNP identification, while weed resistance is often caused by mutations at multiple SNP sites. Existing isothermal amplification technologies are insufficient to meet the needs of identifying multiple SNP sites for weed resistance.

[0005] Based on this, this invention proposes for the first time a rapid, single-tube multiplex SNP detection technique applicable to the field: Comp-rhLAMP. This technique enables rapid analysis and detection of multiplex SNP sites, combining rapid nucleic acid release technology with portable detection equipment to construct a complete field detection platform, providing an efficient tool for scenarios such as agricultural resistance management and gene editing screening. This technology fills the performance gap in existing isothermal amplification techniques for SNP genotyping and has significant application prospects. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a rapid detection method, kit, and application for multiple SNPs based on RNase H2-dependent LAMP, for rapid, highly sensitive, and highly specific identification of weed resistance molecules.

[0007] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a rapid detection method for multiple SNPs based on RNase H2-dependent LAMP (loop-mediated isothermal amplification), comprising the following steps: S1. Design a LAMP primer set and an RNA-modified competitive fluorescent probe set for the target SNP site, wherein the competitive fluorescent probe set contains at least two probes with different fluorescent labels, which are used to identify different alleles of the same SNP site, and the probe contains one or more ribonucleotide modification sites, and the modification sites correspond to the SNP sites on the target. S2. Simultaneously add the LAMP primer set, the RNA-modified competitive fluorescent probe set, a DNA polymerase with strand displacement activity, an RNase H2 enzyme or its variant that has the function of recognizing and cleaving RNA-DNA hybrid chains, and the nucleic acid sample to be tested to a single reaction system to form a tube-type detection system. S3. Perform LAMP reaction (loop-mediated isothermal amplification reaction) under isothermal conditions, using the RNase H2 enzyme or its variant to specifically recognize and cleave the probe-target hybrid strand that matches the target sequence; S4. By detecting signals from different fluorescence channels or combinations thereof, wild-type, heterozygous mutant, homozygous mutant targets, as well as heterozygous targets carrying multiple different mutations, can be identified simultaneously.

[0008] As some specific embodiments of the present invention, in step S1, in the RNA-modified competitive fluorescent probe group, the 5' end of each probe is independently modified with at least one of FAM, ROX, VIC, and Cy5 fluorescent labels, and the 3' end is modified with a quenching group or has a blocking modification, wherein the quenching group is independently selected from at least one of BHQ1, BHQ2, and BHQ3 quenching labels.

[0009] As some specific embodiments of the present invention, in step S1, the length of the RNA-modified competitive probe is 15-30 nt. In step S3, the RNA-modified probe competitively hybridizes with wild-type and mutant alleles, respectively. The RNA-DNA portion of the probe serves as a specific cleavage site for RNase H2, and the modification site of the RNA on the probe corresponds to the mutation site on the target.

[0010] As some specific embodiments of the present invention, in step S2, the DNA polymerase with strand displacement activity is selected from one or more combinations of Bst, Vent, phi29, and Taq DNA polymerase. Bst 3.0 DNA polymerase is preferred.

[0011] As some specific embodiments of the present invention, in step S2, the endonuclease having the function of recognizing and cleaving RNA-DNA hybrid strands is selected from one of RNase H, RNase H2, or a variant thereof. RNase H2 is preferred. Utilizing the specific cleavage activity of RNase H2 on mismatched bases in RNA-DNA hybrid strands, fluorescence signal release is triggered only when the probe matches the target sequence, achieving single-base resolution.

[0012] As some specific embodiments of the present invention, in step S2, the nucleic acid sample to be tested includes one or more of single-stranded DNA, double-stranded DNA, or single-stranded RNA.

[0013] As some specific embodiments of the present invention, in step S1, the LAMP primer set includes forward and reverse external primers and forward and reverse internal primers; In the single-tube detection system of step S2, the concentrations of the forward and reverse external primers are 1.2-1.8 μM, and the concentrations of the forward and reverse internal primers are 0.1-0.3 μM.

[0014] As some specific embodiments of the present invention, in step S1, the LAMP primer set further includes loop primers, which include forward loop primers and / or reverse loop primers; the concentration of the forward loop primers or reverse loop primers in the tube detection system in step S2 is 0.3-0.5 µM.

[0015] As some specific embodiments of the present invention, in the one-tube detection system of step S2, the concentration of each probe in the RNA-modified competitive fluorescent probe group is 50-250 nM. And / or, the concentration of the DNA polymerase with strand displacement activity is 0.32-0.48 U / μL; And / or, the concentration of the endonuclease (RNase H2 or a variant thereof) having the function of recognizing and cleaving RNA-DNA hybrid strands is 0.02-0.05 U / μL; And / or, the single-tube detection system further includes 1-2 mM dNTPs and 8-10 mM Mg. 2+ .

[0016] As some specific embodiments of the present invention, in step S3, the reaction temperature of the LAMP reaction is 55-65℃ and the time is 20-40 min; preferably 57-63.1℃, more preferably 61℃, 40 min.

[0017] As some specific embodiments of the present invention, in step S4, the result is read by any of the following methods: (1) A real-time fluorescence detector was used to collect fluorescence signals in real time; (2) Fluorescence visualization reading under ultraviolet light; (3) The results were read using the lateral chromatographic test strip LFS.

[0018] Secondly, the present invention provides a rapid detection kit for multiple SNPs based on RNase H2-dependent LAMP, for implementing the detection method described in any of the above claims, the kit comprising: (a) A specific set of LAMP primers targeting multiple target SNP sites; (b) Multiple RNA-modified competitive probes with different fluorescent labels are used to identify different alleles at the same SNP site; (c) DNA polymerases with strand displacement activity; (d) Endonucleases that recognize and cut RNA-DNA hybrid chains.

[0019] As some specific embodiments of the present invention, the kit further includes dNTPs, reaction buffer, and Mg. 2+ .

[0020] Thirdly, the present invention provides the application of the detection method or kit as described in any of the above claims in the preparation of any of the following detection reagents: (1) Application in detection reagents for detecting herbicide resistance genotypes in plants; (2) Detection reagents for screening mutations in gene-edited crops; (3) Detection reagents for genotypic identification of horticultural germplasm resources.

[0021] As some specific embodiments of the present invention, in application (1), the plant herbicide resistance gene (target gene) includes the acetyl-CoA carboxylase gene (ACCase), and the detected SNP sites (resistance-related single nucleotide polymorphism sites) include at least two of the sites 1818, 1781, 1999, 2027, 2041, 2078, 2088, and 2096, or two or more mutations at the same site.

[0022] As some specific embodiments of the present invention, the plant is Diospyros kaki (Hypericum hainanense). Digitariaciliarisvar. chrysoblephara The detection reagent is used to identify the target resistance of barnyardgrass to acetyl-CoA carboxylase inhibitor herbicides. The SNP site to be detected is the 2027 site, and the genotypes of this site include wild-type TGG, first mutant TCG and second mutant TGT. The LAMP primer set designed for this SNP site contains primers with sequences as shown in SEQ ID NO. 1-6 or SEQ ID NO. 7-12; RNA modification competitive probes comprise probes with sequences as shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19, used to identify wild-type TGG, first mutant TCG, and second mutant TGT, respectively.

[0023] As some specific embodiments of the present invention, in application (1), the plant is selected from at least one of the following genera and species: Diospyros genus ( Digitariasp .), Barnyardgrass ( Echinochloa sp .), see wheatgrass genus ( Alopecurus sp .), genus *Gnaphalium* ( Beckmannia sp .), genus *Euphorbia* ( Leptochloa sp .), multiflora ryegrass ( Lolium multiflorum Lamk.), Sun-blooming grass ( Fimbristylis littoralis ).

[0024] As some specific embodiments of the present invention, in application (2), the gene-edited crop includes at least one of rice, corn, soybean, rapeseed, wheat, and potato.

[0025] As some specific embodiments of the present invention, the gene-edited crop is rice, and its target gene is the phytochrome B-like protein gene.

[0026] As some specific embodiments of the present invention, in application (3), the horticultural germplasm resources include at least one of loofah, cucumber, melon and Chinese cabbage.

[0027] As some specific embodiments of the present invention, the horticultural germplasm resource is loofah, and its target gene is a flowering regulatory gene.

[0028] The above-mentioned test kits and reagents are suitable for rapid field testing and can be completed within 40 minutes.

[0029] Compared with the prior art, the present invention has the following beneficial effects: 1. Through reaction system optimization, we have achieved ultra-high sensitivity (1.69 copies per reaction) and low mutation frequency multiple SNP detection limit (0.5%), which can meet the needs of rapid detection and identification of SNP genotyping for low concentration and low mutation frequency samples.

[0030] 2. This invention achieves high-precision target identification and multiple SNP analysis capabilities of the Comp-rhLAMP system through multiple fluorescent modifications (FAM, ROX, VIC) and the specific recognition of RNase H2 by RNA-modified probes. It provides an efficient analytical technique for multi-target SNP resistance identification and also improves the detection sensitivity and the ability to identify SNPs in samples with low mutation frequency.

[0031] 3. Through temperature and system compatibility optimization, the LAMP amplification reaction and RNase H2 recognition are integrated into the Comp-rhLAMP one-tube detection, realizing one-tube, rapid detection (40 minutes) and meeting the high-throughput sample analysis of 96 or 384-well plates, thereby greatly improving detection efficiency.

[0032] 4. By combining with rapid nucleic acid release technology, it eliminates the need for expensive instruments, simplifies the operation process, and reduces testing costs, enabling field analysis of multiple resistances in weeds.

[0033] 5. High-specificity, multiplex SNP detection is achieved by combining a competitive one-step reaction (single tube, field) with RNA-modified oligonucleotide probes. This method can be applied to the rapid identification of herbicide-resistant genotypes in field samples, screening of gene-edited crops, and genotyping of germplasm resources. Its single-tube design, compatibility with portable devices, and visualized result reading significantly improve the efficiency of molecular detection in agricultural production, providing a reliable tool for agricultural management. Attached Figure Description

[0034] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram of the workflow of the Comp-rhLAMP single-tube multiple SNP detection method of the present invention; Figure 2 This is a graph showing the amplification efficiency evaluation results of different LAMP primer sets in Example 2; Figure 3 This is a graph showing the detection results at different amplification reaction temperatures in Example 2; Figure 4 Different Mg in Example 2 2+ Graph showing the concentration detection results; Figure 5 This is a graph showing the results of simultaneous detection of wild-type and mutant ACCase gene at the 2027 site in Dioscorea opposita using Comp-rhLAMP in Example 2; Figure 6 This is a graph showing the sensitivity detection results of the wild-type reference plasmid at the 2027 site of the ACCase gene in Dioscorea opposita using Comp-rhLAMP in Example 2. Figure 7 This is a graph showing the detection limit of the frequency of mutations at the 2027 site of the ACCase gene in *Digitalis macrocephala* using Comp-rhLAMP in Example 2. Figure 8 This is a visualization of the results of Comp-rhLAMP analysis of rice phytochrome B-like protein gene editing samples (M1-M4) in Example 3. Figure 9 This is a visualization of the results of Comp-rhLAMP detection of germplasm resource mutations (GR-S1, GR-S2) of the flowering regulatory genes of loofah in Example 4. Detailed Implementation

[0035] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0036] Example 1: Comp-rhLAMP One-Tube Detection System and Method This embodiment provides a rapid detection method for multiple SNPs based on RNase H2-dependent LAMP, including the following steps: (1) Design a set of LAMP primers and a set of RNA-modified competitive fluorescent probes targeting the target SNP site, wherein the competitive fluorescent probe set contains at least two probes with different fluorescent labels, which are used to identify different alleles of the same SNP site.

[0037] The probes are 15-30 nt in length. Each probe has its 5' end independently modified with FAM, ROX, VIC, or Cy5 fluorescent labels, and its 3' end modified with a quencher group or a blocking modification. The quencher group is BHQ1, BHQ2, or BHQ3. The probe contains one or more ribonucleotide modification sites, corresponding to SNP sites. The RNA-modified probes competitively hybridize with wild-type and mutant alleles, respectively. The RNA-DNA portion of the probe serves as a specific cleavage site for RNase H2.

[0038] (2) In a single reaction system, LAMP primer set, RNA-modified competitive fluorescent probe set, DNA polymerase with strand displacement activity, endonuclease with the function of recognizing and cutting RNA-DNA hybrid chains, and nucleic acid sample to be tested are added simultaneously to form a single tube detection system.

[0039] The DNA polymerase is Bst, Vent, phi29, or Taq DNA polymerase, and the restriction enzyme is RNase H or RNase H2. The resulting one-tube detection system includes: 0.32-0.48 U / μL DNA polymerase, 0.02-0.05 U / μL RNase H2, 1-2 mM dNTPs, 250 nM fluorescent probe, 1.2-1.8 μM FIP / BIP primers, 0.1-0.3 µM F3 / B3 primers, and 8-10 mM MgSO4. The one-tube detection system may also include 0.3-0.4 µM LF / LB primers, as well as 20 mM Tris-HCl, 10 mM (NH4)2SO4, and 10 mM KCl.

[0040] (3) The LAMP reaction (loop-mediated isothermal amplification reaction) is carried out under isothermal conditions, and the probe-target hybrid strand that is completely matched with the target sequence is specifically recognized and cut by endonuclease.

[0041] (4) Read the results and simultaneously identify wild-type, heterozygous mutant, homozygous mutant targets as well as heterozygous targets carrying multiple different mutations.

[0042] like Figure 1 The diagram shows the workflow of the Comp-rhLAMP one-tube detection method of the present invention. Based on RNase H2-dependent loop-mediated isothermal amplification (rhLAMP), this method is a rapid, single-tube, on-site multiplex SNP detection method. The detection process includes (1) rapid nucleic acid extraction; and (2) Comp-rhLAMP detection and analysis. The entire process takes as little as 30 minutes, with a detection sensitivity of up to 1.69 copies / reaction and an SNP recognition frequency of 0.5%, making it suitable for one-tube analysis of various SNPs.

[0043] Example 2: Detection of multiple SNP mutations in herbicide resistance genes in Digitaria zebrina leaves using a single-tube detection system. Using field-collected leaves of Dioscorea opposita as samples, a single nucleotide multiple SNP mutation (TGG→TCG / TGT) at the 2027 site of the ACCase gene (SEQ ID NO.20) was detected. This mutation is associated with herbicide resistance.

[0044] The sources of plant samples are shown in Table 1 below: Table 1. Sources of Plant Samples

[0045] The specific steps are as follows: 1. Nucleic acid extraction Referring to Example 1 in patent application CN202411333889, nucleic acids were rapidly extracted from plant leaves. 10 μL of the obtained lysis release solution was diluted 10 times with nuclease-free water and used directly as a template for the Comp-rhLAMP reaction.

[0046] 2. Design primers and probes Three sets of LAMP primers (P1, P2, P3, each containing F3 / B3, FIP / BIP, and possibly LF / LB) targeting the ACCase gene 2027 site were designed using PrimerExplorer V5. Competitive fluorescent probes were designed: a FAM-labeled wild-type probe, a ROX-labeled mutant type 1 (W2027S) probe, and a VIC-labeled mutant type 2 (W2027C) probe. The fluorescent labels were located at the 5' end of all probes, and the 3' end contained quenching groups. The fluorescent probes were modified with RNA bases to adapt for RNase H2 cleavage.

[0047] The primers and probes are shown in Table 2 below: Table 2 Primer and Probe Table

[0048] 3. Comp-rhLAMP single-tube detection Set up a single-tube detection system containing Bst 3.0 DNA polymerase, RNase H2, dNTPs, fluorescent probes, FIP / BIP primers, F3 / B3 primers, and MgSO4. LF / LB primers may also be included, along with Tris-HCl, (NH4)2SO4, and KCl. Place the reaction tube in a portable isothermal amplification instrument and incubate the reaction system at 57.0–65.0°C.

[0049] Optimize the single-tube testing system: (1) Using wild-type samples of Digitaria pilosa as the detection samples, three sets of LAMP primers were used for the experiment. The real-time fluorescence detection results are as follows: Figure 2 As shown. By Figure 2 As can be seen, the P1 primer set has a higher CT value and the highest amplification efficiency for the target. Therefore, the P1 primer set was used for subsequent testing and analysis.

[0050] (2) Temperature optimization experiments were conducted within the range of 57.0-65.0°C. The P1 primer set was used as the LAMP primer set for one-step Comp-rhLAMP detection. The real-time fluorescence detection results are as follows: Figure 3As shown, the results indicate that the amplification and detection system of the present invention can achieve stable amplification effects between 57.0°C and 63.1°C. To ensure optimal specificity, 61.0°C was ultimately selected as the temperature condition for subsequent experiments.

[0051] (3) The concentrations of some components in the one-tube detection system were optimized, with Mg 2+ For example, using wild-type Digitaria pilosa as the test sample, a single-tube detection was performed. The final concentrations of MgSO4 in the reaction system were adjusted to 0 mM, 8 mM, 10 mM, 12 mM, 14 mM, and 16 mM. The real-time fluorescence detection results are as follows: Figure 4 As shown, when Mg 2+ The amplification efficiency is optimal at a concentration of 10 mM, and good amplification efficiency is observed in the range of 8-10 mM.

[0052] The optimal single-tube reaction system was obtained after optimization, with a total volume of 25 μL, containing the following components: 1× reaction buffer final concentration: 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 10 mM MgSO4; 2 mM dNTPs, 0.32 U / μL Bst 3.0 DNA polymerase (purchased from New England Biolabs, USA), 1 U RNase H2; 250 nM of each probe (50 nM probe concentration was used for strip analysis), 1 μL DNA template (20 ng / μL, final concentration 0.8 ng / μL), and 1.6 μM FIP_P1 / BIP_P1 primers, 0.2 µM F3_P1 / B3_P1 primers, and 0.4 µM LF_P1 / LB_P1 primers. The reaction tube was placed in a portable isothermal amplification instrument and incubated at 61°C for 40 minutes.

[0053] 4. Result Interpretation Competitive fluorescent probes bind competitively to their respective targets during the LAMP reaction. When wild-type LAMP products hybridize with wild-type-FAM probes, RNase H2 enzymes specifically recognize and cleave the RNA-DNA mismatch sequence formed at the mutation site. Similarly, mutant LAMP products will form hybrids with corresponding ROX or VIC labeled probes, thereby achieving a precise RNase H2-mediated cleavage reaction and generating a fluorescent signal.

[0054] Comp-rhLAMP technology classifies the detection results into three types: (1) homozygous wild-type samples generate FAM signals through WT probe cleavage; (2) homozygous mutant samples (W2027S or W2027C) generate ROX or VIC signals respectively; (3) heterozygous samples show dual fluorescence signals—FAM and ROX or FAM and VIC signals coexist, indicating the presence of both alleles. Figure 5 As shown, only one set of LAMP primers and the corresponding RNase H2 probe are needed to simultaneously detect two herbicide resistance mutations (W2027S and W2027C) at the W2027 site.

[0055] The following methods can be used to read the results: (1) The fluorescence signals of FAM (wild-type GG), ROX (mutant CC), and VIC (mutant TT) were collected in real time using a real-time fluorescence detector (CFXOpus96); (2) After amplification, the products were observed under a UV lamp: wild-type samples showed green fluorescence (FAM signal), mutant CC showed red fluorescence (ROX signal), and mutant TT showed yellow fluorescence (VIC signal). (3) The results were read using the lateral chromatographic test strip LFS.

[0056] 5. Sensitivity and limit of detection of mutation frequency in Comp-rhLAMP technology (1) Sensitivity of Comp-rhLAMP technology Wild-type (WT) and mutant (CC) reference plasmids were constructed as follows: The pMD18-T empty vector plasmid produced by Takara was used. The plasmid was ampicillin resistant. Wild-type and mutant ACCase fragments (fragment length 1061 bp) were inserted into the HindIII restriction site, respectively. The constructed wild-type (WT) and mutant (CC) reference plasmids were named PMD18-WT and PMD18-Mut1, respectively.

[0057] The constructed reference plasmid was transformed into Dh5. α Plasmids were extracted and quantified from competent cells after shake-flask culture. The quantified plasmids were serially diluted to: 16,900, 1,690, 169, 16.9, 1.69, and 0.169 copies / µL (labeled C1-C6, respectively).

[0058] like Figure 6The image shows the real-time fluorescence detection results of the wild-type reference plasmid using the Comp-rhLAMP method. C1-C6 represent 16900, 1690, 169, 16.9, 1.69, and 0.169 copies per reaction, respectively. The results indicate that in the wild-type detection, fluorescence signals were observed at all dilutions except for 0.169 copies / μL and the negative control. Furthermore, the fluorescence intensity decreased proportionally with plasmid concentration, and the sensitivity of Comp-rhLAMP reached 1.69 copies / reaction. Therefore, the limit of detection (LOD) was determined to be 1.69 copies per reaction.

[0059] (2) Limit of detection for mutation frequency To determine the limit of detection (LOD) for mutation frequency in heterozygous samples, test samples were prepared by mixing mutant and wild-type plasmid DNA, with mutant / wild-type ratios (Mut / WT) ranging from 0%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 50%, 75%, to 100%. Figure 7 As shown, the comparison of fluorescence intensity between the mixed mutant and wild-type plasmids at 30 minutes revealed a significant difference between the two at a mutant concentration of 0.1% and the control, while a mutant concentration of 0.5% was not significantly different, indicating that the detection limit of Comp-rhLAMP is 0.5%. Therefore, Comp-rhLAMP can distinguish mutant allele frequencies of 0.5% (i.e., mutant / wild-type ratio ≥ 1:200 can still detect mutant signals).

[0060] Example 3: Application of Comp-rhLAMP single-tube detection in the detection of gene-edited rice leaves. This embodiment uses Comp-rhLAMP to analyze mutations in gene-edited samples. Plant samples include wild-type (WT) and gene-edited rice leaves (T1 generation). Specific gene-edited sample information is shown in Table 3 below.

[0061] Table 3. Gene Editing Sample Information

[0062] The specific steps are as follows: 1. Rapid nucleic acid extraction was performed on the above plant samples using the same method as in Example 2.

[0063] 2. Using two CRISPR / Cas9 editing target mutation sites in the rice phytochrome B protein (NCBI, LOC4332623) gene (target sites 1 and 2 in SEQ ID NO.39 of Table 4 below, i.e., T1 and T2) as gene editing targets, two sets of LAMP primers were designed using PrimerExplorer V5, as shown in SEQ ID NO.21-26 and SEQ ID NO.27-32, respectively.

[0064] The probes include: a FAM-labeled wild-type probe for gene editing target 1 (SEQ ID NO. 33), a ROX-labeled mutant probe for gene editing target 1 (SEQ ID NO. 34), a ROX-labeled mutant probe for gene editing target 1 (SEQ ID NO. 35), and a ROX-labeled mutant probe for gene editing target 1 (SEQ ID NO. 36). A FAM-labeled wild-type probe for gene editing target 2 (SEQ ID NO. 37) and a ROX-labeled mutant probe for gene editing target 2 (SEQ ID NO. 38) are also included. All probes are modified with RNA. See Table 4 below for details. Table 4. Primer and probe sequences for SNP analysis of rice gene-edited samples

[0065] 3. Four rice gene-edited mutation samples (M1: target 1 mutation 1 and mutation 2, SNP1 / SNP2; M3: target 1 mutation 1, SNP1; M2: target 1 mutation 2, SNP2; M4: target 2 mutation, insA) were detected using the Comp-rhLAMP one-tube detection method described in Example 2 (using the optimal Mg... 2+ (Concentration and reaction temperature), and performed fluorescence visualization analysis, the results are as follows: Figure 8 As shown.

[0066] Figure 8 In the analysis of gene-edited samples, WT (wild type) appeared green, N (blank control) appeared transparent, and all four gene-editing types M1-M4 appeared red. The results showed that Comp-rhLAMP technology can specifically distinguish the four mutation types and accurately identify the gene-editing variants (M1, M2, M3, M4) at four different sites on two editing sites.

[0067] Example 4: Application of Comp-rhLAMP single-tube detection in the detection of germplasm resources of horticultural crops (loofah) In this embodiment, Comp-rhLAMP was used to analyze the germplasm resources of *Luffa cylindrica*. The sources of the plant samples are shown in Table 5 below. Table 5 and germplasm resource sample information

[0068] GR-S1 and GR-S2 are two samples with the same mutant type.

[0069] The specific steps are as follows: 1. Rapid nucleic acid extraction was performed on the above-mentioned loofah germplasm resource samples using the same method as in Example 2.

[0070] 2. Using the flowering regulatory gene of *Luffa cylindrica* (sequence shown in SEQ ID NO. 47) as the target gene, a LAMP primer set was designed using PrimerExplorer V5, as shown in SEQ ID NO. 40 to SEQ ID NO. 44. The FAM-labeled probe for germplasm resource type 1 (wild type) is SEQ ID NO. 45, and the ROX-labeled probe for germplasm resource type 2 (mutant) is SEQ ID NO. 46. The specific primer and probe sequences are shown in Table 6 below: Table 6. Primer and probe sequences for SNP analysis of loofah germplasm resource samples

[0071] 3. The Comp-rhLAMP one-step detection method was used to detect loofah germplasm resource samples. For loofah ( Luffa aegyptiaca Mill Germplasm resources of different genotypes: two samples (GR-S1 and GR-S2) of wild type (GR-WT) and mutant type were tested according to the one-tube detection method in Example 2 (using the optimal Mg... 2+ The concentration and reaction temperature were analyzed, and the results are as follows: Figure 9 As shown.

[0072] Figure 9 In the analysis of germplasm resource samples, WT and negative control (N) were green and transparent, respectively, while the two mutant samples GR-S1 and GR-S2 were red, indicating that Comp-rhLAMP can also quickly identify SNP differences in germplasm resources.

[0073] In summary, Comp-rhLAMP is applicable to rapid analysis of crop resistance, identification of gene-edited crops, and identification of germplasm resources, providing new technical methods and applications for rapid analysis and detection in agricultural fields.

[0074] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A rapid detection method for multiple SNPs based on RNase H2-dependent LAMP, characterized in that, Includes the following steps: S1. Design a LAMP primer set and an RNA-modified competitive fluorescent probe set for the target SNP site, wherein the competitive fluorescent probe set contains at least two probes with different fluorescent labels, which are used to identify different alleles of the same SNP site, and the probe contains one or more ribonucleotide modification sites, and the modification sites correspond to the SNP sites on the target. S2. Simultaneously add the LAMP primer set, the RNA-modified competitive fluorescent probe set, a DNA polymerase with strand displacement activity, an RNase H2 enzyme or its variant that has the function of recognizing and cleaving RNA-DNA hybrid chains, and the nucleic acid sample to be tested to a single reaction system to form a tube-type detection system. S3. Perform the LAMP reaction under isothermal conditions, utilizing the RNase H2 enzyme or its variant to specifically recognize and cleave the probe-target hybrid strand that matches the target sequence; S4. By detecting signals from different fluorescence channels or combinations thereof, wild-type, heterozygous mutant, homozygous mutant targets, as well as heterozygous targets carrying multiple different mutations, can be identified simultaneously.

2. The multiple SNP detection method according to claim 1, characterized in that, In step S1, in the RNA-modified competitive fluorescent probe set, each probe has its 5' end independently modified with at least one of the fluorescent labels FAM, ROX, VIC, and Cy5, and its 3' end modified with a quenching group or a blocking modification. The quenching group is independently selected from at least one of the quenching labels BHQ1, BHQ2, and BHQ3. And / or, the length of the RNA-modified competitive probe is 15-30 nt.

3. The multiple SNP detection method according to claim 1, characterized in that, In step S2, the DNA polymerase with strand displacement activity is selected from one or more combinations of Bst, Vent, phi29, and Taq DNA polymerase. And / or, the nucleic acid sample to be tested includes one or more of single-stranded DNA, double-stranded DNA, and single-stranded RNA.

4. The multiple SNP detection method according to claim 1, characterized in that, In step S1, the LAMP primer set includes forward and reverse external primers and forward and reverse internal primers; in the one-tube detection system of step S2, the concentration of the forward and reverse external primers is 1.2-1.8 μM, and the concentration of the forward and reverse internal primers is 0.1-0.3 μM. And / or, in the one-tube detection system of step S2, the concentration of each probe in the RNA-modified competitive fluorescent probe group is 50-250 nM; And / or, in the one-tube detection system of step S2, the concentration of the DNA polymerase with strand displacement activity is 0.32-0.48 U / μL; And / or, in the one-tube detection system of step S2, the concentration of the RNase H2 enzyme or its variant is 0.02-0.05 U / μL; And / or, in the single-tube detection system of step S2, the single-tube detection system further includes 1-2 mM dNTPs and 8-10 mM Mg. 2+ .

5. The multiple SNP detection method according to claim 1, characterized in that, In step S3, the LAMP reaction is carried out at a temperature of 55-65°C for 20-40 minutes.

6. A rapid detection kit for multiple SNPs based on RNase H2-dependent LAMP, used to implement the detection method as described in any one of claims 1-5, characterized in that, The kit includes: (a) A specific set of LAMP primers targeting multiple target SNP sites; (b) Multiple RNA-modified competitive probes with different fluorescent labels are used to identify different alleles at the same SNP site; (c) DNA polymerases with strand displacement activity; (d) RNase H2 enzymes or variants thereof that have the function of recognizing and cleaving RNA-DNA hybrid chains.

7. The use of a detection method as described in any one of claims 1-5 or a kit as described in claim 6 in the preparation of any one of the following detection reagents: (1) Detection reagents for detecting herbicide resistance genotypes in plants; (2) Detection reagents for screening mutations in gene-edited crops; (3) Detection reagents for genotypic identification of horticultural germplasm resources.

8. The application according to claim 7, characterized in that, In application (1), the plant herbicide resistance gene includes the acetyl-CoA carboxylase gene, and the detected SNP sites include at least two of the sites 1818, 1781, 1999, 2027, 2041, 2078, 2088, and 2096, or two or more mutations at the same site.

9. The application according to claim 8, characterized in that, The plant is *Digitaria sanguinalis*, and the detection reagent is used to identify the target resistance of *Digitaria sanguinalis* to acetyl-CoA carboxylase inhibitor herbicides. The SNP site to be detected is the 2027 site, and the genotypes of this site include wild-type TGG, first mutant TCG, and second mutant TGT. The LAMP primer set designed for this SNP site contains primers with sequences as shown in SEQ ID NO. 1-6 or SEQ ID NO. 7-12; RNA modification competitive probes comprise probes with sequences as shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19, used to identify wild-type TGG, first mutant TCG, and second mutant TGT, respectively.

10. The application according to claim 7, characterized in that, In application (1), the plant is selected from at least one of the following genera and species: Digitaria (… Digitaria sp. ), barnyardgrass ( Echinochloa sp. ), see wheatgrass genus ( Alopecurus sp. ), genus *Amaranthus* ( Beckmannia sp. ), genus *Euphorbia* ( Leptochloa sp. ), multiflora ryegrass ( Lolium multiflorum Lamk. ), Sunlight swaying grass ( Fimbristylis littoralis ); And / or, in application (2), the gene-edited crop includes at least one of rice, corn, soybean, rapeseed, wheat, and potato; And / or, in application (3), the horticultural germplasm resources include at least one of loofah, cucumber, melon, and cabbage.

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