A primer composition, gDNA, probe and method for detecting amino acid mutation sites in amaranthus retroflexus als

Abstract

This invention belongs to the field of molecular biology detection technology, and provides a primer composition, gDNA, probe, and method for detecting amino acid mutation sites in ALS of Amaranthus retroflexus. This invention is the first to construct a method for detecting multiple resistance mutations at multiple sites based on the synergistic effect of multiplex PCR and PfAgo. Through multiplex PCR amplification and PfAgo specific recognition and cleavage, it achieves simultaneous detection and mutation type identification of multiple resistance target sites in the ALS gene of Amaranthus retroflexus. Based on the multiple constraint mechanism of multiplex PCR amplification, PfAgo guided sequence recognition, and fluorescent probe cleavage, it achieves stepwise identification and verification of target sites, exhibiting excellent specificity, sensitivity, and robustness, reducing the risk of false positives, ensuring accurate and reliable results, and requiring no standards for interpretation. It is suitable for rapid and simultaneous multi-site detection of resistance to ALS inhibitors in Amaranthus retroflexus in the field, is simple to operate, and has low cost, providing technical support for resistance monitoring and precision herbicide management.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and in particular to a primer composition, gDNA, probe and method for detecting amino acid mutation sites in ALS of Amaranthus retroflexus. Background Technology

[0002] Acetolactate synthase (ALS) inhibitor herbicides are among the most widely used and effective herbicides for weed management in crops such as soybeans and corn. However, due to the long-term monopolistic use of these herbicides, many noxious weeds, including Amaranthus retroflexus, have gradually evolved resistance, leading to decreased control effectiveness and posing a serious threat to crop production and food security. The resistance evolution of Amaranthus retroflexus is particularly prominent; mutations in amino acids such as Asp-376-Glu (D376E), Trp-574-Leu (W574L), and Ser-653-Tyr (S653Y) in its ALS gene can induce high levels of resistance to ALS inhibitor herbicides.

[0003] Currently, the main detection technologies used to address herbicide resistance caused by mutations at target sites in weeds include Sanger sequencing, CAPS / dCAPS, and loop-mediated isothermal amplification (LAMP). While Sanger sequencing is the gold standard for mutation detection, it has limitations such as long detection cycles, high costs, and strong dependence on professional personnel and equipment, making it difficult to meet the timeliness requirements of large-scale field resistance monitoring. CAPS / dCAPS technology relies on restriction endonuclease digestion and gel electrophoresis, resulting in a cumbersome operation process and difficulty in parallel detection of multiple sites. LAMP, while offering the advantage of isothermal amplification, suffers from complex system design and challenges in multiple-site amplification. These methods generally suffer from low throughput; detecting multiple resistance sites requires independent operation, leading to low efficiency, high costs, and difficulty in simultaneously and rapidly interpreting multiple resistance mutation sites, thus failing to meet the practical needs of real-time assessment of weed resistance status under field conditions.

[0004] PfAgo endonucleases have attracted attention in the field of molecular diagnostics due to their programmed recognition and single nucleotide resolution capabilities, and have been successfully applied to pathogen detection and single nucleotide polymorphism typing. However, existing PfAgo detection methods are rarely used in the field of weed resistance detection, and a detection system targeting resistance mutation sites in the acetolactate synthase gene has not yet been established. Therefore, developing a rapid detection technology for multi-site mutations in resistant weeds that organically combines multiplex PCR amplification with PfAgo cleavage reaction, enabling simultaneous single-tube detection of multiple key resistance sites in the ALS gene of Amaranthus retroflexus, is of great significance for comprehensively monitoring the occurrence and spread of resistant populations and scientifically formulating herbicide application strategies. Summary of the Invention

[0005] To address the problems of low throughput, difficulty in simultaneously distinguishing multiple resistance mutation sites, and unsuitability for rapid on-site detection in existing weed resistance detection methods, this invention provides a primer composition, gDNA, probe, and method for detecting amino acid mutation sites in Amaranthus retroflexus (ALS).

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention designs corresponding PCR amplification primers for multiple preset mutation sites in the ALS gene of Amaranthus retroflexus. By adjusting the amplification fragment length and annealing temperature of each primer, it makes them suitable for simultaneous amplification in a multiplex PCR system. At the same time, by setting the primer positions reasonably, the target mutant base is located in a specific region of the amplification product, which is conducive to the subsequent mutation recognition reaction.

[0007] Therefore, in a first aspect, the present invention provides a primer composition for detecting amino acid mutation sites in Amaranth rotundifolia ALS, wherein the mutation sites include: D376E, W574L, and S653Y; the primer composition includes primer pairs 1 to 3; wherein primer pair 1 includes primers with nucleotide sequences as shown in SEQ ID NO. 1 and 2; primer pair 2 includes primers with nucleotide sequences as shown in SEQ ID NO. 3 and 4; and primer pair 3 includes primers with nucleotide sequences as shown in SEQ ID NO. 5 and 6.

[0008] In this invention, D376E refers to Asp-376-Glu, that is, the amino acid at position 376 of Amaranthus retroflexus ALS is mutated from aspartic acid to glutamic acid.

[0009] In this invention, W574L refers to Trp-574-Leu, that is, the amino acid at position 574 of Amaranthus retroflexus ALS is mutated from tryptophan to leucine.

[0010] In this invention, S653Y refers to Ser-653-Tyr, that is, the amino acid at position 653 of Amaranthus retroflexus ALS is mutated from serine to tyrosine.

[0011] In primer pairs 1 to 3, the amino acid mutation site detected by primer pair 1 is D376E, the amino acid mutation site detected by primer pair 2 is W574L, and the amino acid mutation site detected by primer pair 3 is S653Y.

[0012] Based on the sequence of PCR amplification products, this invention designs specific gDNA corresponding to each mutation site and sets the target mutation base in the seed region of the gDNA to enhance the recognition specificity of mutant and wild-type alleles; on this basis, the gDNA is used to mediate PfAgo to specifically cleave the PCR amplification products to generate secondary gDNA that can be used for signal amplification.

[0013] Therefore, in a second aspect, the present invention provides a gDNA composition for detecting amino acid mutation sites in Amaranth retroflexus ALS, wherein the mutation sites include: D376E, W574L, and S653Y; the gDNA composition includes gDNA1 to gDNA3; wherein gDNA1 includes a nucleotide sequence as shown in SEQ ID NO.7; gDNA2 includes a nucleotide sequence as shown in SEQ ID NO.9; and gDNA3 includes a nucleotide sequence as shown in SEQ ID NO.11.

[0014] Among gDNA1 to gDNA3, the amino acid mutation site detected in gDNA1 is D376E, the amino acid mutation site detected in gDNA2 is W574L, and the amino acid mutation site detected in gDNA3 is S653Y.

[0015] According to the present invention, a gDNA composition for detecting amino acid mutation sites in ALS of Amaranthus retroflexus is provided, preferably, the 5' ends of the gDNA1 to gDNA3 are phosphorylated.

[0016] More preferably, the 5' end of the gDNA1 to gDNA3 is provided with a phosphate group.

[0017] The present invention also designed a fluorescent probe that matches the secondary gDNA, which triggers the output of a fluorescent signal through a PfAgo-mediated cleavage reaction, thereby realizing the simultaneous detection and interpretation of multiple mutations in the ALS gene of Amaranthus retroflexus.

[0018] Therefore, in a third aspect, the present invention provides a probe composition for detecting amino acid mutation sites in Amaranth rotundifolia ALS, wherein the mutation sites include: D376E, W574L and S653Y; the probe composition includes the following probes 1 to 3; wherein, probe 1 includes a nucleotide sequence as shown in SEQ ID NO. 8; probe 2 includes a nucleotide sequence as shown in SEQ ID NO. 10; probe 3 includes a nucleotide sequence as shown in SEQ ID NO. 12; probes 1 to 3 are provided with different fluorescent groups.

[0019] In probes 1 to 3, the amino acid mutation site detected by probe 1 is D376E, the amino acid mutation site detected by probe 2 is W574L, and the amino acid mutation site detected by probe 3 is S653Y.

[0020] According to the present invention, a probe composition for detecting amino acid mutation sites in ALS of Amaranthus retroflexus is provided, preferably wherein the fluorescent group is disposed at the 5' end of probe 1 to probe 3.

[0021] According to the present invention, a probe composition for detecting amino acid mutation sites in ALS of Amaranthus retroflexus is provided, wherein preferably, the fluorescent group is selected from SX670, ROX, FAM, HEX and CY5.

[0022] More preferably, the 5' end of the probe 1 is provided with SX670; the 5' end of the probe 2 is provided with ROX; and the 5' end of the probe 3 is provided with FAM.

[0023] According to the present invention, a probe composition for detecting amino acid mutation sites in ALS of Amaranthus retroflexus is provided, preferably, probe 1 to probe 3 are provided with different quenching groups.

[0024] More preferably, the quenching group is disposed at the 3' end of probe 1 to probe 3.

[0025] More preferably, the quenching group is selected from BHQ1, BHQ2 and BHQ3.

[0026] More preferably, the 3' end of the probe 1 is provided with BHQ1; the 3' end of the probe 2 is provided with BHQ2; and the 3' end of the probe 3 is provided with BHQ3.

[0027] Fourthly, the present invention provides a kit comprising: the primer composition, the gDNA composition, the probe composition, and PfAgo endonuclease.

[0028] The kit is a multiplex PCR-PfAgo kit for rapid detection of multiple key mutation sites in the ALS gene of Amaranthus retroflexus. This kit is suitable for multisite resistance detection under laboratory and field conditions and can efficiently and conveniently complete resistance interpretation.

[0029] According to a kit provided by the present invention, preferably, the kit further includes PCR reaction reagents.

[0030] More preferably, the PCR reaction reagents include any one or more of PerfectStart Taq Buffer, dNTPs, and PerfectStart Taq DNA Polymerase.

[0031] According to a kit provided by the present invention, preferably, the kit further includes PfAgo reaction reagent.

[0032] More preferably, the PfAgo reaction reagent includes Reaction Buffer and / or Mn. 2+ .

[0033] According to a kit provided by the present invention, preferably, the kit further includes nuclease-free water.

[0034] In some specific embodiments, the kit includes a multiplex PCR premix and a PfAgo premix; the multiplex PCR premix includes PerfectStart Taq Buffer, dNTPs, PerfectStart Taq DNA Polymerase, primers with nucleotide sequences as shown in SEQ ID NO. 1–6, and nuclease-free water; the PfAgo premix includes PfAgo endonuclease, Reaction Buffer, and Mn 2+ , gDNA1-3, probe1-3 and nuclease-free water.

[0035] Multiplex PCR premix and PfAgo premix are packaged separately. A nested tube design can be used to physically separate the PfAgo premix from the multiplex PCR premix, which helps ensure reaction stability and reliable fluorescence signal output during subsequent use, reducing the risk of cross-contamination.

[0036] During the use of the kit, the DNA of the target amaranth is added to the multiplex PCR premix, and multiplex PCR amplification reaction is performed under preset reaction conditions to obtain multiplex PCR amplification products containing multiple target sites; then, the above multiplex PCR amplification products are added to the PfAgo premix, and a template-free negative control is set up, and PfAgo cleavage reaction is performed under preset conditions.

[0037] The application of any one or more of the primer composition, the gDNA composition, the probe composition, and the kit in detecting amino acid mutation sites in ALS of Amaranthus retroflexus should also be within the protection scope of this invention.

[0038] Fifthly, the present invention provides the use of any one or more of the primer composition, the gDNA composition, the probe composition, and the kit in detecting amino acid mutation sites in Amaranthus retroflexus ALS and / or identifying resistance of Amaranthus retroflexus to ALS inhibitor herbicides.

[0039] This invention establishes a multi-site mutation detection method based on multiplex PCR-PfAgo technology. By combining specific multiplex PCR amplification with PfAgo endonuclease recognition, it achieves simultaneous detection and fluorescence signal interpretation of multiple target sites and mutations. This method can be extended to the simultaneous detection of multiple targets, enabling the simultaneous detection of multiple target sites closely related to resistance formation in *Amaranthus retroflexus* populations with unknown resistance phenotypes. This allows for rapid differentiation of resistance mutations and determination of specific mutation types. Through this method, the herbicide resistance distribution characteristics of *Amaranthus retroflexus* populations in different regions can be systematically obtained, providing technical support for weed resistance monitoring and scientific control. In addition to its application in *Amaranthus retroflexus* resistance detection, this method can also be extended to multi-site target analysis in other molecular biology fields.

[0040] Therefore, in a sixth aspect, the present invention provides a method for simultaneously detecting amino acid mutation sites in Amaranthus retroflexus ALS, wherein the mutation sites include: D376E, W574L, and S653Y; the method includes: using DNA from the sample to be tested as a template, performing multiplex PCR amplification with the primer composition; performing PfAgo cleavage reaction on the obtained multiplex PCR amplification product with the gDNA composition, the probe composition, and PfAgo endonuclease; detecting the fluorescence signal, and determining whether there is a mutation at the amino acid mutation site in the sample to be tested based on the obtained fluorescence signal.

[0041] Those skilled in the art should understand that conventional DNA extraction techniques, such as DNA extraction kits or automated total nucleic acid extraction, can obtain DNA from the sample to be tested.

[0042] According to the present invention, a method for synchronously detecting amino acid mutation sites in ALS of Amaranthus retroflexus is preferably provided, using genomic DNA of the sample to be tested as a template.

[0043] According to the present invention, a method for simultaneous detection of amino acid mutation sites in ALS of Amaranthus retroflexus is preferably provided in which the PfAgo cleavage reaction system comprises: gDNA at a working concentration of 1.5µM to 2.5µM and probes at a working concentration of 500 nM to 2500 nM; wherein the gDNA is the gDNA described in the gDNA composition and the probes are the probes described in the probe composition.

[0044] More preferably, the PfAgo cleavage reaction system comprises gDNA at a working concentration of 1.8 µM to 2.2 µM.

[0045] More preferably, the PfAgo cleavage reaction system comprises gDNA at working concentrations of 1.8 µM, 1.9 µM, 2.0 µM, 2.1 µM, or 2.2 µM.

[0046] More preferably, the PfAgo cleavage reaction system comprises: gDNA1 at a working concentration of 2.2 µM, gDNA2 at a working concentration of 2.2 µM, and gDNA3 at a working concentration of 2.2 µM.

[0047] More preferably, the PfAgo cleavage reaction system includes probes with working concentrations of 1000 nM to 2500 nM.

[0048] More preferably, the PfAgo cleavage reaction system comprises probes with working concentrations of 1000 nM, 1050 nM, 1100 nM, 1150 nM, 1200 nM, 1250 nM, 1300 nM, 1350 nM, 1400 nM, 1450 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, 1900 nM, 2000 nM, 2100 nM, 2200 nM, 2300 nM, 2400 nM, or 2500 nM.

[0049] More preferably, the PfAgo cleavage reaction system includes: probe 1 with a working concentration of 1000 nM, probe 2 with a working concentration of 1500 nM, and probe 3 with a working concentration of 2500 nM.

[0050] More preferably, the PfAgo cleavage reaction system further includes: PfAgo endonuclease at a working concentration of 0.08 U / μL to 0.12 U / μL.

[0051] Further preferably, the PfAgo cleavage reaction system also includes: PfAgo endonuclease at a working concentration of 0.1 U / μL.

[0052] The PfAgo cleavage reaction system described above can complete the detection without the use of standards, while ensuring the specificity of mutation site interpretation, thus effectively improving the reliability and applicability of the system.

[0053] According to the present invention, a method for simultaneous detection of amino acid mutation sites in ALS of Amaranthus retroflexus is preferably provided in a multiplex PCR reaction system comprising primers with working concentrations of 100 nM to 300 nM, wherein the primers are those described in the primer composition.

[0054] More preferably, the multiplex PCR reaction system includes primers with working concentrations of 150 nM to 250 nM.

[0055] More preferably, the multiplex PCR reaction system includes primers with working concentrations of 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, or 250 nM.

[0056] More preferably, the multiplex PCR reaction system includes: primers with a working concentration of 200 nM and a nucleotide sequence as shown in SEQ ID NO.1, primers with a working concentration of 200 nM and a nucleotide sequence as shown in SEQ ID NO.2, primers with a working concentration of 200 nM and a nucleotide sequence as shown in SEQ ID NO.3, primers with a working concentration of 200 nM and a nucleotide sequence as shown in SEQ ID NO.4, primers with a working concentration of 200 nM and a nucleotide sequence as shown in SEQ ID NO.5, and primers with a working concentration of 200 nM and a nucleotide sequence as shown in SEQ ID NO.6.

[0057] According to the present invention, a method for simultaneous detection of amino acid mutation sites in ALS of Amaranthus retroflexus is preferably provided in the following multiplex PCR reaction program: pre-denaturation at 92℃~96℃ for 1.5 min~2.5 min; denaturation at 92℃~96℃ for 25 s~35 s, annealing at 58℃~62℃ for 40 s~50 s, extension at 63℃~67℃ for 0.5 min~1.5 min, for 30 to 40 cycles; final extension at 63℃~67℃ for 4 min~6 min; and / or, the PfAgo cleavage reaction program includes: reaction at 94℃~96℃ for 40 min~60 min.

[0058] More preferably, the multiplex PCR reaction program includes: pre-denaturation at 94°C for 2 min; followed by 34 cycles, each cycle including denaturation at 94°C for 30 s, annealing at 60°C for 45 s, extension at 65°C for 1 min; and final extension at 65°C for 5 min.

[0059] More preferably, the PfAgo cleavage reaction procedure includes: reacting at 94℃~96℃ for 40 min~60 min.

[0060] More preferably, the PfAgo cleavage reaction program includes: reacting at 94°C, 95°C, or 96°C for 40 min, 45 min, 50 min, 55 min, or 60 min.

[0061] More preferably, the PfAgo cleavage reaction procedure includes: reacting at 95°C for 50 min.

[0062] By combining rapid DNA extraction reagents and nested PCR tubes, rapid, unopened field detection of Amaranthus retroflexus resistant weeds can be achieved. This detection method is simple to operate, provides intuitive results, and can simultaneously interpret multi-site resistance mutations under laboratory or field conditions, significantly improving the applicability and promotional value of the detection method.

[0063] According to the present invention, a method for synchronously detecting amino acid mutation sites in ALS of Amaranthus retroflexus is provided, preferably, the 5' end of the probe 1 is provided with SX670; the 5' end of the probe 2 is provided with ROX; and the 5' end of the probe 3 is provided with FAM.

[0064] More preferably, the mutation status of amino acid mutation sites in ALS of *Amaranthus retroflexus* is determined by combining the fluorescence signals measured by multiplex PCR. The determination criteria include: when the sample only has D>th threshold in the CY5 channel, ROX channel, or FAM channel, then the sample corresponds to only the Asp-376-Glu mutation, Trp-574-Leu mutation, or Ser-653-Tyr mutation; when the sample only has D>th threshold in the CY5 channel and ROX channel, then the sample corresponds to only the Asp-376-Glu mutation and Trp-574-Leu mutation; when the sample only has D>th threshold in the CY5 channel and FAM channel, then the sample corresponds to only the Asp-376-Glu mutation. The Asp-376-Glu and Ser-653-Tyr mutations are identified as follows: When the D values ​​of the ROX and FAM channels in the sample are greater than the threshold, the sample contains only the Trp-574-Leu and Ser-653-Tyr mutations. When the D values ​​of the CY5, ROX, and FAM channels in the sample are greater than the threshold, the sample contains the Asp-376-Glu, Trp-574-Leu, and Ser-653-Tyr mutations. When the D values ​​of the CY5, ROX, and FAM channels in the sample are less than or equal to the threshold, the sample does not contain the Asp-376-Glu, Trp-574-Leu, and Ser-653-Tyr mutations. D is calculated using the following formula: D=FI end -FI 10 ; Among them, FI end The fluorescence signal value at the reaction endpoint; FI 10 This represents the fluorescence signal value corresponding to the 10th fluorescence acquisition. The threshold value of D is 500 au to 600 a.u.

[0065] More preferably, the threshold value of D is 560 au to 570 a.u.

[0066] More preferably, the threshold value of D is 563 au.

[0067] More preferably, when determining the mutation status of amino acid mutation sites in ALS of Amaranthus retroflexus by combining fluorescence signals obtained from multiplex PCR reactions, no negative control is required during the detection process.

[0068] In a seventh aspect, the present invention provides a method for identifying the resistance of Amaranthus retroflexus to ALS inhibitor herbicides, comprising: detecting the mutation status of amino acid mutation sites in the ALS of the test sample using any of the methods described above; if any one or more of D376E, W574L and S653Y are present, the test sample is determined to have resistance to ALS inhibitor herbicides.

[0069] The present invention has the following beneficial effects: This invention is the first to construct a method for detecting multiple resistance mutations based on the synergistic effect of multiplex PCR and PfAgo endonuclease. By combining multiplex PCR amplification with PfAgo specific recognition and cleavage, it enables the simultaneous detection and mutation type identification of multiple resistance target sites in ALS, overcoming the technical limitations of existing detection methods such as low throughput and difficulty in simultaneously distinguishing multiple mutation sites.

[0070] This invention utilizes a multi-constraint mechanism involving multiplex PCR amplification, PfAgo-guided sequence recognition, and fluorescent probe cleavage to achieve stepwise identification and verification of target site sequences. It exhibits excellent specificity, sensitivity, and robustness, effectively reducing the risk of non-specific amplification and misjudgment, ensuring accurate and reliable detection results. It can stably detect mutations at target sites under different template concentrations and can complete interpretation without relying on standards. The detection method established in this invention is suitable for rapid and simultaneous multi-site detection of ALS inhibitor resistance in Amaranthus retroflexus in the field, providing key technical support for monitoring, field distribution surveys, and precision herbicide management. Attached Figure Description

[0071] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0072] Figure 1 The above are the electrophoretic analysis results of the amplification products provided in Example 1 of this invention; A is an agarose gel electrophoresis diagram of single-target PCR amplification products targeting the Asp-376-Glu, Trp-574-Leu and Ser-653-Tyr mutation sites, where M represents a 500 bp DNA Marker, and lanes 1, 2 and 3 correspond to amplification products containing Asp-376-Glu, Trp-574-Leu and Ser-653-Tyr respectively; B is a PAGE electrophoresis diagram of multiplex PCR amplification products containing Asp-376-Glu, Trp-574-Leu and Ser-653-Tyr, where lanes 1 and 2 are the results of two replicate experiments.

[0073] Figure 2 This is a schematic diagram of the specific gDNA screening results for the Asp-376-Glu mutation site provided in Example 2 of the present invention. It is used to evaluate the ability of the designed gDNA to produce a specific fluorescent signal when the mutation occurs at the 376 site (376Mut), while it does not produce a fluorescent signal or only produces a background signal in the sample without mutation at the 376 site (376Wt) and the template-free control (376CK); A to G are 376 gDNA-1 to 376 gDNA-7 in sequence.

[0074] Figure 3 This is a schematic diagram of the specific gDNA screening results for the Trp-574-Leu mutation site provided in Example 2 of the present invention. It is used to evaluate the ability of the designed gDNA to produce a specific fluorescent signal when the 574 site is mutated (574Mut), while it does not produce a fluorescent signal or only produces a background signal in the sample without mutation at the 574 site (574Wt) and the template-free control (574CK); A to G are 574 gDNA-1 to 574 gDNA-7 in sequence.

[0075] Figure 4 This is a schematic diagram of the specific gDNA screening results for the Ser-653-Tyr mutation site provided in Example 2 of the present invention. It is used to evaluate the ability of the designed gDNA to produce a specific fluorescent signal when the 653 site is mutated (653Mut), while it does not produce a fluorescent signal or only produces a background signal in the sample without mutation at the 653 site (653Wt) and the template-free control (653CK). A to F are 653 gDNA-1 to 653 gDNA-6 in sequence.

[0076] Figure 5 This is a schematic diagram of the results obtained by using genomic DNA of *Amaranthus retroflexus* with mutations in Asp-376-Glu, Trp-574-Leu, and Ser-653-Tyr, genomic DNA of wild-type *Amaranthus retroflexus*, and a template-free control (CK) in an established multiplex PCR-PfAgo detection system, as provided in Example 2 of this invention; A represents the detection results of the CY5 channel; B represents the detection results of the ROX channel; and C represents the detection results of the FAM channel.

[0077] Figure 6 This is a schematic diagram showing the results of optimizing the working concentration of the fluorescent probes Asp-376-Glu and Trp-574-Leu according to Embodiment 3 of the present invention; A is a diagram showing the optimized working concentration of the Asp-376-Glu fluorescent probe; B is a diagram showing the optimized working concentration of the Trp-574-Leu fluorescent probe.

[0078] Figure 7This is an evaluation of the optimization effect of the PfAgo cleavage reaction system provided in Example 3 of the present invention; A is a sample of Amaranthus retroflexus with a mutation in Asp-376-Glu; B is a sample of Amaranthus retroflexus with a mutation in Trp-574-Leu; C is a sample of Amaranthus retroflexus with a mutation in Ser-653-Tyr; D is a wild-type Amaranthus retroflexus without mutation; 1 and 2 represent two technically replicated samples.

[0079] Figure 8 This is a schematic diagram showing the sensitivity comparison between the multiplex PCR-PfAgo method provided in Example 4 of the present invention and conventional PCR detection; A shows the detection results of the multiplex PCR-PfAgo method on the genomic DNA of D376E Amaranthus retroflexus using a 3-fold serially diluted genomic DNA solution; B shows the detection results of the genomic DNA of W574L Amaranthus retroflexus using a 3-fold serially diluted genomic DNA solution; C shows the detection results of the multiplex PCR-PfAgo method on the genomic DNA of S653Y Amaranthus retroflexus using a 3-fold serially diluted genomic DNA solution; D shows the sensitivity detection results of conventional PCR.

[0080] Figure 9 This is a schematic diagram of the detection results of a simulation experiment on the coexistence of multiple mutations of Asp-376-Glu, Trp-574-Leu, and Ser-653-Tyr provided in Embodiment 5 of the present invention.

[0081] Figure 10 This is a flowchart of the mutation detection process performed on field-collected Amaranthus retroflexus samples using the multiplex PCR-PfAgo detection system provided in Embodiment 6 of the present invention.

[0082] Figure 11 This is a schematic diagram showing the results of verifying the accuracy of mutation detection in field-collected Amaranthus retroflexus samples using the multiplex PCR-PfAgo detection system provided in Embodiment 6 of the present invention. Detailed Implementation

[0083] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0084] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0085] Unless otherwise specified, the experimental materials and reagents used in the following examples are all conventional products that can be purchased from legitimate channels.

[0086] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. All primer sequences in this invention are in the 5' to 3' orientation.

[0087] The plant materials involved in the following examples are: (1) Amaranthus retroflexus with the Asp-376-Glu mutation (denoted as D376E): collected from a soybean field in Naireng Gacha, Chabugatu Sumu, Zhalute Banner, Tongliao City, Inner Mongolia Autonomous Region (longitude: 120°59′56″E, latitude: 44°16′9″N), and confirmed by PCR amplification and Sanger sequencing to have the Asp-376-Glu mutation; (2) Amaranthus retroflexus with the Trp-574-Leu mutation (denoted as W574L): collected from Baoan Gacha, Chabugatu Sumu (120°57′55″E, latitude: 44°16′9″N). (2) A soybean field with the Trp-574-Leu mutation was found in a soybean field at 121°31′19″E, 43°58′11″N. PCR amplification and Sanger sequencing confirmed the presence of a reverse-branching amaranth with the Ser-653-Tyr mutation (denoted as S653Y reverse-branching amaranth): collected from a soybean field at Maofa Village, Xiaojieji Town, Kailu County, Tongliao City (121°31′19″E, 43°58′11″N). PCR amplification and Sanger sequencing confirmed the presence of a reverse-branching amaranth with the Ser-653-Tyr mutation; (3) Wild-type reverse-branching amaranth (denoted as Wt reverse-branching amaranth): collected from a roadside with no history of herbicide use. The public can obtain the above biological materials from the applicant. Seed and leaf samples of the above-mentioned reverse-branching amaranth plants were collected in the field. The seeds were air-dried and preserved, and the leaves were quick-frozen in liquid nitrogen and then stored in a -80°C freezer.

[0088] Example 1: Design, screening, and construction of a multiplex PCR method for specific multiplex PCR targeting three ALS sites in Amaranthus retroflexus. This embodiment focuses on three known target sites in the ALS gene of Amaranthus retroflexus—Asp-376, Trp-574, and Ser-653—that are associated with anti-ALS inhibitor herbicides. Specific primers suitable for multiplex PCR amplification were designed and screened, and a multiplex PCR reaction system was constructed accordingly.

[0089] 1. Design of PCR primers The ALS gene sequence of *Amaranthus retroflexus* (Gene ID: AF363369.1) was obtained from the NCBI database. The three target sites, Asp-376-Glu, Trp-574-Leu and Ser-653-Tyr, were located using SnapGene software, and corresponding PCR primers were designed accordingly.

[0090] The design of PCR primers follows the following principles: (1) It meets the basic requirements of conventional PCR primer design; (2) By rationally designing the amplification fragment, the target mutation site and its corresponding PfAgo cleavage site are located relatively close to the 3' end of the amplification product, so as to avoid the generation of excessively long secondary gDNA after PfAgo cleavage, which would affect the efficiency of subsequent cleavage reactions; (3) The length of the amplification fragment is controlled at 150-200 bp, and the annealing temperature of each primer is uniformly set at 60℃ to meet the requirements of simultaneous amplification of multiplex PCR.

[0091] Based on the above principles, PCR primers for each target site were designed using SnapGene software, resulting in the PCR primers shown in Table 1.

[0092] Table 1. PCR primers for three target sites of ALS in Amaranthus retroflexus.

[0093] Note: The sequence of the amplified fragment contains " The bases shown in bold are mutation sites, and the bases in bold are mutated bases.

[0094] 2. Screening of PCR primers (1) Obtaining the genomic DNA of Amaranthus retroflexus The TianGen high-efficiency plant genomic DNA extraction kit (catalog number: DP350) was used to extract leaf samples of Amaranthus retroflexus D376E, Amaranthus retroflexus W574L and Amaranthus retroflexus S653Y, respectively, and the genomic DNA of each Amaranthus retroflexus sample was used as a template to establish the PCR reaction system.

[0095] (2) Single-target PCR reaction Using the genomic DNA of *Amaranthus retroflexus* samples as templates, single-target PCR amplification was performed using primers designed in the previous step, and the PCR products were detected by 2% agarose gel electrophoresis.

[0096] The single-target PCR reaction system (25 μL) consists of: 1 μL template (concentration of 30 ng / µL), 1 μL of 10 μM forward primer F, 1 μL of 10 μM reverse primer R, and 12.5 μL of 2×Taq PCR Mix, with ddH2O added to bring the total volume to 25 μL.

[0097] The single-target PCR reaction program was as follows: pre-denaturation at 94℃ for 4 min; followed by 34 cycles, each cycle consisting of denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 10 s; and a final extension at 72℃ for 5 min.

[0098] like Figure 1As shown in A, the amplification reactions of the primer pairs for detecting Asp-376-Glu (forward primer F1 (SEQ ID NO.1) and reverse primer R1 (SEQ ID NO.2)), the primer pairs for detecting Trp-574-Leu (forward primer F1 (SEQ ID NO.3) and reverse primer R1 (SEQ ID NO.4)), and the primer pairs for detecting Ser-653-Tyr (forward primer F1 (SEQ ID NO.5) and reverse primer R1 (SEQ ID NO.6)) all showed single bands of the correct size at the expected positions.

[0099] Subsequently, the corresponding PCR products were sequenced and analyzed. The results showed that the sequencing results were consistent with the target amplified sequence, indicating that the above three sets of PCR primer pairs all had good specificity.

[0100] The other primer pairs had poor specificity and all exhibited non-specific amplification.

[0101] Therefore, Asp-376-Glu MPCR-F (SEQ ID NO.1) and Asp-376-Glu MPCR-R (SEQ ID NO.2) were ultimately selected as the primer pair for detecting Asp-376-Glu, Trp-574-Leu MPCR-F (SEQ ID NO.3) and Trp-574-Leu MPCR-R (SEQ ID NO.4) were selected as the primer pair for detecting Trp-574-Leu, and Ser-653-Tyr MPCR-F (SEQ ID NO.5) and Ser-653-Tyr MPCR-R (SEQ ID NO.6) were selected as the primer pair for detecting Ser-653-Tyr.

[0102] 3. Construction of a multiplex PCR method Using genomic DNA from D376E, W574L, and S653Y amaranth as templates, multiplex PCR amplification was performed in the same reaction tube using the three pairs of PCR primers obtained in the previous step.

[0103] The multiplex PCR reaction system (50 μL) consisted of: 3 μL of Amaranthus retroflexus genomic DNA (concentration 30 ng / µL), 1 µL of 10 µM Asp-376-Glu MPCR-F, 1 µL of 10 µM Asp-376-Glu MPCR-R, 1 µL of 10 µM Trp-574-Leu MPCR-F, 1 µL of 10 µM Trp-574-Leu MPCR-R, 1 µL of 10 µM Ser-653-Tyr MPCR-F, 1 µL of 10 µM Ser-653-Tyr MPCR-R, 5 µL of 10×PerfectStart Taq Buffer, 4 µL of 2.5 mM dNTPs, and 1 µL of PerfectStart Taq DNA Polymerase, with ddH2O added to bring the total volume to 50 µL.

[0104] The multiplex PCR reaction program was as follows: pre-denaturation at 94℃ for 2 min; followed by 34 cycles, each cycle consisting of denaturation at 94℃ for 30 s, annealing at 60℃ for 45 s, extension at 65℃ for 1 min; and final extension at 65℃ for 5 min.

[0105] After the reaction was completed, the amplification products were electrophoresed on a 15% urea-TBE polyacrylamide gel (PAGE) at a constant voltage of 150 V for 3 h, stained with Gel Red, and the results were analyzed using a UV imaging system.

[0106] like Figure 1 As shown in Figure B, the electrophoresis results simultaneously show specific bands corresponding to the sizes of the three target amplification fragments, indicating that the three pairs of PCR primers have good amplification compatibility in the same reaction system, and the multiplex PCR method was successfully constructed. Although two non-specific amplification bands were observed (approximately 700 bp and 500 bp in size, respectively), their generation may be due to all PCR primers targeting the same gene (Amaranthus retroflexus). ALS (Genes), and the reaction procedure is consistent, leading to non-specific pairing between primers and triggering amplification. However, due to Pf Ago exhibits high specificity based on a two-step recognition mechanism, subsequently... Pf Ago can specifically cleave the target sequence containing the three mutation sites without cleaving the amplification products of these two large fragments, thus not affecting the final detection results.

[0107] Example 2: Design and screening of specific gDNA and fluorescent probes for three target sites of the ALS gene in *Amaranthus retroflexus*, and establishment of a multiplex PCR-PfAgo method. 1. Design of gDNA and fluorescent probes The method for designing gDNA in this embodiment is as follows: gDNA sequences are designed using the mutated bases at each target mutation site as the core, and the mutated bases are placed at positions 2 to 8 of the gDNA sequence. When a single mutated base is insufficient to effectively distinguish between mutant and wild-type samples, auxiliary mismatched bases can be introduced into the gDNA sequence to enhance the distinguishing ability. All gDNA sequences are controlled to a length of 16–18 nt as much as possible, and phosphorylation modification is performed at their 5' end.

[0108] This embodiment designs fluorescent probes based on the cleavage characteristics of PfAgo. After PfAgo cleaves the PCR amplification product, it generates secondary gDNA. The fluorescent probe can specifically react with the secondary gDNA, thereby producing a fluorescent signal.

[0109] Based on the above principles, gDNA and fluorescent probes were designed as shown in Table 2.

[0110] Table 2. gDNA and fluorescent probes for three target sites of ALS in Amaranthus retroflexus.

[0111] Note: In the table, "P" represents a phosphate group, SX670 represents a near-infrared fluorescent dye, ROX represents a red fluorescent group, FAM represents a green fluorescent group, and BQH1, BQH2 and BQH3 represent quenching groups.

[0112] 2. Screening of gDNA and fluorescent probes (1) Preparation of reaction template Using genomic DNA from D376E and wild-type Amaranthus retroflexus, and ddH2O as templates, PCR amplification and agarose gel electrophoresis were performed using forward primer F1 (SEQ ID NO.1) and reverse primer R1 (SEQ ID NO.2), respectively. The amplification products were denoted as 376Mut, 376Wt, and 376CK, respectively.

[0113] Using genomic DNA and ddH2O from W574L and wild-type Amaranthus retroflexus as templates, respectively, PCR amplification and agarose gel electrophoresis were performed using forward primer F1 (SEQ ID NO.3) and reverse primer R1 (SEQ ID NO.4). The amplification products were denoted as 574Mut, 574Wt, and 574CK, respectively.

[0114] Using genomic DNA and ddH2O from S653Y and wild-type Amaranthus retroflexus as templates, respectively, PCR amplification and agarose gel electrophoresis were performed using forward primer F1 (SEQ ID NO.5) and reverse primer R1 (SEQ ID NO.6). The amplification products were denoted as 653Mut, 653Wt, and 653CK, respectively.

[0115] The PCR products were stored at 4°C for later use.

[0116] (2) Single-target PCR-PfAgo reaction To screen for gDNA and fluorescent probes that can effectively distinguish between mutant and wild-type alleles, gDNA and their corresponding fluorescent probes designed for Asp-376-Glu, Trp-574-Leu, and Ser-653-Tyr sites were introduced into a single-target PCR-PfAgo reaction system.

[0117] The changes in fluorescence signal during the PfAgo-mediated cleavage reaction were monitored using PCR amplification products of mutant samples (376Mut, 574Mut, and 653Mut), wild-type samples (376Wt, 574Wt, and 653Wt), and template-free control samples (376Ck, 574CK, and 653CK) as reaction templates.

[0118] The single-target PCR-PfAgo reaction system (20µL) consisted of: 7µL PCR amplification product, 4µL 0.1 U / µL PfAgo endonuclease, 2µL 10×Reaction Buffer, and 40 mM Mn. 2+ 1 µL, 4 µL of 10 µM gDNA, and 2 µL of 10 µM fluorescent probe.

[0119] The single-target PCR-PfAgo reaction procedure is as follows: first perform the single-target PCR reaction, then perform the PfAgo reaction. The single-target PCR reaction procedure is the same as in Example 1. The PfAgo cleavage reaction procedure is as follows: in a real-time fluorescence detection PCR instrument, react at 95°C for 50 min, with each cycle lasting 30 s. The fluorescence signal of the target channel (CY5 channel, ROX channel, and FAM channel) is collected once per cycle.

[0120] like Figure 2 As shown in A to G, among the seven gDNA and fluorescent probes targeting the Asp-376-Glu site, 376gDNA-3 (SEQ ID NO.7) and its probe (SEQ ID NO.8) showed the most significant signal difference between mutant and wild-type samples, with the fluorescent signal of the mutant sample being 6413 au and the fluorescent signal of the wild-type sample being 2396 a.u.

[0121] like Figure 3As shown in A to G, among the seven gDNAs and fluorescent probes targeting the Trp-574-Leu site, only 574gDNA-3 (SEQ ID NO.9) and its probe (SEQ ID NO.10) produced a significant fluorescent signal only in the mutant sample, with a fluorescence signal of 3920 au, while maintaining a low background in the wild-type sample and the template-free control, with a fluorescence signal of 1487 a.u.

[0122] like Figure 4 As shown in A-F, six gDNAs and fluorescent probes were selected at the Ser-653-Tyr site. 653 gDNA-2 (SEQ ID NO.11) and its probe (SEQ ID NO.12) exhibited a faster response rate to mutant samples while maintaining low background in wild-type samples. The fluorescence signal of the mutant sample was 710 au, and the fluorescence signal of the wild-type sample was 131 au.

[0123] Therefore, Asp-376-Glu gDNA (SEQ ID NO.7) was ultimately selected as the gDNA for detecting Asp-376-Glu, and the Asp-376-Glu fluorescent probe (SEQ ID NO.8) was selected as the fluorescent probe for detecting Asp-376-Glu; Trp-574-Leug DNA (SEQ ID NO.9) was selected as the gDNA for detecting Trp-574-Leu, and the Trp-574-Leu fluorescent probe (SEQ ID NO.10) was selected as the fluorescent probe for detecting Trp-574-Leu; Ser-653-Tyr gDNA (SEQ ID NO.11) was selected as the gDNA for detecting Ser-653-Tyr, and the Ser-653-Tyr fluorescent probe (SEQ ID NO.12) was selected as the fluorescent probe for detecting Ser-653-Tyr.

[0124] 3. Establishment of the multiplex PCR-PfAgo method (1) Preparation of multiplex PCR products Genomic DNA from D376E, W574L, S653Y, and Wt amaranth, as well as a template-free control (ddH2O), were used as templates for amplification using the multiplex PCR method established in Example 1. The corresponding multiplex PCR amplification products were obtained and were denoted as Asp-376-Glu multiplex product, Trp-574-Leu multiplex product, Ser-653-Tyr multiplex product, Wt multiplex product, and Ck multiplex product, respectively.

[0125] (2) Establishment of multiple PfAgo methods The five multiplex PCR amplification products mentioned above, along with the Asp-376-Glu gDNA (SEQ ID NO.7), Asp-376-Glu fluorescent probe (SEQ ID NO.8), Trp-574-Leu gDNA (SEQ ID NO.9), Trp-574-Leu fluorescent probe (SEQ ID NO.10), Ser-653-Tyr gDNA (SEQ ID NO.11), and Ser-653-Tyr fluorescent probe (SEQ ID NO.12) selected in the previous step, were added to the same PfAgo cleavage reaction system for PfAgo cleavage reaction.

[0126] The PfAgo digestion reaction system (20 µL) consisted of: 10 µL of multiplex PCR amplification product, 4 µL of 0.5 U / µL PfAgo endonuclease, 2 µL of 10×Reaction Buffer, and 40 mM Mn. 2+ 1 µL, 0.4 µL of 100 µM Asp-376-Glu gDNA (SEQ ID NO.7), 0.5 µL of 100 µM Asp-376-Glu fluorescent probe (SEQ ID NO.8), 0.4 µL of 100 µM Trp-574-Leu gDNA (SEQ ID NO.9), 0.5 µL of 100 µM Trp-574-Leu fluorescent probe (SEQ ID NO.10), 0.4 µL of 100 µM Ser-653-Tyr gDNA (SEQ ID NO.11), and 0.5 µL of 100 µM Ser-653-Tyr fluorescent probe (SEQ ID NO.12), and nuclease-free water to a final volume of 20 µL.

[0127] The PfAgo cutting reaction procedure is as follows: react at 95℃ for 50 min in a real-time fluorescence detection PCR instrument, with each cycle lasting 30 s. The fluorescence signal of the target channel (CY5 channel, ROX channel, and FAM channel) is collected once per cycle.

[0128] The detection specificity of different mutation sites and the establishment of the detection system were evaluated by simultaneously monitoring the signal changes of the three fluorescence channels FAM, ROX and CY5.

[0129] like Figure 5As shown in Figure A, the fluorescence signal generated when the Asp-376-Glu multiplex product was added to the CY5 channel (4132 au) was significantly higher than that of the Trp-574-Leu multiplex product (980 au), Ser-653-Tyr multiplex product (833 au), Wt multiplex product (537 au), and Ck multiplex product (146 au), indicating that this channel can specifically recognize the Asp-376-Glu mutation site.

[0130] like Figure 5 As shown in Figure B, the fluorescence signal generated when the Trp-574-Leu multiplex product was added to the ROX channel (3012 au) was significantly higher than that of the Asp-376-Glu multiplex product (748 au), Ser-653-Tyr multiplex product (337 au), Wt multiplex product (541 au), and Ck multiplex product (146 au), indicating that the channel can specifically recognize the Trp-574-Leu mutation site.

[0131] like Figure 5 As shown in C, the fluorescence signal generated when the Ser-653-Tyr multiplex product was added to the FAM channel (2078 au) was significantly higher than that of the Asp-376-Glu multiplex product (273 au), Trp-574-Leu multiplex product (63 au), Wt multiplex product (136 au), and Ck multiplex product (77 au), indicating that the channel can specifically recognize the Ser-653-Tyr mutation site.

[0132] The above results demonstrate that the multiplex PCR-PfAgo method constructed in this invention can achieve channel differentiation and specific identification of different mutation sites, with no interference between the multiple channel signals, indicating a successful detection system construction. Based on the design principles of this system and the success of the above results, this method can theoretically be extended to the simultaneous detection of multiple targets and multiple mutations.

[0133] Example 3: Optimization of PfAgo cleavage reaction system and establishment of interpretation criteria 1. Optimization of the PfAgo cleavage reaction system (1) Optimization principle To enable independent result interpretation without relying on standards when using the detection system for mutation detection, and to provide interpretation criteria for subsequent detection of multiple coexisting mutations, this invention analyzes and optimizes the interpretation thresholds of fluorescence signals corresponding to each mutation site.

[0134] According to the above embodiment 2 Figure 5 The detection results of A to C show that when the Asp-376-Glu site mutates, the corresponding fluorescence signal of the CY5 channel is 4132 au, which is the highest among the three sites; when the Ser-653-Tyr site mutates, the corresponding fluorescence signal of the FAM channel is 2078 au, which is the lowest among the three sites. It can also be observed that the background signal in the FAM channel of the non-target mutant sample and the CK control is at a low level, significantly lower than that in the ROX and CY5 channels.

[0135] Based on the above results, the fluorescence signal of approximately 2000 in the FAM channel of the Ser-653-Tyr mutant sample was used as a standard to optimize the multiplex PCR-PfAgo detection system for the Asp-376-Glu and Trp-574-Leu mutant sites. This ensures that clear, stable and distinguishable results can be achieved for each mutant site without the need for standards.

[0136] Given that the multiplex PCR-PfAgo detection system involves multiple reaction steps such as PCR amplification, PfAgo-mediated cleavage, and fluorescence signal output, the system composition is relatively complex. Furthermore, the aforementioned embodiments have already demonstrated stable and specific identification and interpretation of mutation sites. To ensure the overall stability of the detection system while standardizing and optimizing the fluorescence signal interpretation threshold, this invention does not adjust key functional components such as the gDNA sequence, PfAgo endonuclease, and their reaction conditions. Instead, it optimizes only the fluorescent probe component, which serves as the signal output unit.

[0137] (2) Optimization of Asp-376-Glu fluorescent probe Using the Asp-376-Glu multiplex product as the multiplex PCR amplification product to be tested, while keeping other reaction components and PfAgo cleavage reaction conditions in the PfAgo cleavage reaction system unchanged in Example 2, the working concentration of the Asp-376-Glu fluorescent probe (SEQ ID NO.8) corresponding to the Asp-376-Glu mutation site was optimized by gradient. The working concentrations were set to 500 nM, 1000 nM, 1500 nM, 2000 nM and 2500 nM, and the fluorescence signal intensity under different probe concentration conditions collected by the CY5 channel was compared.

[0138] like Figure 6As shown in Figure A, when the working concentration of the Asp-376-Glu fluorescent probe (SEQ ID NO.8) is 1000 nM, its fluorescence signal is closest to the preset interpretation benchmark value of approximately 2000 au. Therefore, the optimal working concentration of the Asp-376-Glu fluorescent probe (SEQ ID NO.8) is determined to be 1000 nM.

[0139] (3) Optimization of Trp-574-Leu fluorescent probe Based on the previous step, the working concentration of the ROX-labeled fluorescent probe Trp-574-Leu (SEQ ID NO.10) corresponding to the Trp-574-Leu mutation site was further optimized using a gradient method.

[0140] Using the Trp-574-Leu multiplex product as the multiplex PCR amplification product to be tested, while keeping other reaction components and PfAgo cleavage reaction conditions unchanged in the PfAgo cleavage reaction system of Example 2, the working concentration of the Asp-376-Glu fluorescent probe (SEQ ID NO.8) was fixed at 1000 nM, and the working concentrations of the Trp-574-Leu fluorescent probe (SEQ ID NO.10) were set to 1250 nM, 1500 nM, 1750 nM, 2000 nM and 2250 nM, respectively. The fluorescence signal intensity under different probe concentration conditions collected by the ROX channel was compared.

[0141] like Figure 6 As shown in B, when the working concentration of the Trp-574-Leu fluorescent probe (SEQ ID NO.10) is 1500 nM, its fluorescence signal intensity is closest to the interpretation benchmark value of approximately 2000 au, and the signal is stable with low background. Therefore, the final working concentration of the 574-reporter is determined to be 1500 nM.

[0142] In summary, the optimized PfAgo cleavage reaction system (20 µL) is as follows: 10 µL of multiplex PCR amplification product, 4 µL of 0.5 U / µL LPfAgo endonuclease, 2 µL of 10×Reaction Buffer, and 40 mM Mn. 2+1 µL, 0.4 µL of 100 µM Asp-376-Glu gDNA (SEQ ID NO.7), 0.2 µL of 100 µM Asp-376-Glu fluorescent probe (SEQ ID NO.8), 0.4 µL of 100 µM Trp-574-Leu gDNA (SEQ ID NO.9), 0.3 µL of 100 µM Trp-574-Leu fluorescent probe (SEQ ID NO.10), 0.4 µL of 100 µM Ser-653-Tyr gDNA (SEQ ID NO.11), and 0.5 µL of 100 µM Ser-653-Tyr fluorescent probe (SEQ ID NO.12), and nuclease-free water to a final volume of 20 µL.

[0143] (4) Evaluation of the optimization effect of the PfAgo cleavage reaction system Genomic DNA from leaf samples of D376E, W574L, and S653Y Amaranthus retroflexus was used as templates, and detection was performed using the multiplex PCR-PfAgo method established in Example 2 combined with the PfAgo cleavage reaction system optimized in the previous step of this example.

[0144] like Figure 7 As shown in A to C, stable fluorescence signals of approximately 2000 a.u. were obtained in the fluorescence channels corresponding to the respective mutation target sites (Asp-376-Glu mutation corresponds to the CY5 channel, Trp-574-Leu mutation corresponds to the ROX channel, and Ser-653-Tyr mutation corresponds to the FAM channel), while the signals of the remaining non-target fluorescence channels did not exceed 500. Meanwhile, as... Figure 7 As shown in D, the signals of the non-mutant samples were at low background levels in the three fluorescence channels of FAM, ROX, and CY5.

[0145] The above results show that the optimized PfAgo cleavage reaction system can clearly distinguish the three mutation sites without the need for standards. The optimization strategy of this system is effective and the detection results are reliable.

[0146] 2. Criteria for interpreting mutations without the need for standard samples Through the Figure 7Overall analysis of the fluorescence signal change curves shown in A-D revealed that during the multiplex PCR-PfAgo detection process, regardless of whether mutations occurred at the Asp-376-Glu or Trp-574-Leu sites, the fluorescence signals of each channel rapidly increased in the early stages of the multiplex PCR-PfAgo reaction, reaching their initial peak in the 10th acquisition cycle. In the presence of mutations, the fluorescence signal subsequently continued to increase; while in the absence of mutations, the fluorescence signal decreased or remained essentially unchanged.

[0147] Based on the above characteristics, this invention establishes a single-site mutation interpretation rule that does not require standards, specifically: based on the fluorescence signal value at the reaction endpoint (denoted as FI). end The fluorescence signal value corresponding to the 10th fluorescence acquisition (denoted as FI) 10 The difference between FI and F is used as the interpretation index (denoted as D), i.e., D = FI end -FI 10 .against Figure 7 The fluorescence signal measurements corresponding to channels A through D were statistically analyzed, and ROC analysis was performed. The results showed that the area under the ROC curve (AUC) was 1, and the best discrimination effect was achieved when the D value threshold was 987 au. Therefore, when D > 987 au, it is determined that there is a mutation at the target site corresponding to the fluorescence channel in the test sample; when D ≤ 987 au, it is determined that there is no mutation at the target site corresponding to the fluorescence channel in the test sample.

[0148] Specifically, for each target site, the mutation interpretation rules are as follows: When the D value of the CY5 channel in the test sample is greater than 987 au, the test sample contains the Asp-376-Glu mutation; when the D value of the CY5 channel in the test sample is less than or equal to 987 au, the test sample does not contain the Asp-376-Glu mutation; when the D value of the ROX channel in the test sample is greater than 987 au, the test sample contains the Trp-574-Leu mutation; when the D value of the ROX channel in the test sample is less than or equal to 987 au, the test sample does not contain the Trp-574-Leu mutation; when the D value of the FAM channel in the test sample is greater than 987 au, the test sample contains the Ser-653-Tyr mutation; when the D value of the FAM channel in the test sample is less than or equal to 987 au, the test sample does not contain the Ser-653-Tyr mutation.

[0149] Example 4: Evaluation of the detection sensitivity of the multiplex PCR-PfAgo method 1. Template preparation Leaves of D376E, W574L, and S653Y amaranth were extracted according to the method in Example 1 to obtain the genomic DNA of each amaranth plant.

[0150] Quantitative analysis was performed using a UV spectrophotometer, and the initial concentration of each genomic DNA sample was uniformly adjusted to 30 ng / µL. Subsequently, a 3-fold serial dilution method was used to serially dilute each genomic DNA sample to obtain genomic DNA dilutions with concentrations of 30 ng / µL, 10 ng / µL, 3.33 ng / µL, 1.11 ng / µL, 0.37 ng / µL, 0.12 ng / µL, and 0.04 ng / µL, which served as templates. A template-free control (CK) was also included.

[0151] 2. Multiplex PCR-PfAgo detection The template obtained in the previous step was used for detection according to the multiplex PCR-PfAgo method established in Example 2 and the PfAgo cleavage reaction system optimized in Example 3. After the reaction, the difference in fluorescence signal of each channel was counted and the results were analyzed.

[0152] 3. Routine PCR testing PCR detection was performed using specific primer pairs targeting the Asp-376-Glu mutation site, namely Asp-376-Glu MPCR-F (SEQ ID NO.7) and Asp-376-Glu MPCR-R (SEQ ID NO.8), with a diluted genomic DNA solution of D376E Amaranthus retroflexus as a template.

[0153] The PCR reaction system (15 μL) consisted of: 12.5 µL 2×Taq Platinum PCR Mix, 1 µL Asp-376-GluMPCR-F (SEQ ID NO.1) (10 µM), 1 µL Asp-376-Glu MPCR-R (SEQ ID NO.2) (10 µM), 9.5 µL ddH2O, and 1 µL template.

[0154] The PCR reaction procedure was consistent with the single-target PCR reaction conditions in Example 1.

[0155] After the reaction was completed, the amplification products were analyzed by 1% agarose gel electrophoresis.

[0156] 4. Test Results (1) Detection results of multiplex PCR-PfAgo The detection results of the CY5 channel are as follows Figure 8 As shown in A, the limit of detection for the Asp-376-Glu mutation site established in this invention is 0.12 ng / µL.

[0157] The detection results of the ROX channel are as follows: Figure 8 As shown in B, the limit of detection for the Trp-574-Leu mutation site using the multiplex PCR-PfAgo method established in this invention is 0.37 ng / µL.

[0158] The detection results of the FAM channel are as follows Figure 8 As shown in C, the limit of detection for the Ser-653-Tyr mutation site established in this invention is 0.37 ng / µL.

[0159] (2) Results of routine PCR testing like Figure 8 As shown in D, the limit of detection for conventional PCR is 0.12 ng / µL.

[0160] The above results indicate that the multiplex PCR-PfAgo detection system described in this invention is comparable to conventional PCR methods in terms of sensitivity, and both can stably detect target mutation sites under low template concentration conditions.

[0161] Example 5: Simulated detection of multiple mutations under coexisting conditions using multiple PCR-PfAgo method Given that multiple resistance mutation sites may coexist in the same plant or sample during the evolution of herbicide resistance in weeds, and that studies have reported the coexistence of different mutation sites in a single target gene in some weeds.

[0162] However, there are currently no natural samples of *Amaranthus retroflexus* carrying multiple mutation sites simultaneously. To address potential scenarios of multiple mutation coexistence and to verify the detection stability and interpretation reliability of the multiplex PCR-PfAgo method established in this invention under conditions of multiple mutation coexistence, this embodiment constructs simulated samples with multiple mutation coexistence and tests them to evaluate the robustness of the detection system and its ability to simultaneously identify multiple sites.

[0163] 1. Construction of simulated samples with multiple coexisting mutations In this embodiment, genomic DNA from Amaranthus retroflexus plants with different mutations was used to construct a simulated sample with multiple mutations coexisting in a mixed combination manner.

[0164] Genomic DNA of D376E Amaranthus retroflexus with an initial concentration of 30 ng / µL and genomic DNA of W574L Amaranthus retroflexus with an initial concentration of 30 ng / µL were taken in 1.5 μL each, mixed well, and a double mutant simulated sample containing Asp-376-Glu and Trp-574-Leu was obtained, denoted as 376+574.

[0165] Genomic DNA of D376E Amaranthus retroflexus with an initial concentration of 30 ng / µL and genomic DNA of S653Y Amaranthus retroflexus with an initial concentration of 30 ng / µL were taken in 1.5 μL each, mixed well, and a double mutant simulated sample containing Asp-376-Glu and Ser-653-Tyr was obtained, denoted as 376+653.

[0166] Genomic DNA of W574L Amaranthus retroflexus with an initial concentration of 30 ng / µL and genomic DNA of S653Y Amaranthus retroflexus with an initial concentration of 30 ng / µL were taken in 1.5 μL each, mixed well, and a double mutant simulated sample containing Trp-574-Leu and Ser-653-Tyr was obtained, denoted as 574+653.

[0167] One μL of each of the following genomic DNA samples (initial concentrations of 30 ng / µL): D376E, W574L, and S653Y (initial concentrations of 30 ng / µL) were taken and mixed to obtain a triple-mutant simulated sample containing Asp-376-Glu, Trp-574-Leu, and Ser-653-Tyr, denoted as 376+574+653.

[0168] 2. Multiplex PCR-PfAgo detection Using the four simulated samples constructed above as templates, the detection was performed according to the multiplex PCR-PfAgo method established in Example 2 combined with the PfAgo cleavage reaction system optimized in Example 3, and the detection results were interpreted according to the interpretation criteria established in Example 3.

[0169] 3. Test Results The fluorescence signal detection results of the four simulated samples are as follows: Figure 9 As shown, each simulated sample showed a corresponding effective fluorescence signal in its respective fluorescence channel.

[0170] The threshold (987 au) for D obtained in Example 3 cannot simultaneously distinguish mutations at three sites. This is because the threshold (987 au) for D was obtained under single-site mutation detection conditions. While it has extremely high accuracy in single-site mutation interpretation, the overlapping of multiple mutation sites leads to competition among multiple gDNA target cleavage reactions, thereby altering the fluorescence kinetics and causing changes in the real-time fluorescence curve. Simultaneously, the proportion of single-site mutation signal decreases, resulting in a decrease in overall fluorescence intensity. Therefore, it cannot be directly applied to the detection of more complex multi-site mutations.

[0171] To establish a robust and widely applicable interpretation standard, the fluorescence signal obtained from the single-site mutation detection in Example 3 was further compared with that in this example. Figure 9 The fluorescence signal data obtained from the corresponding multi-site mutation detection were merged and ROC analysis was performed. The comprehensive analysis results showed that the optimal threshold for D was 563 au, at which point the AUC was 1.

[0172] Based on the above characteristics, this invention establishes a multi-site mutation interpretation rule without the need for standards. Specifically: when only one fluorescent channel simultaneously satisfies D>563 au, it is determined that the target site corresponding to that fluorescent channel in the sample contains a mutation; when only two fluorescent channels simultaneously satisfy D>563 au, it is determined that multiple target sites corresponding to those two fluorescent channels in the sample contain mutations; when the D of all three fluorescent channels is >563 a.u., it is determined that the sample contains mutations at the Asp-376-Glu, Trp-574-Leu, and Ser-653-Tyr sites; when the D of all three fluorescent channels is ≤563 au, it is determined that the sample does not contain mutations at the above three sites.

[0173] Specifically, for each target site, the mutation interpretation rules are as follows: When the sample only has a D>563 au value in the CY5, ROX, or FAM channels, then the sample corresponds to only the Asp-376-Glu, Trp-574-Leu, or Ser-653-Tyr mutations; when the sample only has a D>563 au value in the CY5 and ROX channels, then the sample corresponds to only the Asp-376-Glu and Trp-574-Leu mutations; when the sample only has a D>563 au value in the CY5 and FAM channels, then the sample corresponds to only the Asp-376-Glu and Ser-653-Tyr mutations; when the sample only has a D>563 au value in the ROX ... Trp-574-Leu mutations; when the sample only has a D>563 au value in the ROX and FAM channels, then the sample corresponds to only the Asp-376-Glu and Trp-574-Leu mutations; when the sample only has a D>563 au value in the ROX and FAM channels, then the sample corresponds to only the Asp-376-Glu and Trp-574-Leu mutations; when the sample only has a D>563 au value If the value of D in the CY5, ROX, and FAM channels is greater than 563 au, then the sample to be tested only contains the Asp-376-Glu, Trp-574-Leu, and Ser-653-Tyr mutations; if the value of D in the CY5, ROX, and FAM channels is less than or equal to 563 a.u., then the sample to be tested does not contain the Asp-376-Glu, Trp-574-Leu, and Ser-653-Tyr mutations.

[0174] This demonstrates that, under conditions of multiple mutations coexisting, the detection system described in this invention can simultaneously identify different mutation sites based on established interpretation criteria. The optimal threshold of D (563 au) has the ability to interpret both single-site and multi-site mutations, thus achieving wider applicability.

[0175] Example 6: Feasibility evaluation of the multiplex PCR-PfAgo method in field samples To evaluate the feasibility of the multiplex PCR-PfAgo detection system described in this invention under field conditions, detection experiments were conducted using real samples.

[0176] 1. Sample Information Seeds of D376E, W574L, S653Y, and wild-type Amaranthus retroflexus were cultured to the 3-4 leaf stage. Whole-plant bioassays were conducted using methoxyfenozide (an ALS-inhibiting herbicide) as the treatment agent, referring to the study "Multiple Resistance to PS II-Inhibiting and ALS-Inhibiting Herbicides in Common Lambsquarters". Chenopodium album The method used in "L.) from China" (https: / / doi.org / 10.3390 / agronomy15061309) was used to assess the resistance level of each plant to the herbicide.

[0177] Subsequently, samples identified as resistant and sensitive were taken and subjected to DNA extraction, PCR amplification, and Sanger sequencing analysis in sequence.

[0178] The results showed that D376E, W574L, and S653Y amaranth were resistant to methoxyfenozide, and sequencing results revealed Asp-376-Glu, Trp-574-Leu, and Ser-653-Tyr mutations in their ALS genes, respectively; while wild-type amaranth was sensitive to methoxyfenozide, and none of the above three mutations were detected in its ALS gene.

[0179] Based on the above results, five plants were randomly selected from each of the three resistant populations containing the mutation and the sensitive population without the mutation, resulting in a total of 20 field samples of Amaranthus retroflexus plants, which were used as the subjects for subsequent experiments.

[0180] 2. Detection Experiment (1) Experimental methods 1) Multiplex PCR-PfAgo method The blind samples obtained in the previous step were tested using the multiplex PCR-PfAgo method established in Example 2 combined with the optimized PfAgo cleavage reaction system obtained in Example 3. The operation procedure is as follows: Figure 10 As shown, the specific method is as follows: After cutting each leaf sample into small pieces, place them in a centrifuge tube, add the total nucleic acid rapid extraction reagent, and mix thoroughly by hand shaking to achieve rapid extraction of sample DNA. The whole process takes about 5 minutes.

[0181] The extracted DNA was added to a pre-configured multiplex PCR reaction system and placed at the bottom of a PCR tube. Meanwhile, all components of the PfAgo cutting reaction system except for the multiplex PCR amplification product were added to a special nested tube (i.e., the fully enclosed integrated reaction tube in the prior art CN214218716U). The nested tube was then embedded into the PCR tube containing the multiplex PCR reaction system to obtain a composite tube.

[0182] Subsequently, the composite tube was placed in a qPCR instrument for multiplex PCR reaction.

[0183] After the multiplex PCR reaction is completed, the PfAgo cleavage reaction system in the nested tube is mixed with the multiplex PCR amplification product by centrifugation or gentle shaking. Then, the PfAgo cleavage reaction is performed at 95°C. Under the mediation of PfAgo endonuclease and gDNA, the target PCR amplification product is specifically cleaved to generate secondary gDNA. The fluorescent probe binds to the secondary gDNA to generate a fluorescent signal.

[0184] After the PfAgo cleavage reaction was completed, the fluorescence signals of each fluorescence channel were collected using a qPCR instrument, and mutation interpretation was performed according to the method in Example 5.

[0185] 2) Sequencing analysis and alignment PCR sequencing analysis was performed on each leaf sample, and the sequencing results were compared with the results of the multiplex PCR-PfAgo method to evaluate the accuracy of the detection.

[0186] (2) Experimental results Table 3. Detection accuracy of the multiplex PCR-PfAgo method

[0187] Note: The three mutations refer to the Asp-376-Glu mutation, the Trp-574-Leu mutation, and the Ser-653-Tyr mutation.

[0188] As shown in Table 3 and Figure 11As shown, when using the multiplex PCR-PfAgo method established in this invention, combined with rapid DNA extraction reagents and a nested PCR tube structure, to detect mutations in actual field samples, the mutation interpretation results for each sample were consistent with the PCR sequencing analysis results, with an accuracy rate of 100%, and no false positives or false negatives occurred. This indicates that the multiplex PCR-PfAgo method established in this invention has excellent detection accuracy and result reliability.

[0189] Furthermore, the entire detection process, from rapid sample processing, DNA extraction, multiplex PCR amplification to PfAgo-mediated fluorescence signal interpretation, is simple to operate and tightly integrated, and can be completed in about 2.5 hours, significantly shortening the time required for traditional laboratory testing procedures.

[0190] Furthermore, by introducing a nested tube structure, the present invention enables the PCR amplification reaction and the PfAgo detection reaction to be completed sequentially in the same closed system, avoiding frequent opening of the cap during the reaction process, effectively reducing the risk of cross-contamination, and significantly improving the stability and reliability of the detection system in field conditions.

[0191] In summary, the multiplex PCR-PfAgo detection system established in this invention, combined with the PCR nested tube structure, can achieve rapid and accurate detection of resistance mutations in Amaranthus retroflexus in non-laboratory environments when further integrated with portable qPCR devices in the future. It has good field applicability and prospects for widespread application.

[0192] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer composition for detecting amino acid mutation sites in ALS of Amaranthus retroflexus, characterized in that, The amino acid mutation sites include: D376E, W574L, and S653Y; The primer composition comprises primer pairs 1 to 3; wherein, Primer pair 1 comprises primers with nucleotide sequences as shown in SEQ ID NO. 1 and 2; Primer pair 2 includes primers with nucleotide sequences as shown in SEQ ID NO. 3 and 4; The primer pair 3 includes primers with nucleotide sequences as shown in SEQ ID NO. 5 and 6.

2. A gDNA composition for detecting amino acid mutation sites in Amaranthus retroflexus ALS, characterized in that, The amino acid mutation sites include: D376E, W574L, and S653Y; The gDNA composition comprises gDNA1 to gDNA3; wherein... The gDNA1 comprises a nucleotide sequence as shown in SEQ ID NO.7; The gDNA2 comprises a nucleotide sequence as shown in SEQ ID NO. 9; The gDNA3 comprises a nucleotide sequence as shown in SEQ ID NO.11; Preferably, the 5' ends of gDNA1 to gDNA3 are phosphorylated.

3. A probe composition for detecting amino acid mutation sites in ALS of Amaranthus retroflexus, characterized in that, The amino acid mutation sites include: D376E, W574L, and S653Y; The probe composition includes the following probes 1 to 3; wherein... The probe 1 comprises a nucleotide sequence as shown in SEQ ID NO. 8; The probe 2 comprises a nucleotide sequence as shown in SEQ ID NO. 10; The probe 3 comprises a nucleotide sequence as shown in SEQ ID NO. 12; Probes 1 to 3 are equipped with different fluorescent groups.

4. A reagent kit, characterized in that, It includes: The primer composition of claim 1, the gDNA composition of claim 2, the probe composition of claim 3, and the PfAgo endonuclease.

5. The use of the primer composition of claim 1, the gDNA composition of claim 2, the probe composition of claim 3, or the kit of claim 4 in detecting amino acid mutation sites in Amaranthus retroflexus ALS and / or identifying resistance of Amaranthus retroflexus to ALS inhibitor herbicides.

6. A method for simultaneously detecting amino acid mutation sites in ALS of Amaranthus retroflexus, characterized in that, The amino acid mutation sites include: D376E, W574L, and S653Y; The method includes: using the DNA of the sample to be tested as a template, performing multiplex PCR amplification with the primer composition of claim 1; performing a PfAgo cleavage reaction on the obtained multiplex PCR amplification product with the gDNA composition of claim 2, the probe composition of claim 3, and PfAgo endonuclease; detecting the fluorescence signal, and determining whether there is a mutation at the amino acid mutation site in the sample to be tested based on the obtained fluorescence signal.

7. The method according to claim 6, characterized in that, The PfAgo cleavage reaction system comprises: gDNA at working concentrations of 1.5µM to 2.5µM, and probes at working concentrations of 500 nM to 2500 nM; the gDNA is the gDNA described in the gDNA composition, and the probes are the probes described in the probe composition. Preferably, the PfAgo cleavage reaction system further includes: PfAgo endonuclease at a working concentration of 0.08 U / μL to 0.12 U / μL.

8. The method according to claim 6 or 7, characterized in that, The multiplex PCR reaction system includes primers with working concentrations of 100 nM to 300 nM, wherein the primers are those described in the primer composition.

9. The method according to any one of claims 6 to 8, characterized in that, The multiplex PCR reaction program includes: pre-denaturation at 92℃~96℃ for 1.5 min~2.5 min; denaturation at 92℃~96℃ for 25 s~35 s, annealing at 58℃~62℃ for 40 s~50 s, extension at 63℃~67℃ for 0.5 min~1.5 min, for 30 to 35 cycles; final extension at 63℃~67℃ for 4 min~6 min; and / or, The PfAgo cleavage reaction procedure includes: reacting at 94℃~96℃ for 40 min~60 min.

10. A method for identifying the resistance of Amaranthus retroflexus to ALS inhibitor herbicides, characterized in that, include: The mutation status of amino acid mutation sites in the ALS of the test sample is detected by the method according to any one of claims 6 to 9; if any one or more of D376E, W574L and S653Y are mutated, the test sample is determined to be resistant to ALS inhibitor herbicides.

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