TdT signal amplification PAM-independent cas12a lateral flow nucleic acid detection method and application

Abstract

The application discloses a TdT signal amplification PAM-independent Cas12a lateral chromatography nucleic acid detection method and application. The method comprises the following steps: performing a first reaction on a first reaction system comprising a to-be-detected nucleic acid, a recombinase polymerase amplification reaction component, a recombinase polymerase amplification primer pair and an exonuclease, so as to obtain a single-stranded target nucleic acid; performing a second reaction on a second reaction system comprising the single-stranded target nucleic acid, a CRISPR / Cas12a reaction component, a reporter probe, a terminal deoxynucleotidyl transferase and a deoxynucleotide triphosphate substrate, so as to obtain an extension product; and contacting and hybridizing the extension product with a gold nanoparticle-labeled detection probe in a lateral chromatography process, and when a visual detection signal is obtained, it is judged that the to-be-detected nucleic acid is a target nucleic acid. The method of the application can significantly improve the detection sensitivity and signal contrast while getting rid of the PAM restriction, the detection process does not need complex instruments, the result can be judged by naked eyes, and the method is suitable for on-site rapid visual detection of pathogenic microorganism nucleic acid.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection and biosensing technology, and relates to a PAM-independent Cas12a lateral chromatography nucleic acid detection method and its application based on TdT signal amplification. Specifically, it relates to a PAM-independent CRISPR / Cas12a lateral chromatography visualization nucleic acid detection method and its application based on terminal deoxynucleotidyl transferase (TdT) extension signal amplification. Background Technology

[0002] Nucleic acid detection technology has significant application value in areas such as rapid screening of foodborne pathogens, public health safety monitoring, and point-of-care testing (POCT). Especially in resource-constrained or field environments, there is an urgent need to develop molecular detection methods that are highly sensitive, easy to operate, and do not require large instruments. In recent years, molecular diagnostic technologies based on clustered regularly spaced short palindromic repeats (CRISPR) and related proteins have developed rapidly. Among them, nucleic acid detection methods utilizing the trans-cleavage activity of CRISPR / Cas12a have attracted widespread attention due to their good programmability, single-base resolution, and good compatibility with isothermal amplification techniques. By combining Cas12a recognition with front-end amplification strategies such as recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), significant progress has been made in the analytical sensitivity and target adaptability of related detection systems.

[0003] However, existing CRISPR / Cas12a nucleic acid detection technologies still face several technical bottlenecks in their transformation into visualized and portable detection methods such as lateral chromatography strips. Firstly, Cas12a strictly relies on specific protospacer adjacent motifs (PAMs) when recognizing double-stranded DNA targets. Its recognition sequence is typically TTTV, which theoretically has a low frequency of occurrence, significantly limiting the range of selectable target regions. For certain conserved gene fragments, mutation hotspots, or highly variable pathogen sequences, suitable PAM sites may be lacking, leading to limitations in detection system design. Although some studies have attempted to relax PAM requirements by modifying Cas proteins or optimizing guide RNA structures, these often result in decreased enzyme activity, reduced specificity, or limited applicability, failing to meet the needs of universal detection platforms. Furthermore, while Cas12a can recognize single-stranded DNA in the absence of PAMs, existing single-stranded DNA preparation strategies typically rely on asymmetric amplification or multi-step enzymatic digestion, which are complex and inefficient, hindering on-site detection applications.

[0004] On the other hand, the signal readout method is also a key factor limiting the performance of CRISPR lateral chromatography. Traditional CRISPR lateral chromatography systems typically achieve detection based on the "signal attenuation" mechanism after the reporter probe is cleaved by the Cas enzyme, which is essentially a signal-off mode. Under low target concentration conditions, this mode has low visual contrast, and strong background signals are easily confused with weak positive results, affecting the accuracy of interpretation. To improve the intuitiveness of the results, some studies have constructed so-called "signal-on" detection modes by adjusting the functional definitions of the T and C lines on the test strip or optimizing the nanoparticle capture strategy. However, this type of design still relies on the signal redistribution caused by the consumption of the reporter molecule, which is not a true signal amplification process. The system is highly sensitive to the concentration and capture capacity of the reporter molecule. It is difficult to completely suppress the background signal under negative conditions, and under positive conditions, the detection sensitivity and dynamic range are affected by the limited amount of reporter molecule, making it difficult to simultaneously meet the requirements of high contrast and high sensitivity.

[0005] In summary, existing CRISPR / Cas12a nucleic acid detection technologies are constrained by PAM-dependent target selection limitations, low signal readout contrast, and insufficient amplification capabilities, making it difficult to meet the practical application requirements of rapid, highly sensitive, and highly visual contrast detection on-site. Summary of the Invention

[0006] The main objective of this invention is to provide a TdT signal amplification PAM-independent Cas12a lateral chromatography nucleic acid detection method and its application, in order to overcome the shortcomings of the prior art.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution.

[0008] According to one aspect of the present invention, a TdT signal amplification PAM-independent Cas12a lateral chromatography method for nucleic acid detection is provided, comprising: A first reaction is performed on a first reaction system containing the nucleic acid to be tested, a recombinase polymerase amplification reaction component, a recombinase polymerase amplification primer pair, and an exonuclease to generate a recombinase polymerase amplification product. The exonuclease selectively digests the amplification product containing the amplification strand with 5' end phosphorylation modification to obtain a single-stranded target nucleic acid. In the primer pair, the 5' end of one primer strand is phosphorylated. A second reaction is performed on a second reaction system comprising the single-stranded target nucleic acid, a CRISPR / Cas12a reaction assembly, a reporter probe, a terminal deoxynucleotidyl transferase, and a deoxynucleoside triphosphate substrate, so that the single-stranded target nucleic acid activates the trans-cleavage activity of Cas12a in a PAM-independent manner and cleaves the reporter probe, generating a cleavage product with a 3'-hydroxy terminus and retaining a 5'-terminal capture label group. The cleavage product is then subjected to template-independent extension under TdT catalysis with the 3'-hydroxy terminus as the starting site, thereby obtaining an extension product, wherein the reporter probe has a 5'-terminal capture label group and a 3'-terminal phosphorylation modification; The extended product is driven by capillary action to migrate along the lateral chromatography support and come into contact with the detection probe labeled with gold nanoparticles and undergo nucleic acid hybridization to form an extended product-detection probe complex. When the extended product-detection probe complex is captured at the detection line and generates a visual detection signal, the nucleic acid to be tested is determined to be the target nucleic acid.

[0009] In one embodiment, the method includes: after the first reaction is completed, performing enzyme inactivation treatment on the obtained first reaction system, taking a certain volume of the enzyme-inactivated first reaction product, and adding a CRISPR / Cas12a reaction component, a reporter probe, a terminal deoxynucleotidyl transferase and a deoxynucleoside triphosphate substrate to it, thereby forming the second reaction system.

[0010] In one embodiment, the recombinase polymerase amplification product is an isothermal amplification reaction product.

[0011] In one embodiment, the exonuclease includes Lambda exonuclease.

[0012] In one embodiment, the reaction temperature of the first reaction is 35–42°C, and the reaction time is 15–40 minutes.

[0013] In one embodiment, the reaction temperature of the second reaction is 35–42°C, and the reaction time is 30–90 minutes.

[0014] In one embodiment, the reporter probe is a reporter probe with a hairpin structure, and the 3' end phosphorylation modification of the reporter probe is used to inhibit the extension of the reporter probe that has not been cleaved by Cas12a by the terminal deoxynucleotidyl transferase.

[0015] In one embodiment, the 5' end capture label group is biotin, and the capture molecule fixed at the detection line is streptavidin or avidin.

[0016] In one embodiment, the deoxynucleoside triphosphate substrate includes any one of dGTP, dATP, dCTP, and dTTP, preferably dGTP.

[0017] In one embodiment, the gold nanoparticle-labeled detection probe comprises a nucleic acid sequence capable of complementary hybridization with the extended product; preferably, when the deoxynucleoside triphosphate substrate is dGTP, the detection probe comprises a polyC sequence. Preferably, the detection probe has the sequence shown in SEQ ID NO. 5.

[0018] In one embodiment, the lateral chromatography carrier is a lateral chromatography test strip. The binding pad of the lateral chromatography test strip is pre-coated with a detection probe labeled with gold nanoparticles, and a capture molecule capable of recognizing and binding to the 5' end capture label group of the extended product is fixed at the detection line. When the nucleic acid to be tested is the target nucleic acid, the extended product binds to the detection probe through nucleic acid hybridization and is captured by the capture molecule through its 5' end capture label group, thereby enriching the gold nanoparticles at the detection line and generating a visual color signal.

[0019] The method described in this invention transforms the Cas12a-mediated transient nuclease cleavage event into a continuously accumulating long-chain extension product by coupling the reporter probe cleavage with TdT template-independent extension. Then, through nucleic acid hybridization between the extension product and the gold nanoparticle-labeled detection probe, signal-enhanced lateral tomography visualization readout is achieved, improving the contrast of the detection signal, the accuracy of visual interpretation, and the detection sensitivity.

[0020] In a more specific implementation, the method includes the following steps: S1, Target Single-Stroke Generation: The extracted nucleic acid from the sample to be tested, the upstream and downstream primers of RPA, the RPA reaction system, and the Lambda exonuclease are added to the same first reaction system for the first reaction. One of the primer strands has a 5' phosphorylation modification, which allows the amplified strand with the 5' phosphorylation modification in the amplification product to be selectively digested by the Lambda exonuclease, while retaining the complementary single-stranded DNA as the Cas12a recognition substrate, thereby directionally converting the double-stranded amplification product into a single-stranded target nucleic acid. The preferred reaction temperature is 35–42℃, and the reaction time is 15–40 minutes.

[0021] S2, PAM-independent CRISPR / Cas12a recognition and TdT signal amplification: The single-stranded target nucleic acid generated in step S1 is incubated at a constant temperature in the same second reaction system with the CRISPR / Cas12a reaction assembly, reporter probe, terminal deoxynucleotidyl transferase, and deoxynucleoside triphosphate substrate. The single-stranded target nucleic acid activates the trans-cleavage activity of Cas12a in a PAM-independent manner. After Cas12a cleaves the reporter probe, it produces a cleavage product with a 3'-hydroxyl terminus and retaining a 5'-terminal capture label group. This 3'-hydroxyl terminus undergoes template-independent extension under TdT catalysis, forming a long-chain extension product capable of complementary hybridization with the detection probe, thereby achieving signal amplification. The preferred reaction temperature is 35–42°C, and the reaction time is 30–90 minutes.

[0022] S3, Lateral tomography visualization inspection: The extended product obtained in step S2 is applied to a lateral chromatography test strip, allowing it to migrate along the strip under capillary action and hybridize with a gold nanoparticle-labeled detection probe pre-coated within the binding pad, forming an extended product-detection probe complex. This complex is captured at the detection line by a capture molecule via the 5' end of the extended product's capillary group, resulting in the enrichment of gold nanoparticles at the detection line and the generation of a visual colorimetric signal. When this visual colorimetric signal is obtained, the nucleic acid to be tested is determined to be the target nucleic acid.

[0023] Furthermore, in step S3, the visualized detection signal is a signal-on readout where the test strip detection line shows enhanced color development, and the signal intensity is positively correlated with the target nucleic acid concentration.

[0024] Furthermore, the method can be used for the detection of pathogenic microorganism nucleic acids in food, environmental, or public health monitoring samples for non-disease diagnostic purposes.

[0025] Compared with existing technologies, the present invention has at least the following beneficial effects: First, by designing 5' phosphorylation primers, the present invention integrates RPA amplification and Lambda exonuclease-mediated strand-selective digestion into the same first reaction system, enabling the double-stranded amplification product to be directionally converted into single-stranded target nucleic acid in the same reaction system, thereby providing Cas12a with a PAM-independent recognition substrate. Compared with conventional stepwise preparation of single-stranded nucleic acid or methods relying on external regulatory strategies, this design reduces intermediate processing steps in the preparation of single-stranded nucleic acids after amplification, reducing operational complexity and the risk of contamination from multi-step transfer. Second, the present invention couples Cas12a reporter probe cleavage and TdT template-independent extension into the same second reaction system, allowing the 3'-hydroxyl terminus generated by Cas12a cleavage to further serve as the TdT extension initiation site, transforming the transient nuclease cleavage event into a continuously accumulating long-chain extension product, thus amplifying the enzymatic cascade signal. Furthermore, this invention utilizes multi-site nucleic acid hybridization between the extended product and the gold nanoparticle-labeled detection probe, enabling the extended product to be captured and enriched with nanoparticles at the lateral chromatography detection line, thereby forming a signal-enhanced visual readout. This method differs from the signal-attenuation readout mode in traditional CRISPR / Cas12a lateral chromatography detection that relies on reporter probe consumption, and also from the apparent signal-enhancing mode achieved solely by changing the T / C line definition and regulating the signal probe distribution. It can reduce negative background signals and improve positive signal contrast and visual interpretation accuracy. Thus, this invention achieves synergistic enhancement between PAM-independent recognition, signal amplification, and lateral chromatography visual detection through RPA-Lambda one-pot substrate reconstruction, Cas12a-TdT one-pot cascade amplification, and extended product-mediated detection line enrichment readout. It has the advantages of simple operation, high sensitivity, high signal contrast, and good applicability for on-site detection. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application 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 only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram illustrating the principle of a PAM-independent Cas12a lateral chromatography nucleic acid detection method with TdT signal amplification in Embodiment 1 of the present invention. Figure 2 This is a visualization analysis of the detection performance of Salmonella in Example 2; Figure 3 This is a graph showing the quantitative detection performance of Salmonella in Example 2; Figure 4 The standard curve and linear fitting analysis diagram for the detection of Salmonella in Example 2 are shown. Figure 5 This is a comparison chart of the detection effects of the method of the present invention in Example 3 with other detection modes; Figure 6 This is a visualization of the detection results of Salmonella in the milk sample in Example 4; Figure 7 This is a graph showing the quantitative detection results of Salmonella in the milk sample in Example 4; Figure 8 The standard curve and linear fitting analysis diagram for the detection of Salmonella in milk samples in Example 4 are shown. Detailed Implementation

[0028] As mentioned above, existing CRISPR / Cas12a lateral chromatography nucleic acid detection technologies suffer from drawbacks such as PAM dependence, low signal contrast, insufficient signal amplification, and complex operation. Therefore, this invention provides a TdT-amplified Cas12a non-PAM-dependent visual nucleic acid detection method. This method constructs a one-pot, dual-module sequential reaction system to achieve cascaded coupling of target single-strand generation, CRISPR-specific recognition, and terminal extension signal amplification, significantly improving detection sensitivity and visual signal contrast while simplifying operation and reducing equipment costs.

[0029] See Figure 1An embodiment of the present invention provides a PAM-independent Cas12a lateral chromatography nucleic acid detection method with TdT signal amplification, comprising: in the upstream homogeneous reaction stage, using RPA amplification to generate double-stranded DNA amplification products. The RPA primers are designed so that one of the amplification strands is phosphorylated at the 5' end, thereby forming double-stranded DNA with chain polarity. After amplification, Lambda exonuclease selectively recognizes and continuously degrades the strand with the 5' phosphate group along the 5'→3' direction, while the complementary strand is retained. Through this chain-selective exonucleation reaction, the double-stranded DNA is efficiently and directionally converted into single-stranded DNA, thereby reconstructing the structural form of the CRISPR recognition substrate at the molecular level. The generated single-stranded DNA can directly bind to the Cas12a / crRNA complex without relying on the PAM sequence, thereby activating the trans-cleavage activity of Cas12a in a PAM-independent manner. The activated Cas12a further cleaves the pre-designed biotin- and phosphate-labeled hairpin reporter probe (Bio-hairpin-P) in the system, destroying its hairpin structure and exposing the 3'-hydroxyl (3'-OH) end. The newly generated 3'-OH terminus serves as the initiation site for the TdT-catalyzed reaction. In the presence of dGTP, TdT performs template-independent extension of the cleaved reporter probe, forming a long-chain polyG sequence structure. This process transforms the transient nuclease cleavage event into a continuously accumulating nucleotide polymerization reaction, thereby constructing a downstream enzymatic signal amplification module and achieving cascade signal enhancement at the molecular level. During the lateral chromatographic signal transduction stage, the extended polyG sequence product can hybridize and bind to the gold nanoparticle recognition probe at multiple sites, achieving high-density nanoparticle loading. During chromatography, the biotin group at the 5' end of the reporter probe is specifically captured by streptavidin immobilized on the detection line (T line), causing the extended product loaded with gold nanoparticles to accumulate at the T line and generate a visible color signal. The unreacted gold nanoparticle recognition probe continues to migrate and is bound to the complementary capture probe at the control line (C line), thereby verifying the effectiveness of the test strip. Therefore, in negative samples, since the target nucleic acid does not trigger Cas12a activation, the reporter probe is not cleaved and does not undergo TdT extension, and the system cannot form an effective nanoparticle enrichment structure; the test strip only displays the C line. In positive samples, however, the target nucleic acid sequentially triggers single-strand generation, Cas12a activation, and TdT extension reactions, ultimately resulting in colored bands appearing simultaneously on both the T and C lines, enabling intuitive signal activation visualization. By integrating the RPA-Lambda exonuclease-mediated substrate reconstruction module with the Cas12a-TdT enzymatic cascade amplification module into a sequential two-stage one-pot reaction system, this invention achieves efficient coupling of amplification, recognition, and signal transduction while significantly reducing operational complexity and the risk of contamination from opening the lid, establishing a robust new PAM-independent CRISPR lateral chromatography ultrasensitive visualization detection mode.

[0030] The technical solution of the present invention will be further described in detail below with reference to several embodiments. These embodiments are implemented on the premise of the technical solution of the invention, and provide detailed implementation methods and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0031] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies. Unless otherwise specified, the experimental methods used in the following examples were performed in accordance with textbooks and reference books in this field (such as *A Concise Guide to Molecular Biology Experiments*, Science Press, January 2025; *A Concise Guide to Molecular Biology Experiments (Fifth Edition)*, Science Press, 2008; *CRISPR Gene Editing Technology*, edited by Liu Shili, Chemical Industry Press, January 2021).

[0032] In this embodiment, a component missing control experiment was conducted to verify the reaction integrity and reliability of the TdT-amplified Cas12a non-PAM-dependent visualization nucleic acid detection method.

[0033] The sequences of the forward primers used in the following examples are shown in SEQ ID NO. 1, specifically: 5'-ACCCATTTGTATTGGTTGTTACGGCTATTTTG-3'; the sequences of the reverse primers are shown in SEQ ID NO. 2, specifically: 5'-GCGGCTGCTCGCCTTTGCTGGTTTTAGGTTTG-3'; Lambda exonuclease, Cas12a and TdT enzyme were purchased from NEB; crRNA was purchased from Bailige Biotechnology Co., Ltd.

[0034] Bio-Focus-P is a reporter probe purchased from GeneBio Inc. The sequence of the reporter probe is shown in SEQ ID NO. 3, specifically: 5'-Bio-CTCTCATTTTTTTTTTAGAGAG-P-3'.

[0035] The sequence of the lateral chromatography reporter probe is shown in SEQ ID NO. 4, specifically: 5'-Bio-CTCTCATTTTTTTTTTAGAGAG-FITC-3'.

[0036] Example 1: Preparation of gold nanoparticles and gold nanoparticle labeled detection probes This embodiment provides a method for preparing gold nanoparticles and gold nanoparticle-labeled detection probes.

[0037] First, gold nanoparticles were prepared using the sodium citrate reduction method. Specifically, under magnetic stirring, 150 mL of deionized water was added to a round-bottom flask and heated to boiling; then 2.55 mL of HAuCl4 solution (5 g / L) was added, and stirring was continued while maintaining a gentle boil. Once the system stabilized at a gentle boil, 1.8 mL of 1% (w / v) trisodium citrate solution was quickly added to initiate the reduction reaction. During the reaction, the solution color gradually changed from light yellow to gray, black, purple, and finally to deep red. After the color stabilized, heating and stirring were continued for 5 minutes; then the heat source was removed, and stirring continued for 10 minutes. After the reaction solution cooled naturally to room temperature, the gold nanoparticle solution was obtained and stored at 4℃ for later use.

[0038] Subsequently, a gold nanoparticle-labeled detection probe was prepared. 1 mL of the above gold nanoparticle solution was added to 4 μL of 10% (v / v) Tween-80 and incubated at room temperature for 30 minutes for pretreatment. The solution was then centrifuged at 12000 rpm for 15 minutes, the supernatant was discarded, and the resulting precipitate was resuspended in 100 μL of PB buffer (10 mM, pH 7.4, containing 0.1 M NaCl and 0.05% Tween-80). Separately, 10 μL of the detection probe solution (10 μM) was mixed with 5 μL of TCEP solution (1 mM) and incubated in the dark for 1 hour to reduce disulfide bonds in the detection probe and expose free thiol groups. The detection probe is a thiol-modified nucleic acid probe, and its nucleotide sequence is shown in SEQ ID NO. 5: 5'-SH-TTTTTTTTTTTTTTTCCCCCCCCCCCCCCC-3'. The TCEP-activated detection probe solution was added to the above gold nanoparticle suspension and incubated at 50 °C for 2 hours, allowing the detection probe to bind to the gold nanoparticle surface via thiol groups, forming a gold nanoparticle-labeled detection probe. Subsequently, 10 μL of dTTP solution (100 μM) was added to the reaction system, and incubation was continued for 1 hour to block the remaining active sites on the gold nanoparticle surface. After the reaction, the mixture was centrifuged at 9000 rpm for 10 minutes, the supernatant was discarded, and the resulting purple-red precipitate was resuspended in 50 μL of resuspension buffer. The resuspension buffer was 10 mM PB buffer, pH 7.4, containing 1% BSA, 0.25% Tween-20, and 10% sucrose. The resulting gold nanoparticle-labeled detection probe was stored at 4 °C for later use.

[0039] Example 2: Sensitivity Evaluation of Salmonella Detection Using a PAM-Independent Cas12a Lateral Chromatography Nucleic Acid Detection Method with TdT Signal Amplification This embodiment is used to evaluate the sensitivity of the established method for detecting Salmonella. The Salmonella bacterial suspension was provided by the Anhui Provincial Institute of Product Quality Supervision and Inspection after culturing and counting using a culture method, with an initial concentration of 10.7 CFU / mL. Subsequently, genomic DNA was extracted from the bacterial culture using a commercially available bacterial genomic DNA extraction kit, and the obtained DNA was used as a detection template. The Salmonella DNA template was serially diluted according to the corresponding bacterial culture concentration, with concentration gradients set at 0, 10, and so on. 0 10 1 50, 10 2 500, 10 3 10 4 10 5 10 6 and 10 7 CFU / mL.

[0040] Under optimized conditions, single-stranded target nucleic acids were first generated via a one-pot reaction coupling RPA and Lambda exonuclease. Specifically, the first reaction system included one lyophilized RPA reaction particle, 18.3 μL of RNase-free ddH2O, 0.6 μL of forward primer (10 μM), 0.6 μL of reverse primer (10 μM), 1.5 μL of Lambda exonuclease (1000 U), 2 μL of DNA template, and 2 μL of MgOAc (250 mM). After thorough mixing, the mixture was incubated at 37 °C for 30 minutes to generate single-stranded target nucleic acids; after the reaction, the mixture was heated at 80 °C for 10 minutes to inactivate the enzymes in the system.

[0041] Subsequently, a PAM-independent Cas12a / TdT-mediated signal amplification system was constructed. The second reaction system consisted of 2 μL NEBuffer™ r2.1 (10×), 2 μL EnGen Lba Cas12a (1 μM), 2 μL crRNA (1 μM), 2 μL Bio-HP-P reporter probe (10 μM), 3 μL TdT buffer (10×), 3 μL CoCl2 (2.5 mM), 1 μL dGTP (25 mM), 1 μL TdT (20000 U), and 4 μL of the prepared single-stranded target nucleic acid, with RNase-free ddH2O added to bring the total volume to 30 μL. The reaction system was incubated at 37 °C for 60 min to allow Cas12a to cleave the reporter probe and further trigger TdT-catalyzed template-independent extension; after the reaction was completed, the reaction was terminated by heating at 80 °C for 10 min.

[0042] For lateral chromatography detection, 2 μL of gold nanoparticle-labeled detection probe, 1 μL of the second reaction product, and 45 μL of chromatography run buffer were mixed and added to the sample pad of the test strip. The chromatography run buffer consisted of 10 mM pH 7.4 Tris-HCl, 5 mM MgCl2, 5 mM KCl, 2×SSC, 1% Tween-20, 0.5% PEG20000, 1 mM CTAB, and 1% BSA. After 5 minutes of chromatography, the test strip image was captured using a smartphone or digital camera, and the gray intensity of the T line was quantitatively analyzed using ImageJ software.

[0043] Test results as follows Figure 2 As shown, with increasing Salmonella concentration, the T-line signal on the test strip gradually increased, exhibiting a clear concentration-dependent change, with the visual detection limit reaching 1 CFU / mL. Quantitative analysis based on the T-line gray intensity yielded the following results: Figure 3 As shown. Further linear fitting was performed on the logarithmic value of Salmonella concentration (X) using the T-line signal intensity (Y), resulting in the linear regression equation Y = 3429.474 + 6167.269X, with a correlation coefficient R0. 2 = 0.973, such as Figure 4 As shown. Based on this, the detection limit was calculated according to LOD = 3σ / k, where σ is the standard deviation of the blank sample signal and k is the slope of the calibration curve. The calculated detection limit for Salmonella by this method was 1.05 CFU / mL.

[0044] The above results demonstrate that the method of this embodiment can achieve rapid, intuitive, and sensitive detection of low concentrations of Salmonella, and can be quantitatively analyzed by the gray intensity of the T-line, verifying the sensitivity and visual interpretation capability of this method in Salmonella nucleic acid detection.

[0045] Example 3: Comparative Analysis of Different Detection Modes This embodiment compares the detection efficacy of different lateral chromatography detection modes for Salmonella. Four detection modes were set up: ① a traditional RPA-CRISPR / Cas12a two-step signal attenuation lateral chromatography detection mode; ② a PAM-independent Cas12a signal attenuation detection mode after one-pot generation of single-stranded target nucleic acid using RPA-Lambda exonuclease; ③ a stepwise TdT signal amplification detection mode based on the reaction principle of this invention; and ④ a PAM-independent Cas12a lateral chromatography nucleic acid detection method based on TdT signal amplification as described in this invention.

[0046] In detection mode ①, RPA amplification is performed first. The total volume of the RPA reaction system is 25 μL, including 1 lyophilized RPA reaction particle, 0.6 μL of forward primer (10 μM), 0.6 μL of reverse primer (10 μM), 2 μL of DNA extraction buffer, and 2 μL of MgOAc (250 mM). The volume is then increased to 25 μL with RNase-free ddH2O. The reaction is carried out at 37℃ for 15 minutes, followed by inactivation at 80℃ for 10 minutes. 4 μL of amplification product was subjected to Cas12a trans-cleavage reaction. The total reaction volume was 20 μL, including 4 μL of amplification product, 2 μL of NEBuffer™ r2.1 (10×), 2 μL of Cas12a (1 μM), 2 μL of crRNA (1 μM), and 0.3 μL of lateral chromatography reporter probe (1 μM). The volume was then brought to 20 μL with RNase-free ddH2O. The reaction was carried out at 37°C for 60 minutes, followed by inactivation at 80°C for 10 minutes. This method achieves interpretation by weakening or eliminating the detection line signal through Cas12a trans-cleavage of the reporter probe.

[0047] In detection mode ②, RPA amplification and Lambda exonuclease digestion were placed in the same reaction system. The total volume of the RPA-Lambda exonuclease reaction system was 25 μL, including 1 lyophilized RPA reaction particle, 0.6 μL of forward primer (10 μM), 0.6 μL of reverse primer (10 μM), 2 μL of DNA extraction buffer, 1.5 μL of Lambda exonuclease (1000 U), and 2 μL of MgOAc (250 mM). The volume was then increased to 25 μL with RNase-free ddH2O. The reaction was carried out at 37°C for 30 minutes, followed by inactivation at 80°C for 10 minutes. 4 μL of the reaction product was used for PAM-independent Cas12a activation. The total reaction volume was 20 μL, including 4 μL of reaction product, 2 μL of NEBuffer™ r2.1 (10×), 2 μL of Cas12a (1 μM), 2 μL of crRNA (1 μM), and 0.3 μL of lateral chromatography reporter probe (1 μM). The volume was then brought to 20 μL with RNase-free ddH2O. The reaction was carried out at 37°C for 60 minutes, followed by inactivation at 80°C for 10 minutes. This method utilizes single-stranded target nucleic acid to achieve PAM-independent Cas12a activation, but still employs signal attenuation readout.

[0048] Detection mode ③ is the stepwise mode of the method of this invention. First, RPA amplification is performed. The total volume of the RPA reaction system is 25 μL, including one lyophilized RPA reaction particle, 0.6 μL of forward primer (10 μM), 0.6 μL of reverse primer (10 μM), 2 μL of DNA template, and 2 μL of MgOAc (250 mM). RNase-free ddH2O is added to bring the volume to 25 μL, and the reaction is carried out at 37°C for 15 minutes, followed by inactivation at 80°C for 10 minutes. Next, Lambda exonuclease digestion is performed. 1.5 μL of Lambda exonuclease (1000 U) is added to the RPA amplification product, and the reaction is carried out at 37°C for 15 minutes, followed by inactivation at 80°C for 10 minutes to generate single-stranded target nucleic acid. Take 4 μL of single-stranded target nucleic acid for PAM-independent Cas12a recognition reaction. The total reaction volume is 20 μL, including 4 μL of single-stranded target nucleic acid, 2 μL of NEBuffer™ r2.1 (10×), 2 μL of Cas12a (1 μM), 2 μL of crRNA (1 μM) and 2 μL of Bio-HP-P reporter probe (10 μM), and make up to 20 μL with RNase-free ddH2O. React at 37℃ for 30 minutes, followed by inactivation at 80℃ for 10 minutes. Finally, a TdT extension and scale-up reaction was carried out. The total volume of the reaction system was 30 μL, including 20 μL of the above Cas12a cleavage reaction product, 3 μL of TdT buffer (10×), 3 μL of CoCl2 (2.5 mM), 1 μL of dGTP (25 mM), and 1 μL of TdT (20000 U). The volume was then brought up to 30 μL with RNase-free ddH2O. The reaction was carried out at 37 °C for 30 minutes, followed by inactivation at 80 °C for 10 minutes.

[0049] Detection mode ④ is the method of this invention. First, a one-pot reaction coupling RPA and Lambda exonuclease is used to generate single-stranded target nucleic acid. The total volume of the first reaction system is 25 μL, including one lyophilized RPA reaction particle, 0.6 μL of forward primer (10 μM), 0.6 μL of reverse primer (10 μM), 1.5 μL of Lambda exonuclease (1000 U), 2 μL of DNA template, and 2 μL of MgOAc (250 mM). The mixture is then brought to 25 μL with RNase-free ddH2O and reacted at 37°C for 30 minutes, followed by inactivation at 80°C for 10 minutes. Subsequently, a PAM-independent Cas12a / TdT signal amplification system was constructed. The total volume of the second reaction system was 30 μL, including 4 μL of the above single-stranded target nucleic acid, 2 μL of NEBuffer™ r2.1 (10×), 2 μL of Cas12a (1 μM), 2 μL of crRNA (1 μM), 2 μL of Bio-HP-P reporter probe (10 μM), 3 μL of TdT buffer (10×), 3 μL of CoCl2 (2.5 mM), 1 μL of dGTP (25 mM), and 1 μL of TdT (20000 U). The volume was then brought up to 30 μL with RNase-free ddH2O. The reaction was carried out at 37 °C for 60 minutes, followed by inactivation at 80 °C for 10 minutes.

[0050] Both detection modes ① and ② utilize signal-attenuating lateral chromatography test strips. The test strip's detection line is coated with 1 mg / mL streptavidin, the control line with 1 mg / mL goat anti-mouse secondary antibody, and the binding pad is loaded with gold nanoparticle-labeled anti-FITC antibody. During detection, 1 μL of the final reaction product is added to the sample pad, followed by 45 μL of chromatography run buffer. The image is acquired after 5 minutes of chromatography.

[0051] Detection modes ③ and ④ both utilize the signal-enhanced lateral chromatography test strip of this invention. The detection line of this test strip is coated with 1 mg / mL streptavidin to capture the biotin-labeled extension product; the control line is coated with the SAV–C line capture probe complex to capture the gold nanoparticle-labeled detection probe and verify the chromatographic effectiveness. The C-line capture probe sequence is SEQ ID NO. 6: 5'-Bio-AAAAAAAAAAAAAAA-3'. During detection, 2 μL of the gold nanoparticle-labeled detection probe, 1 μL of the final reaction product, and 45 μL of chromatography run buffer are mixed and added to the sample pad. Images are acquired after 5 minutes of chromatographic reaction.

[0052] Test results as follows Figure 5 As shown. Detection mode ① is a traditional PAM-dependent Cas12a signal attenuation detection method, with a visual detection limit of 10. 2The detection signal was only CFU / mL, but the signal attenuation was not significant in low-concentration samples, resulting in limited differentiation between positive and negative results. Although detection mode ② achieved PAM-independent Cas12a activation, it still used signal attenuation readout, with a visual detection limit of 10. 3 CFU / mL; Meanwhile, even at higher target concentrations, the detection line did not completely disappear, and the signal attenuation gradient between different concentrations was not obvious, which was not conducive to the identification and differentiation of low-concentration samples. Detection mode ③ employs a stepwise TdT signal amplification strategy, where the detection line signal increases with increasing target concentration, and the visual detection limit is 10. 2 The concentration is CFU / mL, but the reaction process is broken down into multiple independent steps, making the operation complex and involving numerous transfer steps. Detection mode ④, i.e., the method of this invention, exhibits a more obvious enhanced detection line visualization signal, with a visualization detection limit reaching 10. 0 CFU / mL.

[0053] The above results indicate that, compared with the traditional signal attenuation detection mode, the method of the present invention can achieve more intuitive detection line enhancement readout and improve the visualization and identification ability of low concentration target bacteria. Compared with the stepwise TdT signal amplification detection mode, the method of the present invention integrates RPA-Lambda exonuclease reaction, PAM-independent Cas12a recognition and TdT extension amplification into a sequentially connected dual-module reaction system, reducing stepwise operations and sample transfer processes. While improving detection sensitivity and ease of operation, it also reduces the risk of operational contamination.

[0054] Example 4 To verify the applicability of the established platform in real samples, this experiment evaluated its performance in detecting Salmonella in milk samples. Specifically, milk samples confirmed by qPCR to be free of Salmonella contamination were selected, and a known concentration of Salmonella was added to simulate contamination. For rapid extraction of Salmonella genomic DNA from the milk samples, 1 mL of the simulated contaminated milk sample was centrifuged at 10,000 rpm for 1 minute. After discarding the supernatant, 50 μL of lysis buffer (product number B642315, purchased from Sangon Biotech Co., Ltd.) was added to the precipitate. After mixing, the mixture was heated at 80°C for 10 minutes to release the Salmonella genomic DNA. The resulting lysis buffer was used as a template for subsequent detection. Except for sample pretreatment, the detection reaction system, reaction conditions, and lateral chromatography readout method were consistent with detection mode ④ in Example 3. Subsequently, under uniform and rigorous experimental conditions, these milk samples were analyzed by lateral chromatography. The detection results are as follows: Figures 6-8 As shown. Figure 6 The results of the lateral chromatography test strip were visually demonstrated. Observations showed that the T-line signal gradually increased with increasing Salmonella contamination, and the visual detection limit was as low as 10. 1CFU / mL. See also Figure 7 Quantitative analysis can clearly reflect the degree of Salmonella contamination in milk samples. Further analysis showed a good linear relationship between the T-line signal intensity and Salmonella concentration in the milk matrix. (See [reference needed]). Figure 8 The results demonstrate that the detection method described in this embodiment can not only achieve rapid and intuitive visual detection in actual milk samples, but also perform quantitative analysis through signal intensity, fully verifying its reliability and practicality in complex samples.

[0055] It should be understood that the above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A TdT signal amplification PAM-independent Cas12a lateral chromatography method for nucleic acid detection, characterized in that, include: A first reaction is performed on a first reaction system containing the nucleic acid to be tested, a recombinase polymerase amplification reaction component, a recombinase polymerase amplification primer pair, and an exonuclease to generate a recombinase polymerase amplification product. The exonuclease selectively digests the amplification product containing the amplification strand with 5' end phosphorylation modification to obtain a single-stranded target nucleic acid. In the primer pair, the 5' end of one primer strand is phosphorylated. A second reaction is performed on a second reaction system comprising the single-stranded target nucleic acid, a CRISPR / Cas12a reaction assembly, a reporter probe, a terminal deoxynucleotidyl transferase, and a deoxynucleoside triphosphate substrate, so that the single-stranded target nucleic acid activates the trans-cleavage activity of Cas12a in a PAM-independent manner and cleaves the reporter probe, generating a cleavage product with a 3'-hydroxy terminus and retaining a 5'-terminal capture label group. The cleavage product is then subjected to template-independent extension under TdT catalysis with the 3'-hydroxy terminus as the starting site, thereby obtaining an extension product, wherein the reporter probe has a 5'-terminal capture label group and a 3'-terminal phosphorylation modification; The extended product is driven by capillary action to migrate along the lateral chromatography support and come into contact with the detection probe labeled with gold nanoparticles and undergo nucleic acid hybridization to form an extended product-detection probe complex. When the extended product-detection probe complex is captured at the detection line and generates a visual detection signal, the nucleic acid to be tested is determined to be the target nucleic acid.

2. The method according to claim 1, characterized in that, include: After the first reaction is completed, the obtained first reaction system is subjected to enzyme inactivation treatment, and the CRISPR / Cas12a reaction component, reporter probe, terminal deoxynucleotidyl transferase and deoxynucleoside triphosphate substrate are added to form the second reaction system.

3. The method according to claim 1, characterized in that: The recombinase polymerase amplification product is an isothermal amplification reaction product. And / or, the exonuclease includes Lambda exonuclease.

4. The method according to any one of claims 1-3, characterized in that: The reaction temperature of the first reaction is 35–42°C, and the reaction time is 15–40 minutes; And / or, the reaction temperature of the second reaction is 35–42°C, and the reaction time is 30–90 minutes.

5. The method according to claim 1, characterized in that: The reporter probe is a reporter probe with a hairpin structure, and the 3' end phosphorylation modification of the reporter probe is used to inhibit the extension of the reporter probe that has not been cleaved by Cas12a by the terminal deoxynucleotidyl transferase.

6. The method according to claim 1, characterized in that: The 5' end capture label group is biotin, and the capture molecule fixed at the detection line is streptavidin or avidin.

7. The method according to claim 1, characterized in that: The deoxynucleoside triphosphate substrate includes any one of dGTP, dATP, dCTP, and dTTP, preferably dGTP.

8. The method according to claim 1, characterized in that: The gold nanoparticle-labeled detection probe contains a nucleic acid sequence capable of complementary hybridization with the extended product; preferably, when the deoxynucleoside triphosphate substrate is dGTP, the detection probe contains a polyC sequence; preferably, the detection probe has the sequence shown in SEQ ID NO.

5.

9. The method according to claim 1, characterized in that, The lateral chromatography carrier is a lateral chromatography test strip. The binding pad of the lateral chromatography test strip is pre-coated with a detection probe labeled with gold nanoparticles. A capture molecule that can recognize and bind to the 5' end capture label group of the extended product is fixed at the detection line. When the nucleic acid to be tested is the target nucleic acid, the extended product binds to the detection probe through nucleic acid hybridization and is captured by the capture molecule through its 5' end capture label group, thereby enriching the gold nanoparticles at the detection line and generating a visual color signal.

10. The application of the method according to any one of claims 1-9 in the detection of pathogenic microorganism nucleic acids in food, environmental or public health monitoring samples for non-disease diagnostic purposes.

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