Nucleic acid isothermal amplification detection method for reducing product aerosol pollution risk and kit thereof

Abstract

The invention provides a nucleic acid isothermal amplification detection method capable of reducing product aerosol pollution risk, which comprises the following steps: determining a nucleic acid sample containing target nucleic acid to be detected, amplifying a reference time point TtLOD required by isothermal amplification detection limit, and carrying out short-time nucleic acid isothermal amplification reaction on the sample containing the target nucleic acid, the time of the short-time isothermal amplification is TtLOD <-5 > minutes to TtLOD < + 5 > minutes. According to the method disclosed by the invention, a relatively short-time nucleic acid isothermal amplification stage and a subsequent high-temperature-resistant Ago enzyme mediated efficient detection and product fragmentation stage are integrated. According to the present invention, through the well-designed two-stage process, the double functions of the Ago enzyme are utilized, such that the detection is completed while the amplification product is internally and irreversibly converted into the non-pollution potential short fragment;

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid detection, and in particular to a nucleic acid isothermal amplification detection method and a kit thereof for reducing the risk of product aerosol contamination. Background Art

[0002] Isothermal nucleic acid amplification techniques, such as loop-mediated isothermal amplification (LAMP), are widely used in molecular diagnostics, particularly in point-of-care (POCT) settings, due to their rapid, isothermal, and highly sensitive nature. However, the high amplification efficiency of LAMP and other techniques also presents a serious problem of aerosol contamination. The large amount of DNA product generated by amplification can easily diffuse during the procedure, contaminating subsequent experiments and leading to false positives. This severely impacts the accuracy and reliability of the results, limiting their application in routine or resource-limited settings.

[0003] Traditional LAMP detection methods (such as real-time detection based on dyes or turbidity) typically require a long wait for the amplification reaction to proceed until the signal reaches a detectable threshold (such as the Ct value) or enters a plateau phase to ensure the validity of the results. This not only prolongs the detection time but also leads to the accumulation of a large amount of amplification products, increasing the risk of aerosol contamination.

[0004] To address the contamination issue, existing technologies such as the UNG enzymatic method, closed systems, and CRISPR-mediated cleavage during amplification have shown some effectiveness, but they often suffer from limitations such as complex operation, high costs, the need to modify the original amplification system, potential efficiency degradation, or incomplete anti-contamination measures (as mentioned above). These methods essentially "patch" or "set restrictions" outside the standard amplification process, failing to fundamentally address the conflict between high product accumulation and contamination risk.

[0005] Therefore, there is an urgent need to develop a new nucleic acid detection strategy that can not only achieve rapid and sensitive detection, but also inherently and efficiently solve or greatly reduce the risk of aerosol contamination in its design mechanism, and preferably optimize the detection process and improve overall efficiency. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a nucleic acid isothermal amplification detection method for reducing the risk of product aerosol contamination, and the amplification method comprises the following steps: Step 1: determining a nucleic acid sample containing a target nucleic acid to be detected, and a reference amplification time point Tt_LOD required at the isothermal amplification detection limit; Step 2: performing a short-time nucleic acid isothermal amplification reaction on the sample containing the target nucleic acid, and the time of the short-time isothermal amplification is Tt_LOD-5 minutes to Tt_LOD+5 minutes; Step 3: mixing the isothermal amplification product of step 2 with at least one high-temperature resistant gene editing enzyme Ago enzyme and at least two preset initial guide DNAs targeting specific sequences inside the amplification product, and the names of the two initial guide DNA sequences are respectively Respectively, gDNA1 and gDNA2, gDNA1 and gDNA2 form a complex with the Ago enzyme to cut the amplified product column generated in step 2; this step can be simultaneously or subsequently added with a detection probe for signal generation; and, step 4: incubating the mixture of step 3 at the working temperature of the gene editing enzyme Ago enzyme, during which the Ago enzyme cuts the amplified product template under the guidance of gDNA1 and gDNA2, and the nucleic acid fragment gDNA3 is obtained by the simultaneous cutting of the gDNA1-Ago complex and the gDNA1-Ago complex, and the nucleic acid fragment gDNA3-Ago enzyme complex binds to and cuts the detection probe, resulting in the generation or change of a detectable signal, thereby realizing the detection of the target nucleic acid to be detected.

[0007] In one embodiment, the short-term isothermal amplification time is Tt_LOD-3 minutes to Tt_LOD+3 minutes.

[0008] In one embodiment, the high-temperature resistant gene editing enzyme Ago is a PfAgo enzyme, which has an operating temperature of, for example, 90°C-100°C, preferably 95°C.

[0009] In one embodiment, gDNA1 and gDNA2 are 16-18 nt in length, respectively, and their 5' ends are phosphorylated; when gDNA1 and gDNA2 bind to a specific sequence within the amplification product, the two are adjacent and have no gap.

[0010] In one embodiment, specific fluorescence detection is achieved by binding and cleaving the fluorescent probe with the gDNA3-PfAgo complex.

[0011] In one embodiment, a detection kit for a target nucleic acid to be detected used in the above method is provided, the kit comprising: (a) an amplification reagent for isothermally amplifying the target nucleic acid to be detected, the amplification reagent comprising: a primer pair for amplifying the target nucleic acid to be detected, the primer pair being used to perform a specific amplification reaction based on the target nucleic acid to be detected, thereby producing a specific nucleic acid amplification product;

[0012] (b) a cleavage reagent or a cleavage buffer containing the cleavage reagent, wherein the cleavage reagent comprises: two initial guide DNA sequences gDNA1 and gDNA2, a gene editing enzyme Ago, and a detection probe, wherein the detection probe carries a fluorescent group and a quenching group, and when gDNA1 and gDNA2 bind to a specific sequence inside the amplification product, the two are adjacent and unspaced.

[0013] In one embodiment, the kit is a Mycoplasma pneumoniae detection kit, which is a LAMP isothermal amplification kit, and the primer pair sequences included in the kit are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7; and the sequences of the two initial guide DNA sequences gDNA1 and gDNA2 are SEQ ID NO.8 and SEQ ID NO.9, and the detection probe is SEQ ID NO.10.

[0014] In one embodiment, the kit is a novel coronavirus detection kit, which is a LAMP isothermal amplification kit, and the primer pair sequences included in the kit are SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17; and the sequences of the two initial guide DNA sequences gDNA1 and gDNA2 are SEQ ID NO.18 and SEQ ID NO.19, and the detection probe is SEQ ID NO.20.

[0015] The present method integrates a relatively short isothermal nucleic acid amplification phase with a subsequent, highly efficient detection and product fragmentation phase mediated by the thermostable Ago enzyme. This carefully designed two-stage process leverages the dual functions of the Ago enzyme (detection signal triggering and efficient cascade cleavage), enabling detection while the amplified product is intrinsically and irreversibly converted into short fragments with no contamination potential.

[0016] This novel detection method offers the potential for improved efficiency: By integrating short-term amplification with highly efficient Ago enzyme treatment, the amplification process can be shortened without waiting for the amplification to reach a plateau. Ago enzyme-mediated detection may achieve a detectable signal more quickly than traditional methods, potentially shortening overall detection time and improving efficiency.

[0017] The present invention's inherent and thorough anti-contamination mechanism: Anti-contamination is not an additional step, but an inevitable consequence of the method's design. This invention features a short pre-amplification time, leveraging the unique cascade cleavage properties of Ago enzymes, which initiates from gDNA 1 / 2 and generates gDNA 3...n. This ensures extremely efficient and thorough degradation of the amplified product into non-template-active fragments <50 bp. This inherent mechanism fundamentally eliminates the risk of product aerosol contamination, significantly improving the reliability of the results.

[0018] The method of the present invention retains the compatibility of the original amplification system: the standard and mature LAMP isothermal amplification system can be used in the amplification stage without modifying its primers or core components, thus ensuring the stability and ease of use of the technology.

[0019] The method of the present invention is relatively simple to operate: the entire process only requires the addition of Ago enzyme / gDNA (and probe) after a short amplification, and incubation at high temperature to complete detection and inactivation. The process is clear and easy to implement.

[0020] The method of the present invention is a highly integrated dual function, cleverly utilizing the characteristics of Ago enzyme that can be guided by gDNA for cutting (for detection and initiating cascades) and can also produce new gDNA for continuous cutting, highly integrating the two goals of detection and complete product inactivation in the same enzymatic reaction step. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 It is a schematic diagram of the detection principle of the present invention;

[0023] Figure 2 This is a graph showing the reference time point results of the LAMP assay for determining LOD in Example 2;

[0024] Figure 3 1 is a graph showing the LAMP amplification results at different pre-amplification times in Example 2;

[0025] Figure 4 1 is a graph showing the electrophoresis results of LAMP amplification products at different pre-amplification times in Example 2;

[0026] Figure 5 This is a diagram showing the secondary amplification results of the simulated contamination experiment in Example 3;

[0027] Figure 6This is the electrophoresis result of the product after the secondary amplification in Example 3;

[0028] Figure 7 This is a graph showing the LAMP amplification results during the pre-amplification period in Example 4;

[0029] Figure 8 1 is a graph showing the electrophoresis results of LAMP amplification products at different pre-amplification times in Example 4;

[0030] Figure 9 This is the kinetic experimental result of PfAgo enzyme cleavage of LAMP amplification products in Example 5. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the embodiments described are only a part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of this application. In the following embodiments, unless otherwise specified, all are conventional methods in this area.

[0032] Example 1 Basic principle of the present invention

[0033] The present invention provides a nucleic acid isothermal amplification detection method that reduces the risk of product aerosol contamination. Figure 1 As shown, the amplification method comprises the following steps:

[0034] Step 1: Determine the reference amplification time point Tt_LOD required for the isothermal amplification detection limit of the nucleic acid sample containing the target nucleic acid to be detected.

[0035] Step 2: Perform a short-time isothermal nucleic acid amplification reaction on the sample containing the target nucleic acid, wherein the time of the short-time isothermal amplification is Tt_LOD-5 minutes to Tt_LOD+5 minutes.

[0036] The isothermal amplification of this embodiment adopts the LAMP method, which amplifies the template for a short time, and only needs to amplify for a time sufficient to produce the minimum amount of template required for the subsequent Ago enzyme reaction; this short-time amplification product contains target nucleic acid molecules sufficient to initiate the subsequent Ago enzyme detection. In the present invention, the time of short-time isothermal amplification is Tt_LOD-5 minutes to Tt_LOD+5 minutes, which is significantly shorter than the time required for traditional isothermal amplification to reach the detection plateau or standard Ct value; and compared with traditional LAMP amplification detection, this short-time amplification (taking real-time fluorescence LAMP method as an example, the amplification time of short-time amplification is usually before the fluorescence signal reaches the detection threshold) can effectively reduce the accumulation of total product and is a key step in controlling contamination sources.

[0037] Step 3: The isothermal amplification product of step 2 is mixed with at least one high-temperature-resistant gene editing enzyme Ago enzyme and at least two preset initial guide DNAs that target specific sequences within the amplification product. The two initial guide DNA sequences are named gDNA1 and gDNA2, respectively. gDNA1 and gDNA2 form a complex with the Ago enzyme to cut the amplification product column generated in step 2. A detection probe for signal generation can be added simultaneously or afterwards in this step.

[0038] After the shorter amplification time in step 2 is completed, the reaction product is mixed with at least one thermostable Argonaute (Ago) enzyme (preferably PfAgo) and at least two preset initial guide DNAs (gDNA1, gDNA2, and preferably ssDNA phosphorylated at the 5' end) that target specific sequences within the amplification product; a detection probe for signal generation (such as a fluorescent probe, whose signal generation is associated with Ago cleavage activity or cleavage products) can be added simultaneously or subsequently to this step.

[0039] Step 4: Incubate the mixture of step 3 at the working temperature of the gene editing enzyme Ago enzyme. During this incubation process, the Ago enzyme cuts the amplified product template under the guidance of gDNA1 and gDNA2. The nucleic acid fragment gDNA3 is obtained by simultaneous cutting of the gDNA1-Ago complex and the gDNA1-Ago complex. The nucleic acid fragment gDNA3-Ago enzyme complex binds to and cuts the detection probe, resulting in the generation or change of a detectable signal, thereby realizing the detection of the target nucleic acid to be detected.

[0040] The mixture from step 2 is incubated under high temperature conditions (e.g., 90°C-100°C, preferably approximately 95°C). Under these conditions, two synergistic processes occur: a) detection signal generation, as the Ago enzyme cleaves the amplified product template under the guidance of gDNA1 and gDNA2. This cleavage activity directly or indirectly (by releasing fragments capable of interacting with the probe) leads to the generation or change of a detectable signal, enabling detection of the target nucleic acid; b) cascaded, efficient fragmentation, in which the Ago enzyme cleaves the amplified product not only for detection but, more importantly, after the initial guided cleavage of gDNA1 and gDNA2, releases or exposes new DNA fragments that can serve as new guide DNAs (designated gDNA3, gDNA4, gDNA5, ... gDNAn), further guiding the Ago enzyme to cleave the remaining amplified product. This cascade reaction, initiated by the initial gDNA, continuously generates new gDNA and continues cleavage until the enzyme is inactivated or the substrate is exhausted, can extremely efficiently and completely cleave all amplified products produced in step 1 (even if the starting amount is small) into extremely short fragments (≤50bp).

[0041] This example uses a thermostable Ago (PfAgo) enzyme, which is guided by gDNA (a synthetic sequence that requires phosphorylation at the 5' end and is between 16-18 nt in length and needs to be completely complementary to the target sequence). It can cut the complementary target sequence of gDNA at 10-11 nt at a high temperature of 95°C. Based on this working principle, specifically Figure 1 As shown in the figure, we designed two gDNAs (gDNA1 and gDNA2) and a fluorescent probe (fluorescent group at the 5' end and quenching group at the 3' end). The gDNA sequence mainly forms a complex with PfAgo to cut the target sequence. gDNA1 and gDNA2 can be adjacent and without any gap. Under the reaction conditions of 95°C, the two gDNAs form a complex with PfAgo to cut the target sequence. This step can cut the target sequence into three segments. The first segment is cut by the gDNA1-PfAgo complex alone and has an unknown length; the second segment is cut by the gDNA1-PfAgo complex and the gDNA2-PfAgo complex at the same time, with a known length and a phosphorylation mark at the 5' end. We named this segment gDNA3. This segment can continue to form a complex with PfAgo to cut the complementary sequence in the template; the third segment is cut by the gDNA2-PfAgo complex alone, with an unknown length, but a phosphorylation mark at the 5' end. We named this segment gDNA4. This segment can also continue to form a complex with PfAgo to cut the complementary sequence in the template. The sequence of the fluorescent probe (5' end fluorescent group, 3' end quenching group) used for detection is designed to be completely complementary to gDNA3. Through this design method, it is possible to achieve specific fluorescence detection by binding to the fluorescent probe and cutting through the gDNA3-PfAgo complex. While achieving detection, this gDNA-PfAgo reaction is also a cascade cutting reaction. When a new DNA sequence is cut off, if it meets the conditions for becoming gDNA, it will become a new gDNA and combine with PfAgo again to form a new gDNA4, gDNA5, gDNA6...gDNAn-PfAgo complex to cut the sequence until there is no completely matching sequence in the template or the PfAgo enzyme activity is completely lost. Figure 1As shown, based on this cascade cleavage principle, we deduced that gDNA3 and gDNA4 can be stably cleaved through the gDNA1-PfAgo complex and the gDNA2-PfAgo complex. The gDNA3-PfAgo complex is mainly used to cleave the complementary fluorescent probe for detection, but it also cleaves the complementary sequence in the template. The gDNA4-PfAgo complex will directly participate in the subsequent cascade cleavage reaction, cleaving more gDNA. These gDNA sequences will bind to PfAgo and cleave the nucleic acid sequence in the template. Ultimately, we found that the nucleic acid fragments in the template after the cascade cleavage reaction are all less than 100bp in length, while LAMP amplification requires a template of at least 200bp in length. Even if the product becomes an aerosol, it will not interfere with the accuracy of subsequent detection. This shows that the new detection method in this study is a new detection method without the risk of aerosol contamination.

[0042] Example 2: Establishment and verification of a novel detection method combining short-term LAMP and PfAgo treatment and the minimum template concentration for initiating PfAgo reactions

[0043] This example aims to establish and verify a novel nucleic acid detection method that integrates isothermal nucleic acid amplification with PfAgo enzyme-mediated detection and product processing. The specific method is as follows.

[0044] 1. Materials and Reagents

[0045] Artificially synthesized plasmid DNA containing the P1 gene fragment of Mycoplasma pneumoniae (sequence details are shown in Table 1). Before use, it was serially diluted with sterile water to a concentration range of 1×10 3The number of copies / μL to 1 × 100 copies / μL (i.e., 1 copy / μL) was used to determine the limit of detection (LOD) of traditional LAMP. Sterile water was used as a negative control (NTC). A LAMP primer set (F3, B3, FIP, BIP, LF, LB) was designed and synthesized for the P1 gene. Two initial guide DNAs (gDNA1 and gDNA2) (single-stranded DNA, 5'-phosphorylated) were designed to target specific sequences within the P1 gene LAMP amplification product. A fluorescent probe (e.g., labeled with FAM at the 5' end and a quencher group, BHQ1, at the 3' end) was designed. The sequence was designed to interact with the amplicon fragment (gDNA3 or similar) produced after PfAgo cleavage of gDNA1 / gDNA2, or the probe itself was cleaved by the activated PfAgo / gDNA complex, thereby generating a fluorescent signal. Details are shown in Table 2. The LAMP amplification reagent (Fluorescent LAMP / RT-LAMP Master Mix) used was purchased from Nanjing Novozymes Biotechnology Co., Ltd., and a 20 μL system was prepared according to the instructions, as shown in Table 3. The Pyrococcus furiosus Argonaute (PfAgo) enzyme used was purchased from Jiaohong Biotechnology (Shanghai) Co., Ltd., and a 20 μL system was prepared according to the instructions, as shown in Table 4.

[0046] Table 1. Synthetic fragments of the Mycoplasma pneumoniae P1 gene

[0047]

[0048] Table 2. Mycoplasma pneumoniae P1 gene LAMP primers, gDNA and fluorescent probes

[0049]

[0050]

[0051] Table 3. Preparation of LAMP reaction system

[0052] Reagents Volume (μL) 2×Fluorescent LAMP / RT-LAMP Master Mix 10 100 μM FIP / BIP primer 0.32 / 0.32 100 μM F3 / B3 primer 0.04 / 0.04 100 μM LF / LB primer 0.16 / 0.16 Sample DNA 5 Nuclease-free H2O Up to 20μL

[0053] Table 4. Preparation of PfAgo reaction system

[0054] Reagents Volume (μL) 10× reaction buffer 2 PfAgo (200 U / μL) 4 40mM Mg2+ 1 10 μM gDNA1 4 10 μM gDNA2 4 10 μM fluorescent probe (5'-FAM; 3'-BHQ1) 2 Sample DNA (LAMP product) 20 Nuclease-free H2O Up to 20μL

[0055] 2. Experimental Procedure

[0056] 2.1 Determination of the detection limit (LOD) and reference time point (Tt_LOD) of traditional real-time fluorescence LAMP: A standard LAMP reaction system (Table 3, adding the fluorescent dye Evagreen at a final concentration of 0.6×) was used to detect the P1 gene template (10 3To 100 copies / μL, at least 3 replicates for each concentration, and set NTC) for amplification. 65°C, collect fluorescence signals every 60 seconds, and cycle 50 times (i.e., a total of 50 minutes). Analyze the real-time fluorescence amplification curve and the lowest nucleic acid copy number that can be stably detected. This nucleic acid copy number is defined as the LOD of the standard LAMP method. Record the Tt value when the fluorescence signal of the LOD reaches the preset threshold. This time Tt_LOD is used as the reference time point for subsequent experiments. The results are as follows: Figure 2 As shown, the LOD that can be detected is 1×10 1 copies / μl, and the corresponding Tt value was 22.68min.

[0057] 2.2 Perform LAMP amplification at different times to generate Ago detection template: Based on the LOD concentration determined in step 2.1 (1×10 1 copies / μL, corresponding to the initial template amount of 50 copies / reaction) and the reference time point Tt_LOD = 22.68 minutes. This study uses an exponential amplification model to predict the template concentration gradient at different amplification times for subsequent screening of the minimum trigger threshold for Ago detection. The specific model parameters are as follows: 1) Amplification efficiency: 90% (1.9-fold amplification per cycle, referring to the common efficiency range of the LAMP system); 2) Cycle time: 1 minute / cycle (consistent with the fluorescence acquisition frequency of real-time monitoring); 3) Time gradient: set around Tt_LOD ±5, ±10 minutes (ie 12.68, 17.68, 27.68, 32.68 minutes), covering the early amplification (subthreshold), mid-exponential (near the threshold) and plateau (superthreshold) stages. Template concentration calculation, according to the formula N (t) =N0×(1+E) t (N0=50, E=0.9, time t=time), the theoretical concentration corresponding to each time point is shown in Table 5. After reaching the preset time, the reaction tube was immediately placed at 95°C and heated for 5 minutes to inactivate the Bst DNA polymerase.

[0058] Table 5. Results of setting LAMP reaction time based on Tt LOD value

[0059] serial number Sample name Actual reaction time (min) Concentration (copies / reaction) Group 1 Tt LOD 22.68 1.08×108 Group 2 Tt LOD-5min 17.68 4.23×106 Group 3 Tt LOD-10min 12.68 1.69×105 Group 4 Tt LOD+5min 27.68 2.6×109 Group 5 Tt LOD+10min 32.68 6.45×1010

[0060] 2.3 Perform integrated Ago enzyme detection and product fragmentation: Add the pre-prepared Ago reaction mixture (see Table 4) to each of the five LAMP reaction products (volume V_lamp) generated in step 2.2. After mixing, immediately place the reaction tube / plate in a real-time fluorescence quantitative PCR instrument. Set the reaction program to 95°C, collect the fluorescence signal (fluorescence channel FAM) every 10 seconds, and cycle a total of 60 times (i.e., a total duration of 10 minutes). Record the real-time fluorescence data for each reaction well.

[0061] 2.4 Verify the degree of product fragmentation by gel electrophoresis: Take 5 μl of the product from step 2.1 that was amplified by LAMP for 50 minutes at the LOD concentration as a high molecular weight product control. Then take 5 μl of the final product from step 2.3 that was amplified by LAMP for different times and then treated with Ago for 10 minutes, analyze it by 2% agarose gel electrophoresis, and observe and record the DNA bands using a gel imaging system. 3. Result Analysis

[0062] Compare the real-time fluorescence curves of Ago detection of the five groups of samples with different LAMP amplification times recorded in step 2.3. Observe the fluorescence intensity and the time to reach the threshold (Tt at Ago). It is found that except for the sample with very short LAMP amplification (Tt_LOD-10min), no fluorescence signal appears. The samples with other LAMP amplifications (Tt_LOD-5min, Tt_LOD, Tt_LOD+5min, Tt_LOD+10min) all show strong and reliable fluorescence signals. Even the sample with a shorter LAMP amplification time (Tt_LOD-5min) can generate strong and reliable fluorescence signals in the subsequent 10-minute Ago detection stage. The results are shown in the figure. Figure 3 This indicates that Ago detection does not require a very high template concentration during the amplification stage. Even with samples at the LOD concentration, LAMP amplification only requires Tt LOD-5 min (i.e., 17.68 min, corresponding to an amplification product concentration of 4.23 × 10 6 The results show that the short-term LAMP+Ago assay is feasible and efficient, as it can detect the presence of Ago copies of nucleic acids without requiring very high nucleic acid concentrations. Moreover, the signal intensity is superior to that of the traditional 50-min real-time fluorescent LAMP assay.

[0063] In addition, observe the gel electrophoresis results of step 2.4, as shown in Figure 4As shown. The control sample, which underwent a full 50-minute LAMP amplification, showed a typical high-molecular-weight ladder of LAMP products. In contrast, the high-molecular-weight bands in the final samples of Groups 1, 2, 3, and 4, which underwent a short-term LAMP+Ago treatment, completely disappeared from the lanes, with only bands in the low-molecular-weight region (≈50 bp). However, in Group 5, perhaps due to the longer amplification time during the LAMP stage, Ago cleavage was incomplete, resulting in a small number of product bands appearing at 500 bp, while the remaining product band fragments were mostly concentrated between 100 and 200 bp. This demonstrates that this method, using the LAMP amplification time Tt LOD as a reference, can be stably and accurately detected within Tt LOD - 5 min to Tt LOD + 5 min, and that the Ago treatment step can completely fragment the LAMP products.

[0064] Example 3 Verifies the pollution-free performance of the final product of the method of the present invention

[0065] This example directly demonstrates that the final reaction product produced by the novel nucleic acid detection method proposed in this invention, which integrates short-term LAMP amplification with PfAgo enzyme treatment, completely loses its ability to serve as a template to trigger subsequent amplification reactions, and thus inherently lacks the potential to serve as a source of aerosol contamination. The specific method is as follows.

[0066] 1. Materials and Reagents

[0067] The artificially synthesized plasmid DNA containing the P1 gene fragment of Mycoplasma pneumoniae was diluted with sterile water to 1×10 6 copies / μL for the preparation of the "contaminant" source. Sterile water was used for the negative control (NTC). The LAMP reagent preparation method required for contaminant preparation is the same as in Table 3, and the Ago reagent preparation method is the same as in Table 4.

[0068] 2. Experimental Procedure

[0069] 2.1 Pollutant preparation: Take 1×10 6 Copies / μL of P1 gene plasmid DNA was used as a template, and LAMP amplification was performed according to the optimized process established in step 2.3 of Example 1. After the amplification, the Ago reaction mixture prepared in advance was placed at 95°C, and the fluorescence signal was collected every 10 seconds (fluorescence channel was FAM). The cycle was repeated 60 times (i.e., the total time was 10 minutes) to complete the detection and product fragmentation. The final reaction product obtained was contaminant A. In addition, the same concentration (1×10 6The P1 gene plasmid DNA containing 100 copies / μL was used as a template and conventional amplification (65°C, 60 min) was performed using the LAMP reaction system. The final reaction product obtained was contaminant B.

[0070] 2.2 Set up a secondary amplification reaction to simulate contamination: Add 5 μL of "contaminants" to a new, template-free LAMP reaction system (Table 3, add the fluorescent dye Evagreen to a final concentration of 0.6×) according to the following grouping (see Table 6). Place all the set secondary amplification reaction tubes / plates in a real-time fluorescence quantitative PCR instrument at 65°C, collect fluorescence signals every 60 seconds, and cycle a total of 45 times (i.e., a total time of 45 minutes).

[0071] Table 6. Secondary amplification reaction arrangement for simulated contamination

[0072]

[0073] 3. Results Analysis

[0074] The secondary amplification results of this simulated contamination experiment clearly reveal the decisive advantage of the method of the present invention in eliminating the risk of product contamination. Figure 5 As shown in the figure, the negative control (NTC) showed no amplification, and the positive control (PC) exhibited the expected normal amplification curve. When the product (contaminant B) produced by traditional long-term LAMP was introduced as a contaminant, an extremely fast and strong false-positive amplification signal appeared, and its Ct value was much earlier than that of the positive control, fully exposing the serious aerosol contamination risk of the traditional method. In sharp contrast, when the final product (contaminant A) produced by the integrated method of the present invention (short-time LAMP + Ago treatment) was introduced as a contaminant, no amplification signal was detected in the reaction system during the entire amplification period, and its fluorescence curve completely overlapped with the negative control (NTC), showing a flat baseline.

[0075] The products after secondary amplification were analyzed by agarose gel electrophoresis ( Figure 6 The above conclusions were further visually confirmed by the presence of the expected bands in the positive control lane, the absence of any visible bands in the negative control and contaminant group A lanes, and the presence of a clear and intense high-molecular-weight ladder-like band characteristic of LAMP in the contaminant group B lane. Combined real-time fluorescence and electrophoresis results strongly demonstrate that the present method, through its inherent efficient cascade cleavage mechanism of Ago enzymes, completely converts the amplification products into inert short fragments that are incapable of subsequent amplification. This fundamentally eliminates their potential as a source of aerosol contamination, ensuring the safety of the detection process and the reliability of the results.

[0076] Example 4 Verification of the universality and anti-pollution stability of the method for different target genes

[0077] This example aims to verify the effectiveness and universality of the novel nucleic acid detection method proposed in the present invention, which integrates short-term LAMP amplification and PfAgo enzyme treatment, in detecting different target genes, and in particular, the ability to verify product fragmentation to reduce contamination risks while achieving rapid detection.

[0078] 1. Materials and Reagents

[0079] Artificially synthesized plasmid DNA containing the N gene fragment of the new coronavirus (COVID-19) (sequence details are shown in Table 7). Before use, gradient dilution was performed with sterile water to cover a concentration range of 1×10 3 The number of copies / μL to 1×100 copies / μL (i.e., 1 copy / μL) was used to determine the limit of detection (LOD) of traditional LAMP. Sterile water was used as a negative control (NTC). A LAMP primer set (F3, B3, FIP, BIP, LF, LB) was designed and synthesized for the N gene. Two initial guide DNAs (gDNA1 and gDNA2) (single-stranded DNA, 5'-phosphorylated) were designed to target specific sequences within the N gene LAMP amplification product. A fluorescent probe (e.g., labeled with FAM at the 5' end and a quencher group, BHQ1, at the 3' end) was designed. The sequence was designed to interact with the amplicon fragment (gDNA3 or similar) produced after PfAgo cleavage of gDNA1 / gDNA2, or the probe itself was cleaved by the activated PfAgo / gDNA complex, thereby generating a fluorescent signal. See Table 8 for details. The LAMP amplification reagent (Fluorescent LAMP / RT-LAMP Master Mix) used was purchased from Nanjing Novozymes Biotechnology Co., Ltd., and a 20 μL system was prepared according to the instructions, as shown in Table 3. The Pyrococcus furiosus Argonaute (PfAgo) enzyme used was purchased from Jiaohong Biotechnology (Shanghai) Co., Ltd., and a 20 μL system was prepared according to the instructions, as shown in Table 4.

[0080] Table 7. COVID-19N gene synthesis fragments

[0081]

[0082] Table 8. COVID-19N gene LAMP primers, gDNA and fluorescent probes

[0083]

[0084] 2. Experimental Procedure

[0085] 2.1 Determination of the detection limit (LOD) and reference time point (Tt_LOD) of traditional real-time fluorescence LAMP: A standard LAMP reaction system (Table 3, adding the fluorescent dye Evagreen at a final concentration of 0.6×) was used to detect the N gene template (10 3 Amplification was performed at 65°C (10 copies / μL, with at least 3 replicates per concentration and NTC). Fluorescence signals were collected every 60 seconds for 50 cycles (i.e., a total of 50 minutes). The real-time fluorescence amplification curve was analyzed, as was the minimum number of nucleic acid copies that could be stably detected. This number of nucleic acid copies was defined as the LOD of the standard LAMP method. The Tt value at which the fluorescence signal of the LOD reached the preset threshold was recorded. This time, Tt_LOD, served as the reference time point for subsequent experiments. The LOD that could be detected was 1×10 1 copiesl / μl, and the corresponding Tt value was 23.55min.

[0086] 2.2 Perform LAMP amplification at different times to generate Ago detection template: Based on the LOD concentration determined in step 2.1 (1×10 1 copies / μL, corresponding to the initial template amount of 50 copies / reaction) and the reference time point Tt_LOD = 23.55 minutes. This study uses an exponential amplification model to predict the template concentration gradient under different amplification times for subsequent screening of the minimum trigger threshold for Ago detection. The specific model parameters are as follows: 1) Amplification efficiency: 90% (1.9-fold amplification per cycle, referring to the common efficiency range of the LAMP system); 2) Cycle time: 1 minute / cycle (consistent with the fluorescence acquisition frequency of real-time monitoring); 3) Time gradient: set around Tt_LOD ±5, ±10 minutes (ie 13.55, 18.55, 28.55, 33.55 minutes), covering the early amplification (subthreshold), mid-exponential (near the threshold) and plateau (superthreshold) stages. Template concentration calculation, according to the formula N (t) =N0×(1+E) t (N0=50, E=0.9, time t=time), the theoretical concentration corresponding to each time point is shown in Table 9. After reaching the preset time, the reaction tube was immediately placed at 95°C and heated for 5 minutes to inactivate the Bst DNA polymerase.

[0087] Table 9. Experimental results of setting LAMP reaction time based on Tt LOD value

[0088] serial number Sample name Actual reaction time (min) Concentration (copies / reaction) Group 1 Tt LOD 23.55 <![CDATA[1.85×10 8 ]]> Group 2 Tt LOD-5min 18.55 <![CDATA[7.5×10 6 ]]> Group 3 Tt LOD-10min 13.55 <![CDATA[3.0×10 5 ]]> Group 4 Tt LOD+5min 28.55 <![CDATA[4.6×10 9 ]]> Group 5 Tt LOD+10min 33.55 <![CDATA[1.15×10 11 ]]>

[0089] 2.3 Perform integrated Ago enzyme detection and product fragmentation: Add the pre-prepared Ago reaction mix (see Table 4) to each of the five LAMP reaction products (volume V_lamp) generated in step 2.2. After mixing thoroughly, immediately place the reaction tube / plate in a real-time fluorescence quantitative PCR instrument. Set the reaction program to 95°C, collect fluorescence signals (FAM channel) every 10 seconds, and cycle 60 times. Record real-time fluorescence data for each reaction well.

[0090] 2.4 Verify product fragmentation by gel electrophoresis: Take 5 μl of the product from step 2.1, which was amplified at the LOD concentration for 50 minutes, as a high molecular weight control. Also, take 5 μl of the final product from step 2.3, which was amplified for different times with LAMP and then treated with Ago for 10 minutes, and analyze it using 2% agarose gel electrophoresis. DNA bands were visualized using a gel imaging system.

[0091] 3. Results Analysis

[0092] Compare the real-time fluorescence curves of Ago detection of the five groups of samples with different LAMP amplification times recorded in step 2.3. Observe the fluorescence intensity and the time to reach the threshold (Tt at Ago). It is found that except for the sample with very short LAMP amplification (Tt_LOD-10min), no fluorescence signal appears. The samples with other LAMP amplifications (Tt_LOD-5min, Tt_LOD, Tt_LOD+5min, Tt_LOD+10min) all show strong and reliable fluorescence signals. Even the sample with a shorter LAMP amplification time (Tt_LOD-5min) can generate strong and reliable fluorescence signals in the subsequent 10-minute Ago detection stage. The results are shown in the figure. Figure 7 This indicates that Ago detection does not require a very high template concentration during the amplification stage. Even with samples at the LOD concentration, LAMP amplification only requires Tt LOD-5 min (i.e., 18.55 min, corresponding to an amplification product concentration of 7.5 × 10 6 The results show that the short-term LAMP+Ago assay is feasible and efficient, as it can detect the presence of Ago copies of nucleic acids without requiring very high nucleic acid concentrations. Moreover, the signal intensity is superior to that of the traditional 50-min real-time fluorescent LAMP assay.

[0093] In addition, observe the gel electrophoresis results of step 2.4, as shown in Figure 8As shown. The control sample, which underwent a full 50-minute LAMP amplification, displayed a typical high-molecular-weight ladder of LAMP products. In contrast, the high-molecular-weight bands in the final samples from Groups 1, 2, 3, and 4, which underwent a short LAMP+Ago treatment, completely disappeared from the lanes, leaving only bands in the low-molecular-weight region (≈50 bp). However, in Group 5, perhaps due to the longer amplification time during the LAMP stage, incomplete Ago cleavage resulted in a small number of product bands appearing between 500 and 800 bp, while the remaining product bands were mostly concentrated between 100 and 200 bp. This demonstrates that this method, using the LAMP amplification time Tt LOD as a reference, can stably and accurately detect products within a range from Tt LOD - 5 min to Tt LOD + 5 min, and that the Ago treatment step can thoroughly fragment the LAMP products.

[0094] Example 5: Study on the Kinetics and Efficiency of PfAgo-Mediated Cascade Cleavage

[0095] This example aims to quantitatively investigate the kinetics and efficiency of PfAgo enzyme fragmentation under the guidance of gDNA on relatively low-concentration LAMP products generated by short-term isothermal amplification, in order to verify the effectiveness of its cascade cleavage mechanism and further support the inherent anti-contamination ability of the present method by completely inactivating the product template.

[0096] 1. Materials and Reagents

[0097] Take 1×10 6 Copies / μL of P1 gene plasmid DNA was used as a template. The LAMP reaction system and reaction conditions described in Table 3 of Example 1 were used, but the amplification time was fixed at Tt LOD - 5 min ≈ 18 minutes. The LAMP product produced in this step served as the substrate for the subsequent PfAgo enzyme digestion reaction, simulating the situation in the present method where amplification can be terminated early and then proceed to the next stage. The PfAgo system reaction system was prepared according to Table 4 of Example 1. The DNA fragment size distribution was accurately analyzed using the Agilent TapeStation 4200 system.

[0098] 2. Experimental Procedure

[0099] 2.1 PfAgo enzyme digestion reaction setup and time point sampling: As described in Part 1 of this example, 1×10 6The P1 gene template was amplified by LAMP for 18 minutes with 100 copies / μL of the sample to obtain a LAMP product for subsequent research. The LAMP product was added as a template to the PfAgo reaction system prepared according to Table 4 of Example 1. The mixed reaction solution was placed in a 95°C PCR instrument for incubation. During the incubation process, at 5, 10, 15, 20, and 30 minutes after the start of the reaction, 5 μl of the reaction solution was accurately aspirated and immediately placed on ice for quenching to terminate the PfAgo cleavage activity.

[0100] 2.2 High-resolution fragment analysis: The DNA fragment size distribution of samples at each time point was analyzed using Agilent TapeStation.

[0101] 3. Results Analysis

[0102] The results are as follows Figure 9 As shown, at 95°C, PfAgo cleavage of LAMP products is rapid. By 10 minutes, >90% of the high-molecular-weight products may have been cleaved into short fragments <50 bp. The reaction likely reaches a plateau around 15 minutes, indicating that nearly all products are fully fragmented.

[0103] It should be understood that the present invention disclosed is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the term used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0104] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A nucleic acid isothermal amplification detection method for reducing the risk of product aerosol contamination, characterized in that: The amplification method comprises the following steps: Step 1: Determine the reference amplification time point Tt_LOD required for the isothermal amplification detection limit of the nucleic acid sample containing the target nucleic acid to be detected; Step 2: performing a short-term isothermal nucleic acid amplification reaction on the sample containing the target nucleic acid, wherein the time of the short-term isothermal amplification is Tt_LOD-5 minutes to Tt_LOD+5 minutes; Step 3: The isothermal amplification product of step 2 is mixed with at least one thermostable gene editing enzyme, Ago enzyme, and at least two preset initial guide DNAs that target specific sequences within the amplification product. The two initial guide DNA sequences are named gDNA1 and gDNA2. gDNA1 and gDNA2 form a complex with the Ago enzyme to cut the amplification product sequence generated in step 2. A detection probe for signal generation can be added simultaneously or after this step. Step 4: Incubate the mixture of step 3 at the working temperature of the gene editing enzyme Ago enzyme. During this incubation process, the Ago enzyme cuts the amplified product template under the guidance of gDNA1 and gDNA2. The nucleic acid fragment gDNA3 is obtained by simultaneous cutting of the gDNA1-Ago complex and the gDNA1-Ago complex. The nucleic acid fragment gDNA3-Ago enzyme complex binds to and cuts the detection probe, resulting in the generation or change of a detectable signal, thereby realizing the detection of the target nucleic acid to be detected.

2. The nucleic acid isothermal amplification detection method according to claim 1, characterized in that: The time of the short-time isothermal amplification is Tt_LOD-3 minutes to Tt_LOD+3 minutes.

3. The nucleic acid isothermal amplification detection method according to claim 1, characterized in that: The high-temperature resistant gene editing enzyme Ago is PfAgo enzyme, which has an operating temperature of 90°C-100°C, preferably 95°C.

4. The nucleic acid isothermal amplification detection method according to claim 1, characterized in that: gDNA1 and gDNA2 are 16-18 nt in length, and their 5' ends are phosphorylated. When gDNA1 and gDNA2 bind to specific sequences within the amplified product, they are adjacent and unspaced.

5. The nucleic acid isothermal amplification detection method according to claim 1, characterized in that: Specific fluorescence detection is achieved by binding to the fluorescent probe and cutting the gDNA3-PfAgo complex.

6. A detection kit for a target nucleic acid to be detected used in any one of the methods of claims 1 to 5, characterized in that: The kit comprises: (a) an amplification reagent for isothermal amplification of a target nucleic acid to be detected, the amplification reagent comprising: a primer pair for amplifying the target nucleic acid to be detected, the primer pair being used to perform a specific amplification reaction based on the target nucleic acid to be detected, thereby generating a specific nucleic acid amplification product; (b) a cleavage reagent or a cleavage buffer containing the cleavage reagent, wherein the cleavage reagent comprises: two initial guide DNA sequences gDNA1 and gDNA2, a gene editing enzyme Ago, and a detection probe, wherein the detection probe carries a fluorescent group and a quenching group, and when gDNA1 and gDNA2 bind to a specific sequence inside the amplification product, the two are adjacent and unspaced.

7. The detection kit for the target nucleic acid to be detected according to claim 6, characterized in that The kit is a Mycoplasma pneumoniae detection kit, which is a LAMP isothermal amplification kit. The kit includes primer pairs with sequences of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7; and two initial guide DNA sequences gDNA1 and gDNA2 with sequences of SEQ ID NO.8 and SEQ ID NO.9, and the detection probe is SEQ ID NO.

10.

8. The detection kit for the target nucleic acid to be detected according to claim 6, characterized in that: The kit is a Mycoplasma pneumoniae detection kit, which is a LAMP isothermal amplification kit. The kit includes primer pairs with sequences of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7; and two initial guide DNA sequences gDNA1 and gDNA2 with sequences of SEQ ID NO.8 and SEQ ID NO.9, and the detection probe is SEQ ID NO.

10.

9. The detection kit for the target nucleic acid to be detected according to claim 6, characterized in that: The kit is a novel coronavirus detection kit, which is a LAMP isothermal amplification kit. The primer pair sequences included in the kit are SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17; and the sequences of the two initial guide DNA sequences gDNA1 and gDNA2 are SEQ ID NO.18 and SEQ ID NO.19, and the detection probe is SEQ ID NO.20.

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