Nucleic acid testing method, and composition and kit for nucleic acid testing

Abstract

Provided are a nucleic acid testing method, and a composition and kit for nucleic acid testing. According to the nucleic acid testing method, amplification testing of multiple targets can be performed simultaneously, and the amplification system does not require addition of an NFO endonuclease, so that the hybridization efficiency is greatly improved, and good testing sensitivity is thus achieved; moreover, a capture probe only complementarily matches a single-stranded product of a target sequence, ensuring the testing specificity. By means of the testing method, only a simple constant temperature device is required to achieve visual observability of nucleic acid testing results, and there is no need for a complex variable temperature instrument and a fluorescence detection device, so that operation is easy and convenient, results are convenient to read, no professional operation and result analysis are needed, and on-site and grassroots-level rapid nucleic acid testing is facilitated.

Description

Nucleic acid detection method, composition and kit for nucleic acid detection

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on September 11, 2023, with application number 2023111748759 and invention name “Nucleic Acid Detection Method, Composition and Kit for Nucleic Acid Detection”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of nucleic acid detection, and in particular to a nucleic acid detection method, a composition and a kit for nucleic acid detection. Background Art

[0003] Nucleic acid testing has been applied in various fields, including medical diagnosis, epidemic prevention and control, food safety, and species identification. The most commonly used method is PCR-based nucleic acid testing. However, PCR, as a variable-temperature amplification technique, places high demands on instruments, facilities, and personnel, making it unsuitable for rapid on-site testing. Point-of-care (POCT) devices developed based on PCR technology are difficult to widely adopt due to their high cost, technical difficulty, and long operating times. Therefore, isothermal nucleic acid amplification technology has emerged to fill this technological gap.

[0004] Recombinase polymerase amplification (RPA), developed by TwistDx Inc. in the UK, is a novel, rapid, isothermal amplification technology for nucleic acid molecules, hailed as a potential alternative to PCR. This technology uses a recombinase, a single-stranded binding protein, and a DNA polymerase to simultaneously amplify double-stranded DNA under isothermal conditions (35-42°C). The amplified product grows exponentially, typically yielding amplified fragments detectable by agarose gel electrophoresis within 30 minutes. This method has been widely studied because it does not require complex temperature-controlled instrumentation and is suitable for rapid on-site testing. Various RPA-based detection technologies have also been developed. For example, fluorescent RPA incorporates an exo-nuclease and a specifically labeled probe into the system. The presence of the target sequence can be detected by real-time fluorescence monitoring. This method is convenient to use, but the addition of the probe and exonuclease increases system cost, and the requirement for real-time fluorescence detection equipment hinders widespread application.

[0005] Another widely studied detection method combines RPA technology with lateral flow chromatography. NFO endonuclease and a specific labeled probe are added to the RPA system, allowing the amplification product to carry two labels. During lateral flow chromatography, the amplification product specifically binds to the labeled colored nanoparticles and develops color after binding to the specific binder at the detection line. This method does not require expensive temperature-variable amplification instruments and fluorescence detection devices. It is convenient and fast to use, so it has been widely studied. However, this method also has the problem of high cost. The markers and specific binders are often FAM / FAM monoclonal antibody, digoxin / digoxin monoclonal antibody, etc. Each additional target detection requires an additional pair of markers / specific binders, which is expensive. Multiplex detection is also impossible due to the type of markers. For example, Chinese patent CN201810769903.4, "A primer, probe, and kit for dual detection of hepatitis E virus and hepatitis A virus by RT-RPA-lateral flow chromatography," uses this method for dual detection. However, using this method for triple or more detection is very difficult and expensive.

[0006] Summary of the Invention

[0007] In view of this, the present invention provides a nucleic acid detection method, a composition for nucleic acid detection, and a kit. This nucleic acid detection method can simultaneously perform amplification detection of multiple targets, and the amplification system does not require the addition of NFO nuclease, which greatly improves the hybridization efficiency. Therefore, this detection has good sensitivity, and the capture probe only complements the single-stranded product of the target sequence, ensuring the specificity of the detection. This detection method only requires a simple constant temperature device to complete the nucleic acid detection and naked eye observation results, without the need for complex temperature-changing instruments and fluorescence detection devices. It is simple to operate, the results are easy to read, and no professional personnel are required for operation and result analysis, which is convenient for on-site and grassroots rapid nucleic acid detection.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a method for nucleic acid detection, including but not limited to the following steps:

[0010] Asymmetric amplification was performed using RPA;

[0011] Detection was performed by lateral flow chromatography.

[0012] In some specific embodiments of the present invention, the asymmetric amplification using RPA includes: after the limiting primer is consumed, the non-limiting primer with a label is amplified to produce a single-stranded amplification product, and the single-stranded amplification product carries the label and can bind to a colorimetric substance labeled with a specific binding substance to obtain the object to be detected.

[0013] In some specific embodiments of the present invention, the detecting by lateral flow chromatography comprises: the carrier of the lateral flow chromatography is provided with a capture probe, and the capture probe is complementary to the single-stranded amplification product;

[0014] If the single-stranded amplification product is present after amplification, the object to be detected is captured by the capture probe via lateral flow chromatography, and the color-developing substance aggregates and develops color, then the target sequence is present in the sample to be detected;

[0015] On the contrary, if no color is developed, there is no target sequence in the sample to be tested.

[0016] In some specific embodiments of the present invention, the molar ratio of the non-restricting primer to the restricting primer in the nucleic acid detection method includes but is not limited to (2-100):1;

[0017] Preferably, the molar ratio of the non-limiting primer to the limiting primer includes but is not limited to (4-32):1.

[0018] In some specific embodiments of the present invention, the nucleotide sequence of the non-restrictive primer in the nucleic acid detection method is shown as SEQ ID No. 6, 8, 10, 12, 16, 19, 20, 27, 29, 30 or 32.

[0019] In some specific embodiments of the present invention, the nucleotide sequence of the restriction primer in the nucleic acid detection method is shown as SEQ ID No. 5, 7, 9, 11, 17, 18, 21, 26, 28, 31 or 33.

[0020] In some specific embodiments of the present invention, the temperature of the asymmetric amplification in the nucleic acid detection method includes but is not limited to 35-42° C., and the time of the asymmetric amplification includes but is not limited to 10-40 minutes.

[0021] In some specific embodiments of the present invention, the nucleic acid detection method further includes a denaturation step between the asymmetric amplification and the lateral flow chromatography;

[0022] The denaturation temperature includes but is not limited to 80-100° C., and the denaturation time includes but is not limited to 1-5 minutes.

[0023] In some specific embodiments of the present invention, the capture probe in the nucleic acid detection method is partially complementary to the single-stranded amplification product;

[0024] The length of the capture probe includes but is not limited to 10 to 40 bp; preferably, the length of the capture probe includes but is not limited to 20 to 30 bp.

[0025] In some specific embodiments of the present invention, the nucleotide sequence of the capture probe in the nucleic acid detection method is shown as one or more of SEQ ID Nos. 1-4, 13-15, or 22-25.

[0026] In some specific embodiments of the present invention, the color-developing substance in the nucleic acid detection method includes but is not limited to colored nanoparticles;

[0027] The colored nanoparticles include but are not limited to colloidal gold particles or latex microspheres;

[0028] The marker includes but is not limited to one or more of biotin, digoxigenin, and fluorescein;

[0029] The fluorescein includes but is not limited to one or more of FAM, VIC or FITC;

[0030] The conjugate includes but is not limited to one or more of streptavidin, digoxigenin antibody or fluorescein monoclonal antibody.

[0031] In some specific embodiments of the present invention, the carrier in the nucleic acid detection method includes but is not limited to a test strip;

[0032] The test strip includes but is not limited to a sample carrying component, a colorant carrying component, a chromatography capture component, and a water absorbing component;

[0033] The sample carrying component is used to carry the sample to be tested, and the sample to be tested includes but is not limited to a mixture of the single-stranded amplification product and the hybridization solution;

[0034] The color developing substance carrying component is used to carry the color developing substance labeled with the specific binding substance;

[0035] The chromatographic capture component includes, but is not limited to, one or more detection lines, the detection lines carrying the capture probes;

[0036] The water absorbing component causes the sample to be tested to flow through the test strip through a siphon effect.

[0037] Specifically, the nucleic acid detection method provided by the present invention includes:

[0038] 1. Asymmetric amplification of the target sequence is performed using RPA amplification reagent. The primers used for amplification include a non-restricting primer and a restricting primer. The non-restricting primer is labeled with a marker at its 5' end. The marker can be a common labeling group such as biotin, digoxigenin, or fluorescein (FAM, VIC, or FITC). The ratio of the non-restricting primer to the restricting primer is between 2:1 and 100:1, preferably between 4:1 and 32:1. The above primer pair amplifies the target in RPA reagent for 10-40 minutes at a temperature of 35-42°C. During this process, after the restricting primer is consumed, the non-restricting primer can amplify many labeled single-stranded amplification products. The amplification diagram is shown in Figure 1.

[0039] 2. Heat the asymmetric RPA amplified product at 80-100°C for 1-5 minutes to denature the protein for downstream nucleic acid lateral flow hybridization.

[0040] 3. The test strip for downstream nucleic acid chromatography hybridization comprises a sample pad, a chromogenic pad, a chromatography capture membrane, and an absorbent pad. The sample pad and the chromogenic pad can be made of materials such as glass fiber, filter paper, or polyester film. The chromogenic pad contains a modified chromogenic substance such as colored nanoparticles. The modification is labeled with a specific binding substance, and the binding substance is a substance that specifically binds to the label on the non-restricted primer, such as streptavidin, digoxigenin antibody, fluorescein monoclonal antibody, etc. The chromatography capture membrane is made of materials such as nitrocellulose membrane (NC membrane) and nylon membrane, and contains one or more detection lines, and the detection lines contain specific capture probes. The capture probe is complementary to a portion of the single-stranded amplification product amplified by asymmetric RPA. The capture probe is 10 to 40 bp in length, preferably 20 to 30 bp in length. The absorbent pad is a non-woven material. A schematic diagram of the test strip is shown in Figure 2.

[0041] 4. The amplification product and hybridization solution are mixed evenly in a certain ratio. After the mixture is added to the sample pad of the test strip, the liquid will move in the direction of the arrow in Figure 2, first reaching the chromogenic pad. The marker on the single-stranded amplification product will bind to the specific binding substance modified with the chromogenic substance (colored nanoparticles such as colloidal gold particles or colored microspheres) in the chromogenic pad to form a complex, that is, "single-stranded amplification product - marker - specific binding substance - colored nanoparticle." As the solution continues to move, it reaches the detection line of the chromatographic capture membrane. The single-stranded amplification product hybridizes with the capture probe, forming a "capture probe - single-stranded amplification product - marker - specific binding substance - colored nanoparticle" complex and is fixed to the detection line. The fixed complex aggregates and develops color here. The solution continues to move forward and is absorbed by the absorbent pad at the end of the test strip. Finally, the result is determined based on the color development of the detection line (as shown in the schematic diagram in Figure 3).

[0042] When the sample contains the nucleic acid sequence to be tested, asymmetric RPA can amplify the single-stranded target product. During the chromatography process, a complex of 'capture probe-single-stranded amplification product-marker-specific binding substance-colored nanoparticles' will be formed and retained on the detection line, forming a colored band visible to the naked eye, which is a positive result.

[0043] When the nucleic acid sequence to be tested does not exist in the sample, asymmetric RPA cannot amplify the target single-stranded product, so the "capture probe-single-stranded amplification product-marker-specific binding agent-colored nanoparticle" complex cannot be formed. The colorant cannot accumulate on the detection line, and no colored band visible to the naked eye will be formed. This is a negative result.

[0044] In a second aspect, the present invention also provides a composition for nucleic acid detection, comprising a carrier for lateral flow chromatography and a primer set for asymmetric amplification;

[0045] The carrier carries a capture probe and a color developing substance labeled with a specific binding substance;

[0046] The primer set for asymmetric amplification includes but is not limited to a restriction primer and a non-restriction primer with a label;

[0047] After the limiting primer is consumed, the non-limiting primer with the label is amplified to produce a single-stranded amplification product. The single-stranded amplification product carries the label and can bind to the coloring substance labeled by the specific binding substance to obtain the object to be detected;

[0048] The capture probe is complementary to the single-stranded amplification product.

[0049] In some specific embodiments of the present invention, the carrier in the composition for nucleic acid detection includes but is not limited to a test strip;

[0050] The test strip includes but is not limited to a sample carrying component, a colorant carrying component, a chromatography capture component, and a water absorbing component;

[0051] The sample carrying component is used to carry the sample to be tested, and the sample to be tested includes but is not limited to a mixture of the single-stranded amplification product and the hybridization solution;

[0052] The color developing substance carrying component is used to carry the color developing substance labeled with the specific binding substance;

[0053] The chromatographic capture component includes, but is not limited to, one or more detection lines, the detection lines carrying the capture probes;

[0054] The water absorbing component causes the sample to be tested to flow through the test strip through a siphon effect.

[0055] In some specific embodiments of the present invention, the molar ratio of the non-restricting primer to the restricting primer in the composition for nucleic acid detection includes but is not limited to (2-100):1;

[0056] Preferably, the addition amount of the non-limiting primer and the limiting primer includes but is not limited to (4-32):1.

[0057] In some specific embodiments of the present invention, the nucleotide sequence of the non-restrictive primer in the nucleic acid detection composition is shown as SEQ ID No. 6, 8, 10, 12, 16, 19, 20, 27, 29, 30 or 32.

[0058] In some specific embodiments of the present invention, the nucleotide sequence of the restriction primer in the nucleic acid detection composition is shown as SEQ ID No. 5, 7, 9, 11, 17, 18, 21, 26, 28, 31 or 33.

[0059] In some specific embodiments of the present invention, the capture probe in the nucleic acid detection composition is partially complementary to the single-stranded amplification product;

[0060] The length of the capture probe includes but is not limited to 10 to 40 bp; preferably, the length of the capture probe includes but is not limited to 20 to 30 bp.

[0061] In some specific embodiments of the present invention, the nucleotide sequence of the capture probe in the nucleic acid detection composition is shown as one or more of SEQ ID No. 1-4, 13-15 or 22-25.

[0062] In some specific embodiments of the present invention, the material of the sample-carrying component or the color-developing substance-carrying component in the nucleic acid detection composition is independently selected from glass fiber, filter paper or polyester film;

[0063] The material of the chromatography capture component includes but is not limited to nitrocellulose membrane (NC membrane) or nylon membrane;

[0064] The material of the water absorbing component includes but is not limited to non-woven material.

[0065] In some specific embodiments of the present invention, the nucleic acid detection composition does not include NFO endonuclease.

[0066] In a third aspect, the present invention also provides a kit for nucleic acid detection, comprising the composition of the present invention.

[0067] The present invention utilizes RPA amplification technology for asymmetric amplification, that is, the primer with a label at one end is a non-restrictive primer, while the primer at the other end is a restrictive primer. After the restrictive primer is completely consumed during amplification, the non-restrictive primer amplifies to produce a large amount of single-stranded products. The single-stranded amplification products carry a label and can bind to colored nanoparticles (colloidal gold particles or latex microspheres) labeled with a specific binding agent. An oligonucleotide chain (capture probe) complementary to the single-stranded amplification product is fixed at the detection line of the lateral flow chromatography test strip. When the single-stranded amplification product combined with the colored nanoparticles is laterally chromatographed to the detection line, it will be captured by the capture probe, and the colored nanoparticles will aggregate and develop color at the detection line. If there is no single-stranded product after amplification, it means that there is no target sequence in the sample, and no color will be developed.

[0068] The beneficial effects of the present invention include but are not limited to:

[0069] 1) This technology allows for simultaneous amplification and detection of multiple targets. Only primers and capture probes for different targets need to be designed. Primers for different target sequences all carry a marker for binding to colloidal gold / latex microspheres, and the number of marker types is not limited.

[0070] 2) The capture probe does not require any special modification, and the cost is low. The amplification system does not need to add NFO endonuclease, and only the basic amplification system can be used, which further reduces the cost.

[0071] 3) The single-stranded product amplified by asymmetric RPA is used for chromatography hybridization, which greatly improves the hybridization efficiency. Therefore, this detection has good sensitivity, and the capture probe only complements the single-stranded product of the target sequence, ensuring the specificity of the detection.

[0072] 4) This detection method only requires a simple constant temperature device to complete the nucleic acid detection and naked eye observation results, without the need for complex temperature-changing instruments and fluorescence detection devices. It is easy to operate and easy to read the results. It does not require professional personnel to operate and analyze the results, and is convenient for rapid nucleic acid testing on-site and at the grassroots level. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0074] Figure 1 shows a schematic diagram of asymmetric RPA amplification;

[0075] Figure 2 shows a schematic diagram of a test strip;

[0076] FIG3 is a schematic diagram showing the principle of lateral flow chromatography hybridization;

[0077] FIG4 shows the chromatography test results of Example 1;

[0078] FIG5 shows the chromatography test results of Example 2 and Comparative Example 1;

[0079] FIG6 shows the chromatography detection results of Example 3. DETAILED DESCRIPTION

[0080] The present invention discloses a method for nucleic acid detection, a composition and a kit for nucleic acid detection. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted in particular that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0081] The raw materials and reagents used in the nucleic acid detection method, nucleic acid detection composition and kit provided by the present invention can all be purchased from the market.

[0082] The present invention will be further described below in conjunction with the embodiments:

[0083] Example 1: Quadruple Detection of Treponema pallidum, Herpes Simplex Virus Type 1, Herpes Simplex Virus Type 2, and Internal Standard Gene

[0084] 1. Preparation of test strips:

[0085] 1) Spray a solution of colloidal gold particles labeled with streptavidin onto a colorimetric pad using a film sprayer at a spray rate of 1 μL / cm. Place the sprayed colorimetric pad in a 37°C incubator to dry for 1 hour.

[0086] 2) The four capture probes in Table 1 were sequentially streaked onto the chromatographic capture membrane using a film sprayer at a streaking speed of 0.6 μL / cm. The streaked capture membrane was placed in a 60° C. thermostat to dry for 12 hours.

[0087] Table 1 Capture probe sequences and concentrations in Example 1

[0088] 3) Assemble and bond the sample pad, colorimetric pad, chromatography capture membrane, and absorbent pad according to the test paper structure shown in FIG2 . Cut the test paper into 3.08 mm wide strips using a cutting machine and seal them in an aluminum foil bag for future use.

[0089] 2. Asymmetric RPA amplification: Purchase commercially available basic RPA amplification reagents and add specific primers for the four targets to be tested according to Table 2. Perform asymmetric RPA amplification on samples 1-8 at 37°C for 20 minutes.

[0090] Table 2 Example 1 Multiple asymmetric RPA primer formula and concentration

[0091] 3. Chromatography of amplified products: After heating the amplified product at 85°C for 3 minutes, take 10 μL and mix it with 60 μL of hybridization solution (containing 300 mM KCl, 20 mM Tris-HCl, 0.1% SDS, and 5% formamide). After mixing evenly, add all 70 μL dropwise onto the sample pad of the test strip described in step 1. The amplified product of each sample is added dropwise to a test strip for detection. After 10 minutes of chromatography, observe the results. The sample information and chromatography results are recorded in Table 3.

[0092] Table 3 Chromatographic detection results of Example 1

[0093] Note: The degree of color development is recorded as very clear (+++), relatively clear (++), relatively weak (+), and no color development (-).

[0094] The test results showed that asymmetric RPA amplification technology combined with lateral flow hybridization can simultaneously detect four targets: Treponema pallidum, herpes simplex virus type 1, herpes simplex virus type 2, and the human RNaseP internal standard gene. The positive result of the human RNaseP gene as a sample quality control indicates that the amplification and chromatography process of the test is effective, playing a monitoring role for each test. The targets contained in the sample can show positive results on the corresponding test line, and there is no cross-reaction with other test lines, which proves that the method has high accuracy and good specificity. The specific results are shown in Figure 4.

[0095] Example 2 and Comparative Example 1: Triple Detection of Influenza A Virus, Influenza B Virus, and Novel Coronavirus

[0096] 1. Preparation of test strips:

[0097] 1) Spray a solution of red latex microspheres labeled with streptavidin onto a colorimetric pad using a film sprayer at a spray rate of 3 μL / cm. Place the sprayed colorimetric pad in a 37°C incubator to dry for 50 minutes.

[0098] 2) The four capture probes in Table 4 were sequentially streaked onto the chromatographic capture membrane using a film sprayer at a streaking speed of 0.5 μL / cm. The streaked capture membrane was placed in a 55° C. thermostat to dry overnight.

[0099] Table 4 Capture probe sequences and concentrations of Example 2 and Comparative Example 1

[0100] 3) Assemble and bond the sample pad, colorimetric pad, chromatography capture membrane, and absorbent pad according to the test paper structure shown in FIG2 . Cut the test paper into 4 mm wide strips using a cutting machine and seal them in an aluminum foil bag for future use.

[0101] 2. Asymmetric RPA amplification: Purchase commercially available basic RT-RPA amplification reagents and add specific primers for the three targets to be tested according to Table 5. Perform asymmetric RPA amplification on the influenza A / B / COVID-19 sensitivity simulation samples at 39°C for 30 minutes.

[0102] Table 5 Multiple asymmetric RPA primer formulations for Example 2 and Comparative Example 1

[0103] 3. Chromatography of amplified products: After the amplified product was heated at 95°C for 5 minutes, 10 μL was mixed with 70 μL of hybridization solution (containing 300 mM KCl, 20 mM borate buffer, 0.1% SDS, 1% NP-40, and 5% formamide). After mixing evenly, a total of 80 μL was added dropwise to the sample pad of the test strip described in step 1. The results were observed after chromatography for 15 minutes. The sample information and chromatography results are recorded in Table 6.

[0104] Table 6 Chromatographic test results of Example 2 and Comparative Example 1

[0105] Note: The degree of color development is recorded as very clear (+++), relatively clear (++), relatively weak (+), and no color development (-).

[0106] The test results showed that in Example 2, the asymmetric RPA amplification technology combined with lateral flow hybridization was used to detect influenza A virus, influenza B virus and novel coronavirus, and the detection sensitivity was up to 10 3 The results were negative for syncytial virus, adenovirus, parainfluenza virus, Mycoplasma pneumoniae, rhinovirus, and human genome, demonstrating good specificity. In Comparative Example 1, conventional RPA amplification was used instead of asymmetric RPA. The single-stranded content in the amplified product was very low, and the double-stranded product was difficult to be bound by the capture probe for color development. Therefore, the sensitivity of detecting influenza A virus, influenza B virus, and the new coronavirus was poor. See Figure 5 for the specific results.

[0107] Example 3: Detection of transgenic maize MON810 transformants, DBN9936 transformants, and Ruifeng 125 transformants

[0108] 1. Test strip preparation: 1) Spray a solution of blue latex microspheres labeled with fluorescein FITC antibody onto a chromogenic pad using a film sprayer at a spray rate of 1.5 μL / cm. Place the sprayed chromogenic pad in a 37°C incubator to dry for 1 hour.

[0109] 2) The four capture probes in Table 7 were sequentially streaked onto the chromatographic capture membrane using a film sprayer at a streaking speed of 0.8 μL / cm. The streaked capture membrane was placed in a 40° C. thermostat to dry for 16 hours.

[0110] Table 7 Capture probe sequences and concentrations in Example 3

[0111] 3) Assemble and bond the sample pad, colorimetric pad, chromatography capture membrane, and absorbent pad according to the test paper structure shown in FIG2 . Cut the test paper into 3.5 mm wide strips using a cutting machine and seal them in an aluminum foil bag for future use.

[0112] 2. Asymmetric RPA Amplification: Purchase three transformants and non-GM corn kernels, cut them in half along the midline, and incubate half of the kernel in 500 μL of lysis buffer (containing 0.05 M KOH) at room temperature for 1 minute to release nucleic acid for later use. Purchase commercially available basic RPA amplification reagents and add specific primers for the four targets to be tested according to Table 8. Perform asymmetric RPA amplification of the GM corn kernel nucleic acid sample at 37°C for 15 minutes.

[0113] Table 8 Example 3 Multiple Asymmetric RPA Primer Formula

[0114] 3. Chromatography of amplified products: After the amplified product is heated at 90°C for 2 minutes, 5 μL is mixed with 65 μL of hybridization solution (containing 200 mM KCL, 50 mM Tris-HCl, 0.5% Tween 20, 1% NP-40, and 4% DMSO). After mixing evenly, a total of 70 μL is added dropwise to the sample pad of the test strip described in step 1. The amplified product of each sample is added dropwise to a test strip for detection. The results are observed after chromatography for 10 minutes. The sample information and chromatography results are recorded in Table 9.

[0115] Table 9 Chromatographic detection results of Example 3

[0116] Note: The degree of color development is recorded as very clear (+++), relatively clear (++), relatively weak (+), and no color development (-).

[0117] The test results showed that asymmetric RPA amplification combined with lateral flow hybridization could simultaneously detect four targets: transgenic maize MON810, DBN9936, and Ruifeng 125 transformants, as well as a maize internal standard gene. The maize zSSIIb internal standard gene, used as a quality control sample, showed positive results, indicating the effectiveness of the reaction. Kernels from each transgenic variety were observed to be positive at the corresponding transformant detection line, while non-transgenic maize showed negative results for all three transformants. There was no cross-reaction between different transformants, demonstrating the high accuracy and specificity of this method. The total detection time for this method is less than 30 minutes, requiring only a constant temperature device such as a metal bath, making it suitable for rapid field testing. The results are shown in Figure 6.

[0118] The above is a detailed introduction to the nucleic acid detection method, nucleic acid detection composition and kit provided by the present invention. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core ideas. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, the present invention can also be improved and modified in several ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A nucleic acid detection method, characterized in that: Including but not limited to the following steps: Asymmetric amplification was performed using RPA; Detection was performed by lateral flow chromatography.

2. The nucleic acid detection method according to claim 1, characterized in that: The asymmetric amplification using RPA includes: after the limiting primer is consumed, the non-limiting primer with a marker is amplified to produce a single-stranded amplification product, and the single-stranded amplification product carries the marker and can bind to a color developing substance labeled with a specific binding substance to obtain a to-be-detected object.

3. The nucleic acid detection method according to claim 2, characterized in that: The detection by lateral flow chromatography comprises: the carrier of the lateral flow chromatography is provided with a capture probe, and the capture probe is complementary to the single-stranded amplification product; If the single-stranded amplification product is present after amplification, the object to be detected is captured by the capture probe via lateral flow chromatography, and the color developing substance aggregates and develops color, then the target sequence is present in the sample to be detected; On the contrary, if no color is developed, there is no target sequence in the sample to be tested.

4. The nucleic acid detection method according to any one of claims 2 to 4, characterized in that: The molar ratio of the non-limiting primer to the limiting primer includes but is not limited to (2-100):

1.

5. The nucleic acid detection method according to any one of claims 1 to 4, characterized in that: The temperature of the asymmetric amplification includes but is not limited to 35-42° C., and the time of the asymmetric amplification includes but is not limited to 10-40 minutes.

6. The nucleic acid detection method according to any one of claims 1 to 5, characterized in that: A denaturation step is also included between the asymmetric amplification and the lateral flow chromatography; The denaturation temperature includes, but is not limited to, 80 to 100° C., and the denaturation time includes, but is not limited to, 1 to 5 minutes.

7. The nucleic acid detection method according to any one of claims 2 to 6, characterized in that: The capture probe is partially complementary to the single-stranded amplification product; The length of the capture probe includes but is not limited to 10 to 40 bp; preferably, the length of the capture probe includes but is not limited to 20 to 30 bp.

8. The nucleic acid detection method according to any one of claims 2 to 7, characterized in that: The color developing substance includes but is not limited to colored nanoparticles; The colored nanoparticles include but are not limited to colloidal gold particles or latex microspheres; The marker includes but is not limited to one or more of biotin, digoxigenin, and fluorescein; The fluorescein includes but is not limited to one or more of FAM, VIC or FITC; The conjugate includes, but is not limited to, one or more of streptavidin, digoxigenin antibody or fluorescein monoclonal antibody.

9. The nucleic acid detection method according to any one of claims 2 to 8, characterized in that: The carrier includes but is not limited to a test strip; The test strip includes but is not limited to a sample carrying component, a color developing substance carrying component, a chromatography capturing component, Water-absorbing parts; The sample carrying component is used to carry the sample to be tested, and the sample to be tested includes but is not limited to a mixture of the single-stranded amplification product and a hybridization solution; The color developing substance carrying component is used to carry the color developing substance labeled with the specific binding substance; The chromatographic capture component includes, but is not limited to, one or more detection lines, the detection lines carrying the capture probes; The water absorbing component allows the sample to be tested to flow through the test strip through a siphon effect.

10. A composition for nucleic acid detection, characterized in that: Includes vectors for lateral flow chromatography, primer sets for asymmetric amplification; The carrier carries a capture probe and a color developing substance labeled with a specific binding substance; The primer set for asymmetric amplification includes but is not limited to a limiting primer and a non-limiting primer with a label; After the limiting primer is consumed, the non-limiting primer with the marker is amplified to generate a single-stranded amplification product, and the single-stranded amplification product carries the marker and can bind to the color developing substance labeled by the specific binding substance to obtain the object to be detected; The capture probe is complementary to the single-stranded amplification product.

11. The composition for nucleic acid detection according to claim 10, characterized in that: The carrier includes but is not limited to a test strip; The test strip includes but is not limited to a sample-carrying component, a color-developing substance-carrying component, a chromatography capture component, and a water-absorbing component; The sample carrying component is used to carry the sample to be tested, and the sample to be tested includes but is not limited to a mixture of the single-stranded amplification product and a hybridization solution; The color developing substance carrying component is used to carry the color developing substance labeled with the specific binding substance; The chromatographic capture component includes, but is not limited to, one or more detection lines, the detection lines carrying the capture probes; The water absorbing component allows the sample to be tested to flow through the test strip through a siphon effect.

12. The composition according to claim 10 or 11, characterized in that The molar ratio of the non-limiting primer to the limiting primer includes but is not limited to (2-100):

1.

13. The composition according to any one of claims 10 to 12, characterized in that The capture probe is partially complementary to the single-stranded amplification product; The length of the capture probe includes but is not limited to 10 to 40 bp; preferably, the length of the capture probe includes but is not limited to 20 to 30 bp.

14. The composition according to any one of claims 10 to 13, characterized in that The material of the sample-carrying component or the color-developing-substance-carrying component is independently selected from glass fiber, filter paper or polyester film; The material of the chromatography capture component includes but is not limited to nitrocellulose membrane or nylon membrane; The material of the water absorbing component includes but is not limited to non-woven material.

15. The composition according to any one of claims 10 to 14, characterized in that NFO endonuclease is not included.

16. A kit for nucleic acid detection, characterized in that: Comprising the composition according to any one of claims 10 to 15.

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