Method and kit for eliminating false positive result in nucleic acid amplification reaction

Abstract

The invention belongs to the field of nucleic acid detection and molecular biology, and particularly relates to a method and a kit for eliminating false positive results in nucleic acid amplification reaction. According to the method, after a nucleic acid amplification reaction (taking recombinase polymerase amplification, namely RPA, for example) is completed, a target amplification product is subjected to selective enzyme digestion by utilizing restriction endonuclease, non-target products (such as primer dimers and non-specific amplification products) are not cut, and meanwhile, the non-target products which are not subjected to enzyme digestion are removed by combining solid-phase separation, so that the target amplification product is obtained. Therefore, the false positive result is eliminated. The invention also discloses a kit containing the restriction enzyme. According to the method, the specificity and the signal-to-noise ratio of a nucleic acid amplification reaction, especially RPA, are remarkably improved, the cost is low, operation is easy, the method is compatible with an existing technical platform, the method is suitable for detection scenes such as clinical molecular diagnosis, environmental monitoring and food safety, and the problem of misjudgment caused by false positive signals is effectively avoided.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nucleic acid detection and molecular biology, and particularly relates to a method and a kit for eliminating false positive results in nucleic acid amplification. BACKGROUND

[0002] Nucleic acid amplification is a technique based on enzymatic reaction to replicate specific nucleic acid sequences exponentially in vitro. At present, mainstream nucleic acid amplification techniques include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) and the like. In order to achieve high-sensitivity detection of trace amounts of targets, nucleic acid amplification techniques all rely on high-concentration primers, such as RPA primers with a concentration of 0.48 μmmol·L -1 However, factors such as poor primer design and too low reaction temperature may cause erroneous binding and extension of similar sequences between primers or between primers and targets, resulting in non-target products and false positive results.

[0003] Unlike variable-temperature amplification (such as PCR), RPA is a continuous reaction at a constant low temperature (37 ºC). The reduction of reaction temperature leads to more significant non-specific amplification of RPA, which is more susceptible to interference from false positive results in actual applications. The sources of false positive results mainly include two categories:

[0004] 1) Primer interaction: due to low reaction temperature (37 ºC) and high magnesium ion concentration (14 mmol·L -1 ), RPA primers interact to form complex and relatively stable secondary structures, and accumulate a large amount of non-target products under the action of DNA polymerase;

[0005] 2) Non-specific amplification: non-complete complementary binding of primers and similar sequences of target templates leads to amplification of non-target products.

[0006] At present, the main method to solve false positive results is to combine nucleic acid probes to specifically detect target products, including CRISPR technology and artificial nucleic acids and the like. However, these methods have obvious limitations: lack of universality. In order to meet the detection needs of different targets, it is necessary to redesign and optimize the recognition probes.

[0007] Therefore, nucleic acid amplification (especially RPA) detection urgently needs a universal and simple solution to improve the accuracy of nucleic acid amplification detection results. SUMMARY

[0008] The present application aims to overcome the shortcomings of existing nucleic acid amplification (taking RPA as an example) techniques, and provide a method capable of effectively eliminating false positive results in amplification. Another object of the present application is to provide a kit for implementing the method.

[0009] To achieve the above object, the present application adopts the following technical solution: after the completion of nucleic acid amplification reaction (such as RPA), the target amplification product is selectively cleaved by restriction endonuclease, and the non-target product not cleaved by enzyme is removed by solid phase separation, so as to ensure the accuracy of the detection result. The core of the method is that a specific restriction endonuclease is added in the product of conventional nucleic acid amplification reaction (such as RPA), the enzyme specifically binds to the specific sequence site contained in the target product, and is cleaved in or near the recognition site, and the cleavage target does not exist in the upstream primer and downstream primer and non-specific amplification product. In the enzyme incubation stage after the amplification reaction, the restriction endonuclease efficiently recognizes and hydrolyzes the target product existing in the system, and hardly reacts with the non-target product (primer dimer and non-specific amplification product, etc.). Combined with solid phase separation (such as magnetic separation) technology, the non-target product is removed, so as to ensure the accuracy of the positive signal caused by the target product.

[0010] Specifically, the present application provides a method for eliminating false positive results in nucleic acid amplification reaction, the method comprising the following steps:

[0011] (1) designing and synthesizing an upstream primer and a downstream primer for amplifying a target sequence by nucleic acid amplification reaction, wherein one of the upstream primer and the downstream primer comprises a combinable label;

[0012] (2) performing nucleic acid amplification reaction using the upstream primer and the downstream primer to obtain an amplification product carrying the combinable label;

[0013] (3) contacting a nucleic acid amplification reaction system comprising the amplification product with a solid phase carrier capable of specifically combining with the combinable label, so that the amplification product is combined to the solid phase carrier;

[0014] (4) contacting the solid phase carrier with at least one restriction endonuclease, so that the amplification product comprising the target sequence is specifically cleaved by the restriction endonuclease, and then released from the solid phase carrier, wherein the recognition site of the restriction endonuclease is located in the target sequence, and does not exist in the non-target sequence;

[0015] (5) separating the cleaved amplification product released from the solid phase carrier from the solid phase carrier to obtain the cleaved amplification product with false positive results eliminated.

[0016] As used herein, the target sequence is used interchangeably with the target nucleic acid sequence, which refers to the sequence of the region of the target nucleic acid of interest actually amplified by the nucleic acid amplification reaction.

[0017] Methods for designing upstream and downstream primers are well known to those skilled in the art and can be easily performed with the help of software, according to the needs and the target nucleic acid sequence to be amplified.

[0018] In some embodiments, one of the upstream and downstream primers comprises a bindable label. In preferred embodiments, only one of the upstream and downstream primers comprises a bindable label.

[0019] In some embodiments, the bindable label is typically located at the 5' end of the primer and is selected from, but not limited to, a biotin label, a polyhistidine tag, a thiol label, an amino label, a specific oligonucleotide capture sequence label or a fluorescent molecule label.

[0020] In preferred embodiments, the bindable label is a biotin label.

[0021] As used herein, the solid support to which the bindable label specifically binds can be any material, for example, the solid support can be selected from, but not limited to, magnetic particles, agarose or Sepharose affinity resins, silica or silicon-based materials, functionalized microplates, nylon or nitrocellulose membranes, glass or silicon chip microarrays, cellulose or paper-based materials, affinity chromatography resins or microfluidic chip surfaces.

[0022] In preferred embodiments, the solid support is a magnetic particle, such as a magnetic bead.

[0023] The selection or modification of the solid support is within the capabilities of those skilled in the art, depending on the bindable label used. For example, when the label is biotin, the solid support can be a material coated with streptavidin; when the label is a His tag, the solid support can be a chelating support coated with Ni2+or Co2+ions; when the label is a thiol or an amino group, the solid support can be a support with maleimide, carboxyl, epoxy or active ester functional groups; when the label is an oligonucleotide capture sequence, the solid support surface is immobilized with an oligonucleotide probe complementary to the capture sequence.

[0024] In preferred embodiments, when the bindable label is biotin, the solid support is a magnetic bead coated with streptavidin.

[0025] The method of the present application for eliminating false positive results can be used to eliminate false positive results generated in any nucleic acid amplification reaction, for example, the nucleic acid amplification reaction can be a polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) or other in vitro amplification reactions.

[0026] As used herein, a false positive result refers to the occurrence of an amplification signal or product in a system that does not contain the target template, resulting in a false positive result for a negative sample, or the occurrence of an amplification signal or product that is inconsistent with the target amplification product in a system that does contain the target template. False positive results can result from primer interaction amplification products (e.g., primer dimers), or from non-specific amplification resulting from primer mismatch or non-perfect complementarity.

[0027] In some embodiments, the non-target sequences include primer sequences, amplification sequences resulting from primer interaction, and nucleic acid sequences resulting from non-specific amplification.

[0028] The restriction enzyme used in the methods of the present application can be selected from any suitable restriction enzyme, provided that its recognition site is only present in the target amplification sequence of the nucleic acid amplification reaction, but not in the sequence of the non-target product (e.g., primer dimers). For example, the upstream and downstream primers do not contain the recognition site of the restriction enzyme, and the sequence of the specific nucleic acid amplification product contains at least one recognition site of the restriction enzyme. Alternatively, the restriction enzyme is capable of cleaving the target product of the nucleic acid amplification reaction, but not the non-specific target amplification product. The specific selection of the restriction enzyme can be determined by the sequence of the amplification region of the nucleic acid amplification reaction, and a plurality of restriction enzymes can be used to detect the amplification region of the same nucleic acid amplification reaction. The restriction enzyme has a specific recognition sequence, and by reasonably designing the upstream and downstream primers for the nucleic acid amplification reaction, the enzyme can be used to detect nucleic acids of different species. As an example, a suitable restriction enzyme can be selected based on one of the following two strategies: Strategy 1 : Determine the amplification region of the nucleic acid amplification reaction (e.g., RPA) by primers, and use the nucleic acid sequence of the region to screen restriction enzymes that specifically bind and cleave the region; Strategy 2: Determine the RPA amplification region according to the recognition sequence of the restriction enzyme, and then design RPA upstream and downstream primers that do not contain the recognition site.

[0029] In some embodiments, the target sequence can contain at least one recognition site of the restriction enzyme.

[0030] In some embodiments, the restriction enzyme cleavage reaction and subsequent solid phase separation process can be directly performed in the amplification reaction system by adjusting the temperature of the reaction system and / or adding an enzyme reaction buffer to initiate the reaction, or can be performed in an independent reaction system that is separate from the amplification reaction system.

[0031] Therefore, in some embodiments, the step (3) can further include a step of washing the solid phase carrier to remove unbound substances.

[0032] In some embodiments, the cleaved amplification product released from the solid support comprises a portion of the target sequence.

[0033] In some embodiments, after the separation in step (5), another portion of the target sequence and the primer capable of binding a label are attached to the solid support. After the separation step, the amplification product can be transferred to a new dedicated reaction system for detection, or a compartmentalized reaction can be achieved by combining the 3D printed device, thereby avoiding aerosol escape caused by open lid detection.

[0034] In other aspects, the present application also provides a kit for specific nucleic acid amplification, the kit comprising:

[0035] (1) an upstream primer and a downstream primer for amplifying a target sequence, one of which comprises a label capable of binding as described herein;

[0036] (2) at least one restriction enzyme as described herein and its reaction buffer, wherein the recognition site of the restriction enzyme is located in the target sequence but not in the non-target sequence;

[0037] (3) at least one solid support as described herein and its washing buffer, which can specifically bind to the label capable of binding;

[0038] (4) reagents for performing nucleic acid amplification reactions.

[0039] In some embodiments, the nucleic acid amplification reaction is PCR, LAMP, RPA or other in vitro amplification reaction.

[0040] The reagents for performing nucleic acid amplification reactions such as PCR, LAMP or RPA are well known to those skilled in the art, including, for example, dNTPs, DNA polymerase, recombinase complex and buffers, etc.

[0041] As used herein, the reaction buffer for the selected restriction enzyme and the washing buffer for the solid support are well known to those skilled in the art and can be routinely formulated or commercially available as needed.

[0042] The primers in the kit can be designed in advance so that the corresponding amplification region contains the recognition sequence of the selected restriction enzyme, but does not contain the recognition sequence itself, thereby ensuring that the restriction enzyme specifically recognizes only the target product.

[0043] Further, the kit also comprises instructions for performing the method as described herein to eliminate false positive results.

[0044] Advantages of the present application

[0045] Compared with the prior art, the present application has the following remarkable advantages:

[0046] 1) High signal-to-background ratio: reduce the impact of non-target products without affecting the detection of target products, and improve the signal-to-background ratio of nucleic acid amplification detection;

[0047] 2) Universality: the same target product contains recognition sites of multiple restriction enzymes, and a single restriction enzyme can be used to solve the false positive results of nucleic acid amplification detection of different species;

[0048] 3) Easy to operate: only add universal solid phase separation (such as magnetic separation) and enzyme cutting operation after the conventional nucleic acid amplification process, without complex equipment or process optimization, user-friendly;

[0049] 4) Low cost: the restriction enzyme used is cheap, and the cost of a single reaction is much lower than the cost of repeated experiments caused by false positive results;

[0050] 5) High flexibility: according to the characteristics of the target sequence, different restriction enzymes can be flexibly selected and combined. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 is a flowchart of the method of the present application.

[0052] Figure 2 is a gel electrophoresis result graph of Example 1 using restriction enzyme BstYI to eliminate false positive results of RPA detection of bacterial genome (Brucella).

[0053] Figure 3 is a gel electrophoresis result graph of Example 2 using different restriction enzymes to eliminate false positive results of RPA detection of bacterial genome (Brucella).

[0054] Figure 4 is a gel electrophoresis result graph of Example 3 using the same restriction enzyme to eliminate false positive results of RPA detection of nucleic acids of different species.

[0055] Figure 5 is a gel electrophoresis result graph of Example 4 using restriction enzymes with different recognition sequence lengths to eliminate false positive results of RPA detection.

[0056] Figure 6 shows the experimental result graph of Example 5 showing the effect of the continuity of the recognition sequence of the restriction enzyme on its discrimination performance.

[0057] Figure 7 shows the experimental result graph of Example 6 showing the effect of the buffer and reaction temperature of the restriction enzyme on its discrimination performance. DETAILED DESCRIPTION

[0058] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0059] Example 1: Restriction endonuclease eliminates false positive results of RPA

[0060] Technical issue: When using RPA to detect Brucella genomic DNA, the control group ( Figure 2 In the "blank" lane, false positive bands appeared that were inconsistent with the target band, while in the sample group ( Figure 2 The target band and false positive bands appeared simultaneously in the "sample" lane. These bands may be caused by non-specific amplification induced by primer interactions.

[0061] Solution:

[0062] 1) Principle: The restriction endonuclease BstYI is screened based on the nucleic acid sequence of the RPA target amplification region. This enzyme specifically recognizes and cleaves the RPA target product, and has difficulty cleaving non-target products. Furthermore, by combining magnetic separation to remove non-target products, the detection signal of the target product can be obtained.

[0063] 2) Steps:

[0064] a. Design upstream and downstream primers for detecting Brucella RPA, and label one of the primers (upstream or downstream primer) with biotin.

[0065] b. Perform routine RPA responses, including control and sample groups;

[0066] c. Add streptavidin magnetic beads to the above reaction tubes respectively, and separate and wash the magnetic beads;

[0067] d. Add BstYI to the obtained magnetic bead reaction solution, incubate at 37 ºC for 10 min, and separate the magnetic beads;

[0068] e. Analyze the RPA products released by enzymatic cleavage using natural polyacrylamide gel electrophoresis.

[0069] 3) Experimental results: BstYI enzymatic digestion and magnetic separation can effectively eliminate false positive results caused by non-target products of RPA, and have little impact on the judgment of true positive results. Figure 2 (The two rightmost lanes in the middle).

[0070] Example 2: Universality of Restriction Endonuclease Solution

[0071] Technical problem: The recognition sequence of BstYI is single, which is difficult to meet the recognition needs of other RPA target products.

[0072] Solution:

[0073] 1) Principle: There are many types of restriction enzymes, which have multiple recognition and hydrolysis sites. By combining different restriction enzymes, it is expected to solve the problem of false positive results in different RPA detection.

[0074] 2) Steps:

[0075] a. According to the detection area determined by the RPA primer, screen the restriction enzyme which specifically binds and digests with it;

[0076] b. Complete the conventional RPA reaction, including the control group and the sample group;

[0077] c. Refer to steps c ~ d in Example 1, set up 3 groups of parallel analysis samples (including blank group and experimental group), and explore the performance of different restriction enzymes in distinguishing RPA target and non-target products.

[0078] 3) Experimental results: Restriction enzymes 1, 2 and 3 can specifically detect RPA target products, and well solve the problem of false positive results caused by non-target products, indicating that one or more restriction enzymes can be selected according to the needs.

[0079] Example 3: Universality of restriction enzyme scheme

[0080] Technical problem: Non-specific amplification of RPA detection of nucleic acids of different species exists, leading to false positive results.

[0081] Solution:

[0082] 1) Principle: Because the recognition sequence of restriction enzyme is short, it exists in large quantities in nucleic acids of different species. Therefore, the same restriction enzyme is expected to solve the problem of false positive results in different RPA detection.

[0083] 2) Steps:

[0084] a. According to the recognition sequence of restriction enzyme, determine the RPA amplification region in nucleic acids of different species, and design the related RPA upstream and downstream primers. In order to ensure the distinguishing performance of restriction enzyme, the RPA primers do not contain the recognition sequence. At the same time, one of the primers of different species RPA primers is labeled with biotin;

[0085] b. Refer to steps b ~ d in Example 1, add HhaI to the RPA blank group and sample group of different species, so as to explore the universality of the restriction enzyme scheme.

[0086] 3) Experimental results: Restriction enzymes can solve the false positive results of RPA detection of nucleic acids in different species, including viruses (feline herpes virus), bacteria (salmonella) and human genome, with good universality.

[0087] Example 4: Effect of recognition sequence length on restriction enzyme discrimination performance

[0088] Technical problem: The recognition sequence length of restriction enzyme is mainly divided into 4, 6 and 8 bp. The smaller the length, the higher the frequency of its occurrence in the genome, which may interfere with the non-specific amplification of RPA, and then affect the solving performance of false positive results.

[0089] Solution:

[0090] 1) Principle: By comparing the removal effect of non-target products and the number of enzyme digestion of RPA target products, the performance difference of the enzyme can be directly reflected.

[0091] 2) Steps:

[0092] a. Select three restriction enzymes with overlapping recognition sites and the same hydrolysis site. According to the recognition site, determine the RPA amplification region, and design the RPA upstream and downstream primers. At the same time, one of the RPA primers is labeled with biotin;

[0093] b. Refer to steps b ~ d in Example 1, set up 3 groups of parallel analysis samples (including blank group and experimental group), and add three kinds of restriction enzymes respectively, so as to explore the influence of recognition sequence length on the discrimination performance of restriction enzyme.

[0094] 3) Experimental results: The target products released by the three kinds of restriction enzymes are the same in size and quantity, and they all solve the problem of RPA false positive results well, indicating that the recognition sequence length has little effect on the discrimination performance of the enzyme.

[0095] Example 5: Effect of recognition sequence continuity on restriction enzyme discrimination performance

[0096] Technical problem: The discrimination performance of restriction enzyme depends on its recognition sequence. Some enzymes have discontinuous recognition sequences with arbitrary sequences inserted in the middle, which may affect the non-specific amplification of RPA, and then interfere with the discrimination performance of restriction enzyme.

[0097] Solution:

[0098] 1) Principle: Determine the arbitrary sequence base of restriction enzyme to obtain a group of determined recognition sequences, and analyze the discrimination performance of the enzyme. Since random sequences can also be recognized and digested by restriction enzymes, the discrimination performance of the enzyme can be directly explored by the digestion products.

[0099] 2) Steps:

[0100] a. According to the discontinuous recognition sequence of the restriction enzyme, the RPA amplification region is determined, and the RPA upstream and downstream primers are designed. At the same time, one of the RPA primers is labeled with biotin;

[0101] b. Refer to steps b ~ d in Example 1, and add the above restriction enzyme to explore the influence of the continuity of the recognition sequence on the restriction enzyme discrimination performance.

[0102] 3) Experimental results: The restriction enzyme digestion target product is single, and no obvious non-target amplification product is observed, indicating that the length of the recognition sequence has little effect on the enzyme discrimination performance.

[0103] Example 6: Effect of buffer and reaction temperature of restriction enzyme on its discrimination performance

[0104] Problem: The buffer and reaction temperature of some restriction enzymes are different, which may affect the binding of magnetic beads and RPA target nucleic acid, and then interfere with the enzyme discrimination performance.

[0105] Solution:

[0106] 1) Principle: Select the restriction enzyme with NEBuffer™ 1 as the reaction buffer and 55 °C as the reaction temperature to explore the effect of reaction conditions on its discrimination performance.

[0107] 2) Steps:

[0108] a. According to the recognition sequence of the two restriction enzymes, the RPA amplification region is determined, and the RPA upstream and downstream primers are designed. At the same time, one of the RPA primers is labeled with biotin;

[0109] b. Refer to steps b ~ d in Example 1, and add the above restriction enzyme to the two groups of products, respectively, to explore the effect of enzyme reaction conditions.

[0110] 3) Experimental results: The enzyme digestion products of the two enzymes are single target bands, and no obvious non-target bands are observed, indicating that the reaction conditions have no obvious effect on the enzyme digestion reaction and magnetic separation process of the restriction enzyme.

[0111] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

[0112] In summary, the present application provides a flexible and universal solution for nucleic acid amplification false positive results. Taking RPA as an example, the present solution significantly improves the specificity and reliability of RPA detection. Those skilled in the art can make appropriate adjustments to the types of enzymes, reaction conditions, etc. according to the principles of the present application, and these adjustments should all fall within the scope of the present application.

[0113] It should be noted that the preferred embodiments of the present application are given in the specification and drawings of the present application, but the present application can be implemented in many different forms and is not limited to the embodiments described in the specification. These embodiments are not additional limitations on the content of the present application, and the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Furthermore, the above technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present application. Furthermore, those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should be within the scope of the appended claims of the present application.

Claims

1. A method for eliminating false positive results in nucleic acid amplification reactions, characterized in that, The method includes the following steps: (1) Design and synthesize upstream and downstream primers for amplifying a target sequence by a nucleic acid amplification reaction, wherein one of the upstream and downstream primers contains a binding marker; (2) Use the upstream and downstream primers to perform a nucleic acid amplification reaction to obtain an amplification product carrying the binding label; (3) The nucleic acid amplification reaction system containing the amplification product is brought into contact with a solid support that can specifically bind to the bindable label, so that the amplification product binds to the solid support; (4) The solid-phase carrier is contacted with at least one restriction endonuclease, such that the amplification product containing the target sequence is specifically cleaved by the restriction endonuclease and released from the solid-phase carrier, wherein the recognition site of the restriction endonuclease is located in the target sequence and not in a non-target sequence; (5) The cleaved amplification product released from the solid support is separated from the solid support to obtain the cleaved amplification product with the false positive result eliminated.

2. The method according to claim 1, characterized in that, The bindable marker is selected from biotinylate markers, polyhistidine tags, thiol tags, amino tags, specific oligonucleotide capture sequence markers, or fluorescent molecular markers.

3. The method according to claim 1 or 2, characterized in that, The solid support is selected from magnetic particles, agarose or Sepharose affinity resin, silica gel or silicon-based materials, functionalized microplates, nylon or nitrocellulose membranes, glass or silicon microarrays, cellulose or paper-based materials, affinity chromatography resins or microfluidic chip surfaces.

4. The method according to claim 3, characterized in that, When the bindable label is biotin, the solid-phase carrier is coated with streptavidin.

5. The method according to claim 1, characterized in that, The nucleic acid amplification reaction is polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), or other in vitro amplification reactions.

6. The method according to claim 1, characterized in that, The non-target sequences include primer sequences, amplification sequences generated by primer interactions, and nucleic acid sequences generated by non-specific amplification.

7. The method according to claim 1, characterized in that, The target sequence contains at least one recognition site of the restriction endonuclease, and the cleaved amplification product released from the solid-phase carrier contains a portion of the target sequence.

8. The method according to claim 1, characterized in that, Step (3) further includes washing the solid support to remove unbound material.

9. A kit for specific nucleic acid amplification, characterized in that, The kit contains: (1) Upstream and downstream primers for amplifying the target sequence, wherein the upstream and downstream primers are defined as in any one of claims 1-8; (2) At least one restriction endonuclease and its reaction buffer, wherein the restriction endonuclease is defined as in any one of claims 1-8; (3) At least one solid support and its washing buffer, wherein the solid support is defined as in any one of claims 1-8; (4) Reagents used to perform nucleic acid amplification reactions.

10. The reagent kit according to claim 9, characterized in that, The nucleic acid amplification reaction is PCR, LAMP, RPA, or other in vitro amplification reactions; Furthermore, the kit also includes instructions for performing the method according to any one of claims 1-8 to eliminate false positive results.

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