Rapid amplification and side chromatography detection method of virus RNA (Ribonucleic Acid) and application of rapid amplification and side chromatography detection method

Abstract

The invention belongs to the technical field of RNA (Ribonucleic Acid) rapid detection, and particularly relates to a rapid amplification and side chromatography detection method of virus RNA and application. According to the method, a virus nucleic acid crude extract is subjected to reverse transcription of a single-tube compound enzyme reaction system, restriction enzyme digestion and one-way isothermal strand displacement amplification, and then a large number of specific-sequence single-stranded DNA with markers is generated; in the side chromatography process, the single-stranded DNA is captured by a nucleic acid probe fixed in a detection area on the virus RNA detection test paper, meanwhile, avidin marked on the colloidal gold particles is combined with biotin marked on the fixed single-stranded DNA, and the colloidal gold particles are indirectly fixed for developing and indicating a result. The method provided by the invention is simple and convenient to operate, does not need complicated nucleic acid extraction, does not need large equipment in the whole detection process, can interpret the result by naked eyes, can realize on-site rapid detection, and has the advantages of simplicity, rapidness, high sensitivity and low cost.

Description

Technical Field

[0001] The invention belongs to the technical field of RNA rapid detection, and specifically relates to a rapid amplification and lateral chromatography detection method of viral RNA and application thereof. Background Art

[0002] With the development of the economy, animal husbandry has gradually developed towards scale and intensiveness. Frequent outbreaks of animal infectious diseases caused by viruses have caused huge losses to animal husbandry. Most of these viruses are RNA viruses with high mutation rates, which are difficult to achieve rapid on-site detection, diagnosis and control. At present, there is no effective drug treatment for diseases caused by RNA viruses. The most effective means of prevention and control can only be rapid detection and early detection, and early disinfection and treatment. Therefore, the demand for rapid on-site detection of RNA viruses is very urgent.

[0003] Most of the current rapid virus detection technologies are antigen detection, but due to the high variability of RNA viruses, the antigen sites also mutate quickly, and it is difficult for antibodies to stably identify and bind to new variant antigens. Therefore, the accuracy of the test results is low; the target of another rapid detection direction is viral nucleic acid, that is, the virus is monitored by detecting the nucleic acid material of the virus. PCR-related technologies can quickly and efficiently amplify nucleic acid signals and have high detection sensitivity, but the operation process based on nucleic acid detection methods is complicated, requiring professional equipment and venues as well as skilled technicians. Therefore, it is not suitable for low-cost on-site rapid detection, which limits the promotion and popularization of such technical methods in rapid detection scenarios. Direct PCR technology avoids the purification of nucleic acids to a certain extent, but is limited to the detection of some DNA target samples; nucleic acid isothermal amplification technology reduces the equipment requirements for nucleic acid amplification to a certain extent, but its result output still requires corresponding equipment detection signals; nucleic acid chromatography technology expands the detection targets to a certain extent and can greatly simplify the display of test results, but it still requires complex pre-treatment, and the repeatability and stability are poor.

[0004] Therefore, in order to overcome the above technical defects in nucleic acid detection, it is of great significance to develop a simple and stable on-site rapid detection method for viral RNA. Summary of the invention

[0005] In order to solve the deficiencies and shortcomings of the above-mentioned prior art, the primary purpose of the present invention is to provide a method for rapid amplification of viral RNA. The method of the present invention is to use crude target RNA as the detection object, firstly use reverse transcriptase and primers with nicking enzyme recognition sites to initiate the reverse transcription process to synthesize cDNA / RNA hybrid chains; then use extension primers and DNA polymerase to synthesize double-stranded cDNA in the target area; restriction endonucleases cut off the sequence segments related to the extension primers; finally, the nicking endonucleases and DNA polymerases work together to initiate isothermal chain displacement reactions to produce a large number of single-stranded nucleic acids (DNA) of specific sequences. In addition, during the nucleic acid synthesis process, the specifically labeled deoxyribonucleotides added to the system are embedded in the newly synthesized chain to achieve labeling of the product chain; the labeled single-stranded nucleic acid product can be detected using a matching nucleic acid detection test strip, and the results can be read by naked eyes.

[0006] Another object of the present invention is to provide a lateral flow detection method for viral RNA, which first uses a complementary sequence fixed on the lateral flow detection line, a nucleic acid probe, to capture the above-mentioned product single-stranded nucleic acid, and then colloidal gold particles labeled with functional proteins (e.g., SA) recognize and bind to the marker on the fixed DNA, and then are indirectly fixed and display bands, and finally, the results are interpreted by naked eyes. This method has the advantages of simple and rapid operation, high sensitivity and specificity, and can be used for on-site rapid detection of RNA.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for rapid amplification of viral RNA comprises the following steps:

[0009] S1. Lyse the sample containing viral RNA with a lysis buffer to obtain a crude viral nucleic acid extract;

[0010] S2, mixing the crude viral nucleic acid extract obtained in step (1) with a mixed enzyme reaction system, and reacting at 40-45° C. for 20-40 min to obtain an amplification product, i.e., a single-stranded DNA fragment with a marker; wherein the mixed enzyme reaction system comprises the following components: reverse transcriptase buffer, reverse transcriptase, DNA polymerase buffer, DNA polymerase, nicking endonuclease, restriction endonuclease, dNTPs Mix, marker-modified dUTP, reverse transcription primer, extension primer and bovine serum albumin;

[0011] The lysate in step S1 preferably comprises the following components: 0.5% (v / v) Triton X-100, 0.75% (w / v) NaCl;

[0012] When the viral RNA described in step S1 is double-stranded viral RNA, the lysis solution further comprises a reverse transcription primer and an extension primer, and the lysis condition is 60-65°C for 3-5 min; this is because the single-stranded RNA can directly bind to the primer to start the amplification reaction after cleavage, and the double-stranded RNA needs to be heated during cleavage to denature its two RNA chains to open them, so that it can bind to the primer to start the amplification reaction; the concentration of the reverse transcription primer and the extension primer in the lysis solution is preferably 2nM-10μM;

[0013] The lysate in step S1 preferably further comprises exogenous RNA, which can not only occupy the RNase in the system to protect the target viral RNA, but also serve as a target for subsequent quality control line (C line) detection to perform quality control on the entire detection process;

[0014] After the cleavage in step (1) is completed, it is preferred to further mix the system thoroughly;

[0015] The sample to be tested in step S1 may be a nasal swab sample, a throat swab sample, a serum sample or other body fluid sample containing the virus;

[0016] The mixing of the crude viral nucleic acid extract and the mixed enzyme reaction system in step S2 is preferably performed within 5 minutes after the lysis in step (1);

[0017] The volume ratio of the cleavage product and the mixed enzyme reaction system in step S2 is preferably 1:1;

[0018] The nucleic acid product fragment amplified by the reverse transcription primer and the extension primer in step S2 is preferably located in the conserved segment of the target RNA (viral RNA or exogenous RNA to be detected) sequence;

[0019] There is a single restriction endonuclease site inside the nucleic acid product fragment amplified by the reverse transcription primer and the extension primer in step S2 near the 3' end of the extension primer (non-extension primer sequence); the corresponding restriction endonuclease in the mixed enzyme reaction system can completely remove the extension primer segment in the double-stranded cDNA product, thereby avoiding the extension primer causing the single-stranded product to be amplified twice to a double-stranded product;

[0020] The reverse transcription primer described in step S2 is provided with a nicking endonuclease recognition site, and the corresponding nicking endonuclease in the mixed enzyme reaction system can recognize and cleave the nicking endonuclease site on the reverse transcription primer; the nicking enzyme recognition site does not need to exist in the target RNA sequence, and is a sequence added during primer synthesis;

[0021] The DNA polymerase described in step S2 needs to be free of exonuclease activity, and can be klenow fragment (3'-5'exo-) or Bst DNA polymerase, etc., and its corresponding buffer can be klenow fragment buffer, Bst Buffer, etc.;

[0022] In the marker-modified dUTP described in step S2, the marker of the labeled nucleotide is biotin, digoxigenin, fluorescein or other small molecules; the labeled nucleotide can be a single nucleotide or a complex of multiple nucleotides;

[0023] The marker-modified dUTP is preferably Biotin-11-dUTP;

[0024] The single-stranded DNA fragment with the marker described in step S2 has a specific sequence of at least 6 bases inside, and this sequence serves as the capture site of the nucleic acid probe in the viral RNA rapid test strip to ensure the specificity of the test result;

[0025] The mixed enzyme reaction system described in step S2 can change the primer sequence, nicking endonuclease and restriction endonuclease type and dosage according to different target RNA sequences;

[0026] When the viral RNA is single-stranded RNA, the mixed enzyme reaction system (2×) described in step S2 preferably comprises the following components per 20 μL:

[0027]

[0028] When the viral RNA is double-stranded viral RNA, based on the fact that reverse transcription primers and extension primers have been added to the lysate, the mixed enzyme reaction system described in step S2 may not contain reverse transcription primers and extension primers; that is, when the viral RNA is double-stranded viral RNA, the content of reverse transcription primers and extension primers in the mixed enzyme reaction system described in step S2 is 0-5 μM, and the other components are the same as those for RNA detection of single-stranded viral RNA viruses;

[0029] When the mixed enzyme reaction system is a freeze-dried powder premix, it is preferred to add a molding agent, mannitol, in an amount of 5% (w / v) of the freeze-dried powder premix;

[0030] A lateral flow chromatography detection method for viral RNA comprises the following steps:

[0031] S1. Add the amplification product obtained by the above-mentioned rapid amplification method of viral RNA to the sample pad of the viral RNA detection test paper, and add the sample loading buffer to the sample pad; the viral RNA rapid detection test paper is a lateral flow test paper, comprising a back plate, a sample pad (SP), a marker pad (CP), a sample loading pad (LP), a nitrocellulose membrane and absorbent paper; the nitrocellulose membrane is coated with a nucleic acid probe, which can capture the amplification product; the marker pad is coated with a color development conjugate, which can identify the marker of the amplification product and is fixed;

[0032] S2. Interpretation of results: After 10-15 minutes of chromatography, interpret the results;

[0033] The amount of the amplification product in step S1 is preferably 5 μL;

[0034] The color-developing conjugate of the viral RNA detection test paper described in step S1 is a labeled product of colloidal gold, colored microspheres, magnetic microspheres, fluorescent microspheres or quantum dots, and the corresponding labeling method can adopt gold-sulfur bond or covalent bond coupling;

[0035] The back plate of the virus RNA detection test paper described in step S1 is sticky, the nitrocellulose membrane is pasted on the middle part of the back plate, and absorbent paper and a sample loading pad are pasted on both ends thereof, respectively, the absorbent paper and the sample loading pad overlap with the nitrocellulose membrane by 1-2 mm, and the end of the sample loading pad away from the nitrocellulose membrane is coated with a dry SA (streptavidin) gold label conjugate, and this part is called a marker pad; at the sample loading end, except for the sample loading pad part covered with a sample pad, this sample pad completely covers the marker pad and the blank part of the back plate, but does not cover the sample loading pad;

[0036] In the SA gold-labeled conjugate, SA is bound to the surface of colloidal gold particles through electrostatic interaction and gold-sulfur bond labeling. The product is quantitatively sprayed on one end of the sample pad manually or by machine, and becomes a label pad after drying;

[0037] The sample pad is where the sample buffer is added, and its length covers the marker pad to one end of the back plate; it is prepared by the following method: a mixed solution containing 0.2% tween-20 (v / v), 0.2% Triton X-100 (v / v) and 0.2% bovine serum albumin (w / v) is evenly applied to a glass fiber membrane and dried;

[0038] The sample loading pad is where the amplification product is added, and its preparation method is the same as that of the sample pad. The sample loading pad can also be used as a bearing structure of the marker pad;

[0039] The nitrocellulose membrane is fixed with a detection line and a quality control line; the nucleic acid probe coated on the detection line is complementary to a partial sequence of an amplified single-stranded product of the viral RNA to be detected, and the nucleic acid probe coated on the quality control line is complementary to a partial sequence of an amplified product of an exogenous RNA; wherein the quality control line detects an amplified product of an exogenous RNA (for example, rice Actin gene RNA) amplified by the same system, thereby achieving quality control of the entire system.

[0040] The amount of the nucleic acid probe coated on the test line or quality control line is 0.3-1.5 μL / cm, corresponding to a working concentration of the nucleic acid probe of 10-100 μM;

[0041] The specific operation of the coating is preferably: spraying the corresponding nucleic acid probe onto the detection line or quality control line area of ​​the nitrocellulose membrane according to the conventional method, the coated nitrocellulose membrane is firstly subjected to UV glue crosslinking for 10-20 minutes, then dried at 37-50°C for 16-20 hours, and stored in a dark and dry place for later use;

[0042] The viral RNA detection test strip preferably uses the addition of a loading buffer to initiate chromatography of the amplification product and the marker;

[0043] The loading buffer is 4-8×SSC solution, Tris-HCl, boric acid buffer, phosphate buffer or physiological saline;

[0044] The amount of the loading buffer is preferably 3-4 drops of loading buffer (about 75-100 μL);

[0045] When the amplification product is a single-stranded DNA fragment labeled with biotin, and the marker pad of the viral RNA detection test paper is coated with SA gold-labeled conjugate, the result interpretation is:

[0046] When the test line shows red and the quality control line shows normal color, the result is positive, which means the sample contains the corresponding viral RNA and the concentration is higher than the minimum detection limit; when the test line does not show color and the quality control line shows normal color, the result is negative, which means the sample does not contain the corresponding viral RNA or the concentration is lower than the minimum detection limit; when the quality control line does not show color, the result is invalid regardless of whether the detection limit shows color or not, and retesting is required;

[0047] The viral RNA rapid detection test paper can use a portable device to read the grayscale and semi-quantitatively detect the target; it can also use a fluorescent marker to replace colloidal gold and realize semi-quantitative detection of the target by reading the fluorescent signal.

[0048] Application of the above method in the field of viral RNA detection;

[0049] The application described does not include the purpose of treating or diagnosing a disease;

[0050] The working principle of the rapid detection method of viral RNA provided by the present invention is:

[0051] (1) The present invention uses a lysis solution added with exogenous RNA to crudely extract viral RNA, thereby obtaining a lysis crude extract containing viral RNA; wherein the exogenous RNA added to the lysis solution can not only occupy the RNase in the system to protect the target viral RNA, but also serve as a quality control line (C line) target to perform quality control on the entire process.

[0052] (2) The cleavage crude extract containing viral RNA is amplified in a single-tube complex enzyme reaction system. During this process, the RNA is first converted into cDNA / RNA under the action of a reverse transcription (RT) primer with a nicking enzyme recognition site and AMV enzyme; then, the extended primer is synthesized into a double-stranded DNA containing a restriction endonuclease site and a nicking endonuclease site under the action of a DNA polymerase (e.g., klenow fragment); the restriction endonuclease in the system recognizes the synthesized double-stranded DNA and removes the segment associated with the extended primer; finally, the nicking endonuclease and DNA polymerase work together to synthesize a large amount of single-stranded DNA of a specific sequence. During the synthesis process, a specifically labeled deoxynucleotide (e.g., biotin) is embedded in the newly synthesized DNA, which also becomes the binding target of the colloidal gold marker in the subsequent test strip detection process.

[0053] (3) The amplified product obtained in step (2) is then subjected to lateral chromatography, and the single-stranded DNA fragments carrying the marker are captured by the complementary sequence fixed in the detection area of ​​the test paper, namely the nucleic acid probe. Subsequently, the avidin labeled on the colloidal gold particles binds to the biotin embedded in the single-stranded DNA and is indirectly fixed, and then the color is developed and the result is indicated.

[0054] It should be noted that the above method of the present invention is a universal technical platform, and the detection of different target RNAs can be achieved by simply replacing the corresponding primers, nucleic acid probes and enzymes. The present invention takes avian infectious bronchitis virus and influenza A virus as examples, but it does not mean that the embodiments of the present invention are only applicable to these two viruses.

[0055] Similarly, the organic integration of multiple nucleic acid probes and enzymes can realize the joint detection of multiple targets in a single test.

[0056] After the nucleic acid is amplified by the method of the present invention, it can also be used for fluorescence detection, enzyme-linked adsorption experiment, chemiluminescence method, etc.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] (1) The present invention provides a method for rapid amplification of viral RNA, which method does not require complicated nucleic acid extraction. In a mixed enzyme system containing reverse transcriptase, DNA polymerase (e.g., klenow fragment (3'-5'exo-)), nicking endonuclease, restriction endonuclease and marker-modified dUTP, a large amount of labeled single-stranded nucleic acid products can be synthesized.

[0059] (2) The present invention provides a lateral chromatography detection method for viral RNA based on the rapid amplification method of viral RNA. This method can realize on-site rapid detection of crude RNA extracts, avoids the previous method being restricted by the nicking enzyme sequence, and also avoids the consumption of the target single strand by the extended primer, thereby improving the flexibility and sensitivity of the method. The operation is simple, no large equipment is required throughout the detection process, and the results can be interpreted by the naked eye, which can realize on-site multiple rapid detection. It has the advantages of multi-channel, simplicity, rapidity, high sensitivity and low cost.

[0060] (3) The present invention also provides a rapid viral RNA detection test paper, wherein the use of a single immune-labeled product simplifies the structure of the test paper and improves the stability of the test paper. If the colloidal gold is replaced with a fluorescent marker, it can be used in conjunction with a portable device to achieve semi-quantitative detection of the target, thereby achieving on-site rapid and convenient detection of nucleic acids.

[0061] (4) The present invention is suitable for rapid detection of viral RNA in nasal swab samples, throat swab samples, serum samples and body fluid samples, and has good and broad application prospects and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 It is a schematic diagram of the RNA rapid detection method of the present invention, wherein the inverted cup is a plastic transfer consumable with a cup-shaped structure at the head, and the droplets are transferred by surface tension.

[0063] Figure 2 This is a schematic diagram of primers and probes related to the detection of avian infectious bronchitis virus.

[0064] Figure 3 It is a diagram of the detection result of avian infectious bronchitis virus of the present invention, wherein B: loading buffer solution (loading buffer solution has no amplification product, so no band color development), N1-N4: negative samples, P1-P4: positive simulation samples.

[0065] Figure 4This is a graph of the specificity verification results of the test paper for detecting infectious bronchitis virus of chickens of the present invention, wherein: 1: IBV (H120) live vaccine; 2: negative control (ddH2O); 3: recombinant avian influenza H5+H7 trivalent inactivated vaccine (H5-R13, H5-R14, H7-R4); 4: NDV (LaSota) live vaccine; 5: bursal disease (B87) live vaccine; 6: laryngeal disease (K317) live vaccine; 7: fowlpox live vaccine (quail attenuated strain); 8: influenza B virus nucleic acid; 9: novel coronavirus nucleic acid. In this test, the amount of live vaccine used is not less than 20 doses / test, and the amount of purified nucleic acid is not less than 200 ng / test.

[0066] Figure 5 It is a sensitivity test result diagram of the test paper for detecting infectious bronchitis virus of chickens of the present invention, wherein, from left to right, the IBV virus RNA copy numbers are: 0, 5, 10, 20, 40, 80, 200, 500, 1500 copies / test.

[0067] Figure 6 This is a schematic diagram of primers and probes related to influenza A virus detection.

[0068] Figure 7 It is a diagram of the detection result of influenza A virus detected by the present invention, wherein N: negative sample, P: positive simulation sample. DETAILED DESCRIPTION

[0069] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0070] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0071] Example 1 On-site rapid detection method of infectious bronchitis virus (IBV) RNA in swab samples

[0072] 1. Reagent Preparation

[0073] 1. Primer and probe synthesis

[0074] (1) According to the conserved region of infectious bronchitis virus (IBV), a reverse transcription primer, an extension primer and a nucleic acid probe on a subsequent lateral flow detection test strip are designed, wherein the reverse transcription primer contains a nicking endonuclease recognition site, the first cDNA strand obtained after reverse transcription is used as a template, and then a double-stranded cDNA product is obtained by amplification and extension through an extension primer. The restriction endonuclease BsrDI site exists on the sequence of the double-stranded cDNA product (non-extension primer sequence), and the extension primer segment sequence can be removed by the corresponding enzyme. The nucleic acid probe is complementary to the specific sequence near the 5' end of the single-stranded product, thereby realizing lateral flow detection (Table 1, Figure 2 ).

[0075] (2) Referring to the conserved region of rice Actin gene according to method (1), reverse transcription primer, extension primer and nucleic acid probe on subsequent lateral flow test strip were designed (Table 1).

[0076] (3) The above primers and probes were synthesized by a synthesis company.

[0077] Table 1 Primers and probes for rapid on-site detection of IBV viral RNA

[0078] Primer name Sequence (5'-3') describe IBV-rtE <![CDATA[TCAA GAGTC GGATCATCACG]]> IBV virus reverse transcription primers IBV-SP GGCGTGTTACAGC IBV Virus Extension Primer IBV-cap TTACTACTGTGGTTC IBV virus capture nucleic acid probe CON-rtE <![CDATA[GATCCTC GAGTC CAGACAC]]> Rice Actin reverse transcription primers CON-SP ACCACTATGTTCCC Rice Actin extension primer CON-cap TCCTGAAAGGAAGTAC Rice Actin Capture Nucleic Acid Probe

[0079] Note: The horizontal line part is the recognition site of the nicking endonuclease (Nt.BstNBI enzyme).

[0080] 2. Preparation of Reagents

[0081] (1) Sample lysis buffer: 0.5% (v / v) Triton X-100, 0.75% (w / v) NaCl.

[0082] (2) 2× mixed enzyme reaction system (20 μL): 4 μL of 5× AMV buffer, 3 μL of 10× klenow fragment buffer (3'-5' exo-), 5 U of AMV enzyme, 10 U of klenow fragment (3'-5' exo-), 5 U of Nt.BstNBI enzyme, 5 U of BsrD I, 2 μL of 0.5 mM / each dNTPs, 1 μL of 10 μM Biotin-11-dUTP, the final concentration of the reverse transcription primer and the extension primer were both 200 nM, 2 mg / mL of bovine serum albumin, and 5% mannitol (added in the lyophilized reagent).

[0083] (3) Label resuspension solution: 20 mM Tris (pH 8.0), 0.3% sodium chloride (w / v), 0.3% bovine serum albumin (w / v), 0.2% Casein (w / v), 0.15% tween-20 (v / v), 10% sucrose (w / v), of sodium azide (w / v).

[0084] (4) Loading buffer (8×SSC): 70.13 g of sodium chloride and 31.00 g of sodium citrate were dissolved and made up to 1 L (pH 7.0);

[0085] (5) Sample pad treatment solution: 0.2% (v / v) tween-20, 0.2% (v / v) Triton X-100 and 0.2% (w / v) bovine serum albumin.

[0086] 3. Preparation of test strips

[0087] (1) Place the nitrocellulose membrane and absorbent paper in accordance with Figure 1 The diagram of B was pasted on the backboard, and 50μM IBV-cap and CON-cap probes diluted in 20mM PB were coated on the test line (T line) and the quality control line (C line) at a dosage of 0.7μL / cm using a film scribing instrument. The samples were treated with UV light for 15min, and then dried in an oven at 50℃ for more than 16h, and stored in a dry and dark place for later use.

[0088] (2) Apply 25cm per ml of sample pad treatment solution. 2 The amount of glass fiber membrane is treated and then used as a sample pad and a loading pad after drying.

[0089] (3) Label streptavidin (SA) according to the conventional colloidal gold labeling method. After SA is labeled with 4 parts per ten thousand colloidal gold, it is concentrated 20 times and resuspended in a label resuspension solution to obtain a SA gold-labeled conjugate; 2-8 μL / cm2 of the conjugate is evenly sprayed on one end of the sample pad, and the label pad is obtained after drying.

[0090] (4) Mix the above components according to Figure 1 The diagram of B is pasted to make a large detection plate, which is further cut into detection test papers of corresponding width by a strip cutting machine for later use.

[0091] 2. Rapid on-site detection of viral RNA

[0092] S1. Add rice RNA (exogenous RNA) to the sample lysate to obtain a sample lysate containing 10 ng / μL rice RNA, take 200 μL of the sample lysate containing 10 ng / μL rice RNA, lyse the virus sample to be tested at room temperature and mix thoroughly, wherein the lysis time is 5 min, and obtain a crude viral nucleic acid extract;

[0093] S2. Within 5 minutes after lysis, transfer 20 μL of the lysate to an equal volume of 2× enzyme mixture reaction system, mix well, and amplify at 40-45°C for 30 minutes;

[0094] S3. Take about 5 μL of the amplified product and add it to the sample pad of the test strip. Add 4 drops of sample buffer (about 100 μL of 8×SSC) to the outermost end of the sample pad. After 10-15 minutes of chromatography, read the result.

[0095] S4. Result interpretation: When the test line shows red and the quality control line shows normal color, the result is positive; when the test line does not show color and the quality control line shows normal color, the result is negative; when the quality control line does not show color, regardless of whether the detection limit shows color or not, the result is invalid and needs to be retested.

[0096] The samples were loaded with sample buffer, negative samples (saline eluate of chicken oral swab with negative PCR test results), and positive simulated samples (positive samples containing 2 doses of IBV (H120) live vaccine) were tested according to the above method. The test results are shown in the table. Figure 3 :The product amplified with lysate has no detection bands in the corresponding test; the negative sample (N) only shows the quality control line (C line), indicating a negative result, while the four positive simulation sample test lines (T line) and the quality control line (C line) are colored, indicating a positive result. The above results show that the test results of different samples are in line with expectations.

[0097] Example 2 On-site rapid detection method for infectious bronchitis virus (IBV) RNA Specificity and sensitivity detection 1. Specificity detection

[0098] Using IBV (H120) live vaccine, recombinant avian influenza H5+H7 trivalent inactivated vaccine (H5-R13, H5-R14, H7-R4), NDV (LaSota) live vaccine, bursal (B87) live vaccine, larynx (K317) live vaccine, fowlpox live vaccine (quail attenuated strain), influenza B virus nucleic acid (Vicroria) and novel coronavirus nucleic acid (Omicron) as the objects, the on-site rapid detection of RNA was performed according to the method of Example 1;

[0099] 2. Sensitivity test

[0100] The IBV (H120) live vaccine was diluted in a gradient manner, and the on-site rapid detection of RNA was performed according to the method of Example 1, wherein the virus dosage during the detection was 0, 5, 10, 20, 40, 80, 200, 500, and 1500 copies / test, respectively.

[0101] Figure 4 The results showed that positive results were only displayed when the target virus sample (IBV) was detected, and negative results were displayed when the negative control and the other 7 virus samples (specific virus types are shown in the description of the attached figure), indicating that this detection method has good specificity.

[0102] Figure 5It is the test result of gradient dilution samples. When the corresponding 5 copies / test target is detected, the test result is negative. When the 10-1500 copies / test target is detected, the test results are all positive. And the results are enhanced successively, indicating that the minimum detection limit of the reagent is about 5 copies / test, and the detection line intensity has good linearity.

[0103] Example 3 On-site rapid detection method of influenza A virus (IVA) RNA in swab samples

[0104] 1. Reagent Preparation

[0105] 1. Primer and probe synthesis

[0106] (1) According to the conserved region of influenza A virus (IVA), the reverse transcription primer, the extension primer and the nucleic acid probe on the subsequent lateral flow test strip were designed with reference to Example 1, wherein the reverse transcription primer contains a nicking endonuclease recognition site, the first strand of cDNA obtained after reverse transcription is used as a template, and the extension primer is used as an amplification primer to extend a double-stranded cDNA product, and the restriction endonuclease PstI exists on the sequence of the double-stranded cDNA product (non-extension primer sequence), which can bind to the nucleic acid probe, that is, it contains a sequence complementary to the nucleic acid probe (Table 1, Figure 6 ).

[0107] (2) Referring to the conserved region of rice Actin gene according to method (1), reverse transcription primer, extension primer and nucleic acid probe on subsequent lateral flow test strip were designed (Table 2).

[0108] Table 2 Primers and probes for rapid on-site detection of IVA virus RNA

[0109]

[0110]

[0111] Note: The horizontal line part is the recognition site of the nicking endonuclease (Nt.BstNBI enzyme).

[0112] 2. Preparation of Reagents

[0113] (1) Sample lysis buffer: 0.5% Triton X-100 (v / v), 0.75% NaCl (w / v), 100 nM reverse transcription primer (IVA-rtE) and 100 nM extension primer (IVA-SP);

[0114] (2) 2× mixed enzyme reaction system (20 μL): 5 U of PstI was added to the mixture in Example 1, and the remaining components were the same as in Example 1.

[0115] (3) The remaining reagents are the same as those in Example 1.

[0116] 3. Preparation of test strips

[0117] The IBV-cap in Example 1 was replaced by 50 μM IVA-cap diluted in 20 mM PB, and the remaining operations were the same as in Example 1.

[0118] II. Rapid on-site detection of IVA virus RNA

[0119] Simulated swab samples containing 0.01, 0.05, 0.3, and 1.0 doses of IVA live vaccine (recombinant avian influenza H5+H7 trivalent inactivated vaccine (H5-R13, H5-R14, H7-R4)) were used as detection objects, respectively.

[0120] S1. Add rice RNA to the sample lysate to obtain a sample lysate containing 10 ng / μL rice RNA, take 200 μL of the sample lysate to lyse the virus sample to be tested and mix thoroughly, wherein the lysis conditions are: treat at 65°C for 3 minutes to obtain a crude viral nucleic acid extract;

[0121] S2. Within 5 minutes after lysis, transfer 20 μL of the lysate to an equal volume of 2× enzyme mixture reaction system, mix well, and amplify at 40-45°C for 30 minutes;

[0122] S3. Same as Example 1;

[0123] S4. Same as Example 1.

[0124] Test results see Figure 7 .from Figure 7 It can be seen that when this reagent detects 4 negative samples, the results are all negative, and when it detects 4 positive simulation samples of different concentrations, the results are all positive, which is in line with expectations. This reagent works normally.

[0125] In the present invention, the crude nucleic acid extract containing viral RNA is reverse transcribed, digested by restriction endonuclease and amplified by unidirectional isothermal strand displacement by a single-tube complex enzyme reaction system, thereby generating a large amount of single-stranded DNA of a specific sequence labeled with biotin; during the lateral chromatography process, the amplified product is captured by a complementary sequence nucleic acid probe fixed in the detection area on the test paper, and at the same time, the avidin labeled on the colloidal gold particles binds to the biotin labeled on the fixed single-stranded DNA, and the colloidal gold particles are indirectly fixed to develop a color to indicate the result. The method is easy to operate, does not require complicated nucleic acid extraction, does not require large-scale equipment throughout the detection process, and the results can be interpreted by naked eyes, can realize on-site rapid detection, and has the advantages of simplicity, rapidity, high sensitivity and low cost.

[0126] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for rapid amplification of viral RNA, characterized in that The following steps are included: S1. Lyse the sample containing viral RNA with a lysis buffer to obtain a crude viral nucleic acid extract; S2, mixing the crude viral nucleic acid extract obtained in step (1) with a mixed enzyme reaction system, and reacting at 40-45° C. for 20-40 min to obtain an amplification product, i.e., a single-stranded DNA fragment with a marker; wherein the mixed enzyme reaction system comprises the following components: reverse transcriptase buffer, reverse transcriptase, DNA polymerase buffer, DNA polymerase, nicking endonuclease, restriction endonuclease, dNTPs Mix, marker-modified dUTP, reverse transcription primer, extension primer and bovine serum albumin; There is a single restriction endonuclease site inside the nucleic acid product fragment amplified by the reverse transcription primer and the extension primer in step S2, near the 3' end of the extension primer; the corresponding restriction endonuclease in the mixed enzyme reaction system can recognize and cut the site; The reverse transcription primer described in step S2 is provided with a nicking endonuclease recognition site, and the corresponding nicking endonuclease in the mixed enzyme reaction system can recognize and cleave the nicking endonuclease site on the reverse transcription primer.

2. The method according to claim 1, characterized in that: The lysis solution described in step S1 contains the following components: 0.5% (v / v) Triton X-100, 0.75% (w / v) NaCl.

3. The method according to claim 1, characterized in that: When the viral RNA described in step S1 is double-stranded viral RNA, the lysis solution further comprises a reverse transcription primer and an extension primer, and the lysis condition is 60-65° C. for 3-5 min.

4. The method according to claim 1, characterized in that: In the marker-modified dUTP described in step S2, the marker of the labeled nucleotide is biotin, digoxigenin, fluorescein or other small molecules.

5. The method according to claim 1, characterized in that: When the viral RNA is single-stranded RNA, the mixed enzyme reaction system described in step S2 contains the following components per 20 μL: When the viral RNA is double-stranded viral RNA, the content of the reverse transcription primer and the extension primer in the mixed enzyme reaction system described in step S2 is 0-5 μM, and the other components are the same as the mixed enzyme reaction system for single-stranded viral RNA virus detection.

6. A lateral chromatography detection method for viral RNA, characterized in that The following steps are included: S1. Add the amplification product obtained by the rapid amplification method of viral RNA according to any one of claims 1 to 5 to the sample pad of the viral RNA detection test paper, and add the sample loading buffer to the sample pad; the viral RNA rapid detection test paper is a lateral flow test paper, comprising a back plate, a sample pad, a marker pad, a sample loading pad, a nitrocellulose membrane and absorbent paper; the nitrocellulose membrane is coated with a nucleic acid probe, which can capture the amplification product; the marker pad is coated with a color developing conjugate, which can identify the marker of the amplification product and is fixed; S2. Interpretation of results: After 10-15 minutes of chromatography, interpret the results.

7. The lateral flow detection method for viral RNA according to claim 6, characterized in that: The color-developing conjugate of the viral RNA detection test paper described in step S1 is a labeled product of colloidal gold, colored microspheres, magnetic microspheres, fluorescent microspheres or quantum dots, and the corresponding labeling method can adopt gold-sulfur bond or covalent bond coupling.

8. The lateral flow detection method for viral RNA according to claim 6, characterized in that: The back plate of the virus RNA detection test paper described in step S1 is sticky, the nitrocellulose membrane is pasted on the middle of the back plate, and absorbent paper and a sample loading pad are pasted on both ends, respectively. The absorbent paper and the sample loading pad overlap with the nitrocellulose membrane by 1-2 mm, and the end of the sample loading pad away from the nitrocellulose membrane is coated with a dry SA gold-labeled conjugate, which is called a marker pad; at the sample loading end, except for the sample loading pad, the sample pad is covered with a sample pad, which completely covers the marker pad and the blank part of the back plate, but does not cover the sample loading pad.

9. The lateral flow detection method for viral RNA according to claim 8, characterized in that: When the amplification product is a single-stranded DNA fragment labeled with biotin, and the marker pad of the viral RNA detection test paper is coated with SA gold-labeled conjugate, the result interpretation is: When the test line shows red and the quality control line shows normal color, the result is positive, which means the sample contains the corresponding viral RNA and the concentration is higher than the minimum detection limit; when the test line does not show color and the quality control line shows normal color, the result is negative, which means the sample does not contain the corresponding viral RNA or the concentration is lower than the minimum detection limit; When the quality control line does not show color, the result is invalid regardless of whether the detection limit shows color or not, and retesting is required.

10. Use of the method according to any one of claims 1 to 9 in the field of virus detection.

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