A rapid amplification and lateral flow detection method for viral RNA and application thereof

Abstract

The application belongs to the technical field of RNA rapid detection, and particularly relates to a rapid amplification and lateral chromatographic detection method for virus RNA and application. The crude virus nucleic acid extract is subjected to a single-tube composite enzyme reaction system reverse transcription, restriction endonuclease digestion and one-way isothermal strand displacement amplification, and then a large amount of specific sequence single-stranded DNA with a marker is generated; in the lateral chromatographic process, the single-stranded DNA is captured by a nucleic acid probe fixed on a detection area of a virus RNA detection test paper, at the same time, avidin labeled on a colloidal gold particle is combined with biotin labeled on the fixed single-stranded DNA, and the colloidal gold particle is indirectly fixed to show a color indication result. The method provided by the application is simple to operate, does not need complicated nucleic acid extraction, does not need large equipment in the whole detection process, and the result can be judged by naked eyes, so that on-site rapid detection can be realized, and the method has the advantages of simplicity, rapidness, high sensitivity and low cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapid RNA detection, and in particular relates to a rapid amplification and lateral flow chromatography detection method of viral RNA and its application. Background Art

[0002] With economic development, the livestock industry has gradually become more large-scale and intensive. Frequent outbreaks of viral animal diseases have caused significant losses to the industry. These viruses, many of which are RNA viruses with high mutation rates, are difficult to rapidly detect, diagnose, and control on-site. Currently, there are no effective medications for diseases caused by RNA viruses. The most effective prevention and control measures are rapid testing for early detection and prompt disinfection. Therefore, the need for rapid on-site detection of RNA viruses is urgent.

[0003] Current rapid virus detection technologies mostly rely on antigen detection. However, due to the high variability of RNA viruses, antigenic sites also mutate rapidly, making it difficult for antibodies to stably recognize and bind to newly mutated antigens. Consequently, test results are less accurate. Another rapid detection approach targets viral nucleic acids, meaning that the virus can be monitored by detecting viral nucleic acid. PCR-related technologies can rapidly and efficiently amplify nucleic acid signals, resulting in high sensitivity. However, nucleic acid-based detection methods are complex to operate, requiring specialized equipment, facilities, and skilled technicians. Therefore, they are not suitable for low-cost, on-site rapid testing, which limits their widespread adoption in rapid testing scenarios. Direct PCR technology partially avoids the need for nucleic acid purification but is limited to testing certain DNA target samples. Nucleic acid isothermal amplification technology reduces the equipment requirements for nucleic acid amplification, but the output still requires corresponding equipment to detect the signal. Nucleic acid chromatography technology expands the detection targets to a certain extent and can greatly simplify the display of test results, but still requires complex pre-processing and lacks reproducibility and stability.

[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 address 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, first using reverse transcriptase and a primer with a nicking enzyme recognition site to initiate the reverse transcription process to synthesize a cDNA / RNA hybrid chain; then using an extension primer and a DNA polymerase to synthesize a double-stranded cDNA in the target region; a restriction endonuclease cuts off the sequence segment related to the extension primer; finally, the nicking endonuclease and the DNA polymerase work together to initiate an isothermal chain displacement reaction, generating a large amount of single-stranded nucleic acid (DNA) with a specific sequence. 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 paper, and the results can be interpreted with the naked eye.

[0006] Another object of the present invention is to provide a lateral flow detection method for viral RNA. This method first uses a complementary sequence-immobilized nucleic acid probe on the lateral flow detection line to capture the aforementioned single-stranded nucleic acid product. Subsequently, colloidal gold particles labeled with a functional protein (e.g., SA) recognize and bind to the marker on the immobilized DNA, thereby indirectly immobilizing it and displaying a band. Finally, the results are interpreted by the naked eye. This method has the advantages of simple and rapid operation, high sensitivity and specificity, and can be used for rapid on-site 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 amplified 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 lysis solution described 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 contains a reverse transcription primer and an extension primer, and the lysis conditions are 60-65°C for 3-5 minutes; this is because single-stranded RNA can directly bind to the primer to initiate the amplification reaction after lysis, while double-stranded RNA needs to be heated during lysis to denature its two RNA strands and open them, thereby allowing them to bind to the primer to initiate 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 described in step S1 preferably further contains 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 cracking is completed in step (1), 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 a conserved region of the target RNA (viral RNA to be detected or exogenous RNA) sequence;

[0019] The nucleic acid product fragment amplified by the reverse transcription primer and the extension primer in step S2 has a single restriction endonuclease site 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 preventing the extension primer from causing the single-stranded product to be amplified into a double strand.

[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 be present 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 for 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. This sequence serves as the capture site of the nucleic acid probe in the rapid viral RNA detection test strip to ensure the specificity of the test results;

[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 contains the following components per 20 μL:

[0027]

[0028] When the viral RNA is double-stranded viral RNA, based on the addition of reverse transcription primers and extension primers 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 amplified 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 loading buffer to the sample pad; the viral RNA rapid detection paper is a lateral flow test paper, comprising a back plate, a sample pad (SP), a marker pad (CP), a loading pad (LP), a nitrocellulose membrane and absorbent paper; the nitrocellulose membrane is coated with a nucleic acid probe that can capture the amplified product; the marker pad is coated with a chromogenic conjugate that can recognize the marker of the amplified product and is fixed;

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

[0033] The amount of the amplified 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 use gold-sulfur bond or covalent bond coupling;

[0035] The back plate of the viral RNA detection test paper described in step S1 is sticky, and the nitrocellulose membrane is attached to the middle of the back plate. The absorbent paper and the sample loading pad are attached to both ends, respectively. The absorbent paper and the sample loading pad overlap with the nitrocellulose membrane by 1-2 mm. The end of the sample loading pad away from the nitrocellulose membrane is coated with a dry SA (streptavidin) gold label conjugate, which is called the marker pad. At the sample loading end, except for the sample loading pad, the sample pad is covered. 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 by hand or machine, and becomes a label pad after drying;

[0037] The sample pad is where the loading 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 used to add the amplified product. Its preparation method is the same as that of the sample pad. The sample loading pad can also serve as a supporting structure for the marker pad.

[0039] A detection line and a quality control line are fixed on the nitrocellulose membrane; the nucleic acid probe coated on the detection line is complementary to a partial sequence of the amplified single-stranded product of the viral RNA to be tested, and the nucleic acid probe coated on the quality control line is complementary to a partial sequence of the amplified product of the exogenous RNA; wherein the quality control line realizes quality control of the entire system by detecting the amplified product of the exogenous RNA (for example, rice Actin gene RNA) amplified in the same system.

[0040] The amount of 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 10-100 μM nucleic acid probe;

[0041] The specific operation of the coating is preferably as follows: spraying the corresponding nucleic acid probe onto the detection line or quality control line area of ​​the nitrocellulose membrane according to conventional methods, firstly crosslinking the coated nitrocellulose membrane under ultraviolet light for 10-20 minutes, then drying at 37-50°C for 16-20 hours, and storing in a dark and dry place for future use;

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

[0043] The loading buffer is 4-8×SSC solution, Tris-HCl, borate 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 amplified 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, regardless of whether the detection limit shows color or not, the result is invalid and needs to be retested;

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

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

[0049] The application 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 and thus 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 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 an AMV enzyme. Subsequently, 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 with 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 fragment with the marker is captured by the complementary sequence fixed in the detection area of ​​the test paper - 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 to develop color and indicate the result.

[0054] It should be noted that the above-described method of the present invention is a universal technology platform that can detect different target RNAs by simply replacing the corresponding primers, nucleic acid probes, and enzymes. While the present invention uses avian infectious bronchitis virus and influenza A virus as examples, this does not mean that the present invention is only applicable to these two viruses.

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

[0056] After the nucleic acid is amplified and produced 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 does not require complicated nucleic acid extraction. In a mixed enzyme system containing components such as 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 target single strands by extended primers, thereby improving the flexibility and sensitivity of the method. The operation is simple, no large equipment is required for the entire detection process, and the results can be interpreted by naked eyes, which can realize on-site multiple rapid detection and 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 immunolabeled 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 realizing 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 This is a schematic diagram of the principle of the RNA rapid detection method of the present invention, wherein the inverted cup is a plastic transfer consumable with a cup-shaped head that transfers droplets through 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 This is a diagram showing the detection results of avian infectious bronchitis virus according to the present invention, wherein B: loading buffer solution (the loading buffer solution does not contain amplification products, so no bands are developed), N1-N4: negative samples, and P1-P4: positive simulation samples.

[0065] Figure 4This is a graph showing 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. The amount of live vaccine used in this test is not less than 20 doses / test, and the amount of purified nucleic acid is not less than 200 ng / test.

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

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

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

[0069] The present invention will be described in further detail below with reference to the 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 well 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 for infectious bronchitis virus (IBV) RNA in swab samples

[0072] 1. Reagent Preparation

[0073] 1. Primer and probe synthesis

[0074] (1) Based on the conserved region of infectious bronchitis virus (IBV), a reverse transcription primer, an extension primer and a nucleic acid probe on the subsequent lateral flow detection test paper were designed. The reverse transcription primer contained a nicking endonuclease recognition site. The first strand of cDNA obtained after reverse transcription was used as a template, and then a double-stranded cDNA product was amplified and extended by the extension primer. The restriction endonuclease BsrDI site existed on the sequence of the double-stranded cDNA product (non-extension primer sequence). The extension primer segment sequence could be removed by the corresponding enzyme. The nucleic acid probe was 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 the rice Actin gene according to method (1), reverse transcription primers, extension primers and nucleic acid probes for subsequent lateral flow cytometry test strips 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 viral 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 underlined 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 are both 200 nM, 2 mg / mL of bovine serum albumin, and 5% mannitol (added to the lyophilized reagent).

[0083] (3) Marker 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): Dissolve 70.13 g of sodium chloride and 31.00 g of sodium citrate 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 the Figure 1 The diagram shown in Figure B was pasted on the backboard. 50 μM IBV-cap and CON-cap probes diluted in 20 mM PB were coated on the test line (T line) and the quality control line (C line) at a concentration of 0.7 μL / cm using a film streaker. The samples were then treated with UV light for 15 minutes, dried in a 50°C oven for more than 16 hours, and stored dry and away from light for later use.

[0088] (2) Apply 25cm2 of sample pad treatment solution per ml 2 The glass fiber membrane is treated and then dried before use as a sample pad and loading pad.

[0089] (3) Label streptavidin (SA) according to the traditional colloidal gold labeling method. After 40,000 parts per million of colloidal gold is used to label SA, the SA gold-labeled conjugate is concentrated 20 times and resuspended in the label resuspension solution to obtain the SA gold-labeled conjugate; the conjugate is evenly sprayed on one end of the sample pad at a volume of 2-8 μL / cm. After drying, the label pad is obtained.

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

[0091] 2. On-site rapid 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 and lyse the virus sample to be tested at room temperature and mix thoroughly. The lysis time is 5 minutes to 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 mix, mix thoroughly, and amplify at 40-45°C for 30 minutes.

[0094] S3. Approximately 5 μL of amplified product was loaded onto the sample pad of the test strip. Four drops of loading buffer (approximately 100 μL of 8× SSC) were added to the outermost end of the sample pad. After 10–15 minutes of chromatography, the results were analyzed.

[0095] S4. Interpretation of results: 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 simulation samples (positive samples containing 2 doses of IBV (H120) live vaccine) as the objects, and the test results were shown in the above method. Figure 3 The product amplified using the lysate showed no detection bands. The negative sample (N) showed only the control line (C line), indicating a negative result. However, the four positive mock samples showed both the test line (T line) and the control line (C line), indicating a positive result. These results indicate that the test results for the different samples were consistent with expectations.

[0097] Example 2 On-site Rapid Detection Method for Infectious Bronchitis Virus (IBV) RNA Specificity and Sensitivity Detection I. Specificity Detection

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

[0099] 2. Sensitivity test

[0100] The IBV (H120) live vaccine was serially diluted, and on-site rapid RNA detection was performed according to the method of Example 1, wherein the virus dosage during 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 accompanying figure description), indicating that this detection method has good specificity.

[0102] Figure 5This is the test result of the gradient dilution sample. When the corresponding test target is 5 copies / test, the test result is negative. When the test target is 10-1500 copies / test, the test results are all positive. The results are successively enhanced, 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) Based on the conserved region of influenza A virus (IVA), a reverse transcription primer, an extension primer, and a nucleic acid probe on a subsequent lateral flow test strip were designed with reference to Example 1, wherein the reverse transcription primer contained a nicking endonuclease recognition site, the first strand of cDNA obtained after reverse transcription was used as a template, and the extension primer was used as an amplification primer to extend a double-stranded cDNA product, and the restriction endonuclease PstI existed on the sequence of the double-stranded cDNA product (non-extension primer sequence), which could bind to the nucleic acid probe, that is, it contained a sequence complementary to the nucleic acid probe (Table 1, Figure 6 ).

[0107] (2) Referring to the conserved region of the rice Actin gene according to method (1), reverse transcription primers, extension primers and nucleic acid probes for subsequent lateral flow cytometry test strips were designed (Table 2).

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

[0109]

[0110]

[0111] Note: The underlined 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 reaction system 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] 2. 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.

[0120] S1. Add rice RNA to the sample lysate to obtain a sample lysate containing 10 ng / μL rice RNA. Take 200 μL of this sample lysate to lyse the virus sample to be tested and mix thoroughly. The lysis conditions are: incubate 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 mix, mix thoroughly, and amplify at 40-45°C for 30 minutes.

[0122] S3. Same as Example 1;

[0123] S4. Same as Example 1.

[0124] Test results are shown in 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 and the reagent works normally.

[0125] In the present invention, a crude nucleic acid extract containing viral RNA undergoes reverse transcription, restriction endonuclease digestion, and unidirectional isothermal strand displacement amplification in a single-tube complex enzyme reaction system, thereby generating a large amount of single-stranded DNA with a specific biotin-labeled sequence. During lateral chromatography, the amplified product is captured by a complementary sequence nucleic acid probe fixed in the detection zone on the test paper. Simultaneously, 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. This method is simple 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 the naked eye, enabling rapid on-site detection. It has the advantages of simplicity, speed, 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 considered as equivalent replacement methods and are included in the scope of protection 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. Mix the crude viral nucleic acid extract obtained in step (1) with a mixed enzyme reaction system, and react at 40-45°C for 20-40 minutes to obtain an amplified product, which is 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; The nucleic acid product fragment amplified by the reverse transcription primer and the extension primer in step S2 has a single restriction endonuclease site near the 3' end of the extension primer; the corresponding restriction endonuclease in the mixed enzyme reaction system can recognize and cut this 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, wherein: The lysis buffer 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, wherein: When the viral RNA in step S1 is double-stranded viral RNA, the lysis solution further comprises a reverse transcription primer and an extension primer, and the lysis conditions are 60-65° C. for 3-5 minutes.

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

5. The method according to claim 1, wherein: 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 method for lateral flow detection of viral RNA, wherein the method is for non-disease diagnosis purposes, characterized in that The following steps are included: S1. Add the amplified product obtained by the rapid amplification method of viral RNA according to any one of claims 1 to 5 to the sample pad of a viral RNA detection test paper, and add the sample loading buffer to the sample pad; the viral RNA 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 that can capture the amplified product; the marker pad is coated with a chromogenic conjugate that can recognize the marker of the amplified 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 uses gold-sulfur bond or covalent bond coupling.

8. The lateral flow chromatography detection method for viral RNA according to claim 6, characterized in that: The back plate of the viral RNA detection test paper described in step S1 is sticky, and the nitrocellulose membrane is attached to the middle of the back plate. The absorbent paper and the sample loading pad are attached to both ends. The absorbent paper and the sample loading pad overlap with the nitrocellulose membrane by 1-2 mm. The end of the sample loading pad away from the nitrocellulose membrane is coated with a dry SA gold label conjugate, which is called the marker pad. At the sample loading end, the sample pad 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 amplified 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, wherein the use is for non-disease diagnosis purposes.

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