LSDV detection kit and method based on colloidal gold immunochromatography nucleic acid detection test paper

Abstract

The invention relates to the technical field of animal epidemic disease detection, in particular to an LSDV detection kit and method based on colloidal gold immunochromatography nucleic acid detection test paper. All mainstream genotypes of poxvirus GPCR genes are compared, specific primers are designed aiming at a conserved region of the GPCR genes of LSDV as a diagnosis target, the primer pair capable of specifically detecting LSDV nucleic acid is finally obtained through screening and optimization, and rapid detection of the LSDV can be achieved by combining with nucleic acid lateral chromatography test paper for detecting digoxin markers. A product obtained by isothermal amplification is detected by a nucleic acid detection test strip and then directly subjected to result interpretation, expensive instruments and equipment are not needed in the whole detection process, and a detection result can be obtained only through naked eye interpretation. The method has a wide application prospect in the aspect of basic and on-site rapid detection of LSDV infection.

Description

Technical Field

[0001] This invention relates to the field of animal disease detection technology, specifically to a LADV detection kit and method based on colloidal gold immunochromatographic nucleic acid detection test strips. Background Technology

[0002] LSD (Bovine Nodular Dermatitis) was first discovered in Zambia, Africa in 1929. LSD is listed as a notifiable animal disease in the World Organisation for Animal Health (OIE) Animal Disease List. The GPCR (G protein-coupled receptor) gene is an important gene in the LSDV genome and a crucial target for studying LSDV's genetic characteristics and phylogenetic analysis. The GPCR gene is frequently used for the molecular detection and identification of LSDV, revealing its genetic relationships with other poxviruses (such as sheep poxvirus SPV and goat poxvirus GPV), and constructing phylogenetic trees to understand LSDV's epidemiology and support the establishment of disease control programs.

[0003] Currently, molecular biology methods such as PCR, real-time quantitative PCR, high-resolution melting curve analysis (HRM), and loop-mediated isothermal amplification (LAMP) are mainly used domestically and internationally for the detection and diagnosis of LSDV infection. While serological methods such as virus neutralization assays, enzyme-linked immunosorbent assays, and indirect immunofluorescence can also detect sheep pox virus, they cannot differentiate between different species within the same genus. Although these detection methods play an important role in the detection and diagnosis of LSDV, they generally suffer from drawbacks such as complex operation, long processing time, high requirements for equipment, or overly complex primer design, making it difficult to meet the practical needs of clinical practice for rapid and visual on-site detection of LSDV-infected pigs.

[0004] In recent years, recombinase-mediated isothermal nucleic acid amplification (RAA) technology has shown great potential in disease diagnosis and detection, and has been widely used worldwide. Pathogen nucleic acid detection methods based on RAA and lateral flow immunochromatography have been successfully applied to the rapid diagnosis of SARS-CoV-2 and African swine fever virus (ASFV).

[0005] Since the entire RAA amplification process does not require complex and expensive instruments and equipment, and can be completed with just a thermostat or even body temperature, RAA technology is very suitable for development and application in rapid on-site testing. Summary of the Invention

[0006] The purpose of this invention is to provide a LADV detection kit and detection method based on RAA-colloidal gold test strips to solve the technical problem of rapid and accurate on-site detection of LSDV infection of bovine nodular dermatosis virus. This invention combines digoxigenin-labeled nucleic acid lateral chromatography test strips with nucleic acid isothermal amplification to achieve LSDV detection within 20 minutes at 37°C. It features rapid detection, sensitivity, simple operation, and suitability for rapid on-site detection.

[0007] The technical solution of the present invention is as follows: The RAA-colloidal gold test strip for detecting nucleic acids includes a T line coated with avidin and a C line coated with Staphylococcus aureus protein A, wherein the conjugate pad of the test strip is coated with a colloidal gold-labeled anti-DIG monoclonal antibody.

[0008] The LADV detection kit based on colloidal gold immunochromatographic nucleic acid detection test strip includes the colloidal gold immunochromatographic nucleic acid detection test strip, primer pairs, and RAA isothermal amplification reaction reagent. The primer pairs include the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2. The 5' end of the upstream primer is labeled with DIG, and the 5' end of the downstream primer is labeled with Biotin. Their sequences are as follows: SEQ ID NO: 1: 5'-TAGCGCAACCATGTATAATAGTAGCAGTAAT-3'; SEQ ID NO: 2: 5'-CTATGCTCACTTCATAATCATCATAATCATCGC-3'.

[0009] Further optimization includes a positive plasmid control and a negative plasmid control, wherein the positive plasmid control contains the target sequence of the LSDVGPCR gene.

[0010] Further optimization involves adding magnesium acetate solution to the RAA isothermal amplification reaction reagent.

[0011] A method for detecting LADV includes the following steps: S1. Amplify the sample DNA using the primer pairs in the kit and via RAA isothermal amplification reaction; S2. Use colloidal gold immunochromatographic nucleic acid test strips to detect the amplification products, and interpret the results based on the color development of the T and C lines. The interpretation criteria are as follows: If the test line is red, the result is positive; If the test line does not show color, the result is negative.

[0012] Further optimization was performed by conducting the RAA isothermal amplification reaction at 37°C for 10 min.

[0013] Further optimization involved diluting the amplified product before detection using lateral flow immunochromatography.

[0014] Further optimization was performed by conducting the RAA isothermal amplification reaction at 37°C for 10 min.

[0015] Further optimization involves diluting the amplified product before detection using colloidal gold immunochromatographic nucleic acid test strips.

[0016] The beneficial effects of this technical solution are: 1. This invention comprehensively considers factors such as target region conditions, RAA primer length and its special structure, GC content, and amplification product length. After three rounds of primer screening, RAA primers that can be used for rapid detection of LSDV are obtained. They have good specificity and high sensitivity, comparable to traditional PCR detection methods, but can be completed within 30 minutes, far exceeding the speed of traditional PCR methods. 2. Based on RAA, this invention combines a self-developed colloidal gold immunochromatographic nucleic acid test strip for detecting digoxigenin-labeled nucleic acid. It uses a sandwich method of "anti-DIG monoclonal antibody-colloidal gold" label and "avidin-T line" to detect DIG and Biotin dual-labeled nucleic acid amplification products. It can quickly achieve visual interpretation of LSDV detection results without the need for complicated professional background. 3. The LSDV detection using the kit of this invention mainly includes two steps: isothermal amplification and test strip interpretation. The detection of LSDV can be completed within 20 minutes, which can meet the needs of rapid and high-throughput screening on site. The detection process is convenient and quick, and the detection results are safe and reliable. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the LSDV detection process in an embodiment of the present invention; Figure 2 This is a diagram showing the RAA-specific primer screening results in an embodiment of the present invention; Figure 3 This is a diagram showing the optimization results of RAA primers in an embodiment of the present invention; Figure 4 The figures show the results of the sensitivity comparison experiment between the RAA primers before optimization (A), after optimization (B), and the OIE recommended PCR method (C) in the embodiments of the present invention. Figure 5 This is a graph showing the optimization results of the reaction time based on the RAA-LFA detection method in an embodiment of the present invention; Figure 6 This is a graph showing the sensitivity test results of the RAA-LFA detection method in an embodiment of the present invention; Figure 7This is a diagram showing the specificity experimental results of the RAA-LFA detection method in an embodiment of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments. The components of the embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0019] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0020] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0021] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0022] LSDV Detection Kit Based on Colloidal Gold Immunochromatographic Nucleic Acid Test Strip: A kit for detecting bovine nodular dermatovirus, comprising RAA primer pairs, DNA extraction reagent, RAA microspheres, colloidal gold immunochromatographic nucleic acid test strip, positive control, and negative control. The RAA primers were designed and optimized to target the specific conserved region of the LSDV GPCR gene. DIG and Biotin were added to the 5' ends of the upstream and downstream primers, respectively. The colloidal gold immunochromatographic nucleic acid detection test strip was independently developed. The DIG-BSA conjugate (a conjugate of the hapten digoxigenin and BSA) was used as an artificial complete antigen to immunize BALB / c mice to screen for hybridoma cell lines that stably secrete anti-DIG monoclonal antibodies and to prepare ascites fluid. The ascites fluid was then labeled with colloidal gold for specific detection of DIG-labeled nucleic acid molecules. Avidin (SA) was immobilized on the T line to capture the amplification products, and Staphylococcus aureus protein A (SPA) was immobilized on the C line as a quality control. Qualitative detection of the target nucleic acid was achieved by using a sandwich method of DIG-labeled antibody and avidin. The positive control was a standard positive plasmid containing the full length of the LSDV GPCR gene; the negative control was an empty vector plasmid without the LSDV genome. The RAA beads and DNA extraction reagents were customized by Suzhou Keer Life Science Technology Co., Ltd.

[0023] The primer pair includes the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2, with the following sequences: SEQ ID NO: 1: 5'-TAGCGCAACCATGTATAATAGTAGCAGTAAT-3'; SEQ ID NO: 2: 5'-CTATGCTCACTTCATAATCATCATAATCATCGC-3'.

[0024] A method for detecting LSDV using a colloidal gold immunochromatographic nucleic acid test strip-based LSDV detection kit includes the following steps: S1. Amplify the sample DNA using the primer pairs from the kit and the RAA isothermal amplification reaction; S2. Use colloidal gold immunochromatographic nucleic acid test strips to detect the amplification products, and interpret the results based on the color development of the T and C lines. The interpretation criteria are as follows: If the test line is red, the result is positive; If the test line does not show color, the result is negative.

[0025] The specific detection method is as follows: Dissolve one unit of RAA reaction ball in 20.75 μL of deionized water, add 1.00 μL each of upstream and downstream primers and 1.00 μL of template positive plasmid, and finally add 1.25 μL of 280 mM magnesium acetate solution. Mix the RAA amplification system thoroughly and react in a 37℃ water bath for 10 min. After the reaction, obtain the RAA amplification product. Dilute the obtained RAA amplification product with 175 μL of buffer and add it to the sample wells of colloidal gold-labeled colloidal gold immunochromatographic nucleic acid test strip. Incubate at room temperature for 5-10 min and observe the results. If the test line is red, it is positive; if the test line is not colored, it is negative.

[0026] Primer design and condition optimization The key to RAA amplification lies in primer design. However, RAA differs from conventional PCR reactions. This invention selects conserved regions as RAA amplification targets. Taking into account various factors such as (1) GC content (40-60%), (2) length (30-35 nt), (3) amplification product size (100-300 bp), etc., cross-pairing was performed on the designed upstream and downstream RAA primers, and the best primer pairing was screened in three rounds to obtain the optimal primers. See Table 1 for details.

[0027] Table 1. RAA primers designed for the conserved region of the LSDV GPCR gene.

[0028] After RAA amplification using the RAA primer pairs designed in Table 1, the GPCR-RAA-3F / GPCR-RAA-2R primer pairs showed the best amplification effect by agarose gel electrophoresis, as shown in the figure. Figure 2 As shown, Figure 2 A shows the nucleic acid gel electrophoresis results of different primer combinations in Table 1 after amplification of 105 copies / μL of positive plasmid for 10 min. The bands are highly specific and there is no non-specific amplification, while other primer pairs have problems such as non-specific amplification and low amplification efficiency. Figure 2 B represents the grayscale analysis of specific bands in nucleic acid gel electrophoresis; therefore, primer pairs GPCR-RAA-3F / GPCR-RAA-2R were selected for subsequent primer optimization.

[0029] RAA primers were screened and optimized. Based on the screened RAA primers, following the design principles of RAA primers, single base shifts and base additions / reductions were performed, using different primer combinations to target 10 bases. 3After amplification of positive plasmids at copies / μL for 10 min, the results of nucleic acid gel electrophoresis and the gray values ​​of specific bands were analyzed. The results are shown in the figure. Figure 3 As shown, Figure 3 A represents different primer combinations during the base translation optimization process, using GPCR-RAA-3F as the upstream primer and GPCR-RAA-2R as the downstream primer for translation. Among them, GPCR-RAA-3F / GPCR-RAA-2R has the highest amplification efficiency. Figure 3 B represents different primer combinations during the base translation optimization process, using GPCR-RAA-2R as the downstream primer and GPCR-RAA-3F as the upstream primer for translation. Among them, GPCR-RAA-3F-5 / GPCR-RAA-2R has the highest amplification efficiency. Figure 3 C represents different primer combinations during the base addition / reduction optimization process, using GPCR-RAA-2R as the downstream primer and GPCR-RAA-3F-5 as the upstream primer with additions / reductions. Among them, GPCR-RAA-3F5-5 / GPCR-RAA-2R has the highest amplification efficiency. Figure 3 D represents different primer combinations used in the base addition / reduction optimization process, with GPCR-RAA-3F-5 as the upstream primer and GPCR-RAA-2R as the downstream primer. Among them, GPCR-RAA-3F5-5 / GPCR-RAA-2R-6 had the highest amplification efficiency. After screening and optimization, GPCR-RAA-3F5-5 / GPCR-RAA-2R-6 was selected as the final primer combination for RAA amplification.

[0030] Sensitivity analysis of RAA primers After optimization, the RAA primers further improved the sensitivity of the RAA reaction, such as... Figure 4 As shown, Figure 4 A shows the amplification results of positive plasmids with different copy numbers diluted before optimization using GPCR-RAA-3F / GPCR-RAA-2R. The efficiency of the primer combination before optimization can reach 105 copies / μL. Figure 4 B shows the amplification results of positive plasmids with different copy numbers diluted using the optimized GPCR-RAA-3F5-5 / GPCR-RAA-2R-6 primer combination. The efficiency of the primer combination before optimization reached 103 copies / μL, which is higher than that of the previous combination. Figure 4 The amplification efficiency of PCR recommended by OIE in C was used; the final optimized GPCR-RAA-3F5-5 / GPCR-RAA-2R-6 were labeled with DIG and Biotin at the 5' end, respectively.

[0031] Optimization of reaction time for RAA-LFA (RAA amplification product binding lateral flow chromatography strip) detection method Following the basic isothermal reaction procedure described above, with negative controls substituted for templates, the reaction was carried out in a 37°C water bath for 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min. After adding diluent, the solution was added dropwise to the sample wells of the colloidal gold-labeled lateral chromatography test strip. Results showed... Figure 5 , Figure 5 A shows the effect of primer dimers on the color development of the lateral flow chromatography strip under different amplification times. When the amplification time is 15 min, the negative template has a longer RAA primer, and the primer dimer formed after amplification causes the colloidal gold-labeled lateral flow chromatography strip to show a positive result. Therefore, the optimal amplification time is 10 min. Figure 5 B represents the colorimetric effect of amplifying standard positive and negative templates at an amplification time of 10 min.

[0032] Sensitivity and specificity experiments of the RAA-LFA detection method Using a standard LSDV positive plasmid sample as a template, the plasmid was serially diluted 10-fold with double-distilled water to a concentration of 10. 8 Standard plasmid templates were prepared in the form of 10 copies / μL to 1 copy / μL, with double-distilled water as a blank control. The detection was performed under optimized reaction conditions according to the RAA-LFA detection method described above. The result showed a minimum detectable template amount of 10. 3 copies / μL (see Figure 6 It has high sensitivity, and the detection method of the present invention is simpler and faster, especially suitable for on-site detection, and has more relaxed requirements for experimental conditions.

[0033] Nucleic acid samples extracted from positive samples were used as templates, and RAA-LFA detection was performed under optimized reaction conditions according to the RAA-LFA detection method. Detection was performed using gel electrophoresis and lateral chromatography. Experimental results are as follows: Figure 7 As shown, Figure 7 A represents the PCR identification results of each virus in the sample tray; Figure 7 B shows the RAA amplification results of each virus in the sample dish using the GPCR-RAA-3F5-5 / GPCR-RAA-2R-6 primer combination. Figure 7 C represents the RAA-LFA detection results of each virus in the sample dish using the labeled primer pairs. Only LSDV was positive, while the others were negative, indicating that the primer combination for RAA amplification of the present invention has extremely high specificity and the RAA-LFA detection system is extremely specific.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the description and drawings of the present invention shall also be included within the scope of protection of the present invention.

Claims

1. A colloidal gold immunochromatographic nucleic acid detection test strip, characterized in that, The test strip includes a T line coated with avidin and a C line coated with Staphylococcus aureus protein A, and the conjugate pad of the test strip is coated with colloidal gold-labeled anti-DIG monoclonal antibody.

2. An LSDV detection kit based on colloidal gold immunochromatographic nucleic acid detection test strips, characterized in that, The test kit includes the colloidal gold immunochromatographic nucleic acid detection strip, primer pair, and RAA isothermal amplification reaction reagent as described in claim 1. The primer pair comprises the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO:

2. The 5' end of the upstream primer is labeled with DIG, and the 5' end of the downstream primer is labeled with Biotin. Their sequences are as follows: SEQ ID NO: 1: 5'-TAGCGCAACCATGTATAATAGTAGCAGTAAT-3'; SEQ ID NO: 2: 5'-CTATGCTCACTTCATAATCATCATAATCATCGC-3'.

3. The LSDV detection kit based on colloidal gold immunochromatographic nucleic acid detection test strip according to claim 2, characterized in that, It also includes a positive plasmid control and a negative plasmid control, wherein the positive plasmid control contains the target sequence of the LSDV GPCR gene.

4. The LSDV detection kit based on colloidal gold immunochromatographic nucleic acid detection test strip according to claim 2, characterized in that, The reagents for the RAA isothermal amplification reaction include magnesium acetate solution.

5. A method for detecting LSDV using the kit described in any one of claims 2-4, characterized in that, Includes the following steps: S1. Amplify the sample DNA using the primer pairs in the kit and via RAA isothermal amplification reaction; S2. Use colloidal gold immunochromatographic nucleic acid test strips to detect the amplification products, and interpret the results based on the color development of the T and C lines. The interpretation criteria are as follows: If the test line is red, the result is positive; If the test line does not show color, the result is negative.

6. The method for detecting LSDV according to claim 5, characterized in that, The RAA isothermal amplification reaction was carried out at 37°C for 10.5-20 min.

7. The method for detecting LSDV according to claim 5, characterized in that, The amplified product was diluted before detection using colloidal gold immunochromatographic nucleic acid test strips.

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