Detection system and method of betel nut yellowing-related virus based on RPA-LFD technology

Abstract

The present invention relates to the technical field of plant virus detection, and in particular to a detection system and method for betel nut yellowing-related viruses based on RPA-LFD technology. A primer set for detecting betel nut yellowing virus APV1 is characterized in that: it includes a specific primer pair; the nucleotide sequence of the upstream primer in the specific primer pair is shown in the sequence table SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown in the sequence table SEQ ID No.2; it also includes a probe; the probe is modified by biotin or FAM fluorescent group labeling; the modified probe nucleotide sequence is shown in the sequence table SEQ ID No.3. The advantages are: it has high specificity and low detection limit in detection, and has no cross reaction with other viruses such as betel nut necrotic ringspot virus; the primer probe combination is suitable for RPA-LFD detection, and the time required for the amplification reaction is short; the reaction result will be displayed on the test strip and can be observed with the naked eye.

Description

Technical Field

[0001] The present invention relates to the technical field of plant virus detection, and in particular to a detection system and method for betel nut yellowing-related virus based on RPA-LFD technology. Background Art

[0002] Betel nut ( Areca catechu L.) is a species of Areca genus in the family Palmae. Areca ) is a tropical perennial evergreen tree and the largest specialty cash crop in Hainan Province. Currently, yellow leaf disease (YLD) is a bottleneck restricting the betel nut cultivation industry. Areca palm velarivirus 1 (APV1) is highly correlated with YLD and can be transmitted by the two-striped mealybug ( Ferrisia virgata ) and Pseudococcus citri ( Pseudococcus cryptus ) was transmitted to areca nut in a non-cyclic semi-persistent manner and caused YLD symptoms. The infectious clone of APV1 was successfully inoculated into areca nut and caused YLD yellowing symptoms, completing the Koch's postulate verification of APV1 as the pathogen of yellowing disease, proving that APV1 is one of the key pathogens that infect areca nut and cause YLD.

[0003] The spread of betel nut chlorosis has seriously hindered the development of the betel nut industry. The construction of a healthy seedling guarantee system is insufficient, especially the lack of fast, accurate and portable betel nut chlorosis detection products in the field. Many farmers are very enthusiastic about betel nut planting, but due to the lack of corresponding detection products for betel nut chlorosis on the market, the cultivated betel nut seedlings have not been tested, and it is impossible to prove whether the cultivated betel nut seedlings are healthy, which brings hidden dangers to the outbreak and spread of betel nut chlorosis.

[0004] Traditional detection methods rely on laboratory PCR platforms, which have defects such as strong equipment dependence and long detection cycle (>4 hours), making it difficult to meet the needs of real-time field detection. The traditional immunocolloidal gold method has limited detection accuracy (detection limit of 10 4 -10 5 The RPA-LFD method, however, does not require large equipment, can be tested in under half an hour, and has a detection limit of 100 copies / reaction, enabling accurate, rapid, and convenient testing in the field.

[0005] Recombinase polymerase isothermal amplification (RPA) is a novel nucleic acid amplification technology developed in recent years based on recombinase polymerases. The recombinase first binds to the primer to form a complex, scanning the double-stranded DNA and mediating the melting of the target region, allowing for precise primer pairing. Subsequently, single-stranded DNA binding proteins (SSBs) stabilize the single-stranded structure, and the polymerase extends along the template to synthesize a new strand. This reaction is carried out at a constant temperature (typically 37-42°C), eliminating the need for thermal cycling equipment and completing amplification within 10-30 minutes. Its advantages include speed, portability, and low resource consumption, making it particularly suitable for point-of-care testing.

[0006] Lateral flow dipstick (LFD) is an endpoint detection technology for visual observation of amplification products. The double-labeled product amplified by RPA forms a "fluorescein antibody-double-labeled nucleic acid amplification product-colloidal gold complex" at the detection line through antigen-antibody binding. The results can be directly observed with the naked eye after 10 minutes.

[0007] RPA-LFD combines nucleic acid amplification with immunoassays to achieve highly sensitive target identification. In the assay process, RPA primers are pre-labeled with molecules such as biotin or FAM, resulting in a dual-labeled amplification product. The test line of the LFD strip is coated with streptavidin (to capture biotin), while the control line contains an anti-FAM antibody. The amplified product is diluted and loaded onto the sample. The dual-labeled product is captured and displayed by the test line, while unbound material migrates to the control line. This combined technique, which also allows for crude sample extraction, is completed within half an hour, making it suitable for on-site screening. Summary of the Invention

[0008] In order to solve the above problems, the present invention provides a detection system and method for betel nut yellowing-related virus based on RPA-LFD technology.

[0009] The present invention aims to provide a method for rapidly detecting betel nut yellows virus APV1 based on RPA-LFD technology, which uses a detection system for rapidly detecting betel nut yellows virus APV1, specifically comprising the following steps:

[0010] S1. Crude sample extraction: 50-100 mg of betel nut leaf tissue was placed in a mortar and pestle, and then ground with sample release solution to obtain a crude extract. Viral RNA was then extracted from the sample.

[0011] S2. Isothermal Amplification: Using the RNA obtained in step S1 as a template, add the RPA reaction system containing a specific primer pair. Insert the centrifuge tube into the centrifuge tube rack in the constant temperature amplification device and adjust the temperature using the temperature control button to perform the isothermal amplification reaction at 37-44°C.

[0012] S3. Chromatographic testing: Dilute the amplified product with pure water and add it dropwise to the sample area of ​​the test strip. Observe the color development within 10-15 minutes. The test results are determined based on the following criteria:

[0013] When the control line of the test strip shows color and the test line shows no color, it is judged as APV1 negative;

[0014] When the control line and the detection line of the test strip show color at the same time, it is judged as APV1 positive.

[0015] Preferably, the detection system includes a specific primer pair; the nucleotide sequence of the upstream primer in the specific primer pair is shown in SEQ ID No. 1 in the sequence listing, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2 in the sequence listing;

[0016] It also includes a probe; the probe is modified by labeling with a biotin or FAM fluorescent group; the fluorescent group is selected from one of FAM, HEX, TET, cyanine dyes, ROX, and Texas Red; the alkyl carbon spacer in the probe is selected from one of C2 Spacer, C3 Spacer, and C4 Spacer.

[0017] Preferably, the 5' end of the downstream primer is modified with a Biotin group;

[0018] The modified probe nucleotide sequence is shown in SEQ ID No. 3 in the sequence listing.

[0019] In the detection system, each 50 μL reaction system contains 21±3 pmoL of upstream primer, 21±3 pmoL of downstream primer and 6±1 pmoL of probe;

[0020] Reaction conditions: The RPA reaction temperature was constant at 37-44°C, and the reaction time was 10±2 min. The lateral flow immunoassay test temperature was room temperature, and the reaction time was 10±1 min.

[0021] Preferably, the reaction system includes the following components and amounts:

[0022]

[0023] The reaction time of the RPA-LFD reaction was 10 min.

[0024] Preferably, the constant temperature amplification device has a split cavity structure, including an upper cavity and a lower cavity; the upper cavity is provided with an insulation inner tank and a centrifuge tube rack, and the lower cavity is provided with a heating plate for heating the upper cavity; a temperature display screen and a temperature adjustment button are provided on the outside of the constant temperature amplification device; the constant temperature amplification device is provided with a charging interface, which realizes wireless temperature control and heating after charging.

[0025] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0026] The present invention optimizes a conserved sequence of the gene encoding the capsid protein of the betel nut APV1 virus to obtain the RPA primer pair and RPA primer probe combination, which have high specificity and low detection limit in detection, have no cross-reaction with other viruses such as the betel nut necrotic ringspot virus, and have a detection limit of up to 100 copie template amounts. The detection system and detection method of the present invention have a detection sensitivity that is 3 orders of magnitude higher than that of the traditional immunocolloidal gold method. At the same time, the primer pair and primer probe combination are suitable for RPA-LFD detection, and the reaction temperature can be set using a small constant temperature device, and the time required for the amplification reaction is short; the reaction results will be displayed on the test strip and can be observed with the naked eye, which is very suitable for on-site rapid detection of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the RPA-LFD detection result of the betel nut yellowing virus (APV1) provided in an embodiment of the present invention; in the figure, N is a negative control; P is a positive control, that is, the total RNA of the betel nut yellowing virus plant.

[0028] Figure 2 Specificity determination results provided according to an embodiment of the present invention; in the figure, N is a negative control; 1 is ANRSV; 2 is ANSSV.

[0029] Figure 3 The detection limit determination results provided by the embodiment of the present invention; in the figure, N: negative control; 1: template amount 10 1 copies; 2: template amount 10 2 copies; 3: template amount 10 3 copies; 4: template amount 10 4 copies; 5: template amount 10 5 copies; 6: template amount 10 6 copies; 7: template amount 10 7 copies; 8: template amount 10 8 copies.

[0030] Figure 4 The performance comparison results of the RPA-LFD detection system of the present invention and RT-PCR detection are shown; (A) is the result of RT-PCR detection of 32 samples; (B) is the result of RPA-LFD detection of 32 samples in the present invention.

[0031] Figure 5The detection limit test results provided by the comparative example of the present invention are shown in FIG. 1 ; (A) shows the detection limit test results of the eliminated primer set 1; (B) shows the detection limit test results of the eliminated primer set 2; N: negative control; 1: template amount 10 1 copies; 2: template amount 10 2 copies; 3: template amount 10 3 copies; 4: template amount 10 4 copies; 5: template amount 10 5 copies; 6: template amount 10 6 copies; 7: template amount 10 7 copies; 8: template amount 10 8 copies. DETAILED DESCRIPTION

[0032] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, identical modules are denoted by identical reference numerals. In the case of identical reference numerals, their names and functions are also identical. Therefore, their detailed description will not be repeated.

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0034] The present invention provides a primer set for rapid detection of betel nut yellowing virus APV1 based on RPA-LFD technology, comprising a specific primer pair and a probe;

[0035] The nucleotide sequences of the specific primer pairs are:

[0036] Upstream primer: 5'-CTGCTGGAACTAACATTGATAGATCATTTT-3' (as shown in SEQ ID No. 1 in the sequence listing);

[0037] Downstream primer: 5'-ACATCAGCAGCAGTTGGTCGATTTGCGGAA-3' (as shown in SEQ ID No. 2 in the sequence listing);

[0038] The nucleotide sequence of the probe is:

[0039] (As shown in the sequence listing SEQ ID No. 3).

[0040] In some embodiments, the 5' end of the downstream primer is modified with a Biotin group.

[0041] The probe is modified by labeling with a biotin or FAM fluorescent group, which can achieve specific capture and visual detection of the amplified product; in some embodiments, the fluorescent group is selected from one of FAM (Fluorescein amidite), HEX (Hexach loro-fluorescein), TET (Tetrachlorofluorescein), cyanine dye (Cyanine dye), ROX (Carboxy-X-rhodamine), and Texas Red (Texas Red); in a specific embodiment, the fluorescent group is FAM;

[0042] In some embodiments, the alkyl carbon spacer in the probe is selected from one of C2 Spacer, C3 Spacer, and C4 Spacer; in a specific embodiment, the alkyl carbon spacer is C3 Spacer.

[0043] In a specific embodiment, the nucleotide sequence of the modified probe is as follows:

[0044] (As shown in the sequence listing SEQ ID No. 3).

[0045] A kit for rapidly detecting betel nut yellows virus APV1 comprises a primer set for rapidly detecting betel nut yellows virus APV1 based on RPA-LFD technology.

[0046] A rapid detection system for betel nut yellowing virus APV1, the RPA-LFD reaction system comprises:

[0047] Upstream primer 21±3 pmoL

[0048] Downstream primer 21±3 pmoL

[0049] Probe 6±1 pmoL

[0050] Template 2 μL

[0051] Activator 2 μL

[0052] Water Residual

[0053] The total reaction system was 50 μL;

[0054] The reaction temperature of the RPA reaction was a constant 37–44°C, and the reaction time was 10 ± 2 min;

[0055] The temperature for testing using lateral flow immunoassay strips is room temperature and the time is 10 ± 1 min;

[0056] As a preferred embodiment, the final concentrations of the upstream primer and the downstream primer in the RPA-LFD reaction are 0.4 μM, respectively;

[0057] As a preferred embodiment, the final concentration of the probe in the RPA-LFD reaction is 0.12 μM;

[0058] As a preferred embodiment, the reaction time of the RPA-LFD reaction is 10 min;

[0059] The detection system also contains lyophilized RPA enzyme microspheres, which contain recombinase, single-stranded binding protein (SSB), and DNA polymerase. These are core components of the RPA reaction known in the art. In some embodiments, individual components of the RPA enzyme mixture can also be provided separately.

[0060] The detection system also contains: nucleic acid detection test strips;

[0061] In a specific embodiment, the nucleic acid detection test strip is a lateral flow chromatography test strip;

[0062] In a specific embodiment, the detection system further contains a stabilizer and a preservative to ensure the stability of the detection system during storage and use;

[0063] In a specific embodiment, the detection system further contains a detergent to help reduce inhibitors in the sample and improve amplification efficiency;

[0064] In a specific embodiment, the detection system further comprises a positive control and / or a negative control for verifying the accuracy and specificity of the RPA reaction;

[0065] In a specific embodiment, the detection system also contains instructions for providing detailed operating steps, reaction conditions, usage guidelines and precautions.

[0066] Other components in the detection system mentioned in the present invention can be obtained through commercial channels, and can be provided individually or in combination in multiple or one reagent.

[0067] A method for rapidly detecting betel nut yellows virus APV1 based on RPA-LFD technology specifically comprises the following steps:

[0068] S1. Crude sample extraction: 50-100 mg of betel nut leaf tissue was placed in a mortar and pestle, and then ground with sample release solution to obtain a crude extract. Viral RNA was then extracted from the sample.

[0069] S2. Isothermal Amplification: Using the RNA obtained in step S1 as a template, add the RPA reaction system containing a specific primer pair. Insert the centrifuge tube into the centrifuge tube rack in the constant temperature amplification device and adjust the temperature using the temperature control button to perform the isothermal amplification reaction at 37-44°C.

[0070] S3. Chromatographic testing: Dilute the amplified product with pure water and add it dropwise to the sample area of ​​the test strip. Observe the color development within 10-15 minutes. The test results are determined based on the following criteria:

[0071] When the control line (C line) of the test strip shows color and the test line (T line) does not show color, it is judged as APV1 negative;

[0072] When the control line (C line) and the test line (T line) of the test strip show color at the same time, it is judged as APV1 positive.

[0073] The constant temperature amplification device has a split cavity structure, including an upper cavity and a lower cavity; the upper cavity is equipped with an insulation liner and a centrifuge tube rack, and the lower cavity is equipped with a heating plate to heat the upper cavity; a temperature display screen and temperature adjustment buttons are set on the outside of the device; after charging the constant temperature amplification device, wireless temperature control and heating can be achieved, which is suitable for field testing.

[0074] The method of the present invention can achieve better amplification results in 10 minutes. The upper limit mentioned here is more based on considerations of detection efficiency. In actual detection, the reaction time here can also be extended.

[0075] By optimizing the reaction system and reaction conditions, the specificity of the method of the present invention is enhanced, with no cross-reaction with other viruses and no nonspecific amplification; the detection limit is reduced to 100 copies of the template; and the target gene can be detected in just 20 minutes, while traditional nested PCR requires about 3 to 4 hours and fluorescent PCR requires 1 to 1.5 hours.

[0076] A primer set for rapid detection of betel nut yellows virus APV1 based on RPA-LFD technology or a kit for rapid detection of betel nut yellows virus APV1 is used in the detection or auxiliary detection of betel nut yellows virus APV1.

[0077] Example 1

[0078] Using a conserved sequence (SEQ ID No. 4) of the gene encoding the capsid protein of Areca nut yellowing virus (APV1) as a template, a primer set for rapid detection of Areca nut yellowing virus (APV1) based on RPA-LFD technology was designed and screened, including specific primer pairs and probes.

[0079] Upstream primer: 5′-CTGCTGGAACTAACATTGATAGATCATTTT-3′ (SEQ ID No .1);

[0080] Downstream primer: 5′-ACATCAGCAGCAGTTGGTCGATTTGCGGAA-3′ (SEQ ID No .2);

[0081] Probe:

[0082] (SEQ ID No .3);

[0083] Its target gene (542bp):

[0084] (SEQ ID No.4).

[0085] After further optimization and exploration, the reaction system and specific detection method are as follows:

[0086] The method for rapid detection of betel nut yellows virus APV1 specifically comprises the following steps:

[0087] S1. Crude sample extraction: 50-100 mg of betel nut leaf tissue was placed in a mortar and pestle, and then ground with sample release solution to obtain a crude extract. Viral RNA was then extracted from the sample.

[0088] S2. Isothermal Amplification: Using the RNA obtained in step S1 as a template, add the RPA reaction system containing a specific primer pair. Insert the centrifuge tube into the centrifuge tube rack in the constant temperature amplification device and adjust the temperature using the temperature control button to perform the isothermal amplification reaction at 37-44°C.

[0089] S3. Chromatographic detection: Dilute the amplified product with pure water and add it dropwise to the sample area of ​​the test strip. Observe the color development within 10-15 minutes.

[0090] The constant temperature amplification device has a split chamber structure, consisting of an upper chamber and a lower chamber. The upper chamber is equipped with an insulation liner and a centrifuge tube rack, while the lower chamber is equipped with a heating plate to heat the upper chamber. A temperature display and temperature adjustment buttons are set on the outside of the device. After charging, the constant temperature amplification device can achieve wireless temperature control and heating, making it suitable for field testing.

[0091] The reaction system was prepared using the test paper-based nucleic acid amplification kit (ERA method) from Suzhou Xianda Technology Co., Ltd., as shown in Table 1:

[0092] Table 1 Reaction system

[0093]

[0094] A metal bath apparatus was used, and the reaction conditions were: constant temperature 37-44°C, and reaction time 10 min.

[0095] After the reaction is complete, extract 5 μL of the reaction product and dilute it with pure water, using a 40-120-fold dilution factor. Place 80 μL of the diluted reaction product on the sample area of ​​the test strip. Incubate at room temperature for 10 minutes and observe the results. If only the C line (control band) appears on the test strip, the test result is negative for APV1 nucleic acid. If both the C line and the T line (test band) appear, the test result is positive for APV1 nucleic acid.

[0096] Water was used as negative control and APV1 virus was used as template (positive control). The test results are shown in Figure 1 From the results, it can be seen that the reaction system and reaction method of the present invention can better detect the betel nut APV1 virus.

[0097] Example 2

[0098] The reaction system and detection method in Example 1 were used, and RPA-LFD amplification was performed using areca necrotic ringspot virus (ANRSV) and areca necrotic spindle spot virus (ANSSV) nucleic acids as templates, APV1 viral nucleic acid as a positive control, and water as a negative control; a specificity experiment was performed.

[0099] Test results are shown in Figure 2 The results show that the detection method established by the present invention is positive only when APV1 virus RNA is used as a template, and has no cross reaction with other viruses.

[0100] Example 3

[0101] The crude extract containing APV1 virus was diluted 10-fold in a gradient manner, and the crude extract of each dilution was used as a template for RPA-LFD amplification, with water as a negative control; a detection limit experiment was performed.

[0102] Test results are shown in Figure 3 The results showed that: at a template amount of 100 copies, the test result was still positive; at a template amount of 10 copies, no T line was visible, indicating a negative result. Therefore, the detection limit of the RPA-LFD detection method for APV1 virus associated with betel nut yellowing disease established in this invention is 100 copies of template.

[0103] Example 4

[0104] The performance of the rapid detection system for betel nut yellowing virus APV1 (RPA-LFD detection system) of the present invention was compared with that of the traditional RT-PCR detection system. 32 samples were collected from the Zhongyuan Agricultural Experiment Demonstration Base in Zhongyuan Town, Qionghai City, Hainan Province, and tested using RT-PCR and the detection system of the present invention respectively. The results showed that the detection results of the present invention were consistent with the RT-PCR detection results. Figure 4 .

[0105] Comparative Example 1

[0106] This comparative example provides some of the primers screened out by the present invention and their detection limit effect data. The specific primer probe sequences are shown in Table 2; the specific determination method of the detection limit is the same as in Example 3, and the detection limit test results are shown in Table 2. Figure 5 The results show that the primer pair screened by the present invention has the best detection effect.

[0107] Table 2 Eliminated primer sets

[0108]

[0109] It should be understood that the various forms of the processes shown above can be used to reorder, add, or delete steps. For example, the steps described in the present disclosure can be performed in parallel, sequentially, or in a different order, as long as the desired results of the technical solutions disclosed in the present disclosure can be achieved. This is not limited herein.

[0110] The above specific embodiments do not limit the scope of protection of the present invention. Those skilled in the art will appreciate that various modifications, combinations, sub-combinations, and substitutions may be made based on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A method for rapid detection of betel nut yellows virus APV1 based on RPA-LFD technology, using a detection system for rapid detection of betel nut yellows virus APV1, characterized in that: The specific steps include: S1. Crude sample extraction: 50-100 mg of betel nut leaf tissue was placed in a mortar and pestle, and then ground with sample release solution to obtain a crude extract. Viral RNA was then extracted from the sample. S2. Isothermal Amplification: Using the RNA obtained in step S1 as a template, add the RPA reaction system containing a specific primer pair and probe. Insert the centrifuge tube into the centrifuge tube rack in the constant temperature amplification device and adjust the temperature using the temperature control button to perform the isothermal amplification reaction at 37-44°C. The nucleotide sequence of the upstream primer in the specific primer pair is shown in SEQ ID No. 1 in the sequence listing, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2 in the sequence listing; the nucleotide sequence of the modified probe is shown in SEQ ID No. 3 in the sequence listing; the 5' end of the downstream primer is modified with a Biotin group; The reaction system includes the following components and amounts: The reaction time of RPA reaction was 10 ± 2 min; the reaction temperature of RPA reaction was constant at 37~44℃; S3. Chromatographic testing: Dilute the amplified product with pure water and add it dropwise to the sample area of ​​the test strip. Observe the color development within 10-15 minutes. The test results are determined based on the following criteria: When the control line of the test strip shows color and the test line shows no color, it is judged as APV1 negative; When the control line and the test line of the test strip show color at the same time, it is determined to be APV1 positive; The temperature for detection using lateral flow immunoassay strips is room temperature and the detection time is 10±1 min.

2. A method for rapid detection of betel nut yellowing virus APV1 based on RPA-LFD technology according to claim 1, characterized in that: In the detection system, each 50 μL reaction system contains 21±3 pmoL of upstream primer, 21±3 pmoL of downstream primer and 6±1 pmoL of probe.

3. A method for rapid detection of betel nut yellowing virus APV1 based on RPA-LFD technology according to claim 2, characterized in that: The reaction time of the RPA-LFD reaction was 10 min.

4. A method for rapid detection of betel nut yellowing virus APV1 based on RPA-LFD technology according to claim 1, characterized in that: The constant temperature amplification device has a split cavity structure, including an upper cavity and a lower cavity; the upper cavity is provided with an insulation inner tank and a centrifuge tube rack, and the lower cavity is provided with a heating plate for heating the upper cavity; a temperature display screen and a temperature adjustment button are provided on the outside of the constant temperature amplification device; the constant temperature amplification device is provided with a charging interface, which realizes wireless temperature control and heating after charging.

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