Kit for dual visual detection of CGMMV and / or WSMoV based on RT-RPA-LFD, detection method and application

Abstract

The invention belongs to the technical field of molecular diagnosis of plant pathogens, and particularly relates to a kit for dual visual detection of CGMMV and / or WSMoV based on RT-RPA-LFD, a detection method and application. The detection method can simultaneously distinguish two pathogens within 15 minutes, and the time is obviously shortened compared with that of RT-PCR (Reverse Transcription-Polymerase Chain Reaction); the operation temperature is low: the RPA is carried out under the constant temperature condition of 42 DEG C, and is suitable for detection in a field or a resource limited environment in combination with portable equipment; dual detection: a WSMoV (DIG tag) and a CGMMV (FAM tag); the sensitivity is high, and the detection limit reaches about 2.58 * 10 <-5 > ng / L; high specificity: no cross reaction with CMV, WMV, ZYMV and other related viruses exists; the method is high in applicability, and can be used for rapid diagnosis of various plant tissues such as seeds and leaves of cucurbitaceae crops; and a technical means is provided for rapid, high-specificity and portable field instant diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology for plant pathogens, and specifically relates to a kit for dual visualization detection of CGMMV and / or WSMoV. Background Technology

[0002] Cucurbitaceae crops (including watermelon, cucumber, and melon) have significant economic value globally; however, they are susceptible to various viruses, particularly Watermelon silver mottle virus (WSMoV) and Cucumber green mottle mosaic virus (CGMMV). These two viruses often cause similar symptoms in the field, such as leaf mottling, yellowing, and wrinkling, making accurate differentiation based on field appearance difficult and prone to misdiagnosis.

[0003] Currently, among the detection methods for the aforementioned viruses, ELISA, RT-PCR, and RT-qPCR, while possessing high sensitivity, all rely on specialized laboratory conditions and instruments, and have long detection cycles, failing to meet the needs of rapid field diagnosis. Isothermal amplification techniques (such as LAMP) improve some of the requirements, but still require relatively high temperatures (60-65℃) and complex primer design. For example, patent 202510778944.X discloses a primer set, kit, and application for detecting WSMoV, employing SYBR Green dye-based quantitative PCR technology, which can achieve efficient and accurate quantitative detection of the WSMoV genome, but still does not overcome the limitations of laboratory detection. Patent 202511517237.1 discloses a kit, detection method, and application for detecting CGMMV based on enzyme-mediated double amplification nucleic acid amplification. This technology can achieve RNA detection at the aM concentration level, and its high sensitivity can meet the needs of early CGMMV screening, but it also fails to solve the problem of rapid field detection. Currently, there is a lack of multiplex detection systems for the simultaneous rapid detection of WSMoV and CGMMV in the field. Summary of the Invention

[0004] To address the aforementioned issues, this invention proposes a kit, detection method, and application for dual visualization detection of CGMMV and / or WSMoV based on RT-RPA-LFD.

[0005] The technical solution of this invention is implemented as follows: This invention establishes a dual RT-RPA-LFD method by optimizing primer and probe design and reaction system, which can simultaneously distinguish two pathogens within 15 minutes, providing a technical means for rapid, highly specific, and portable field-based real-time diagnosis. Specifically, it involves a dual visualization detection method and related kit based on reverse transcription-recombinase polymerase amplification (RT-RPA) and lateral flow detection strips (LFD) for rapid field detection of watermelon silver spot virus (WSMoV) and cucumber green mottle mosaic virus (CGMMV).

[0006] Based on this, the present invention proposes a primer-probe set for dual visualization detection of CGMMV and / or WSMoV based on RT-RPA-LFD: The forward primer for detecting CGMMV is CGCP5 as shown in SEQ ID NO:1, and the reverse primer is CGCP6 as shown in SEQ ID NO:2, with biotin labeled at its 5' end; the probe is CG56P as shown in SEQ ID NO:3, with FAM fluorescent group modified at its 5' end, C3-spacer group modified at its 3' end, and THF modified between the 35th base G and the 36th base T starting from the 5' end. The forward primer for detecting WSMoV is WS280F, as shown in SEQ ID NO:4; the reverse primer is WS280R, as shown in SEQ ID NO:5, with biotin labeled at its 5' end; the probe is WS280P, as shown in SEQ ID NO:6, with digoxigenin labeled at its 5' end, a C3-spacer group modified at its 3' end, and THF modified between the 36th and 37th bases starting from the 5' end.

[0007] Secondly, the present invention provides a kit for dual visualization detection of CGMMV and / or WSMoV based on RT-RPA-LFD, comprising the aforementioned primer and probe set.

[0008] Preferably, the above kit also includes RT-RPA amplification reagent, LFD test strips, RNA extraction reagent, positive control, and negative control.

[0009] Preferably, the LFD test strip contains an anti-biotin C line, an anti-FAM T1 line, and an anti-digoxin T2 line.

[0010] The kit may include: lyophilized RT-RPA system; primers and probes (SEQ ID NO:1-6); LFD test strips; sample lysis / extraction buffer; positive / negative controls; and instructions for use.

[0011] Thirdly, the present invention also provides a method for dual visualization detection of CGMMV and / or WSMoV based on RT-RPA-LFD, the steps of which are: (1) Extract RNA from the sample to be tested; (2) The above-mentioned kit was used to perform an RT-RPA reaction to obtain the amplification product; (3) Load the amplification product onto the LFD test strip; (4) Visually interpret the presence of CGMMV and / or WSMoV based on the test lines on the LFD test strip; If the C line and T1 line appear, then only CGMMV exists in the sample being tested; If the C line and T2 line appear, then only WSMoV is present in the sample being tested; If the C line + T1 + T2 line appears, then the sample to be tested contains both CGMMV and WSMoV. If only the C line appears, then CGMMV and WSMoV are not present in the sample being tested.

[0012] Preferably, the above RT-RPA reaction system is 50 μL, including 29.4 μL of Buffer A; 2 μL of each primer in the above primer-probe set; 0.6 μL of each probe in the above primer-probe set; 5 μL of RNA template; 3.9 μL of nuclease-free water; and 2.5 μL of Buffer B.

[0013] Preferably, the above RT-RPA reaction is carried out at 42°C for 15 minutes, and the amplification product is diluted 1:10 and then loaded onto the LFD test strip. Preferably, the sample to be tested is the leaf, stem, fruit, or seed tissue of a Cucurbitaceae plant.

[0014] Preferably, the detection limit of the above method is 2.58 × 10⁻⁶. -5 ng / µL (based on total RNA concentration).

[0015] Thirdly, the application of the aforementioned primer-probe set, kit, or method in the field visualization detection of CGMMV and / or WSMoV.

[0016] The present invention has the following beneficial effects: This invention establishes a dual visualization detection method and related kit based on reverse transcription-recombinase polymerase amplification (RT-RPA) and lateral flow detection strips (LFD) by optimizing primer and probe design and reaction system. This dual RT-RPA-LFD method can simultaneously distinguish two pathogens within 15 minutes, significantly shorter than RT-PCR; it operates at a low temperature: RPA is performed at around 42℃, suitable for portable devices or field use; it provides dual detection: simultaneously distinguishing between WSMoV (DIG-tagged) and CGMMV (FAM-tagged); and it has high sensitivity: the detection limit reaches approximately 2.58 × 10⁻⁶. -5 ng / µL (based on total RNA concentration); High specificity: no cross-reactivity with other related viruses such as CMV, WMV, and ZYMV; Wide applicability: can be used for rapid on-site diagnosis of various plant tissues such as seeds and leaves; ultimately achieving rapid, highly specific, and portable real-time field diagnosis. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Results of RT-RPA reaction temperature optimization.

[0019] Figure 2 To optimize amplification time.

[0020] Figure 3 This is a specificity test.

[0021] Figure 4 For sensitivity testing.

[0022] Figure 5 The results of LFD band analysis for field samples.

[0023] Figure 6 This is the overall flowchart of dual RT-RPA-LFD.

[0024] Figure 7 Additional data related to primer screening experiments. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0027] This invention employs specific primers and probes designed for conserved sequences of two viruses, combined with reverse transcription-RPA amplification, and distinguishes between the two viruses using an LFD test strip based on different tags: FAM (6-Carboxyfluorescein) and digoxigenin. The system completes amplification at an isothermal temperature of 42°C for 15 minutes, and the signal is then directly read using the test strip, making interpretation simple and rapid. The overall flowchart of the dual RT-RPA-LFD process is shown below. Figure 6 .

[0028] Example 1: Primer and Probe Design Primers and probes were designed for the WSMoV S fragment (GenBank: AF133128.1) and the CGMMV capsid protein gene (GenBank: NC_001801.1). Each primer set was approximately 30-35 bp in length, targeting a fragment of 100-500 bp; the probes contained a 5′ marker, an internal THF site, and a 3′ C3 end block.

[0029] Table 1 Primers and probes used in this invention CGMMV: Forward primer: CGCP5 (SEQ ID NO:1) Reverse primer: CGCP6 (SEQ ID NO:2, 5′ biotinylated) Probe: CG56P (SEQ ID NO:3, 5′ end FAM tag) WSMoV: Forward primer: WS280F (SEQ ID NO:4) Reverse primer: WS280R (SEQ ID NO:5, 5′ biotinylated) Probe: WS280P (SEQ ID NO: 6, 5′ end digoxigenin tag) Figure 7Additional data related to primer screening experiments, provided by Figure 7 It can be seen that by screening multiple candidate primers by agarose gel electrophoresis, the primer sets CG56 (for CGMMV) and WS280 (for WSMoV) can achieve specific and consistent amplification of the target fragment, showing the best amplification effect. Therefore, they were selected as the optimal primer combination for subsequent dual RT-RPA reaction.

[0030] Example 2: RT-RPA reaction system and conditions The RT-RPA reaction system is as follows: The 50 μL reaction mixture included: 29.4 μL Buffer A; 2 μL each of the four primers; 0.6 μL each of the two probes; 5 μL RNA template (optimized ratio WSMoV:CGMMV = 1:3, total amount 128.75 ng total RNA); 3.9 μL nuclease-free water; and 2.5 μL Buffer B. Amplification was completed by incubating at 42℃ for 15 min. Figure 1 As shown, the preferred reaction temperature of this invention is 42°C. Figure 2 As shown, the optimal reaction time is 15 minutes.

[0031] Example 3: LFD detection The amplification product was diluted 1:10 and then loaded onto the side-flow test strip.

[0032] The LFD test strip (sideflow chromatography test strip) has three functional lines, C line (control line, avidin). T1 line (CGMMV detection line, anti-FAM); T2 line (WSMoV detection line, anti-digoxin).

[0033] Interpretation rules: Positive: If C line + T1 line appears, only CGMMV is present in the sample; if C line + T2 line appears, only WSMoV is present in the sample; if C line + T1 + T2 line appears, both CGMMV and WSMoV are present in the sample. Negative: If only the C line appears, then CGMMV and WSMoV are not present in the sample.

[0034] Invalid: No C line.

[0035] Application example: Specificity and sensitivity verification (1) Specificity A dual RT-RPA-LFD detection system was established to detect a series of common cucurbit viruses, including cucumber mosaic virus (CMV), watermelon mosaic virus (WMV), zucchini yellow mosaic virus (ZYMV), tomato leaf curl New Delhi virus (ToLCNDV), melon yellowspot virus (MYSV), and cucurbit aphid-borne yellows virus (CABYV). Each virus sample was tested three times, and the band patterns on the test strips were observed to verify whether the system cross-reacted with non-target viruses.

[0036] The results are as follows Figure 3 As shown, no test lines appeared, indicating no cross-reaction with other non-target viruses.

[0037] (2) Sensitivity The extracted WSMoV (71.2 ng / µL) and CGMMV (10.6 ng / µL) RNA templates were mixed at a 1:3 ratio, resulting in an initial concentration of approximately 25.75 ng / µL. Subsequently, the mixture was serially diluted 10-fold with nuclease-free water to prepare RNA templates with concentrations ranging from 10... -1 Up to 10⁻ 7 A series of diluted samples were analyzed. The samples at each dilution were tested using the optimized RT-RPA-LFD system, with each concentration tested three times.

[0038] The results are as follows Figure 4 As shown, the sample was diluted at a concentration of 10... -6 Specific bands can still be detected at 10 hours, while at 10 hours... -7 No bands were observed at dilution. The calculated limit of detection (LOD) for this method is 2.58 × 10⁻⁶. -5 ng / µL (based on total RNA concentration). On the other hand, parallel assays were performed on the same series of diluted samples using an RT-PCR system. The RT-PCR method was used at a dilution of 10⁻ 5 Specific amplification bands appeared at times, while 10 -6 No amplification bands appeared. Based on calculations and analysis, the limit of detection for CGMMV at this point was approximately 1.06 × 10⁻⁶. -4 The limit of detection for WSMoV is approximately 7.12 × 10⁶ ng / μL (based on total RNA concentration). -4ng / μL (based on total RNA concentration). A comparison of the results from the two methods revealed that the dual RT-RPA-LFD method of this invention has higher sensitivity, shorter detection time, and is simpler to operate compared to the traditional RT-PCR method.

[0039] (3) Field sample validation Sample source and collection: During the 2023-2024 growing season, leaf samples of watermelon and cucumber exhibiting typical viral infection symptoms (such as mottling and chlorosis) were collected from major production areas in China (Shandong, Zhejiang, Henan, Hainan and Xinjiang), totaling 40 suspected infected samples; at the same time, 22 leaves of asymptomatic healthy plants were collected as negative controls, for a total of 62 samples.

[0040] Sample processing: Total RNA was extracted from all collected samples using a plant total RNA extraction kit according to the manufacturer's instructions and stored for later use. Detection procedure: All 62 samples were subjected to the dual RT-RPA-LFD detection described in this invention, and RT-PCR was used for comparative detection. Each sample was tested three times to verify the accuracy of this method in field screening.

[0041] Table 2. Field RT-PCR and RT-RPA-LFD detection results Figure 5 For LFD band results from field samples, such as Figure 5 As shown in Table 2, the WSMoV detection results of the 62 randomly collected samples were almost identical to those of multiplex RT-PCR. However, for CGMMV detection, RT-RPA-LFD detected 32 positive samples, while RT-PCR detected 30. This result indicates that for CGMMV detection, the RT-RPA-LFD technology is superior to the RT-PCR technology.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer-probe set for dual visualization detection of CGMMV and / or WSMoV based on RT-RPA-LFD, characterized in that: The forward primer for detecting CGMMV is CGCP5 with the sequence shown in SEQ ID NO:1, and the reverse primer is CGCP6 with the sequence shown in SEQ ID NO:2, whose 5' end is labeled with biotin; the probe is CG56P with the sequence shown in SEQ ID NO:3, whose 5' end is labeled with the FAM fluorescent group, whose 3' end is labeled with C3 spacer, and whose 3' end is modified with THF between the 35th base G and the 36th base T starting from the 5' end; The forward primer for detecting WSMoV is WS280F with the sequence shown in SEQ ID NO:4, and the reverse primer is WS280R with the sequence shown in SEQ ID NO:5, whose 5' end is labeled with biotin. The probe is WS280P with the sequence shown in SEQ ID NO:6, whose 5' end is labeled with digoxigenin, whose 3' end is modified with a C3 spacer, and whose 36th base C and 37th base C from the 5' end are modified with THF.

2. A kit for dual visualization detection of CGMMV and / or WSMoV based on RT-RPA-LFD, characterized in that: It includes the primer and probe set as described in claim 1.

3. The reagent kit according to claim 2, characterized in that: The kit also includes RT-RPA amplification reagent, LFD test strips, RNA extraction reagent, positive control, and negative control.

4. The reagent kit according to claim 3, characterized in that: The LFD test strip contains an anti-biotin C line, an anti-FAM T1 line, and an anti-digoxin T2 line.

5. A method for detecting CGMMV and / or WSMoV based on dual visualization using RT-RPA-LFD, characterized in that, The steps are as follows: (1) Extract RNA from the sample to be tested; (2) Perform an RT-RPA reaction using the kit described in any one of claims 2-4 to obtain the amplification product; (3) Load the amplification product onto the LFD test strip; (4) Visually interpret the presence of CGMMV and / or WSMoV based on the test lines on the LFD test strip; If the C line and T1 line appear, then only CGMMV exists in the sample being tested; If the C line and T2 line appear, then only WSMoV is present in the sample being tested; If the C line + T1 + T2 line appears, then the sample to be tested contains both CGMMV and WSMoV. If only the C line appears, then CGMMV and WSMoV are not present in the sample being tested.

6. The method for dual visualization detection of CGMMV and / or WSMoV according to claim 5, characterized in that: The RT-RPA reaction system is 50 μL, including 29.4 μL of Buffer A; 2 μL of each primer; 0.6 μL of each probe; 5 μL of RNA template; and 3.9 μL of nuclease-free water. 2.5 μL of Buffer B.

7. The method for dual visualization detection of CGMMV and / or WSMoV according to claim 6, characterized in that: The RT-RPA reaction was carried out at 42°C for 15 minutes, and the amplification product was diluted 1:10 and then loaded onto the LFD test strip.

8. The method for dual visualization detection of CGMMV and / or WSMoV according to claim 7, characterized in that: The samples to be tested are leaves, stems, fruits, or seed tissues of Cucurbitaceae plants.

9. The method for dual visualization detection of CGMMV and / or WSMoV according to any one of claims 5-8, characterized in that: The limit of detection for this method, based on total RNA concentration, is 2.58 × 10⁻⁶. -5 ng / µL.

10. The application of the primer and probe set of claim 1, the kit of any one of claims 2-4, or the method of any one of claims 5-8 in the rapid visual detection of CGMMV and WSMoV.

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