Primer, probe, kit and method for detecting tomato brown rugose fruit virus based on mira technology

Abstract

This invention discloses primers, probes, a kit, and a method for detecting TobRFV brown wrinkled fruit virus based on MIRA technology, belonging to the field of plant virus detection technology. The primers and probes include an upstream primer ToBRFV-ORF1-3F3, a downstream primer ToBRFV-ORF1-3R6, and a probe ToBRFV-ORF1-P3. The nucleotide sequence of the upstream primer ToBRFV-ORF1-3F3 is shown in SEQ ID NO:15, the nucleotide sequence of the downstream primer ToBRFV-ORF1-3R6 is shown in SEQ ID NO:21, and the nucleotide sequence of the probe ToBRFV-ORF1-P3 is shown in SEQ ID NO:2. The kit of this invention can achieve highly sensitive and specific rapid amplification of ToBRFV nucleic acid under isothermal conditions, and is suitable for rapid on-site detection in non-laboratory environments such as fields and greenhouses.

Description

Technical Field

[0001] This invention relates to the field of plant virus detection technology, specifically to primers, probes, kits, and methods for detecting tomato brown wrinkled fruit virus based on MIRA technology. Background Technology

[0002] Tomato brown rugose fruit virus (ToBRFV) is a plant pathogen that has posed a serious threat to global tomato and pepper production in recent years. After infecting its host, the virus causes symptoms such as pustules, narrow leaves, and mosaic patterns on leaves; necrosis of petioles, pedicels, and calyxes; brown, wrinkled, or necrotic spots on fruits; and widespread stunting of plants. ToBRFV is highly resilient, able to survive for extended periods in infected plant material and seeds, and can spread over long distances through seedling transportation and agricultural operations. Once an outbreak occurs, especially in greenhouses and polytunnels, it can easily infect all plants, causing severe yield losses and reduced fruit quality, significantly impacting the entire tomato industry chain. Therefore, establishing rapid, accurate, and sensitive ToBRFV detection technology is crucial for early warning and scientific control.

[0003] Currently, the detection of ToBRFV mainly relies on the following technologies: (1) Symptom diagnosis: relies on experience and is easily confused with other viruses or physiological diseases; it is only applicable to the middle and late stages of the disease and cannot be used to detect the early or asymptomatic infection stages. (2) Transmission electron microscopy: can directly observe the morphology of virus particles, but the equipment is expensive, the operation is complicated, and the sample pretreatment is time-consuming; it requires a high level of professional expertise from the operators and is not suitable for rapid screening of large batches of samples. (3) Serological detection (such as ELISA): commercial kits are available, but the sensitivity is limited and it is difficult to detect low concentrations of virus samples; antibodies may cross-react with other members of the Tobacco Mosaic Virus genus, affecting specificity. (4) Molecular biological detection (such as RT-PCR, qPCR): has high sensitivity and strong specificity and is currently the mainstream method. However, it relies on laboratory instruments and has a long detection cycle; RNA extraction is easily degraded, and there is a risk of contamination during the operation; it requires high technical expertise from facilities and personnel and is difficult to apply rapidly in the field or on-site. (5) High-throughput sequencing technology: It has high throughput and can be used for the discovery of new pathogens and the monitoring of mutations, but it is costly, data analysis is complex and the cycle is long. It is not suitable for routine testing or production scenarios that require rapid judgment. It is mostly used for research and source tracing analysis.

[0004] Multienzyme isothermal rapid amplification (MIRA) is a novel isothermal nucleic acid amplification technology that utilizes the synergistic action of multiple enzymes, including recombinases, single-stranded binding proteins, and DNA polymerases, to achieve efficient and rapid amplification of target nucleic acid sequences at a constant temperature (typically 37-42℃) without relying on sophisticated thermal cycling instruments. This technology boasts significant advantages such as ease of operation, rapid reaction, high sensitivity, good specificity, and low equipment requirements, making it particularly suitable for rapid on-site pathogen detection and applications in grassroots laboratories. Therefore, developing a ToBRFV rapid detection method based on MIRA technology that combines high sensitivity, high specificity, and ease of on-site operation to overcome the shortcomings of existing technologies in terms of applicability, timeliness, and convenience is particularly urgent. Summary of the Invention

[0005] This invention addresses the aforementioned problems by providing primers, probes, a kit, and a method for detecting ToBRFV (Tomato Brown Curly Fruit Virus) based on MIRA technology. This overcomes the shortcomings of existing detection technologies in terms of field applicability, detection speed, operational complexity, and equipment dependence. The kit enables highly sensitive and specific rapid amplification of ToBRFV nucleic acid under isothermal conditions, making it suitable for rapid on-site detection in non-laboratory environments such as fields and greenhouses.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides primers and probes for detecting tomato brown wrinkled fruit virus based on MIRA technology. The primers and probes include an upstream primer ToBRFV-ORF1-3F3, a downstream primer ToBRFV-ORF1-3R6, and a probe ToBRFV-ORF1-P3. The nucleotide sequence of the upstream primer ToBRFV-ORF1-3F3 is shown in SEQ ID NO:15, the nucleotide sequence of the downstream primer ToBRFV-ORF1-3R6 is shown in SEQ ID NO:21, and the nucleotide sequence of the probe ToBRFV-ORF1-P3 is shown in SEQ ID NO:2.

[0007] The present invention also provides a kit for detecting tomato brown wrinkled fruit virus based on MIRA technology, the kit comprising the aforementioned primers and probes.

[0008] Furthermore, the kit also contains buffer A, water, buffer B, and template.

[0009] This invention also provides a method for detecting tomato brown wrinkled fruit virus based on MIRA technology, specifically including the following steps: S1: Sample processing: Take the plant tissue sample to be tested, add the sample pretreatment solution, mix well and obtain the sample extract. S2: Nucleic acid release: Take the sample extract, add it to the nucleic acid release agent, let it stand, and obtain the nucleic acid solution to be tested; S3: MIRA isothermal amplification reaction: The nucleic acid solution to be tested, the primers and probes, and other components required for the MIRA isothermal amplification reaction are mixed to form a reaction system, and an isothermal amplification reaction is carried out. S4: Result Interpretation: Determine whether the sample contains tomato brown wrinkled fruit virus by detecting the real-time fluorescence signal during the amplification process.

[0010] Further, in step S1, the plant tissue sample is a tomato leaf, the mass ratio of the plant tissue sample to the sample pretreatment solution is 1:10, and the mixing treatment is manual shaking for 30 seconds.

[0011] Further, in step S2, the volume ratio of the sample leachate to the nucleic acid release agent is 1:10, and the nucleic acid release agent is an RNA-II type nucleic acid release agent; the standing time is 5 minutes.

[0012] Furthermore, in step S3, the amount of the nucleic acid solution to be tested loaded into the reaction system is 50 μL.

[0013] Furthermore, in step S3, the temperature of the isothermal amplification reaction is 42°C, and the reaction time is 20 minutes.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes specific primers and probes designed based on conserved regions of the ToBRFV genome, combined with the high-efficiency amplification capability of MIRA technology, to achieve single-copy-level virus detection. It is also less prone to cross-reaction with other tobacco mosaic viruses, achieving highly sensitive and specific detection of Tomato Brown Fruit Virus (ToBRFV). Sample pretreatment is simple, requiring no RNA purification step, reducing operational difficulty and the risk of cross-contamination. It eliminates the need for complex instruments and specialized laboratory environments, completing the entire process from sample processing to result output in approximately 30 minutes. The isothermal reaction conditions are compatible with portable fluorescence detection equipment, reducing detection costs and site limitations. With low equipment dependence, it is particularly suitable for rapid screening and diagnosis in field, greenhouse, and other on-site environments, thus providing an accurate, reliable, and easily deployable complete solution for the early detection and control of ToBRFV. Attached Figure Description

[0015] Figure 1 It is a negative test using the ToBRFV-ORF1-P2+2F1 probe combination; Figure 2It is a positive test for the ToBRFV-ORF1-P2 probe combination (downstream screening); Figure 3 It is a positive test for the ToBRFV-ORF1-P2 probe combination (upstream screening); Figure 4 It is a negative test using the ToBRFV-ORF1-P3+3F1 probe combination; Figure 5 It is a positive test for the ToBRFV-ORF1-P3 probe combination (downstream screening); Figure 6 It is a positive test for the ToBRFV-ORF1-P3 probe combination (upstream screening); Figure 7 It is a negative test of the ToBRFV-ORF1-P4+4F1 probe combination (1); Figure 8 It is a negative test of the ToBRFV-ORF1-P4+4F1 probe combination (2); Figure 9 It is a positive test for the ToBRFV-ORF1-P4 probe combination (downstream screening); Figure 10 It is a positive test for the ToBRFV-ORF1-P4 probe combination (upstream screening); Figure 11 This is the optimal probe sensitivity verification (routine). Figure 12 Suboptimal probe sensitivity verification (routine); Figure 13 This is a verification of the second-best probe sensitivity (mix well); Figure 14 This is the optimal probe sensitivity verification (mix well); Figure 15 These are test results graphs for different pretreatment and grinding methods; Figure 16 The graph shows the test results for different types of pretreatment solutions (sample: pretreatment solution = 1:10). Figure 17 The graph shows the test results for different types of pretreatment solutions (sample: sample pretreatment solution = 1:20). Figure 18 This is a graph showing the test results for different ratios of nucleic acid release agents and a sample loading volume of 10 μL. Figure 19 This is a graph showing the test results for different ratios of nucleic acid release agents and a sample loading volume of 50 μL. Detailed Implementation

[0016] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to 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.

[0017] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0018] Example 1 1. Primer and probe design Tomato brown rugose fruit virus (ToBRFV) comprises a 74-nt 5'-UTR, a 201-nt 3'-UTR, and four ORFs. ORF1 and ORF2 encode proteins related to viral replication. ORF1 encodes a 126 kD methyltransferase and a helicase (p126). ORF1 and ORF2 can be translated together into a 183 kD RNA-dependent RNA polymerase (RdRp). ORF3 encodes a movement protein (MP), and ORF4 encodes a capsid protein (CP). Based on the above sequence analysis, within the conserved region of ORF1, according to primer and probe design principles and sequence specificity requirements, six fluorescent probe positions and primer combinations were initially selected. After extensive comparison and confirmation, and considering factors such as sequence specificity, uniformity of base distribution, and GC content, three probes and matching primers were finally synthesized (see Tables 1 and 2), totaling more than 30 primer and probe combinations, for subsequent project screening.

[0019] Table 1 Probe Sequences

[0020] Table 2 Primer Sequences

[0021] 2. Primer and probe screening 2.1 Basic Process of Primer and Probe Screening in the Early Stage (1) Perform all negative verification to screen out negative and normal primer-probe combinations; exclude primer-probe combinations that cause false positives due to non-specific amplification.

[0022] (2) Use synthetic plasmids to perform positive experiments, and comprehensively consider the combination of fluorescence value and TT value to screen for the best performance.

[0023] (3) Verify the sensitivity of primers and probes and screen out primer-probe combinations that meet the performance requirements.

[0024] Table 3 Basic Process of Primer and Probe Screening

[0025] Table 4. Standard Procedures

[0026] Table 5 Mixing Procedure

[0027] Table 6 RNA Isothermal Rapid Amplification Kit (Fluorescent Type) - II Reagent System

[0028] 2.2 Screening of Introducing and Probing Combinations 2.2.1 First group of probe combinations (1) Negative test Reagent: RNA isothermal rapid amplification kit (fluorescent type)-II Template: Water Program: Mixing program The primer-probe combination ToBRFV-ORF1-P2+2F1 used in this screening experiment showed negative results for all normal results (see [link]). Figure 1 ).

[0029] (2) Positive test Reagent: RNA isothermal rapid amplification kit (fluorescent type)-II Template: ToBRFV-ORF1 plasmid Program: Mixing program The results of this screening were as follows: The optimal combination ToBRFV-ORF1-P2 +2R1 +2F3 was selected. Based on development experience, this combination has a low fluorescence value, so further screening and comparison with subsequent results will be conducted.

[0030] Table 7. ToBRFV-ORF1-P2 Probe Combination (Downstream Screening) Positive Test TT Values

[0031] Table 8. TT values ​​for positive test of ToBRFV-ORF1-P2 (upstream screening) priming combination

[0032] 2.2.2 Second set of probe combinations (1) Negative test Reagent: RNA isothermal rapid amplification kit (fluorescent type)-II Template: Water Program: Mixing program The primer-probe combination ToBRFV-ORF1-P3+3F1 used in this screening experiment showed negative results for all normal results (see [link]). Figure 4 ).

[0033] (2) Positive test Reagent: RNA isothermal rapid amplification kit (fluorescent type)-II Template: ToBRFV-ORF1 plasmid Program: Mixing program The results of this screening were as follows: ToBRFV-ORF1-P3+3F3+3R6 showed the best results.

[0034] Table 9. ToBRFV-ORF1-P3 Probe Combination (Downstream Screening) Positive Test TT Values

[0035] Table 10. TT values ​​for positive test of ToBRFV-ORF1-P3 (upstream screening) priming combination

[0036] 2.2.3 Third group of probe combinations (1) Negative test Reagent: RNA isothermal rapid amplification kit (fluorescent type)-II Template: Water Program: Mixing program In this screening experiment, the primer-probe combination ToBRFV-ORF1-P4+4F1 showed some negative results, and the abnormal combinations were discarded (see...). Figure 7 , 8 ).

[0037] (2) Positive test Reagent: RNA isothermal rapid amplification kit (fluorescent type)-II Template: ToBRFV-ORF1 plasmid Program: Mixing program Based on a comprehensive evaluation of both TT and fluorescence values, ToBRFV-ORF1-P4+4F4+4R1 yielded the best results.

[0038] Table 11. TT values ​​for positive test of ToBRFV-ORF1-P4 probe combination

[0039] 2.3 Summary of Primer and Probe Screening By combining upstream and downstream approaches, negative and positive tests were conducted, and the optimal probe results were ultimately selected as follows: (1) The fluorescence value of the P2 combination is generally low. As a candidate for the project, other combinations should be given priority.

[0040] (2) Optimal probe: ToBRFV-ORF1-P3 (6 pmol) + ToBRFV-ORF1-3F3 (20 pmol) + ToBRFV-ORF1-3R6 (20 pmol) (3) Suboptimal probe: ToBRFV-ORF1-P4 (6 pmol) + ToBRFV-ORF1-4R1 (20 pmol) + ToBRFV-ORF1-4F4 (20 pmol) (4) To facilitate subsequent multiple detection, the following probe combinations are provided as alternative options: ToBRFV-ORF1-P3+ToBRFV-ORF1-3F1+ToBRFV-ORF1-3R6 ToBRFV-ORF1-P3+ToBRFV-ORF1-3F3+ToBRFV-ORF1-3R3 ToBRFV-ORF1-P3+ToBRFV-ORF1-3F2+ToBRFV-ORF1-3R3 2.4 Sensitivity Verification 2.4.1 Sensitivity Verification 1 Reagent: RNA isothermal rapid amplification kit (fluorescent type)-II Primer probes: optimal probes, suboptimal probes Template: ToBRFV-ORF1 plasmid Procedure: Standard procedure The optimal probe and the second-best probe were both able to stably detect 0.1 fg / μL with conventional procedures, while the probes were unable to detect 0.01 fg / μL.

[0041] Table 12 Sensitivity Verification (Routine)

[0042] 2.4.2 Sensitivity Verification 2 Reagent: RNA isothermal rapid amplification kit (fluorescent type)-II Primers and probes: Optimal probes Template: ToBRFV-ORF1 plasmid Program: Mixing program Both primer-probe combinations could be detected at 0.1 fg / μL (24.5 copies / μL) and 0.01 fg / μL (2.45 copies / μL) using the mixing procedure.

[0043] Note: Copy number is calculated based on plasmid concentration and fragment size. The calculation formula is as follows: (6.02×10^23) × (ng / μL × 10^-9) / (DNA length × 660) = copies / μL The plasmid length (including the target gene and vector length) is approximately 3723 bp.

[0044] Table 13 Sensitivity Verification (Mixed)

[0045] 2.4.3 Summary of Sensitivity Verification Optimal induction: ToBRFV-ORF1-P3 (6 pmol) + ToBRFV-ORF1-3F3 (20 pmol) + ToBRFV-ORF1-3R6 (20 pmol) Suboptimal probe: ToBRFV-ORF1-P4 (6 pmol) + ToBRFV-ORF1-4R1 (20 pmol) + ToBRFV-ORF1-4F4 (20 pmol) The optimal and second-optimal probes showed stable detection of the ToBRFV-ORF1 plasmid at 0.1 fg / μL (24.5 copies / μL) and were detectable at 0.01 fg / μL (2.45 copies / μL). Preliminary screening has reached the expected level for laboratory development.

[0046] Example 2: MIRA-based fluorescence detection kit and detection method 1. Development of positive leaf treatment methods 1 - Pretreatment and grinding method testing Extraction reagents: Magnetic bead-based viral RNA extraction kit, nucleic acid release agent (RNA-II type) Reagent: Fluorescent reagent for isothermal detection of tomato brown wrinkled fruit virus Template: 0.05g of positive leaf Sample loading volume: 5 μL Procedure: Standard procedure Table 14 Pretreatment and Grinding Method Tests

[0047] Results: Comparing scheme 5 with the other schemes, water pretreatment resulted in better nucleic acid extraction. Comparing schemes 2 and 4, manual shaking for 30 seconds and grinding yielded consistent extraction results. Considering the convenience of on-site operation, it was determined that manual shaking for 30 seconds would be used for sample processing in the future.

[0048] Note: The nucleic acid release agent (RNA-II type) was purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd.

[0049] 2. Test of positive leaf treatment method 2 - Pretreatment solution test Extraction reagents: Nucleic acid release agent (RNA-II type), sample pretreatment solution Reagent: Fluorescent reagent for isothermal detection of tomato brown wrinkled fruit virus Template: Nucleic acid extracted from positive samples, nucleic acid:releasing agent = 1:4 Procedure: Standard procedure Table 15 Pretreatment and Grinding Method Tests

[0050] Results: Comparing schemes 1 and 2, the sample pretreatment solution showed better extraction performance. Comparing schemes 2 and 4, considering all factors, the optimal pretreatment scheme was sample:sample pretreatment solution = 1:10.

[0051] Note: Sample pretreatment solution: purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd.

[0052] 3. Test of positive leaf treatment method: 3-Nucleic acid release agent and sample loading amount test. Extraction reagent: Nucleic acid release agent (RNA-II type) Reagent: Fluorescent reagent for isothermal detection of tomato brown wrinkled fruit virus Template: Nucleic acid extraction from positive samples, loading volume: 10μL, 50μL Procedure: Standard procedure Table 16 Nucleic Acid Release Agent and Sample Loading Volume Test

[0053] Results: When loading samples at 10 μL, schemes 2 and 3 showed the best extraction results. When loading samples at 50 μL, schemes 3 and 4 showed the best extraction results. Considering the TT value and operating procedures, scheme 3 (sample leachate: release agent = 1:10) and 50 μL loading were determined to be the optimal extraction scheme.

[0054] 4. Summary Based on actual positive samples, a nucleic acid release agent (RNA-II type) was developed. After testing the pretreatment method, nucleic acid release agent ratio, sample loading volume, and operational procedures, the final extraction scheme for isothermal detection of tomato brown wrinkled fruit virus was confirmed as follows: Table 17 Final Extraction Scheme for Isothermal Detection of Tomato Brown Wrinkled Fruit Virus

[0055] 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. Primers and probes for the detection of Tomato brown rugose fruit virus based on the MIRA technology, characterized in that: The primers and probes include an upstream primer ToBRFV-ORF1-3F3, a downstream primer ToBRFV-ORF1-3R6, and a probe ToBRFV-ORF1-P3; the nucleotide sequence of the upstream primer ToBRFV-ORF1-3F3 is shown in SEQ ID NO:15, the nucleotide sequence of the downstream primer ToBRFV-ORF1-3R6 is shown in SEQ ID NO:21, and the nucleotide sequence of the probe ToBRFV-ORF1-P3 is shown in SEQ ID NO:

2.

2. A kit for the detection of Tomato brown rugose fruit virus based on the MIRA technology, characterized in that: The kit contains the primers and probes as described in claim 1.

3. The kit of claim 2, wherein: The kit also contains A buffer, water, B buffer, and template.

4. A method for detecting Tomato brown rugose fruit virus based on the MIRA technology, characterized in that: Specifically, the following steps are included: S1: Sample processing: Take the plant tissue sample to be tested, add the sample pretreatment solution, mix well and obtain the sample extract. S2: Nucleic acid release: Take the sample extract, add it to the nucleic acid release agent, let it stand, and obtain the nucleic acid solution to be tested; S3: MIRA isothermal amplification reaction: The nucleic acid solution to be tested, the primers and probes as described in claim 1, and other components required for the MIRA isothermal amplification reaction are mixed to form a reaction system, and an isothermal amplification reaction is performed; S4: Result Interpretation: Determine whether the sample contains tomato brown wrinkled fruit virus by detecting the real-time fluorescence signal during the amplification process.

5. The method of claim 4, wherein: In step S1, the plant tissue sample is a tomato leaf, the mass ratio of the plant tissue sample to the sample pretreatment solution is 1:10, and the mixing process is manual shaking for 30 seconds.

6. The method of claim 4, wherein: In step S2, the volume ratio of the sample leachate to the nucleic acid release agent is 1:10, and the nucleic acid release agent is an RNA-II type nucleic acid release agent; the standing time is 5 minutes.

7. The method of claim 4, wherein: In step S3, the amount of the nucleic acid solution to be tested loaded into the reaction system is 50 μL.

8. The method of claim 4, wherein: In step S3, the isothermal amplification reaction is carried out at a temperature of 42°C for 20 minutes.

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