Rice virus multiplex RT-PCR detection kit, detection system and application

Abstract

The invention discloses a rice virus multiplex RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit, a detection system and application. In particular discloses a composition for simultaneously detecting three rice viruses RRSV, SRBSDV and RSMV, the composition comprises three primer pairs, and the nucleotide sequences of the three primer pairs are shown as SEQ ID NO: 1-SEQ ID NO: 6. The invention also discloses a multiplex RT-PCR detection method for simultaneously detecting the three rice viruses. The primer and the detection method thereof are sensitive, specific and good in stability, an efficient tool can be provided for early diagnosis of rice virus diseases, pathogen composition of field diseases can be rapidly and clearly determined by simultaneously detecting the three viruses, and precious time is gained for formulating targeted prevention and control measures. The method is easy and convenient to operate, can be completed only through conventional PCR equipment, is suitable for being popularized in primary laboratories and is beneficial to improving the virus monitoring capacity of main rice producing areas in China.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a rice virus multiplex RT-PCR detection kit, detection system, and applications. Background Technology

[0003] The key to controlling rice viral diseases lies in early diagnosis and accurate monitoring. Traditional virus detection methods mainly include electron microscopy, serological testing (such as ELISA), and molecular biology techniques (such as RT-PCR). While electron microscopy provides intuitive results, the equipment is expensive and the operation is complex, hindering its widespread adoption. ELISA relies on antibody quality and specificity and has limited sensitivity, failing to meet the detection needs of samples with low viral loads. Singleton RT-PCR technology has become a commonly used method for virus detection due to its high sensitivity and specificity; however, it can only detect one virus per reaction, resulting in lower efficiency when facing complex mixed infections in the field.

[0004] In recent years, multiplex RT-PCR technology has shown great potential in the field of plant virus detection due to its high efficiency, cost-effectiveness, and high throughput. By optimizing primer design and reaction conditions, this technology can simultaneously amplify specific fragments of multiple viruses in a single tube, significantly improving detection efficiency. Currently, multiplex RT-PCR has been successfully applied to the detection of viruses in various crops, including citrus and potato viruses, but its application in rice virus detection is still in the development stage.

[0005] Rice ragged stunt virus (RRSV) infection can lead to serrated leaf margins and stunted plants; Southern rice black-streaked dwarf virus (SRBSDV) causes nodular growths and black streaks on stem nodes, affecting not only water and nutrient transport but also the appearance and quality of rice; Rice stripe mosaic virus (RSMV) manifests as striped chlorosis on leaves and stunted plant growth, reduced tillering, and decreased effective panicle number, posing a serious threat to rice yield. These three viruses often occur together in the same region, increasing the complexity of control and posing significant challenges to field diagnosis and control.

[0006] Therefore, establishing an efficient and specific multiplex RT-PCR detection system, through primer design, reaction condition optimization, and field validation, to achieve simultaneous detection of RRSV, SRBSDV, and RSMV, will provide important technical support for early monitoring, epidemic warning, and integrated control of rice viral diseases. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to sensitively, specifically, and efficiently detect multiple rice viruses simultaneously. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0008] To address the aforementioned technical problems, the present invention first provides a composition comprising three primer pairs, the nucleotide sequences of which may be as shown in SEQ ID NO:1-SEQ ID NO:6.

[0009] The primer composition can be a composition for the simultaneous detection of three rice viruses.

[0010] The three rice viruses mentioned are rice ragged stunt virus (RRSV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV).

[0011] The three primer pairs are used to detect the three rice viruses, respectively: The primer pair used to detect rice spur dwarf virus (RRSV) consists of two single-stranded DNA molecules as shown in SEQ ID NO:1 and SEQ ID NO:2; The primer pair used to detect Southern Rice Black-Streaked Dwarf Virus (SRBSDV) consists of two single-stranded DNA molecules as shown in SEQ ID NO:3 and SEQ ID NO:4. The primer pair used to detect rice stripe mosaic virus (RSMV) consists of two single-stranded DNA molecules, as shown in SEQ ID NO:5 and SEQ ID NO:6.

[0012] The present invention also provides a kit that may include the composition.

[0013] The kit can be used to simultaneously detect the three rice viruses described herein.

[0014] Furthermore, the kit may also include reagents required for reverse transcription PCR (RT-PCR) detection.

[0015] Furthermore, the kit may also include RNA extraction reagents, RNA purification reagents, reverse transcriptase, RNase inhibitors, reverse transcription buffer, DNA polymerases (such as Taq DNA polymerase, Tth DNA polymerase, Vent DNA polymerase, and Pfu DNA polymerase), dNTPs, and Mg2+.2+ One or more of the following: solution (such as MgSO4 or MgCl2 solution), PCR buffer (such as Tris-HCl), positive control, negative control, and electrophoresis analysis reagent.

[0016] Furthermore, the test samples for the kit include, but are not limited to, plant samples (such as plants, plant tissues or plant seeds), microbial samples (such as plant pathogenic fungi) and environmental samples (such as water, soil, air and objects).

[0017] Furthermore, the plant sample may be a sample of a rice species (such as rice grains, field rice, rice seeds, etc.).

[0018] The various reagent components of the kit may be present in separate containers, or may be pre-assembled into a reagent mixture, either wholly or partially.

[0019] The components of the kit may be provided in solution form, such as an aqueous solution. When present in aqueous solution, the concentration or content of these components can be readily determined by those skilled in the art according to different needs. For example, for storage purposes, the components may be present at a higher concentration, which can be reduced to the working concentration by diluting the higher concentration solution when in operation or for use.

[0020] Furthermore, the kit may also include a readable carrier describing the method of the present invention for simultaneously detecting three rice viruses. The readable carrier may be a kit instruction manual (e.g., a printed instruction manual) on which information is recorded (e.g., a floppy disk, CD, etc.) regarding the practice of the method of the present invention.

[0021] The kit described in this article may be a detection kit based on multiplex RT-PCR.

[0022] The present invention also provides a DNA chip, which may include the composition.

[0023] The carriers used in the DNA chips described in this article may include glass chips, silicon chips, membrane chips (such as nitrocellulose membranes and nylon membranes), and ceramic chips, etc.

[0024] The DNA chip described herein can be fabricated using methods known to those skilled in the art. For example, the primers of this invention can be spotted onto the chip carrier using a fully automated spotting instrument. After spotting, the DNA chip is placed in a desiccator and kept at room temperature in the dark to allow the primers and the chip to covalently bind, thus forming a DNA chip that enables parallel detection.

[0025] The present invention also provides the application of the composition, the kit, or the DNA chip in the detection of rice viruses, including rice serrated leaf dwarf virus, southern rice black-streaked dwarf virus, and rice stripe mosaic virus.

[0026] The present invention also provides the use of the composition in the preparation of products for detecting the rice virus described herein.

[0027] The products described in this article can be selected from reagents, kits, chips, test strips, and test cards.

[0028] The present invention also provides a method for simultaneously detecting three rice viruses, the method comprising the following steps: A1) Extract total RNA from the sample to be tested; A2) Using the total RNA as a template, perform multiplex reverse transcription PCR (multiplex RT-PCR) using the composition, kit, or DNA chip described herein, and determine the presence of three rice viruses in the sample based on the PCR results; The three rice viruses are rice serrated leaf dwarf virus, southern rice black-streaked dwarf virus, and rice stripe mosaic virus.

[0029] In the above method, the annealing temperature of the multiplex reverse transcription PCR can be 55°C.

[0030] In the above method, the reaction conditions for the multiplex reverse transcription PCR can be as follows: reverse transcription at 50℃ for 30 min; pre-denaturation at 94℃ for 2 min; followed by 35 cycles of amplification: 94℃ for 30 s, 55℃ for 30 s, 72℃ for 5 min; and finally, final extension at 72℃ for 7 min.

[0031] In the above method, the reaction system of the multiplex reverse transcription PCR may include: the composition described herein, 2×OneStep MIX, RNase-free dH2O, One-Step Enzyme MIX, and total RNA described herein.

[0032] In the above method, the molar ratio of the primers shown in SEQ ID NO:1, 2, 3, 4, 5 and 6 in the multiplex reverse transcription PCR reaction system can be 3:3:2:2:3:3.

[0033] Furthermore, the reaction system (10 μL) for the multiplex reverse transcription PCR can be: 5 μL 2×One Step MIX, 1.6 μL primer mixture, 2.2 μL RNase Free dH2O, 0.2 μL One-Step Enzyme MIX and 1 μL template RNA.

[0034] Furthermore, the primer mixture comprises three pairs of primers with nucleotide sequences as shown in SEQ ID NO:1-SEQ ID NO:6, wherein: The concentrations of both the forward primer RRSV-F2 (SEQ ID NO:1) and the reverse primer RRSV-R2 (SEQ ID NO:2) in the reaction system were 0.3 µM. The concentrations of the forward primer SRBSDV-S10-F2 (SEQ ID NO:3) and the reverse primer SRBSDV-S10-R (SEQ ID NO:4) in the reaction system were both 0.2 µM; The concentrations of the forward primer RSMV-N-F1 (SEQ ID NO:5) and the reverse primer RSMV-N-R1 (SEQ ID NO:6) in the reaction system were both 0.3 µM.

[0035] Furthermore, the situation where the test sample is determined to contain three rice viruses based on PCR results can be as follows: If the amplification product contains a DNA fragment of 394 bp, the sample is considered to contain RRSV; if the amplification product does not contain a DNA fragment of 394 bp, the sample is considered not to contain RRSV.

[0036] If the amplification product contains a DNA fragment of 798 bp, the sample is determined to contain SRBSDV; if the amplification product does not contain a DNA fragment of 798 bp, the sample is determined not to contain SRBSDV.

[0037] If the amplification product contains a DNA fragment of 1006 bp, the sample is considered to contain RSMV; if the amplification product does not contain a DNA fragment of 1006 bp, the sample is considered not to contain RSMV.

[0038] The samples to be tested described in this article include, but are not limited to, plant samples (such as plants, plant tissues or plant seeds), microbial samples (such as plant pathogenic fungi), and environmental samples (such as water, soil, air and objects).

[0039] This article describes multiplex reverse transcription PCR (multiplex RT-PCR), a method that combines reverse transcription (RT) and multiplex polymerase chain reaction (PCR). It utilizes multiple primer pairs to simultaneously reverse transcribe and amplify multiple target RNA sequences, enabling the detection of various RNA molecules. Primer design and method establishment for multiplex RT-PCR are far more complex than for single PCR. It is not simply a matter of combining multiple pairs of specific primers; the composition of the reaction system and reaction conditions require repeated adjustments and refinements to suit the simultaneous amplification of multiple fragments.

[0040] Through extensive and in-depth research, this invention has successfully constructed a highly efficient and specific multiplex RT-PCR detection system capable of simultaneously identifying three important rice viral diseases: rice serrata dwarf virus (RRSV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV).

[0041] By comparing and screening primer types, and systematically optimizing key parameters such as primer concentration, enzyme dosage, and annealing temperature, this system achieved specific amplification of three viruses in a 10 µL reaction system. The target bands (394 bp, 798 bp, and 1006 bp) were clearly distinguishable, and the system exhibited high sensitivity, making it suitable for detecting samples with low viral load. The optimal primer combinations determined through primer screening were: the second set of primers for RRSV (394 bp) (SEQ ID NO:1 and SEQ ID NO:2), the second set of primers for SRBSDV (798 bp) (SEQ ID NO:3 and SEQ ID NO:4), and the first set of primers for RSMV (1006 bp) (SEQ ID NO:5 and SEQ ID NO:6). In a 10 µL reaction system, the optimized key parameters were: primer concentrations of RRSV 0.3 µM, SRBSDV 0.2 µM, and RSMV 0.3 µM; the minimum enzyme volume of 0.2 µL was sufficient for amplification; the annealing temperature was 55℃; and the annealing time was 30 s. Through systematic optimization of reaction conditions, the multiplex RT-PCR reaction system constructed in this invention clarified the types and optimal concentration ratios of primers, the appropriate enzyme concentration, and the optimal annealing temperature. Electrophoretic analysis of the amplified products showed significant differences in the size of the virus-specific bands (100-400 bp intervals), facilitating accurate interpretation.

[0042] Field rice sample testing demonstrated that this reaction system can analyze the infection status of the corresponding virus, effectively distinguish between single and multiple infected samples, and exhibit significant virus-specific amplification. Compared with traditional PCR, this technology significantly improves detection efficiency, reduces reagent consumption and operation time, and is particularly suitable for rapid screening of large-scale field samples, providing reliable technical support for the early diagnosis and precise control of rice viral diseases.

[0043] This invention establishes a multiplex RT-PCR detection method by carefully designing primers targeting three important rice viral diseases: Rice Saw Leaf Dwarf Virus (RRSV), Southern Rice Black-Streaked Dwarf Virus (SRBSDV), and Rice Stripe Mosaic Virus (RSMV) and optimizing reaction conditions. This method simultaneously amplifies specific conserved fragments of the three rice viral diseases, enabling simultaneous detection of all three. The method is sensitive, specific, and stable, demonstrating three key values ​​in practical applications: First, it provides an efficient tool for the early diagnosis of rice viral diseases. Simultaneous detection of three viruses allows for rapid identification of the pathogen composition of diseases in the field, saving valuable time for developing targeted control measures. For example, in field sample testing in South China, this system successfully identified single and mixed infections, providing data support for disease epidemic trend analysis. Second, the technology is highly scalable; by adjusting primer combinations, it can be further integrated to detect more rice viruses (such as RSV or RBSDV) in the future, forming a more comprehensive detection system. Third, the system is easy to operate and can be completed with only conventional PCR equipment, making it suitable for promotion in grassroots laboratories and helping to improve virus monitoring capabilities in my country's main rice-producing areas. Attached Figure Description

[0044] Figure 1 The results of the screening of two sets of primers for RRSV in Example 3 are shown. Among them, 1, 2, 3, 4, 5, 6, and 7 correspond to the virus detection samples Guangxi 1, Guangxi 2, Guangxi 3, Guangxi 4, Guangxi 5, Guangxi 6, and Guangxi 7, respectively.

[0045] Figure 2 The results of screening two sets of primers for SRBSDV in Example 3 are shown. Wherein: 1, 2, 3, 4, 5, 6, and 7 correspond to samples Guangxi 1, Guangxi 2, Guangxi 3, Guangxi 4, Guangxi 5, Guangxi 6, and Guangxi 7, respectively, for virus detection.

[0046] Figure 3 The results of the screening of the two sets of primers for RSMV in Example 3 are shown. Among them, 1, 2, 3, 4, 5, 6, and 7 correspond to the virus detection samples Guangdong 1, Guangdong 2, Guangdong 3, Guangdong 4, Guangdong 5, Guangdong 6, and Guangdong 7, respectively.

[0047] Figure 4 This is a gel electrophoresis of the PCR products amplified by the three rice virus-specific primers in Example 3. Wherein: 1, 3, and 5 correspond to healthy rice, and 2, 4, and 6 correspond to samples Guangxi 1, Guangxi 5, and Guangdong 5, respectively, for virus detection.

[0048] Figure 5 This section describes the optimization of different concentrations of RRSV, SRBSDV, and RSMV primers in Example 3. The concentrations of 0.2, 0.3, 0.4, 0.5, and 0.6 correspond to the primer concentrations used in virus detection.

[0049] Figure 6 This is an optimization of different enzyme concentrations for RRSV, SRBSDV, and RSMV in Example 3. Wherein: 0.2, 0.3, 0.4, 0.5, and 0.6 correspond to the volumes (µL) of enzyme added for virus detection.

[0050] Figure 7 This is an optimization of the different annealing temperatures for RRSV, SRBSDV, and RSMV in Example 3. Wherein: 52, 55, 58, and 61 correspond to different annealing temperatures (°C).

[0051] Figure 8 The results show the sensitivity and stability analysis of the RT-PCR detection in Example 4. Where: 1-5 correspond to the dilution factors of the mixed template, respectively.

[0052] Figure 9 The results are from multiplex RT-PCR detection of field rice samples in Example 5. Wherein: 1: negative control (healthy rice); 2-10: field rice virus samples with mixed infection. Detailed Implementation

[0053] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0055] The 2×One Step MIX in the following examples was purchased from Novizan, product number P612.

[0056] The One-Step Enzyme MIX used in the following examples was purchased from Novizan, product number P612.

[0057] Example 1: Primer Design This invention selects three rice viruses that cause serious damage in rice-growing areas of southern my country—Rice ragged stunt virus (RRSV), Southern rice black-streaked dwarf virus (SRBSDV), and Rice stripe mosaic virus (RSMV)—as targets. For these three rice viruses, the whole genome sequences of the target rice viruses were obtained from the NCBI database, and primers were designed using Primer Premier 5.0 software. Primer parameters were rigorously optimized: the Tm values ​​(Nearest-Neighbor method) of both forward and reverse primers were controlled at 60-62℃, the GC content was 40%-60%, the length was 18-24 bp, and 3' end stability was ensured. Primer specificity was verified by NCBI BLAST whole genome alignment to eliminate the risk of non-specific binding and dimer formation. The verified primers were synthesized by a professional company; specific sequence information is shown in Table 1.

[0058]

[0059] Example 2: Establishment of a multiplex RT-PCR method for simultaneous detection of three rice viruses 1. RNA extraction and detection Total RNA was extracted from the apical 3 cm tissue of rice leaves using the Trizol method. After flash freezing in liquid nitrogen, the samples were ground and lysed in centrifuge tubes containing glass beads, followed by 500 μL of Trizol Reagent. After chloroform extraction, the aqueous phase was separated by centrifugation at 12,000 rpm for 15 min at 4°C. 200 μL of the supernatant was collected, and RNA was precipitated with isopropanol, washed twice with 75% ethanol, and then dissolved in DEPC water. RNA purity (A260 / A280) and concentration were determined using a Nanodrop ONE analyzer. Qualified samples were stored at -20°C for later use.

[0060] 2. Multiplex RT-PCR system This invention establishes a multiplex reverse transcription PCR (mRT-PCR) detection system with a total reaction volume of 10 μL, containing 5 μL of 2×One Step MIX, 0.5 μL each of 0.5 μmol / L forward and reverse primers, 2.5 μL of RNase-free dH2O, 0.5 μL of One-Step Enzyme MIX, and 1 μL of template RNA. The reaction program is as follows: reverse transcription at 50℃ for 30 min; pre-denaturation at 94℃ for 2 min; followed by 35 cycles of amplification (94℃ for 30 s, 58℃ for 30 s, 72℃ for 5 min); and finally, final extension at 72℃ for 7 min. This system enables the simultaneous detection of multiple viruses.

[0061] The multiplex RT-PCR system is shown in Table 2.

[0062]

[0063] The multiplex RT-PCR reaction procedure is shown in Table 3.

[0064]

[0065] 3. Result Interpretation The multiplex RT-PCR method of this invention can simultaneously detect three rice viruses.

[0066] If the amplification product contains a DNA fragment of 394 bp, the sample is considered to contain RRSV; if the amplification product does not contain a DNA fragment of 394 bp, the sample is considered not to contain RRSV.

[0067] If the amplification product contains a DNA fragment of 798 bp, the sample is determined to contain SRBSDV; if the amplification product does not contain a DNA fragment of 798 bp, the sample is determined not to contain SRBSDV.

[0068] If the amplification product contains a DNA fragment of 1006 bp, the sample is considered to contain RSMV; if the amplification product does not contain a DNA fragment of 1006 bp, the sample is considered not to contain RSMV.

[0069] Example 3: Optimization of the multiplex RT-PCR reaction system This embodiment systematically optimizes the established multiplex RT-PCR method capable of simultaneously detecting three rice viruses. By optimizing key parameters such as primer concentration, enzyme concentration, and annealing temperature, a multiplex RT-PCR detection method with good specificity and sensitivity was established. After repeated experimental verification, the optimal conditions for each reaction parameter were finally determined.

[0070] 1. Primer screening This invention designs specific primers based on the conserved sequences of rice saw leaf dwarf virus (RRSV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV) (see Table 1). The experimental materials consist of 14 samples (Guangxi No. 1-7 and Guangdong No. 1-7) collected from major rice-producing areas in Guangxi Zhuang Autonomous Region and Guangdong Province.

[0071] In RRSV (400 bp) detection, the method described in Example 2 was used to perform double primer alignment analysis on samples 1-7 from Guangxi. The first set of primers consisted of RRSV-F1 and RRSV-R1; the second set consisted of RRSV-F2 and RRSV-R2. The results showed ( Figure 1 Both primer sets effectively amplified the target fragment, but the second primer set showed significantly better band clarity and specificity than the first primer set. Sample No. 1 from Guangxi demonstrated the best amplification efficiency and was selected as the standard template for subsequent experiments. These results validate the superior performance of the second primer set in RRSV detection and provide experimental evidence for establishing a standardized detection method.

[0072] For the detection of SRBSDV (approximately 700 bp), the method described in Example 2 was used, and the samples were also from Guangxi province. The first set of primers was SRBSDV-S10-F1 and SRBSDV-S10-R1; the second set of primers was SRBSDV-S10-F2 and SRBSDV-S10-R2. The results are as follows: Figure 2 As shown, all seven samples exhibited valid amplification products. In primer screening, the second set of primers demonstrated superior performance, exhibiting high band clarity and minimal non-specific amplification, and was therefore selected for the experiments. Further analysis of the sample amplification results revealed that sample 5 from Guangxi produced the brightest band with the strongest specificity, and was thus prioritized for subsequent experiments.

[0073] When detecting RSMV (around 1000 bp), the method in Example 2 was used, and the samples used were Guangdong No. 1-7. The first set of primers was RSMV-N-F1 and RSMV-N-R1; the second set of primers was RSMV-N-F2 and RSMV-N-R2. The results are as follows: Figure 3 As shown, the first set of primers could detect samples 1, 3, 5, 6, and 7 from Guangdong, with sample 5 showing the clearest amplification. The second set of primers produced blurry bands and insufficient effective product, only clearly amplifying samples 3 and 5 from Guangdong. Comparing the two sets of primers, the first set exhibited superior specificity, and the band for sample 5 from Guangdong was clearer. Therefore, the first set of RSMV primers and sample 5 from Guangdong were selected as the materials for subsequent experiments.

[0074] The optimal primer combinations for each virus were determined after system optimization, as shown in Table 4.

[0075]

[0076] 2. Specificity verification Samples selected were Guangdong No. 1, Guangxi No. 5, and Guangdong No. 5. Primers were used corresponding to the second group of RRSV (394 bp), the second group of SRBSDV (798 bp), and the first group of RSMV (1006 bp), respectively, i.e., the primers in Table 4. Specificity was verified using the method described in Example 2. Results are as follows: Figure 4 As shown, PCR amplification and 1.0% agarose gel electrophoresis results indicated that all target viruses were successfully detected as specific bands in the corresponding rice samples. The bands were exactly the same size as the expected amplified fragments, and the amplified bands were clear and free of interference. In the negative control experiment, total RNA extracted from healthy rice leaves was used as a template. After the same amplification procedure, no non-specific amplification products were observed, verifying the relative specificity of the designed primer set. Specificity experiments showed that the amplified bands of the three viruses were highly specific, with RRSV, SRBSDV, and RSMV corresponding to specific bands of approximately 400 bp, 700 bp, and 1000 bp, respectively. This demonstrates that the method established in this invention can effectively distinguish between different rice viruses.

[0077] 3. Optimization of primer concentrations for RT-PCR of three rice viruses During the optimization of the reaction system, except for the primer concentration gradient settings, all other components were as described in Example 2: template RNA 10 µL, 2×One Step MIX 5 µL, One-Step Enzyme MIX 0.5 µL, and the annealing temperature was set to 58℃. For the detection primers of three rice viruses—RRSV (second set of primers), SRBSDV (second set of primers), and RSMV (first set of primers)—the samples corresponded to Guangdong No. 1, Guangxi No. 5, and Guangdong No. 5, respectively. Five concentration gradients of 0.2, 0.3, 0.4, 0.5, and 0.6 µM were set for optimization of each primer.

[0078] The results are as follows Figure 5 As shown, gel electrophoresis analysis revealed that the RRSV (394 bp) detection primers exhibited a bright and clear specific amplification band at 0.3 µM, which became blurred with increasing concentration, thus determining 0.3 µM as the optimal concentration. SRBSDV (798 bp) showed effective amplification at all gradients (0.2-0.6 µM) without significant differences, and 0.2 µM was chosen for its economic efficiency. RSMV (1006 bp) showed specific amplification at all test concentrations; although band brightness was not significantly correlated with concentration, 0.3 µM showed slightly better amplification, therefore 0.3 µM was selected as the most suitable concentration.

[0079] 4. Optimization of enzyme concentration Based on the determination of the optimal concentrations of upstream and downstream primers, the dosage of the one-step enzyme MIX was further optimized. The second set of primers for RRSV (394 bp), the second set of primers for SRBSDV (798 bp), and the first set of primers for RSMV (1006 bp) were used, corresponding to samples Guangdong No. 1, Guangxi No. 5, and Guangdong No. 5. Five dosage gradients of 0.2, 0.3, 0.4, 0.5, and 0.6 µL were set, and other conditions were as described in Example 2 for systematic evaluation.

[0080] The results are as follows Figure 6 As shown, enzyme dosages of 0.2–0.6 µL (in increments of 0.1 µL) were tested for the detection of RRSV (394 bp), SRBSDV (798 bp), and RSMV (1006 bp). The results showed that all enzyme concentrations effectively amplified the target fragments, but increasing the enzyme dosage did not significantly enhance amplification efficiency or product yield. Based on considerations of amplification stability and cost-effectiveness, 0.2 µL was ultimately determined to be the optimal enzyme dosage for the detection of the three viruses.

[0081] 5. Optimization of primer annealing temperature A series of gradient annealing temperatures were designed for the RT-PCR reaction, namely 52℃, 55℃, 58℃, and 61℃. The optimal upstream and downstream primer concentrations and enzyme concentrations, as defined above, were selected for the RT-PCR reaction (all other components were as described in Example 2). The RT-PCR reaction program was as follows: reverse transcription at 50℃ for 30 min; pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s; annealing at four gradients for 30 s; extension at 72℃ for 5 min; 35 cycles; final extension at 72℃ for 7 min.

[0082] The results are as follows Figure 7 As shown in the results, the gradient annealing temperature optimization experiment (52-61℃) revealed that: RRSV amplification efficiency showed no significant difference within the test temperature range; SRBSDV exhibited the highest amplification efficiency (brightest 798 bp band) at 55℃, with a weak signal at 52℃, and a slight decrease at 58-61℃; RSMV showed the clearest 1006 bp band at 55℃, with amplification efficiency decreasing as the temperature increased. Considering the amplification performance of the three viruses, 55℃ was ultimately determined to be the optimal annealing temperature, simultaneously ensuring both amplification efficiency and specificity.

[0083] The optimized multiplex RT-PCR reaction system (10 μL) consisted of: 5 μL 2×One Step MIX, 1.6 μL primer mixture, 2.2 μL RNase-free dH2O, 0.2 μL One-Step Enzyme MIX, and 1 μL template RNA.

[0084] The primer mixture contains three pairs of primers with nucleotide sequences as shown in SEQ ID NO:1-SEQ ID NO:6, wherein: The concentrations of both the forward primer RRSV-F2 (SEQ ID NO:1) and the reverse primer RRSV-R2 (SEQ ID NO:2) in the reaction system were 0.3 µM. The concentrations of the forward primer SRBSDV-S10-F2 (SEQ ID NO:3) and the reverse primer SRBSDV-S10-R (SEQ ID NO:4) in the reaction system were both 0.2 µM; The concentrations of the forward primer RSMV-N-F1 (SEQ ID NO:5) and the reverse primer RSMV-N-R1 (SEQ ID NO:6) in the reaction system were both 0.3 µM.

[0085] The optimized multiplex RT-PCR reaction conditions were as follows: reverse transcription at 50℃ for 30 min; pre-denaturation at 94℃ for 2 min; followed by 35 cycles of amplification (94℃ for 30 s, 55℃ for 30 s, 72℃ for 5 min); and finally, final extension at 72℃ for 7 min.

[0086] Example 4: Sensitivity and stability analysis of the multiplex RT-PCR method This embodiment is based on the optimized multiplex RT-PCR reaction system and conditions described in Example 3. It uses a balanced mixture of RRSV (Guangxi No. 4), SRBSDV (Guangxi No. 6), and RSMV (Guangdong No. 5) RNA samples (RNA concentration adjusted to the same level, 400 ng / μL for experiments), combined with a mixture of three specific primers, and is validated at 55℃ annealing temperature and 30 s annealing time. The results show ( Figure 8 Even under conditions of 1-5 fold dilution of the mixed RNA template (corresponding RNA concentrations of 400 ng / μL, 200 ng / μL, 100 ng / μL, 50 ng / μL, and 25 ng / μL, respectively), the one-step multiplex RT-PCR method could still stably and accurately detect the specific amplification bands of the three viruses, and the bands showed significant differences in size, making them easy to identify. This result fully demonstrates that the one-step multiplex RT-PCR detection system established in this invention has good sensitivity and stability, fully meeting experimental expectations.

[0087] Example 5: Field detection using multiplex RT-PCR method The multiplex RT-PCR reaction system established above (i.e., the optimized multiplex RT-PCR reaction system and reaction conditions in Example 3) was used to test 10 rice field samples from South China. The results are as follows: Figure 9 As shown, nine rice samples were infected with the virus. The first sample was healthy rice. The second and third samples were infected with a combination of three viruses: RRSV, SRBSDV, and RSMV. The fourth, fifth, and sixth samples were infected with a single virus: RRSV, SRBSDV, and RSMV, respectively. The seventh sample was infected with a combination of RRSV and SRBSDV. The eighth and tenth samples were infected with a combination of RRSV and RSMV. The ninth sample was infected with a single virus: RSMV.

[0088] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A composition, characterized in that, The composition comprises three primer pairs, the nucleotide sequences of which are shown in SEQ ID NO:1-SEQ ID NO:

6.

2. A reagent kit, characterized in that, The kit comprises the composition of claim 1.

3. A DNA chip, characterized in that, The DNA chip comprises the composition of claim 1.

4. The application of the composition of claim 1, the kit of claim 2, or the DNA chip of claim 3 in the detection of rice viruses, wherein the rice viruses include rice serrated leaf dwarf virus, southern rice black-streaked dwarf virus, and rice stripe mosaic virus.

5. The use of the composition of claim 1 in the preparation of a product for detecting the rice virus of claim 4.

6. A method for simultaneously detecting three rice viruses, characterized in that, The method includes the following steps: A1) Extract total RNA from the sample to be tested; A2) Using the total RNA as a template, perform multiple reverse transcription PCR using the composition of claim 1, the kit of claim 2, or the DNA chip of claim 3, and determine the presence of three rice viruses in the sample based on the PCR results. The three rice viruses are rice serrated leaf dwarf virus, southern rice black-streaked dwarf virus, and rice stripe mosaic virus.

7. The method according to claim 6, characterized in that, The annealing temperature for the multiplex reverse transcription PCR is 55°C.

8. The method according to claim 6 or 7, characterized in that, The reaction conditions for the multiplex reverse transcription PCR were as follows: reverse transcription at 50℃ for 30 min; pre-denaturation at 94℃ for 2 min; followed by 35 cycles of amplification: 94℃ for 30 s, 55℃ for 30 s, 72℃ for 5 min; and finally, extension at 72℃ for 7 min.

9. The method according to any one of claims 6-8, characterized in that, The reaction system of the multiplex reverse transcription PCR includes: the composition of claim 1, 2×One Step MIX, RNase Free dH2O, One-Step Enzyme MIX, and total RNA as described in claim 6.

10. The method according to any one of claims 6-9, characterized in that, In the reaction system of the multiplex reverse transcription PCR, the molar ratio of the primers shown in SEQ ID NO:1, 2, 3, 4, 5 and 6 is 3:3:2:2:3:3.

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