Primer and method for detecting southern tomato virus
By designing STV148 and STV287 primers for one-step RT-PCR amplification, the problem of rapid and accurate virus detection in southern tomatoes was solved, achieving efficient and reliable virus detection, which is suitable for plant virus detection and quarantine.
Patent Information
- Application Number
- CN202511229240.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2025-11-28
AI Technical Summary
Existing technologies are insufficient for the rapid and accurate detection of viruses in southern tomatoes, especially in cases of multiple viral infections, leading to difficulties in disease monitoring and control.
Two primer pairs, STV148 and STV287, were designed for one-step RT-PCR amplification, amplifying specific fragments of 148bp and 287bp, respectively. Combined with electrophoresis detection, rapid and accurate virus detection was achieved.
It has achieved highly sensitive and specific virus detection in southern tomatoes, shortening the detection time, reducing operational risks, and improving the accuracy and efficiency of detection.
Smart Images
Figure CN121023097A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biological detection technology for plant viruses, and in particular to a primer and method for detecting Southern Tomato Virus. Background Technology
[0002] Southern tomato virus (STV) is a virus discovered in tomato crops in recent years. In most cases, this disease is caused by a combination of 2-3 viruses (Yang Cuirong et al., 1992), and may be related to symptoms such as chlorosis, yellowing, senescence, and smaller fruit size in tomatoes. The disease was first discovered in California, USA in 1984, followed by similar symptoms in Mexico and Mississippi, USA, and was named in 2005. In my country, STV-infected plants were first discovered in Xinjiang in 2011 (Su Haidi, 2013), and STV has since been detected in other parts of the country.
[0003] STV belongs to the genus Amalgavirus in the family Amalgaviridae. Its genome consists of double-stranded RNA (dsRNA) and is approximately 3.5 kb in length. Its genome structure is highly similar to viruses in the family Totiviridae. The positive strand contains two partially overlapping open reading frames (ORFs), each encoding a major functional protein. Currently, complete viral particle structures of STV have not been observed. This virus exhibits strict species-to-species transmission characteristics and cannot be transmitted through sap-rubbed inoculation, grafting, or other mechanical means. Given the potential for serious harm caused by STV, especially when co-infecting with other viruses, establishing rapid and highly sensitive detection technologies for its monitoring and control is of great significance.
[0004] Molecular biology detection works by specifically recognizing the nucleic acids (DNA or RNA) of viruses to determine their presence. Based on the high specificity of the viral genome, this method offers advantages such as high detection sensitivity, high throughput, and ease of operation, and has become a core technology for plant virus detection and quarantine. It mainly includes polymerase chain reaction (PCR), double-stranded RNA electrophoresis, and molecular hybridization. For STVs (RNA viruses), a one-step RT-PCR method is typically used for detection. This technique integrates reverse transcription and PCR amplification into a single reaction system, which not only shortens the detection time but also reduces the risk of contamination from tube opening (Zhou, 2003). Summary of the Invention
[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide primers and methods for detecting Southern Tomato Virus, aiming to provide a more accurate method for detecting whether plants or seeds carry Southern Tomato Virus.
[0006] The technical solution of the present invention is as follows:
[0007] In a first aspect, the present invention provides primers for detecting Southern Tomato Virus, said primers comprising: STV148 or STV287;
[0008] The sequence of the upstream primer of STV148 is shown in SEQ ID NO.1, and the sequence of the downstream primer of STV148 is shown in SEQ ID NO.2.
[0009] The sequence of the upstream primer of STV287 is shown in SEQ ID NO.3, and the sequence of the downstream primer of STV287 is shown in SEQ ID NO.4.
[0010] In a second aspect, the present invention provides the application of the above-described primers in the detection of Southern Tomato Virus.
[0011] A third aspect of the present invention provides a kit for detecting Southern Tomato Virus, the kit comprising the primers described above.
[0012] A fourth aspect of the present invention provides a method for detecting Southern Tomato Virus, the method comprising the following steps:
[0013] Total RNA was extracted from tomato leaves to be tested;
[0014] Using the total RNA from the leaves of the southern tomato to be tested as a template, one-step RT-PCR amplification was performed using the STV148 or STV287 pair to obtain the first amplification product and the second amplification product, which were then detected.
[0015] If the first amplification product is a 148bp fragment or the second amplification product is a 287bp fragment, then the tomato being tested is infected with Southern Tomato Virus.
[0016] If the first amplification product does not detect a 148bp fragment or the second amplification product does not detect a 287bp fragment, then the tomato being tested is not infected with Southern Tomato Virus.
[0017] Preferably, the reaction system for the one-step RT-PCR amplification is as follows: 2 μL total RNA extract, 12.5 μL 2×SuperRTOneStep Buffer, 0.5 μL SuperRT OneStep EnzymeMix, 1 μL each of upstream and downstream primers, and 8 μL RNase-Free Water.
[0018] Preferably, the reaction conditions for the one-step RT-PCR amplification are as follows: preheat PCR to 45°C, reverse transcription at 45°C for 30 min, pre-denaturation at 95°C for 2 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 35 cycles, and final extension at 72°C for 5 min.
[0019] Preferably, the first amplification product or the second amplification product is detected by electrophoresis using 2% agarose gel or 8% polyacrylamide gel.
[0020] The present invention has the following beneficial effects:
[0021] 1. The primers for detecting Southern Tomato Virus provided by this invention are primer sequences developed based on previously reported primers that identified the specific sequence of Southern Tomato Virus. These primer sequences have smaller fragments and more markers, exhibiting high specificity and accuracy. 2. Any pair of primers provided by this invention can be used to identify whether tomato seeds or plants are infected with Southern Tomato Virus. 3. This invention designs two pairs of primers for detecting Southern Tomato Virus. If RT-PCR amplification is performed using these two pairs of primers, fragment lengths of 148bp and 287bp can be obtained, consistent with the predicted fragments. No further sequencing is required, thus confirming that the sample is infected with Southern Tomato Virus. This method not only features accuracy, reliability, and high specificity, but also completes one-step RT-PCR reverse transcription and PCR amplification within a single reaction tube and system, significantly reducing detection time and saving time and effort. Attached Figure Description
[0022] Figure 1 The images shown are electrophoresis diagrams of positive samples and negative controls amplified with STV148 and STV287 respectively in this embodiment of the invention (Note: DNA Marker is 2000bp, 1-4 are positive samples to be tested, 5-8 are negative controls). Detailed Implementation
[0023] This invention provides primers and a method for detecting Southern Tomato Virus. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.
[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
[0025] This invention provides primers for detecting Southern Tomato Virus, as shown in Table 1 below. The primers include STV148 or STV287.
[0026] The sequence of the upstream primer of STV148 is shown in SEQ ID NO.1, and the sequence of the downstream primer of STV148 is shown in SEQ ID NO.2.
[0027] The sequence of the upstream primer of STV287 is shown in SEQ ID NO.3, and the sequence of the downstream primer of STV287 is shown in SEQ ID NO.4.
[0028] Table 1
[0029]
[0030] The primers were synthesized at Hangzhou Shangyasai Biotechnology Co., Ltd.
[0031] This invention utilizes the reported Southern Tomato Virus-specific primers STVdF / STVdR to perform RT-PCR detection on Southern Tomato Virus-positive samples. The amplified products are then TA cloned and sequenced. The sequencing results are analyzed using Blastx from the NCBI database to identify Southern Tomato Virus-specific sequences. Two pairs of high-efficiency primers, STV148F / STV148R and STV287F / STV287R, are designed using Primer5 software. The objective of this invention is to design more specific primers based on the conserved sequences of Southern Tomato Virus, enabling more accurate detection of whether plants or seeds carry the virus. Furthermore, these primer fragments are shorter, have higher amplification efficiency, and better specificity.
[0032] In some implementations, the above-described primers are provided for use in detecting Southern Tomato Virus.
[0033] The two primer pairs provided in this invention can specifically amplify the Southern Tomato Virus, which can identify whether tomato seeds or plants are infected with the Southern Tomato Virus. The RT-PCR products can be detected by electrophoresis, with clear amplification bands. The two primers can be repeated, resulting in high accuracy.
[0034] In some embodiments, a kit for detecting Southern Tomato Virus is provided, the kit comprising the primers described above.
[0035] Specifically, the kit includes 2×SuperRT OneStep Buffer, SuperRT OneStep EnzymeMix, the aforementioned upstream and downstream primers, and RNase-Free Water.
[0036] This invention provides a method for detecting Southern Tomato Virus, the method comprising the following steps:
[0037] Total RNA was extracted from tomato leaves to be tested;
[0038] Using the total RNA from the leaves of the southern tomato to be tested as a template, one-step RT-PCR amplification was performed using the STV148 or STV287 pair to obtain the first amplification product and the second amplification product, which were then detected.
[0039] If the first amplification product is a 148bp fragment or the second amplification product is a 287bp fragment, then the tomato being tested is infected with Southern Tomato Virus.
[0040] If the first amplification product does not detect a 148bp fragment or the second amplification product does not detect a 287bp fragment, then the tomato being tested is not infected with Southern Tomato Virus.
[0041] This invention designs two pairs of primers for detecting Southern Tomato Virus. If RT-PCR amplification is performed using these two primer pairs, fragment lengths of 148 bp and 287 bp can be obtained, consistent with the predicted fragments. Without further sequencing, it can be confirmed that the sample is infected with Southern Tomato Virus. This method not only features accuracy, reliability, and high specificity, but also completes one-step RT-PCR reverse transcription and PCR amplification in a single reaction tube and system, significantly reducing detection time and saving time and effort.
[0042] The following detailed description uses specific examples. The two pairs of primers and methods provided by this invention were used to detect STV-positive plants, blank plants, and healthy plants.
[0043] Example
[0044] Total RNA extraction from plant samples
[0045] Positive samples were selected from tomato plants exhibiting slow growth, slight yellowing or curling of leaves, and those confirmed to carry Southern Tomato Virus by metagenomic sequencing. Healthy tomato plants served as negative controls. Total RNA was extracted from tomato leaves using the Novizan RNA extraction kit.
[0046] One-step RT-PCR amplification
[0047] The RT-PCR reaction system consisted of: 2 μL total RNA, 12.5 μL 2×SuperRT OneStep Buffer, 0.5 μL SuperRT OneStep EnzymeMix, 1 μL each of the upstream and downstream primers, and 8 μL of LNase-Free Water to bring the total volume to 25 μL.
[0048] The RT-PCR reaction conditions were as follows: preheat PCR to 45℃, reverse transcription at 45℃ for 30 min, PCR pre-denaturation at 95℃ for 2 min, denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, 35 cycles, and final extension at 72℃ for 5 min.
[0049] Electrophoretic detection
[0050] Take 5 μL of the above RT-PCR amplification product, add 3 μL of loading buffer (6× loading buffer), and perform detection using a 2% agarose gel with a 2000bp loading marker. Observe the results using a gel imaging system.
[0051] Analysis of test results
[0052] Test results as follows Figure 1 As shown, the negative control did not amplify the target fragment; the positive control used primers...
[0053] STV148 amplified a target fragment of approximately 148 bp, and primer STV287 amplified a target fragment of approximately 287 bp. The test sample also amplified a fragment of the same size as the positive sample.
[0054] sequencing
[0055] To further verify the reliability of the final results, the target product amplified by primers STV148 and STV287 was sequenced by Hangzhou Shangyasai Biotechnology Co., Ltd. The obtained target product sequence was compared and analyzed using BLAST in the NCBI database. The comparison revealed that the target sequence was 100% consistent with the Southern tomato virus (STV) sequence number MG808383.1 reported in GenBank. Furthermore, the corresponding vector-free sequences SEQ ID NO.5 and SEQ ID NO.6 were identical to the primer amplification fragments 148bp (SEQ ID NO.5) and 287bp (SEQ ID NO.6), respectively, proving that the sample carried Southern tomato virus (its sequence is shown in SEQ ID NO.7).
[0056] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.
Claims
1. Primers for detecting Southern Tomato Virus, characterized in that, The primers include: STV148 or STV287; The sequence of the upstream primer of STV148 is shown in SEQ ID NO.1, and the sequence of the downstream primer of STV148 is shown in SEQ ID NO.
2. The sequence of the upstream primer of STV287 is shown in SEQ ID NO.3, and the sequence of the downstream primer of STV287 is shown in SEQ ID NO.
4.
2. The application of the primer for detecting Southern Tomato Virus as described in claim 1 in the detection of Southern Tomato Virus.
3. A kit for detecting Southern Tomato Virus, characterized in that, The kit includes the primers as described in claim 1.
4. A method for detecting Southern Tomato Virus, characterized in that, The method includes the following steps: Total RNA was extracted from tomato leaves to be tested; Using the total RNA from the leaves of the southern tomato to be tested as a template, one-step RT-PCR amplification was performed using STV148 or STV287 as described in claim 1 to obtain the first amplification product and the second amplification product, which were then detected. If the first amplification product is a 148bp fragment or the second amplification product is a 287bp fragment, then the tomato being tested is infected with Southern Tomato Virus. If the first amplification product does not detect a 148bp fragment or the second amplification product does not detect a 287bp fragment, then the tomato being tested is not infected with Southern Tomato Virus.
5. The method for detecting Southern Tomato Virus according to claim 4, characterized in that, The reaction system for the one-step RT-PCR amplification is as follows: 2 μL total RNA extract, 12.5 μL 2×SuperRT OneStep Buffer, 0.5 μL SuperRT OneStep Enzyme Mix, 1 μL each of the upstream and downstream primers of STV148 or STV287, and 8 μL RNase-Free Water.
6. The method for detecting Southern Tomato Virus according to claim 4, characterized in that, The reaction conditions for the one-step RT-PCR amplification were as follows: preheating the PCR to 45°C, reverse transcription at 45°C for 30 min, pre-denaturation at 95°C for 2 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for 35 cycles, and final extension at 72°C for 5 min.
7. The method for detecting Southern Tomato Virus according to claim 4, characterized in that, The first or second amplification product was detected by electrophoresis using 2% agarose gel or 8% polyacrylamide gel.