A primer and probe combination, kit and application thereof for detecting Fusarium fujikuroi species carrying bacteria on rice seedling blight pathogen by recombinase polymerase amplification - lateral flow dipstick
By designing the RPA-LFD primer and probe combination of rice seedling bacteria-Fuscius Fusaria sprout bacteria, combined with RPA-LFD technology, the problem of long detection time and low accuracy in the existing technology is solved, and efficient and simple rice seedling bacteria detection is achieved, and rapid visual detection in the field is supported.
Patent Information
- Application Number
- CN202411562136.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-05
- Publication Date
- 2025-07-01
- Estimated Expiration
- 2044-11-05
AI Technical Summary
The prior art has problems with long detection time, low accuracy and the need for expensive and precise instruments when detecting the rice seedling bacteria Fusarium fumarus fumarus species. It is urgently needed for a convenient, specific and efficient detection method.
A primer and probe combination of RPA-LFD, a rice seedling bacteria-Fuscius Fusaria sprout bacteria, was designed. Combined with RPA nucleic acid amplification technology and lateral flow test strip (LFD) technology, a kit and corresponding detection method were developed to achieve rapid and visual detection results.
This method has high sensitivity, strong specificity and simple operation. It can quickly realize the field rapid visual detection of the rice seedling bacteria Fusarium fumarus species, supporting the effective prevention and treatment of rice seedling disease.
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Figure CN119061193B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a primer and probe combination, a kit and an application thereof for Fusarium fujikuroi strain with bacteria on rice seed belt by RPA-LFD. Background Art
[0002] Rice bakanae disease is an important disease on rice, which occurs almost in all rice planting areas in the world and is one of the important diseases affecting rice production. Rice bakanae disease is widely distributed in China and causes different degrees of damage in each rice-growing area. Generally, rice bakanae disease can cause a yield reduction of 10% - 20%, and in severe cases, the yield reduction is more than 50%. Fusarium fujikuroi Fusarium fujikuroi ) is the main pathogenic bacterium causing rice bakanae disease.
[0003] Research shows that the seed with bacteria is the main initial source of bacteria for bakanae disease, which not only affects the seed germination rate, but also causes the seedlings to get sick after the infected rice seeds are sown. Therefore, it is of great significance to detect the bacteria on seeds for the prevention and control of rice bakanae disease. The traditional methods for detecting bacteria on seeds include: isolation and culture method, washing test method, blotting paper moisturizing test method, agar plate test method, as well as conventional PCR method, LAMP and real-time fluorescence quantitative PCR detection methods, etc. Although these detection methods can reflect the bacteria-carrying situation of seeds, they also have certain defects, such as long detection time, low accuracy of detection results, or the need for expensive and precise instruments, etc. Therefore, it is urgent to establish a convenient, specific and efficient detection method for Fusarium fujikuroi causing rice bakanae disease.
[0004] Recombinase Polymerase Amplification (RPA) is a novel isothermal (37 - 42°C) nucleic acid amplification technology invented by Piepenburg et al. in 2006. Its amplification template can be either DNA or RNA, and the entire reaction process proceeds quite rapidly. Generally, detectable levels of amplified products can be obtained within 20 minutes. Compared with PCR, the RPA reaction does not require high-temperature denaturation and low-temperature annealing and does not rely on a precise temperature control device, the PCR instrument. The requirements for instruments in RPA are greatly reduced, the reaction time is significantly shortened, it is simple, rapid, efficient, highly sensitive, and highly specific. Therefore, it is considered a nucleic acid detection technology that can replace PCR. The RPA-LFD technology, which combines RPA with a lateral flow dipstick (LFD), can achieve visual detection of amplified products and has good application prospects in rapid on-site nucleic acid detection of pathogens. RPA-LFD detection is simple and rapid, the equipment is portable, and the results can be read with the naked eye, truly enabling rapid on-site nucleic acid detection of pathogens. So far, the RPA-LFD detection technology has been widely used in the detection of food, the human body, and pathogenic microorganisms. Summary of the Invention
[0005] Aiming at the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for a primer and probe combination, a kit, and its application for detecting the presence of Fusarium fujikuroi on the surface of rice seeds.
[0006] The present invention specifically adopts the following technical solutions to achieve:
[0007] In the first aspect of the present invention, a primer and probe combination for detecting the presence of Fusarium fujikuroi on the surface of rice seeds by RPA-LFD is provided, which includes primer Ff-RPA-F1, primer Ff-RPA-R2, and probe Ff-RPA-Probe. The nucleotide sequence of primer Ff-RPA-F1 is as shown in SEQ ID NO.1, the nucleotide sequence of primer Ff-RPA-R2 is as shown in SEQ ID NO.5, and the nucleotide sequence of probe Ff-RPA-Probe is as shown in SEQ ID NO.7.
[0008] Furthermore, the 5'-end of primer Ff-RPA-R2 is labeled with biotin; the 5'-end of probe Ff-RPA-Probe is labeled with FAM, the 3'-end is labeled with C3 Spacer, and a tetrahydrofuran abasic site is inserted between the 30th and 31st bases at the 5'-end of probe Ff-RPA-Probe.
[0009] In the second aspect of the present invention, a kit containing the above primer and probe combination is provided.
[0010] Furthermore, the RPA amplification detection system of the kit is as follows: 29.4 μl of Buffer A, 2.5 μL of Buffer B, 2 μl of primer Ff-RPA-F1, 2 μl of primer Ff-RPA-R2, 0.6 μl of probe Ff-RPA-Probe, 5 μl of template DNA, and made up to 50 μL with ddH2O.
[0011] The third aspect of the present invention provides the application of the above primer and probe combination or the above kit in detecting the presence of Fusarium fujikuroi on the surface of rice seeds.
[0012] The fourth aspect of the present invention provides an RPA-LFD detection method for the presence of Fusarium fujikuroi on the surface of rice seeds, which includes the following steps:
[0013] 1) Extract the DNA to be detected;
[0014] 2) Perform RPA-LFD reaction using the primer and probe combination as described above to obtain an amplification product;
[0015] 3) Visually determine the result of the amplification product in step 2), and observe whether colored bands appear on the control line and the detection line. If bands appear on both the control line and the detection line, it is a positive result; if a band appears on the control line but not on the detection line, it is determined as a negative result.
[0016] Furthermore, the RPA-LFD reaction conditions are: incubate at 39°C for 20 min and then at 75°C for 5 min.
[0017] Based on the H3 gene of Fusarium fujikuroi as the target gene, the present invention screened a primer and probe combination for RPA-LFD detection of Fusarium fujikuroi on the surface of rice seeds and established an RPA-LFD detection method. The present invention has high sensitivity, good specificity, and simple operation, providing important technical support for the rapid visual detection of bakanae disease of rice in the field. Description of the Drawings
[0018] Figure 1 Schematic diagram for screening of primer-probe combination;
[0019] Figure 2 Schematic diagram for specific detection of RPA-LFD;
[0020] Figure 3 Schematic diagram for sensitivity detection of RPA-LFD;
[0021] Figure 4 Schematic diagram for real-time fluorescence PCR detection of the presence of Fusarium fujikuroi on the surface of rice seeds;
[0022] Figure 5 Schematic diagram of detecting the presence of *Fusarium fujikuroi* on the surface of rice seeds by RPA-LFD Specific implementation mode
[0023] The present invention will be further described below in conjunction with embodiments.
[0024] Strains and rice seeds
[0025] The information of the tested strains in this embodiment is shown in Table 1. All the strains are existing strains, collected and preserved by the applicant's laboratory. A total of 11 kinds of tested rice seeds are shown in Table 2, namely, Hanyou 73, Y Liangyou 689, Y Liangyou 957, Qianyou 1, Shenliangyou 5814, Longliangyou 1686, Y Liangyou 900, Yuanliangyou, Yongyou 7861, Jiayou Zhongke 13-1 and Yongyou 7872.
[0026] Table 1 Strains used in the present invention
[0027]
[0028] Table 2 Rice seeds used in the present invention
[0029]
[0030] Main reagents
[0031] The RPA nucleic acid amplification kit and the lateral flow chromatography strip detection kit are both purchased from Changzhou Anpu Future Biotechnology Co., Ltd. The E.Z.N.A.® Fungal DNA Mini Kit is purchased from Hangzhou Omega Bio-Tek Co., Ltd. The fluorescence quantitative PCR kit is the Premix Ex (Probe gPCR) quantitative detection kit of Takara Company.
[0032] Example 1: Preparation of fungal DNA
[0033] Extract the mycelial genomic DNA of each strain in Table 1 using the OMEGA fungal DNA extraction kit.
[0034] Example 2: Preparation of DNA of rice seeds with bacteria
[0035] Rice seeds in Table 2 were used to rapidly extract DNA by alkaline lysis method. Take 50 seeds each, place the paddy grains in sterile 15 ml centrifuge tubes respectively, add 4 ml of 0.5 M NaOH solution, and shake vigorously for 1 min. Take 1 ml of the supernatant and transfer it to a new 1.5 ml centrifuge tube, and centrifuge at 10000 g / min for 3 min. Then aspirate 700 μl of the supernatant into a new 1.5 ml centrifuge tube, add 700 μl of isopropanol, invert and mix several times, and centrifuge at 12000 g / min for 10 min. Discard the supernatant, add 1 ml of 70% ethanol to wash once, and centrifuge at 10000 g / min for 2 min. Carefully pour out the supernatant, dry at room temperature, and add 50 μl of ddH2O to fully dissolve the DNA. Obtain the rice seed DNA solution.
[0036] Example 3: Establishment of RPA-LFD reaction system
[0037] The design of RPA-LFD detection primers and probes is the key to the success of amplification. The primer length is between 28 and 35 bp, the amplified fragment size is between 100 and 400 bp, and the probe design length is 46 to 52 bp. Based on the histone H3 gene of Fusarium fujikuroi (GenBank: KF466361) as the target gene, sequence alignment analysis was performed on the sequences of the same genus in the GenBank database, and specific regions were selected to design RPA primers and probes.
[0038] Use the RPA nucleic acid amplification kit for gene amplification. The RPA amplification system is 50 μL: First, fully mix 29.4 μl of A Buffer, 2 μl of Ff-F (5 μM), 2 μl of Ff-R (5 μM), 0.6 μl of Ff-Probe (5 μM) and 8.5 μl of sterile water, add them to the reaction tube until the solid particles dissolve, and then add 5 μL of the DNA template to be detected; then add 2.5 μL of B Buffer to the lid of the detection unit tube, cover the tube lid, invert and gently shake 5 - 6 times, and centrifuge at low speed for 10 sec. Place the detection unit tube in a constant temperature metal bath, incubate at 39 °C for 20 min and then at 75 °C for 5 min.
[0039] Take out the corresponding test strips according to the number of detections, and marks can be made on the cassette to distinguish the experimental groups. Take 10 μl of the nucleic acid amplification product and transfer it to a 1.5 ml centrifuge tube, dilute it 10 times with sterile water, and mix well. Take 80 μl of the diluted reaction product and drop it into the sample loading hole, and record the detection results in the reading area within 15 min. Observe whether colored bands appear in the quality control line and the detection line. If bands appear in both the quality control line and the detection line, it is a positive result; if a band appears in the quality control line but not in the detection line, it is determined as a negative result. After recording the detection results, seal the test strip and discard it in a safe place.
[0040] Results and analysis:
[0041] Primer screening
[0042] Based on the histone H3 gene sequence of Fusarium fujikuroi, a total of 3 forward primers, 3 reverse primers and 1 probe were designed (Table 3). The forward primers and reverse primers were combined, and there were 9 primer combinations in total (Table 4). Using sterile water, a mixed DNA sample composed of non-target fungi such as Fusarium oxysporum, Fusarium graminearum, Fusarium equiseti and Verticillium tricorpus, and the DNA sample of the target fungus Fusarium fujikuroi as templates, in order to screen out primer-probe combinations that showed positive results only in the target fungus samples. The results are as Figure 1 , only combination 1 met the expected effect and could be used for subsequent RPA-LFD detection of Fusarium fujikuroi.
[0043] Table 3 Primers and probes designed based on the histone H3 gene of Fusarium fujikuroi
[0044]
[0045] Table 4 Different primer and probe combinations
[0046]
[0047] Specific detection of RPA-LFD
[0048] Using the primer pair Ff-RPA-F1 / Ff-RPA-R2 and the probe Ff-RPA-Probe, the DNA samples of 10 common fungi on rice in Table 1 were detected by RPA-LFD, using sterile water as the negative control and the positive control of the kit as the positive control. The detection results are as Figure 2 , only the LFD test line results of Fusarium fujikuroi and the positive sample were positive among the above samples. The results showed that RPA-LFD using the primer pair Ff-RPA-F1 / Ff-RPA-R2 and the probe Ff-RPA-Probe combination could specifically detect Fusarium fujikuroi.
[0049] Sensitivity detection of RPA-LFD
[0050] The DNA sample of Fusarium fujikuroi was successively diluted to 100 pg / μl, 10 pg / μl, 1 pg / μl, 100 fg / μl, 10 fg / μl and 1 fg / μl, and the sensitivity of the above primer-probe group for RPA-LFD detection of Fusarium fujikuroi was determined. The results showed that the DNA samples of 100 pg / μl, 10 pg / μl and 1 pg / μl were all positive ( Figure 3 ). The results indicated that the RPA-LFD reaction system could detect at least the DNA concentration of Fusarium fujikuroi at 100 fg / μl.
[0051] Example 4: Detection of Fusarium fujikuroi on the Surface of Rice Seeds
[0052] Real-time fluorescence quantitative PCR detection: The detection of Fusarium fujikuroi, the causal agent of bakanae disease in rice, was performed using the method of Carneiro et al. (Carneiro et al. Phytopathology. 2017 107(7):885-892.). The primer and probe sequences were PCR Forward Primer: 5’-GGCGCGTTTTGCCCTTTCCT-3’ (SEQ ID NO.8), PCR Reverse Primer: 5’-AGCGGCTTCCTATTGTCGAA-3’ (SEQ ID NO.9), and Probe: 5’-FAM-TCACGTGTCAAACTAAA-TEMRA-3’ (SEQ ID NO.10). DNA from different rice seeds was used as a template, 1 ng / μl of Fusarium fujikuroi DNA was used as a positive control, and sterile water was used as a negative control. The PCR system was prepared using the Premix Ex (Probe qPCR) quantitative detection kit from Takara. The system consisted of 10 μl of Premix Ex Taq (2×), 0.5 μl of PCR Forward Primer, 0.5 μl of PCR Reverse Primer, 0.4 μl of Probe, 0.2 μl of ROX Reference Dye (50×), 2 μl of template, and 6.4 μl of ddH2O, for a total volume of 20 μl. The specific method can be found in Carneiro et al. Phytopathology. 2017 107(7):885-892. The mixed system was placed in a real-time ABI 7500 fluorescence quantitative PCR instrument for reaction. The PCR reaction program was: 95°C for 30 s; 95°C for 10 s, 62°C for 35 s (40 cycles). The presence of the pathogen on commercial seeds was detected.
[0053] The RPA-LFD detection was the same as in Example 3.
[0054] Results and Analysis
[0055] The surface DNA of the 11 rice seeds in Table 2 was extracted and subjected to real-time fluorescence quantitative PCR and RPA-LFD detection, respectively. Using the real-time fluorescence quantitative PCR detection method of Carneiro et al. (Phytopathology, 2017, 107:885-892), with 1 ng / μl of Fusarium fujikuroi DNA sample as a positive control, 11 rice samples were detected. The results showed that the Ct values of the 11 rice samples were between 31.97 and 36.66, indicating that the above-mentioned rice seeds were all infected with Fusarium fujikuroi (Table 5, Figure 4). The above rice seed samples were detected by RPA-LFD, and the results showed that all 11 rice seeds were positive ( Figure 5 ), which was consistent with the results of real-time fluorescence quantitative PCR. The results indicated that the RPA-LFD detection method of the present invention could be used for the visual detection of Fusarium fujikuroi in rice seeds.
[0056] Table 5 Ct values of real-time fluorescence PCR detection of Fusarium fujikuroi in rice seeds
[0057]
[0058] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A primer and probe combination of RPA-LFD for rice seedling pathogen-Fusarium fujikura species-banded bacteria in detecting rice seedling pathogen-Fusarium fujikura species-banded bacteria, the primer and probe combination comprising primer Ff-RPA-F1, primer Ff-RPA-R2 and probe Ff-RPA-Probe, the nucleotide sequence of the primer Ff-RPA-F1 is shown in SEQ ID NO.1, the nucleotide sequence of the primer Ff-RPA-R2 is shown in SEQ ID NO.5, and the nucleotide sequence of the probe Ff-RPA-Probe is shown in SEQ ID NO.
7.
2. The use according to claim 1, characterized in that: The 5' end of the primer Ff-RPA-R2 is labeled with biotin; the 5' end of the probe Ff-RPA-Probe is labeled with FAM, the 3' end is labeled with C3 Spacer, and a tetrahydrofuran abasic site is inserted between the 30 bp and 31 bp bases at the 5' end of the probe Ff-RPA-Probe.
3. Use of a kit containing the primer and probe combination according to claim 1 or 2 in detecting the rice seedling pathogen - Fusarium fujikura species band.
4. The kit according to claim 3, characterized in that The RPA amplification detection system of this kit is: Buffer A29.4μl, Buffer B2.5 μL, primer Ff-RPA-F1 2μL, primer Ff-RPA-R2 2μL, probe Ff-RPA-Probe0.6μL, template DNA 5μL, ddH2O is added to 50μL.
5. A method for detecting rice seedling pathogen-Fusarium fujikura species surface bacteria by RPA-LFD, characterized in that: The following steps are involved: 1) Extract the DNA to be tested; 2) performing an RPA-LFD reaction using the primer and probe combination of claim 1 or 2 to obtain an amplified product; 3) Visually determine the results of the amplified products in step 2) by observing whether colored bands appear on the quality control line and the test line. If bands appear on both the quality control line and the test line, it is a positive result; if bands appear on the quality control line but not on the test line, it is a negative result.
6. The method according to claim 5, characterized in that The RPA-LFD reaction conditions are: incubation at 39° C. for 20 min and then incubation at 75° C. for 5 min.