Grapevine botryosphaeria dothidea lamp visual detection primer group, kit and detection method

Abstract

The application belongs to the technical field of biology, and discloses a LAMP visual detection primer group of Coniella vitis, a kit and application thereof. The LAMP primer group comprises a pair of outer primers CV-F3 and CV-B3, a pair of inner primers CV-FIP and CV-BIP, and a pair of loop primers CV-LF and CV-LB. The color presented by a nucleic acid dye solution after a LAMP amplification reaction or the result of LAMP product agarose gel electrophoresis is judged. The application further provides a kit for detecting Coniella vitis and application of the LAMP primer group in identifying Coniella vitis. The method for detecting Coniella vitis by using the LAMP primer group has the advantages of fast detection speed, high sensitivity, strong specificity, simple operation and intuitive detection result, and provides a convenient, fast and reliable method for field detection of Coniella vitis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to primer sets, reagent kits, and detection methods for LAMP visualization detection of grape white rot fungus. Background Technology

[0002] Grapes (Vitis Vinifera L.) are an important fruit tree widely cultivated worldwide. China is the world's largest grape producer. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), in 2022, China's grape production accounted for 46% of the world's total fresh grape production, ranking first globally. Grapes can be eaten directly or processed into various products such as wine, raisins, and grape juice. Grapes are rich in vitamins C and K, as well as various antioxidants such as resveratrol, which are beneficial to cardiovascular health and boost immunity. They are highly popular with consumers and have significant economic value.

[0003] Based on their place of origin, grapes can be divided into three groups: East Asian, North American, and Eurasian. Eurasian grapes, due to their superior quality and high yield, are the main cultivated varieties in my country and worldwide. However, Eurasian grapes generally suffer from poor resistance to fungal diseases, leading to severe losses in both quality and yield. White rot is currently a major fungal disease in grape production, affecting multiple parts of the grapevine, including leaves, young shoots, and bunches, and in severe cases, can cause a 20-30% reduction in grape yield annually.

[0004] Different grape fungal diseases have similar symptoms, making it difficult for non-professionals to accurately judge and identify the disease type. Traditional disease identification methods include disease isolation, purification, Koch's postulates verification, molecular identification, and sequencing verification. These methods are generally time-consuming and require certain experimental instruments, which is not conducive to rapid disease diagnosis and may cause the best time for disease control to be missed.

[0005] Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technique proposed by Japanese scholar Notomi. This detection technique utilizes a Bst DNA polymerase with high strand displacement activity, employing 4-6 amplification primers. Under isothermal conditions, nucleic acid amplification can be completed in a short time, without the need for expensive precision instruments and special reagents. It possesses advantages such as high specificity, high sensitivity, visualized reaction results, and simple operation, and has been widely used in medical and health, plant, and other microbial detection fields. Currently, there are no reports on LAMP detection techniques for *C. vitis*, the causal agent of grape white rot. Therefore, the purpose of this invention is to provide a rapid LAMP primer set and detection method for *C. vitis*, providing a powerful tool for the scientific control of grape white rot. Summary of the Invention

[0006] The purpose of this invention is to provide a LAMP primer set, kit, and application for detecting Coniella vitis, the pathogen of grape white rot. This LAMP primer set has advantages such as high specificity, high sensitivity, short detection time, and direct visual observation of detection results. It does not rely on professional technicians and detection equipment, and provides a reliable detection tool for the prevention and control of grape white rot at the grassroots level.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] The LAMP primer set for detecting grape white rot (Coniella vitis) includes one pair of outer primers, one pair of inner primers, and one pair of loop primers. The specific primers and sequences are shown in Table 1.

[0009] Table 1. LAMP and conventional PCR primers and primer sequences of the present invention.

[0010]

[0011] The outer primer pair consists of a forward outer primer with a nucleotide sequence as shown in CV-F3 and a reverse outer primer with a nucleotide sequence as shown in CV-B3; the inner primer pair consists of a forward inner primer with a nucleotide sequence as shown in CV-FIP and a reverse inner primer with a nucleotide sequence as shown in CV-BIP; the circular primer pair consists of a forward circular primer with a nucleotide sequence as shown in CV-LF and a reverse circular primer with a nucleotide sequence as shown in CV-LB.

[0012] This invention also provides the application of the above-mentioned LAMP detection primer set in the preparation and identification of products containing grape white rot pathogens.

[0013] The present invention also provides a LAMP detection kit for detecting Coniella vitis, the causal agent of grape white rot, comprising the aforementioned LAMP detection primer set;

[0014] Furthermore, the kit also includes: LAMP reaction tubes, LAMP reaction mixture, and Bst 2.0 WarmStart DNA polymerase.

[0015] Furthermore, the LAMP reaction mixture comprises: OG orange-green color-changing tubes, 10×Isothermal Amplification Buffer, MgSO4 solution, dNTP Mix, and sterile distilled water.

[0016] This invention also provides a method for detecting Coniella vitis, the causal agent of grape white rot, using LAMP primer sets, specifically comprising the following steps:

[0017] 1. Extraction of genomic DNA from the sample to be tested;

[0018] (1) Extraction of fungal genomic DNA: Extract genomic DNA from the sample to be tested according to the instructions of the fungal genomic DNA extraction kit (Beijing Tiangen Biochemical);

[0019] (2) Extraction of DNA from grape samples: DNA was extracted from grape samples according to the instructions of the Plant Genome Extraction Kit (Beijing Tiangen Biochemical).

[0020] 2. LAMP amplification reaction: Using the genomic DNA of the sample to be tested and / or the DNA of the grape sample to be tested as templates, the LAMP detection primer set described above is used to perform the LAMP amplification reaction;

[0021] 3. After the LAMP reaction is complete, gently invert the OG orange-green color-changing reaction tube (Harbin Xinhai Gene) to dissolve the OG dye in the tube cap. If the solution turns green, the reaction result is positive; if the solution remains orange, the reaction result is negative.

[0022] Alternatively, LAMP amplification products can be analyzed by agarose gel electrophoresis. If the electrophoresis result shows a waterfall-like trapezoidal pattern, the reaction result is considered positive; if the electrophoresis result shows no bands, the reaction result is considered negative.

[0023] The preferred 25 μL LAMP reaction system is as follows: 2.5 μL 10×Isothermal Amplification Buffer, 1.5 μL MgSO4 (100 mM), 3.5 μL dNTPs (10 mM), 1 μL Bst 2.0 WarmStart DNA Polymerase (8000 U / ml), 2.5 μL 10×LAMP primer mixture including 2 μM forward and reverse outer primers CV-F3 / CV-B3, 16 μM forward and reverse inner primers CV-FIP / CV-BIP, 4 μM forward and reverse circular primers CV-LF / CV-LB, 1 μL DNA template, and 13 μL ddH2O.

[0024] The optimized LAMP amplification reaction conditions are: isothermal reaction at 63℃ for 45 min.

[0025] The present invention also provides the application of the above-described LAMP detection primer set or the above-described LAMP detection kit in the preparation of reagents for detecting grape fungal diseases.

[0026] Furthermore, the fungal disease of grapes is grape white rot.

[0027] Beneficial effects

[0028] 1. High Specificity: LAMP primers are designed based on multiple regions of the target gene DNA sequence, ensuring that only the target sequence can be effectively amplified. The LAMP detection technology of this invention can effectively distinguish grape white rot fungus from other plant pathogenic fungi, exhibiting extremely high specificity.

[0029] 2. High sensitivity: The detection sensitivity of this invention against grape white rot fungus DNA can reach 100 fg / μL, which is 10 times the sensitivity of conventional PCR. It can detect grape white rot fungus in extremely low concentrations and has extremely high sensitivity.

[0030] 3. Rapid and simple detection: The LAMP detection of this invention only requires a constant temperature water bath or other constant temperature equipment, without the need for an expensive PCR instrument. The operation is simple, and the result can be obtained after reacting at a constant temperature of 63°C for 45 minutes, which greatly shortens the reaction time compared with conventional PCR reaction.

[0031] 4. The test results are intuitive and can be judged by the naked eye: the positive reaction of LAMP amplification in this invention is green and the negative reaction is orange, and the test results can be judged directly by the naked eye.

[0032] In summary, the LAMP detection method for grape white rot pathogen described in this invention has advantages such as high specificity, high sensitivity, low detection cost, simple operation process, and visualized detection results. It can provide technical support for rapid detection of grape white rot pathogen for grassroots plant protection and other relevant departments, and provide a basis for early diagnosis and forecasting of the disease and timely formulation of disease control strategies. Attached Figure Description

[0033] Figure 1 Figure A shows the results of the LAMP feasibility experiment in this embodiment of the invention; A: Visual detection of LAMP amplification reaction; B: Gel electrophoresis of LAMP amplification reaction; M: DL2000 DNA marker; 1-7 in Figures A and B are grape white rot pathogens, and 8 is the negative control.

[0034] Figure 2 Figure 1 shows the experimental results of LAMP amplification reactions at different temperatures in this embodiment of the invention; A: Visualization results of LAMP reactions at different reaction temperatures; B: Agarose gel electrophoresis results of LAMP reactions at different reaction temperatures; M: DL2000 DNA marker. Figure 2 A and Figure 2 In section B, 1-9 represent different reaction temperatures, namely 55℃, 57℃, 60℃, 63℃, 65℃, 67℃, 70℃, 73℃, and 75℃.

[0035] Figure 3The figure shows the experimental results of LAMP amplification reaction at different reaction times in the embodiments of the present invention; M: DL2000 DNA marker, 1-6 represent different reaction times, namely 15min, 30min, 45min, 60min, 75min and 90min, respectively, and 7 is the negative control;

[0036] Figure 4 The following diagram shows the sensitivity measurement results of the LAMP method in this embodiment of the invention; A: Visualization results of LAMP amplification reaction, B: LAMP amplification agarose gel electrophoresis results, C: Conventional PCR amplification results. M: DL2000 DNA marker; 1-7 represent 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and ddH2O, respectively.

[0037] Figure 5 Figure 1 shows the LAMP specificity assay results in this embodiment of the invention; A: Visualization of LAMP amplification reaction; B: LAMP amplification agarose gel electrophoresis results; M: DL2000 DNA marker; strains 1-7 are C. vitis, C. granati, Botrytis cinerea, Alternaria alternate, A. tenuisima, Colletotrichumgloeosporioides, and C. fructicola, respectively; 8 is ddH2O.

[0038] Figure 6 The following diagram shows the detection results of LAMP on diseased grape samples in this embodiment of the invention: A: Visualization results of LAMP amplification reaction; B: LAMP amplification agarose gel electrophoresis results; M: DL2000 DNA marker; 1-8 represent: DNA of white rot pathogen, diseased leaf 1, diseased leaf 2, healthy leaf 1, diseased fruit 1, diseased fruit 2, and ddH2O, respectively. Detailed Implementation

[0039] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments, but this does not limit the present invention.

[0040] Main materials and reagents

[0041] The tested strains were: *C. vitis*, *C. granati*, *Botrytis cinerea*, *Alternaria alternate*, *A. tenuisima*, *Colletotrichum gloeosporioides*, and *C. fructicola*, all of which were propagated and preserved by the Grape Innovation Team of the Fruit Tree Research Institute of Jiangsu Academy of Agricultural Sciences.

[0042] Main reagents: Fungal genome extraction kit and plant genome DNA extraction kit were purchased from Tiangen Biotech (Beijing) Co., Ltd., Bst2.0 WarmStart DNA polymerase was purchased from New England Biotech, dNTP Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd., OG orange-green color-changing reaction tubes were purchased from Harbin Xinhai Gene Testing Co., Ltd., DL2000 DNA Marker was purchased from Takara, and routine PCR amplification reagents were purchased from Yijia Biotechnology Co., Ltd. (Nanjing). Primers were synthesized by General Biotech (Anhui) Co., Ltd.

[0043] Example 1 Primer Design

[0044] Using the TEF1 gene sequence (Genbank: KX890065) of *Coniella vitis*, the pathogen of grape white rot, as the target, six LAMP amplification primers were designed online using the NEB LAMP Primer Design Tool (https: / / lamp.neb.com). The primer set included one pair of outer primers CV-F3 / CV-B3, one pair of inner primers CV-FIP / CV-BIP, and one pair of circular primers CV-LF / CV-LB. Conventional PCR primers CV-F / CV-R were designed using Primer Premier5. The specific primer sequences are as follows:

[0045]

[0046] Example 2: Establishment of the LAMP reaction system

[0047] System setup: The total reaction volume was 25 μL, including 2.5 μL of 10×Isothermal Amplification Buffer, 3.5 μL of dNTP Mix (10 mM), 1.5 μL of MgSO4 (100 mM), 2.5 μL of 10×LAMP primer mixture including 2 μM forward and reverse outer primers CV-F3 / CV-B3, 16 μM forward and reverse inner primers CV-FIP / CV-BIP, 2.5 μL of 4 μM forward and reverse circular primers CV-LF / CV-LB, 1 μL of Bst2.0 WarmStart DNA Polymerase (8000 U / mL), 1 μL of DNA template, and ddH2O to a final volume of 25 μL. The reaction was incubated at 60 °C for 45 min. After the LAMP reaction is complete, take 3 μL of the amplification product for 1% agarose gel electrophoresis. The result is determined by whether a typical waterfall-like band is produced, indicating a negative or positive reaction. Alternatively, after the reaction is complete, gently invert the reaction tube to allow the reaction solution and the dye on the tube cap to react fully. A positive amplification reaction will turn green, while a negative amplification product will turn orange.

[0048] The results are as follows Figure 1 As shown, the templates for amplifications 1-7 were all DNA from *Grape White Rot*, and the color reaction was green in all cases, while the negative control was orange. Figure 1 A); Gel electrophoresis results showed the same outcome: the amplification of grape white rot fungus produced typical waterfall-like bands, while negative reactions showed no amplification bands. Figure 1 B).

[0049] Example 3: Optimization of the LAMP reaction system

[0050] To improve the efficiency of LAMP amplification, two key factors, reaction temperature and reaction time, were optimized.

[0051] Optimization of reaction temperature: Using grape white rot pathogen DNA as a template, 25 μL of the same reaction system was incubated at 55℃, 57℃, 60℃, 63℃, 65℃, 67℃, 70℃, 73℃, and 75℃ for 45 min, respectively. The results were analyzed by agarose gel electrophoresis and visualization.

[0052] The reaction results are as follows Figure 2 As shown, temperature has a significant impact on the LAMP amplification reaction. Amplification cannot be completed under low temperature (55℃ and 57℃) and high temperature (73℃ and 75℃) conditions, and the reaction tube turns orange (…). Figure 2 A), no bands were observed during gel electrophoresis ( Figure 2 B) Amplification products were successfully obtained at 60℃, 63℃, 65℃, 67℃ and 70℃. Considering both color reaction and electrophoresis band brightness, the optimal reaction temperature for LAMP was set to 63℃.

[0053] Optimization of reaction time: Under the optimal reaction temperature of 63℃, 25 μL of the same reaction system was reacted for 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, respectively. The results were analyzed by agarose gel electrophoresis. Figure 3 As shown, a clear amplification band can be obtained at a reaction time of 15 min. With the extension of time, the electrophoresis bands at 30 min and 45 min gradually become brighter, but the reaction time exceeding 45 min does not increase the amplification amount of the product. Therefore, the optimal reaction time is set to 45 min. That is, the method described in this invention can obtain results in only 45 min, which greatly shortens the reaction time compared to the reaction time of several hours for conventional PCR.

[0054] Example 3: LAMP Sensitivity Detection

[0055] The extracted DNA (100 ng / μL) of the grape white rot pathogen was serially diluted 10-fold with sterile water to obtain nine concentrations. Seven of these concentrations (1 ng / μL to 1 fg / μL) were used as templates, with 1 μL of each concentration. LAMP amplification was performed at the optimal reaction temperature of 63℃ and reaction time of 45 min. The sensitivity was compared with that of conventional PCR. The conventional PCR 25 μL reaction system consisted of: 12.5 μL of 2×Es Taq Master Mix, 1 μL each of CV-F / CF-R (10 μM), 1 μL of template DNA, and ddH2O to a final volume of 25 μL. The PCR program was: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, followed by a 2 min extension at 72℃, and finally cooling to 4℃ to terminate the reaction. The experimental results are as follows: Figure 4 As shown in A and 4B, under optimal conditions, the lowest detection threshold of LAMP is 100 fg / μL, demonstrating very high sensitivity; the detection limit of conventional PCR is 1 pg / μL. The LAMP detection sensitivity provided by this invention is 10 times that of conventional PCR, demonstrating the advantage of LAMP technology in terms of sensitivity and providing reliable technical support for the rapid detection of grape white rot pathogens.

[0056] Example 4: LAMP-specific detection

[0057] DNA extracted from *Coniella vitis*, the causal agent of grape white rot, and other tested strains such as *C. granati*, *Botrytis cinerea*, *Alternaria alternate*, *A. tenuisima*, *Colletotrichum gloeosporioides*, and *C. fructicola* were used as templates, with ddH2O as a negative control, for LAMP-specific detection. Results are as follows: Figure 5 As shown in A and 5B, only the LAMP reaction using grape white rot fungus DNA as a template is green, and the gel electrophoresis result shows a waterfall-like band. The other strains are negative orange and no electrophoresis bands are formed, indicating that the LAMP method established in this invention has extremely high specificity.

[0058] Example 5: Detection of grape white rot disease samples using LAMP

[0059] Leaves and fruits suspected of being infected with grape white rot were collected from the field, with healthy grape leaves and fruits used as negative controls. Genomic DNA was extracted from each sample as a template for LAMP reaction, and the infected samples were tested using optimized LAMP amplification conditions. The test results are as follows: Figure 6 As shown, diseased leaves and fruits all showed positive reactions, while healthy leaves and fruits all showed negative reactions, indicating that the LAMP detection method of the present invention can be successfully used for the detection of grape samples.

Claims

1. White rot spores ( Coniella vitis The LAMP visualization detection primer set is characterized by, This includes outer primer pairs, inner primer pairs, and loop primer pairs; The outer primer pair consists of a forward outer primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse outer primer with a nucleotide sequence as shown in SEQ ID NO:2; the inner primer pair consists of a forward inner primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse inner primer with a nucleotide sequence as shown in SEQ ID NO:4; the circular primer pair consists of a forward circular primer with a nucleotide sequence as shown in SEQ ID NO:5 and a reverse circular primer with a nucleotide sequence as shown in SEQ ID NO:

6.

2. The application of the LAMP visualization detection primer set according to claim 1 in the preparation and identification of products containing *Cyclocarya paliurus*.

3. A LAMP assay kit for identifying white-rot causal fungi, characterized in that, The LAMP detection kit comprises the LAMP visualization detection primer set as described in claim 1.

4. The kit according to claim 3, characterized in that, The LAMP detection kit also includes: 10×Isothermal Amplification Buffer, MgSO4 solution, dNTP Mix and OG orange-green color-changing reaction tubes.

5. A LAMP detection method of Gliocladium virens, characterized by, Includes the following steps: DNA is extracted from the sample to be tested, and LAMP amplification is performed using the LAMP visualization detection primer set described in claim 1. The presence of *Tetranychus leucopus* in the sample is determined based on the electrophoretic detection results and / or colorimetric results of the LAMP amplification products.

6. The LAMP detection method of Trichoderma alboniger according to claim 5, wherein, If the electrophoresis result shows a waterfall-like trapezoidal characteristic band and / or the color development result is green, the sample is determined to contain *Cytospora albopictus*. If the electrophoresis result does not show a waterfall-like trapezoidal characteristic band and / or the color development result is orange, the sample is determined not to contain *Cytospora albopictus*.

7. The LAMP detection method of Trichoderma alboniger according to claim 5, characterized by, The reaction conditions for LAMP amplification were incubation at 63°C for 45 min.

8. The LAMP detection method according to claim 5, characterized in that, The detection system comprises: 2.5 μL 10 × Isothermal Amplification Buffer, 1.5 μL 100 mM MgSO4, 3.5 μL 10 mM dNTPs, 1 μL 8000 U / ml Bst 2.0 WarmStart DNA Polymerase, 2.5 μL 10 × LAMP primer mixture, 1 μL DNA template and 13 μL ddH2O; the 10 × LAMP primer mixture comprises 2 μM forward / reverse outer primers, 16 μM forward / reverse inner primers and 4 μM forward / reverse loop primers.

9. The application of the LAMP visualization detection primer set as described in claim 1 or the LAMP detection kit as described in claim 3 or 4 in the preparation of a reagent for detecting *Cyclocarya paliurus*.

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