LAMP (Loop-Mediated Isothermal Amplification) visual detection primer group, kit and detection method for grape white rot fungi

Abstract

The invention belongs to the technical field of biology, and discloses a grape white rot pathogen LAMP (loop-mediated isothermal amplification) visual detection primer group, a kit and application of the grape white rot pathogen LAMP visual detection primer group. 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, and the color presented by a nucleic acid dye solution after an LAMP amplification reaction or an agarose gel electrophoresis result of an LAMP product is judged. The invention further provides a kit for detecting the grape white rot fungi, application of the LAMP primer group in identifying the grape white rot fungi and a method for detecting the grape white rot fungi by utilizing the LAMP primer group, and the method is high in detection speed, high in sensitivity, high in specificity, simple to operate and visual in detection result; and a convenient, rapid and reliable method is provided for field detection of grape white rot.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP visualization detection primer set, a kit and a detection method for grape white rot pathogens. Background Art

[0002] Grapes (Vitis Vinifera L.) are important fruit trees that are widely cultivated in the world. China is the world's largest grape producer. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), China's grape production accounted for 46% of the world's total fresh grape production in 2022, ranking first in the world. Grapes can be eaten directly or processed into a variety of 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 contribute to cardiovascular health and enhance immunity. They are deeply loved by consumers and have important economic value.

[0003] According to the origin, grapes can be divided into three populations: East Asian population, North American population and Eurasian population. The Eurasian population is the main cultivated variety in my country and around the world due to its excellent quality and high yield. However, Eurasian grapes generally have the disadvantage of poor resistance to fungal diseases, which causes serious losses in grape quality and yield. White rot is the main fungal disease in grape production at present. The disease can harm multiple parts of grape leaves, young branches and bunches, and in severe cases can cause a 20-30% reduction in grape production each year.

[0004] The symptoms of different grape fungal diseases are similar, and it is difficult for non-professionals to accurately judge and identify the type of disease. Traditional disease identification methods include disease isolation, purification, Koch's postulates verification, molecular identification and sequencing verification. The above methods are generally time-consuming and require the assistance of certain experimental instruments. They are not conducive to the rapid diagnosis of diseases and are prone to miss the best prevention and control period of diseases.

[0005] Loop-mediated isothermal amplification (LAMP) is a new type of nucleic acid amplification technology proposed by Japanese scholar Notomi. This detection technology uses a Bst DNA polymerase with high chain displacement activity and 4 to 6 amplification primers to complete nucleic acid amplification under constant temperature conditions and in a short time. It does not require expensive precision instruments and special reagents. It has the advantages of strong specificity, high sensitivity, visualization of reaction results and simple operation. It has been widely used in the fields of medical and health care, plants and other microbial detection. At present, there is no report on the LAMP detection technology of grape white rot fungus C.vitis. Therefore, the purpose of the present invention is to provide a LAMP primer set and detection method for rapid detection of grape white rot fungus, which provides a powerful tool for the scientific prevention and control of grape white rot. Summary of the invention

[0006] The present invention aims to provide a LAMP detection primer set, a kit and an application thereof for Coniella vitis, a pathogen of grape white rot. The LAMP primer set has the advantages of strong specificity, high sensitivity, short detection time, and the detection results can be directly observed by naked eyes. It does not rely on professional technicians and detection equipment, and provides a reliable detection tool for grassroots grape white rot prevention and control.

[0007] In order to solve the above-mentioned technical problems, the technical solution adopted in the present invention is as follows:

[0008] The LAMP detection primer set for grape white rot fungus (Coniella vitis) includes a pair of outer primers, a pair of inner primers and a 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 loop primer pair consists of a forward loop primer with a nucleotide sequence as shown in CV-LF and a reverse loop primer with a nucleotide sequence as shown in CV-LB.

[0012] The present invention also provides application of the above-mentioned LAMP detection primer set in the preparation of a product for identifying grape white rot pathogens.

[0013] The present invention also provides a LAMP detection kit for detecting grape white rot fungus (Coniella vitis), comprising the LAMP detection primer set;

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

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

[0016] The present invention also provides a method for detecting grape white rot fungus (Coniella vitis) using a LAMP primer set, which specifically comprises the following steps:

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

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

[0019] (2) Extraction of DNA from grape samples: DNA from grape samples was extracted 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 a template, and using the LAMP detection primer set to perform a LAMP amplification reaction;

[0021] 3. After the LAMP reaction is completed, gently turn the OG orange-green color-changing reaction tube (Harbin Xinhai Gene) upside down to dissolve the OG dye in the tube cap. If the solution turns green, the reaction result is positive. If the solution still turns orange, the reaction result is negative.

[0022] Alternatively, the LAMP amplification product is subjected to agarose gel electrophoresis analysis. If the electrophoresis detection result is a waterfall-like trapezoid, the reaction result is judged to be positive. If the electrophoresis detection result has no bands, the reaction result is negative.

[0023] The preferred 25 μL LAMP reaction system is: 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 loop primers CV-LF / CV-LB, 1 μL DNA template, 13 μL ddH2O.

[0024] The optimized LAMP amplification reaction conditions were: isothermal reaction at 63°C for 45 min.

[0025] The present invention also provides the use of the above-mentioned LAMP detection primer set or the above-mentioned LAMP detection kit in preparing a reagent for detecting grape fungal diseases.

[0026] Furthermore, the grape fungal disease 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 to ensure that only the target sequence can be effectively amplified. The LAMP detection technology of the present invention can effectively distinguish grape white rot fungi from other plant pathogenic fungi and has extremely high specificity.

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

[0030] 3. Rapid and easy detection: The LAMP detection of the present invention only requires a constant temperature water bath or other constant temperature equipment, and does not require an expensive PCR instrument. The operation is simple, and the result can be obtained after 45 minutes of reaction at a constant temperature of 63°C, which greatly shortens the reaction time compared with conventional PCR reactions;

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

[0032] In summary, the LAMP detection method for grape white rot pathogens described in the present invention has the advantages of strong specificity, high sensitivity, low detection cost, simple operation process, and visualization of detection results. It can provide technical support for the rapid detection of grape white rot pathogens by grassroots plant protection and other relevant departments, and provide a basis for the early diagnosis and prediction of the disease and the timely formulation of disease prevention and control strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of the LAMP feasibility experiment in the embodiment of the present invention are shown in FIG. A: a visualization detection diagram of the LAMP amplification reaction, B: a gel electrophoresis diagram of the LAMP amplification reaction; M: DL2000 DNA marker, 1-7 in FIG. A and FIG. B are all grape white rot fungi, and 8 is a negative control;

[0034] Figure 2 The experimental results of LAMP amplification reaction at different temperatures in the embodiment of the present invention are shown in FIG. A: visualization results of LAMP reaction at different reaction temperatures, B: agarose gel electrophoresis results of LAMP reaction at different reaction temperatures; M: DL2000 DNA marker, Figure 2 A and Figure 2 In B, 1-9 all have different reaction temperatures, namely 55°C, 57°C, 60°C, 63°C, 65°C, 67°C, 70°C, 73°C and 75°C;

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

[0036] Figure 4 The results of the sensitivity determination of the LAMP method in the embodiment of the present invention are shown in the figure; A: visualization result of LAMP amplification reaction, B: agarose gel electrophoresis result of LAMP amplification, C: conventional PCR amplification result. M: DL2000 DNA marker; 1-7 are 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, ddH2O respectively;

[0037] Figure 5 The results of LAMP specificity determination in the embodiment of the present invention are shown in Figure 1; A: visualization results of LAMP amplification reaction, B: results of LAMP agarose gel electrophoresis. M: DL2000 DNA marker; strains 1-7 are C. vitis, C. granati, Botrytis cinerea, Alternaria alternate, A. tenuisima, Colletotrichum gloeosporioides, C. fructicola, and 8 is ddH2O;

[0038] Figure 6 The results of the detection of diseased grape samples by LAMP in the embodiment of the present invention; A: Visualization results of LAMP amplification reaction, B: Agarose gel electrophoresis results of LAMP amplification. M: DL2000 DNA marker; 1-8 are: DNA of white rot pathogen, diseased leaf 1, diseased leaf 2, healthy leaf 1, diseased fruit 1, diseased fruit 2, ddH2O. DETAILED DESCRIPTION

[0039] In order to enable ordinary technicians in the field to better understand the technical solution of the present invention, the present invention is further described below in conjunction with the accompanying drawings and implementation cases, but the present invention is not limited thereby.

[0040] Main materials and reagents

[0041] Test strains: Grape white rot fungus (C.vitis), granular cushion spore (C.granati), grape gray mold fungus (Botrytis cinerea), alternaria alternate, tenuisima, grape anthracnose fungus Colletotrichum gloeosporioides and grape anthracnose fungus C.fructicola. All of the above strains were propagated and preserved by the Grape Innovation Team of the Fruit 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 Biochemical Technology (Beijing) Co., Ltd., Bst2.0 WarmStart DNA polymerase was purchased from New England Company, dNTP Mix was purchased from Nanjing Novozymes Biotechnology Co., Ltd., OG orange-green color change reaction tube was purchased from Harbin Xinhai Gene Testing Co., Ltd., DL2000 DNA Marker was purchased from Takara Company, conventional PCR amplification reagents were purchased from Yijia Biotechnology Co., Ltd. (Nanjing), and primers were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0043] Example 1 Primer Design

[0044] Taking the TEF1 gene sequence (Genbank: KX890065) of grape white rot pathogen (scientific name: Coniella vitis) 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 a pair of outer primers CV-F3 / CV-B3, a pair of inner primers CV-FIP / CV-BIP and a pair of loop primers CV-LF / CV-LB; conventional PCR primers CV-F / CV-R were designed using Primer Premier5. The specific sequences of the primers are as follows:

[0045]

[0046] Example 2 Establishment of LAMP reaction system

[0047] System establishment: total reaction volume 25μL, including 10×Isothermal Amplification Buffer 2.5μL, dNTP Mix (10mM) 3.5μL, MgSO4 (100mM) 1.5μL, 10×LAMP primer mixture including 2μM forward and reverse outer primers CV-F3 / CV-B3, 16μM forward and reverse inner primers CV-FIP / CV-BIP, 4μM forward and reverse loop primers CV-LF / CV-LB 2.5μL, Bst2.0 WarmStart DNA Polymerase (8000U / mL) 1μL, DNA template 1μL, ddH2O supplemented to 25μL. Reaction conditions are incubation at 60℃ for 45min. After the LAMP reaction is completed, take 3 μL of the amplified product for 1% agarose gel electrophoresis, and judge whether the reaction result is a negative reaction or a positive reaction by whether a typical waterfall-like band is produced; or after the reaction is completed, gently turn the reaction tube upside down to allow the reaction solution and the dye on the tube cap to fully react. Positive amplification reactions turn green, and negative amplification products turn orange.

[0048] The results are as follows Figure 1 As shown, the amplification templates 1-7 are all grape white rot fungus DNA, and the color reaction is green, while the negative control is orange ( Figure 1 A); The gel electrophoresis results showed the same result. The amplification of grape white rot bacteria produced a typical waterfall-like band, while the negative reaction had no amplification band ( Figure 1 B).

[0049] Example 3 Optimization of LAMP reaction system

[0050] In order to improve the efficiency of LAMP amplification reaction, two key factors, reaction temperature and reaction time, were optimized;

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

[0052] The reaction results are as follows Figure 2 As shown in the figure, temperature has a significant effect 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 in gel electrophoresis ( Figure 2 B); Amplification products were successfully obtained under 60℃, 63℃, 65℃, 67℃ and 70℃ conditions. Considering the color reaction and the brightness of the electrophoresis band, the optimal reaction temperature of LAMP was set at 63℃.

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

[0054] Example 3 LAMP sensitivity detection

[0055] The extracted DNA of grape white rot pathogen (100ng / μL) was diluted with sterile water in a 10-fold series gradient to obtain 9 concentrations in succession. Seven concentrations ranging from 1ng / μL to 1fg / μL were used as reaction templates, and 1μL of each concentration was taken for LAMP amplification at the optimal reaction temperature of 63℃ and reaction time of 45min. At the same time, the sensitivity was compared with that of conventional PCR. The conventional PCR 25μL reaction system was: 2×Es Taq Master Mix 12.5μL, 1μL of CV-F / CF-R (10μM), 1μL of template DNA, and ddH2O was added to make up to 25μL. The PCR reaction program was: 94℃ pre-denaturation for 2min, 94℃ deformation for 30s, 58℃ annealing for 30s, 72℃ extension for 30s, 35 cycles, 72℃ extension for 2min, and then the reaction was terminated by cooling to 4℃. The experimental results are as follows: Figure 4 A and 4B show that under the optimal conditions, the minimum threshold of LAMP detection is 100 fg / μL, showing a very high sensitivity; the detection limit of conventional PCR is 1 pg / μL, and the LAMP detection sensitivity provided by the present invention is 10 times that of conventional PCR, showing the advantage of LAMP technology in sensitivity and providing reliable technical support for the rapid detection of grape white rot fungi.

[0056] Example 4 LAMP specific detection

[0057] The extracted DNA of grape white rot pathogen Coniella vitis and other test strains C. granati, Botrytis cinerea, Alternaria alternate, A. tenuisima, Colletotrichum gloeosporioides, and C. fructicola were used as templates, and ddH2O was used as negative control to perform LAMP specificity detection. The results are shown in Figure 5 As shown in Figures A and 5B, only the LAMP reaction using grape white rot pathogen DNA as template was green, and the gel electrophoresis result formed waterfall-like bands, while the other strains were negative orange, and no electrophoresis bands were formed, indicating that the LAMP method established in the present invention has extremely strong specificity.

[0058] Example 5 Detection of Grape White Rot Samples by LAMP

[0059] Leaves and fruits suspected of being infected with grape white rot were collected from the field, and healthy grape leaves and fruits were used as negative controls. Genomic DNA of each sample was extracted as a template for LAMP reaction, and the diseased samples were tested using the optimized LAMP amplification conditions. Figure 6 As shown, the diseased leaves and fruits showed positive reactions, while the healthy leaves and fruits 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. A LAMP visualization detection primer set for grape white rot fungus (Coniella vitis), characterized in that: It 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 loop primer pair consists of a forward loop primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a reverse loop primer with a nucleotide sequence as shown in SEQ ID NO:

6.

2. Use of the LAMP visualization detection primer set according to claim 1 in the preparation and identification of grape white rot pathogen products.

3. A LAMP detection kit for identifying grape white rot pathogens, characterized in that: The LAMP detection kit comprises the LAMP detection primer set according to 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 tube.

5. A LAMP detection method for grape white rot pathogens, characterized in that: The following steps are involved: Extract DNA from the sample to be tested, perform LAMP amplification using the LAMP visualization detection primer set described in claim 1, and determine whether the sample to be tested contains grape white rot pathogens based on the electrophoresis detection result and / or color development result of the LAMP amplification product.

6. The LAMP detection method for grape white rot pathogens according to claim 5, characterized in that: If the electrophoresis test result shows a waterfall-shaped trapezoidal characteristic band and / or the color development result is green, it is judged that the sample to be tested contains grape white rot fungi; if the electrophoresis test result does not have a waterfall-shaped trapezoidal characteristic band and / or the color development result is orange, it is judged that the sample to be tested does not contain grape white rot fungi.

7. The LAMP detection method for grape white rot pathogens according to claim 5, characterized in that: The reaction conditions of the 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 of LAMP amplification includes: 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 2.0WarmStart DNA Polymerase, 2.5 μL 10×LAMP primer mixture including 2 μM forward / reverse outer primers, 16 μM forward / reverse inner primers, 4 μM forward / reverse loop primers, 1 μL DNA template, and 13 μL ddH2O.

9. Use of the LAMP visualization detection primer set according to claim 1 or the LAMP detection kit according to claim 3 or 4 in preparing a reagent for detecting grape fungal diseases.

10. The use according to claim 9, characterized in that: The grape fungal disease is grape white rot.

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