A method for rapid detection of apple ring rot pathogen based on LAMP

Abstract

The present invention discloses a LAMP detection primer for apple ring rot pathogen and its application. A primer composition for detecting apple ring rot pathogen based on LAMP technology is composed of 6 specific primers F3, B3, FIP, BIP, LF and LB. The present invention also provides a LAMP detection method for apple ring rot pathogen, comprising the following steps: 1) extracting DNA of a sample to be tested; 2) using the primer composition provided by the present invention to perform loop-mediated isothermal amplification on the DNA of the sample to be tested; 3) detecting the result, judging that the color development result shows green fluorescence or the appearance of ladder bands in electrophoresis is positive, and judging that the color development result shows orange or orange-yellow or the appearance of ladder bands is negative. The present invention provides a new molecular detection method and primer composition for apple ring rot pathogen, which can detect apple ring rot pathogen quickly, conveniently, efficiently, highly specifically and sensitively, and provides a reliable technical and theoretical basis for early diagnosis, detection and rapid import and export detection of apple ring rot pathogen.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop disease detection, identification and prevention, and specifically relates to a LAMP detection primer for apple ring rot pathogen and its application. The primer can be used for rapid, sensitive and specific molecular detection of apple ring rot pathogen, and can also be used for early diagnosis of apple ring rot disease and monitoring and identification of pathogens. Background Art

[0002] apple( Malus sieversii (Led.) Roem. ) The fruit is rich in nutrients, including zinc, and is known as the "fruit of wisdom" and "fruit of memory". Apples are also rich in nutrients such as protein, polyphenols, and vitamin C. In addition, apples have a fragrant smell and sweet taste, making them a high-value crop that is deeply loved by producers and consumers.

[0003] Apple ring rot ( Botryosphaeria dothidea ) has become an important factor restricting the development of the industry in the high-yield and high-quality cultivation of apples. In recent years, with the large-scale planting of susceptible Fuji apples, the incidence of apple ring rot has increased year by year, and the affected area has continued to expand. It has become an important disease in China's apple production, seriously threatening the sustainable development of the apple industry. The ring rot bacteria mainly harm branches and fruits. After infecting branches, the bacteria rapidly spread in the cortical tissue, causing the bark of the main trunk and large branches to be rough, forming "rough bark disease" and weakening the tree. The fruit is affected and produces concentric ring-shaped lesions of different depths and lightness, with obvious water stains on the outer edge. The fruit becomes sour and rotten, which leads to the reduction of apple quality and loss of commercial value. However, there is no good disease-resistant variety for this disease, and the incubation period of the ring rot bacteria is long, so it is difficult to detect the symptoms by naked eye in the early stage. Therefore, it is of great significance to quickly and accurately monitor the infection dynamics of the bacteria before or in the early stage of the disease, and take effective prevention and control measures in time to control the occurrence of the disease in the field and during storage and transportation, as well as the rapid detection of imported and exported apples.

[0004] The classification and identification of traditional ring spot pathogens are mainly based on morphological characteristics and pathogenicity determination. This identification method is time-consuming, cumbersome, low-sensitivity and highly empirical. It takes several days to isolate and identify pathogens from diseased plants, making it difficult to monitor the occurrence of diseases in a timely manner and effectively control the spread and epidemic of pathogens. With the continuous development of molecular biology technology, there are more and more successful examples of using PCR, serum immunology and other technologies for specific and rapid molecular detection of pathogens, but these molecular biology technologies also have some shortcomings to a certain extent. For example, serum immunology detection technology is time-consuming and labor-intensive in the preparation of serum, is often affected by the quality of antiserum, and may have cross-reactions, poor specificity, and easily cause false positives; PCR rapid detection technology mainly includes conventional PCR, nested PCR and real-time fluorescence quantitative PCR, but these PCR technologies have a long detection time and need to rely on expensive instruments and equipment such as PCR instruments and gel imaging systems, which is not conducive to the promotion and application of grassroots production. The above shortcomings limit the promotion and application of these advanced methods.

[0005] Loop-mediated isothermal amplification (LAMP) is a simple, rapid, accurate and inexpensive nucleic acid amplification technology developed by Japan Rongyan Co., Ltd. This technology can be used at a constant temperature of 60℃-65℃ to amplify nucleic acids using highly active strand displacement DNA polymerase ( Bst DNA polymerase is used to specifically amplify the target DNA fragment. A small number of copies of the target gene can be amplified to 10 within 1 hour. 9 -10 10 The LAMP reaction product can be detected not only by turbidity meter, real-time PCR instrument and gel electrophoresis instrument, but also by naked eye after staining with SYBR Green I, calcein and hydroxynaphthol blue. Since the LAMP reaction is simple, rapid, efficient, economical, and does not require special equipment, the test results can be judged by naked eye, which is very suitable for promotion and application in grassroots production departments, and thus has a very broad application prospect. At present, LAMP detection is mainly used in the detection of human and animal pathogens, food safety and environmental hygiene, and there are few reports on the detection of plant pathogens. There are no reports on LAMP detection of apple ring rot pathogens at home and abroad.

[0006] The present invention is based on the principle of loop-mediated isothermal amplification (LAMP) technology, selects the ITS gene sequence of apple ring rot pathogen as the detection target, designs 6 apple ring rot pathogen-specific primers in 6 regions of the ITS gene sequence, and establishes a visualized LAMP detection technology for apple ring rot pathogen using SYBR Green I as a fluorescent color indicator by optimizing the reaction system and reaction conditions. The technology is simple to operate, has high sensitivity and specificity, and does not require expensive instruments and equipment. It is suitable for early diagnosis of apple ring rot in the field and detection and identification of pathogens. It is of great significance for timely and effective prevention and control of apple ring rot, and at the same time provides a simple and easy detection and initial screening method for quarantine departments such as ports. Summary of the invention

[0007] The purpose of the present invention is to provide a LAMP technology detection method for apple ring rot pathogen and a primer combination used therein to solve the problems that the detection and identification of apple ring rot pathogen in the prior art is mainly based on morphological characteristics, the method is time-consuming, the procedure is cumbersome, the experience is strong, the accuracy is low, and it is difficult to timely monitor the occurrence of the disease and control the spread and prevalence of pathogens, and the existing PCR molecular detection needs to rely on expensive instruments such as amplification instruments and the detection time is long. The detection method of the present invention is easy to operate, has strong specificity, high sensitivity, and accurate and reliable results.

[0008] To achieve the above purpose, the present invention adopts the following technical solution:

[0009] 1. Apple ring rot pathogen LAMP detection primers:

[0010] Psora malariae Botryosphaeria dothidea The ITS gene sequences of the main apple pathogens such as apple rot, apple anthracnose and apple rot were compared and analyzed, and the sequences with the lowest homology between the ring rot pathogen and other pathogens were screened for LAMP primer design. A set of apple ring rot-specific LAMP primers was designed using the online LAMP primer design software Primer software Explorer V5 (http: / / primerexplorer.jp / lampv5e / index.html), including 6 primers F3, B3, FIP, BIP, LF and LB, with the sequences as follows:

[0011] F3: 5'-TGGGTGTTGGAGGGTGAT-3'

[0012] B3: 5'- TGGGAGGTGAAGGTTGAGAA-3'

[0013] FIP: 5'-GAGGGGGGAGGTGTTTGAGGTAGAAGGGTGAAGGTGTGGT-3'

[0014] BIP: 5'- GGGTGTTGGGTGAAGGGTAGTGTTGAGAAGGTTGGTGGGG-3'

[0015] LF:5'-GGAGGGTGGGGAATAGGA-3'

[0016] LB: 5'- AGAAGATAGATGTGGGTTGGGAG-3'

[0017] 2. The LAMP detection method for apple ring rot pathogen includes the following steps:

[0018] (1) Extracting genomic DNA from the sample to be tested:

[0019] When used to detect pure cultures of pathogenic bacteria, the CTAB method is used to extract genomic DNA. The specific method is as follows: take a small amount of mycelium powder in a 1.5 mL centrifuge tube (it is best if the mycelium powder just covers the semicircular bottom), add 900 μL of 2% CTAB (hexadecyltrimethylammonium bromide) extraction solution (2% CTAB; 100 mmol / L Tris-HCl, pH 8.0; 20 mmol / L EDTA, pH8.0; 1.4 mol / L NaCl) and 90 μL SDS (sodium dodecylbenzene sulfonate) [Note: CTAB, SDS need to be preheated at 60°C], use an oscillator to mix, place in a 60°C water bath for 20 min (DNA is released into the buffer), and centrifuge at 12000 r / min for 15 min; take 700 μL of the supernatant, add equal volumes of phenol, chloroform, and isoamyl alcohol (25:24:1), gently shake to mix, and centrifuge at 12000 r / min for 10 Take 500 μL of the supernatant, add an equal volume of chloroform to extract again, and centrifuge at 12000 r / min for 5 min; take 350 μL of the supernatant, add 1 / 10 volume of 3 mol / L NaAc and 2 times volume of anhydrous ethanol, precipitate at -20℃ for 15 min, and centrifuge at 12000 r / min for 5 min; discard the supernatant, add 700 μL of ice 70% ethanol for washing (centrifuge slightly; pour off the supernatant), dry on the clean bench until there is no alcohol smell, add 30-60 μL TE (10 mmol / L Tris-HCl, 0.1 mmol / L EDTA, pH 8.0) solution to dissolve, obtain DNA solution, detect DNA concentration with UV spectrophotometer and dilute to 100 ng / μL for standby use;

[0020] When used to detect the presence of apple ring rot pathogens in plant tissues, the NaOH rapid lysis method is used to extract DNA. The specific process is as follows: add 10 μL of 0.5 mol / L NaOH to each milligram of plant tissue, grind the tissue into a paste in a mortar, transfer it to a 1.5 mL centrifuge tube, centrifuge at 12000 r / min for 6 min, take 5 μL of the supernatant, add 495 μL of 0.1 mol / L Tris-HCl (pH=8.0), mix well, and take 1.0 μL as a PCR template for amplification;

[0021] (2) Establishment of LAMP reaction system: Using the DNA extracted in step (1) as the template, LAMP amplification was performed using primers F3, B3, FIP, BIP, LF, and LB. The LAMP detection reaction system was 25 μL, including 0.5 μL each of 10 μM F3 and B3, 2.0 μL each of 10 μM FIP and BIP, 1.0 μL each of 10 μM LF and LB, 12.5 μL of 2X LAMP Master Mix reaction mixture, and 8 U Bst DNA polymerase 0.5 μL, DNA template 1.0 μL, add sterile ultrapure water to 25 μL, and add 1.0 μL of color developer SYBR Green I into the lid of the reaction tube;

[0022] (3) LAMP reaction conditions: incubation at 65°C for 60 min and inactivation at 85°C for 10 min;

[0023] (4) Determination of reaction results: The determination was performed by visual observation with fluorescent dye or agarose gel electrophoresis. When the fluorescent dye visual observation method was used, after the LAMP reaction was completed, the tube was centrifuged instantly to allow the color developer SYBR Green I on the lid of the reaction tube to be added to the reaction solution. Green fluorescence was observed as a positive result, while orange or yellow fluorescence was a negative result. When the agarose gel electrophoresis method was used, 5.0 μL of the LAMP amplification product was tested by 2% agarose gel electrophoresis. If a ladder-shaped band appeared, it was judged as a positive result, and if no band appeared, it was judged as a negative result.

[0024] Significant advantages of the present invention

[0025] The invention has the beneficial effects that the apple ring rot pathogen LAMP detection primers provided by the invention have strong specificity; and the use of the primers for apple ring rot pathogen LAMP detection overcomes the problems of long cycle, time-consuming, labor-intensive, cumbersome, and poor specificity required by the biological detection methods in the prior art, and the PCR detection technology requires expensive equipment such as thermal cyclers and cannot be applied to the grassroots. The detection method of the invention can detect apple ring rot pathogens quickly, conveniently, efficiently, highly specifically, and highly sensitively under isothermal conditions of 65°C, does not require complex instruments, can better meet the on-site detection of apple ring rot pathogens, provides a new technical platform for the detection of apple ring rot pathogens, and can be used for early diagnosis of apple ring rot in the field and monitoring of pathogens.

[0026] Compared with the prior art, the present invention has the following technical advantages and positive effects:

[0027] 1. Strong specificity and reliable results: The LAMP primers provided by the present invention are based on the Botryosphaeria dothidea Six specific primers were designed for six different regions in the ITS gene sequence. If any region in the six regions does not match the primer, nucleic acid amplification cannot be performed. Compared with the two independent regions of the target sequence recognized by the PCR primer, the specificity and sensitivity are relatively high. The present invention uses the designed LAMP primers to establish a LAMP detection method for apple ring rot fungus. The apple ring rot fungus and other apple pathogenic fungi are specifically tested. Only the apple ring rot fungus can be detected, and other pathogens are not detected. The results of multiple tests are consistent, indicating that the LAMP detection method of the present invention has strong specificity and reliable results.

[0028] 2. High sensitivity: The detection sensitivity of the present invention to apple ring rot bacteria can reach 1fg / μL at the DNA level, which has a very high sensitivity.

[0029] 3. Good practicability: Unlike the PCR method, the LAMP detection method of the present invention does not require expensive instruments and equipment such as thermal cyclers (PCR instruments), so it is free from dependence on expensive equipment such as thermal cyclers; as long as there is a stable heat source, the LAMP reaction can occur, greatly expanding the detection range of apple ring rot bacteria. At the same time, the LAMP reaction of the present invention only needs to be carried out in a constant temperature water bath, and the result can be directly judged by color change after the reaction is completed, thereby increasing its application value in detecting bacteria-carrying plants.

[0030] 4. Simple and fast operation: The detection method of the present invention can detect apple ring rot pathogen quickly, conveniently, efficiently, highly specifically and sensitively under isothermal conditions of 65°C. It does not require complex instruments and only requires a constant temperature device, which can better meet the on-site detection of apple ring rot pathogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the detection result of apple ring rot pathogen using the LAMP technology of the present invention. Figure 1 The above figure shows the agarose gel electrophoresis detection results after LAMP amplification; Figure 1 The figure below shows the fluorescent dye visualization results after LAMP amplification. In the figure, tubes 1-2 are green, and tubes 3-6 are orange; among them: M is DL 2000 DNA marker, 1-2 are apple ring rot pathogens, 3 is apple anthracnose pathogens, 4 is apple rot pathogens, 5 is apple rot pathogens, and 6 is a negative control.

[0032] Figure 2 The results are the sensitivity test results of the LAMP technology of the present invention for detecting apple ring rot pathogens. Figure 2 The above figure shows the agarose gel electrophoresis detection results after LAMP amplification; Figure 2 The figure below shows the fluorescent dye color development result after LAMP amplification. In the figure, tubes 1-6 are green, and tubes 7-8 are orange; among them: M is DL 2000 DNA marker, and the concentrations of template DMA in 1-7 are 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL, and 100ag / μL, respectively, and 8 is a negative control.

[0033] Figure 3 The present invention is a method for detecting apple ring rot pathogens in diseased plants. Figure 3 The above figure shows the agarose gel electrophoresis detection results after LAMP amplification; Figure 3 The figure below shows the fluorescent dye color development result after LAMP amplification. In the figure, tubes 1-3 are green, and tubes 4-6 are orange: Among them: M is DL 2000 DNA marker, 1 is the mycelium DNA control of the pure culture of apple ring rot pathogen, 2 and 3 are leaves with ring rot disease, 4 and 5 are healthy leaves, and 6 is the negative control. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific examples, but is not intended to limit the scope of the present invention. The following examples are all based on conventional experimental conditions, or the operating technical procedures described in the relevant published literature, or the experimental conditions recommended by the manufacturer.

[0035] Example 1: Design of a specific primer composition for loop-mediated isothermal amplification (LAMP) detection of apple ring rot pathogen and verification of the primer specificity for apple ring rot pathogen.

[0036] 1. Extraction of genomic DNA from test strains

[0037] The CTAB method was used to extract the genomic DNA of the test strains (apple ring rot pathogen, apple rot pathogen, apple anthracnose pathogen, and apple rot pathogen). The specific method is as follows: Take a small amount of mycelium powder in a 1.5 mL centrifuge tube (it is best if the mycelium powder just covers the semicircular bottom), add 900 μL 2% CTAB (hexadecyltrimethylammonium bromide) extraction solution (2% CTAB; 100 mmol / L Tris-HCl, pH 8.0; 20 mmol / L EDTA, pH 8.0; 1.4 mol / L NaCl) and 90 μL SDS (sodium dodecylbenzene sulfonate) [Note: CTAB and SDS need to be preheated at 60°C], use an oscillator to mix, place in a 60°C water bath for 20 min (DNA is released into the buffer), and centrifuge at 12000 r / min for 15 min; take 700 μL of the supernatant, add an equal volume of phenol, chloroform, and isoamyl alcohol (25:24:1), gently shake to mix, centrifuge at 12000 r / min for 10 min, take 500 μL of the supernatant, add an equal volume of chloroform to extract again, and centrifuge at 12000 r / min for 5 min; take 350 μL of the supernatant, add 1 / 10 volume of 3 mol / L NaAc and 2 times volume of anhydrous ethanol, precipitate at -20℃ for 15 min, and centrifuge at 12000 r / min for 5 min; discard the supernatant, add 700 μL of ice 70% ethanol for washing (centrifuge slightly; pour off the supernatant), dry on the clean bench until there is no alcohol smell, add 30-60 μL TE (10 mmol / LTris-HCl, 0.1 mmol / L EDTA, pH 8.0) solution to dissolve, obtain DNA solution, detect DNA concentration with a UV spectrophotometer and dilute to 100 ng / μL for standby use.

[0038] 2. Design of specific primers for loop-mediated isothermal amplification (AMP) of apple ring rot pathogen

[0039] Psora malariae Botryosphaeria dothidea The ITS gene sequences of the main apple pathogens such as apple rot, apple anthracnose and apple rot were compared and analyzed, and the sequences with the lowest homology between the ring rot pathogen and other pathogens were screened for LAMP primer design. A set of apple ring rot-specific LAMP primers was designed using the online LAMP primer design software Primer software Explorer V5 (http: / / primerexplorer.jp / lampv5e / index.html), including 6 primers F3, B3, FIP, BIP, LF and LB, with the sequences as follows:

[0040] F3: 5'-TGGGTGTTGGAGGGTGAT-3'

[0041] B3: 5'- TGGGAGGTGAAGGTTGAGAA-3'

[0042] FIP: 5'-GAGGGGGGAGGTGTTTGAGGTAGAAGGGTGAAGGTGTGGT-3'

[0043] BIP: 5'- GGGTGTTGGGTGAAGGGTAGTGTTGAGAAGGTTGGTGGGG-3'

[0044] LF:5'-GGAGGGTGGGGAATAGGA-3'

[0045] LB: 5'- AGAAGATAGATGTGGGTTGGGAG-3'

[0046] 3. Establishment of LAMP detection method for apple ring rot pathogen and verification of primer specificity

[0047] The DNA of several test strains was used as template, and the primer combination F3, B3, FIP, BIP, LF, and LB were used for LAMP amplification. The reaction system included 2X LAMP Master Mix reaction mixture 12.5 μL, 8U Bst DNA polymerase 0.5μL, DNA template 1.0μL, add sterile ultrapure water to 25μL; LAMP reaction conditions: incubate at 65℃ for 60 min, inactivate at 85℃ for 10 min; Determination of reaction results: determination by fluorescent dye visual observation method or agarose gel electrophoresis method. Using fluorescent dye visual observation method, after the LAMP reaction is completed, centrifuge instantly to add the color developer SYBR GreenI on the lid of the reaction tube to the reaction solution. The color development result shows green fluorescence as positive, and orange or orange-yellow as negative; using agarose gel electrophoresis method, take 5.0μL of LAMP amplification product and use 2% agarose gel electrophoresis to detect. If a ladder-shaped band appears, it is judged as positive, and if no band appears, it is judged as negative.

[0048] 4. Primer specificity verification results

[0049] The results of LAMP amplification showed that the color development results of the tested apple ring rot pathogen (lanes 1 and 2) showed green fluorescence or a ladder-shaped band with LAMP characteristics in agarose gel electrophoresis, while the color development results of the other pathogens and negative controls (lanes 3-6) were orange or no amplification bands appeared in agarose gel electrophoresis (see Appendix Figure 1), indicating that the designed apple ring rot pathogen-specific primer combination F3, B3, FIP, BIP, LF, and LB can distinguish apple ring rot pathogen from other pathogens, has species specificity, and can be used for rapid and reliable detection and identification of apple ring rot pathogen.

[0050] Example 2: Loop-mediated isothermal amplification (LAMP) detection sensitivity determination of apple ring rot pathogen

[0051] 1. Preparation of Genomic DNA at Different Concentrations

[0052] Dilute the genomic DNA of apple ring rot pathogen with sterile ultrapure water and prepare a series of concentrations of 10 times the order of magnitude for later use;

[0053] 2. Sensitivity determination of LAMP detection method and observation of results

[0054] Using different concentrations of apple ring rot pathogen genomic DNA as template, LAMP amplification was performed using primer combinations F3, B3, FIP, BIP, LF, and LB. The LAMP detection reaction system included 2XLAMP Master Mix reaction mixture 12.5μL, 8U Bst DNA polymerase 0.5μL, DNA template 1.0μL, add sterile ultrapure water to 25μL; LAMP reaction conditions: incubate at 65℃ for 60 min, inactivate at 85℃ for 10 min; Determination of reaction results: determination by fluorescent dye visual observation method or agarose gel electrophoresis method. Using fluorescent dye visual observation method, after the LAMP reaction is completed, centrifuge instantly to add the color developer SYBR GreenI on the lid of the reaction tube to the reaction solution. The color development result shows green fluorescence as positive, and orange or orange-yellow as negative; using agarose gel electrophoresis method, take 5.0μL of LAMP amplification product and use 2% agarose gel electrophoresis to detect. If a ladder-shaped band appears, it is judged as positive, and if no band appears, it is judged as negative.

[0055] 3. LAMP amplification sensitivity test results

[0056] The results of LAMP amplification sensitivity test showed that the genomic DNA of apple ring rot pathogen at concentrations of 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, and 1fg / μL (lanes 1-6) showed green fluorescence or a ladder-shaped band with LAMP characteristics appeared in agarose gel electrophoresis, while the 100ag / μL concentration and negative control (lanes 7-8) showed orange color or no amplification band appeared in agarose gel electrophoresis, indicating that the designed primer combination F3, B3, FIP, BIP, LF, and LB for apple ring rot pathogen can be detected with a sensitivity of 1fg / μL (attached) through LAMP amplification. Figure 2 )

[0057] Example 3: LAMP detection of apple ring rot pathogen in diseased tissues

[0058] Sample collection: Branches and trunks with typical symptoms of apple ring rot and healthy branches were collected from the experimental field of Yantai Academy of Agricultural Sciences and brought back to the laboratory for use. Branch DNA extraction: DNA was extracted using the NaOH rapid lysis method. The specific process is as follows: 10 μL 0.5 mol / L NaOH was added to each milligram of plant tissue, the tissue was fully ground into a paste in a mortar and transferred to a 1.5 mL centrifuge tube, centrifuged at 12,000 rpm for 6 min, 5 μL of the supernatant was added to 495 μL 0.1 mol / L Tris-HCl (pH=8.0) and mixed evenly, and 1.0 μL was taken as a PCR template for amplification.

[0059] Amplification detection and observation: Using the above-extracted DNA as a template, LAMP amplification was performed using primer combination F3, B3, FIP, BIP, LF, and LB. The LAMP detection reaction system was 25 μL, including 2X LAMP Master Mix reaction mixture 12.5 μL, 8U Bst DNA polymerase 0.5μL, DNA template 1.0μL, add sterile ultrapure water to 25μL; LAMP reaction conditions: incubate at 65℃ for 60 min, inactivate at 85℃ for 10 min; Determination of reaction results: determination by fluorescent dye visual observation method or agarose gel electrophoresis method. Using fluorescent dye visual observation method, after the LAMP reaction is completed, centrifuge instantly to add the color developer SYBR GreenI on the lid of the reaction tube to the reaction solution. The color development result shows green fluorescence as positive, and orange or orange-yellow as negative; using agarose gel electrophoresis method, take 5.0μL of LAMP amplification product and use 2% agarose gel electrophoresis to detect. If a ladder-shaped band appears, it is judged as positive, and if no band appears, it is judged as negative.

[0060] Test results: Test results (attached Figure 3) showed that the mycelial DNA of the pure culture of apple ring rot pathogen (lane 1) and the genomic DNA of the branches and trunks with typical symptoms of apple ring rot (lanes 2 and 3) were amplified by LAMP, and the color development results showed green fluorescence or the ladder-shaped bands with LAMP characteristics appeared in agarose gel electrophoresis, indicating the presence of apple ring rot pathogen, while the color development results of healthy branches and trunks (lanes 4 and 5) and the negative control (lane 6) were orange or no amplification bands appeared in agarose gel electrophoresis, indicating that apple ring rot pathogen was not present, indicating that this set of technology can be used for rapid molecular detection of apple ring rot pathogen in plant tissues.

[0061] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention. Sequence Listing <110> Yantai Academy of Agricultural Sciences, Shandong Province <120> A method for rapid detection of apple ring rot pathogen based on LAMP <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Apple ring rot (Botryosphaeria dothidea) <400> 1 tgggtgttcc agcctgat 18 <210> 2 <211> 20 <212> DNA <213> Apple ring rot (Botryosphaeria dothidea) <400> 2 tgggacctga aggttcagaa 20 <210> 3 <211> 40 <212> DNA <213> Apple ring rot (Botryosphaeria dothidea) <400> 3 gaggggccag gtgtttgagg tagaaccgtg aaggtctggt 40 <210> 4 <211> 40 <212> DNA <213> Apple ring rot (Botryosphaeria dothidea) <400> 4 gggtgttccg tgaaccgtag tgttgagaag gttggtccgg 40 <210> 5 <211> 18 <212> DNA <213> Apple ring rot (Botryosphaeria dothidea) <400> 5 ggagggtccg gaatacga 18 <210> 6 <211> twenty three <212> DNA <213> Apple ring rot (Botryosphaeria dothidea) <400> 6 agaacataca tgtgggttcc gag 23

Claims

1. A method for detecting apple ring rot pathogen using LAMP primers, characterized in that: The specific steps include: The apple ring rot pathogen LAMP detection primer is characterized in that its nucleotide sequence is: F3: 5′-TGCCTGTTCGAGCGTCAT-3′ B3: 5'-TCCGAGGTCAACCTTGAGAA-3' FIP: 5'-CACCGCCGAGGTCTTTGAGG TACAACCCTCAAGCTCTGCT-3'BIP: 5'-GCGTCTTGCCTCAAGCGTAGT GTTCAGAAGGTTCGTCCGG-3' LF: 5′-GGACGGTGCCCAATACCA-3′ LB: 5'-AGAACATACATCTCGCTTCGGAG-3'; (1) extracting genomic DNA of the sample to be tested; (2) Establishment of LAMP reaction system: The LAMP detection reaction system is 25 μL, with the DNA extracted in step (1) as the template. The reaction system includes 0.5 μL each of 10 μM F3 and B3, 2.0 μL each of 10 μM FIP and BIP, 1.0 μL each of 10 μM LF and LB, 12.5 μL of 2×LAMP Master Mix reaction mixture, 0.5 μL of 8UBst DNA polymerase, 1.0 μL of DNA template, and the mixture is made up to 25 μL with sterile ultrapure water. Then, 1.0 μL of the color developer SYBR Green I is added to the lid of the reaction tube; (3) LAMP reaction conditions: incubation at 65°C for 60 min and inactivation at 85°C for 10 min; (4) Determination of reaction results: determination by fluorescent dye visual observation or agarose gel electrophoresis; In the step (4), the fluorescent dye visual observation method is used. After the LAMP reaction is completed, the reaction tube is centrifuged instantly to allow the color developer SYBR Green I on the lid of the reaction tube to be added to the reaction solution. The color development result shows that green fluorescence is positive, and orange or orange-yellow fluorescence is negative.

2. The LAMP detection method for apple ring rot pathogen according to claim 1, characterized in that: In the step (4), agarose gel electrophoresis is used to detect 5.0 μL of the LAMP amplification product by 2% agarose gel electrophoresis. If a ladder-shaped band appears, it is judged as positive, and if no band appears, it is judged as negative.

3. The LAMP detection method for apple ring rot pathogen according to claim 1, characterized in that: The 2×LAMPMasterMix reaction mixture consists of the following components: 40 mM Tris-HCl, 20 mM (NH4)2SO4, 20 mM KC1, 16 mM MgSO4, 2.0 mM dNTPs, and 0.2% Trion X-100.

4. Application of the LAMP detection method for apple ring rot pathogen described in claim 1 in early diagnosis of apple ring rot pathogen and monitoring and identification of the pathogen.

Images