Primer group, kit and method for detecting cladosporium cucumerinum based on RPA-LFD technology and application

Abstract

The invention belongs to the technical field of plant disease detection, and particularly relates to a primer group, a kit and a method for rapidly detecting cladosporium cucumerinum based on an RPA-LFD technology and application, a forward primer sequence of the primer group is as shown in SEQ ID No.1, and a reverse primer sequence of the primer group is as shown in SEQ ID No.2; the sequence of the modified reverse primer is as shown in SEQ ID No.3, and the sequence of the probe is as shown in SEQ ID No.4. The RPA-LFD method is adopted for detecting the apple scab for the first time, and the method has the advantages of being high in specificity, high in sensitivity, simple, convenient and rapid, independent of large instruments and equipment and the like and can be used for field actual sample detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease detection, and particularly relates to a primer set, a kit, a method and an application for rapidly detecting Venturia inaequalis by RPA-LFD technology. Technical Background

[0002] Apple (Malus domestica Borkh.) is one of the most important and common fruits in the world, ranking fourth in the global fruit production. Apple scab is caused by the plant fungal pathogen Venturia inaequalis (Cooke) Wint., and is one of the most destructive diseases affecting apple production, which can cause a loss of 70% or more in the value of apple fruits. The conidia transmitted through the air infect the fruits to produce typical "scab" lesions, seriously damaging the appearance of apple fruits and losing their commercial value.

[0003] It is estimated that apple producers in the eastern United States alone spend more than $18.6 million annually to control this disease. V. inaequalis is an object of quarantine in China. It can overwinter in the form of perithecia on fallen leaves or apple fruits, and overwinter as mycelium and conidia in bud scales or tree branch ulcers. The primary infection sources are relatively wide, and infections can occur during key growth periods such as the germination period, flower bud opening period, and petal shedding period of fruit trees. It spreads quickly and is difficult to control. Therefore, rapid detection of Venturia inaequalis plays an important role in disease control.

[0004] For the identification of Venturia inaequalis, traditional methods require the use of single-spore isolation or tissue isolation methods to isolate and purify the pathogen, and then identify it through morphology and molecular biology. However, the growth of this pathogen is extremely slow and it is easily interfered by other bacteria, making it difficult to isolate and obtain. Therefore, the traditional isolation and identification methods are time-consuming and laborious. For Venturia inaequalis, ordinary PCR, Real-time PCR, and loop-mediated isothermal amplification detection methods have been developed. The ordinary PCR method has a long detection time, the Real-time PCR method is costly and requires expensive equipment; loop-mediated isothermal amplification (LAMP) requires a dedicated heating device, and the existing detection methods cannot meet the requirements of efficient field detection.

[0005] Recombinase polymerase amplification (RPA) is a newly emerging nucleic acid amplification technology in recent years. The reaction process is less restricted by the environment, easy to operate, and user-friendly for testers. The visual detection method of lateral flow dipstick (LFD) can be completed in only 10 minutes without the need for other colorimetric equipment. Combining it with RPA simplifies the detection procedure. This method meets the conditions for rapid detection of pathogenic bacteria, does not require a complex thermal cycler, and is suitable for on-site tests.

[0006] Chinese invention CN202311516904.5 describes in its specification a primer, kit, and detection method for detecting Venturia inaequalis, including RPA primers and CRISPR / Cas12a primers for detecting V. inaequalis; a kit for detecting V. inaequalis, containing the above primers, Cas12 protein, T7 transcriptase, DNA polymerase, and a Cas12 / 13 specific nucleic acid detection dipstick. The detection method includes: establishing a CRISPR / Cas12a detection reaction system using RPA amplification products, in vitro transcribed guide RNA, and FB probe; after the reaction, inserting the Cas12 / 13 specific nucleic acid detection dipstick into the reaction tube and observing the change of the test line on the dipstick; if there is a color change on the test strip of the dipstick, it is a positive reaction, indicating the presence of V. inaequalis. The present invention has strong specificity, high sensitivity, accurate results, and high practicality.

[0007] In terms of specificity, the above patent method did not test whether it can distinguish between V. inaequalis and its closely related species such as V. nashicola, V. asperata, and other pathogens such as brown rot and gray mold that often occur on fruits during port quarantine and field detection. Therefore, it cannot be determined whether the method is specific to V. inaequalis. In terms of detection sensitivity, the above patent method set a detection concentration range from 1 pg to 0.01 fg. The detection results showed that all tested concentrations except the blank control showed positive reactions, and the detection intensity differences were not obvious. Therefore, it is impossible to determine the true detection sensitivity of the method. Summary of the Invention

[0008] To solve the problems existing in the above technologies, the present invention provides a primer set, a kit, a method and an application for rapidly detecting Venturia inaequalis based on the RPA-LFD technology. Based on the RPA-LFD technology, a new method for rapidly detecting Venturia inaequalis is established for the identification of quarantine plant pathogens and the rapid detection of field plant diseases. The present invention uses the RPA-LFD method to detect Venturia inaequalis, which has the characteristics of strong specificity, high sensitivity, simplicity, rapidity, and independence from large-scale instrument equipment, and can be used for the detection of actual field samples.

[0009] A primer set for rapidly detecting Venturia inaequalis (V. inaequalis) based on the RPA-LFD technology to solve the above technical problems, which is a PCR amplification primer. The forward primer sequence is as shown in SEQ ID No. 1, and the reverse primer sequence is as shown in SEQ ID No. 2.

[0010] Specifically: Forward primer V.in-LFD-F1: 5′-CATTTATTCACTTTGTTATCACCCTCACTG-3′; (SEQ ID NO.1);

[0011] Reverse primer V.in-LFD-R1: CTATTGTAATCGTTAGCGTCGTCATAGT-3′; (SEQ ID NO.2).

[0012] The application of the above primers in the detection of Venturia inaequalis.

[0013] The application of the above primers in the preparation of detection reagents or kits for Venturia inaequalis.

[0014] The present invention also provides a kit for detecting Venturia inaequalis based on the RPA-LFD technology, which at least includes the primer and probe combination described in claim 1 for more than 1 dosage. The primer and probe combination, the forward primer sequence is as shown in SEQ ID No. 1, the reverse primer sequence is as shown in SEQ ID No. 2, and the primer sequence after biotin (Biotin) labeling at the 5′ end of the reverse primer sequence is as shown in SEQ ID No. 3; the probe sequence is as shown in SEQ ID No. 4, the 5′ end is modified with a FAM antigen group, a tetrahydrofuran (THF) is used as a dSpacer to replace a base at the 31st base position from the 5′ end, and the 3′ end is labeled with a C3-spacer modification group for blocking.

[0015] Specifically: The modified reverse primer V.in-LFD-R1B:

[0016] 5′-Biotin-CTATTGTAATCGTTAGCGTCGTCATAGT-3′; (SEQ ID NO.3);

[0017] Probe sequence V.in-LFD-probe1:

[0018] 5′-FAM-CGCTATTCACGTACCGCCACTCAAGGCAGC(THF)CAACTTTCTCCGGTCC3-spacer-3′; (SEQ ID NO.4).

[0019] The kit further includes: reaction tubes filled with lyophilized powder, A Buffer, B Buffer, deionized water, and LFD lateral flow test strips.

[0020] Application of the kit for detecting Venturia inaequalis in the detection of Venturia inaequalis.

[0021] This application also provides a method for detecting Venturia inaequalis based on RPA-LFD technology, including the following steps:

[0022] (1) Extract DNA from the sample to be tested;

[0023] (2) Using the DNA as a template, perform RPA amplification with the primer and probe combination or the kit for detecting Venturia inaequalis; use a lateral flow test strip to detect the amplification product; when two bands (Control line and Test line) appear on the test strip, one in the quality control area and one in the detection area, the result is positive, indicating that the sample contains Venturia inaequalis; when only one band (Control line) appears in the quality control area and no band appears in the detection area, the result is negative, indicating that the sample does not contain Venturia inaequalis.

[0024] The present invention first targeted the EF gene of Venturia inaequalis as the target sequence, designed a series of RPA primers using the primer 3plus software, and screened the designed primers through experiments to obtain a pair of special primers and probes for RPA detection of Venturia inaequalis.

[0025] The beneficial effects of the present invention are as follows:

[0026] A. The primer-probe used in the present invention has good amplification effect, strong specificity, and high sensitivity. By forming a primer-probe heterodimer in the detection area, the test strip shows a positive reaction. The detection method established by the present invention can detect 6.116×10 - 6 ng / μL of Venturia inaequalis genomic DNA.

[0027] B. The method for detecting Venturia inaequalis of the present invention by combining the RPA technology with the lateral flow chromatography technology combines the high sensitivity of molecular biology detection and the high specificity of immunological detection, and is easy to operate without the need for complex instruments, making it suitable for rapid screening and detection of Venturia inaequalis in grass-roots laboratories and quarantine sites.

[0028] C. Compared with conventional PCR, the method of the present invention has a fast detection speed, does not require changing the reaction temperature throughout the process, does not require denaturation, gets rid of the dependence on thermal cycling instruments, and can complete amplification in about 9 minutes at 41°C. It does not require complex instrument equipment and is suitable for on-site detection.

[0029] D. The present invention can be used for rapid detection of Venturia inaequalis in diseased plant tissues, and it only takes about 12 minutes to complete the detection, which is an effective means for detecting Venturia inaequalis. The detection method of the present invention can also be used for pre-onset detection of Venturia inaequalis, which is of great significance for predicting and forecasting diseases, determining the appropriate control period, and effectively controlling diseases.

[0030] The present invention uses the RPA-LFD technology to establish a method for rapid detection of Venturia inaequalis, which has strong specificity and high sensitivity, can be used for the detection of actual samples, and provides a sensitive, reliable and convenient new method for on-site detection of Venturia inaequalis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the specificity result of using the common PCR detection primers F1 / R1 in Example 3 of the present invention. In the figure, M is DL2000, and the templates in lanes 1-8 are ddH2O, Colletotrichum gloeosporioides, Botryosphaeria dothidea, Botrytis cinerea, Monilinia fructicola, Venturia asperata, Venturia nashicola, and Venturia inaequalis respectively.

[0032] Figure 2 This is the specificity result of using the RPA kit detection primers F1 / R1 in Example 4 of the present invention.

[0033] (In the figure, M is DL2000, and the templates in lanes 1-8 are ddH2O, Colletotrichum gloeosporioides (C.gloeosporioides), Botryosphaeria dothidea (B.dothidea), Botrytis cinerea (B.cinerea), Monilinia fructicola (M.fructicola), Venturia asperata, Venturia nashicola, and Venturia inaequalis, respectively).

[0034] Figure 3 This is the specific verification result of the RPA-LFD detection of Venturia inaequalis in Example 6 of the present invention.

[0035] (In the figure, the templates of test strips 1-8 are ddH2O, Colletotrichum gloeosporioides (C.gloeosporioides), Botryosphaeria dothidea (B.dothidea), Botrytis cinerea (B.cinerea), Monilinia fructicola (M.fructicola), Venturia asperata, Venturia nashicola, and Venturia inaequalis, respectively).

[0036] Figure 4 This is the sensitivity verification result of the RPA-LFD detection of Venturia inaequalis in Example 7 of the present invention.

[0037] (In the figure, the template concentrations of test strips 1-10 are 6.116 ng / μL, 6.116×10 -1 ng / μL, 6.116×10 -2 ng / μL, 6.116×10 -3 ng / μL, 6.116×10 -4 ng / μL, 6.116×10 -5 ng / μL, 6.116×10 -6 ng / μL, 6.116×10 -7 ng / μL, 6.116×10 -8 ng / μL, 6.116×10 -9 ng / μL, and lane 12 is the negative control).

[0038] Figure 5 This is the detection result of the RPA-LFD for apple scab plant samples in Example 8 of the present invention. In the figure, the templates of test strips 1-8 are ddH2O, healthy apple leaves, healthy apple fruits, genomic DNA of Venturia inaequalis, diseased apple leaves-1, diseased apple leaves-2, diseased apple fruits-1, and diseased apple fruits-2, respectively. Detailed implementation manners

[0039] The embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples, the operations are carried out under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer can be obtained as conventional products commercially available.

[0040] Example 1

[0041] Extract the genomic DNA of the test pathogen strain. The specific extraction process is as follows:

[0042] (1) Scrape the mycelium with the tip of a sterilized pipette. Try not to scrape the culture medium during the operation. Collect the mycelium into a 2 mL disruption tube containing disruption beads, quickly freeze it in liquid nitrogen, disrupt it twice in a disruptor at 45 Hz for 1 min, and then add 500 μL of CTAB.

[0043] (2) Then place the disruption tube in a water bath at 65 °C for 30 min, invert and mix the sample every 10 min, and centrifuge at 10,000 rpm for 10 min.

[0044] (3) Take 450 μL of the supernatant after centrifugation into a new 1.5 mL centrifuge tube, add 450 μL of chloroform:isoamyl alcohol (24:1), mix well, shake thoroughly until there is no layering, centrifuge at 4 °C, 10,000 rpm for 10 min, and extract twice.

[0045] (4) Pipette 300 μL of the supernatant into a new 1.5 mL centrifuge tube, add 210 μL of isopropanol (0.7 times the volume of the supernatant), and centrifuge at 12,000 rpm for 10 min.

[0046] (5) Discard the supernatant, wash the precipitate with 300 μL of 75% ethanol. During this process, blow up the precipitate at the bottom or on the wall of the tube, and centrifuge at 12,000 rpm for 5 min.

[0047] (6) After removing the supernatant, dry the residual liquid in the centrifuge tube in a laminar flow hood, add 30 μL of ddH2O, measure the DNA concentration of the extracted sample and the ratio of A 260 / A 280 with a Nanodrop 2000 in the public laboratory, and then store it in a refrigerator at -34 °C.

[0048] Example 2

[0049] Extract the genomic DNA of the test plant tissue. The specific extraction process is as follows: (Use the kit from Tiangen Biotech Co., Ltd. for plant genomic DNA extraction).

[0050] (1) Weigh 0.1 g of plant leaf tissue (cut about 1 / 4 of the leaf, two square peels of about 1.5 cm × 1.5 cm for nucleic acid extraction) into a crushing tube, then add 700 μL of buffer GP 1 (containing β-mercaptoethanol with a final concentration of 0.1% and preheated in a 65°C water bath), and place it in a grinder at 60 Hz for grinding for 2 min;

[0051] (2) Place the crushing tube in a 65°C water bath for 20 min, invert the sample every 5 min, and unscrew the lid of the crushing tube to release gas and then tighten it again;

[0052] (3) Directly add 700 μL of phenol:chloroform (1:1) to the crushed tube after water bath for equal-volume extraction, and centrifuge at 12,000 rpm for 5 min;

[0053] (4) Take about 700 μL of the supernatant into a new 2 mL centrifuge tube, add 700 μL of chloroform and mix well, then centrifuge at 12,000 rpm for 5 min;

[0054] (5) Aspirate the upper aqueous phase obtained in (4) into a new 2 mL centrifuge tube, add 700 μL of buffer GP 2, and mix well;

[0055] (6) Transfer the solution obtained in (5) to the adsorption column CB 3 in portions of 700 μL each for centrifugation (12,000 rpm, 30 s), and discard the waste liquid;

[0056] (7) Add 500 μL of buffer GD (anhydrous ethanol has been added in the appropriate proportion as required by the instruction manual before use) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, pour out the waste liquid, and place the adsorption column in the collection tube;

[0057] (8) Add 600 μL of wash buffer PW (anhydrous ethanol has been added in the appropriate proportion as required by the instruction manual before use) to the adsorption column CB 3 and then centrifuge (12,000 rpm, 30 s), and discard the waste liquid in the tube;

[0058] (9) Repeat step (8);

[0059] (10) Centrifuge the collection tube with the adsorption column (12,000 rpm, 2 min), discard the collection tube, transfer the adsorption column to a new 1.5 mL centrifuge tube, and place it at room temperature for 10 min to fully dry the residual wash buffer in the column;

[0060] (11) Add 50 μL of elution buffer TE to the middle of the adsorption membrane, place it at room temperature for 5 min, and then centrifuge at 12,000 rpm for 5 min; measure the DNA concentration and the ratio of A260 / A280 of the liquid in the centrifuge tube using NanoDrop 2000, and then store it in a -34 °C refrigerator.

[0061] Example 3

[0062] Design of RPA primers for specific amplification of Venturia inaequalis, screening by conventional PCR, and design of probes. Using the V. inaequalis EF gene sequence as the target fragment, 7 pairs of primers were designed through the online design tool primer 3plus, as shown in Table 1 and Table 2:

[0063] Table 1 Sequence information of seven pairs of primers

[0064]

[0065]

[0066] After screening by conventional PCR amplification, primers F1 / R1, F2 / R1, F5 / R5, and F7 / R7 only amplified the V. inaequalis samples, while other primers also amplified non-target samples. Therefore, the primers for subsequent RPA detection will be selected from the above four pairs of primers. By comparing the EF gene sequences of V. inaequalis with other Venturia fungi, and limited by the lengths of the synthetic EF gene fragments of Venturia asperata and Venturia nashicola, F1 / R1 was finally selected for subsequent research ( Figure 1 ). When performing specific detection, primer F1 / R1 only amplified the EF gene fragment of Venturia inaequalis, and the background was clean ( Figure 2 ), and this primer will be used in subsequent experiments. A probe was designed referring to the amplification product of primer pair F1 / R1, and the specific sequence is shown in Table 2.

[0067] Table 2 Primers and sequences for PCR amplification and RPA amplification of Venturia inaequalis

[0068]

[0069] Example 4

[0070] RPA amplification:

[0071] (1) Extraction of target DNA: Extract the genomic DNA of Venturia inaequalis using the CTAB method.

[0072] (2) Using the genomic DNA of Venturia inaequalis as a template, and setting a blank control at the same time, RPA amplification was carried out using the primers in Example 3. The RPA amplification system is as follows: Add 29.4 μL of A Buffer, 2 μL of each upstream and downstream primer (10 μM), 2 μL of template to the RPA reaction tube containing freeze-dried powder, and finally add 2.5 μL of B Buffer. The total reaction system is 50 μL. The reaction conditions of RPA: Manually invert the above RPA reaction system up and down to mix well, and amplify at 39 °C for 15 min. Electrophoresis was carried out using 1.5% agarose gel to observe the amplification of the selected primers. It can be seen that the primers F1 / R1 only amplified the EF gene fragment of Venturia inaequalis, and the background was relatively clean, which was suitable for subsequent experiments( Figure 3 ). Select the reverse primer sequence V.in-LFD-R1 and label its 5′ end with biotin (Biotin), as shown in V.in LFD-R1B. Design a probe V.in-LFD-probe1 according to the amplification sequences of the forward and reverse primers, label its 5′ end with fluorescein (FAM), use tetrahydrofuran (THF) as a dSpacer to replace a base at the position 30 bases away from the 5′ end, and block the 3′ end with C3-spacer; the specific sequence is shown in Table 2.

[0073] Example 5

[0074] Establishment of the RPA-LFD detection method:

[0075] Using the extracted DNA as a template, and using the designed RPA primers, carry out RPA reaction in the following reaction system: Sample detection: Add 29.4 μL of A Buffer, 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 0.5 μL of 100 nM probe, 2.0 μL of DNA, 2.5 μL of B Buffer to a 0.2 mL reaction tube (Anpu Future Biotechnology Co., Ltd., DNA rapid amplification (colloidal gold test strip type) kit) containing freeze-dried powder, and make up to 50 μL with deionized water. After adding B Buffer, invert the reaction tube up and down 10 times to mix well, then centrifuge with a bench-top centrifuge, and immediately place the reaction tube in a 41 °C metal bath for a constant temperature reaction for 9 min. Take 10 μL of the products after the reaction of each treatment group and the control group, add them to a 1.5 mL EP tube containing 190 μL of ddH2O, mix well, and then insert the single-target HybriDetect lateral flow test strip into the dilution solution and wait for 3 min to observe the reaction result.

[0076] When two bands appear on the test strip, one in the control region and one in the test region, the result is positive, indicating that the sample contains Venturia inaequalis; when only one band (Control line) appears in the control region and no band appears in the test region on the test strip, the result is negative, indicating that the sample does not contain Venturia inaequalis.

[0077] Example 6

[0078] Specificity verification of RPA-LFD for detecting Venturia inaequalis:

[0079] To verify the specificity of the RPA-LFD method for detecting Venturia inaequalis, Venturia inaequalis strains and other common pathogenic fungi on apples and its closely related species Venturia nashicola were used as test materials as shown in Table 3, and experiments were carried out according to the reaction system of Example 5. The results of the RPA lateral flow chromatography test strip detection method showed that two bands (Control line and Test line) appeared on the test strip of Venturia inaequalis, one showing blue in the control region and the other showing red in the test region, then the result was positive (+), and only one band appeared in the control region and no band appeared in the test region on the test strips of the other common pathogenic fungi on apples and its closely related species Venturia nashicola, then the result was negative (-), indicating that the sample did not contain Venturia inaequalis. As Figure 3 shown, only the test line of the SZY-1 test strip of the positive sample of V. inaequalis showed red, and no bands appeared in the test line of other fungal samples and the negative control, proving that the specificity of this system is good and can be used for subsequent detection work.

[0080] Table 3 Samples and test results for RPA primer specificity detection Pathogenic bacteria

[0081]

[0082] The EF gene fragments of Mycosphaerella pomi and Venturia nashicola were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0083] Example 7

[0084] Sensitivity determination of RPA-LFD for detecting Venturia inaequalis:

[0085] The concentration of the Venturia inaequalis DNA extracted in Example 1 was measured by NanoDrop 2000 to be 611.6 ng / μL, and it was successively diluted to 6.116 ng / μL, 6.116×10 -1 ng / μL, 6.116×10 -2 ng / μL, 6.116×10 -3 ng / μL, 6.116×10 -4 ng / μL, 6.116×10-5 ng / μL, 6.116×10 -6 ng / μL, 6.116×10 -7 ng / μL, 6.116×10 -8 ng / μL, 6.116×10 -9 ng / μL. According to the primers, reaction system and reaction conditions used in Example 3 and Example 5, RPA-LFD detection was performed on DNA with different concentrations.

[0086] The results are as Figure 4 shown. The test strips in the reaction system containing 6.116 ng / μL, 6.116×10 -1 ng / μL, 6.116×10 -2 ng / μL, 6.116×10 -3 ng / μL, 6.116×10 -4 ng / μL of Venturia inaequalis DNA showed two bands (Control line and Test line), showing a strong positive reaction. The test strips in the reaction system containing 6.116×10 -5 ng / μL, 6.116×10 -6 ng / μL of Venturia inaequalis DNA also showed two bands, and the red color of the test line became lighter, showing a weak positive reaction. The test strips in the reaction system containing 6.116×10 -7 ng / μL, 6.116×10 -8 ng / μL, 6.116×10 -9 ng / μL of Venturia inaequalis DNA showed only one band (Control line), showing a negative reaction; the results showed that the sensitivity of the RPA-LFD technology reached 6.116×10 -6 ng / μL; the RPA-LFD detection time only takes 12 min, including 9 min of RPA amplification time and 3 min of waiting for the test results. The reaction process does not require expensive instrument equipment such as a PCR instrument, and the operation procedure is simple, which is more conducive to popularization and application in production.

[0087] Example 8

[0088] Detection of Venturia inaequalis plant samples by RPA-LFD:

[0089] To verify whether RPA-LFD can detect apple scab plant samples, diseased plant leaves and fruits, as well as healthy apple leaves and fruits, were used as test materials, and experiments were carried out according to the reaction systems of Example 2 and Example 5. The results of the RPA lateral flow chromatographic strip detection method showed that the test strips of the plant materials infected with Venturia inaequalis had two bands (Control line and Test line), one in the quality control area and the other in the test area, and the result was positive (+). For the remaining healthy apple leaves, fruits, and the test results using ddH2O as a template, only a blue band appeared in the quality control area of the test strip, and no band appeared in the test area, and the result was negative (-), indicating that the sample did not contain Venturia inaequalis. As Figure 5 shown, RPA-LFD tests were performed using genomic DNA of healthy plants and diseased plants as templates. The detection results of the DNA of diseased plants were consistent with those using genomic DNA of pure-cultured Venturia inaequalis as a template, and the depth of the color of the test line was the same, indicating that good detection results could be obtained using genomic DNA of diseased plant materials as a template, and no test line was produced when detecting healthy plants.

[0090] The above-mentioned embodiments / tests are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

[0091]

[0092]

[0093]

[0094]

[0095]

Claims

1. A primer set for detecting apple black spot fungus based on RPA-LFD technology, characterized in that: The forward primer sequence is shown as SEQ ID No.1, and the reverse primer sequence is shown as SEQ ID No.

2.

2. A kit for detecting apple black spot pathogen based on RPA-LFD technology, characterized in that: The method comprises the primer set and probe combination as described in claim 1, wherein the forward primer sequence of the primer set is shown as SEQ ID No. 1, the reverse primer sequence is shown as SEQ ID No. 2, the modified reverse primer sequence is shown as SEQ ID No. 3, and the probe sequence is shown as SEQ ID No.

4.

3. The kit according to claim 2, characterized in that: The kit also includes a reaction tube containing freeze-dried powder, A Buffer, B Buffer, deionized water, and LFD lateral flow chromatography test strips.

4. Use of the primer set according to claim 1, or the kit according to claim 2 or 3 in detecting apple scab pathogen.

5. A method for detecting apple black spot pathogen based on RPA-LFD technology, characterized in that: The following steps are involved: (1) Extracting DNA from the sample to be tested; (2) Using the DNA of the sample to be tested as a template, conventional PCR amplification is performed using the primers described in claim 1, and then a band with a fragment size of 204 bp is detected by agarose gel electrophoresis, indicating that the sample does not contain apple black spot pathogen; or RPA amplification is performed using the kit for detecting apple black spot pathogen described in claims 2, 3, and 4, and then the RPA amplification product is detected using a lateral flow chromatography test strip; when two bands appear on the test strip, one in the quality control area and the other in the detection area, the result is positive, indicating that the sample contains apple black spot pathogen; when only one band appears in the quality control area of ​​the test strip and there is no band in the detection area, the result is negative, indicating that the sample does not contain apple black spot pathogen.

6. The method for detecting apple black spot pathogen based on RPA-LFD technology according to claim 5, characterized in that: The RPA amplification system includes: 29.4 μL of A Buffer, 2 μL of each of 10 μM upstream and downstream primers, 0.5 μL of 100 nM probe, 2 μL of template, 2.5 μL of B Buffer, and a total reaction system of 50 μL.

7. The method for detecting apple black spot pathogen based on RPA-LFD technology according to claim 6, characterized in that: The reaction conditions of the RPA were as follows: the RPA reaction system was inverted and mixed thoroughly, and amplified at 39° C. for 15 min.

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