Rpa-exo product for detecting walnut anthracnose and application thereof

Abstract

The present application relates to the technical field of molecular biology, in particular to RPA-EXO product for detecting walnut anthracnose and application thereof. A group of walnut anthracnose specific primer probe combinations specially suitable for RPA-EXO isothermal amplification system is screened and optimized for the first time, which takes ITS conserved region as a target and is specially designed. The primer probe combination has good specificity and sensitivity, can complete nucleic acid amplification and detection under isothermal conditions at 39 DEG C, and only needs 10 min. The minimum detection limit can reach 10 ‑2 ng / μL. Through the introduction of RPA-EXO system, the detection speed is greatly improved (from several hours to 10 min), the operation process is greatly simplified (without complex thermal cycling), and the rapid diagnosis suitable for non-laboratory environment is realized. The overall detection scheme has significant synergistic optimization effect and practical application advantage.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to the RPA-EXO product for detecting walnut anthracnose and its applications. Background Technology

[0002] Walnuts, as an important economic forest tree species in China, are now cultivated on a large scale in many provinces, autonomous regions, and municipalities. Walnut kernels are rich in nutrients, possess antioxidant properties, and can scavenge free radicals, effectively preventing cardiovascular diseases and benefiting human health. However, with the expansion of concentrated walnut cultivation areas and the lack of disease-resistant varieties, walnut anthracnose has become a major disease in walnut production, severely hindering the healthy development of the walnut industry.

[0003] Fungal pathogen taxonomy studies have shown that walnut anthracnose is caused by infection with various anthracnose fungi, the main pathogens including *Colletotrichum fioriniae* (C. fioriniae), *Colletotrichum fructicola* (C. fructicola), *Colletotrichum gloeosporioides* (C. gloeosporioides), *Colletotrichum godetiae* (C. godetiae), *Colletotrichum karsti* (C. karsti), *Colletotrichum nymphaeae* (C. nymphaeae), and *Colletotrichum siamense* (C. siamense). Traditional detection techniques can only detect a single pathogen in walnut anthracnose, and have significant limitations such as low sensitivity and long detection time. Therefore, there is an urgent need to develop a method that is highly sensitive, has a short detection time, and can detect multiple pathogens. Summary of the Invention

[0004] The purpose of this invention is to provide an RPA-EXO product for detecting walnut anthracnose and its applications, thereby addressing the problems existing in the prior art. This invention provides an optimal primer-probe combination for RPA-EXO detection of walnut anthracnose, and based on this primer-probe combination, develops a rapid detection system for walnut anthracnose based on recombinase polymerase amplification-exonuclease technology (RPA-EXO), enabling efficient, accurate, and simultaneous detection of multiple anthracnose fungi. This invention provides an efficient solution for the early monitoring and control of walnut anthracnose, and is of great significance for ensuring the sustainable development of the walnut industry.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides an RPA-EXO universal primer and probe set for detecting walnut anthracnose, the RPA-EXO universal primer and probe set comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.5, and a probe with a nucleotide sequence as shown in SEQ ID NO.11;

[0007] The pathogens causing the aforementioned walnut anthracnose include *Colletotrichum fioriniae*, *Colletotrichum fructicola*, *Colletotrichum gloeosporioides*, *Colletotrichum godetiae*, *Colletotrichum karsti*, *Colletotrichum nymphaeae*, and *Colletotrichum siamense*.

[0008] This invention provides the application of the above-mentioned RPA-EXO universal primer and probe set in the preparation of products for detecting walnut anthracnose. The pathogens causing walnut anthracnose include *Anthracnose pinenei*, *Anthracnose caecifolius*, *Anthracnose glomeratus*, *Anthracnose spp.*, *Anthracnose karstii*, *Anthracnose lilyii*, and *Anthracnose siamensis*.

[0009] Preferably, the product includes reagents, reagent kits, or chips.

[0010] This invention provides a product for detecting walnut anthracnose, the product comprising the aforementioned RPA-EXO universal primer and probe set.

[0011] Preferably, the product includes reagents, reagent kits, or chips.

[0012] This invention provides the application of the above-mentioned RPA-EXO universal primer and probe set or the above-mentioned product in the detection of walnut anthracnose. The pathogens causing walnut anthracnose include *Anthracnose pinenei*, *Anthracnose caecifolius*, *Anthracnose glomeratus*, *Anthracnose galbana*, *Anthracnose kastyrax*, *Anthracnose lily*, and *Anthracnose siamensis*.

[0013] This invention provides a method for detecting walnut anthracnose, comprising using the DNA of the sample to be tested as a template, performing RPA-EXO detection using the aforementioned RPA-EXO universal primer and probe set to obtain a fluorescence value; and determining whether the sample to be tested suffers from walnut anthracnose based on the fluorescence value.

[0014] The pathogens causing the aforementioned walnut anthracnose include *Anthracnose pineneus*, *Anthracnose fruitensis*, *Anthracnose collodion*, *Anthracnose gorgoniana*, *Anthracnose karstii*, *Anthracnose water lily*, and *Anthracnose siamensis*.

[0015] Preferably, the RPA-EXO detection temperature is 39°C and the time is 10 minutes.

[0016] Preferably, the RPA-EXO detection system is 50 μL, comprising 32.9 μL A Buffer, 2.5 μL B Buffer, 2 μL upstream primer, 2 μL downstream primer, 2 μL DNA template, 8 μL ddH2O and 0.6 μL probe.

[0017] Preferably, the working concentrations of the upstream primer, the downstream primer, and the probe are all 10 μM.

[0018] The present invention discloses the following technical effects:

[0019] This invention is the first to screen and optimize a set of walnut anthracnose-specific primer-probe combinations specifically suitable for the RPA-EXO isothermal amplification system. Targeting the conserved region of the ITS (intracytoplasmic thyroid hormone), and specially designed, this primer-probe combination exhibits excellent specificity and sensitivity, enabling nucleic acid amplification and detection to be completed in just 10 minutes under isothermal conditions at 39°C, with a detection limit of 10. -2 ng / μL. This invention, through the introduction of the RPA-EXO system, achieves a significant increase in detection speed (reduced from several hours to 10 minutes), a greatly simplified operation process (eliminating the need for complex thermal cycling), and rapid diagnosis suitable for non-laboratory environments. The overall detection scheme exhibits significant synergistic optimization effects and practical application advantages. Simultaneously, this invention can detect multiple pathogens causing walnut anthracnose, avoiding misdiagnosis caused by the inability to detect multiple pathogens, and enabling early detection of walnut anthracnose.

[0020] Therefore, this invention not only provides a highly efficient and effective detection method for walnut anthracnose, but more importantly, it constructs a complete detection system based on isothermal rapid amplification technology. This system is characterized by its speed, simplicity, accuracy, ease of promotion, and low dependence on equipment, filling a gap in existing rapid detection technologies for walnut anthracnose and possessing significant practical value and promising prospects for widespread application. The proposal of this invention provides an efficient solution for the early monitoring and control of walnut anthracnose, which is of great significance for ensuring the sustainable development of the walnut industry. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a diagram showing the specificity results of primer pair F1 / R1; where 1-7 are positive templates, namely *Anthracis chinensis*, *Anthracis oryzae*, *Colletotrichum gloeosporioides*, *Anthracis chinensis*, *Anthracis calcareosa*, *Anthracis karstii*, *Anthracis nephrolepis*, and *Anthracis siamensis*; 8 is the blank control, double-deionized water; 9-16 are negative templates, namely *Botrytis cinerea*, *Ceratophyllum demersum*, *Diplostomum tarda*, *Discostomium walnutum*, *Ceratophyllum demersum*, *Clostridium difficile*, *Pseudomonas citrinum*, and *Trichoderma purpureus*.

[0023] Figure 2 The image shows the fluorescence measurement results of the optimal primer pair F1 / R1 reaction conditions for RPA-EXO (amplification for 10 min); where 1-8 represent Anthracnose of Pine Needle, Anthracnose of Fruit, Anthracnose of Colloidal Sporozoa, Anthracnose of High-Generation Flower, Anthracnose of Karst, Anthracnose of Water Lily, Anthracnose of Siam, and double deionized water, respectively. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] The strains used in this invention and their sources:

[0030] *Colletotrichum fioriniae* (C. fioriniae) is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Colletotrichum fructicola* (C. fructicola) is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Colletotrichum gloeosporioides* (C. gloeosporioides) is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Colletotrichum godetiae* (C. godetiae) is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Colletotrichum karsti* (C. karsti) is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Colletotrichum nymphaeae* (C. * *Nymphaeae* is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Colletotrichum siamense* (C. siamense) is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Botryosphaeria dothidea* is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Diaporthe eres* is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Diplodia mutila* is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Juglanconis juglandina* is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Nectria pseudotrichia* is listed in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Neofusicoccum* *Parvum* is published in the literature "List of Pathogenic Fungi of Walnut Diseases in China"; *Paraeutypella citricola* is published in the literature "Paraeutypella guizhouensis gen. etsp. nov. and Diatrypella longiasca sp. nov. (Diatrypaceae) from China"; *Trichothecium roseum* is published in the literature "Systema Mycologicum" (Fries, EM.1832. Systema Mycologicum).3(2): 261-524); The applicant promises to issue the publication for 20 years from the date of publication.

[0031] Example 1: Genomic DNA extraction using the CTAB method

[0032] (1) Use a sterilized pipette tip to scrape the hyphae of the aforementioned strain, collect the hyphae into a 1.5 mL centrifuge tube, add 4-10 sterilized small steel balls, add 200 μL of CTAB to the centrifuge tube, and grind and shake evenly in a grinder;

[0033] (2) After grinding, add 800 μL CTAB and 100 μL SDS to the centrifuge tube and heat in a water bath at 60°C for 40 min;

[0034] (3) After water bath, centrifuge at 13000rpm for 10min, take 800μL of the clear liquid into a new 2mL centrifuge tube, add 800μL of chloroform, shake vigorously, and centrifuge at 13000rpm for another 10min.

[0035] (4) Take 600 μL of the supernatant into a new 1.5 mL centrifuge tube, add 600 μL of isopropanol, mix by inverting, let stand at 4 °C for 20 min, and centrifuge at 13000 rpm for 10 min.

[0036] (5) Discard the supernatant, add 800 μL of 75% ethanol, and centrifuge at 13000 rpm for 7 min;

[0037] (6) Drain the DNA precipitate, add 50 μL of pure water to dissolve it, and store it in a -20℃ refrigerator.

[0038] Example 2: Genomic DNA was extracted using a plant sample nucleic acid rapid release agent (DNA type) (using a product from Beijing Huakang Innovation Technology Co., Ltd.)

[0039] (1) Take a 0.5mm×0.5mm walnut anthracnose sample tissue, place it in a 1.5mL centrifuge tube, and grind it with a disposable grinding stick;

[0040] (2) Add 50 μL of plant sample nucleic acid rapid release agent (DNA type), invert and mix well to ensure that the sample and reagent are fully mixed;

[0041] (3) Let stand for 5-10 minutes. The liquid in the tube is the extracted and purified genomic DNA of the sample.

[0042] This invention rapidly extracts DNA from walnut anthracnose samples using the CTAB method, and performs RPA-EXO amplification using the optimal primer-probe combination screened in Example 5. Ultimately, the RPA-EXO method successfully detected diseased walnut anthracnose samples. If a plant sample nucleic acid rapid release agent (DNA type) is used to extract DNA, the entire process takes only about 15 minutes, is simple to operate, requires no complex instruments, and provides real-time field detection capabilities.

[0043] Example 3 Primer and probe design

[0044] To specifically amplify RPA primers and probes for walnut anthracnose, conserved ITS genes from the above-mentioned *Anthracnose pinenei*, *Anthracnose caecifolius*, *Colletotrichum gloeosporioides*, *Anthracnose spp.*, *Anthracnose karstii*, *Anthracnose lilyii*, and *Anthracnose siamensis* strains were selected as target sequences. Using the Primer 3 Plus online design tool, a total of 10 primer pairs were designed, including 4 upstream primers and 6 downstream primers, as shown in Table 1.

[0045] Table 1 Primer and probe sequences for RPA amplification in walnut anthracnose.

[0046]

[0047] Example 4: Establishment of the RPA-EXO detection method

[0048] (1) Using the genomic DNA extracted in Example 1 as DNA templates (Anthracis pineensis, Anthracnoseus caecifolius, Anthracnoseus collodionus, Anthracnoseus caecifolius, Anthracnoseus karstii, Anthracnoseus lilyii, and Anthracnoseus siamensis), blank controls (double deionized water) and negative templates (Botrytis cinerea, Prunus cerasifera, Diplostomum tarda, Discostomum walnutis, Pseudomonas erythroderma, Clostridium difficile, Pseudomonas citrinum, and Trichoderma purpureus), RPA amplification was performed by combining primers from Example 3 in pairs. The RPA amplification system is as follows (total reaction volume 50 μL): Add 32.9 μL of Buffer A, 2.5 μL of Buffer B, 2 μL of upstream primer (10 μM), 2 μL of downstream primer (10 μM), 2 μL of DNA template, and 8 μL of ddH2O to an RPA reaction tube containing lyophilized powder. Finally, add 0.6 μL of probe EJT-RPA-P (10 μM). Buffer A and Buffer B were purchased from Suzhou Geneno Biomedical Technology Co., Ltd. RPA reaction conditions: Mix the above RPA reaction system thoroughly and amplify at 39℃ for 20 min.

[0049] The primer pairs with the best amplification effect can be screened by observing the fluorescence amplification curve and peak time. Successful amplification of a positive sample is indicated by a gradual increase in fluorescence signal during the reaction process.

[0050] Example 5: Validation of the specificity of walnut anthracnose detection

[0051] To verify the specificity of the RPA-EXO method for detecting walnut anthracnose, the main pathogen of walnut anthracnose and other common pathogenic fungi on walnuts were used as test materials, and experiments were conducted according to the RPA reaction system in Example 4. The experimental results are shown in Tables 2-5. Figure 1 As shown. The results indicate that only primer F1 / R1 amplified only *Anthracis walnutensis* (including *Anthracis pineinae*, *Anthracis caesarea*, *Colletotrichum gloeosporioides*, *Anthracis chinensis*, *Anthracis kastriata*, *Anthracis lily*, and *Anthracis siamensis*), and the signal was strong, making it suitable for subsequent experiments. No amplification was observed in other negative samples. Figure 1 ).

[0052] Table 2. Pathogens and Detection Results Specific to Primer F1 / R Combinations

[0053]

[0054] Note: + indicates good amplification and can be detected; - indicates no amplification and cannot be detected; the same applies to the following table.

[0055] Table 3. Pathogens and Detection Results Specific to Primer F2 / R Combinations

[0056]

[0057] Table 4. Pathogens and Detection Results Specific to Primer F3 / R Combination

[0058]

[0059] Table 5. Pathogens and Detection Results Specifically Detected by Primer F4 / R Combination

[0060]

[0061] Example 6: Optimization of RPA-EXO Detection Conditions

[0062] The optimal primer pair F1 / R1 was used to identify *Anthracis pineensis*, *Anthracis fruitensis*, *Colletotrichum gloeosporioides*, *Anthracis chinensis*, *Anthracis moniliformis*, *Anthracis karstii*, *Anthracis lilyii*, and *Anthracis siamensis*. The RPA reaction system was prepared according to the method described in Example 4. The RPA reaction conditions were as follows: the RPA reaction system was thoroughly mixed and amplified at a constant temperature of 39°C for 5 min, 10 min, 15 min, and 20 min, respectively, with double-deionized water as a blank control. The optimal reaction time was determined based on the Ct value and fluorescence intensity (the results were interpreted based on the presence or absence of fluorescence and the rising fluorescence curve). The results are as follows: Figure 2As shown, after 10 minutes of amplification, the F1 / R1 primer pair exhibited the fastest amplification rate and fluorescence signal peak. Therefore, the optimal amplification time for the RPA-EXO detection method constructed in this invention is 10 minutes.

[0063] Example 7: Sensitivity determination of RPA-EXO for detecting walnut anthracnose

[0064] Using genomic DNA of walnut anthracnose pathogens (Pine needle anthracnose, Fruit anthracnose, Colletotrichum anthracnose, High-generation flower anthracnose, Karst anthracnose, Water lily anthracnose, and Siamese anthracnose) as templates, sterile water was used for 10... 0 ~10 -3 The DNA was serially diluted to a concentration of ng / μL and then subjected to RPA-EXO amplification. The reaction system and conditions were the same as in Example 6 (amplification at 39℃ for 10 min). Double deionized water was used as a blank control. The amplification results were observed by fluorescence curves to explore the sensitivity of RPA-EXO in detecting walnut anthracnose samples. The experiment was repeated three times. The results showed that the DNA concentration at 10 ng / μL was optimal. 0 ~10 -2 Within the ng / μL range, increasing curves and high fluorescence intensity were observed (Table 6). Experimental results show that the established RPA-EXO method can detect concentrations of 10... 0 ~10 -2 The detection limit of this method is 10 ng / μL for genomic DNA of the walnut anthracnose pathogen. -2 ng / μL genomic DNA.

[0065] Table 6. Sensitivity detection results of primer F1 / R1

[0066]

[0067] Note: + indicates good amplification and can be detected; - indicates no amplification and cannot be detected.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of RPA-EXO universal primer and probe set or product in the detection of walnut anthracnose, characterized in that, The RPA-EXO universal primer and probe set includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.5, and a probe with a nucleotide sequence as shown in SEQ ID NO.11; The pathogens causing the aforementioned walnut anthracnose include *Colletotrichum fioriniae*, *Colletotrichum fructicola*, *Colletotrichum gloeosporioides*, *Colletotrichum godetiae*, *Colletotrichum karsti*, *Colletotrichum nymphaeae*, and *Colletotrichum siamense*. The product includes the RPA-EXO universal primer and probe set; The application includes using the DNA of the sample to be tested as a template, performing RPA-EXO detection using the RPA-EXO universal primer and probe set, and obtaining fluorescence values; The fluorescence value is used to determine whether the sample to be tested has walnut anthracnose. The RPA-EXO was used to detect a temperature of 39°C for 10 minutes.

2. The application according to claim 1, characterized in that, The products include reagents, reagent kits, or chips.

3. A method for detecting walnut anthracnose, characterized in that, The method includes using the DNA of the sample to be tested as a template, performing RPA-EXO detection using the RPA-EXO universal primer and probe set described in claim 1, and obtaining fluorescence values; determining whether the sample to be tested suffers from walnut anthracnose based on the fluorescence values; The pathogens that cause the walnut anthracnose include *Anthracnose pineneus*, *Anthracnose fruitensis*, *Anthracnose collodion*, *Anthracnose high-generation flower*, *Anthracnose karstii*, *Anthracnose water lily*, and *Anthracnose siamensis*. The RPA-EXO was used to detect a temperature of 39°C for 10 minutes.

4. The detection method according to claim 3, characterized in that, The RPA-EXO detection system is 50 μL, including 32.9 μL A Buffer, 2.5 μL B Buffer, 2 μL upstream primer, 2 μL downstream primer, 2 μL DNA template, 8 μL ddH2O and 0.6 μL probe.

5. The detection method according to claim 3, characterized in that, The working concentrations of the upstream primer, the downstream primer, and the probe are all 10 μM.

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