RPA-EXO product for detecting walnut anthracnose and application of RPA-EXO product

Abstract

The invention relates to the technical field of molecular biology, in particular to an RPA-EXO product for detecting walnut anthracnose and application of the RPA-EXO product. The walnut anthracnose specific primer probe combination specially suitable for an RPA-EXO isothermal amplification system is screened and optimized for the first time, an ITS conserved region serves as a target, through special design, the primer probe combination has good specificity and sensitivity, nucleic acid amplification and detection can be completed only within 10 min under the isothermal condition of 39 DEG C, and the primer probe combination has the advantages of being high in specificity, high in sensitivity, high in specificity and high in specificity and sensitivity. And the lowest detection limit can reach 10 <-2 > ng / mu L. The RPA-EXO system is introduced, so that the detection speed is greatly increased (shortened from several hours to 10 minutes), the operation process is greatly simplified (no complex thermal cycle is needed), the method is suitable for rapid diagnosis in a non-laboratory environment, and the whole detection scheme has a remarkable collaborative optimization effect and practical application advantages.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to an RPA-EXO product for detecting walnut anthrax and applications thereof. Background Art

[0002] Walnut, an important economic tree species in China, is cultivated on a large scale in many provinces (autonomous regions, and municipalities). Walnut kernels are rich in nutrients, have antioxidant and free radical scavenging properties, and are effective in preventing cardiovascular disease and benefiting human health. However, with the expansion of concentrated walnut cultivation and the lack of disease-resistant varieties, walnut anthracnose has become a major disease in walnut production, seriously hindering the healthy development of the walnut industry.

[0003] Fungal pathogen taxonomic studies have shown that walnut anthracnose is caused by a variety of anthracnose fungi, 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 of walnut anthracnose, resulting in significant limitations such as low sensitivity and time-consuming testing. Therefore, there is an urgent need to develop a method with high sensitivity, short detection time and the ability to detect multiple pathogens. Summary of the Invention

[0004] The present invention aims to provide an RPA-EXO product and its application for detecting walnut anthracnose, thereby addressing the aforementioned problems of the prior art. This invention provides an optimized primer-probe combination for RPA-EXO detection of walnut anthracnose. Based on this primer-probe combination, a rapid walnut anthracnose detection system based on recombinase polymerase amplification-exonuclease technology (RPA-EXO) has been developed. This system enables efficient, accurate, and simultaneous detection of multiple anthracnose fungi. This invention provides a highly effective solution for the early monitoring and prevention of walnut anthracnose and is of great significance for ensuring the sustainable development of the walnut industry.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The application provides a RPA-EXO universal primer probe set for detecting walnut anthracnose, the RPA-EXO universal primer 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 pathogenic bacteria causing the walnut anthracnose include Colletotrichum fioriniae, Colletotrichum fructicola, Colletotrichum gloeosporioides, Colletotrichum godetiae, Colletotrichum karsti, Colletotrichum nymphaeae and Colletotrichum siamense.

[0008] The application provides application of the RPA-EXO universal primer probe set in preparation of a product for detecting walnut anthracnose, and the pathogenic bacteria causing the walnut anthracnose include Colletotrichum fioriniae, Colletotrichum fructicola, Colletotrichum gloeosporioides, Colletotrichum godetiae, Colletotrichum karsti, Colletotrichum nymphaeae and Colletotrichum siamense.

[0009] Preferably, the product comprises a reagent, a kit or a chip.

[0010] The application provides a product for detecting walnut anthracnose, and the product comprises the RPA-EXO universal primer probe set.

[0011] Preferably, the product comprises a reagent, a kit or a chip.

[0012] The application provides application of the RPA-EXO universal primer probe set or the product in detection of walnut anthracnose, and the pathogenic bacteria causing the walnut anthracnose include Colletotrichum fioriniae, Colletotrichum fructicola, Colletotrichum gloeosporioides, Colletotrichum godetiae, Colletotrichum karsti, Colletotrichum nymphaeae and Colletotrichum siamense.

[0013] The application provides a detection method for walnut anthracnose, comprising the following steps: taking DNA of a sample to be detected as a template, performing RPA-EXO detection on the sample to be detected by using the RPA-EXO universal primer probe set, and obtaining a fluorescence value; and determining whether the sample to be detected is infected with walnut anthracnose according to the fluorescence value.

[0014] The pathogens causing the walnut anthracnose include pine needle anthracnose, fruit anthracnose, gloeosporium anthracnose, high-flower anthracnose, karst anthracnose, water lily anthracnose and Siamese anthracnose.

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

[0016] Preferably, 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.

[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] The present invention screened and optimized a set of walnut anthracnose-specific primer-probe combinations specifically suitable for the RPA-EXO isothermal amplification system for the first time. The primer-probe combination targets the conserved ITS region and is specially designed to have good specificity and sensitivity. It can complete nucleic acid amplification and detection in just 10 minutes under isothermal conditions at 39°C, and its minimum detection limit reaches 10 -2 ng / μL. By introducing the RPA-EXO system, this invention significantly improves detection speed (from several hours to 10 minutes), greatly simplifies the operational process (no complex thermal cycling required), and makes it suitable for rapid diagnosis in non-laboratory environments. The overall detection solution has significant synergistic optimization effects and practical application advantages. Furthermore, this invention can detect multiple pathogens that cause walnut anthracnose, avoiding misdiagnosis caused by the inability to detect multiple pathogens and enabling early detection of walnut anthracnose.

[0020] This shows that the present invention not only provides an efficient and effective detection method for walnut anthrax, but more importantly, it constructs a complete detection system based on isothermal rapid amplification technology, which is fast, simple, accurate, easy to promote, and has low dependence on instruments and equipment. It fills the gap in existing walnut anthrax rapid detection technology and has significant practical value and promotion prospects. The proposal of the present invention provides an efficient solution for the early monitoring and prevention and control of walnut anthrax, which is of great significance for ensuring the sustainable development of the walnut industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to illustrate the technical solutions in the embodiments of the present application or the prior art more clearly, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without any creative effort.

[0022] Figure 1 Figure 4 is a specific result diagram of the primer pair F1 / R1; wherein 1-7 are positive templates, which are respectively G. juniperi, G. fructuolum, G. glosporum, G. subalpinum, G. karstii, G. nasturtium, and G. siamense; 8 is a blank control double deionized water; 9-16 are negative templates, which are respectively V. uva-cris, D. sacubinum, D. destructor, P. juglandis, C. globoidea, N. novoschwarzii, and P. citricarpa;

[0023] Figure 2 Figure 5 is a fluorescence determination result diagram of the RPA-EXO optimal primer pair F1 / R1 reaction condition optimization (amplification for 10 min); wherein 1-8 are respectively G. juniperi, G. fructuolum, G. glosporum, G. subalpinum, G. karstii, G. nasturtium, G. siamense, and double deionized water. DETAILED DESCRIPTION

[0024] The various illustrative embodiments of the present application will now be described in detail below. This detailed description is not intended to limit the present application, but rather to provide a more full understanding of certain aspects, features and embodiments of the present application.

[0025] It should be understood that the terms used in the present application merely describe particular embodiments, and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within any stated range or within any stated intermediate value, as well as any other stated value or intermediate value within the stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated by reference, the content of the specification prevails.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

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

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

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

[0031] Example 1: Extraction of genomic DNA 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, and add 200 μL CTAB to the centrifuge tube, place it in a grinder and grind it evenly;

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

[0034] (3) After water bath, centrifuge at 13000 rpm for 10 min, remove 800 μL of the supernatant and place it in a new 2 mL centrifuge tube, add 800 μL of chloroform, shake vigorously, and centrifuge again at 13000 rpm for 10 min;

[0035] (4) Take 600 μL of the supernatant and place it in a new 1.5 mL centrifuge tube. Add 600 μL of isopropanol, mix thoroughly by inverting the tube, let it stand at 4°C for 20 min, and centrifuge at 13,000 rpm for 10 min.

[0036] (5) Discard the supernatant, add 800 μL of 75% alcohol, and centrifuge at 13,000 rpm for 7 minutes;

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

[0038] Example 2 Extraction of genomic DNA using a plant sample nucleic acid rapid release agent (DNA type) (product of Beijing Huakang Innovation Technology Co., Ltd.)

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

[0040] (2) Add 50 μL of plant sample nucleic acid rapid release reagent (DNA type) and mix thoroughly by inverting the tube to ensure that the sample and reagent are fully mixed;

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

[0042] The present invention uses the CTAB method to rapidly extract DNA from walnut anthracnose samples. RPA-EXO amplification is then performed using the optimal primer-probe combination selected in Example 5. Ultimately, the RPA-EXO method successfully detects walnut anthracnose-susceptible samples. Using a DNA-based rapid nucleic acid release reagent for plant sample extraction, the entire process takes only approximately 15 minutes, is simple to operate, and requires no complex instrumentation, enabling instant field detection.

[0043] Example 3 Primer and probe design

[0044] RPA primers and probes for specific amplification of walnut anthracnose were designed. The conserved genes of the ITS of the above-mentioned pine needle anthracnose, fruit anthracnose, gloeosporium anthracnose, high-generation flower anthracnose, karst anthracnose, water lily anthracnose and siam anthracnose strains were selected as target sequences. A total of 10 primer pairs were designed using the Primer 3 Plus online design tool, including 4 upstream primers and 6 downstream primers, as shown in Table 1.

[0045] Table 1 Primer and probe sequences used for RPA amplification of walnut anthracnose

[0046]

[0047] Example 4 Establishment of RPA-EXO Detection Method

[0048] (1) The genomic DNA extracted in Example 1 was used as a DNA template (Coleotrichum pine needles, Coleotrichum fruit, Coleotrichum gloeosporioides, Coleotrichum karstii, Coleotrichum water lily and Coleotrichum siamese), and a blank control (double deionized water) and a negative template (Coleotrichum botrytis, C. sweet cherry, C. damaged color, C. walnut black disc, C. pseudo-hairy red shell, C. small new shell, C. citrus black cortex and C. pink) were set up at the same time. The primers in Example 3 were combined in pairs to perform RPA amplification. The RPA amplification system (total reaction system, 50 μL) was as follows: 32.9 μL of A Buffer, 2.5 μL of B Buffer, 2 μL of upstream primer (10 μM), 2 μL of downstream primer (10 μM), 2 μL of DNA template, 8 μL of ddH₂O, and finally 0.6 μL of the probe EJT-RPA-P (10 μM) were added to an RPA reaction tube containing lyophilized powder. Buffer A and Buffer B were purchased from Suzhou Kinno Biomedical Technology Co., Ltd. RPA reaction conditions: The RPA reaction system was thoroughly mixed and amplified at 39°C for 20 min.

[0049] By observing the fluorescence amplification curve and peak time, you can screen for primer pairs with optimal amplification effects. Successful amplification of positive samples is indicated by a gradual increase in the fluorescence signal during the reaction.

[0050] Example 5 Verification of specificity for detecting walnut anthrax

[0051] To verify the specificity of the RPA-EXO method for detecting walnut anthracnose, the main pathogens of walnut anthracnose and other common pathogenic fungi on walnuts were used as test materials and the RPA reaction system in Example 4 was used for experiments. The experimental results are shown in Tables 2-5 and Figure 1 The results showed that only primers F1 / R1 amplified walnut anthracnose (pine needle anthracnose, fruit anthracnose, gloeosporium anthracnose, high-generation flower anthracnose, karst anthracnose, water lily anthracnose and Siamese anthracnose), and the signal was strong, suitable for subsequent tests. Other negative samples did not show amplification ( Figure 1 ).

[0052] Table 2 Pathogens specifically detected by primer combination F1 / R and detection results

[0053]

[0054] Note: +, good amplification, detectable; -, no amplification, undetectable; same as the following table.

[0055] Table 3 Pathogens specifically detected by primer combination F2 / R and detection results

[0056]

[0057] Table 4 Pathogens specifically detected by primer combination F3 / R and detection results

[0058]

[0059] Table 5 Pathogens specifically detected by primer combination F4 / R and detection results

[0060]

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

[0062] The screened optimal primer pair F1 / R1 was used to identify pine needle anthracnose, fruit anthracnose, gloeosporium anthracnose, high-generation flower anthracnose, karst anthracnose, water lily anthracnose, and siam anthracnose. The RPA reaction system in Example 4 was configured. The RPA reaction conditions were as follows: the above RPA reaction system was thoroughly mixed and amplified at a constant temperature of 39°C for 5 minutes, 10 minutes, 15 minutes, and 20 minutes, respectively. Double deionized water was used 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 value and rising fluorescence curve). The results are shown in Figure 4. Figure 2As shown, after 10 minutes of amplification, the F1 / R1 primer pair showed the fastest amplification speed and fluorescence signal peak. Therefore, the optimal amplification time of the RPA-EXO detection method constructed by the present invention is 10 minutes.

[0063] Example 7 Sensitivity Determination of RPA-EXO for Detecting Walnut Anthracnose

[0064] The genomic DNA of walnut anthracnose pathogens (Colletotrichum pine needles, Colletotrichum maltophilum, Colletotrichum gloeosporioides, Colletotrichum karstii, Colletotrichum waterlily and Colletotrichum siamese) was used as template and the samples were purified by filtration with sterile water for 10 min. 0 ~10 -3 ng / μL concentration gradient dilution was performed for RPA-EXO amplification. The reaction system and conditions were the same as those in Example 6 (10 min at 39°C constant temperature). Double deionized water was used as a blank control. The amplification results were observed by fluorescence curve to explore the sensitivity of RPA-EXO in detecting walnut anthracnose samples. The experiment was repeated 3 times. The results showed that the DNA mass concentration was 10 0 ~10 -2 ng / μL, an upward curve was observed with high fluorescence intensity (Table 6). The experimental results showed that the established RPA-EXO method can detect the concentration of 10 0 ~10 -2 ng / μL of genomic DNA of the pathogen of walnut anthracnose, the detection limit of this method is 10 -2 ng / μL genomic DNA.

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

[0066]

[0067] Note: +, good amplification and detectable; -, no amplification and undetectable.

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

Claims

1. An RPA-EXO universal primer probe set for detecting walnut anthrax, characterized in that: The RPA-EXO universal primer 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 walnut anthracnose include Colletotrichum fioriniae, Colletotrichum fructicola, Colletotrichum gloeosporioides, Colletotrichum godetiae, Colletotrichum karsti, Colletotrichum nymphaeae and Colletotrichum siamense.

2. Use of the RPA-EXO universal primer probe set according to claim 1 in preparing a product for detecting walnut anthrax, characterized in that, The pathogens causing the walnut anthracnose include pine needle anthracnose, fruit anthracnose, gloeosporium anthracnose, high-flower anthracnose, karst anthracnose, water lily anthracnose and Siamese anthracnose.

3. The use according to claim 2, characterized in that The product includes a reagent, a kit or a chip.

4. A product for detecting walnut anthrax, characterized in that: The product includes the RPA-EXO universal primer probe set according to claim 1.

5. The product according to claim 4, characterized in that The product includes a reagent, a kit or a chip.

6. Use of the RPA-EXO universal primer probe set according to claim 1 or the product according to claim 4 or 5 in detecting walnut anthracnose, characterized in that: The pathogens causing the walnut anthracnose include pine needle anthracnose, fruit anthracnose, gloeosporium anthracnose, high-flower anthracnose, karst anthracnose, water lily anthracnose and Siamese anthracnose.

7. A method for detecting walnut anthrax, characterized in that: The method comprises using the DNA of the sample to be tested as a template and performing RPA-EXO detection using the RPA-EXO universal primer probe set of claim 1 to obtain a fluorescence value; and determining whether the sample to be tested has walnut anthracnose according to the fluorescence value; The pathogens causing the walnut anthracnose include pine needle anthracnose, fruit anthracnose, gloeosporium anthracnose, high-flower anthracnose, karst anthracnose, water lily anthracnose and Siamese anthracnose.

8. The detection method according to claim 7, characterized in that The temperature of the RPA-EXO detection was 39° C. and the detection time was 10 min.

9. The detection method according to claim 7, 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.

10. The detection method according to claim 7, characterized in that: The working concentrations of the upstream primer, the downstream primer and the probe were all 10 μM.

Images