Plant pathogen nucleic acid rapid extraction reagent, use method and molecular detection kit thereof

Abstract

This invention relates to a rapid nucleic acid extraction reagent for plant pathogens, its usage method, and a molecular detection kit. The invention provides a nucleic acid extraction reagent for plant pathogens and a lysis reagent containing this extraction reagent. On one hand, PVP-40 is added to the extraction reagent to improve cell permeability and enhance the nucleic acid extraction efficiency for plant pathogens, thereby improving detection efficiency and enabling rapid detection of plant pathogens. Simultaneously, the addition of SDS and CTAB to the lysis reagent synergistically disrupts the cell walls of plant pathogens, further enhancing the extraction efficiency of plant pathogen nucleic acids. Combined with magnetic bead technology, this enables batch extraction and detection analysis of plant pathogens. This invention provides reagents and methods for rapid and batch detection of plant pathogen nucleic acids, meeting the needs of different scenarios and suitable for rapid molecular detection of pathogens in field-exposed diseased plant tissues and pure cultured pathogens.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid extraction technology, and relates to a rapid extraction reagent for plant pathogen nucleic acids, its usage method, and its molecular detection kit. Specifically, it is a rapid extraction reagent for plant pathogen nucleic acids that combines direct extraction and magnetic bead extraction functions, its usage method, and its molecular detection kit. Background Technology

[0002] Rapid detection of plant pathogens is a crucial foundation for plant disease control. Pathogen identification and diagnosis based on molecular biology techniques such as PCR and LAMP all require obtaining high-quality pathogen nucleic acids first. Therefore, establishing a simple, rapid, and efficient nucleic acid extraction method suitable for plant pathogens has significant practical value.

[0003] Currently, methods for extracting nucleic acids from plant pathogens, such as the SDS method and the CTAB method, have been reported. While these methods can obtain nucleic acids with high purity, the procedures are complex, involving multiple steps such as centrifugation and washing, making it difficult to achieve high-throughput rapid preparation. Furthermore, some steps use toxic chemical reagents, posing safety hazards to the operating environment and personnel. In recent years, some biotechnology companies have developed genomic nucleic acid extraction kits, but existing kits still involve numerous procedures, resulting in high nucleic acid extraction costs. Moreover, they are mostly designed for single-type samples such as pure cultured mycelia or plant tissues, making them unsuitable for the rapid processing of both field-borne diseased tissues and pure laboratory culture samples, thus their versatility needs improvement.

[0004] Chinese patent CN119162165A discloses a one-tube rapid plant DNA extraction reagent, its usage method and application. Although the extraction reagent can rapidly extract plant DNA, it cannot effectively extract plant pathogen nucleic acids, and therefore cannot be used for rapid detection of plant pathogens.

[0005] Therefore, there is an urgent need for a rapid nucleic acid extraction reagent for plant pathogens, which requires no equipment or consumables such as centrifuges or centrifuges, and has an extraction method that is easy to operate. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a rapid nucleic acid extraction reagent for plant pathogens, its usage method, and a molecular detection kit. The extraction reagent can be used directly for one-step rapid nucleic acid extraction, or it can be used in conjunction with the accompanying reagents for nucleic acid extraction based on magnetic beads. The reagent combination of this invention meets the needs of detection technologies such as PCR, qPCR, LAMP, and RPA, and is particularly suitable for the molecular detection of pathogens in field-exposed plant tissues and pure cultures.

[0007] The objective of this invention can be achieved through the following methods: In a first aspect, the present invention provides a rapid nucleic acid extraction reagent for plant pathogens, comprising a lysis buffer I and a dilution and neutralization solution II; wherein the components of the lysis buffer I include: a strong base, disodium ethylenediaminetetraacetate (EDTA-2Na), potassium acetate, guanidine hydrochloride, NaCl, CTAB, PVP-40, and Triton X-100, and the components of the dilution and neutralization solution II include: Tris-HCl and EDTA.

[0008] As one embodiment of the present invention, the strong alkali includes one of potassium hydroxide and sodium hydroxide.

[0009] As one embodiment of the present invention, the lysis buffer I comprises: a strong base of 0.3-0.5 mol / L, disodium ethylenediaminetetraacetate of 8-12 mmol / L, potassium acetate of 1-3 mol / L, guanidine hydrochloride of 0.05-0.2 mol / L, NaCl of 0.5-1.4 mol / L, CTAB of 1%-4%, PVP-40 of 1-2%, and Triton X-100 of 1%-2%.

[0010] Preferably, the lysis buffer I comprises: 0.4 mol / L strong base, 10 mmol / L disodium ethylenediaminetetraacetate, 2 mol / L potassium acetate, 0.1 mol / L guanidine hydrochloride, 1.4 mol / L NaCl, 4% CTAB, 1% PVP-40, and 2% Triton X-100.

[0011] In one embodiment of the present invention, the pH of Tris-HCl in the dilution and neutralization solution II is 7.5-8.0, and the concentration of EDTA is 0.3-0.6 mM. Preferably, the pH of Tris-HCl is 7.8, and the concentration of EDTA is 0.5 mM.

[0012] Secondly, the present invention provides a method for rapidly extracting plant pathogen nucleic acid in one step using the extraction reagent, comprising the following steps: adding lysis buffer I to the isolated culture sample of the plant pathogen, taking the lysed nucleic acid sample, and then adding dilution and neutralization solution II to obtain the analytical test sample.

[0013] As one embodiment of the present invention, the plant pathogen originates from the roots, stems, leaves, or pure cultured plant pathogen hyphae of the diseased plant; the plant includes one of field crops, horticultural crops, and weeds; the pathogen includes fungi or bacteria; wherein, the field crop is selected from one of rice, wheat, corn, soybean, rapeseed, cotton, and peanut; the horticultural crop is selected from one of cabbage, cucumber, radish, tomato, strawberry, pear, and peach; the weed is selected from one of barnyard grass, Echinochloa crus-galli, Alopecurus aequalis, Alopecurus japonicus, Alopecurus nigra, Gnaphalium affine, Stellaria media, Stellaria dichotoma, Eleusine indica, and Alopecurus thunbergii; the fungus is selected from one of Fusarium graminearum, Fusarium pseudograminearum, and Fusarium oxysporum strawberry-specific strain; and the bacteria is selected from Xanthomonas oryzae, a pathogen causing rice bacterial blight.

[0014] As one embodiment of the present invention, the preparation process of the isolated culture sample is as follows: when using a pure culture of plant pathogen, the mycelium is picked up with a pipette tip to obtain the sample; when using the roots, stems, and leaves of diseased plants, they are cut into strips of 1-2 mm to obtain the sample.

[0015] Specifically, a sterile 10 μL pipette tip is used to pick up the isolated culture sample of plant pathogens and place it in a 2 mL centrifuge tube; then 50-200 μL of lysis buffer I is added to the centrifuge tube and incubated at room temperature for 5-10 min; 10 μL of the above lysis buffer is transferred to a new 1.5 mL centrifuge tube and 90 μL of dilution and neutralization solution II is added. The resulting nucleic acid sample can be used for subsequent amplification and detection.

[0016] Thirdly, the present invention provides a high-throughput rapid extraction and lysis reagent for plant pathogen nucleic acids comprising the extraction reagent, including lysis buffer A, neutralization buffer B, washing buffer C, and elution buffer D; wherein the components of lysis buffer A include: strong base, disodium ethylenediaminetetraacetate, potassium acetate, guanidine hydrochloride, NaCl, CTAB, PVP-40, Triton X-100, and SDS; the components of neutralization buffer B include: EDTA, acetic acid, and Tris-HCl; the components of washing buffer C include: LiCl, Tris-HCl, EDTA, and ethanol; and the components of elution buffer D include: Tris-HCl and EDTA.

[0017] As one embodiment of the present invention, the strong alkali includes one of potassium hydroxide and sodium hydroxide.

[0018] As an embodiment of the present invention, the components of said lysis buffer A comprise: strong alkali 0.3-0.5 mol / L, disodium ethylenediaminetetraacetate 8-12 mmol / L, potassium acetate at a concentration of 1-3 mol / L, guanidine hydrochloride 0.05-0.2 mol / L, NaCl 0.5-1.4 mol / L, CTAB 1%-4%, PVP-40 1-2%, Triton X-100 1-2%, SDS 0.5%-1%.

[0019] As an embodiment of the present invention, the components of said neutralization buffer B comprise: EDTA 0.5-2 mmol / , acetic acid 10-15 mmol / L, Tris-HCl 8-12 mmol / L, with a pH of 7.5-8.0.

[0020] As an embodiment of the present invention, the components of said washing buffer C comprise: LiCl 0.2-1 mmol / L, Tris-HCl 10 mM-20 mM, EDTA 0.1-1 mmol / L, ethanol 90-100%.

[0021] As an embodiment of the present invention, the components of said elution buffer D comprise: Tris-HCl 10-15 mmol / L, pH 7.5-8.0, EDTA at a concentration of 0.5-1 mol / L.

[0022] Preferably, the concentration of the strong alkali is 0.5 mol / L, the concentration of disodium ethylenediaminetetraacetate is 10 mmol / L, the concentration of potassium acetate is 1 mol / L, the concentration of guanidine hydrochloride is 0.1 mol / L, and the concentration of SDS is 1%; in the neutralization solution B for rapid extraction of plant pathogens by magnetic bead method, the concentration of EDTA is 1 mmol / , the concentration of acetic acid is 10 mmol / L, and the concentration of Tris-HCl is 10 mmol / L (with a pH of 7.5). In the magnetic bead method washing buffer C, the concentration of LiCl is 0.5 mmol / L, the concentration of Tris-HCl is 10 mM mM, the concentration of EDTA is 1 mmol / L, and the concentration of ethanol is 95%. In the elution buffer, the concentration of Tris-HCl is 10 mmol / L (pH 7.8), and the concentration of EDTA is 1 mol / L.

[0023] In a fourth aspect, the present invention provides a method for batch extraction of plant pathogen nucleic acid by magnetic bead method matched with said lysis reagent, comprising the following steps: S1, placing the isolated and cultured sample of plant pathogen in a centrifuge tube, adding lysis buffer A and neutralization buffer B sequentially, mixing and neutralizing, then adding magnetic bead suspension to allow nucleic acids to bind to the surfaces of magnetic beads; S2. Place the centrifuge tube on the magnetic rack and let it stand until the magnetic beads are completely attracted. Discard the supernatant. S3. Remove the centrifuge tube from the magnetic rack, add washing buffer C, and resuspend the magnetic beads by blowing or vortexing. Then put it back into the magnetic rack to adsorb the magnetic beads and discard the supernatant. S4. Remove the centrifuge tube from the magnetic rack, add elution buffer D, and put it back into the magnetic rack to adsorb the magnetic beads. After the solution becomes clear, aspirate the supernatant to obtain the nucleic acid sample.

[0024] As one embodiment of the present invention, in step S1, 10 μL of the reagent is added for every 1 mg of sample, that is, the weight-to-volume ratio of sample to reagent is 1:10 (mg: μL).

[0025] Fifthly, the present invention provides the application of the extraction reagent or lysis reagent in the preparation of a nucleic acid molecular detection kit for plant pathogens.

[0026] As one embodiment of the present invention, the molecular detection kit employs one of the following amplification methods: PCR amplification, qPCR amplification, LAMP amplification, and RPA amplification. The extraction or lysis reagent can also be used for Sanger sequencing.

[0027] The reagents of this invention not only enable rapid preparation of plant pathogen nucleic acids in the field without equipment, but also provide a rapid method for preparing plant pathogen nucleic acids in the laboratory using magnetic beads. The extracted nucleic acid samples have good integrity and high concentration, and can be used as templates for routine analyses such as PCR amplification, qPCR amplification, LAMP amplification, and RPA amplification. In addition, this method has a short extraction time, low cost, and high safety for operators.

[0028] The applicant's prior patent CN119162165A discloses a one-tube rapid plant DNA extraction reagent, its usage method, and its applications. The active ingredients of this reagent consist of sodium hydroxide, disodium EDTA, potassium acetate, guanidine hydrochloride, and Tween-80. While this extraction reagent can rapidly extract plant DNA, it cannot extract nucleic acids from plant pathogens. This invention improves cell permeability by adding PVP-40 to the rapid extraction reagent, thereby enhancing the nucleic acid extraction efficiency for plant pathogens and improving detection efficiency, enabling rapid detection of plant pathogens. Simultaneously, the addition of SDS and CTAB to the rapid lysis reagent synergistically disrupts the cell walls of plant pathogens, further enhancing the extraction efficiency of plant pathogen nucleic acids and improving detection efficiency, enabling batch detection of plant pathogens.

[0029] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a rapid nucleic acid extraction reagent for plant pathogens and a lysis reagent containing the extraction reagent. On one hand, adding PVP-40 to the extraction reagent improves cell permeability and enhances the nucleic acid extraction efficiency for plant pathogens, thereby improving detection efficiency and enabling rapid detection of plant pathogens. Simultaneously, adding SDS and CTAB to the lysis reagent synergistically disrupts the cell walls of plant pathogens, further enhancing the extraction efficiency of plant pathogen nucleic acids. Combined with magnetic bead technology, this enables batch detection of plant pathogens. This invention provides reagents and methods for rapid and batch detection of plant pathogen nucleic acids, meeting the needs of various scenarios.

[0030] 2. This invention further improves the nucleic acid extraction efficiency of plant pathogens by adjusting the amount of PVP-40 used. The obtained samples can be directly connected to PCR, qPCR, LAMP, RPA and other amplification detection methods.

[0031] 3. The rapid extraction reagent or rapid lysis reagent of this invention has a long shelf life; the extracted nucleic acid can be stored at -20℃ for 30 days and is not easily degraded. Furthermore, the extracted nucleic acid, whether amplifying genes or SSR molecular markers, exhibits high amplification efficiency, clear bands, high extraction rate, good quality, and strong molecular stability.

[0032] 4. The nucleic acid extraction method of this invention is simple to operate, requiring no liquid nitrogen grinding, no toxic chemical reagents, and no repeated extractions. It only requires a single reagent addition, eliminating the need for complex pretreatment such as heating or freezing, and eliminating the need for equipment and consumables such as centrifuges and columns. It uses fewer reagents and has lower costs. Furthermore, it is applicable to the extraction of nucleic acids from various plant pathogens, satisfying both on-site nucleic acid preparation for small sample volumes and batch nucleic acid preparation. Attached Figure Description

[0033] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram of a rapid nucleic acid extraction method for plant pathogens. Figure 2 The results of PCR electrophoresis of strawberry-specific nucleic acid extraction from Fusarium graminearum, Fusarium pseudograminearum, and Fusarium oxysporum in Example 1; Figure 3 The results of PCR electrophoresis of nucleic acid extraction from Xanthomonas oryzae pathogenic species causing rice bacterial blight in Example 1; Figure 4 The sequencing results of the PCR amplification product of the Fusarium graminearum ITS1 gene in Example 1; Figure 5 The sequencing results of the PCR amplification product of the Fusarium pseudobulb ITS1 gene in Example 1; Figure 6 The sequencing results of the PCR amplification product of the strawberry-specific ITS1 gene of Fusarium oxysporum in Example 1; Figure 7 The sequencing results of the PCR amplification product of the 16S rRNA gene of Xanthomonas oryzae in the rice white leaf blight pathogen species in Example 1; Figure 8 The qPCR amplification curves of Fusarium graminearum and Xanthomonas oryzae, the pathogens causing bacterial blight in rice, are shown in Example 1. Figure 9 The LAMP amplification curves of Fusarium graminearum and Xanthomonas oryzae, the pathogens causing bacterial blight in rice, are shown in Example 1. Figure 10 The RPA amplification electrophoresis results of Fusarium graminearum and Xanthomonas oryzae in Example 1, which caused rice bacterial blight. Figure 11 The results of PCR electrophoresis of strawberry-specific nucleic acid extraction from Fusarium graminearum, Fusarium pseudograminearum, and Fusarium oxysporum in Example 2; Figure 12 The results of PCR electrophoresis of nucleic acid extraction from Xanthomonas oryzae pathogenic species causing rice bacterial blight in Example 2; Figure 13 The qPCR amplification curves of Fusarium graminearum and Xanthomonas oryzae, the pathogens causing white leaf blight in rice, are shown in Example 2. Figure 14 The LAMP amplification curves of Fusarium graminearum and Xanthomonas oryzae, the pathogens causing bacterial blight in rice, are shown in Example 2. Figure 15 The RPA amplification electrophoresis results of Fusarium graminearum and Xanthomonas oryzae in rice bacterial blight pathogens in Example 2; Figure 16 The qPCR amplification curves of Fusarium graminearum in Example 1 and Comparative Example 1 are shown. Figure 17 The qPCR amplification curves of Fusarium graminearum in Example 1 and Comparative Example 2 are shown. Figure 18 The qPCR amplification curves of Fusarium graminearum in Example 1 and Comparative Example 3 are shown. Figure 19 The qPCR amplification curves of Fusarium graminearum in Example 2 and Comparative Example 4 are shown. Figure 20 The qPCR amplification curves of Fusarium graminearum in Example 2 and Comparative Example 5 are shown. Detailed Implementation

[0034] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0035] Example 1: One-step rapid extraction of plant pathogen nucleic acids and testing with PCR, qPCR, and isothermal amplification 1. Experimental Materials Pure culture of fungal pathogen: Fusarium graminearum ( Fusarium graminearum Fusarium pseudograss () Fusarium pseudograminearum Fusarium oxysporum strawberry-specific strain ( Fusarium oxysporum Schl. f.sp. fragariae Winks et Willams.). Pure culture of bacterial pathogen hyphae: Xanthomonas oryzae, a pathogen causing bacterial blight in rice ( Xanthomonas oryzae pv. oryzae).

[0036] The pathogen purification process is as follows: Take the bacterial strain to be purified and aseptically inoculate it onto the surface of the corresponding solid culture medium. Incubate it upside down at a suitable temperature. After the fungal pathogen has grown visible mycelia, use a sterile inoculation needle to pick the tips of the vigorously growing mycelia at the edge of the colony and transfer them to fresh culture medium. Repeat this tip picking and transfer operation 2-3 times until microscopic examination confirms that uniform pure mycelia have been obtained. For bacterial pathogens, pure cultures are obtained by picking a single isolated bacterial colony and repeatedly streaking it 2-3 times using the same method.

[0037] Culture parameters for fungal pathogens: Potato dextrose agar (PDA) was used as the culture medium, with a pH of 5.4-6.0, a culture temperature of 25-28℃, aerobic culture, and an ambient humidity of 65%-70%.

[0038] Bacterial pathogen culture parameters: Potato semi-synthetic agar (PSA) medium was used, with a pH of 6.8-7.2, a culture temperature of 25-28℃, aerobic culture, and an ambient humidity of 65%-70%.

[0039] The reagent composition for the one-step extraction of plant pathogen nucleic acids is as follows: 1) Lysis buffer I: potassium hydroxide concentration is 0.4 mol / L, disodium ethylenediaminetetraacetate concentration is 10 mmol / L, potassium acetate concentration is 2 mol / L, guanidine hydrochloride concentration is 0.1 mol / L, NaCl concentration is 1.4 mol / L, CTAB concentration is 4%, PVP-40 concentration is 1%, and Triton X-100 concentration is 2%. The pH of the one-step plant pathogen dilution and neutralization solution II is 7.8 with Tris-HCl and 0.5 mM with EDTA.

[0040] 2. Experimental Methods 2.1 One-step rapid extraction of nucleic acids from plant pathogens refer to Figure 1 The procedure is as follows: Use a sterile 10 μL pipette tip to pick up the isolated culture sample of plant pathogens and place it in a 2 mL centrifuge tube; then add 100 μL of lysis buffer I to the centrifuge tube and let it stand at room temperature for 10 min; 3) Pipette 10 μL of the above lysis buffer into a new 1.5 mL centrifuge tube and add 90 μL of dilution and neutralization solution II, which is the DNA template.

[0041] 2.2 PCR detection PCR amplification was performed using 2 × Rapid Taq Master Mix reagent (purchased from Novozymes). The specific system configuration is shown in Table 1. Table 1. PCR amplification reaction components

[0042] Amplification program: The PCR reaction program consisted of 34 cycles of pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 25 sec, annealing at 56℃ for 25 sec, and extension at 72℃ for 30 sec, ending with a final extension at 72℃ for 10 min. Agarose gel electrophoresis analysis was performed after amplification.

[0043] The nucleotide sequences of the primers FgITS1-F / R for amplifying the Fusarium graminearum ITS1 gene fragment (515 bp), Fusarium pseudograminearum ITS1 gene fragment (512 bp), and Fusarium oxysporum strawberry-specific ITS1 gene fragment (516 bp) are shown in SEQ ID NO.1 and SEQ ID NO.2. The nucleotide sequences of the primers Xo16s_rRNA-F / R (i.e., primers 7F and 1540R in Table 5) for amplifying the Xanthomonas xanthomonas oryzae 16S rRNA gene fragment (1385 bp) are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0044] 2.3 qPCR detection The qPCR amplification reaction used SGExcel universal dye qPCR premix (purchased from Sangon Biotech). The specific system configuration is shown in Table 2. Table 2. Components of qPCR amplification reaction

[0045] Amplification program: The PCR reaction program was 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 sec, 60℃ annealing for 30 sec, for 40 cycles, with fluorescence signal collected once per cycle.

[0046] The primer sequences for quantitative real-time PCR used for Fusarium graminearum are shown in SEQ ID NO.20 and SEQ ID NO.21, and the primer sequences for quantitative real-time PCR used for Xanthomonas oryzae, a pathogenic species of rice, are shown in SEQ ID NO.22 and SEQ ID NO.23.

[0047] 2.4 Isothermal amplification (LAMP) detection The LAMP amplification reaction used the Bst II Fluorescent LAMP Kit (DNA & RNA) (purchased from TransGen). The specific system configuration is shown in Table 3. Table 3. LAMP amplification reaction components

[0048] The LAMP amplification procedure is as follows: amplify at 65℃ for 60 minutes, and collect the fluorescence signal once per cycle.

[0049] The LAMP primer sets F3, B3, FIP, BIP, and LB sequences used for Fusarium graminearum are shown in SEQ ID NO.5-SEQ ID NO.9, respectively; the LAMP primer sets F3, B3, FIP, BIP, LF, and LB sequences used for Xanthomonas oryzae causing rice bacterial blight are shown in SEQ-ID NO.10-SEQ-ID NO.15, respectively.

[0050] 2.5 Isothermal amplification (RPA) detection RPA amplification was performed using the RPA / RAA DNA isothermal amplification kit (EZassay). The specific system configuration is shown in Table 4. Table 4. RPA Amplification Reaction Components

[0051] The procedure for RPA amplification is as follows: amplification at 39℃ for 40 minutes.

[0052] The F1 and R1 sequences of the RPA primer sets used for Fusarium graminearum are shown in SEQ ID NO.16-SEQ ID NO.17, respectively; the F1 and R1 sequences of the RPA primer sets used for Xanthomonas oryzae causing bacterial blight in rice are shown in SEQ-ID NO.18-SEQ-ID NO.19, respectively.

[0053] 3. Experimental Results: 3.1 Amplification Result Testing and Analysis Fungal PCR amplification results as follows Figure 2 As shown: 1-3 represent the strawberry-specific strains of *Fusarium graminearum*, *Fusarium pseudograminearum*, and *Fusarium oxysporum*, respectively, with Marker (M) sizes of 2000, 1500, 750, 500, 250, and 100 bp. The amplification results were as expected, with sizes of 515 bp, 512 bp, and 516 bp, respectively. The bacterial PCR amplification results are as follows... Figure 3 As shown: 1 represents *Xanthomonas oryzae*, a pathogen causing bacterial blight in rice, with Marker (M) sizes of 2000, 1500, 750, 500, 250, and 100 bp. The amplification results were as expected, with sizes of 1385 bp. This indicates that a one-step rapid extraction method for plant pathogen nucleic acids can be used for the extraction of fungal and bacterial genes. The PCR products were sent to Sangon Biotech (Shanghai) for sequencing; the fungal results are as follows... Figure 4-6 As shown, the PCR amplification results are as expected. Bacterial results are as follows... Figure 7 As shown, the PCR amplification results were as expected. This indicates that the quality of nucleic acids extracted using the one-step rapid extraction method for plant pathogens meets the requirements for sequencing.

[0054] qPCR amplification detection results are as follows Figure 8 As shown in the figure. The results indicate that the one-step rapid extraction method for plant pathogen nucleic acids is highly effective in qPCR amplification and can be applied to qPCR amplification and detection reactions.

[0055] LAMP amplification detection results are as follows: Figure 9 As shown in the figure. The results indicate that the one-step rapid extraction method for plant pathogen nucleic acids is highly effective in LAMP amplification and can be applied to LAMP amplification detection reactions. RPA amplification detection results are shown in the figure. Figure 10 As shown in the figure. The results indicate that the one-step rapid extraction method for plant pathogen nucleic acids is highly effective in RPA amplification and can be applied to RPA amplification detection reactions.

[0056] The primer sequences are shown in Table 5.

[0057] Table 5 Primer List

[0058] Example 2: Rapid extraction of plant pathogen nucleic acids using magnetic beads and testing with PCR, qPCR, and isothermal amplification. 1. Experimental Materials Pure culture of fungal pathogen hyphae: Same as in Implementation Case 1.

[0059] Pure culture of bacterial pathogen hyphae: Same as in Implementation Case 1.

[0060] Extraction reagents: The composition of the reagent for extracting plant pathogen nucleic acids using magnetic beads is as follows: The rapid extraction and lysis buffer A for plant pathogen nucleic acids using the magnetic bead method contains 0.5 mol / L sodium hydroxide, 10 mmol / L disodium EDTA, 1 mol / L potassium acetate, 0.1 mol / L guanidine hydrochloride, 1.4 mol / L NaCl, 4% CTAB, 1% PVP-40, 2% Triton X-100, and 1% SDS. The neutralization buffer B for the rapid extraction and lysis buffer B contains 1 mmol / L EDTA, 10 mmol / L acetic acid, and 10 mmol / L Tris-HCl (pH 7.5). The washing buffer C contains 0.5 mmol / L LiCl, 10 mM Tris-HCl, 1 mmol / L EDTA, and 95% ethanol. The elution buffer contains 10 mmol / L Tris-HCl (pH 7.8) and 1 mol / L EDTA.

[0061] 2. Experimental Methods The procedure for rapid extraction of plant pathogen nucleic acids using magnetic beads is as described above. The PCR, qPCR, LAMP, and RPA amplification methods are the same as in Case Study 1.

[0062] 3. Experimental Results: 3.1 PCR test results Fungal results such as Figure 11 As shown: 1-3 represent the strawberry-specific morphologies of *Fusarium graminearum*, *Fusarium pseudograminearum*, and *Fusarium oxysporum*, respectively, with Marker (M) sizes of 2000, 1500, 750, 500, 250, and 100 bp. The amplification results were as expected, with sizes of 515 bp, 512 bp, and 516 bp, respectively. Bacterial results are as follows... Figure 12 As shown: 1 represents *Xanthomonas oryzae*, a pathogen causing bacterial blight in rice, with Marker (M) sizes of 2000, 1500, 750, 500, 250, and 100 bp. The amplification results were as expected, with sizes of 1385 bp. This indicates that the rapid extraction of plant pathogen nucleic acids using magnetic beads can be applied to the extraction of fungal and bacterial genes.

[0063] qPCR amplification detection results are as follows Figure 13As shown in the figure. The results indicate that the rapid extraction of plant pathogen nucleic acids using magnetic beads is highly effective in qPCR amplification and can be applied to qPCR amplification detection reactions. LAMP amplification detection results are shown in the figure. Figure 14 As shown in the figure. The results indicate that the rapid extraction of plant pathogen nucleic acids using magnetic beads is highly effective in LAMP amplification and can be applied to LAMP amplification detection reactions. RPA amplification detection results are shown in the figure. Figure 15 As shown in the figure. The results indicate that the rapid extraction of plant pathogen nucleic acids using magnetic beads is highly effective in RPA amplification and can be applied to RPA amplification detection reactions.

[0064] Comparative Example 1 The difference between this comparative example and Example 1 is that PVP-40 was omitted from the one-step extraction reagent for plant pathogen nucleic acids. The specific composition is as follows: 1) Lysis buffer I: potassium hydroxide concentration is 0.4 mol / L, disodium ethylenediaminetetraacetate concentration is 10 mmol / L, potassium acetate concentration is 2 mol / L, guanidine hydrochloride concentration is 0.1 mol / L, NaCl concentration is 1.4 mol / L, CTAB concentration is 4%, and Triton X-100 concentration is 2%. The one-step plant pathogen dilution and neutralization solution II has a Tris-HCl pH of 7.8 and an EDTA concentration of 0.5 mM. Figure 16 As shown, the detection efficiency of Example 1 is higher than that of Comparative Example 1, meaning that Example 1 has a higher extraction efficiency for plant pathogen nucleic acids than Comparative Example 1. The results indicate that PVP-40 can enhance the nucleic acid extraction efficiency for plant pathogen nucleic acids by increasing cell permeability, thereby improving detection efficiency and enabling rapid detection of plant pathogens.

[0065] Comparative Example 2 The difference between this comparative example and Example 1 is that the concentration of components in the one-step extraction reagent for plant pathogen nucleic acids is too high. The specific composition is as follows: 1) Lysis buffer I: potassium hydroxide concentration is 0.6 mol / L, disodium ethylenediaminetetraacetate concentration is 15 mmol / L, potassium acetate concentration is 5 mol / L, guanidine hydrochloride concentration is 0.4 mol / L, NaCl concentration is 1.4 mol / L, CTAB concentration is 4%, PVP-40 concentration is 4%, and Triton X-100 concentration is 4%. The one-step plant pathogen dilution and neutralization solution II has a Tris-HCl pH of 7.8 and an EDTA concentration of 0.8 mM. Figure 17 As shown, the detection efficiency of Example 1 is higher than that of Comparative Example 2, proving that the extraction efficiency of plant pathogen nucleic acid by Example 1 is higher than that of Comparative Example 2.

[0066] Comparative Example 3 The difference between this comparative example and Example 1 is that the concentrations of the components in the one-step extraction reagent for plant pathogen nucleic acids are too low. The specific composition is as follows: 1) Lysis buffer I: potassium hydroxide concentration is 0.1 mol / L, disodium ethylenediaminetetraacetate concentration is 6 mmol / L, potassium acetate concentration is 0.5 mol / L, guanidine hydrochloride concentration is 0.01 mol / L, NaCl concentration is 1.4 mol / L, CTAB concentration is 4%, PVP-40 concentration is 0.5%, and Triton X-100 concentration is 0.5%. The one-step plant pathogen dilution and neutralization solution II has a Tris-HCl pH of 7.8 and an EDTA concentration of 0.1 mM. Figure 18 As shown, the detection efficiency of Example 1 is higher than that of Comparative Example 3, proving that the extraction efficiency of plant pathogen nucleic acid by Example 1 is higher than that of Comparative Example 3.

[0067] According to Comparative Examples 2 and 3, both excessively high and low PVP-40 concentrations can lead to a decrease in the extraction efficiency of plant pathogen nucleic acids, thereby reducing the detection efficiency.

[0068] Comparative Example 4 The difference between this comparative example and Example 2 is that CTAB was omitted from the magnetic bead extraction reagent for plant pathogen nucleic acids. The specific composition is as follows: Lysis buffer A contains 0.5 mol / L sodium hydroxide, 10 mmol / L disodium EDTA, 1 mol / L potassium acetate, 0.1 mol / L guanidine hydrochloride, 1.4 mol / L NaCl, 1% PVP-40, 2% Triton X-100, and 1% SDS; Magnetic bead-based rapid extraction neutralization buffer B contains 1 mmol / L EDTA, 10 mmol / L acetic acid, and 10 mmol / L Tris-HCl (pH 7.5); Magnetic bead-based washing buffer C contains 0.5 mmol / L LiCl, 10 mM Tris-HCl, 1 mmol / L EDTA, and 95% ethanol. The elution buffer contained 10 mmol / L Tris-HCl (pH 7.8) and 1 mol / L EDTA. Figure 19 As shown, the detection efficiency of Example 2 is higher than that of Comparative Example 4, proving that the extraction efficiency of plant pathogen nucleic acid by Example 2 is higher than that of Comparative Example 4.

[0069] Comparative Example 5 The difference between this comparative example and Example 2 is that SDS was omitted from the magnetic bead extraction reagent for plant pathogen nucleic acids. The specific composition is as follows: Lysis buffer A contains 0.5 mol / L sodium hydroxide, 10 mmol / L disodium EDTA, 1 mol / L potassium acetate, 0.1 mol / L guanidine hydrochloride, 1.4 mol / L NaCl, 4% CTAB, 1% PVP-40, and 2% Triton X-100; Magnetic bead-based rapid extraction neutralization buffer B contains 1 mmol / L EDTA, 10 mmol / L acetic acid, and 10 mmol / L Tris-HCl (pH 7.5); Magnetic bead-based washing buffer C contains 0.5 mmol / L LiCl, 10 mM Tris-HCl, 1 mmol / L EDTA, and 95% ethanol. The elution buffer contained 10 mmol / L Tris-HCl (pH 7.8) and 1 mol / L EDTA. Figure 20 As shown in the figure. The results indicate that the detection efficiency of Example 2 is higher than that of Comparative Example 5, proving that the extraction efficiency of plant pathogen nucleic acids by Example 2 is higher than that of Comparative Example 5.

[0070] According to Comparative Examples 4 and 5, SDS and CTAB can synergistically disrupt cell walls, enhance the extraction efficiency of plant pathogen nucleic acids, thereby improving detection efficiency and enabling batch detection of plant pathogens.

[0071] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A rapid nucleic acid extraction reagent for plant pathogens, characterized in that, It includes lysis buffer I and dilution neutralization solution II; wherein, the components of lysis buffer I include: strong base, disodium ethylenediaminetetraacetate, potassium acetate, guanidine hydrochloride, NaCl, CTAB, PVP-40, and Triton X-100, and the components of dilution neutralization solution II include: Tris-HCl and EDTA.

2. The extraction reagent according to claim 1, characterized in that, The strong base includes either potassium hydroxide or sodium hydroxide.

3. The extraction reagent according to claim 1, characterized in that, The lysis buffer I comprises: a strong base of 0.3-0.5 mol / L, disodium ethylenediaminetetraacetate of 8-12 mmol / L, potassium acetate of 1-3 mol / L, guanidine hydrochloride of 0.05-0.2 mol / L, NaCl of 0.5-1.4 mol / L, CTAB of 1%-4%, PVP-40 of 1%-2%, and Triton X-100 of 1%-2%; the dilution neutralization buffer II has a Tris-HCl pH of 7.5-8.0 and an EDTA concentration of 0.3-0.6 mM.

4. A method for rapid one-step extraction of plant pathogen nucleic acids using the extraction reagent as described in any one of claims 1-3, characterized in that, Includes the following steps: Add lysis buffer I to the isolated culture sample of plant pathogens, take the lysed nucleic acid sample, and then add dilution and neutralization solution II to obtain the analytical test sample.

5. The method according to claim 4, characterized in that, The plant pathogen originates from the roots, stems, leaves, or pure cultured mycelia of the plant pathogen; the plant includes one of field crops, horticultural crops, and weeds; the pathogen includes fungi or bacteria; wherein, the field crops are selected from one of rice, wheat, corn, soybean, rapeseed, cotton, and peanut; the horticultural crops are selected from one of cabbage, cucumber, radish, tomato, strawberry, pear, and peach; the weeds are selected from one of barnyard grass, Echinochloa crus-galli, Alopecurus aequalis, Alopecurus japonicus, Alopecurus nigra, Gnaphalium affine, Chickweed, Chickweed maxima, Eleusine indica, and Alopecurus thunbergii; the fungi are selected from one of Fusarium graminearum, Fusarium pseudograminearum, and Fusarium oxysporum strawberry-specific strain; the bacteria include Xanthomonas oryzae, a pathogen causing white leaf blight in rice.

6. The method according to claim 4, characterized in that, The preparation process of the isolated culture sample is as follows: when using pure cultured plant pathogens, the mycelium is picked up with a pipette tip to obtain the sample; when using the roots, stems, and leaves of diseased plants, they are cut into strips of 1-2 mm to obtain the sample.

7. A high-throughput rapid extraction and lysis reagent for plant pathogen nucleic acids comprising the extraction reagent according to any one of claims 1-3, characterized in that, The buffer comprises lysis buffer A, neutralization buffer B, washing buffer C, and elution buffer D. The components of lysis buffer A include: a strong base, disodium EDTA, potassium acetate, guanidine hydrochloride, NaCl, CTAB, PVP-40, Triton X-100, and SDS. The components of neutralization buffer B include: EDTA, acetic acid, and Tris-HCl. The components of washing buffer C include: LiCl, Tris-HCl, EDTA, and ethanol. The components of elution buffer D include: Tris-HCl and EDTA.

8. The pyrolysis reagent according to claim 7, characterized in that, The lysis buffer A comprises: a strong base (0.3-0.5 mol / L), disodium ethylenediaminetetraacetate (8-12 mmol / L), potassium acetate (1-3 mol / L), guanidine hydrochloride (0.05-0.2 mol / L), NaCl (0.5-1.4 mol / L), CTAB (1%-4%), PVP-40 (1-2%), Triton X-100 (1-2%), and SDS (0.5%-1%). The neutralization buffer B comprises: EDTA (0.5-2 mmol / L), acetic acid (10-15 mmol / L), Tris-HCl (8-12 mmol / L), and pH 7.5-8.

0. The washing buffer C comprises: LiCl (0.2-1 mmol / L), Tris-HCl (10 mM-20 mM), and EDTA (0.1-1 mmol / L). The elution buffer D contains 90-100% ethanol and 10-15 mmol / L Tris-HCl, pH 7.5-8.0, and 0.5-1 mol / L EDTA.

9. A method for mass-extracting nucleic acids of plant pathogens by a magnetic bead method matched with the lysis reagent according to claim 7 or 8, characterized in that, Includes the following steps: S1. Place the isolated culture sample of plant pathogens in a centrifuge tube, add lysis buffer A and neutralization buffer B in sequence, mix well and neutralize, then add magnetic bead suspension to allow nucleic acid to bind to the surface of magnetic beads. S2. Place the centrifuge tube on the magnetic rack and let it stand until the magnetic beads are completely attracted. Discard the supernatant. S3. Remove the centrifuge tube from the magnetic rack, add washing buffer C, and resuspend the magnetic beads by blowing or vortexing. Then put it back into the magnetic rack to adsorb the magnetic beads and discard the supernatant. S4. Remove the centrifuge tube from the magnetic rack, add elution buffer D, and put it back into the magnetic rack to adsorb the magnetic beads. After the solution becomes clear, aspirate the supernatant to obtain the nucleic acid sample.

10. The use of the extraction reagent as described in any one of claims 1-3, or the lysis reagent as described in claim 7 or 8, in the preparation of a molecular detection kit for plant pathogen nucleic acids.

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