Method for detecting soybean grey leaf spot germs based on RPA (recombinase polymerase amplification)

Abstract

The invention relates to the technical field of soybean gray leaf spot pathogen detection, and particularly discloses a method for detecting soybean gray leaf spot pathogen based on RPA (recombinase polymerase amplification), which comprises the following steps: designing three pairs of forward and reverse primers by using Pr immer 5.0 according to a specific section of a soybean gray leaf spot pathogen gene KC888798.1 downloaded from NCBI (National Center of Biotechnology Information), and screening; on the basis, optimization of temperature and time and verification of sensitivity and specificity are carried out. Results show that under the condition of constant temperature of 38 DEG C, reaction is performed for 15-20 min, 90 fg / mu L of soybean gray leaf spot germs can be detected at least, reaction with fusarium oxysporum, fusarium oxysporum, peronospora northeast and the like is avoided, a rapid detection system of the soybean gray leaf spot germs based on the RPA technology is successfully established, and technical support is provided for rapid detection of the soybean gray leaf spot germs.

Description

Technical Field

[0001] The invention belongs to the technical field of soybean gray leaf spot detection, and specifically relates to a detection method for soybean gray leaf spot based on RPA. Background Art

[0002] Soybean gray spot pathogen (CerCospora sojina Hara) is one of the main hazards that affect the quality of crops and their total yield. The soybean gray spot pathogen (CerCospora sojina Hara) mainly overwinters with diseased residues in the form of fruiting bodies or hyphae, becoming the source of infection at the beginning of the next year, invading the leaves, causing serious death, and affecting the quality of crops and their total yield. According to a comprehensive analysis by the National Agricultural Technology Center, soybean gray spot disease is distributed in major soybean producing areas across the country, and is most likely to occur in the northeast soybean area. The general gray spot disease rate is 10% to 15%, and in severe plots it is as high as more than 30%. The affected bean plants shed leaves early, the grain weight decreases, the rate of blighted pods and green beans increases, and the protein and oil content decreases, which seriously affects the quality of soybeans. Therefore, the detection and prevention of crop diseases are urgent. In actual production, in order to effectively block the further invasion of the pathogen, chemical control methods are often used to block it. Although the control effect has been improved, in the long run, the pesticide content in the crop will exceed the standard, thereby affecting its economic benefits.

[0003] At present, the detection method of soybean gray spot pathogen mainly relies on morphological observation and biological characteristics identification of the pathogen, but this method is cumbersome, labor-intensive, time-consuming, and has a certain degree of error. The molecular diagnosis of plant pathogens at home and abroad is mainly based on amplification technologies represented by polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR, but due to the dependence on thermal cyclers and high-quality templates, it is difficult to apply to rapid on-site detection.

[0004] Isothermal amplification technology has been widely used for virus detection due to its advantages such as no need for thermal cycling steps and suitability for on-site detection. Among them, loop-mediated isothermal amplification technology LAMP is the earliest technology studied, but the focus on soybean diseases is mainly on the detection of soybean phytophthora, and the primer design is cumbersome. Although the recombinase polymerase amplification (RPA) technology appeared later, its high detection sensitivity, no need for expensive supporting instruments and equipment, high sample tolerance, and diversified detection methods have gradually become a research hotspot in recent years. At present, this technology has been applied in many aspects, such as the detection of Fusarium oxysporum that causes Astragalus root rot, citrus Huanglongbing pathogen, and tomato yellow leaf curl virus, indicating that there is already a lot of experience in RPA detection technology.

[0005] This study selected the soybean gray spot pathogen as the research object and successfully established an early, accurate and rapid diagnosis technology that does not rely on large-scale equipment, namely, a rapid detection system for soybean gray spot pathogen based on RPA technology. Currently, there are few molecular detection methods for soybean gray spot pathogen. Summary of the invention

[0006] The object of the present invention is to provide a detection method for soybean gray spot pathogen based on RPA to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for detecting soybean gray spot pathogen based on RPA, comprising the following steps:

[0009] S1. Sample preparation and genomic DNA extraction:

[0010] After taking the sample fungal strain out of the -80°C freezer, it was activated on a PDA plate and cultured in an incubator at 25°C for genomic DNA extraction;

[0011] The extraction method includes one or more of CTAB method, SDS method, magnetic bead method, sodium lauroyl sarcosinate method and CTAB magnetic bead method;

[0012] S2. RPA reaction system configuration

[0013] Primer Pairs:

[0014] Forward primer F75: 5'-TCCAGCTCAGCAAGGCTCGCATCACTCCGT-3'

[0015] Reverse primer R213: 5′-ACTGGAAAGAAGCCTCAAACTAAACCTAAG-3′

[0016] Reaction system (50 μL)

[0017] 2.5 μL each of forward and reverse primers

[0018] 20 μL RPA Lysis Reagent

[0019] 2μL genomic DNA (10ng / μL)

[0020] 2 μL activator

[0021] Add ddH2O to 50μL;

[0022] S3. RPA amplification conditions

[0023] Temperature: 38℃ constant temperature; time: 20-25min

[0024] Operation: After centrifugation and mixing, place in a constant temperature water bath immediately. After the reaction is completed, add 6× loading buffer and incubate at 56℃ for 5 minutes;

[0025] S4. Electrophoresis detection

[0026] Prepare 3.5% agarose gel (containing nucleic acid dye), and run the electrophoresis at 80 V constant voltage for 1 h.

[0027] When observed under a gel imager, the presence of 100-200bp is considered positive.

[0028] Preferably, the CTAB method in S1 comprises the following steps: firstly grind the fungus into powder with liquid nitrogen, put it into a tube, add 800 μL of 2% preheated CTAB lysis solution (add 40 μL of β-mercaptoethanol in advance), shake and mix in a 56°C water bath for 40 min, shake and mix for 1 min every 5 min, centrifuge at 12000 rpm and 4°C for 10 min, take the supernatant into a new tube, add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), invert several times to mix, and then centrifuge at 12000 rpm and 4°C for 10 min. 00rpm, centrifuge at 4℃ for 10min, take the supernatant and put it in a new tube, add 2 times the volume of pre-cooled anhydrous ethanol, and let it stand at -20℃ refrigerator for 20min. A translucent flocculent precipitate will appear in the centrifuge tube, which is the fungal genome. Centrifuge at 12000rpm, 4℃ for 8min, discard the supernatant, wash the precipitate with pre-cooled 70% ethanol, and centrifuge at 10000rpm for 5min (repeat 2 times), discard the supernatant, put it on ice in an ultra-clean workbench to dry, then add 50μLTE to dissolve and 3μL RNaseA to remove RNA, and store it in a -20℃ refrigerator for later use after measuring the concentration.

[0029] Preferably, the SDS method comprises the following steps: grinding the fungus with liquid nitrogen to powder in advance, quickly filling the tube and immediately adding 1 mL of preheated SDS lysis solution, shaking and mixing in a 56°C water bath for 40 minutes, and shaking and mixing for 1 minute every 5 minutes, and the subsequent steps are the same as the CTAB method.

[0030] Preferably, the magnetic bead method comprises the following steps: grinding the fungus with liquid nitrogen to powder, quickly filling the tube, adding 800 μL of preheated lysis solution, 20 μL of proteinase K, shaking and mixing in a 56°C water bath for 40 minutes, and shaking and mixing for 1 minute every 5 minutes, taking the supernatant into a new tube, adding an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), inverting several times to mix, centrifuging at 12000rpm, 4°C for 10 minutes, taking the supernatant and adding an equal volume of isoamyl alcohol (precooled in advance) and an appropriate amount of magnetic bead suspension to a new tube, mixing and letting stand for 15 minutes, transferring to a magnetic stand and letting stand for 1 minute, discarding the waste liquid, washing with deproteinized solution and 70% ethanol (2 times), drying at room temperature for 5 minutes, adding 50 μL TE to dissolve and letting stand on a magnetic stand for 1 minute, and the resulting solution is genomic DNA.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] In this study, Gray Spot Pathogen of Soybean was selected as the research object, and a rapid detection method for the fungus based on RPA technology was established for the first time. According to the specific segment of the Gray Spot Pathogen gene KC888798.1 downloaded from NCBI, three pairs of forward and reverse primers were designed using Primer 5 and experimental screening was carried out. F75 / R213 was confirmed to be the best primer pair. On this basis, the temperature and time were optimized, and the sensitivity and specificity were verified. Finally, under the constant temperature of 38℃, the reaction time was 20-25min, and the minimum detectable concentration of Gray Spot Pathogen of Soybean was 90fg / μL, and it did not react with other fungi such as Fusarium oxysporum, Fusarium acutum, and Peronospora manchuria. A rapid detection system for Gray Spot Pathogen of Soybean based on RPA technology was successfully established.

[0033] Compared with conventional detection methods such as conventional PCR and fluorescent quantitative PCR, this method is faster and more convenient, breaking through the limitations of expensive instruments and time-consuming problems, making this detection method have higher sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The electrophoresis diagrams of DNA extracted from soybean gray leaf spot pathogen using different methods of the present invention;

[0035] Figure 2 This is the electrophoresis diagram of the whole genome of soybean gray spot fungus amplified by PCR using the TF primers of the present invention;

[0036] Figure 3 Agarose gel electrophoresis diagram for screening the best primer pair of the present invention;

[0037] Figure 4 Agarose gel electrophoresis diagram for optimal reaction temperature screening of the present invention;

[0038] Figure 5Agarose gel electrophoresis diagram for screening the optimal reaction time of the present invention;

[0039] Figure 6 Agarose gel electrophoresis diagram for sensitivity verification of the present invention;

[0040] Figure 7 Agarose gel electrophoresis diagram for specific verification of the present invention;

[0041] Figure 8 The agarose gel electrophoresis diagram is verified by the actual sample of the present invention. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Embodiment 1:

[0044] A rapid detection method for soybean gray spot pathogen based on RPA, comprising the following steps:

[0045] Sample preparation and genomic DNA extraction: After taking the sample fungal strain out of the -80°C freezer, activate it on a PDA plate and culture it in a 25°C incubator for genomic DNA extraction; the extraction method includes the CTAB method;

[0046] First, the fungus was ground into powder with liquid nitrogen, and then placed in a tube and 800 μL of 2% preheated CTAB lysis solution was added (40 μL of β-mercaptoethanol was added in advance), and the mixture was shaken in a 56°C water bath for 40 min, and shaken for 1 min every 5 min. The mixture was centrifuged at 12000 rpm and 4°C for 10 min. The supernatant was taken into a new tube, and an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was added. After inverting several times to mix, the tube was centrifuged at 12000 rpm and 4°C. Centrifuge for 10 minutes, take the supernatant and add 2 times the volume of pre-cooled anhydrous ethanol to a new tube, and let it stand at -20℃ refrigerator for 20 minutes. A translucent flocculent precipitate will appear in the centrifuge tube, which is the fungal genome. Centrifuge at 12000rpm, 4℃ for 8 minutes, discard the supernatant, wash the precipitate with pre-cooled 70% ethanol, and centrifuge at 10000rpm for 5 minutes (repeat 2 times), discard the supernatant, put it on ice in the clean bench to dry, then add 50μLTE to dissolve and 3μL RNaseA to remove RNA, measure the concentration and store it in a -20℃ refrigerator for later use.

[0047] RPA reaction system configuration

[0048] Primer Pairs:

[0049] Forward primer F75: 5'-TCCAGCTCAGCAAGGCTCGCATCACTCCGT-3'

[0050] Reverse primer R213: 5′-ACTGGAAAGAAGCCTCAAACTAAACCTAAG-3′

[0051] Reaction system (50 μL)

[0052] 2.5 μL each of forward and reverse primers

[0053] 20 μL RPA Lysis Reagent

[0054] 2μL genomic DNA (10ng / μL)

[0055] 2 μL activator

[0056] Add ddH2O to 50μL;

[0057] RPA amplification conditions: Temperature: 38°C; Time: 20-25 min: After centrifugation and mixing, immediately place in a constant temperature water bath. After the reaction, add 6× loading buffer and incubate at 56°C for 5 min.

[0058] Electrophoresis detection: prepare 3.5% agarose gel (containing nucleic acid dye), electrophoresis conditions: 80V constant voltage electrophoresis for 1 hour. Observe under the gel imager, the appearance of 100-200bp is positive.

[0059] Please refer to the following experimental steps for details:

[0060] The main reagents and instruments include DL 15000bp DNA Marker, 10× Loading buffer (Baoriyi Biotechnology Co., Ltd.), DL 500bp DNA Marker, sodium dodecyl sulfate (SDS) (Shanghai Bioengineering Co., Ltd.), nucleic acid extraction magnetic beads (universal type, Maitu Pharmaceutical Technology Co., Ltd.), polyvinyl pyrrolidone (PVP) purchased from Feike Biotechnology Co., Ltd., hexadecyltrimethylammonium bromide (CTAB), sodium lauroyl sarcosine (Tianjin Fuchen Chemical Reagent Co., Ltd.), β-mercaptoethanol (Tianjin Damao Chemical Technology Co., Ltd.), isopropanol and anhydrous ethanol (Beijing Solaibao Biotechnology Co., Ltd.), basic nucleic acid amplification kit (Suzhou Xianda Gene Technology Co., Ltd.); high pressure sterilizer (MVS~83, Beijing Guanpujia Technology Co., Ltd.), ultraviolet spectrophotometer (Thermo Scientific NanoDrop One, Thermo Fisher Scientific China Co., Ltd.), desktop high-speed refrigerated centrifuge (Centrifuge 5430R, Eppendorf, Germany), electrophoresis instrument (DYY-60, Beijing Liuyi Biotechnology Co., Ltd.), gel imager (GenoSens 2000, Shanghai Qinxiang Scientific Instrument Co., Ltd.), constant temperature water bath (HH-12468, Changzhou Langyue Instrument Manufacturing Co., Ltd.), and the primers used in the experiment were synthesized by Shenggong (Shanghai) Bioengineering Co., Ltd.

[0061] Genomic DNA extraction

[0062] The extraction method includes one or more of the following: CTAB method, SDS method, magnetic bead method, sodium lauroyl sarcosinate method and CTAB magnetic bead method.

[0063] Sodium Lauroyl Sarcosinate Method

[0064] Grind the fungus with liquid nitrogen to powder in advance, quickly put it into a tube, add 1 mL of preheated sodium lauroyl sarcosinate lysis solution, shake and mix in a 56°C water bath for 40 minutes, and shake and mix for 1 minute every 5 minutes. The subsequent steps are the same as the CTAB method.

[0065] CTAB magnetic beads method

[0066] Grind the fungus with liquid nitrogen to powder in advance, quickly put it into a tube, add 800μL of preheated 2% CTAB lysis solution, 20μL of proteinase K, mix thoroughly, incubate at 60℃ for 10min, centrifuge at 12000rpm at room temperature for 5min, take the supernatant and place it in a new centrifuge tube and add an equal volume of isopropanol (pre-cooled in advance), mix well, add magnetic beads, mix well again and let stand for 5min, transfer to a magnetic stand and let stand for 2min, discard the waste liquid and add rinse solution, mix well and place on a magnetic stand for 2min, discard the waste liquid and repeat this step. Finally, add 50μLTE to dissolve and mix well, then bathe in 65℃ water for 5min, and adsorb on the magnetic stand for 5min to obtain genomic DNA.

[0067] Identification of soybean gray leaf spot pathogen and screening of specific fragments

[0068] Conventional PCR amplification and identification of soybean gray spot pathogen were performed using known primers TF (primer sequences are shown in Table 1). The PCR amplification system was 10×Taq Buffer 2.5μL, dNTPmix 2μL, 0.3μL of each of the forward and reverse primers 10mmol / L, 7μL of genome with a final concentration of 10ng / μL, 0.25μL of Taq enzyme, and water was added to 25μL. The program was set as pre-denaturation at 94℃ for 3min, denaturation at 94℃ for 30s, annealing at 57℃ for 30s, extension at 72℃ for 1min, 30 cycles, and post-extension at 72℃ for 10min. After agarose gel electrophoresis detection, the PCR reaction product was sent to Shanghai Bioengineering Co., Ltd. for sequencing and sequence alignment with soybean gray spot pathogen on NCBI.

[0069] The genome sequences of soybean gray spot pathogen and the fungus used in this experiment were selected and downloaded from NCBI, and the SnapGene software and NCBI website were used for comparison and analysis. Finally, the gene sequence KC888798.1 was screened and determined as the specific fragment and primers were designed.

[0070] RPA primer design

[0071] According to the RPA primer design principle, three pairs of forward and reverse primers were designed on the specific fragment using Primer 5.0 software to obtain 9 primer combinations, as shown in Table 1

[0072] Table 1 Primer sequences used in this study

[0073]

[0074] 1.1 Establishment of basic RPA reaction system and screening of optimal primer pairs

[0075] RPA amplification was performed using the basic ERA kit of Suzhou XinDa Gene Technology Co., Ltd. The reaction system was as follows: 2.5 μL each of forward and reverse primers, 21 μL of ddH2O, 20 μL of dissolving agent, 2 μL of 10 ng / μL genome, and 2 μL of activating agent were added to the inside of the tube cap, centrifuged and mixed, and immediately incubated in a 39°C water bath for 20 min. When the time was up, it was immediately taken out and 6× loading buffer was added to incubate at 56°C for 5 min. At the same time, a negative control was prepared with ddH2O. After the reaction, 3.5% agarose gel electrophoresis was performed for identification at 80 V for 1 h. After the electrophoresis, the electrophoresis results of the 9 primer combinations were analyzed.

[0076] Optimization of basic RPA reaction conditions

[0077] Determination of optimal reaction temperature

[0078] Set 6 reaction temperatures of 37, 38, 39, 40, 41, and 42°C, configure the same reaction system according to the best primer pair selected in 1.1, and use ddH2O to prepare the negative control, incubate at the corresponding 6 temperatures for 20 minutes, take out immediately after the time is up, add 6× loading buffer and incubate at 56°C for 5 minutes, perform 3.5% agarose gel electrophoresis at 80V for 1h after the reaction, and analyze the electrophoresis results.

[0079] Determine the optimal response time

[0080] Set 7 reaction times of 10, 15, 20, 25, 30, 35, and 40 min. Configure the same reaction system according to the best primer pair screened in 1.1, and use ddH2O to prepare the negative control. Incubate at the corresponding 7 time periods. Use the optimal reaction temperature to determine the optimal reaction temperature in the step. When the time is up, immediately take out and add 6× loading buffer to incubate at 56°C for 5 min. After the reaction, perform 3.5% agarose gel electrophoresis at 80V for 1 h. After the electrophoresis, analyze the electrophoresis results.

[0081] Test verification

[0082] Sensitivity Verification

[0083] Set 8 template concentrations of 9×107, 9×106, 9×105, 9×104, 9×103, 9×102, 9×101, and 9fg / μL. Configure the reaction system according to the best primer pair screened in 1.1. Add the templates diluted to the corresponding concentrations in advance into different reaction tubes respectively, and use ddH2O to prepare the negative control. Incubate at the optimal reaction temperature and optimal reaction time. When the time is up, take out and add 6×loading buffer immediately and incubate at 56℃ for 5min. After the reaction, perform 3.5% agarose gel electrophoresis for identification at 80V for 1h. After the electrophoresis, analyze the electrophoresis results.

[0084] Specificity verification

[0085] Six fungi, including gray leaf spot pathogen, Fusarium oxysporum, Fusarium solani, Phytophthora, Fusarium acuminate, and Peronospora manchuria, were set up for specific verification. The same reaction system was configured according to the best primer pair screened in 1.1, and negative control was prepared using ddH2O. The cells were incubated under the optimal reaction temperature and optimal reaction time conditions. After the time was up, the cells were immediately taken out and added with 6× loading buffer and incubated at 56°C for 5 minutes. After the reaction, 3.5% agarose gel electrophoresis was performed for identification at 80V for 1 hour. After the electrophoresis was completed, the electrophoresis results were analyzed.

[0086] Actual sample verification

[0087] DNA was extracted and diluted to a uniform concentration from soybean plants infected with gray leaf spot pathogen, mycelium of gray leaf spot pathogen, soybean plants not infected with gray leaf spot pathogen, soybean plants infected with Fusarium oxysporum, soybean plants infected with Phytophthora sojae, mycelium of Fusarium acutum, and mycelium of Peronospora mandshurica. Negative controls were prepared using ddH2O. The cultures were incubated under the optimal reaction temperature and optimal reaction time. After the time was up, the cultures were immediately taken out and 6× loading buffer was added and incubated at 56°C for 5 min. After the reaction was completed, 3.5% agarose gel electrophoresis was performed at 80V for 1 h. After the electrophoresis was completed, the electrophoresis results were analyzed.

[0088] Results and Analysis

[0089] Genomic DNA extraction results

[0090] The DNA of soybean gray spot fungus extracted by different methods was detected by ultraviolet spectrophotometer. The identification results are shown in Table 2. The electrophoresis diagrams of genomic DNA extracted by different methods are shown in Figure 1Combining the data of the two, it can be obtained that the concentration of genome extracted by SDS method and sodium lauroyl sarcosinate method is low, and the values ​​of OD260 / 280 and OD260 / 230 are lower than 1.8, and there is contamination by macromolecules such as proteins. The concentration of genome extracted by magnetic bead method tends to be medium, and the electrophoresis bands appear diffuse. Combining the two sets of data, CTAB method and CTAB magnetic bead method are better. CTAB method is inexpensive, while CTAB magnetic bead method relies on specific reagents and has a high cost. CTAB method can be selected for small-scale laboratories, basic research, and those that are sensitive to detection costs and have no urgent time requirements. For laboratories that require high precision and rapid detection and need to quickly obtain high-purity DNA and have conditions for field detection, CTAB magnetic bead method can be selected.

[0091] Table 2 DNA UV identification results of different extraction methods

[0092]

[0093] in Figure 1 :M.DL 15000bp DNA Marker; 1. CTAB method; 2. SDS method; 3. Magnetic bead method; 4. Sodium lauroyl sarcosinate method; 5. CTAB magnetic bead method

[0094] Identification of the pathogen of soybean gray leaf spot

[0095] Electrophoresis of the whole genome of soybean gray leaf spot pathogen amplified by PCR using known primers TF ( Figure 2 ), the result showed that the band was consistent with the expected size, and it was sent to Shanghai Biotechnology Co., Ltd. for sequencing and sequence comparison. The homology with the soybean gray spot fungus on NCBI reached more than 99%, which met the needs of designing RPA primers.

[0096] in Figure 2 The complete genome of soybean gray spot fungus was amplified by PCR using primers M.DL 2000bp DNA Marker 1 and 2.TF.

[0097] Screening of optimal primer pairs

[0098] Depend on Figure 3 It can be seen that all 9 primer combinations can amplify the genome of soybean gray spot pathogen, but non-specific amplification bands appear except for lanes 3 and 6, and the amplification effect is poor, and the bands are shallow. Therefore, lanes 3 and 6 are the best among the 9 primer combinations, and the band in lane 3 is brighter under the same conditions. In summary, lane 3, i.e., the primer combination of F75 and R213, is the best primer pair.

[0099] Figure 3Medium M.DL 500bp DNA Marker 1: Negative control 2: F67 / R213 3: F75 / R213 4: F46 / R213 5: F67 / R247 6: F75 / R247 7: F46 / R247 8: F67 / R215 9: F75 / R215 10: F46 / R21.

[0100] Determination of optimal reaction temperature

[0101] Depend on Figure 4 It can be seen that within the temperature range of 37-42°C, the target bands can be amplified without non-specific bands and diffusion. The bands are lighter at 37°C, which may be due to the poor activity of the enzyme under lower temperature conditions. The brightness of the bands in the range of 40-42°C shows a downward trend, which may be due to the decrease in enzyme activity as the temperature increases. In summary, 38°C and 39°C are better temperatures. Since the target bands are lighter at 39°C, 38°C is the best reaction temperature.

[0102] in Figure 4 M.DL 500bp DNA Marker; 1: negative control; 2: 37℃; 3: 38℃; 4: 39℃; 5: 40℃; 6: 41℃; 7: 42℃.

[0103] Determine the optimal response time

[0104] Depend on Figure 5 It can be seen that the target band can be amplified at different reaction times. The band is very light at 10 minutes. It is speculated that the reaction time is insufficient, only a small amount of template is amplified, and the amount of amplified product is small. The band is bright at 25 minutes, and the amplification is complete. The brightness of the band in the range of 30 to 40 minutes becomes lighter than that at 25 minutes, and non-specific bands appear at around 300bp. It is speculated that under long-term reaction conditions, the primers may bind to some non-target sequences and amplify under the action of the enzyme. As the proportion of the target product in the total product decreases, its band brightness will become lighter. In summary, 25 minutes is the optimal reaction time.

[0105] in Figure 5 M.DL 500bp DNA Marker; 1: negative control; 2: 10min; 3: 15min; 4: 20min; 5: 25min; 6: 30min; 7: 35min; 8: 40min.

[0106] Sensitivity Verification

[0107] Sensitivity test results are as follows Figure 6As shown in the figure, the target bands were amplified in the concentration range of 9×107 to 9×101fg / μL, and the brightness of the bands tended to become lighter as the template concentration decreased. When the concentration was 9×101fg / μL, the detection amount was significantly reduced. When the concentration continued to drop to 9fg / μL, no bands could be detected, indicating that the detection sensitivity can reach the concentration range of 9×101fg / μL. In summary, the detection sensitivity of gray spots is relatively high, which meets the needs of on-site detection.

[0108] in Figure 6 M.DL 500bp DNA Marker 1: Negative control 2: 9×107fg / μL 3: 9×106fg / μL 4: 9×105fg / μL 5: 9×104fg / μL 6: 9×103fg / μL 7: 9×102fg / μL 8: 9×101fg / μL 9: 9fg / μL.

[0109] Specificity verification

[0110] The specificity verification results are as follows Figure 7 As shown, under the same amplification conditions, only the soybean gray spot pathogen in lane 2 could be detected, while no target bands appeared for Fusarium oxysporum, Fusarium solani, Phytophthora sojae, Fusarium acuminate, and Peronospora manchuriae, proving that the detection system has high detection specificity.

[0111] in Figure 7 M.DL 500bp DNA Marker; 1: negative control; 2: Gray leaf spot pathogen; 3: Fusarium oxysporum; 4: Fusarium solani; 5: Phytophthora sojae; 6: Fusarium acuminate; 7: Peronospora mandshurica.

[0112] Actual sample verification

[0113] The actual sample verification results are as follows Figure 8 As shown, the target bands appeared in the plants infected with gray leaf spot pathogen in lane 2 and gray leaf spot pathogen in lane 3, while no bands were amplified in normal soybean plants, Fusarium oxysporum, Phytophthora sojae, Fusarium acuminate and Peronospora manchuriae, which was the same as the negative results, proving that it still had strong specificity when verified in actual samples.

[0114] in Figure 8 M.DL 500bp DNA Marker; 1: negative control; 2: soybean plant infected with gray leaf spot pathogen; 3: gray leaf spot pathogen; 4: soybean plant; 5: Fusarium oxysporum; 6: Phytophthora sojae; 7: Fusarium acuminata; 8: Downy mildew of soybean.

[0115] In summary, this study selected soybean gray spot pathogen as the research object, and established a rapid detection method for the fungus based on RPA technology for the first time. The improved SDS method independently developed was used to extract genomic DNA with good concentration and purity. According to the specific segment of the soybean gray spot pathogen gene KC888798.1 downloaded from NCBI, three pairs of forward and reverse primers were designed using Primer 5 and experimental screening was carried out. F75 / R213 was confirmed to be the best primer pair. On this basis, the temperature and time were optimized, and the sensitivity and specificity were verified. Finally, under the constant temperature of 38℃, the reaction time was 20-25min, and the lowest detection of soybean gray spot pathogen was 90fg / μL, and it did not react with other fungi such as Fusarium oxysporum, Fusarium acutum, and Downy Mildew of Northeast China. A rapid detection system for soybean gray spot pathogen based on RPA technology was successfully established.

[0116] It will be appreciated that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will be a routine task of design, fabrication, and production for those of ordinary skill having the benefit of this disclosure without undue experimentation.

[0117] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A rapid detection method for soybean gray spot pathogen based on RPA, characterized in that: The following steps are involved: S1. Sample preparation and genomic DNA extraction: After taking the sample fungal strain out of the -80°C freezer, it was activated on a PDA plate and cultured in an incubator at 25°C for genomic DNA extraction; The extraction method includes one or more of CTAB method, SDS method, magnetic bead method, sodium lauroyl sarcosinate method and CTAB magnetic bead method; S2. RPA reaction system configuration Primer Pairs: Forward primer F75: 5'-TCCAGCTCAGCAAGGCTCGCATCACTCCGT-3' Reverse primer R213: 5′-ACTGGAAAGAAGCCTCAAACTAAACCTAAG-3′ Reaction system (50 μL) 2.5 μL each of forward and reverse primers 20 μL RPA Lysis Reagent 2μL genomic DNA (10ng / μL) 2 μL activator Add ddH2O to 50μL; S3. RPA amplification conditions Temperature: 38℃ constant temperature; time: 20-25min Operation: After centrifugation and mixing, place in a constant temperature water bath immediately. After the reaction is completed, add 6× loading buffer and incubate at 56℃ for 5 minutes; S4. Electrophoresis detection Prepare 3.5% agarose gel (containing nucleic acid dye), and run the electrophoresis at 80 V constant voltage for 1 h. When observed under a gel imager, the presence of 100-200bp is considered positive.

2. The method for detecting soybean gray spot pathogen based on RPA according to claim 1, characterized in that: The CTAB method in S1 comprises the following steps: firstly, the fungus is ground into powder with liquid nitrogen, and then 800 μL of 2% preheated CTAB lysis solution (40 μL of β-mercaptoethanol is added in advance) is added after being put into a tube, and then the mixture is shaken and mixed in a 56°C water bath for 40 min, and shaken and mixed for 1 min every 5 min, and then centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant is taken into a new tube, and an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) is added, and after being mixed by inverting several times, the mixture is centrifuged at 12000 rpm and 4°C for 10 min. in, take the supernatant and add 2 times the volume of pre-cooled anhydrous ethanol to a new tube, let it stand in a -20 ℃ refrigerator for 20 minutes, a translucent flocculent precipitate will appear in the centrifuge tube, which is the fungal genome, centrifuge at 12000rpm, 4 ℃ for 8 minutes, discard the supernatant, wash the precipitate with pre-cooled 70% ethanol, and centrifuge at 10000rpm for 5 minutes (repeat 2 times), discard the supernatant, put it on ice in an ultra-clean workbench to dry, then add 50μLTE to dissolve and 3μLRNaseA to remove RNA, measure the concentration and store it in a -20 ℃ refrigerator for later use.

3. The method for detecting soybean gray spot pathogen based on RPA according to claim 1, characterized in that: The SDS method comprises the following steps: grinding the fungus with liquid nitrogen to powder in advance, quickly putting it into a tube, and immediately adding 1 mL of preheated SDS lysis solution, shaking and mixing in a 56° C. water bath for 40 minutes, and shaking and mixing for 1 minute every 5 minutes, and the subsequent steps are the same as the CTAB method.

4. The method for detecting soybean gray spot pathogen based on RPA according to claim 1, characterized in that: The magnetic bead method comprises the following steps: grinding the fungus with liquid nitrogen to powder, quickly putting it into a tube, adding 800 μL of preheated lysis solution and 20 μL of proteinase K, shaking and mixing in a 56°C water bath for 40 minutes, and shaking and mixing for 1 minute every 5 minutes, taking the supernatant into a new tube, adding an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), inverting several times to mix, centrifuging at 12000 rpm and 4°C for 10 minutes, taking the supernatant and adding an equal volume of isoamyl alcohol (precooled in advance) and an appropriate amount of magnetic bead suspension into a new tube, mixing and standing for 15 minutes, transferring to a magnetic stand and standing for 1 minute, discarding the waste liquid, washing with deproteinized solution and 70% ethanol (twice), drying at room temperature for 5 minutes, adding 50 μL TE to dissolve and standing on a magnetic stand for 1 minute, and the resulting solution is genomic DNA.

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