CN117683933A - Primer group, kit and method for detecting fusarium oxysporum on basis of RPA isothermal amplification technology - Google Patents

Primer group, kit and method for detecting fusarium oxysporum on basis of RPA isothermal amplification technology Download PDF

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CN117683933A
CN117683933A CN202410004794.2A CN202410004794A CN117683933A CN 117683933 A CN117683933 A CN 117683933A CN 202410004794 A CN202410004794 A CN 202410004794A CN 117683933 A CN117683933 A CN 117683933A
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primer
fusarium oxysporum
sample
detecting
amplification
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徐亚维
范文忠
王俊玲
王欢
楚海娇
魏健
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Jilin Agricultural Science and Technology College
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Jilin Agricultural Science and Technology College
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Abstract

The invention provides a primer group, a kit and a method for detecting soybean fusarium oxysporum based on an RPA isothermal amplification technology, which belong to the technical field of soybean fusarium oxysporum detection, and the primer group for detecting soybean fusarium oxysporum based on the RPA isothermal amplification technology comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2. The method of the invention comprises the following steps: 1) Extracting genomic DNA of a sample; 2) Carrying out recombinase polymerase amplification on the genomic DNA of the sample by utilizing the primer group to obtain an amplified product; 3) Detecting the amplified product, and determining whether the soybean fusarium oxysporum exists in the sample according to the detection result. The method provided by the invention can display the result in a short time under convenient conditions, has high sensitivity and strong specificity, and can realize on-site rapid detection.

Description

Primer group, kit and method for detecting fusarium oxysporum on basis of RPA isothermal amplification technology
Technical Field
The invention belongs to the technical field of detection of soybean fusarium oxysporum, and particularly relates to a primer group, a kit and a method for detecting soybean fusarium oxysporum based on an RPA isothermal amplification technology.
Background
Fusarium sojae (Fusarium oxysporum) has been considered as a plant vascular bundle soil-borne pathogenic fungus distributed worldwide as a member of the Fusarium genus (Zhang Yaduo, liu Jia, huang Wenkun, etc.. Molecular characterization of Fusarium sojae, hebei gallery, molecular characterization of Fusarium sojae, J. Plant pathogeny, 2018,48 (06): 738-747.). The host range of the soybean fusarium oxysporum is particularly wide, and the soybean fusarium oxysporum can cause more than 100 plants such as vegetables and fruits such as leguminous plants, melons and bananas, flowers and the like to be infected, and is also a facultative parasitic fungus which can infect plants and survive in soil. Infection of soybean by fusarium oxysporum causes soybean wilt, and the soybean wilt may be affected in various growth periods, and the affected vegetation symptoms appear dwarfing, pod bearing reduction, root mildew and rot of serious people and finally wilt until death. Therefore, under the urgent demands of agricultural production, in order to effectively prevent and control soybean diseases caused by fusarium oxysporum, a method capable of rapidly and accurately detecting the soybean fusarium oxysporum is urgently needed to be established.
Regarding fungi of fusarium, identification is usually performed according to morphology, namely growth morphology, spore structure and the like of the fungi, but the method is time-consuming and labor-consuming, and as the types of fungi found are more and more, fungi of different genus also show similar characteristics, and the fungi are difficult to accurately distinguish by morphology alone. At present, molecular detection technology is developed rapidly, has higher sensitivity, specificity and accuracy compared with the traditional morphological identification method, and the molecular detection technology for fungi has the conventional PCR and real-time quantitative PCR detection technology, but both rely on large and expensive instruments, and has long time consumption, and needs professional technicians, thereby being unfavorable for rapid and convenient detection (Cui Linkai, shihua, kang Ye and the like). Recombinase polymerase amplification (recombinase polymerase amplification, RPA) is an isothermal nucleic acid amplification technique developed in recent years that accomplishes in vitro amplification of nucleic acid target sequences by the interaction of a recombinase, a recombinase loading factor, and a single-stranded binding protein with primers and target sequences (Wang Shuai, yang Yange, wu Zhanwen, etc., research advances in rapid detection of food-borne pathogenic bacteria for recombinase polymerase amplification, recombinase-mediated isothermal amplification, and enzymatic recombination isothermal amplification techniques [ J ]. Food science, 2023,44 (09): 297-305.). The method not only maintains the characteristics of sensitive and efficient reaction and high cost performance, but also has the advantages of isothermal amplification, no need of special equipment, short time consumption and the like, and is more suitable for on-site detection. At present, RPA has been gradually applied to detection of plant diseases and insect pests such as Cherry Virus A (CVA) (Chen Ling, duan Xuwei, zhang Kaichun, etc.. A detection method based on Recombinase Polymerase Amplification (RPA) technology [ J ]. Gardening theory, 2020,47 (02): 390-398.), bean pod mottle virus (BPMV for short) (Zhang Yongjiang, wei Shuang, yuan Junjie, etc.. A one-step reverse transcriptase polymerase normal temperature amplification (RT-RPA) technology detects bean pod mottle virus [ J ]. Jiangsu agricultural science, 2018,46 (21): 96-98), tomato yellow leaf curl virus (TYLCV for short) (WangTM, yangJT.Visual DNA diagnosis of tomato yellow leafcurl virus with integrated recombinase polymerase amplification and a gold-nanoparticle probe [ J ]. Scientific Reports,2019,9 (1): 1-8). However, the detection of fusarium oxysporum has not been reported.
Disclosure of Invention
In view of the above, the invention aims to provide a primer group, a kit and a method for detecting fusarium oxysporum on the basis of an RPA isothermal amplification technology; the primer group and the specific detection method provided by the invention have the characteristics of high sensitivity and good specificity.
The invention provides a primer group for detecting soybean fusarium oxysporum based on an RPA isothermal amplification technology, which comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2.
The invention provides a kit for detecting soybean fusarium oxysporum based on an RPA isothermal amplification technology, which comprises the primer group and a detection reagent.
The invention also provides a method for detecting soybean fusarium oxysporum based on the RPA isothermal amplification technology, which comprises the following steps:
1) Extracting genomic DNA of a sample;
2) Carrying out recombinase polymerase amplification on the sample genome DNA obtained in the step 1) by utilizing the primer group to obtain an amplified product;
3) Detecting the amplified product, and determining whether the soybean fusarium oxysporum exists in the sample according to the detection result.
Preferably, the method for extracting genomic DNA from a sample in step 1) comprises SDS method or modified CTAB.
Preferably, the system for recombinase polymerase amplification described in step 2) comprises the following components in 50 μl: 20. Mu.L of lytic reagent, 2.5. Mu.L of forward primer, 2.5. Mu.L of reverse primer, and 2. Mu. L, ddH of sample genomic DNA 2 O21. Mu.L and activator 2. Mu.L.
Preferably, the concentration of genomic DNA of the sample is greater than 9 fg/. Mu.L.
Preferably, the temperature of the recombinase polymerase amplification is 39-40 ℃, and the time of the recombinase polymerase amplification is 15-20 min.
Preferably, the method for detecting the amplification product in step 3) is a gel electrophoresis method or an EXO probe method.
Compared with the prior art, the invention has the following beneficial effects: the primer group for screening and obtaining the soybean fusarium oxysporum according to the specific gene design of the fusarium oxysporum has the advantages of good specificity and high detection sensitivity in the amplification procedure of RPA isothermal amplification.
The method for detecting the soybean fusarium oxysporum based on the RPA isothermal amplification technology provided by the invention can show the result in a short time under a convenient condition, has high sensitivity, can reach 90 fg/mu L in lower detection limit, is convenient to operate, can realize on-site rapid detection, provides a new direction for detecting the soybean fusarium oxysporum, and has a great practical significance.
Drawings
FIG. 1 is a diagram showing DNA electrophoresis of Fusarium oxysporum extracted by different methods, wherein: DL 15000bp DNAMaroker; SDS method; 2. the CTAB method is improved; 3. a urea process; 4. sodium lauroyl sarcosinate process; CTAB magnetic bead method;
FIG. 2 is a diagram of the EF primer PCR amplification of whole genome of F.sojae, wherein: DL 500bp DNAMaroker; 1. the EF primer PCR amplifies the whole genome product of the fusarium oxysporum;
FIG. 3 is a diagram of primer screening agarose gel electrophoresis, wherein: DL 500bp DNAMaroker; F274/R459; F274/R500; F274/R466; F237/R459; F237/R500; F237/R466; F180/R466; F180/R459; F180/R500;10. a negative control;
FIG. 4 is a diagram of an optimal reaction temperature determination agarose gel electrophoresis in which M.DL 500bp DNA markers; 1.37 ℃;2.38 ℃;3.39 ℃;4.40 ℃;5.41 ℃;6.42 ℃;7. a negative control;
FIG. 5 is a graph of agarose gel electrophoresis for optimal reaction time determination, wherein M.DL 500bp DNA Marker;1.15min;2.20min;3.25min;4.30min;5.35min;6.40min;7. a negative control;
FIG. 6 is a sensitivity verification agarose gel electrophoresis diagram in which M.DL 500bp DNA markers; 1.9X10 7 fg/μL；2.9×10 6 fg/μL；3.9×10 5 fg/μL；4.9×10 4 fg/μL；5.9×10 3 fg/μL；6.9×10 2 fg/. Mu.L; 7.9X10 fg/. Mu.L; 8.9 fg/. Mu.L; 9. a negative control;
FIG. 7 is a diagram of specificity-verifying agarose gel electrophoresis, wherein: DL 500bp DNA Marker;1. fusarium oxysporum genome; 2. phytophthora sp; 3. gray plaque bacteria; 4. fusarium solani; 5. botrytis cinerea; 6. northeast downy mildew; 7. a negative control;
FIG. 8 is an actual sample-validated agarose gel electrophoresis, wherein: DL 500bpDNA Marker;1. fusarium oxysporum mycelium of soybean; 2. soybean plants not infected with fusarium oxysporum; 3. infection of fusarium oxysporum soybean plants; 4. soybean plants infected with phytophthora sojae; 5. a soybean gray plaque mycelium; 6. fusarium solani hyphae; 7. a downy mildew hypha; 8. negative control.
Detailed Description
The invention provides a primer group for detecting soybean fusarium oxysporum based on an RPA isothermal amplification technology, which comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; the method comprises the following steps:
F274：5’-GACTTCTGGTATCGTCGTATCTCACGGCGA-3’(SEQ ID No.1)；
R466：5’-GCTTCCAAGGCTGGTAAGTCTTCACCGCGA-3’(SEQ ID No.2)。
the primer group provided by the invention has no nonspecific amplification, no dispersive strip, good repetition stability, and the length of an amplification product is 193bp, so that the detection is convenient.
The invention provides a kit for detecting soybean fusarium oxysporum based on an RPA isothermal amplification technology, which comprises the primer group and a detection reagent. In the present invention, the detection reagent comprises a basic nucleic acid amplification kit, preferably purchased from Soviet gene technologies Inc.
The invention also provides a method for detecting soybean fusarium oxysporum based on the RPA isothermal amplification technology, which comprises the following steps: 1) Extracting genomic DNA of a sample; 2) Carrying out recombinase polymerase amplification on the sample genome DNA obtained in the step 1) by utilizing the primer group to obtain an amplified product; 3) Detecting the amplified product, and determining whether the soybean fusarium oxysporum exists in the sample according to the detection result.
In the invention, the sample genomic DNA is firstly extracted, and the method for extracting the sample genomic DNA preferably comprises an SDS method or modified CTAB method, and the purity and the concentration of the genomic DNA extracted by the two methods are both better.
The invention uses the primer group to obtain the sample genome after obtaining the sample genome DNACarrying out recombinase polymerase amplification on DNA to obtain an amplified product; the system for amplifying the recombinase polymerase preferably comprises the following components in 50 mu L: 20. Mu.L of lytic reagent, 2.5. Mu.L of forward primer, 2.5. Mu.L of reverse primer, and 2. Mu. L, ddH of sample genomic DNA 2 O21. Mu.L and activator 2. Mu.L. The preferred concentration of genomic DNA in the sample is greater than 9 fg/. Mu.L, more preferably greater than 9X 10 fg/. Mu.L. In the present invention, the genomic DNA sample is preferably diluted to 8 to 12 ng/. Mu.L, more preferably 10 ng/. Mu.L, before amplification. The concentration of the forward and reverse primers is preferably 10. Mu.M. In the present invention, the temperature of the recombinase polymerase amplification is preferably 39-40 ℃, and the time of the recombinase polymerase amplification is preferably 15-20 min.
In the invention, after the amplification product is obtained, the amplification product is detected, and whether fusarium sojae is present in the sample is determined according to the detection result. In the present invention, the method for detecting an amplification product is preferably a gel electrophoresis method or an EXO probe method. The specific operation steps of the gel electrophoresis method or the EXO probe method are not particularly limited, and the gel electrophoresis method or the EXO probe method which are conventional in the art can be adopted. When the detection is carried out by using a gel electrophoresis method, when only one specific band appears at 193bp, the soybean fusarium oxysporum is considered to exist in the sample, otherwise, the soybean fusarium oxysporum is considered to not exist in the sample.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Main materials
Fusarium oxysporum (Fusarium oxysporum), phytophthora sojae (Phytophthora megasperma f.sp.glyconeae Kuan & Erwin), leuconostoc sojae (Cercospora sojina Hara), fusarium solani (Fusarium sorghi Henn.), botrytis cinerea Pers, and downy mildew (Peronospora manschurica) are all gifts from the Jilin agricultural sciences and sciences agricultural college; the above biological material can be replaced by corresponding commercial strains.
Main reagent
Basic nucleic acid amplification kits were purchased from suda gene technologies ltd; sodium Dodecyl Sulfate (SDS), DL 500bpDNA Marker were purchased from Shanghai Biotechnology Co., ltd; cetyl trimethylammonium bromide (CTAB), sodium sarcosinate laurate were purchased from Tianjin Fuchen chemical reagent Co., ltd; polyvinylpyrrolidone (PVP) was purchased from fei biotechnology limited; beta-mercaptoethanol was purchased from Tianjin metallocene chemical technology Co., ltd; nucleic acid-extracting magnetic beads (general purpose) were purchased from michiz pharmaceutical technologies, inc; DL 15000bp DNA Marker, 10×Loading buffer were purchased from Takara doctor materials technologies Co.
Main instrument
A tabletop high-speed refrigerated Centrifuge (Centrifuge 5430R, germany Ai Bende); thermostatic water bath (HH-12468, changzhou Lang Yue instruments Co., ltd.); electrophoresis apparatus (DYY-60, beijing six biotechnology Co., ltd.); ultraviolet spectrophotometry (BioSpec-nano, shimadzu corporation); autoclave (MVS-83, beijing guanpula technologies Co., ltd.); gel imager (GenoSens 2000, shanghai Saint scientific instruments Co., ltd.).
Example 1
Genome extraction method
SDS method
To the powder of Fusarium oxysporum, pre-heated SDS lysate [2% SDS, 100mmol/L Tris-HCl (pH 8.0), 20mmol/L EDTA (pH 8.0), 1.4mol/LNaCl, 6% PVP-40, 40. Mu.L beta-mercaptoethanol, 0.5% Tween 20] 800. Mu.L and 40. Mu.L beta-mercaptoethanol were added, and the mixture was mixed, and the mixture was subjected to a water bath in a thermostatic water bath at 55℃for 50 minutes, and gently mixed every 10 minutes. Centrifuging at 12000rpm and 4deg.C for 10min, collecting supernatant, adding equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), and mixing. Centrifuge at 12000rpm,4℃for 10min. (cf. The following: wang Haiying, liu aviation, comparison of different methods of microextraction of tomato genomic DNA and suitability analysis [ J ]. Proc. North-West agriculture, 2022,31 (12): 1560-1567.). Adding 2 times of pre-cooled isopropanol into the supernatant, mixing, and standing at-20deg.C for 30min to obtain floccule. Centrifuging at 10000rpm at 4deg.C for 10min, discarding supernatant, washing the precipitate with 70% ethanol for 2 times, discarding supernatant, and air drying. Add 50. Mu.L of LTE buffer and 3. Mu.L of RNaseA for solubilization and store in a refrigerator at-20 ℃.
2. Improved CTAB process
Adding preheated 2% CTAB lysate [0.05g PVP-40 powder, 100mmol/L Tris-HCl, 20mmol/L EDTA-Na ] into fungal powder 2 1.4mol/L NaCl, 2% CTAB, 5mmol/L diethyl dithiocarbamate, 40. Mu.L beta-mercaptoethanol (added before use)]mu.L and 20 mu.L proteinase K solution (see Liang Meidan, shoujian, chen Jie, etc. for magnetic bead method for extracting plant protein beverage DNA [ J ]]Food safety quality inspection school report, 2021,12 (13): 5363-5368.). Mixing, keeping temperature in a water bath kettle at 55deg.C for 50min, shaking and mixing once every 10min, centrifuging at 12000rpm for 10min, adding equal volume of phenol-chloroform-isoamyl alcohol into supernatant, and mixing. Centrifuging at 12000rpm and 4deg.C for 10min, collecting supernatant, adding 2 times of pre-cooled absolute ethanol, mixing, and precipitating in-20deg.C refrigerator for 30min to obtain flocculent precipitate. Centrifuging at 10000rpm at 4deg.C for 10min, discarding supernatant, washing the precipitate with 70% ethanol for 2 times, discarding supernatant, and air drying. Add 50. Mu.L of LTE buffer and 3. Mu.L of RNaseA for solubilization and store in a refrigerator at-20 ℃.
3. Urea extraction method
Adding preheated urea extract (7 mol/L CO (NH) 2 ) 2 、50mmol/L Tris-HCl(pH 8.0)、1％SDS、62.5mmol/L NaCl]1mL and 40. Mu.L of beta-mercaptoethanol, in a water bath at 65 ℃ for 50min, were mixed for 30s every 10min by vortex oscillation (see Luo Sumei, guo Chongyan, liu Xiao et al, comparative study on the extraction method of genome DNA of. 5 kinds of daphne giraldii [ J ]]Anhui agricultural science, 2023,51 (05): 74-77+88.). 0.1 times of 3mol/L potassium acetate solution is added, and the mixture is kept stand for 30min at the temperature of minus 20 ℃. The subsequent procedure was the same as that of SDS.
4. Sodium lauroyl sarcosinate extraction
The fungal powder is added with preheated sodium lauroyl sarcosinate lysate [1% SLS, 100mmol/L Tris-HCl (pH 8.0), 20mmol/L EDTA (pH 8.0), 100mm NaCl ]1mL and mixed in a 55 ℃ water bath for 30min, and gently inverted and evenly mixed every 5min (see the following documents: cai Wenjiao, xu Dabin, lan Xia, etc.. A novel method for extracting fungal genome DNA [ J ]. Agricultural research and application, 2014 (03): 1-5'), the subsequent extraction steps are the same as 2. The modified CTAB method.
CTAB magnetic bead method
To the fungal powder, add 1mL of preheated 2% CTAB lysate and 40. Mu.L proteinase K solution, in a 65℃water bath for 30min, vortex several times during, centrifuge at 4℃and transfer the supernatant to a new centrifuge tube. Equal amounts of isopropanol were added and DNA was adsorbed using magnetic beads as medium. Adding 15 μl of magnetic bead suspension, and mixing (refer to the following documents: shouxingyu, zhang Yongzhe, zhang Kaihua, etc.. The method for rapidly extracting DNA from meat products based on the magnetic bead method is described in J. Food safety journal, 2022 (17): 65-67.). And the mixture is placed at room temperature for 5min, the magnetic bracket is placed for about 3min, the solution becomes transparent, and the magnetic beads are adsorbed completely. The supernatant was discarded, washed 2 times with 70% ethanol and air-dried, 70. Mu.L of TE eluent was added, the beads were removed from the wall of the centrifuge tube by vortexing, water-bath at 65℃for 5min, and allowed to stand on a magnetic rack for 5min. The eluate was aspirated and placed in a fresh centrifuge tube and stored in a refrigerator at-20 ℃.
Results:
the DNA of Fusarium sojae obtained by different methods was detected by ultraviolet spectrophotometry, and the identification results are shown in Table 1. The method has the advantages that the method can be obtained by synthesizing the following data, if the extraction result of the improved CTAB method is best according to the concentration and purity data analysis, but the traditional extraction method has complicated operation and long extraction time from the time consideration, and the CTAB magnetic bead method has high cost, can realize the rapid extraction of genome DNA, is more suitable for on-site detection, and has stronger superiority.
TABLE 1 ultraviolet identification results of DNA from different extraction methods
And (3) performing 0.7% gel electrophoresis verification on the DNA obtained by the different extraction methods, and determining a genome extraction result. The electrophoresis results of the different extraction methods are shown in FIG. 1. Analysis of the electrophoresis results revealed that: lanes 2-6 are all striped, which indicates that 5 different extraction methods can extract fusarium oxysporum DNA; lanes 2 and 3 are bright and have fewer impurities, which indicates that the SDS method and the modified CTAB method can obtain DNA with better extraction concentration and purity; lanes 4 and 5 are darker than the other lanes, indicating that the urea and sodium lauroyl sarcosinate extraction concentrations are lower than the other methods. Lane 6 is bright, indicating that CTAB bead extraction concentration is better, but there is little DNA degradation.
Example 2
Primer screening
Identification of Fusarium oxysporum
The whole genome of Fusarium oxysporum is amplified by conventional PCR using known primer EF primer (primer sequence is shown in Table 2), buffer 2.5 mu L, dNTPM 2 mu L, forward and reverse primer are diluted to 10mmol/L and added with 0.3 mu L respectively, final concentration is 7 mu L of genome with 10 ng/mu L, taq enzyme is 0.25 mu L, water is used for balancing to 25 mu L, two tubes are arranged together to form mutual comparison, the reaction is carried out for 30 cycles under the procedures of denaturation at 94 ℃, annealing at 58 ℃ for 30 seconds and extension at 72 ℃ for 1min, after agarose gel electrophoresis detection, the result is sent to the Shanghai bioengineering limited company for sequencing and sequence comparison with the Fusarium oxysporum on NCBI.
The PCR amplification of the whole genome electrophoresis diagram (figure 2) of the soybean fusarium oxysporum by using the known primer EF primer shows that the result accords with the expected size, and the result is sent to the Shanghai biological engineering Limited company for sequencing and sequence comparison, the homology with the soybean fusarium oxysporum on NCBI reaches more than 99%, and the requirement of designing the RPA primer is met.
RPA primer design
The basic RPA reaction system comprises two kinds of primers, namely an upstream Primer (F) and a downstream Primer (R), according to the conserved sequence of the soybean fusarium oxysporum in NCBI (National Center for Biotechnology Information), the whole genome of the soybean fusarium oxysporum is amplified by using the defined EF Primer, a known sequence is obtained through sequencing, the known sequence is designed by Primer 5.0 Primer design software, three kinds of upstream and downstream primers are selected after preliminary screening and are all synthesized by the biological engineering company of the Shanghai, and the Primer sequence is shown in table 2.
TABLE 2 primer sequence listing
The genome extracted by the optimal method of example 1 was subjected to Ultraviolet (UV) identification to give a final concentration of 10 ng/. Mu.L, and diluted to 2.5. Mu.L of genome according to 20. Mu.L of lytic reagent, 2.5. Mu.L of forward and reverse primers, and 2. Mu. L, ddH of genome after dilution 2 O21. Mu.L of the premix was prepared in a total volume of 48. Mu.L, and the mixture was stirred and centrifuged briefly.
Before the experiment starts, taking out the freeze-dried powder reaction tube in the basic nucleic acid amplification kit from a refrigerator at the temperature of minus 20 ℃, balancing and placing for 10min at room temperature, transferring the uniformly mixed premix into a basic reaction reagent, centrifuging briefly, dripping 2 mu L of activator into the inner side of a reaction tube cover, slightly covering the reaction tube cover, and preventing the activator from entering the reaction tube in advance to start reaction in advance to influence the experiment.
9 kinds of combinations are respectively paired with the designed primers, the corresponding primers are respectively added into a body u system according to a specified quantity to prepare a premix liquid, a reaction system is established, and ddH is simultaneously utilized 2 O negative control was prepared, the reaction tube was placed in a thermostat and incubated at 39℃for 20min, 7.5. Mu.L of 6 Xlocking buffer was added to the reaction tube immediately after the completion of the reaction and incubated at 56℃for 5min, and 10. Mu.L of the sample was taken and identified by 3.5% agarose gel electrophoresis.
According to the optimal primer screening electrophoresis chart (figure 3), all primer pairs amplify specific bands, but non-specific bands are amplified except for a third channel and an eighth channel, wherein the third channel and the eighth channel are optimal primers, but partial dispersion bands exist in the eighth channel. And the third primer pair is a better primer pair after multiple times of verification, which shows that the repeated stability is good, namely F274/R466 is the optimal primer pair, and the amplification length is 193bp.
Basic RPA reaction condition optimization
Determination of optimal reaction temperature
The optimal primer (F274/R466) determined above was selected to prepare a reaction system (in terms of 50. Mu.L, comprising the following components: 20. Mu.L of a lytic reagent, 2.5. Mu.L of a forward primer at a concentration of 10. Mu.M, 2.5. Mu.L of a reverse primer at a concentration of 10. Mu.M, and 2. Mu. L, ddH of genomic DNA of the sample) 2 O21. Mu.L and 2. Mu.L of activator) to prepare a 6-tube complete phaseSame amplification reagent and using ddH 2 O negative control was prepared, 7-tube amplification reagent was numbered, incubated at 37℃at 38℃at 39℃at 40℃at 41℃for 20min, the reaction tube was immediately removed after the end of the reaction time, 7.5. Mu.L of 6 Xlobing buffer was added thereto and incubated at 56℃for 5min, and 10. Mu.L of the sample was identified by 3.5% agarose gel electrophoresis.
The determination of the electrophoresis pattern (FIG. 4) based on the optimal reaction temperature shows that all temperatures amplify specific bands, but that at 37℃a lower molecular weight of non-specific bands and a diffuse band are present, probably because at low temperatures enzymes are poorly active at low temperatures, at 41℃a higher molecular weight of non-specific bands are present and further increases in temperature to 42℃a non-specific band and a diffuse band is present at both high and low molecular weights, probably because when the temperature is too high an enzyme specificity may be reduced and thus a non-specific band may be present. In summary, the electrophoresis pattern showed that 39℃and 40℃are preferable reaction temperatures, and 39℃is preferable reaction temperature because the dispersion band is shallow compared with 40℃at 39 ℃.
Determination of optimal reaction time
The same amplification reagents as 6 tubes were used with ddH 2 O preparing negative control, numbering the 7-tube amplification reagent, placing the sample in a thermostat for incubation for 15min, 20min, 25min, 30min, 35min and 40min respectively, immediately taking out the reaction tube after the reaction is finished, adding 7.5 mu L of 6 Xlodingbuffer into the reaction tube, incubating at 56 ℃ for 5min, freezing at-20 ℃, waiting for the reaction of other samples to be finished, taking 10 mu L of sample after the reaction of all the samples is finished, and carrying out 3.5% agarose gel electrophoresis identification.
As can be seen from the electrophoresis results (FIG. 5), the overall difference is smaller under different reaction times, but when the time begins to exceed 20min, nonspecific amplification starts to appear, and the nonspecific bands gradually become more and deeper along with the time, so that the cause of the nonspecific bands may be that when the reaction time is too long, the mismatch probability in the reaction system is increased, so that a large amount of nonspecific amplification appears in the system, and the optimal reaction time is 15-20 min, so that the experiment can meet the requirement of rapid detection.
Sensitivity detection
Before the amplification reagents are prepared, the amplification reagents are prepared according to the ratio of 9 to 10 7 fg/μL、9×10 6 fg/μL、9×10 5 fg/μL、9×10 4 fg/μL、9×10 3 fg/μL、9×10 2 Fg/. Mu.L, 9X 10 Fg/. Mu.L, 9 Fg/. Mu.L as final concentration dilution of F.oxysporum genome [15] Amplification systems were prepared at different concentrations, respectively, while using ddH 2 O negative control was prepared and incubated at 39℃for 20min, after the sample reaction was completed, 6 Xlocking buffer was mixed, and 10. Mu.L of the sample was taken and identified by 3.5% agarose gel.
From the sensitivity-verifying electrophoretogram (FIG. 6), it was found that the presence of the specific bands except for 9 fg/. Mu.L gave a gradual increase in concentration from 9X 10 7 Specific bands exist when the fg/mu L is diluted to 9×10fg/mu L, the bands become shallow along with the continuous decrease of the concentration, and no specific amplified bands are detected when the fg/mu L is further diluted to 9 fg/mu L, which means that the detection limit level is the detection limit level when the detection concentration is lower than 9 fg/mu L, and whether the detection limit level contains fusarium oxysporum DNA can be detected when the concentration is continuously increased. In conclusion, the experiment has higher sensitivity, can detect fg grade, and can meet most of demands in production and life.
Specific detection
Respectively diluting the genome of common plant disease fungi such as soybean fusarium oxysporum, soybean phytophthora, soybean gray plaque bacteria, eggplant fusarium, gray botrytis cinerea, northeast downy mildew and the like to a final concentration of 10 ng/mu L, preparing the same amplification reagent, and simultaneously utilizing ddH 2 O negative control was prepared and incubated at 39℃for 20min, after the sample reaction was completed, 6 Xlocking buffer was mixed, and 10. Mu.L of the sample was taken and identified by 3.5% agarose gel.
The result is shown in a specificity verification electrophoresis chart (figure 7), and the result shows that the characteristic of high specificity of the reaction can be demonstrated by the fact that the specific bands are out of the electrophoresis chart, namely the amplification system of the fusarium oxysporum genome, and the other bands are the same as negative bands.
Actual sample detection
Extracting DNA from Fusarium oxysporum mycelium, non-infected soybean plants, infected soybean phytophthora, gray plaque mycelium, fusarium solani mycelium, and downy mildew wire respectively, diluting to uniform concentration, and using ddH 2 O negative control was prepared, incubated at 39℃for 20min, 6 Xlocking buffer was mixed after the sample reaction was completed, 10. Mu.L of sample was assayed by 3.5% agarose gel to verify whether the experiment could be used in life.
In the practical sample verification electrophoresis chart (figure 8), the first lane and the third lane show specific bands, and the first lane and the third lane are fusarium oxysporum hyphae and soybean plants infected with soybean fusarium oxysporum respectively, and the second lane shows no specific bands, so that the experiment can be used for specifically detecting soybean fusarium oxysporum and cannot be interfered by the genome of the plants, and the fourth lane to the seventh lane in the chart are the same as the negative lanes and have no bands, so that the experiment has stronger specificity and practical application value.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (8)

1. The primer group for detecting the soybean fusarium oxysporum based on the RPA isothermal amplification technology is characterized by comprising an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2.
2. The kit for detecting soybean fusarium oxysporum based on the RPA isothermal amplification technology is characterized by comprising the primer group and the detection reagent as claimed in claim 1.
3. The method for detecting the soybean fusarium oxysporum based on the RPA isothermal amplification technology is characterized by comprising the following steps of:
1) Extracting genomic DNA of a sample;
2) Performing recombinase polymerase amplification on the sample genomic DNA obtained in the step 1) by using the primer set of claim 1 to obtain an amplified product;
3) Detecting the amplified product, and determining whether the soybean fusarium oxysporum exists in the sample according to the detection result.
4. The method of claim 3, wherein the method of extracting genomic DNA from the sample of step 1) comprises SDS method or modified CTAB.
5. A method according to claim 3, characterized in that the system for recombinase polymerase amplification in step 2) comprises the following components in 50 μl: 20. Mu.L of a lytic reagent, 2.5. Mu.L of a forward primer at a concentration of 10. Mu.M, 2.5. Mu.L of a reverse primer at a concentration of 10. Mu.M, and 2. Mu. L, ddH of genomic DNA of a sample 2 O21. Mu.L and activator 2. Mu.L.
6. The method of claim 5, wherein the concentration of genomic DNA in the sample is greater than 9fg/μl.
7. The method of claim 5 or 6, wherein the temperature of the recombinase polymerase amplification is 39-40 ℃, and the time of the recombinase polymerase amplification is 15-20 min.
8. The method according to claim 5, wherein the method for detecting the amplification product in step 3) is a gel electrophoresis method or an EXO probe method.
CN202410004794.2A 2024-01-02 2024-01-02 Primer group, kit and method for detecting fusarium oxysporum on basis of RPA isothermal amplification technology Pending CN117683933A (en)

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