CN116083640A - Primer probe set, kit and method for detecting or identifying verticillium alfa by RPA and application - Google Patents

Primer probe set, kit and method for detecting or identifying verticillium alfa by RPA and application Download PDF

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CN116083640A
CN116083640A CN202310232692.1A CN202310232692A CN116083640A CN 116083640 A CN116083640 A CN 116083640A CN 202310232692 A CN202310232692 A CN 202310232692A CN 116083640 A CN116083640 A CN 116083640A
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verticillium
primer
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张园园
林克剑
陈正强
吴杰
王乐
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Grassland Research Institute of Chinese Academy of Agricultural Sciences
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Abstract

The invention provides a primer probe set, a kit and a method for detecting or identifying verticillium alfa by RPA and application thereof, belonging to the technical field of molecular biology detection. The primer probe group can accurately identify and detect the verticillium medicago. Proved by verification, the primer probe group has no cross reaction with other 11 types of verticillium fungi, has strong specificity, and has the lowest detection limit of 10fg total DNA content and high detection sensitivity.

Description

Primer probe set, kit and method for detecting or identifying verticillium alfa by RPA and application
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a primer probe set, a kit and a method for detecting or identifying verticillium medicago and application thereof.
Background
Verticillium alfalfa verticillium is a destructive disease caused by verticillium alfalfa (Verticillium alfalfae). Once the disease occurs, it is difficult to eradicate, and can significantly reduce alfalfa chlorophyll, nitrogen, phosphorus and starch content, resulting in 15% -50% and 50% reductions in alfalfa yield and seed yield, respectively. In addition, the occurrence of verticillium wilt of alfalfa can accelerate the premature senility of the cultivated alfalfa grasslands to a certain extent, shortens the utilization period to 2-3 years, and causes a great amount of economic loss for the alfalfa industry.
In the early stage of verticillium wilt, the upper leaf blades of plants are drooping or curling upwards/inwards and wilting, and V-shaped chlorosis spots gradually enlarged along the veins from the leaf tips of the leaves. After the infected plants are mowed, the newly grown seedlings show normal asymptomatic conditions, the symptoms of verticillium wilt are not shown until the full bloom stage or the mature stage, and the whole leaves of the plants turn yellow, wilt and dry from bottom to top. The verticillium wilt of alfalfa has the symptom of cryptogamy, and the field cryptogamy rate is 11.11% -30.55%. In general, the verticillium wilt of alfalfa is distributed in the field at intervals, and only individual plants in a plurality of plants infected by verticillium alfa show symptoms of the verticillium alfa, and the field identification accuracy rate of the V-shaped symptoms according to the leaves is 22.22-83.33%.
The verticillium medicago can survive in alfalfa plants, disease residues, soil and seeds, and can be transmitted through various ways, wherein alfalfa hay and seed carrier transmission are the main modes of long-distance transmission. Quarantine measures for prohibiting infected alfalfa seeds and hay produced by imported or regulated alfalfa verticillium wilt disease areas at home and abroad are one of the most effective means for effectively preventing pathogenic bacteria from being transmitted.
Verticillium meliloti, non-Verticillium meliloti (V.nonnalfalfae) and Verticillium black and white (V.albo-atrum) have very similar morphological features on PDA medium and lack multiple gene phylogenetic identification classification techniques, and the etiology of verticillium meliloti has been considered to be verticillium black and white until 2011, inderbitzin et al identified verticillium meliloti as Verticillium meliloti (V.alfalfae).
The recombinant enzyme polymerase amplification technology (recombinase ploymerase amplication, RPA) is a novel isothermal amplification technology, and target fragments can be amplified by reacting for 10-50 min at the constant temperature of 37-42 ℃. The technology has the characteristics of quick response, sensitivity, high efficiency, high sensitivity, strong specificity and the like, does not need an expensive variable-temperature experimental instrument, and can be used for dripping an amplified product on a lateral flow chromatography test strip after the RPA is combined with a lateral flow chromatography detection technology, so that the detection result can be observed visually for about 5 min.
The RPA reaction system is established based on the Twitter Amp nfo kit reported by Gaige et al, twitter Dx, UK. The total volume was 10. Mu.L, wherein the upstream and downstream primers F/R (10. Mu.M) were each 0.42. Mu.L, the probe (10. Mu.M) was 0.12. Mu.L, the buffer was 5.9. Mu. L, DNA, the template was 1. Mu. L, ddH2O 1.64. Mu.L, the magnesium acetate (2.8. Mu.M) was 0.5. Mu.L, the reaction temperature was 37℃and the reaction time was 20 minutes, and the minimum detection limit was 800fg (see [ Gaige A R, dung J K S, weiland J E.A rapid, sensitive and field-deployable isothermal assay for the detection of Verticillium alfalfae [ J ]. Canadian Journal ofPlantPathology,2018 ]).
At present, the sensitivity of detecting the verticillium meliloti by utilizing RPA is still required to be further improved.
Disclosure of Invention
In view of the above, the invention aims to provide a primer probe set, a kit and a method for detecting or identifying verticillium alfa by using RPA, and application of the primer probe set, the kit and the method, and the method for detecting or identifying verticillium alfa by using the RPA have high sensitivity.
The invention provides a primer probe group for detecting or identifying verticillium medicago, which is characterized by comprising an upstream primer with a nucleotide sequence shown as SEQ ID NO.1, a downstream primer with a nucleotide sequence shown as SEQ ID NO.2 and a probe with a nucleotide sequence shown as SEQ ID NO. 3; the 5' end of the probe contains a fluorescence reporter gene; the 3' end of the probe contains a quenching group.
The invention also provides a kit for detecting or identifying the verticillium medicago, which comprises the primer probe group, the reagent for RPA reaction and the lateral flow chromatography test strip.
The invention also provides application of the primer probe group or the kit in detecting or identifying verticillium medicago or assisting in identifying alfalfa verticillium resistance traits.
Preferably, said detecting or identifying verticillium medicago comprises detecting or identifying verticillium medicago from the genus verticillium.
The invention also provides a method for detecting or identifying the verticillium medicago through the combination of RPA and current measurement chromatography, which comprises the following steps:
1) Extracting genome DNA of a sample to be detected;
2) Taking genomic DNA of the sample to be detected as a template, and carrying out RPA amplification on the template by adopting the primer probe set according to the scheme to obtain an RPA amplification product;
3) And dripping the RPA amplification product on a lateral flow chromatography test strip, wherein if a positive detection zone is detected, the sample to be detected contains the verticillium alfa, and if no positive detection zone is observed, the sample to be detected does not contain the verticillium alfa.
Preferably, the procedure for RPA amplification is: reacting for 10-50 min at 34-42 ℃.
Preferably, the procedure for RPA amplification is: reacting for 20-30 min at 37 ℃.
Preferably, the reaction system for RPA amplification comprises the following components in 10 mu L: upstream primer 0.8. Mu.L, downstream primer 0.8. Mu.L, probe 0.4. Mu.L, buffer 5.9. Mu.L, template 1. Mu. L, ddH 2 0.6. Mu.L of O and 0.5. Mu.L of magnesium acetate;
the working of the upstream primer and the downstream primer is respectively 0.8-1.0 mu M; the concentration of the probe is 0.4-0.5 mu M.
Preferably, the concentration of magnesium acetate is 2.8. Mu.M.
The invention provides a primer probe group for detecting or identifying verticillium medicago, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1, a downstream primer with a nucleotide sequence shown as SEQ ID NO.2 and a probe with a nucleotide sequence shown as SEQ ID NO. 3; the 5' end of the probe contains a fluorescence reporter gene; the 3' end of the probe contains a quenching group. The primer probe group can accurately identify and detect the verticillium medicago. Proved by verification, the primer probe group has no cross reaction with other 11 types of verticillium fungi, has strong specificity, and has the lowest detection limit of 10fg total DNA content and high detection sensitivity.
Drawings
FIG. 1 is a schematic diagram of a primer probe design;
FIG. 2 shows the results of the RPA cross-reaction assay, where 1-7: verticillium dahliae strain PD327, V.tricorpus strain PD685, V.nubilum strain PD621, V.nonalfalfae strain PD592, V.zaregenmsianum strain PD736, V.isaacii strain PD341, V.nigrescens strain PD710, water (negative control); 8-11: verticillium meliloti strains Ms197, ms198, PD338 and PD683;12-16: longisporum A1/D1 strain PD348, V.longisporum A1/D2 strain PD356, V.albo-atrum strain PD693 and PD747 and V.klebahnii strain PD347;
FIG. 3 shows the RPA sensitivity test results; wherein, 1 to 8 are respectively the template concentration of 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L and 1 fg/. Mu.L, and NTC is water (negative control);
FIG. 4 shows the RPA test results of the inoculated alfalfa plants; wherein NTC is water (negative control), 2-3 is non-inoculated alfalfa plants, and 4-8 is inoculated alfalfa plants.
Detailed Description
The invention provides a primer probe group for detecting or identifying verticillium medicago, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1, a downstream primer with a nucleotide sequence shown as SEQ ID NO.2 and a probe with a nucleotide sequence shown as SEQ ID NO. 3; the 5' end of the probe contains a fluorescence reporter gene; the 3' end of the probe contains a quenching group.
In the invention, the upstream primer Va-RPA-F with the nucleotide sequence shown as SEQ ID NO.1 specifically comprises the following components: 5'-CCACTTTTCTCACAACACCACAAC-3'.
In the invention, the downstream primer Va-RPA-R with the nucleotide sequence shown as SEQ ID NO.2 is specifically: 5'-AAGGGTTTGCATAATCTG-3'.
In the invention, the probe Va-RPA-P with the nucleotide sequence shown as SEQ ID NO.3 specifically comprises: 5'-CCGTCCACTCCATCACCACAATCCTTCTCAACTTG-THF-CTCCATCTC TTGTAT-3'.
In the present invention, the fluorescent reporter gene preferably includes a FAM fluorescent reporter gene; the quenching group does not fluoresce, preferably the quenching group comprises a Spacer C3 quenching group.
In the present invention, the primer probe set designs specific primers based on conserved sequences of the Verticillium meliloti elongation factor (translation elongation factor. Alpha., TEF-1. Alpha.) genome. The method specifically comprises the following steps: a pair of specific primers with an amplification length of 134bp was designed in this section by Beacon Designer 8.20 software according to the RPA primer design principle, and the 5' end of the downstream primer contained a biotin (biotin) tag. Meanwhile, an RPA probe was designed at a site near the upstream primer, which contained a fluorescent group "-FAM" at the 5 'end, a quenching group "-Spacer C3" at the 3' end, and a tetrahydrofuran group (THF) was designed between the 35 th and 36 th bases.
In the present invention, the primers and probes are synthesized by Beijing thick Botai Biotechnology Co.
The invention also provides a kit for detecting or identifying the verticillium medicago, which comprises the primer probe group, the reagent for RPA reaction and the lateral flow chromatography test strip.
In the present invention, the lateral flow chromatographic test strip is preferably purchased from the biological technology company of Botai, beijing.
In the present invention, the reagent for RPA reaction preferably further includes a positive control and a negative control.
The invention also provides application of the primer probe group or the kit in detecting or identifying verticillium medicago or assisting in identifying alfalfa verticillium resistance traits.
In the present invention, the detecting or identifying verticillium alfalfa preferably includes detecting or identifying verticillium alfalfa from the genus verticillium.
In the present invention, detecting or identifying Verticillium meliloti from Verticillium preferably includes distinguishing Verticillium meliloti from other Verticillium fungi; the other genus Verticillium preferably includes Verticillium dahliae (Verticillium dahliae), verticillium longum A1/D1 (Verticillium longisporum A/D1), verticillium longum A1/D2 (Verticillium longisporumA/D2), verticillium isaacii, verticillium klebahnii, verticillium trisomy (Verticillium tricorpus), verticillium black and white (Verticillium albo-atrum), verticillium non-alfalfa (Verticillium nonalfalfae), verticillium zaregamsianum, and Verticillium cloud (Verticillium nubilum) and Verticillium nigrescens.
The invention also provides a method for detecting or identifying the verticillium medicago through the combination of RPA and current measurement chromatography, which comprises the following steps:
1) Extracting genome DNA of a sample to be detected;
2) Taking genomic DNA of the sample to be detected as a template, and carrying out RPA amplification on the template by adopting the primer probe set according to the scheme to obtain an RPA amplification product;
3) And dripping the RPA amplification product on a lateral flow chromatography test strip, wherein if a positive detection zone is detected, the sample to be detected contains the verticillium alfa, and if no positive detection zone is observed, the sample to be detected does not contain the verticillium alfa.
The invention firstly extracts the genome DNA of the sample to be detected.
The method for extracting the genome DNA of the sample to be detected is not particularly limited, and a fungus genome DNA extraction method conventional in the art is adopted. In the implementation process of the invention, the invention adopts a fungus DNA extraction kit (EE 101) to extract the genome DNA of a sample to be detected.
After obtaining the genome DNA of a sample to be detected, the invention uses the genome DNA of the sample to be detected as a template, and adopts the primer probe set in the scheme to carry out RPA amplification on the template to obtain an RPA amplification product.
After the RPA amplification product is obtained, the RPA amplification product is dripped on a lateral flow chromatography test strip, if a positive detection zone is detected, the sample to be detected contains the verticillium alfa, and if the positive detection zone is not observed, the sample to be detected does not contain the verticillium alfa.
In the present invention, the procedure for RPA amplification is preferably: reacting for 10-50 min at 34-42 ℃.
In the present invention, the procedure for RPA amplification is preferably: reacting for 20-30 min at 37 ℃.
In the present invention, the reaction system for RPA amplification preferably comprises the following components in 10. Mu.L: upstream primer 0.8. Mu.L, downstream primer 0.8. Mu.L, probe 0.4. Mu.L, buffer 5.9. Mu.L, template 1. Mu. L, ddH 2 O0.6. Mu.L and magnesium acetate 0.5. Mu.L.
In the present invention, the works of the upstream primer and the downstream primer are preferably 0.8 to 1.0. Mu.M, respectively; the concentration of the probe is preferably 0.4 to 0.5. Mu.M.
In the present invention, the concentration of magnesium acetate is preferably 2.8. Mu.M.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention.
Test materials, reagents and apparatus
Test strain: verticillium alfalfae strains Ms197, ms198, PD338 and PD683, V.dahliae strain PD327, V.tricorpus strain PD685, V.nubilum strain PD621, V.longisporum A1/D1 strain PD348, V.longisporum A1/D2 strain PD356, V.albo-atrum strains PD693 and PD747, V.nonalfalfae strain PD592, V.zaregenmium strain PD736, V.isaacii strain PD341, V.klebahnii strain PD347 and V.nigrescens strain PD710, etc. 16 strains of 12 Verticillium are each given by Subbarao professor, V.alfalfae strain 197 and Ms Li Yanzhong, all of which are grown on potato dextrose agar (potato dextrose agar) by activation.
Test reagents and apparatus: fungus DNA extraction kit (EE 101), beijing full gold biotechnology Co., ltd; 2×TaqPCR Mix (KT 201), 100bp DNALader, invitrogen, USA; taqMan PCR Mix (HS 0615) was purchased from Beijing thick raw Botai biotechnology Co., ltd; the experimental primers and probes were synthesized by Beijing thick Botai Biotechnology Co. Other reagents are all of domestic analytical purity; 5810R centrifuge, eppendorf, germany; UVP Biospectrum310Manual Platform gel imaging System, tianmei science instruments Co., ltd.
Primer and probe design
The elongation factor gene (translation elongation factor. Alpha. Gene, TEF-1. Alpha.) sequences of the 14 strains of 12 Verticillium, such as V.alfalfae strains PD338 and PD683, V.dahliae strain PD327, V.tricorlus strain PD685, V.nubilum strain PD621, V.longisporum A1/D1 strain PD348, V.longisporum A1/D2 strain PD356, V.albo-atrum strains PD693 and PD747, V.nonalfalfae strain PD592, V.zaregenmsian strain PD736, V.isaacii strain PD341, V.klebahnii strain PD347 and V.nigrescens strain PD710, were downloaded in NCBI database. All the test sequences were then introduced into BioEdit (v 7.0.9.0) for sequence alignment, phylogenetic trees were constructed using the adjacency method (Neighbor Joining Tree) in MEGA 11.0 software, and the Bootstrap test method was used for 1000 times. From the sequence alignment and the construction result of phylogenetic tree, the relatedness between V.alfalfae and the strains such as V.longisporum 1/D1, V.longisporum 1/D2, V.nonalfalfae is closer. To avoid cross-reactions, a relatively specific nucleotide site was selected as the target, and a pair of specific primers (see FIG. 1) with an amplification length of 134bp were designed in this section by Beacon Designer 8.20 software according to the RPA primer design principle, and the 5' end of the downstream primer contained a biotin (biotin) tag. Meanwhile, an RPA probe was designed at a site near the upstream primer, which contained a fluorescent group "-FAM" at the 5 'end, a quenching group "-Spacer C3" at the 3' end, and a tetrahydrofuran group (THF) was designed between the 35 th and 36 th bases. The upstream primer is Va-RPA-F:5'-CCACTTTTCTCACAACACCACAAC-3', the downstream primer is Va-RPA-R:5'-AAGGGTTTGCATAATCTG-3'; the probe is Va-RPA-P: p5'-CCGTCCACTCCATCACCACAATCCTTCTCAACTTG-THF-CTCCATCTC TTGTAT-3'. Primers and probes were synthesized by Beijing thick Biotechnology Botai Co.
Test example 1 specific determination of primers and probes
Test strain DNA extraction: mycelium of the strain to be tested is respectively frozen by liquid nitrogen and then is put into a grinder for grinding, and fungus DNA extraction kit is used for extracting and separating genome DNA of the strain, and the genome DNA is stored in a refrigerator at-20 ℃ for standby.
Dissolving and diluting the primer and the probe dry powder: taking out a tube of primer or probe dry powder, placing the tube of primer or probe dry powder in a centrifugal machine, centrifuging at 5000rpm/min for 1min, lightly opening the tube cover of the centrifugal tube, and adding deionized water into the tube by using a pipetting gun to ensure that the concentration of the primer or probe reaches 100 mu M, namely a storage solution. Then, 20. Mu.L of the solution was removed and added to 180. Mu.L of deionized water, and the solution was vortexed sufficiently and centrifuged to obtain a primer or probe working solution having a concentration of 10. Mu.M for use.
Reaction system (10 μl system) according to the RPA kit (beggar's biotech limited) instructions: 0.8. Mu.L of each of the upstream and downstream primers at a concentration of 10. Mu.M, 0.4. Mu.L of the probe at a concentration of 10. Mu.M, and 1. Mu. L, ddH of the template in buffer 5.9. Mu. L, DNA 2 0.6 mu L of O and 0.5 mu L of magnesium acetate with the concentration of 2.8 mu M, fully oscillating and uniformly mixing, instantly centrifuging for 10s, and placing in a metal bath at 37 ℃ for reaction for 30min. The results of the primer and probe specific assays are shown in FIG. 2, and the results of the negative control (water) no-detection zones were detected for 11 strains of the genus Verticillium, such as V.dahliae strain PD327, V.tricorlus strain PD685, V.nubilum strain PD621, V.longisporum A1/D1 strain PD348, V.longisporum A1/D2 strain PD356, V.albo-atrum strains PD693 and PD747, V.nonalfalfae strain PD592, V.zaregenmsianum strain PD736, V.isaacii strain PD341, V.klebahnii strain PD347 and V.nigrocens strain PD710, except that the results of the primer and probe specific assays were clear-detection zones. The results illustrate: the primers and probes designed by the research have stronger specificity.
Test example 2RPA reaction System
1) The final concentrations of the primers were 0.2, 0.4, 0.8, 1.0 and 1.2. Mu.M, respectively, i.e., 0.2, 0.4, 0.8, 1.0 and 1.2. Mu.L of each of the upstream and downstream primers F/R (10. Mu.M) were added to the reaction system, respectively, and similarly, the final concentrations of the probes were 0.1, 0.2, 0.3, 0.4 and 0.5. Mu.M, respectively, i.e., 0.1, 0.2, 0.3, 0.4 and 0.5. Mu.L of each of the probes (10. Mu.M) were added, respectively, and the reaction was carried out at a constant temperature of 39℃for 30 minutes, and the amplified products were dropped on a lateral flow chromatographic strip, and after 5 minutes, the detection results were observed, thereby screening the optimal concentrations of the primers and probes. Setting the reaction time to be 30min under the optimal primer and probe concentration, and setting the reaction gradient temperature: 34. 35, 36, 37, 38, 39, 40, 41 and 40 ℃, the optimum reaction temperature is selected. Finally, setting a reaction time gradient under the optimal primer and probe concentration and optimal temperature conditions: 10. and 20, 30, 40 and 50min, and selecting the optimal reaction time so as to finally determine the optimal reaction system.
2) The final probe concentrations were set at 37℃for 30min at 0.1, 0.2, 0.3, 0.4 and 0.5. Mu.M, respectively. The RPA test result showed that the resolution of the positive test strip was gradually increased with the increase of the primer concentration when the final probe concentration was 0.4. Mu.M, and the clear test strip was observed at the final primer concentration of 0.8. Mu.M, and the resolution of the test strip was not significantly different at the final primer concentrations of 0.8 and 1.0. Mu.M, and the final probe concentrations of 0.4 and 0.5. Mu.M, so that the optimal primer and probe concentrations were 0.8 and 0.4. Mu.M for reagent saving. That is, 0.8. Mu.L of each of the upstream and downstream primers (10. Mu.M) and 0.4. Mu.L of each of the probes (10. Mu.M) were added to the detection system.
3) Under the optimal primer and probe concentrations, the reaction time was set to 30min, and the reaction gradient temperatures 34, 35, 36, 37, 38, 39, 40, 41 and 42 ℃ were set, and the detection results showed that the detection bands were observed at all 9 reaction temperature gradients, but with increasing temperature, the detection bands were progressively more clear at 37 ℃ and the color of the detection bands began to fade as the temperature continued to increase. Thus, the optimum reaction temperature is 37 ℃.
4) The reaction time gradients were set at the optimal primer and probe concentrations and the optimal reaction temperature for 10, 20, 30, 40 and 50min. The detection result shows that the detection zone can be observed under 5 reaction time gradients, the definition of the detection zone gradually increases along with the increase of the reaction time, but the definition of the detection zone is not obviously different beyond 20 minutes, so the optimal reaction time is 20 minutes.
Combining the results, determining the RPA detection system of the verticillium medicago: the total volume of the reaction system was 10. Mu.L, wherein the upstream and downstream primers F/R (10. Mu.M) were each 0.8. Mu.L, the probe (10. Mu.M) was 0.4. Mu.L, and the buffer was 5.9. Mu. L, DNA template 1. Mu. L, ddH 2 O0.6 mu L, magnesium acetate (2.8 mu M) 0.5 mu L, reaction temperature 37 ℃ and reaction time 20min.
Test example 3 sensitivity test
DNA of Verticillium meliloti strain Ms198 was used as template with RNase Free ddH 2 The relative sensitivity of the DNA was examined by diluting the DNA with O, and the DNA concentrations were respectively 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L and 1 fg/. Mu.L for a total of 8 concentration gradients. And (3) performing RPA reaction by using the optimized reaction system, dripping the amplified product on a lateral flow chromatography test strip, and observing a sensitivity test result after 5min (figure 3), wherein when the DNA content of the verticillium alfalfa strain Ms198 is below 10fg, no detection band is observed, which shows that the detection limit is 10fg. As the concentration of template increases, the detection zone becomes clearer.
Test example 4RPA detection of the condition of a grafted and non-grafted alfalfa plant carrying Verticillium meliloti
Strain Ms198 was activated on PDA medium for 10d, the colony surface was rinsed with a pipette adding 10mL of sterile water to each dish, the conidium suspension was collected with a spreader, and the concentration of the conidium suspension was adjusted to 1X 10 with a hemocytometer 7 The sample was kept at one/mL. The conidiophore suspension disease concentration of the test strain is adjusted to 1 multiplied by 10 by adopting a root dipping inoculation method (without root cutting) 7 individual/mL, in preparation for infestation.
Selecting alfalfa variety 'gongnong No. 5' seeds with plump seeds, sterilizing the surfaces of the seeds with 3% sodium hypochlorite for 2min, washing the seeds with sterile water for 5 times, and sowing the sterile seeds into seedling raising trays of 4X 8 holes filled with sterile soil. The plug is placed in a greenhouse with the temperature of 24-26 ℃ and the relative air humidity of 30-40% for cultivation, and water is poured every 3-5 d after seedlings come out of soil. In the 3-4-leaf stage of alfalfa, water is poured into the plug tray, the root system of the seedling is removed lightly, soil on the root system is flushed with tap water, then the whole root system is soaked in the prepared conidium suspension, 30 seedlings (5-pot) of the conidium suspension of the single strain Ms198 are inoculated, and 30 seedlings (5-pot) inoculated with sterile water are used as a control. After 30min of inoculation, all treated seedlings were re-planted in a nutrient bowl (10 cm. Times.10 cm) filled with sterile soil and continued to be cultivated in a greenhouse.
And after 2 weeks of inoculation, respectively taking stem basal tissues of the inoculated and non-inoculated plants, subpackaging in a 1.5mL centrifuge tube, grinding after quick freezing by liquid nitrogen, extracting genome DNA by using a DNA extraction kit, performing RPA reaction by using an optimized reaction system, dripping an amplified product on a lateral flow chromatography test strip, and observing a detection result after 5 minutes. As a result, as shown in FIG. 4, a positive detection band could be detected in alfalfa plants after inoculation, the detection rate was 100%, and no detection band was observed in non-inoculated plants.
Test example 5
To further verify the accuracy of the RPA assay, all plants tested were subjected to pathogen re-isolation. Collecting all inoculated plants, separating and purifying pathogenic bacteria by adopting a conventional tissue separation method under indoor conditions, and carrying out common PCR detection on DNA of the separated and purified product by utilizing a specific primer pair of the verticillium alfa. The reaction system was 25. Mu.L, including 12.5. Mu.L of 2 XTaqPCRMastermix, 2.5. Mu.L of primer mix AlfF/AlfD1r, 1. Mu.L of genomic DNA, 9. Mu.L ddH at a final concentration of 5. Mu.mol/L 2 O, annealing temperature is 64 ℃, and gel electrophoresis detection is carried out on the PCR product. The detection result shows that: all test DNA can amplify a single target band of approximately 1060bp in size.
In summary, the invention takes 12 types of verticillium meliloti and similar types thereof as research objects, and develops the research of detecting the verticillium meliloti by combining a recombinase polymerase amplification technology with a current measurement chromatography detection technology. The method for detecting the verticillium alfa by utilizing the recombinase polymerase amplification technology has the characteristics of quick response, sensitivity, high efficiency, high sensitivity, strong specificity and the like, does not need an expensive variable temperature experimental instrument, combines RPA with a current measurement chromatography detection technology, is simple and quick in detection, convenient to carry, can judge and read the result by naked eyes, can truly realize the visual quick on-site nucleic acid detection of the verticillium alfa, and has good application prospects in the aspects of port entrance and exit plant inspection and quarantine, monitoring and early warning of verticillium wilt of alfalfa and the like.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, according to which one can obtain other embodiments without inventiveness, these embodiments are all within the scope of the invention.

Claims (9)

1. The primer probe group for detecting or identifying the verticillium medicago by RPA is characterized by comprising an upstream primer with a nucleotide sequence shown as SEQ ID NO.1, a downstream primer with a nucleotide sequence shown as SEQ ID NO.2 and a probe with a nucleotide sequence shown as SEQ ID NO. 3; the 5' end of the probe contains a fluorescence reporter gene; the 3' end of the probe contains a quenching group.
2. A kit for detecting or identifying Verticillium meliloti, which comprises the primer probe set of claim 1, an RPA reaction reagent and a lateral flow chromatography test strip.
3. Use of the primer-probe set of claim 1 or the kit of claim 2 for detecting or identifying verticillium alfalfa or for aiding in the identification of alfalfa verticillium resistance traits.
4. The use according to claim 3, wherein said detecting or identifying verticillium alfalfa comprises detecting or identifying verticillium alfalfa from the genus verticillium.
5. A method for detecting or identifying verticillium medicago by combining RPA and current measurement chromatography, comprising the steps of:
1) Extracting genome DNA of a sample to be detected;
2) Taking genomic DNA of the sample to be detected as a template, and carrying out RPA amplification on the template by adopting the primer probe set of claim 1 to obtain an RPA amplification product;
3) And dripping the RPA amplification product on a lateral flow chromatography test strip, wherein if a positive detection zone is detected, the sample to be detected contains the verticillium alfa, and if no positive detection zone is observed, the sample to be detected does not contain the verticillium alfa.
6. The method of claim 5, wherein the procedure for RPA amplification is: reacting for 10-50 min at 34-42 ℃.
7. The method of claim 6, wherein the procedure for RPA amplification is: reacting for 20-30 min at 37 ℃.
8. The method of claim 5, wherein the reaction system for RPA amplification comprises the following components in 10 μl: upstream primer 0.8. Mu.L, downstream primer 0.8. Mu.L, probe 0.4. Mu.L, buffer 5.9. Mu.L, template 1. Mu. L, ddH 2 0.6. Mu.L of O and 0.5. Mu.L of magnesium acetate;
the working of the upstream primer and the downstream primer is respectively 0.8-1.0 mu M; the concentration of the probe is 0.4-0.5 mu M.
9. The method of claim 8, wherein the concentration of magnesium acetate is 2.8 μm.
CN202310232692.1A 2023-03-13 2023-03-13 Primer probe set, kit and method for detecting or identifying verticillium alfa by RPA and application Pending CN116083640A (en)

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