CN116516047A - Primer group, primer probe group, kit and method for detecting or identifying verticillium alfa and application - Google Patents

Primer group, primer probe group, kit and method for detecting or identifying verticillium alfa and application Download PDF

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CN116516047A
CN116516047A CN202310232590.XA CN202310232590A CN116516047A CN 116516047 A CN116516047 A CN 116516047A CN 202310232590 A CN202310232590 A CN 202310232590A CN 116516047 A CN116516047 A CN 116516047A
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verticillium
primer
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detected
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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 group, a primer probe group, a kit and a method for detecting or identifying verticillium meliloti and application thereof, belonging to the technical field of molecular biology detection. The primer group disclosed by the invention can accurately identify and detect the verticillium medicago. In addition, the invention utilizes the TaqMan fluorescent probe method to quantitatively detect the amplification of the target gene of the verticillium alfa in real time, realizes the simultaneous performance of PCR amplification and probe detection, completely closes the tube in the whole detection process, does not need to carry out post-PCR treatment, completely avoids the pollution of PCR products, simultaneously reduces the detection steps, saves the time and solves the defects of the traditional detection method of the verticillium alfa. Has better application prospect in the aspects of port entrance and exit plant inspection and quarantine, alfalfa verticillium wilt monitoring and early warning, alfalfa variety disease resistance evaluation and the like.

Description

Primer group, primer probe group, kit and method for detecting or identifying verticillium alfa and application
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a primer group, a primer probe group, a kit, a method and application for detecting or identifying verticillium medicago.
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).
Currently, for the identification of verticillium meliloti, the existing traditional morphological method is difficult to accurately identify.
Disclosure of Invention
In view of the above, the invention aims to provide a primer group, a primer probe group, a kit and a method for detecting or identifying verticillium alfa and application thereof, and the invention can accurately detect or identify verticillium alfa.
The invention provides a primer group for detecting or identifying verticillium medicago, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2.
The invention also provides a primer probe group for detecting or identifying the verticillium medicago, which comprises the primer group 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 alfa, which comprises the primer group and the reagent for PCR reaction; alternatively, the kit comprises the primer probe set and the reagent for real-time fluorescence quantitative PCR reaction.
The invention also provides application of the primer group, the primer probe group or the kit in detecting or identifying verticillium meliloti or assisting in identifying alfalfa verticillium wilt resistance.
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 based on PCR, 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 PCR reaction on the template by adopting the primer group according to the scheme to obtain a PCR reaction product;
3) And carrying out agarose gel electrophoresis on the PCR reaction product, if a single target strip appears and the size is 104bp, judging that the sample to be tested contains the verticillium medicago or the sample to be tested is the verticillium medicago, and if no target strip appears, judging that the sample to be tested does not contain the verticillium medicago or the sample to be tested is not the verticillium medicago.
Preferably, the reaction system of the PCR reaction comprises the following components in 25 mu L: 12.5. Mu.L of 2X Taq PCR Mastermix, 2.5. Mu.L of a mixture of upstream and downstream primers, 1. Mu.L of genomic DNA and 9. Mu.L of ddH 2 O; the working concentration of the upstream primer and the downstream primer is 5 mu mol/L respectively;
the reaction program of the PCR reaction is as follows: pre-denaturation at 95℃for 1min; denaturation at 95℃for 15s, annealing at 51℃for 20s, extension at 72℃for 20s,40 cycles, and finally extension at 72℃for 7min.
The invention also provides a method for detecting or identifying the verticillium medicago based on the TaqMan probe, which comprises the following steps:
s1, extracting genome DNA of a sample to be detected;
s2, taking genomic DNA of the sample to be detected as a template, carrying out real-time fluorescent quantitative PCR reaction on the template by adopting the primer probe set according to the scheme, if a fluorescent signal is detected, judging that the sample to be detected contains the verticillium alfa or the sample to be detected is the verticillium alfa, and if no fluorescent signal is detected, judging that the sample to be detected does not contain the verticillium alfa or the sample to be detected is not the verticillium alfa.
Preferably, the primer probe set according to the above scheme is adopted to perform real-time fluorescent quantitative PCR reaction on the template to obtain Ct value, and further comprises calculating genomic DNA concentration of the sample to be detected according to the formula shown in formula 1,
y＝-3.1563x+40.189(R 2 = 0.9933) formula 1, wherein x represents genomic DNA and y represents a corresponding Ct value.
Preferably, the inverse of the real-time fluorescent quantitative PCR reactionThe reaction system comprises the following components in 25 mu L: 2. Mu.L each of the upstream primer and the downstream primer, 1. Mu.L each of the probe, 12.5. Mu.L each of TaqMan PCR Mix, 1. Mu.L each of genomic DNA and ddH 2 O 6.5μL；
The working concentration of the upstream primer and the downstream primer is 0.8 mu mol/L respectively; the working concentration of the probe is 0.4 mu mol/L;
the reaction program of the real-time fluorescence quantitative PCR reaction is as follows: pre-denaturation at 95 ℃ for 60s; denaturation at 95℃for 15s, annealing at 51 or 52℃for 20s, elongation at 72℃for 20s,40 cycles.
The invention provides a primer group for detecting or identifying verticillium medicago, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2. The primer group disclosed by the invention can accurately identify and detect the verticillium medicago. Through verification, the pathogen re-separation is carried out on the test plants inoculated with the verticillium medicago, the PCR detection is carried out on the DNA of the re-separated purified product by utilizing the primer group, and all the test DNA can be amplified into a single target strip with the size of about 104bp.
Drawings
FIG. 1 shows the results of primer-specific PCR assays;
FIG. 2 is a Taqman fluorescent quantitative PCR amplification curve at different annealing temperatures;
FIG. 3 shows the results of PCR assay of primer sensitivity;
FIG. 4 is a real-time fluorescent quantitative PCR relative sensitivity detection of Verticillium meliloti;
FIG. 5 is a real-time fluorescence quantitative PCR detection standard curve of Verticillium meliloti;
FIG. 6 shows real-time fluorescent quantitative PCR detection of the root, stem and leaf of the alfalfa;
FIG. 7 is a real-time fluorescent quantitative PCR assay in inoculated alfalfa roots;
FIG. 8 shows the PCR detection results of the re-isolated Verticillium alfalfa wherein: m is DNA molecular weight standard; 1-8 is the DNA of the separated Verticillium meliloti strain; -water (negative control).
Detailed Description
The invention provides a primer group for detecting or identifying verticillium medicago, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2.
In the present invention, the upstream primers Va1-F with the nucleotide sequence shown as SEQ ID NO.1 are specifically: 5'-CACTCCATCACCACAATC-3'.
In the present invention, the downstream primers Va1-R with the nucleotide sequence shown as SEQ ID NO.2 are specifically: 5'-AAGGGTTTGCATAATCTG-3'.
The invention also provides a primer probe group for detecting or identifying the verticillium medicago, which comprises the primer group 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 present invention, probes Va1-P having nucleotide sequences shown in SEQ ID NO.3 are specifically: 5'-TCTCAACTTGCTCCATCTCTTGTATGAAC-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 BHQ1 quenching group.
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 alfa, which comprises the primer group and the reagent for PCR reaction; alternatively, the kit comprises the primer probe set and the reagent for real-time fluorescence quantitative PCR reaction.
In the present invention, the reagents for PCR reaction preferably include 2X Taq PCR Mastermix and ddH 2 O. In the present invention, the reagent for PCR reaction preferably further comprises a positive control and a negative control.
In the present invention, the reagents for the real-time fluorescent quantitative PCR reaction preferably include TaqMan PCRMix 12.5. Mu.L and ddH 2 O. In the present invention, the reagent for real-time fluorescent quantitative PCR reaction preferably further includes a positive control and a negative control.
The invention also provides application of the primer group, the primer probe group or the kit in detecting or identifying verticillium meliloti or assisting in identifying resistance characters of alfalfa germplasm resources to alfalfa verticillium.
In the present invention, the detection or identification includes qualitative and/or quantitative detection or identification.
In the present invention, the detecting or identifying of the Verticillium meliloti preferably comprises detecting or identifying of the Verticillium meliloti from Verticillium, more preferably comprises 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 based on PCR, 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 PCR reaction on the template by adopting the primer group according to the scheme to obtain a PCR reaction product;
3) And carrying out agarose gel electrophoresis on the PCR reaction product, if a single target strip appears and the size is 104bp, judging that the sample to be tested contains the verticillium medicago or the sample to be tested is the verticillium medicago, and if no target strip appears, judging that the sample to be tested does not contain the verticillium medicago or the sample to be tested is not the verticillium medicago.
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 uses the primer group of the scheme to carry out PCR reaction on the template to obtain a PCR reaction product.
In the present invention, the reaction system of the PCR reaction preferably comprises the following components in 25. Mu.L: 12.5. Mu.L of 2X Taq PCR Mastermix, 2.5. Mu.L of a mixture of upstream and downstream primers, 1. Mu.L of genomic DNA and 9. Mu.L of ddH 2 O。
In the present invention, the working concentrations of the upstream primer and the downstream primer are preferably 5. Mu. Mol/L, respectively.
In the present invention, the reaction procedure of the PCR reaction is preferably: pre-denaturation at 95℃for 1min; denaturation at 95℃for 15s, annealing at 51℃for 20s, extension at 72℃for 20s,40 cycles, and finally extension at 72℃for 7min.
After the PCR reaction product is obtained, agarose gel electrophoresis is carried out on the PCR reaction product, if a single target strip appears and the size is 104bp, the sample to be detected is judged to contain the verticillium medicago or is the verticillium medicago, and if no target strip appears, the sample to be detected is judged to not contain the verticillium medicago or is not the verticillium medicago.
The invention also provides a method for detecting or identifying the verticillium medicago based on the TaqMan probe, which comprises the following steps:
s1, extracting genome DNA of a sample to be detected;
s2, taking genomic DNA of the sample to be detected as a template, carrying out real-time fluorescent quantitative PCR reaction on the template by adopting the primer probe set according to the scheme, if a fluorescent signal is detected, judging that the sample to be detected contains the verticillium alfa or the sample to be detected is the verticillium alfa, and if no fluorescent signal is detected, judging that the sample to be detected does not contain the verticillium alfa or the sample to be detected is not the verticillium alfa.
The invention firstly extracts the genome DNA of the sample to be detected.
After obtaining 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 real-time fluorescence quantitative PCR reaction on the template, if a fluorescence signal is detected, the sample to be detected is judged to contain the verticillium medicago or the sample to be detected is the verticillium medicago, and if the fluorescence signal is not detected, the sample to be detected is judged to not contain the verticillium medicago or the sample to be detected is not the verticillium medicago.
In the invention, the reaction system of the real-time fluorescence quantitative PCR reaction preferably comprises the following components in 25 mu L: 2. Mu.L each of the upstream primer and the downstream primer, 1. Mu.L each of the probe, 12.5. Mu.L each of TaqMan PCR Mix, 1. Mu.L each of genomic DNA and ddH 2 O 6.5μL。
In the present invention, the working concentrations of the upstream primer and the downstream primer are preferably 0.8. Mu. Mol/L, respectively; the working concentration of the probe is preferably 0.4. Mu. Mol/L.
In the present invention, the reaction procedure of the real-time fluorescent quantitative PCR reaction is preferably: pre-denaturation at 95 ℃ for 60s; denaturation at 95℃for 15s, annealing at 51 or 52℃for 20s, elongation at 72℃for 20s,40 cycles.
The primer probe set of the scheme is adopted to carry out real-time fluorescence quantitative PCR reaction on the template to obtain Ct value, the invention preferably also comprises the step of calculating the concentration of genome DNA of a sample to be detected according to a formula shown in a formula 1,
y＝-3.1563x+40.189(R 2 = 0.9933) formula 1, wherein x represents genomic DNA and y represents a corresponding Ct value.
The invention utilizes the TaqMan fluorescent probe method to quantitatively detect the amplification of the target gene of the verticillium alfa in real time, realizes the simultaneous performance of PCR amplification and probe detection, completely closes the tube in the whole detection process, does not need to carry out post-PCR treatment, completely avoids the pollution of PCR products, simultaneously reduces the detection steps, saves the time and solves the defects of the traditional detection method of the verticillium alfa. Has better application prospect in the aspects of port entrance and exit plant inspection and quarantine, alfalfa verticillium wilt monitoring and early warning, alfalfa variety disease resistance evaluation and the like.
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×Taq PCR Mix (KT 201), 100bp DNA Ladder, 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
TEF-1 alpha sequences of 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 A1/D1, V.longisporum A1/D2, V.nonalfalfae is closer. In order to avoid cross reaction, a relatively specific nucleotide site, namely, the verticillium meliloti TEF-1 alpha gene, is selected as a target, and primers and probes are respectively designed by using primer design software Beacon Designer 8.20. The forward primers were Va1-F:5'-CACTCCATCACCACAATC-3' (SEQ ID NO. 1), reverse primer Va1-R:5'-AAGGGTTTGCATAATCTG-3' (SEQ ID NO. 2); probes Va1-P: P5'-TCTCAACTTGCTCCATCTCTTGTATGAAC-3' (SEQ ID NO. 3), the 5 'end of the probe contains FAM fluorescence reporter gene, and the 3' end contains non-fluorescing BHQ1 quenching group. Primers and probes were synthesized by Beijing thick Biotechnology Botai Co.
In the present invention, the nucleotide sequence (SEQ ID NO. 4) of the Verticillium meliloti TEF-1. Alpha. Gene, and the primer pair and probe binding sites are shown below:
wherein only the bolded sequence is the forward primer binding site, only the underlined sequence is the probe binding site, bolded and double-underlined sequence is the reverse primer binding site.
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.
The reaction system of the PCR reaction was 25. Mu.L: 12.5. Mu.L of 2X Taq PCR Mastermix, 2.5. Mu.L of a mixture of upstream and downstream primers, 1. Mu.L of genomic DNA and 9. Mu.L of ddH 2 O. The PCR reaction comprises the following steps: pre-denaturation at 95℃for 1min; denaturation at 95℃for 15s, annealing at 51℃for 20s, extension at 72℃for 20s,40 cycles, and finally extension at 72℃for 7min. The primer-specific PCR reaction results are shown in FIG. 1, and the results of the detection of 11 strains of 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 strain PD693 and PD747, V.nonalfalfae strain PD592, V.zaregenmsian strain PD736, V.isaacii strain PD341, V.klebahnii strain PD347 and V.nigrocecens strain PD710 did not amplify the target bands, except that the single target band of about 104bp size was amplified by the Verticillium strains Ms197, ms198, PD338 and PD 683. The results illustrate: the primer designed by the research has stronger specificity.
The real-time fluorescence quantitative PCR reaction system is a 25 mu L system: forward and reverse primers (10. Mu.M) each 2. Mu.L, probe (10. Mu.M) 1. Mu.L, taqMan PCRMix 12.5. Mu. L, DNA template 1. Mu. L, ddH 2 O6.5. Mu.L. Pre-denaturation at 95℃for 60s, denaturation at 95℃for 15s, annealing at 52℃for 20s, elongation at 72℃for 20s,40 cycles. The results of the primer and probe specificity assays are shown in Table 1, and the results of the assays for 12 strains of 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 strain PD693 and PD747, V.nonalfalfae strain PD592, V.zaregenmsianum strain PD736, V.isaacii strain PD341, V.klebahnii strain PD347 and V.nigrescens strain PD710 are negative, except for the positive results for the Verticillium alfalfa strains Ms197, ms198, PD338 and PD 683. The results illustrate: the primers and probes designed by the research have stronger specificity.
TABLE 1 fluorescence intensity and Ct values of different strains in real-time fluorescent quantitative PCR
Test example 2 real-time fluorescent quantitative PCR reaction system
To test and select the optimal primer and probe concentrations, 20 different primer and probe concentration ratios were tested one by one using the DNA of the Verticillium meliloti strain Ms198 (1 pg/. Mu.L concentration) as a template, and the specific concentration ratios are shown in Table 2, and the PCR reaction system and reaction conditions were the same.
The optimized results of the real-time fluorescence quantitative PCR reaction system are shown in Table 2, and the concentration proportioning scheme of 12 primers and probes in groups 1-4, 6-9 and 11-14 is feasible. And determining the optimal concentration ratio scheme of the 12 th group primer and the probe according to the principle that the concentration ratio scheme of the primer and the probe with the fluorescence intensity of more than 4000 units and the lowest Ct value is optimal. Namely, when the final concentration of the upstream and downstream primers is 0.8 mu mol/L, the final concentration of the probe is 0.4 mu mol/L, the fluorescence intensity is 4000 units or more, the Ct value is the smallest, and finally, the optimal concentrations of the primers and the probes are respectively determined as 0.8 mu mol/L and 0.4 mu mol/L.
TABLE 2 concentration ratio of Taqman Probe method fluorescence quantitative PCR primers and probes
After the optimal primer and probe concentrations were determined, 11 annealing temperature gradients were set in the temperature range 47-57 ℃, i.e., 47 ℃, 48 ℃, 49 ℃, 50 ℃,51 ℃,52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃ and 57 ℃. The optimal annealing temperature is determined by fluorescence intensity and Ct value at different annealing temperatures. The results are shown in FIG. 2: at an annealing temperature of 51 ℃, the fluorescence intensity was the strongest and the Ct value was the smallest, so that the annealing temperature was determined to be the optimum annealing temperature at 51 ℃.
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 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 7 concentration gradients. The PCR reaction comprises the following steps: pre-denaturation at 95℃for 1min; denaturation at 95℃for 15s, annealing at 51℃for 20s, extension at 72℃for 20s,40 cycles, and finally extension at 72℃for 7min. As shown in FIG. 3, the result of the primer-specific PCR reaction was that a single target band of about 104bp was amplified at each of the template concentrations of 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L and 1 pg/. Mu.L, and that the target bands were not amplified at each of 100 fg/. Mu.L, 10 fg/. Mu.L and 1 fg/. Mu.L with decreasing template concentration. The result showed that the detection limit was 1 pg/. Mu.L.
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 and 10 fg/. Mu.L for a total of 7 concentration gradients. And carrying out real-time fluorescence quantitative PCR reaction by using the optimized reaction system.
As a result of the sensitivity test, as shown in FIG. 4, when the DNA content of the Verticillium alfalfa strain Ms198 was 100fg or less, no fluorescent signal was detected, indicating that the detection limit was 100fg. As the template concentration increases, ct values become smaller and smaller, and the construction result of the standard curve shows that the DNA template concentration is extremely correlated with the Ct value, y= -3.1563x+40.189 (R) 2 = 0.9933), x represents DNA template concentration, y represents the corresponding Ct value (fig. 5).
Example 1
TaqMan fluorescent probe method for real-time quantitative detection of condition that grafted and non-grafted alfalfa plants carry verticillium alfalfa
Strain Ms198 was activated on PDA medium for 10d and 10mL of sterile water was added to each dish with a pipette to rinse the colony surfaceThe conidium suspension was collected by a coater and the concentration of the conidium suspension was adjusted to 1X 10 by 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.
After 2 weeks of inoculation, respectively taking three plant tissues of roots, stems and leaves of the inoculated and non-inoculated plants, subpackaging in a centrifuge tube with the volume of 1.5mL, grinding after quick freezing by liquid nitrogen, extracting genome DNA by using a DNA extraction kit, performing real-time fluorescence quantitative PCR reaction by using an optimized reaction system, and calculating the variation coefficient in a group based on a variation coefficient calculation formula: coefficient of variation cv= (standard deviation/average value) ×100%. As a result, as shown in FIG. 4, fluorescence signals were detected in the roots, stems and leaves of alfalfa plants after inoculation, the fluorescence intensity in the roots was strongest, the fluorescence intensity in the leaves was weakest, and no fluorescence signals were detected in the roots, stems and leaves of non-inoculated plants (FIG. 6).
The three PCR variation coefficients of root, stem and leaf are:
CV root of Chinese character ＝(0.370589/26.235)×100％＝1.41％；
CV Stems of plants ＝(0.419011426/30.14866667)×100％＝1.38％；
CV Leaves of the plant ＝(0.474643121/32.40966667)×100％＝1.46％；
The CV values of the three groups are less than 5%, which shows that the reaction system of the study has better repeatability.
Example 2
TaqMan fluorescent probe method for quantitatively detecting condition of alfalfa plant carrying verticillium medicago after inoculation in real time
Selecting disease-resistant alfalfa variety 'WL354HQ' with plump seeds and disease-resistant alfalfa variety 'gongnong No. 5' 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 culture 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. And (3) pouring enough water into the plug tray in the 3-4-leaf stage of alfalfa, slightly removing the root system of the seedling, flushing the soil on the root system with tap water, and then soaking the whole root system in the prepared conidium suspension, wherein 30 seedlings (5 pot) are respectively and independently suspended in the Ms198 conidium suspension. After inoculation for 30min, after water planting for 7d, root tissues of 'WL354HQ' and 'gong nong No. 5' alfalfa plants are respectively taken, and are respectively packaged in a centrifuge tube with the volume of 1.5mL, ground after quick freezing by liquid nitrogen, genomic DNA is extracted by using a DNA extraction kit, and real-time fluorescence quantitative PCR reaction is carried out by using an optimized reaction system. As a result, as shown in FIG. 7, both the disease-resistant alfalfa variety `WL 354 HQ` and the disease-sensitive alfalfa variety `Gongnong No. 5` were able to detect fluorescence signals in root tissues after inoculation for 7d, but the fluorescence intensity in the root of the disease-sensitive alfalfa variety `Gongnong No. 5` was stronger than that of the disease-resistant alfalfa variety `WL 354 HQ`.
Example 3
Pathogen re-isolation was performed on all the plants tested in example 2. 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 XTaqPCR Mastermix, 2.5. Mu.L of a mixture of the upstream primer and the downstream primer, 1. Mu.L of genomic DNA and 9. Mu.L of ddH2O. PCR reaction procedure: pre-denaturation at 95℃for 1min; denaturation at 95℃for 15s, annealing at 51℃for 20s, extension at 72℃for 20s,40 cycles, and finally extension at 72℃for 7min. The result of taking 15. Mu.L of PCR product and detecting by 1.5% agarose gel electrophoresis at 100V for 30min, and then placing the gel plate under a gel imaging system for observation is shown in FIG. 8. The detection result shows that: all test DNA can amplify a single target band, and the size is about 104bp.
In conclusion, the method for quantitatively detecting the verticillium alfa in real time by utilizing the TaqMan fluorescent probe method reduces detection steps, saves time, and has good application prospects in the aspects of monitoring and early warning of verticillium wilt of alfalfa, disease resistance evaluation of alfalfa varieties 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 (10)

1. A primer group for detecting or identifying verticillium meliloti is characterized by comprising an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2.
2. A primer probe set for detecting or identifying verticillium meliloti, which is characterized by comprising the primer set according to claim 1 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.
3. A kit for detecting or identifying verticillium medicago, comprising the primer set of claim 1 and a reagent for PCR reaction; alternatively, the kit comprises the primer probe set according to claim 2 and a reagent for real-time fluorescent quantitative PCR reaction.
4. Use of the primer set of claim 1, the primer probe set of claim 2 or the kit of claim 3 for detecting or identifying verticillium alfalfa or for aiding in the identification of resistance traits of alfalfa germplasm resources to verticillium alfalfa.
5. The use according to claim 4, wherein said detecting or identifying verticillium alfalfa comprises detecting or identifying verticillium alfalfa from the genus verticillium.
6. A method for detecting or identifying verticillium medicago based on PCR, 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 PCR reaction on the template by adopting the primer group of claim 1 to obtain a PCR reaction product;
3) And carrying out agarose gel electrophoresis on the PCR reaction product, if a single target strip appears and the size is 104bp, judging that the sample to be tested contains the verticillium medicago or the sample to be tested is the verticillium medicago, and if no target strip appears, judging that the sample to be tested does not contain the verticillium medicago or the sample to be tested is not the verticillium medicago.
7. The method according to claim 6, wherein the reaction system of the PCR reaction comprises the following components in 25. Mu.L: 12.5. Mu.L of 2 XTaqPCRMastermix, 2.5. Mu.L of a mixture of upstream and downstream primers, 1. Mu.L of genomic DNA and 9. Mu.LddH 2 O; the working concentration of the upstream primer and the downstream primer is 5 mu mol/L respectively;
the reaction program of the PCR reaction is as follows: pre-denaturation at 95℃for 1min; denaturation at 95℃for 15s, annealing at 51℃for 20s, extension at 72℃for 20s,40 cycles, and finally extension at 72℃for 7min.
8. A method for detecting or identifying verticillium medicago based on a TaqMan probe, comprising the steps of:
s1, extracting genome DNA of a sample to be detected;
s2, taking genomic DNA of the sample to be detected as a template, carrying out real-time fluorescent quantitative PCR reaction on the template by adopting the primer probe set according to claim 2, judging that the sample to be detected contains the verticillium alfa or the sample to be detected is the verticillium alfa if a fluorescent signal is detected, and judging that the sample to be detected does not contain the verticillium alfa or is not the verticillium alfa if the fluorescent signal is not detected.
9. The method according to claim 8, wherein the primer probe set according to claim 2 is used to perform a real-time fluorescent quantitative PCR on the template to obtain a Ct value, further comprising calculating the genomic DNA concentration of the sample to be tested according to the formula shown in formula 1,
y＝-3.1563x+40.189(R 2 = 0.9933) formula 1, wherein x represents genomic DNA and y represents a corresponding Ct value.
10. The method according to claim 8, wherein the reaction system of the real-time fluorescent quantitative PCR reaction is 25 μl, comprising the following components: 2. Mu.L each of the upstream primer and the downstream primer, 1. Mu.L each of the probe, 1. Mu.L each of the TaqManPCRMix12.5. Mu.L each of the genomic DNA, 1. Mu.L each of the genomic DNA and ddH each of the genomic DNA 2 O6.5μL；
The working concentration of the upstream primer and the downstream primer is 0.8 mu mol/L respectively; the working concentration of the probe is 0.4 mu mol/L;
the reaction program of the real-time fluorescence quantitative PCR reaction is as follows: pre-denaturation at 95 ℃ for 60s; denaturation at 95℃for 15s, annealing at 51 or 52℃for 20s, elongation at 72℃for 20s,40 cycles.
CN202310232590.XA 2023-03-13 2023-03-13 Primer group, primer probe group, kit and method for detecting or identifying verticillium alfa and application Pending CN116516047A (en)

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