CN115044696A - Rapid early molecular detection method for vegetable sclerotiniose and application thereof - Google Patents

Rapid early molecular detection method for vegetable sclerotiniose and application thereof Download PDF

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CN115044696A
CN115044696A CN202210495096.8A CN202210495096A CN115044696A CN 115044696 A CN115044696 A CN 115044696A CN 202210495096 A CN202210495096 A CN 202210495096A CN 115044696 A CN115044696 A CN 115044696A
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sclerotinia sclerotiorum
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张敬泽
商庆华
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Shanghai Academy of Agricultural Sciences
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Abstract

The invention discloses a method for quickly detecting early molecules of vegetable sclerotinia sclerotiorum and application thereof, wherein a primer combination for quickly detecting comprises a specific primer pair for identifying sclerotinia sclerotiorum and a specific primer pair for identifying sclerotinia sclerotiorum; wherein the specific primer pair for identifying the sclerotinia sclerotiorum is Ss-F/Ss-R (SEQ ID NO:1/SEQ ID NO:2), and the amplification product is about 400 bp; the specific primer pair for identifying sclerotinia sclerotiorum is Sm-F/Sm-R (SEQ ID NO:3/SEQ ID NO:4), and the amplification product is about 600 bp. By adopting the method, the PCR amplification detection can be carried out on the fungal genome DNA or the field sample DNA without the separation and culture of pathogenic bacteria, the amplification efficiency is high, the specificity is good, and the sclerotinia sclerotiorum causing the vegetable sclerotiniose can be quickly detected and identified; provides basis for the comprehensive prevention and treatment of vegetable sclerotinia rot.

Description

Rapid early molecular detection method for vegetable sclerotiniose and application thereof
Technical Field
The invention relates to the technical field of DNA molecular detection, in particular to a detection method of sclerotinia sclerotiorum and the technical field of application of the detection method to early detection, prevention, control and the like of vegetable sclerotiniose.
Background
Sclerotinia is a worldwide group of fungal diseases caused by pathogenic bacteria that produce sclerotia. These pathogenic bacteria include Sclerotinia (Sclerotinia), Botrytis (Botrytis), Rhizoctonia (Rhizoctonia) and Sclerotinia (Sclerotium) fungi of the phylum Ascomycota, and the like.
The main pathogenic bacteria causing vegetable sclerotiniose are Sclerotinia sclerotiorum (sclerotiotinia sclerotiorum) and Sclerotinia sclerotiorum (sclerotiotinia minor). Wherein sclerotinia sclerotiorum caused sclerotinia sclerotiorum is widely occurred in the world, and can infect more than 400 kinds of plants including 75 families and 278 genus all over the world, including many important crops, most dicotyledonous plants, such as vegetables, lettuce and lettuce; sclerotinia sclerotiorum also has a wide host range, and can infect more than 100 plants according to statistics.
When sclerotinia sclerotiorum and sclerotinia sclerotiorum infect a host, brown lesions are generated on the surface of the host, and further serious tissue degradation is caused, and the host is in a soft rot shape. Most infected hosts often show brown scabs formed on the surfaces of leaf tissues or caulicle tissues in the initial infection stage, and then the scabs are gradually enlarged to form water-soaked soft rot; in addition to infecting leaves and young stem tissues, sclerotinia sclerotiorum and sclerotinia sclerotiorum can infect stems or twigs of hosts, and as scabs develop, diseased stem parts form annular scabs, so that terminal leaves turn yellow and then brown until the whole plant dies.
Although both pathogens are of the same genus, the two pathogens infect the host in different ways. The primary infection source of sclerotinia sclerotiorum is mainly that sclerotium germinates to form an ascospore, and ascospores are released. After the ascospores are strongly ejected from the ascospores of the ascodisc, they are transmitted to the whole field or the adjacent field by wind and infect the above-ground plants. Sclerotinia sclerotiorum is mainly produced by sclerotium germination to produce mycelium which infects hosts. The strategy for disease control is also different due to the differences in the biological properties of the two pathogens and the infecting host. However, at the initial stage of infecting the host, the symptoms of the host are very similar, which causes difficulty in selecting a disease control strategy.
At present, the detection and identification of pathogenic bacteria of vegetable sclerotinia sclerotiorum mainly rely on the traditional pathogenic bacteria separation and identification, and mainly comprise field symptom observation, pathogenic bacteria separation, sclerotium, molecular identification and the like. These methods are time consuming and are not well suited for early diagnosis of vegetable sclerotinia. In addition, sclerotinia is caused by two pathogenic bacteria, and there has been no technique for simultaneously detecting two pathogenic bacteria so far. In order to effectively control diseases, the production needs to provide early disease diagnosis information urgently. Therefore, the development of accurate and rapid early disease diagnosis technology has important significance for effective prevention and control of sclerotinia sclerotiorum.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a specific detection primer and a detection method for rapidly detecting sclerotinia sclerotiorum and sclerotinia sclerotiorum. Can be used for detecting vegetable sclerotiniose (in leaves, stem segments and soil) and has important significance for the early rapid detection of vegetable sclerotiniose diseases, the population distribution of soil germs and the prevention and treatment of the diseases.
A primer combination for rapidly detecting Sclerotinia sclerotiorum and Sclerotinia sclerotiorum comprises a specific primer pair for identifying Sclerotinia sclerotiorum (sclerotiotinia sclerotiorum):
Ss-F(SEQ ID NO:1):5’GCATGGGATATCCTCAATC 3’；
Ss-R(SEQ ID NO:2):5’ATGGACGAGTGCCCGCTCTA3’；
and a specific primer pair for identifying Sclerotinia sclerotiorum (sclerotiotia minor):
Sm-F(SEQ ID NO:3):5’TTCACTACCTAGTCTAGTAC-3’；
Sm-R(SEQ ID NO:4):5’TTGGAAGAGAGATAGGAGGG 3’。
the invention also provides application of the primer combination in rapid detection of sclerotinia sclerotiorum and/or sclerotinia sclerotiorum.
The invention also provides application of the primer combination in early rapid detection of vegetable sclerotinia rot.
Optionally, the vegetable is lettuce, lettuce or rape. Preferably lettuce.
Optionally, the sclerotinia sclerotiorum is caused by sclerotinia sclerotiorum and/or sclerotinia sclerotiorum infection. The primer combination can rapidly identify that the sclerotinia rot of vegetables is caused by the infection of sclerotinia sclerotiorum and/or sclerotinia sclerotiorum.
The invention also provides a reagent or a kit for rapidly detecting the sclerotinia sclerotiorum and/or the sclerotinia sclerotiorum, which comprises the primer combination.
The invention also provides a method for rapidly detecting sclerotinia sclerotiorum and sclerotinia sclerotiorum, which comprises the following steps:
taking the genome DNA of a sample to be detected as a template, and carrying out PCR amplification by using the primer combination;
and judging whether the sample to be detected is infected by sclerotinia sclerotiorum and/or sclerotinia sclerotiorum according to the size of the fragment of the amplification product.
The size of a corresponding fragment amplified by taking the sclerotinia sclerotiorum DNA as a template and using the specific primer pair Ss-F, Ss-R is about 400 bp; the corresponding fragment amplified by using the specific primer pair Sm-R, Sm-R with sclerotinia sclerotiorum DNA as a template is about 600bp in size. Based on this, when judging whether the sample to be tested is infected by sclerotinia sclerotiorum and/or sclerotinia sclerotiorum, the judgment rule is as follows: (1) if fragments with the size of about 400bp appear in the amplification product, the sample to be detected is infected by sclerotinia sclerotiorum; (2) if fragments with the size of about 600bp appear in the amplification product, the sample to be detected is infected by sclerotinia sclerotiorum; (3) if fragments with the size of about 400bp and fragments with the size of about 600bp simultaneously appear in the amplification product, the sample to be detected is infected by sclerotinia sclerotiorum and sclerotinia sclerotiorum simultaneously; (4) if the amplification product has no band, the sample to be detected does not contain sclerotinia sclerotiorum and sclerotinia sclerotiorum.
Optionally, the sample to be tested is from vegetable leaf tissue, vegetable stem segment, soil or pathogenic bacteria DNA. Preferably, the vegetable is lettuce, lettuce or rape; most preferably lettuce.
Optionally, the PCR amplification system is: 2 x Hieff TM PCR Master Mix(With Dye)，12.5μl；ddH 2 O, 8 μ l; a mixed primer pair (primer Ss-F, 0.5. mu.l; primer Sm-F, 0.5. mu.l; primer Ss-R, 0.5. mu.l; primer Sm-R, 0.5. mu.l); DNA template, 2.5. mu.l; the PCR amplification procedure was: 3min at 95 ℃; 30sec at 95 ℃, 30sec at 56 ℃, 30sec at 72 ℃ and 35 cycles; 10min at 72 ℃.
Compared with the prior art, the invention has at least one of the following beneficial effects:
(1) the invention designs two pairs of specific primers by utilizing the difference of highly conserved laccase 2(laccase2) gene sequences in different species, and amplifies DNA fragments by taking the genomic DNA of sclerotinia sclerotiorum and sclerotinia sclerotiorum as templates. In a PCR reaction system of two pairs of specific primers, sclerotinia sclerotiorum can amplify a specific band of about 400bp, sclerotinia sclerotiorum can amplify a specific band of about 600bp, and DNA templates of other strains and clear water contrast have no amplification product. And specific primers can be used for amplifying specific bands from leaf blades, stem segments and soil samples containing sclerotinia sclerotiorum and sclerotinia sclerotiorum. The specific primer can be used for molecular detection of vegetable sclerotinia rot and early diagnosis of diseases.
(2) By utilizing the primer combination, the PCR amplification detection can be carried out on the fungal genome DNA or the field sample DNA without the separation and culture of pathogenic bacteria, the amplification efficiency is high, the specificity is good, and the sclerotinia sclerotiorum causing the vegetable sclerotiniose can be quickly detected and identified; by quickly detecting the diseases, the vegetable sclerotiniose can be prevented and treated in advance, even avoided, the negative effects caused by pesticide use are reduced, and a basis is provided for the comprehensive prevention and treatment of the vegetable sclerotiniose.
(3) The method of the present invention can identify sclerotinia sclerotiorum and sclerotinia sclerotiorum causing vegetable sclerotiniose by only one-time PCR.
Drawings
FIG. 1 is an electrophoretogram of the products of specific PCR amplification of Sclerotinia sclerotiorum and Sclerotinia sclerotiorum with two pairs of primers Ss-F/R and Sm-F/R. Wherein M is marker, lanes 1-5 are Sclerotinia sclerotiorum, Sclerotinia sclerotiorum and Sclerotinia sclerotiorum, Botrytis cinerea and control (ddH) 2 O)。
FIG. 2 is an electrophoresis diagram of PCR amplification products of Sclerotinia sclerotiorum and Sclerotinia sclerotiorum strains of different sources using a mixed primer set. M is marker; 1-2 is sclerotinia sclerotiorum; sclerotinia sclerotiorum No. 3-16; no. 17 as control (ddH) 2 O)。
FIG. 3 is an electrophoresis diagram of PCR amplification products of different source diseased leaf samples by using a mixed primer group. Wherein M is Maker; no. 1-2 is diseased leaves infected by sclerotinia sclerotiorum; no. 3 is infected diseased leaves of sclerotinia sclerotiorum and sclerotinia sclerotiorum; no. 3-16 is diseased leaves infected by sclerotinia sclerotiorum; reference No. 17 (ddH) 2 O)。
FIG. 4 is an electrophoresis picture of PCR amplification products of lettuce leaves using a mixed primer group under different infection times. Wherein lanes 1-3 are healthy lettuce leaves; lanes 4-6The diseased leaves are inoculated with sclerotinia sclerotiorum for 24 hours; lane 7-9 is diseased leaves inoculated with sclerotinia sclerotiorum for 48 h; lane 10-12 is diseased leaves inoculated with sclerotinia sclerotiorum for 72 h; lane 13-15 is diseased leaves inoculated with sclerotinia sclerotiorum for 24 h; lanes 16-18 are diseased leaves inoculated with sclerotinia sclerotiorum for 48 h; lanes 19-21 are lettuce leaf samples inoculated with sclerotinia sclerotiorum for 72 h; lane 22 is a control (ddH) 2 O)。
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The media referred to in the examples below:
potato dextrose agar medium (PDA medium): 200g of potato, 20g of glucose, 12g of agar powder and 1L of deionized water, and sterilizing at 121 ℃ for 20min, wherein the pH value is natural.
Example 1 primer design for rapid detection of Sclerotinia sclerotiorum and Sclerotinia sclerotiorum
PCR detection is a common molecular biology detection method, can realize rapid detection of pathogenic bacteria by rapidly amplifying specific nucleic acid sequences of pathogenic microorganisms, has been widely applied in the fields of infectious diseases, food-borne pathogenic bacteria pollution, environmental pollution, tumor genetic markers and the like in recent years, and is a mature detection technology with the widest application range.
In PCR detection, how to select a proper target gene for primer design is the key. Studies have shown that the laccase 2(laccase2) gene is widely present in plant, insect, bacterial and fungal species and shows relative conservation among different organisms. Vegetable sclerotiniose and gray mold are usually caused by different pathogenic bacteria. For example, lettuce sclerotinia is caused by sclerotinia sclerotiorum and sclerotinia sclerotiorum, and gray mold is caused by botrytis cinerea. Although the laccase2 gene sequence differs greatly between sclerotinia and sclerotinia botrytis, it differs less within sclerotinia. In addition, there is a variation in the base of the gene sequence among strains of the same species and different origins. Therefore, the design of primers requires sequencing or analysis of strains from different sources. The research designs specific primers aiming at conserved regions of laccase2 gene sequences of sclerotinia sclerotiorum and sclerotinia sclerotiorum from different sources, can quickly detect the sclerotinia sclerotiorum and the sclerotinia sclerotiorum in the early vegetable pathogenesis, and provides a powerful tool for preventing and treating vegetable sclerotiniose and early warning the vegetable sclerotiniose.
Specifically, the method comprises the following steps:
in the first step, primer design is carried out by taking the downloading sequence as a template to obtain primers lacF and lacR.
There may be a base mutation problem in the same gene due to different source strains. Thus, the laccase2 isogenes of Sclerotinia sclerotiorum and Sclerotinia viticola (GenBank: EF050080.1), Sclerotinia minor (GenBank: EF050079.1), Borrytis cinerea (GenBank: CP009818.1), Borrytis fragariae (GenBank: XM-037333897.1), Borrytis deweyae (GenBank: XM-038955775.1), Borrytis byssoidea (GenBank: XM-038874818.1), Borrytis sinoallii (GenBank: XM-038901467.1), Borrytis porri (GenBank: XM-038918558.1) were downloaded in GenBank. Wherein the annotated northern sclerotinia sclerotiorum (s. borealis) genome (AYSA01000041.1) is free of laccase2 homologous gene. Primers were designed using software Primer5.0 based on their conserved sequence regions, where the forward primer was lacF (SEQ ID NO:5):5 'ATGAAGTGTTTTACATCACT 3', and the downstream primer was lacR (SEQ ID NO:6):5 'TCGAATTTCTTACCACCACC 3'.
And secondly, performing PCR amplification and sequencing on 3 sclerotinia sclerotiorum, 3 sclerotinia sclerotiorum and 1 botrytis cinerea laccase2 (lacase 2) genes separated from Chinese lettuce in Shanghai and Zhejiang by taking lacF and lacR obtained by the first step as primers (Hangzhou Populaceae Biotech company).
The gene sequence analysis result shows that (the partial sequence of the sclerotinia sclerotiorum laccase2 gene is shown as SEQ ID NO:7, the partial sequence of the sclerotinia sclerotiorum laccase2 gene is shown as SEQ ID NO:8, and the partial sequence of the botrytis cinerea laccase2 gene is shown as SEQ ID NO: 9) is from the same strains in Zhejiang and Shanghai, the laccase2 gene sequence is completely consistent, but has the difference of individual bases with the homologous genes in GenBank.
And thirdly, designing a special primer pair by taking SEQ ID NO 7-9 as a target sequence.
On the basis of the second step, a PCR specific primer is designed according to the difference of the conserved region sequences of the laccase2 gene of the sclerotinia sclerotiorum shown in SEQ ID NO. 7, the laccase2 gene of the sclerotinia sclerotiorum shown in SEQ ID NO. 8 and the laccase2 gene of the botrytis cinerea shown in SEQ ID NO. 9.
In order to save cost and time in the identification, a one-time PCR amplification technique with a mixture of two pairs of primer pairs was established. In the design of the primers, not only the sequence difference among different species is considered, but also the dimer problem among the designed primers is considered, so that the established reaction has high efficiency and sensitivity.
After repeated verification, two pairs of primers were obtained. Wherein the specific primer pair for identifying Sclerotinia sclerotiorum is Ss-F (SEQ ID NO:1) (5 'GCATGGGATATCCTCAATC 3')/Ss-R (SEQ ID NO:2) (5 'ATGGACGAGTGCCCGCTCTA 3'); the specific primer set for identifying Sclerotinia sclerotiorum was Sm-F (SEQ ID NO:3) (5 'TTCACTACCTAGTCTAGTAC 3')/Sm-R (SEQ ID NO:4) (5 'TTGGAAGAGAGATAGGAGGG 3'), and was synthesized by Oncodinaceae Biotech, Hangzhou.
The specificity of the primer pair is verified by using the DNA of the sclerotinia sclerotiorum, the sclerotinia sclerotiorum and the botrytis cinerea as templates and using a one-time PCR amplification technology.
The PCR validation procedure was as follows:
1. DNA of samples to be detected (sclerotinia sclerotiorum, sclerotinia sclerotiorum and botrytis cinerea) is respectively extracted, and a commercialized kit (Ezup column type fungus genome DNA extraction kit, biological engineering (Shanghai) corporation) can be directly adopted for DNA extraction.
2. Respectively taking (a) sclerotinia sclerotiorum DNA, (b) sclerotinia sclerotiorum DNA, (c) mixed DNA of sclerotinia sclerotiorum and (d) Botrytis cinerea DNA as templates, and carrying out PCR amplification by adopting a specific primer pair of the invention, wherein the reaction system is as follows:
2×Hieff TM PCR Master Mix(With Dye)，12.5μl；ddH 2 o, 8 μ l; primer Ss-F, 0.5. mu.l; primer Sm-F, 0.5. mu.l; primers Ss-R, 0.5. mu.l, Sm-R, 0.5. mu.l; DNA template, 2.5. mu.l. The PCR amplification procedure was: 3min at 95 ℃; 95 ℃ 30sec, 56 ℃ 30sec, 72 ℃ 30sec, 35 cycles; 10min at 72 ℃.
3. And (3) carrying out agarose gel electrophoresis analysis on the amplification product: mu.L of the PCR product was electrophoresed through 1% agarose gel in 1 XTBE buffer for 30min at 110V, and after completion, the image was observed under UV lamp.
The electrophoresis imaging result is shown in FIG. 1, wherein lane 1 is an amplification product using Sclerotinia sclerotiorum DNA as a template, and the size of the amplification product is about 400 bp; lane 2 is an amplification product using a DNA of sclerotinia sclerotiorum as a template, and the size of the amplification product is about 600 bp; lane 3 is an amplification product using a mixed DNA of Sclerotinia sclerotiorum and Sclerotinia sclerotiorum as a template, and the amplification product has two obvious bands with the sizes of about 400bp and 600bp respectively; lane 4 is an amplification product using Botrytis cinerea DNA as a template; lane 5 is a control (ddH) 2 O)。
Test results prove that the two pairs of specific primers Ss-F/R and Sm-F/R can specifically amplify the DNA of the sclerotinia sclerotiorum and can be used for detecting the sclerotinia sclerotiorum and the sclerotinia sclerotiorum.
Example 2 detection of Sclerotinia sclerotiorum and Sclerotinia sclerotiorum samples isolated in different regions
Collectingsclerotium of sclerotinia sclerotiorum and sclerotinia sclerotiorum separated from 16 different sites in Shanghai city. The sclerotium is treated with 75% ethanol for 5min, and then washed with sterile water for 3min, and repeated 3 times. The sterilized sclerotia were placed in the center of the PDA plate, and after the plate was filled with the germinated hyphae, DNA was extracted according to the method of example 1.
The 16 samples were tested rapidly using the two pairs of specificity primers, Ss-F/R and Sm-F/R, designed in example 1. The detection method was the same as in example 1.
The results of electrophoretic imaging of the amplification products of 16 DNA samples are shown in FIG. 2, where M is Maker, respectively; the amplification products of the samples No. 1 and No. 2 are about 600bp and are identified as sclerotinia sclerotiorum; the amplification product of the sample No. 3-16 is about 400bp and is identified as sclerotinia sclerotiorum; lane 17 is a control (ddH) 2 O). Experiments show that the two pairs of specific primers provided by the invention can efficiently detect sclerotinia sclerotiorum and sclerotinia sclerotiorum samples in different areas, and can be used for identifying pathogenic bacteria of vegetable sclerotinia sclerotiorum.
Example 3 Rapid detection of field sclerotinia sclerotiorum samples
The method comprises the steps of collecting 9 sclerotinia sclerotiorum disease-causing leaves in vegetable bases in Shanghai, and collecting 16 field sclerotinia sclerotiorum leaf samples in total, wherein the collection places and the sample numbers are shown in the following table 1.
TABLE 1 vegetable sclerotinia onset leaf samples of 9 vegetable bases in Shanghai
Figure BDA0003632608310000091
The method for rapidly detecting the field vegetable sclerotinia sclerotiorum disease-causing leaves comprises the following steps:
1. the genomic DNA of the diseased leaf sample is extracted by directly adopting a commercial kit (plant genomic DNA extraction kit, TIANGEN company).
2. The PCR amplification was performed using the DNA of the sample to be tested as a template and the specific primer pairs Ss-F/R and Sm-F/R (designed in example 1) in the same manner as in example 1.
3. And (5) carrying out agarose gel electrophoresis analysis on the amplification product.
The collected 16 diseased leaf samples are subjected to PCR amplification through specific primers Ss-F/R and Sm-F/R, the electrophoresis results of amplification products are shown in figure 3, and M is marker; the No. 1 and No. 2 samples are amplified to form bands with the size of about 600bp, which indicates that two lettuce diseased leaves are infected by sclerotinia sclerotiorum; two bands are amplified from the No. 3 sample, the sizes of the two bands are about 400bp and 600bp respectively, and the result shows that the lettuce leaves are infected by sclerotinia sclerotiorum and sclerotinia sclerotiorum together; DNA extracted from the leaves of the lettuce No. 4-16 is amplified to form a strip with the size of about 400bp, which indicates that the sample is infected by sclerotinia sclerotiorum; lane 17 is a control (ddH) 2 O)。
Example 4 detection of lettuce leaves artificially inoculated with Sclerotinia sclerotiorum and Sclerotinia sclerotiorum
Fresh lettuce leaves with similar growth vigor are taken, washed clean by sterile water and dried, and the leaves are placed in a culture dish with the bottom padded with wet filter paper. A piece of sclerotinia or sclerotinia sclerotiorum with a diameter of 6mm was inoculated on lettuce leaves, and the moist petioles were covered with moist cotton balls, and the test was repeated 3 times with healthy lettuce leaves as a control. The leaf is placed in an illumination incubator at 25 ℃ for 12h for culture, the DNA of the lettuce leaf is extracted after 24h, 48h and 72h of culture respectively, and two pairs of specific primers of Ss-F/R and Sm-F/R are used for rapidly detecting the lettuce leaf. The detection method was the same as in example 3.
The test results are shown in FIG. 4, lanes 1-3 are healthy lettuce leaves, and no band is amplified; lane 4-6 is the leaf sample of lettuce after 24h inoculation with sclerotinia sclerotiorum, lane 7-9 is the leaf sample of lettuce after 48h inoculation with sclerotinia sclerotiorum, lane 10-12 is the leaf sample of lettuce after 72h inoculation with sclerotinia sclerotiorum, lane 13-15 is the leaf sample of lettuce after 24h inoculation with sclerotinia sclerotiorum, lane 16-18 is the leaf sample of lettuce after 48h inoculation with sclerotinia sclerotiorum, lane 19-21 is the leaf sample of lettuce after 72h inoculation with sclerotinia sclerotiorum, and lane 22 is the control (ddH) 2 O)。
Test results show that after the sclerotinia sclerotiorum is infected with healthy lettuce leaves for 24 hours, a leaf sample can be amplified to form a strip of about 400bp, and after the sclerotinia sclerotiorum is infected with healthy lettuce leaves for 48 hours, a leaf sample can be amplified to form a strip of about 600 bp. Therefore, the specific primer group provided by the invention can be used for molecular detection of vegetable sclerotinia sclerotiorum and early diagnosis of diseases.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
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<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 6
tcgaatttct taccaccacc 20
<210> 7
<211> 772
<212> DNA
<213> Sclerotinia minor
<400> 7
tgcctcctgt gcacactcgg ctacttcccg atcctgctgg ggagcttact ccatcgatac 60
cgactggtat gatgttatac cacacactgg agttaccaga ggtatgcgat atcacattct 120
cattcactac ctagtctagt actaatttat gcatagaata ctggctttcg gttgagaact 180
ccactataac ccctgatggt tatacccgct cagccatgac catcaatgga accgtccctg 240
gccctgcaat tatcgcggac tggggcgaca atcttgtaat ccgtaagtct ccgatagcgc 300
accgaatctc aaaagtcctc gatagcatgg gatatcctca atcatccttt tgcatgtttc 360
tcttcactaa ctacgagtca cagatgtcac caacaatctt caacacaatg gaacagccat 420
tcacttccac ggtattcgtc agaagggaag cttagagtac gatggtgtgc ccggtgttac 480
tcaatgtcct atcgctcctg gagatacctt gacctacaag ttccaagcta ctcaatatgg 540
aactacttgg tatcactctc acttctctct tcaatacgct gatgggctct tcggccctct 600
catcattaac ggtcctgcca ctgcagacta cgatgaggat cttggagtaa tgtttttggg 660
tgattgggca cacgaaaccg tcttcgatct ttgggatcgc gctagatccg gccctcctat 720
ctctcttcca aacactttga tgaacggaac caacagttgc gagtgcgata ct 772
<210> 8
<211> 777
<212> DNA
<213> Sclerotinia sclerotiorum
<400> 8
tgcctcctgt gtacactcgg ctacttctag atcctgctgg ggagattact ctatcgatac 60
tgactggtat gatgttattc ctcacactgg agtcactaga ggtatgcgat atcacattct 120
caatttacta gtctaatact aatttatgca tagaatactg gctttcggtt gagaactcca 180
ctataacccc tgacgggtat acccgctcag ccatgacctt taatggaacc gcccctggcc 240
ctgcaattat cgccgactgg ggcgacaatc ttgtcatccg taagtcttcg atagcgcgcc 300
gaatctcaat gtcctccata agatcagaaa ttttcatatc atccttttgc atgattcttc 360
ttactaacca cgggtcatag atgtcaccaa taatcttcaa cataatggaa cagccattca 420
tttccacggt attcgtcaga agggaagctt agagtacgat ggagtgcccg gtgttactca 480
gtgtcctatc gctcctggag ataccttgac ctacaagttc caagctactc aatatggaac 540
tacttggtat cactctcact tctctcttca atacgctgat ggactctttg gccctcttat 600
catcaatggt cctgccactg cagactacga tgaggatctt ggagtcatgt tcttgggaga 660
ttgggcacac caaaccgtct tcgatatttg ggatgccgct agagcgggca ctcgtccatc 720
tcttcaaaac actttgatga acggaaccaa tagttgcgag tgcgatactt ctgatcc 777
<210> 9
<211> 915
<212> DNA
<213> Botrytis cinerea
<400> 9
tgcctcttgt gcaaactcgg ctacttccag atcttgctgg ggagagtatt ccattgatac 60
caactggtat gatgttactc ctaatactgg agtcaccaga ggtatgtgat atcgaattcc 120
tagtctctag tctatcacta actcgatata cagaatactg gctttcagtt gagaactcca 180
ccatcacacc tgatggttat actcgctcag ccatgacctt caatggaact gttccaggac 240
ctgcaattac agcagactgg ggtgacaatc ttataatccg taggtctcca gtcactcaga 300
gagcctcttt gtcctcacaa cttgaggccc tcaaatatca ctgtttttaa gctttgattg 360
tcattgctaa ctacctgatt acagacgtta ccaacaatct ccaacacaat ggtacatcta 420
ttcattggca tggaattcgt caactaggaa gtctcgaata cgacggcgta cccggtatgt 480
ctttaaaaga ttgtgaccat gcgatcacaa gaatattccc ccttttccct cccttttcgt 540
cccatatttc ctcctcccta catccataac tcttgccagt cttctactga aaaattacta 600
acataaatta caggtgtaac tcaatgtcct atcgctcctg gagatacctt gacctacaaa 660
ttccaagcta ctcaatatgg aaccacctgg tatcactctc acttctctct tcaatacgct 720
gatggactct ttggaccctt gatcattaat ggtcccgcta ctgcggacta tgatgaagat 780
gttggtgcaa ttttcctcca agattgggca cataaatccg ttttcgaaat ttgggactcc 840
gctagacaag gtgctcctcc agcacttgaa aacactttga tgaatggaac caacatctac 900
gattgctcag cttct 915

Claims (9)

1. A primer combination for rapidly detecting Sclerotinia sclerotiorum and Sclerotinia sclerotiorum is characterized by comprising a specific primer pair for identifying Sclerotinia sclerotiorum (sclerotiotium sclerotiorum):
Ss-F(SEQ ID NO:1):5’GCATGGGATATCCTCAATC 3’；
Ss-R(SEQ ID NO:2):5’ATGGACGAGTGCCCGCTCTA 3’；
and a specific primer pair for identifying Sclerotinia sclerotiorum (sclerotiotia minor):
Sm-F(SEQ ID NO:3):5’TTCACTACCTAGTCTAGTAC-3’；
Sm-R(SEQ ID NO:4):5’TTGGAAGAGAGATAGGAGGG 3’。
2. use of the primer combination of claim 1 for rapid detection of sclerotinia sclerotiorum and/or sclerotinia sclerotiorum.
3. Use of the primer combination of claim 1 for the rapid detection of vegetable sclerotiniose in an early stage.
4. Use according to claim 3, characterized in that the vegetable is lettuce, lettuce or rape.
5. Use according to claim 3, characterized in that the sclerotinia sclerotiorum is caused by an infection with sclerotinia sclerotiorum and/or sclerotinia sclerotiorum.
6. A reagent or a kit for rapid detection of sclerotinia sclerotiorum and/or sclerotinia sclerotiorum, comprising the primer combination according to claim 1.
7. A method for rapidly detecting Sclerotinia sclerotiorum and Sclerotinia sclerotiorum is characterized by comprising the following steps:
performing PCR amplification by using the primer combination of claim 1 by using the genomic DNA of a sample to be detected as a template;
and judging whether the sample to be detected is infected by sclerotinia sclerotiorum and/or sclerotinia sclerotiorum according to the size of the fragment of the amplification product.
8. The method of claim 7, wherein the sample to be tested is derived from vegetable leaf tissue, vegetable stem segments, soil or pathogen DNA.
9. The method according to claim 7, wherein the PCR amplification system is: 2 x Hieff TM PCR Master Mix(With Dye)，12.5μl；ddH 2 O, 8 μ l; primer Ss-F, 0.5. mu.l; primer Sm-F, 0.5 mu l of the solution; primer Ss-R, 0.5. mu.l; primer Sm-R, 0.5. mu.l; DNA template, 2.5. mu.l; the PCR amplification procedure was: 3min at 95 ℃; 30sec at 95 ℃, 30sec at 56 ℃, 30sec at 72 ℃ and 35 cycles; 10min at 72 ℃.
CN202210495096.8A 2022-05-07 2022-05-07 Rapid early molecular detection method for vegetable sclerotiniose and application thereof Pending CN115044696A (en)

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