CN113897453A - Primer group for rapidly detecting cercospora apiacea and detection method - Google Patents
Primer group for rapidly detecting cercospora apiacea and detection method Download PDFInfo
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Abstract
The invention discloses a primer group and a detection method for rapidly detecting pathogenic cercospora apiacea of cercospora leaf spot of celery. The invention designs and screens a set of specific detection primer group aiming at the specific cercosporin facetting protein (cfp) gene sequence of the cercosporin facetting protein of the cercosporin celerians, the primer group consists of 2 specific primers cfp-F, cfp-R, and a detection method of the cercosporin facetting protein cfp gene is established, and the detection result can judge whether the cfp gene exists in a sample to be detected. The primer screened by the invention has high sensitivity and strong specificity, and the established detection method has the advantages of high accuracy, short detection time, simple operation and high specificity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection primer group and a detection method for rapidly detecting cercospora apiacea.
Background
Tail spore of celery (Cercospora apii) Is an important plant pathogenic fungus, which is found parasitic on tropical zone andumbrella-shaped vegetable of subtropical zone. It mainly harms the leaves of plants, forms typical scabs, and can also infect stems, causing phenomena such as fallen leaves and premature senility in severe cases. In recent years, the harm of cercospora leaf spot diseases of celery and the like in China is increased year by year, the cercospora leaf spot diseases can be attacked in both seedling stage and adult stage, the victimization in the adult stage is serious, celery leaves and petioles and stems can be harmed, the morbidity is generally 20-40%, the serious disease rate is over 60%, the celery yield and quality are seriously reduced, and the cercospora leaf spot diseases become an important limiting factor for celery production development. The primary infection source of the cercospora apiacea is mainly seeds or residual disease strains, germs live through the winter by hypha attached to the surfaces of the seeds and invaded into seed skins, the hypha and spores in the residual disease strains and soil, when the environmental conditions are proper, the hypha generates conidia which are spread to host plants through air flow, and the conidia directly invade from the host epidermis under the condition of the existence of water drops to cause primary infection. Sowing seeds with diseases, infecting diseases after emergence of seedlings, generating a new generation of conidia at the damaged part, carrying out repeated re-infection by airflow wind and rain transmission, and continuing to the end of autumn repeatedly, wherein the harm is gradually increased.
The cercospora apiacea leaf spot caused by the cercospora apiacea has various symptoms on leaves, is very easy to be confused with diseases caused by other pathogenic bacteria, and has similar symptoms with the alternaria apiacea leaf spot, the spot blight and the bacterial leaf spot when the pathogenic bacteria are infected in different periods. In the field, because many farmers misdiagnose by mistake and misdiagnose, diseases are not effectively controlled in time, and huge economic loss is caused. Therefore, the establishment of a quick detection technology for the alternaria leaf spot has important significance for prediction and forecast of the occurrence of diseases.
At present, the detection of the cercospora apiacea in plant tissues mainly adopts the traditional methods of tissue isolation culture, morphological characteristic observation, pathogenicity identification and the like, has long detection period and low sensitivity, is easily interfered by a plurality of factors such as human factors, environment factors and the like, and is not easy to make timely and correct diagnosis in the disease incubation period and the initial period.
In recent years, with the development of molecular biology, detection methods based on PCR technology have been successfully applied to the detection of various pathogenic bacteria. At present, the PCR detection method is successfully applied to detection of various plant pathogenic microorganisms, has the advantages of strong specificity and high sensitivity, such as tomato yellow leaf curl virus, citrus canker, fusarium, wheat leaf rust and the like, can quickly diagnose diseases at the early stage of disease attack, and timely guides farmers to take effective prevention and control measures. However, the detection of the cercosporin-based promoting protein cfp gene is not reported at present. Can be popularized and applied to the base layer as an early detection technology of plant diseases.
The invention designs 2 specific primers by utilizing the specific toxin protein cfp gene sequence characteristics of the alternaria apiacea, and establishes a specific molecular detection method of the alternaria apiacea on the basis.
Disclosure of Invention
The invention aims to establish a PCR molecular detection technology which is good in specificity, high in sensitivity, rapid and simple for cercospora apiacea, and relates to a specific primer pair of cercospora apiacea-cercospora pigment promoting protein cfp gene and a molecular detection method established by using the primer pair.
In order to achieve the purpose, the invention discloses the following technical contents:
a method for quickly detecting pathogenic cercospora apiacea (Cercospora apiacea, Cercospora leaf spot disease)Cercospora apii) The cercosporin promotes the specificity primer of the protein cfp gene, characterized in that the primer pair is a forward primer cfp-F and a reverse primer cfp-R, the nucleic acid sequence of the primer is cfp-F (5 'to 3'): TAGAAATGACTGGCATAGTC, respectively; cfp-R (5 'to 3'): GTCATAGCCAATAAGCTAGC are provided.
The invention further discloses a method for detecting pathogen cercospora apiacea of cercospora leaf spot disease by using the specific primer, which comprises the following steps: extracting DNA of a sample to be detected, and then performing specific amplification on the DNA of the sample to be detected by using a detection primer group through a polymerase chain reaction amplification method to confirm whether an amplification product exists or not. The sample to be detected is celery leaf tissue or pure fungus culture. The DNA of the cercospora brassicae is specifically amplified by a PCR amplification method, the total volume of a specific amplification system is 25 mu L, and the specific amplification system comprises 2 × taq Master Mix: 12.5 mu L; primer cfp-F: 1 mu L of the solution; primer cfp-R：1μL；ddH2O: 9.5. mu.L and 1. mu.L of DNA template; wherein the 2 × taq Master Mix comprises the following components: 20 mmol/L Tris-HCl, 0.1mmol/L EDTA, 50mmol/L KCl, 1.5mmol/L MgCl2200 mu mol/L dNTPs, and the balance being sterilized ddH2O。
The invention also discloses a rapid detection method of the pathogenic cercospora apiacea of the cercospora leaf spot of the celery, which adopts specific primers; the amplification reaction system is formulated according to claim 5; carrying out PCR amplification on the prepared reaction solution under the following reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 53 ℃ for 45s, and extension at 72 ℃ for 45s, after 32 cycles; extending for 10min at 72 ℃; and after the amplification reaction is finished, performing 1% agarose gel electrophoresis under the electrophoresis condition of 100-110V for 20-30 min. Specific bands are generated at the position of 817bp and are used as result judgment, the generation of the specific bands indicates that the detection result is positive, and the pathogenic cercospora apiacea (A) of cercospora apiacea purpurea existsC. apii) The absence of specific bands at the position of 817bp indicates that the detection result is negative and the pathogenic cercospora apiacea of the cercospora leaf spot of celery is absent ((C. apii) The sample to be detected can be tissues such as celery leaves and the like or pure culture of fungi.
The invention further discloses a method for rapidly detecting pathogenic cercospora apiacea of cercospora leaf spot of celery (C. apii) The application of the cercosporin-promoting protein cfp gene specific primer in the aspect of quickly detecting the cercospora apiacea is provided. The experimental results show that: the detection result can accurately judge whether the sample to be detected contains the cercospora apiacea (C. apii) The cercosporal bacteriocin promotes protein cfp gene, thereby realizing the rapid detection of the celery cercosporal. The group of specific primers screened by the invention has high sensitivity and strong specificity, and the established detection method has the characteristics of high accuracy, short detection time, simple operation and the like.
The invention is described in more detail below:
the invention extracts the DNA of a sample to be detected, and then utilizes a detection primer group to perform specific amplification on the DNA of the sample to be detected by a Polymerase Chain Reaction (PCR) amplification method to confirm whether an amplification product exists or not. The sample to be detected is celery leaf tissue or fungusA pure culture. Specifically amplifying DNA of the cercospora apiacea by a PCR amplification method, wherein the total volume of a specific amplification system is 25 mu L, and the specific amplification system comprises 2 × taq Master Mix: 12.5 mu L; primer cfp-F: 1 mu L of the solution; primer cfp-R: 1 mu L of the solution; ddH2O: 9.5. mu.L and 1. mu.L (2.5 ng/. mu.L-7.5 ng/. mu.L) of DNA template; wherein the 2 × taq Master Mix comprises the following components: 20 mmol/L Tris-HCl, 0.1mmol/L EDTA, 50mmol/L KCl, 1.5mmol/L MgCl2200 mu mol/L dNTPs, and the balance being sterilized ddH2And O. Carrying out PCR amplification on the prepared reaction solution under the following reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 53 ℃ for 45s, and extension at 72 ℃ for 45s, after 32 cycles; extension is carried out for 10min at 72 ℃ and the amplification reaction is finished.
The detection of the amplification product may be carried out by electrophoresis. And (3) performing 1% agarose gel electrophoresis under the electrophoresis condition of 100V-110V for 20min-30min, generating a specific band at a position of 817bp as result judgment, wherein the generated specific band indicates that the detection result is positive and the pathogenic cercospora apiacea of the cercospora leaf spot exists, and the absence of the specific band at the position of 817bp indicates that the detection result is negative and the pathogenic cercospora apiacea of the cercospora apiacea exists.
The 2 XTaq Master Mix reaction buffer solution is a substance in the prior art, can be purchased from a reagent company and comprises the following components: 20 mmol/L Tris-HCl, EDTA 0.1mmol/L, 50mmol/L KCl, 1.5mmol/L Mg Cl2200 mu mol/L dNTPs, and the balance being sterilized ddH2O。
Compared with the prior art, the primer group and the detection method for rapidly detecting the cercospora apiacea disclosed by the invention have the positive effects that:
(1) the kit and the method perform specific detection on the target gene of the cercospora apiacea specific cercospora pigment promoting protein cfp, and have the advantages of strong specificity and high sensitivity.
(2) The method has the greatest advantage that the specificity detection is carried out aiming at the specific cercospora apiacea purpurea hormone promoting protein cfp gene sequence, the method is simple and easy to implement, and the method is related to pathogenicity.
(3) The molecular detection method of the pathogenic cercospora apiacea of the cercospora leaf spot of the celery, which is established by the invention, is quick, simple and convenient, meets the requirements of quick and early diagnosis of the cercospora apiacea leaf spot, and realizes the purposes of detection, early diagnosis and monitoring of the latent period of the disease.
The specific primer and the amplification system of the invention are adopted to treat the celery tail spore: (C. apii) Alternaria gracilis (A) and (B)Alternaria tenuissima) Septoria apiacea (A. apiacea)Septoria apii) Fusarium oxysporum (F.), (Fusariumn oxysporum) Botrytis cinerea (A. cinerea)Botrytis cinerea) Penicillium griseovii (A) and (B)Penicillium glaucum) Trichotheca pinicola (A) and (B)Trichothecium roseum) Aspergillus (A), (B), (C)Aspergillus sp.) Trichoderma harzianum (a)Trichoderma harzianum) Rhizopus (A) and (B)Rhizopus sp.) Mucor: (A), (B)Mucor sp.) The DNA is subjected to PCR specific amplification, and through detection, only celery strain amplification is positive, clear bands can be seen in 817bp, and other non-target strains are negative. The PCR detection method established by the invention can be used for detecting alternaria apiacea and specifically detecting the target pathogen, namely the cercospora apiacea.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.
Example 1
And (3) carrying out PCR detection on the cercospora apiacea.
In order to verify the feasibility of the PCR detection method, a pure culture of the green grass carpospora apiiformis QC-1 which is separated from a green grass carpospora apiiformis specimen and identified by morphology and molecular biology is selected as a target strain, and the genome DNA of the pure culture is used as a template. The total volume of the PCR reaction system was 25. mu.L, including 2 × taq Master Mix:12.5 mu L; primer cfp-F: 1 mu L of the solution; primer cfp-R: 1 mu L of the solution; ddH 2O: 9.5uL and 1. mu.L of DNA template. To add 1. mu.L of sterile ddH2O as a negative control. Carrying out PCR amplification under the following reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 53 ℃ for 45s, and extension at 72 ℃ for 45s, after 32 cycles; extension at 72 ℃ for 10 min.
The detection result judging method comprises the following steps: and (3) carrying out electrophoresis on the amplification product by using 1% agarose gel under the electrophoresis condition of 110V for 20min, generating a specific band at a position of 817bp as a result for judgment, wherein the specific band indicates that the detection result is positive, the pathogenic cercospora apiacea of the cercospora leaf spot exists, and the absence of the specific band at the position of 817bp indicates that the detection result is negative and the pathogenic cercospora apiacea of the cercospora apiacea does not exist. The PCR detection method established by the invention can be applied to detection of the pathogen cercospora apiacea of the cercospora apiacea purpurea.
Example 2
PCR primer specificity amplification test of the cercospora apiacea.
11 strains (the test strains are shown in table 1) of target pathogen cercospora apiacea, non-target pathogen and common mould are collected, and the genome DNA of the 11 strains is used as the template DNA to be detected. The total volume of the PCR reaction system was 25. mu.L, including 2 × taq Master Mix: 12.5 mu L; cfp-F: 1 mu L of the solution; primer cfp-R: 1 mu L of the solution; ddH 2O: 9.5uL and 1. mu.L of DNA template. A negative control was prepared by adding 1. mu.L of sterile ddH 2O. Carrying out PCR amplification under the following reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 53 ℃ for 45s, and extension at 72 ℃ for 45s, after 32 cycles; extension at 72 ℃ for 10 min. The detection result judging method comprises the following steps: and (3) carrying out electrophoresis on the amplification product by using 1% agarose gel under the electrophoresis condition of 100V for 30min, generating a specific band at a position of 817bp as a result for judgment, wherein the specific band indicates that the detection result is positive, the pathogenic cercospora apiacea of the cercospora leaf spot is present, and the absence of the specific band at the position of 817bp indicates that the detection result is negative and the pathogenic cercospora apiacea of the cercospora apiacea is absent.
This example demonstrates that the PCR method of the present invention has good specificity, only the Cercospora apiacea strain is positive in amplification, and other non-target strains are negative. The PCR detection method established by the invention can distinguish target pathogen cercospora apiacea and non-target pathogen in cercospora apiacea detection.
TABLE 1 strains used in the tests and test results
+: the amplification reaction is positive; and (2) preparing: amplification reaction is negative
Target pathogen cercospora apiacea, non-target pathogen and common mould are separated, purified and stored (survive well) by plant protection research institute of agricultural academy of Tianjin, and the strains used in the experiment can be freely obtained by contacting the plant protection research institute of agricultural academy of Tianjin, and the addresses are as follows: the plant protection research institute of agricultural science institute of Tianjin city at 17 km of Jingjing highway of Xiqing of Tianjin city contacts telephone: 022-86433374. Or separating and purifying target pathogenic bacteria and common mold in the disease sample by conventional disease sample tissue separation culture method.
The specific method comprises the following steps:
the required materials are as follows: 75% alcohol, tweezers, scissors, sterile water, PDA culture medium, sterile inoculating needle and marking pen.
The method comprises the following operation steps:
1. cutting the interface of the affected part into pieces of 5mm × 5mm, and sterilizing the surface in 75% ethanol for 30-45 s.
2. The diseased tissue was picked up with sterile forceps, rinsed 3 times in sterile water and air dried.
3. Transferring the air-dried diseased tissues into a PDA culture medium for culture at the temperature of 25 ℃ under the condition of alternating 12h light/12 h dark.
4. After hyphae grow around the diseased tissue, picking out marginal hyphae by using a sterile inoculating needle, and transferring the marginal hyphae to a new PDA culture medium for purification.
5. The purified strains were numbered, colony growth was recorded and culture characteristics were photographed.
6. The purified strain was stored in a PDA slant tube at 4 ℃.
As can be seen from Table 1, the PCR method of the invention has good specificity, only the Cercospora apiacea strain is positive in amplification, and other non-target pathogenic bacteria and common pollution bacteria are negative.
Example 3
Sensitivity test of PCR primer of cercospora celebrati
In order to determine the sensitivity of the PCR detection method of the cercospora apiacea, the concentration of the extracted DNA of the pure culture of the cercospora apiacea is measured by a spectrophotometer, adjusted to be a DNA solution with the concentration of 1 mu g/mu L, and then sterilized ddH is used2O10-fold gradient dilution was performed to prepare standard DNA solutions at concentrations of 1. mu.g/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 0.1 ng/. mu.L, 0.01 ng/. mu.L and 0.001 ng/. mu.L, and 1. mu.L of each concentration of the standard solution was taken as a template DNA, and PCR reaction and observation of the results were performed in the same manner as in example 1. The result shows that the established PCR molecular detection method can detect the DNA of the cercospora apiacea with the concentration of 0.1 ng/. mu.L.
Example 4
PCR detection based on cercosporin promoting protein cfp gene and conventional pathogen identification comparison:
and (4) conclusion: by adopting the detection method based on the cercosporal apiacea essence promoting protein cfp gene, detection and diagnosis can be carried out in the stage and the initial stage of plant disease infection and in the absence of symptoms, and whether the plant is infected by the cercosporal apiacea can be judged. The whole method does not need separation and purification of pathogenic bacteria and microscopic morphology observation, can complete detection within 2 hours, and has detection sensitivity of 0.1 ng/mu L. In the conventional detection method for the pathogen of the cercospora apiacea, when the disease sample has obvious symptoms in the middle and later stages of plant diseases, the disease sample is subjected to surface disinfection, then the pathogen is separated and purified, and the morphological identification is completed by further microscopic observation, so that the conclusion whether the plant is infected by the cercospora apiacea can be obtained.
SEQUENCE LISTING
<110> Tianjin City academy of agricultural sciences
<120> primer group for rapidly detecting cercospora apiacea and detection method
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
tagaaatgac tggcatagtc 20
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
gtcatagcca ataagctagc 20
Claims (6)
1. A method for quickly detecting pathogenic cercospora apiacea (Cercospora apiacea, Cercospora leaf spot disease)Cercospora apii) The cercosporin promotes the specificity primer of the protein cfp gene, characterized in that the primer pair is a forward primer cfp-F and a reverse primer cfp-R, the nucleic acid sequence of the primer is cfp-F (5 'to 3'): TAGAAATGACTGGCATAGTC, respectively; cfp-R (5 'to 3'): GTCATAGCCAATAAGCTAGC are provided.
2. The kit for rapidly detecting the pathogen cercospora apiacea of cercospora leaf spot of celery as claimed in claim 1 (C. apii) The application of the cercosporin-promoting protein cfp gene specific primer in the aspect of quickly detecting the cercospora apiacea is provided.
3. The method for detecting the pathogen of the alternaria leaf spot of celery cercospora by using the specific primer in claim 1 is characterized by extracting the DNA of a sample to be detected, and then performing specific amplification on the DNA of the sample to be detected by using the detection primer group in claim 1 by using a polymerase chain reaction amplification method to confirm whether an amplification product exists.
4. The detection method as claimed in claim 3, wherein the sample to be detected is celery leaf tissue or a pure culture of fungus.
5. The detection method according to claim 3, wherein the DNA of the cercospora species is specifically amplified by the PCR amplification method, and the specific amplification system has a total volume of 25 μ L and comprises 2 × taq Master Mix: 12.5 mu L; primer cfp-F: 1 mu L of the solution; primer cfp-R: 1 mu L of the solution; ddH2O: 9.5. mu.L and 1. mu.L of DNA template; wherein the 2 × taq Master Mix comprises the following components: 20 mmol/L Tris-HCl, 0.1mmol/L EDTA, 50mmol/L KCl, 1.5mmol/L MgCl2200 mu mol/L dNTPs, and the balance being sterilized ddH2O。
6. A rapid detection method for pathogenic cercospora apiacea of cercospora leaf spot of celery is characterized in that the specific primer of claim 1 is utilized; the amplification reaction system is formulated according to claim 5; carrying out PCR amplification on the prepared reaction solution under the following reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 53 ℃ for 45s, and extension at 72 ℃ for 45s, after 32 cycles; extending for 10min at 72 ℃; after the amplification reaction is finished, carrying out 1% agarose gel electrophoresis under the electrophoresis condition of 100-110V for 20-30 min; specific bands are generated at the position of 817bp and are used as result judgment, the generation of the specific bands indicates that the detection result is positive, and the pathogenic cercospora apiacea (A) of cercospora apiacea purpurea existsC. apii) The absence of specific bands at the position of 817bp indicates that the detection result is negative and the pathogenic cercospora apiacea of the cercospora leaf spot of celery is absent ((C. apii）。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101821409A (en) * | 2007-08-29 | 2010-09-01 | 孟山都技术公司 | Methods and compositions for breeding for preferred traits |
| CN108913806A (en) * | 2018-08-13 | 2018-11-30 | 天津市植物保护研究所 | A kind of plant botrytis molecular detecting method based on grey mold toxin sesquiterpene synthase BcBOT2 gene |
| CN113789406A (en) * | 2021-09-26 | 2021-12-14 | 海南大学 | Visualized mung bean leaf spot disease bacteria loop-mediated isothermal amplification detection method |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101821409A (en) * | 2007-08-29 | 2010-09-01 | 孟山都技术公司 | Methods and compositions for breeding for preferred traits |
| US20180044697A1 (en) * | 2007-08-29 | 2018-02-15 | Monsanto Technology Llc | Methods and Compositions for Goss' Wilt Resistance in Corn |
| CN108913806A (en) * | 2018-08-13 | 2018-11-30 | 天津市植物保护研究所 | A kind of plant botrytis molecular detecting method based on grey mold toxin sesquiterpene synthase BcBOT2 gene |
| CN113789406A (en) * | 2021-09-26 | 2021-12-14 | 海南大学 | Visualized mung bean leaf spot disease bacteria loop-mediated isothermal amplification detection method |
Non-Patent Citations (2)
| Title |
|---|
| MARÍA GABRIELA LATORRE RAPELA等: "Early Detection of Cercospora Species in Soybean Plants: Immunologic and Molecular Methods", 《AMERICAN JOURNAL OF PLANT SCIENCES》 * |
| 李晓颖;宋莉;田宇;谢华;: "芹菜腐烂病原菌QC02的鉴定及其生物学特性", 农业生物技术学报 * |
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