CN113025744A - Nested PCR (polymerase chain reaction) specific primer for loquat colletotrichum gloeosporioides as well as detection method and application thereof - Google Patents
Nested PCR (polymerase chain reaction) specific primer for loquat colletotrichum gloeosporioides as well as detection method and application thereof Download PDFInfo
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Abstract
The invention provides nested PCR (polymerase chain reaction) specific primers of loquat anthrax, which are respectively used as inner primer pairs of colletotrichum gloeosporioides and colletotrichum gloeosporioides, wherein a genomic DNA (deoxyribonucleic acid) of a sample to be detected is used as a template to perform two-round PCR amplification, an amplification product is subjected to electrophoresis detection, and whether the sample to be detected carries colletotrichum gloeosporioides or colletotrichum gloeosporioides is judged through a target fragment. The method disclosed by the invention has the advantages of high detection sensitivity, strong specificity and good practicability on genomic DNA of colletotrichum gloeosporioides and colletotrichum oxysporum, can be used for quickly, accurately and sensitively detecting trace colletotrichum gloeosporioides and colletotrichum oxysporum in loquat leaves, and has important significance on early diagnosis and accurate prevention and control of loquat anthracnose.
Description
Technical Field
The invention belongs to the field of molecular biology, relates to detection of loquat anthracnose pathogen, and particularly relates to a nest type PCR (polymerase chain reaction) specific primer of loquat anthracnose pathogen and a detection method and application thereof.
Background
Loquat (Eriobotrya japonica Lindl) is a subtropical evergreen fruit tree originated from China, is deeply favored by consumers due to unique flavor, rich nutrition and medicinal value, but has prominent disease problems along with the increase of planting area, wherein loquat anthracnose is the disease with highest incidence and widest incidence range, and the industrial development is seriously restricted. The disease mainly damages loquat leaves and fruits to cause leaf spot and fruit soft rot symptoms, has long damage time, can be attacked from growth and development to postharvest storage period, and brings huge economic loss for fruit growers and dealers.
Loquat anthracnose is mainly caused by infection of the fungi Colletotrichum gloeosporioides and Colletotrichum oxysporum, conidia of which generally invade from wounds, stomata and the like and sometimes can directly penetrate through epidermis. The conidium is transmitted by medium such as wind, rain, insects and the like, the suitable temperature for the spore germination is 20-30 ℃, wherein the germination rate is the highest at 25 ℃; the relative humidity is more than 90%, and the germination rate in saturated humidity and water drops is the highest, and is 72.35%. The spore can generate a germ tube after germination, the top end of the germ tube expands after contacting with the host surface to form an attachment cell attached to the host surface, and hyphae generated from the lower part of the attachment cell infect the host cell through a cell gap.
The content of chlorogenic acid in the immature loquat is high, and meanwhile, some bacteriostatic substances contained in the immature loquat can passivate hydrolase secreted by pathogenic bacteria, so that attached cells of the pathogenic bacteria cannot generate invasion hyphae immediately after invading the loquat through skin holes, but are buried in the loquat epidermis or cutin layer and can be dormant for months, which shows that the anthracnose pathogen has the characteristic of latent infection, and if disease symptoms are observed in the field and then prevented and controlled, the situation is too late. Therefore, it is necessary to establish a rapid and accurate molecular detection technique for colletotrichum gloeosporioides.
Disclosure of Invention
In order to solve the problems in the prior art, according to the first aspect of the invention, the invention provides nested PCR specific primers for colletotrichum eriobotrys, which are respectively used as inner primer pairs for colletotrichum gloeosporioides and colletotrichum oxysporum, wherein the amplified products are subjected to electrophoresis detection after two rounds of PCR amplification by taking the genomic DNA of a sample to be detected as a template, and whether the sample to be detected carries colletotrichum gloeosporioides or colletotrichum oxysporum is judged through a target fragment.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a nest PCR specific primer of loquat anthrax is characterized in that: the said colletotrichum Eriobotryae is colletotrichum Eriobotryae (C.gloeosporioides) or colletotrichum Eriobotryae (C.acutatum);
an upstream primer Cg1F: 5'-GAAGGAAGGGTCTCCGCGAC-3' of an internal side specific primer of the ITS zone of the colletotrichum gloeosporioides (C.gloeosporioides);
the downstream primer Cg1R: 5'-TCCTAGTGCGAGACGTAA-3' of the internal side specific primer of the ITS zone of the colletotrichum gloeosporium (C.gloeosporioides).
An upstream primer Ca1F: 5'-ACCCTTTGTGACATACCTAACC-3' of an ITS zone inside specific primer of the loquat leaf tip anthracnose (C.acutatum);
the downstream primer Ca1R: 5'-GAGCCGAGTTCAACCTGTAA-3' of the internal side specific primer of the ITS zone of the loquat-shaped spore anthrax (C.acutatum).
In one embodiment of the present invention, during the detection process of nested PCR,
the upstream primer of the universal primer outside the ITS zone of the loquat anthrax:
ITS1：5'-TCCGTAGGTGAACCTGCGG-3'；
the downstream primer of the universal primer outside the ITS zone of the loquat anthrax is as follows:
ITS4：5'-TCCTCCGCTTATTGATATGC-3'。
according to the second aspect of the invention, the invention provides a nested PCR detection method for colletotrichum eriobotryae, which has high detection sensitivity, strong specificity and good practicability on genomic DNA of colletotrichum eriobotryae and colletotrichum eriobotryae.
A nest type PCR high-efficiency detection method for loquat colletotrichum gloeosporioides (colletotrichum gloeosporioides and colletotrichum oxysporum), which is characterized in that: firstly extracting the genome DNA of a sample to be detected, taking the obtained genome DNA as a template, carrying out first round PCR amplification by using the universal primer as a first round reaction primer, then carrying out second round PCR amplification by using the first round PCR reaction product as a template for second round PCR amplification and the Cg1F/R or Ca1F/R as a second round PCR amplification primer of the specific primer pair, finally carrying out agarose gel electrophoresis detection on the amplification product, and judging whether the sample to be detected carries colletotrichum gloeosporioides or colletotrichum oxysporum gloeosporum through a target fragment.
In the method, the first round of PCR amplification procedure is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 40s, annealing at 56 ℃ for 40s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
In the method, the second round of PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C.
In the method, if a 392bp specific band is amplified by using a primer pair Cg1F/R, the detected sample is determined to be colletotrichum gloeosporioides or loquat tissue infected by colletotrichum gloeosporioides; if a specific band of 490bp is amplified by utilizing a primer pair Ca1F/R, the tested sample is determined to be the colletotrichum oxysporum or the loquat tissue infected by the colletotrichum oxysporum.
Specifically, the nested PCR detection method for loquat anthrax comprises the following steps:
firstly, extracting the genome DNA of a sample to be detected
Extracting genome DNA of loquat anthrax (colletotrichum gloeosporioides and colletotrichum oxysporum) strains and other test strains by adopting a fungus-improved CTAB method; by using
The soil microorganism total DNA extraction kit extracts genome DNA of healthy leaves, leaf samples with typical anthracnose disease in fields, and loquat leaf samples with symptoms or non-symptoms after artificial inoculation of anthrax bacteria.
② first round PCR amplification
Using the genomic DNA obtained in the first step as a template and using the universal primers ITS1/ITS4 as a first roundThe reaction system of the reaction primer is as follows: 2 XPCR Mastermix (dye-containing) (Shanghai Biotech) 20. mu.L, primers ITS1 and ITS4 each 2. mu.L, DNA template (50ng/mL) 1. mu.L, plus dd H2O to 40 mu L, and the amplification program is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 40s, annealing at 56 ℃ for 40s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
Third round of PCR amplification
Taking 1 mu L of the first round PCR reaction product diluted by 1000 times as a template for the second round PCR amplification, taking a specific primer pair Cg1F/R or Ca1F/R as a second round PCR amplification primer, wherein the reaction system is 20 mu L of 2 XPCR Mastermix (containing dye) (Shanghai work), 1 mu L of each primer, 1 mu L of the template and ddH2O to 40. mu.L. The amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C.
Detection of PCR amplification product
10 μ L of the amplification product was subjected to agarose gel electrophoresis, detected on a gel imaging system and photographed. If a 392bp specific band is amplified by using the primer pair Cg1F/R, determining that the sample is colletotrichum gloeosporioides or loquat tissue infected by colletotrichum gloeosporioides; if a specific band of 490bp is amplified by utilizing a primer pair Ca1F/R, the tested sample is determined to be the colletotrichum oxysporum or the loquat tissue infected by the colletotrichum oxysporum.
According to a third aspect of the invention, the invention provides an application of the nested PCR specific primer for loquat anthrax in loquat anthrax detection, wherein the loquat anthrax is C.gloeosporioides or C.acutatum.
Has the advantages that:
the invention provides nested PCR (polymerase chain reaction) specific primers Cg1F/R and Ca1F/R of loquat anthrax (colletotrichum gloeosporioides and colletotrichum oxysporum), which are respectively used as inner primer pairs of colletotrichum gloeosporioides and colletotrichum oxysporum, wherein after two-round PCR amplification is carried out by taking the genome DNA of a sample to be detected as a template, the amplification product is subjected to electrophoresis detection, and whether the sample to be detected carries colletotrichum gloeosporioides or colletotrichum oxysporum is judged through a target fragment. The method disclosed by the invention has the advantages of high detection sensitivity, strong specificity and good practicability on the genomic DNA of colletotrichum gloeosporioides and colletotrichum gloeosporioides, can be used for quickly, accurately and sensitively detecting trace colletotrichum gloeosporioides and colletotrichum gloeosporioides in loquat leaves, and has important significance on early diagnosis and accurate prevention and control of colletotrichum gloeosporioides.
The method has the advantages of high sensitivity, strong specificity and good practicability, and is embodied as follows:
(1) the sensitivity is high: sensitivity detection is carried out by diluting DNA standard products (100 ng/mu L, 10 ng/mu L, 1 ng/mu L, 100 pg/mu L, 10 pg/mu L, 1 pg/mu L and 100 fg/mu L) in a double proportion, the lowest concentration of colletotrichum gloeosporioides can be detected to be 1 pg/mu L, and the detection is improved by 10 pg/mu L compared with the conventional PCR3Doubling; the minimum concentration of the bacillus anthracis which aims at the bacillus anthracis can be detected to be 10 ag/mu L, which is 10 g/mu L higher than that of the conventional PCR6Doubling;
(2) the specificity is strong: gel electrophoresis results of the primer pairs Cg1F/R or Ca1F/R show that no amplification band is seen in 21 common pathogenic bacteria (including 6 anthrax fungi) on loquat and 5 common pathogenic fungi on other hosts, and only the anthrax gloeosporioides or the anthrax oxysporum generate amplification bands.
(3) The practicability is good: the invention can be used for disease detection in the latent period or the early disease stage of loquat anthracnose, can also be used for identifying different varieties of loquat anthracnose, and provides technical support for early diagnosis, timely prevention and accurate medication of loquat anthracnose.
Drawings
FIG. 1 shows nested PCR amplification results of C.gloeosporioides specific primer pair Cg1F/R of colletotrichum gloeosporioides, shown in lane M: DL1000 DNAmarker; lanes 1-28: test strains numbered correspondingly in table 1; 29: and (5) negative control.
FIG. 2 is a graph showing the sensitivity detection of C.gloeosporioides specific primer pair Cg1F/R in conventional PCR and nested PCR, wherein M: DL1000 DNAmarker; lanes 1-7: the genomic DNA concentrations of colletotrichum gloeosporioides are respectively 100 ng/muL, 10 ng/muL, 1 ng/muL, 100 pg/muL, 10 pg/muL, 1 pg/muL and 100 fg/muL.
FIG. 3 shows the amplification results of the DNA Cg1F/R of loquat tissue carrying C.gloeosporioides under different conditions, wherein M: DL1000 DNAmarker; lanes 1-9 are healthy loquat leaf tissue DNA; lanes 10-12 are typical anthrax-like loquat leaf DNA in the field; lanes 13-16 are C.gloeosporioides inoculated artificially but no significant loquat leaf DNA; lanes 17-18 are C.gloeosporioides inoculated artificially and show loquat leaf DNA; lane 19 is a negative control.
FIG. 4 shows the result of C.eutataum specific primer Ca1F/R nested-PCR amplification of C.eutataum of loquat-shaped spore bacillus, wherein the lane M: DL1000 DNA marker; lanes 1-28: test strains numbered correspondingly in table 1; 29: and (5) negative control.
FIG. 5 is a comparison graph of sensitivity detection of conventional PCR and nested PCR of C.aculeatus colletotrichum specific primer pair Ca1F/R, wherein M: DL1000 DNA marker; lanes 1-12: the genomic DNA concentrations of colletotrichum gloeosporioides are respectively 100 ng/muL, 10 ng/muL, 1 ng/muL, 100 pg/muL, 10 pg/muL, 1 pg/muL, 100 fg/muL, 10 fg/muL, 1 fg/muL, 100 ag/muL, 10 ag/muL and 1 ag/muL
FIG. 6 shows the results of amplification of tissue DNA Ca1F/R from loquat carrying C.acutatum of B.oxysporum under different conditions, wherein M: DL1000 DNA marker; lanes 1-9 are healthy loquat leaf tissue DNA; lanes 10-12 are typical anthrax-like loquat leaf DNA in the field; lanes 13-16 are DNA from loquat leaves that were artificially inoculated with C.acutum but not significantly diseased; lanes 17-18 are DNA from artificial inoculated c.acutatum and overt loquat leaf; lane 19 is a negative control.
Detailed Description
The present invention is described in detail below with reference to specific examples, which are given for the purpose of further illustrating the invention and are not to be construed as limiting the scope of the invention, and the invention may be modified and adapted by those skilled in the art in light of the above disclosure. The raw materials and reagents used in the invention are all commercial products.
Example 1: primer specificity verification for loquat anthrax PCR detection
The specificity of primer pairs Cg1F/R and Ca1F/R for detecting colletotrichum gloeosporioides and colletotrichum oxysporum gloeosporum was verified by selecting common anthrax strains on loquat and common fungi on other species (Table 1).
TABLE 1 test loquat anthrax and other pathogenic bacterial strains of fungal diseases
The method comprises the following specific steps:
(1) extraction of genomic DNA of anthrax and other test strains (fungus-modified CTAB method)
Transferring purified loquat anthrax to a PDA culture medium, scraping a proper amount of hyphae after bacterial colonies grow over a whole flat plate, putting the hyphae into a 1.5mL sterilizing centrifuge tube, and grinding the hyphae into powder by using a Coyote G200 high-throughput tissue grinder;
adding 500 mu L of DNA extracting solution [ Tris-HCl 0.2mol/L (pH 7.5), NaCl 0.5mol/L, EDTA 0.01mol/L and SDS 10g/L ] into the ground hypha, and standing on ice for 10 min;
③ adding 500. mu.L chloroform/isoamyl alcohol (24:1), fully vortexing, centrifuging at 12000rpm for 10min, and taking the supernatant (about 400. mu.L);
adding 400 μ L phenol (pH8.0), chloroform and isoamyl alcohol (25:24:1), and vortexing for 2 min;
adding 200 mu L of alkaline phenol, vortexing for 2min, adding 200 mu L of chloroform, and uniformly mixing (vortexing);
sixthly, centrifuging at 13000rpm for 10min, and taking supernatant (about 300 mu L);
adding 300 mu L chloroform/isoamyl alcohol (24:1), fully whirling, and centrifuging at 13000rpm for 10 min;
eighthly, taking the supernatant (about 200 mu L) into a 1.5mL centrifuge tube, adding 500 mu L of precooled absolute ethyl alcohol, and standing at-80 ℃ for precipitation for more than 30 min;
ninthly, centrifuging at 6000rpm for 1min, discarding supernatant, and adding 300 mu L of 75% ethanol for washing;
performing centrifugation at 6000rpm for 1min to remove supernatant;
(2) A first round of PCR products are obtained by taking strain genome DNA as a template and a fungus universal primer pair ITS1/ITS4(ITS1:5'-TCCGTAGGTGAACCTGCGG-3'/ITS4:5'-TCCTCCGCTTATTGATATGC-3') as an outer primer. The reaction system is as follows: 2 XPCR Mastermix (dye-containing) (Shanghai Biotech) 20. mu.L, primers ITS1 and ITS4 each 2. mu.L, DNA template (50ng/mL) 1. mu.L, plus dd H2O to 40 mu L, and the amplification program is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 40s, annealing at 56 ℃ for 40s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
Taking 1 mu L of the first round PCR reaction product diluted by 1000 times as a template for the second round PCR amplification, and taking a specific primer pair Cg1F/R (Cg1F: 5'-GAAGGAAGGGTCTCCGCGAC-3'/Cg 1R: 5'-TCCTAGTGCGAGACGTAA-3') or Ca1F/R (Ca1F: 5'-ACCCTTTGTGAC ATACCTAACC-3'/Ca 1R: 5'-GAGCCGAGTTCAACCTGTAA-3') as an inner primer to obtain a second round amplification product. The reaction system was 2 XPCR Mastermix (with dye) (Shanghai Prov.) 20. mu.L, primers 1. mu.L each, template 1. mu.L, plus ddH2O to 40. mu.L. The amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C.
(3) Agarose gel electrophoresis and imaging observation
10 μ L of the amplified product was electrophoretically detected on 1.2% agarose gel (0.5 XTBE running buffer, 120V voltage), and the image was taken under BIO-RAD gel imaging system to see if there was a bright band and the image was saved. The result shows that only one 392bp specific band appears in colletotrichum gloeosporioides in Cg1F/R (figure 1), and only one 490bp specific band is amplified in colletotrichum gloeosporioides in Ca1F/R (figure 4), which indicates that the primer pair Cg1F/R, Ca1F/R has strong specificity to colletotrichum gloeosporioides and colletotrichum gloeosporioides respectively, and can be used for identification and detection of the two kinds of colletotrichum gloeosporioides.
Example 2: sensitivity detection of conventional PCR (polymerase chain reaction) and nested PCR (polymerase chain reaction) of loquat anthrax
(1) Preparation of DNA Standard
The colletotrichum gloeosporioides genome DNA is diluted from 100 ng/mu L to 1 pg/mu L with 10-fold concentration as an order of magnitude, namely 100 ng/mu L, 10 ng/mu L, 1 ng/mu L, 100 pg/mu L, 10 pg/mu L and 1 pg/mu L; the genomic DNA of anthrax conidioides was diluted from 100 ng/. mu.L to 10 ag/. mu.L at a 10-fold concentration of one order of magnitude, i.e., 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 100 pg/. mu.L, 10 pg/. mu.L, 1 pg/. mu.L, 100 fg/. mu.L, 10 fg/. mu.L, 1 fg/. mu.L, 100 ag/. mu.L, 10 ag/. mu.L.
(2) Routine PCR sensitivity detection
Taking 1 mu L of diluted standard as a template for conventional PCR sensitivity detection, and respectively carrying out conventional PCR reaction on colletotrichum gloeosporioides and colletotrichum oxysporum gloeosporum by taking Cg1F/R and Ca1F/R as primers. The reaction system is as follows: 2 XPCRMastermix (containing dye) (Shanghai Prov.) 20. mu.L, 1. mu.L of each primer, 1. mu.L of template, and dd H2O to 40. mu.L. The amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C.
(3) Nested PCR sensitivity detection
Taking 1 mu L of diluted standard as a first round PCR template, and taking a fungus universal primer pair ITS1/ITS4(ITS1:5'-TCCGTAGGTGAACCTGCGG-3'/ITS4:5'-TCCTCCGCTTATTGATATGC-3') as an outer primer to obtain a first round PCR product. The reaction system is as follows: 2 XPCR Mastermix (dye-containing) (Shanghai Biotech) 20. mu.L, primers ITS1 and ITS4 each 2. mu.L, DNA template (50ng/mL) 1. mu.L, plus dd H2O to 40 mu L, and the amplification program is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 40s, annealing at 56 ℃ for 40s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
Taking 1 mu L of the first round PCR reaction product diluted by 1000 times as a template for the second round PCR amplification, and taking a specific primer pair Cg1F/R (Cg1F: 5'-GAAGGAAGGGTCTCCGCGAC-3'/Cg 1R: 5'-TCCTAGTGCGAGACGTAA-3') or Ca1F/R (Ca1F: 5'-ACCCTTTGTGACATACCTAACC-3'/Ca 1R: 5'-GAGCCGAGTTCAACCTGTAA-3') as an inner primer to obtain a second round amplification product. The reaction system was 2 XPCR Mastermix (with dye) (Shanghai Prov.) 20. mu.L, primers 1. mu.L each, template 1. mu.L, plus ddH2O to 40. mu.L. The amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C.
(4) Agarose gel electrophoresis and imaging observation
10 μ L of the amplified product was electrophoretically detected on 1.2% agarose gel (0.5 XTBE running buffer, 120V voltage), and the image was taken under BIO-RAD gel imaging system to see if there was a bright band and the image was saved. The results show that the conventional PCR can detect that the minimum concentration of the genomic DNA of the colletotrichum gloeosporioides is 1 ng/mu L, the nested PCR can amplify a specific band of about 392bp from a template with the concentration of more than 1 pg/mu L (figure 2), and the sensitivity of the colletotrichum gloeosporioides to the nested PCR is improved by 10 compared with the conventional PCR3Doubling; the conventional PCR can detect that the minimum concentration of the genome DNA of the colletotrichum eriobotryae is 10 pg/mu L, the nested PCR can amplify a specific band with about 490bp from a template with the concentration of more than 10 ag/mu L (figure 5), and the sensitivity of the colletotrichum eriobotryae to the nested PCR is improved by 10 compared with the conventional PCR6And (4) doubling.
Example 3: loquat leaf detection method for carrying loquat anthrax under different conditions
(1) Preparation of sample to be tested
Taking colletotrichum gloeosporioides and colletotrichum oxysporum as research objects, samples to be detected comprise four types: 9 healthy loquat leaf samples, 3 field loquat leaf samples with typical anthracnose, 4 loquat leaf samples which were not shown after inoculation of loquat anthrax by man, and 2 loquat leaf samples which were shown after inoculation of loquat anthrax by man. The operation of artificially inoculating loquat anthrax is as follows: culturing colletotrichum gloeosporioides and colletotrichum oxysporum on potato glucose agar medium for 6 days, washing with sterile water to obtain spore with concentration of 1 × 106And (3) uniformly spraying 3mL of spore suspension liquid on healthy and sterile loquat leaves respectively, placing the healthy and sterile loquat leaves in a white porcelain dish paved with wet filter paper, covering a preservative film for preserving moisture, taking the undeveloped loquat leaves after 3 days, and taking the undeveloped loquat leaves after 5 days. All the above leaf samples are according to
In the soil microorganism total DNA extraction kitThe method for extracting the genome DNA is slightly improved, and comprises the following steps:
accurately weighing 0.5G of loquat diseased leaf sample, grinding hyphae into powder by using a Coyote G200 high-throughput tissue grinder, adding 1mL of SLX-Mlus buffer solution, and fully swirling on a swirling machine for 3-5 min to dissolve cells;
② adding 100 mu LDS buffer solution, whirling, fully mixing, putting the centrifuge tube in a 70 ℃ water bath kettle to incubate for 10min, and reversing the centrifuge tube for several times in the water bath process to fully mix the sample. Centrifuging at room temperature at 3000g for 3 min;
③ take 800. mu.L of supernatant, transfer to a new 2mL centrifuge tube, add 270. mu. L P2 buffer solution, vortex, mix well. Incubating on ice for 5 min;
13000g was centrifuged for 5min at 4 ℃ and the supernatant was then carefully transferred to a new 2mL centrifuge tube to which was added 0.7 times the volume of isopropanol of the supernatant from the previous step. The tube was inverted for 30min to mix well. 13000g is centrifuged for 10min at 4 ℃;
fifthly, carefully sucking out the supernatant by using a micropipette, and paying attention to avoid damaging DNA. Inverting the centrifuge tube on clean absorbent paper, and standing for 1min to discharge liquid in the tube;
sixthly, adding 200 mu L of lysate by using a micropipette, and vortexing for 10 sec. Then, carrying out water bath in a water bath kettle at 70 ℃ for 20min to fully dissolve the DNA;
adding 100 mu LHTR reagent, fully whirling, and standing at room temperature for 2 min. Then transferring the supernatant into a new 2mL centrifuge tube;
adding XP1 buffer solution with the same amount as the supernatant into the mixture, fully mixing the mixture, and transferring the mixture into a container
Centrifuging the DNA filter column at room temperature at 10000g for 1 min;
ninthly, after the filtrate is poured out, repeating the previous step;
⑩
the DNA filter column was inserted into a new 2mL collection tube and 700. mu.L was addedSPW washing buffer, at room temperature, 10000g centrifugation for 1 min;
(2) Nested PCR amplification was performed according to step (2) in example 1;
(3) agarose gel electrophoresis and imaging observations were performed according to step (3) of example 1. The results show that the loquat leaf-like, artificially inoculated colletotrichum gloeosporioides (figure 3) or colletotrichum oxysporum (figure 6) with typical anthracnose in the field amplified a 392bp or 490bp large band, while the healthy leaves and the negative control (ddH2O) failed to amplify any band (figures 3 and 6). The primer can be used for detecting the loquat leaf samples carrying trace colletotrichum gloeosporioides or colletotrichum oxysporum.
Claims (8)
1. A nest PCR specific primer of loquat anthrax is characterized in that: the said colletotrichum Eriobotryae is colletotrichum Eriobotryae (C.gloeosporioides) or colletotrichum Eriobotryae (C.acutatum);
an upstream primer Cg1F: 5'-GAAGGAAGGGTCTCCGCGAC-3' of an internal side specific primer of the ITS zone of the colletotrichum gloeosporioides (C.gloeosporioides);
a downstream primer Cg1R: 5'-TCCTAGTGCGAGACGTAA-3' of an internal side specific primer of the ITS zone of the colletotrichum gloeosporioides (C.gloeosporioides);
an upstream primer Ca1F: 5'-ACCCTTTGTGACATACCTAACC-3' of an ITS zone inside specific primer of the loquat leaf tip anthracnose (C.acutatum);
the downstream primer Ca1R: 5'-GAGCCGAGTTCAACCTGTAA-3' of the internal side specific primer of the ITS zone of the loquat-shaped spore anthrax (C.acutatum).
2. The nested PCR specific primer for loquat anthrax of claim 1, which is characterized in that: in the detection process of nested PCR, the upstream primer of the universal primer outside the ITS zone of the loquat anthrax: ITS1:5'-TCCGTAGGTGAACCTGCGG-3', respectively; the downstream primer of the universal primer outside the ITS zone of the loquat anthrax is as follows: ITS4:5'-TCCTCCGCTTATTGATATGC-3' are provided.
3. A nest PCR high-efficiency detection method for loquat anthrax is characterized in that: firstly extracting the genomic DNA of a sample to be detected, taking the obtained genomic DNA as a template, carrying out first round PCR amplification by using the universal primer as a first round reaction primer according to claim 2, then carrying out second round PCR amplification by using the first round PCR reaction product as a template for second round PCR amplification and the specific primer as a second round PCR amplification primer Cg1F/R or Ca1F/R according to claim 1, finally carrying out agarose gel electrophoresis detection on the amplification product, and judging whether the sample to be detected carries colletotrichum gloeosporioides or colletotrichum oxysporum through a target fragment.
4. The method of claim 3, wherein: the first round of PCR amplification procedure is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 40s, annealing at 56 ℃ for 40s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
5. The method of claim 3 or 4, wherein: the second round of PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C.
6. The method of claim 3 or 4, wherein: if a 392bp specific band is amplified by using the primer pair Cg1F/R, determining that the sample is colletotrichum gloeosporioides or loquat tissue infected by colletotrichum gloeosporioides; if a specific band of 490bp is amplified by utilizing a primer pair Ca1F/R, the tested sample is determined to be the colletotrichum oxysporum or the loquat tissue infected by the colletotrichum oxysporum.
7. The method of claim 3, comprising the steps of:
firstly, extracting the genome DNA of a sample to be detected
Extracting genome DNA of loquat anthrax bacterial strains and other test strains; by using
Extracting genome DNA of healthy leaves, leaf samples with typical anthracnose disease in fields, and symptomatic or undisplayed loquat leaf samples after artificial inoculation of anthrax bacteria by using the soil microorganism total DNA extraction kit;
② first round PCR amplification
Taking the genomic DNA obtained in the first step as a template, and taking universal primers ITS1/ITS4 as first round reaction primers, wherein the reaction system is as follows: 2 XPCR Mastermix 20. mu.L, primers ITS1 andITS4 2. mu.L each, 50ng/mL DNA template 1. mu.L, plus dd H2O to 40 mu L, and the amplification program is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 40s, annealing at 56 ℃ for 40s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃;
third round of PCR amplification
Taking 1 mu L of the first round PCR reaction product diluted by 1000 times as a template for the second round PCR amplification, taking a specific primer pair Cg1F/R or Ca1F/R as a second round PCR amplification primer, wherein the reaction system is 2 x PCRMastermix 20 mu L, 1 mu L of each primer, 1 mu L of the template and ddH2O to 40 μ L; the amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 7min at 72 ℃, and storing at 4 ℃;
detection of PCR amplification product
Taking 10 mu L of the amplification product to carry out agarose gel electrophoresis, detecting on a gel imaging system and taking a picture; if a 392bp specific band is amplified by using the primer pair Cg1F/R, determining that the sample is colletotrichum gloeosporioides or loquat tissue infected by colletotrichum gloeosporioides; if a specific band of 490bp is amplified by utilizing a primer pair Ca1F/R, the tested sample is determined to be the colletotrichum oxysporum or the loquat tissue infected by the colletotrichum oxysporum.
8. Use of nested PCR specific primers for loquat anthrax according to claim 1 or 2 in the detection of loquat anthrax, wherein the loquat anthrax is C.gloeosporioides or C.acutatum.
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