CN114703310A - Monilinia fructicola LAMP (loop-mediated isothermal amplification) detection primers and application thereof - Google Patents
Monilinia fructicola LAMP (loop-mediated isothermal amplification) detection primers and application thereof Download PDFInfo
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Abstract
The invention discloses a Monilinia fructicola LAMP detection primer and application thereof, wherein an ITS gene of Monilinia fructicola is used as a detection target, and a set of primer group for specific detection is designed and screened, and the primer group consists of 4 specific primers F3, B3, FIP and BIP. The invention also provides an LAMP detection method suitable for the primer group, which comprises the following steps: 1) extracting DNA of a sample to be detected; 2) performing loop-mediated isothermal amplification on the DNA of a sample to be detected by using the primer composition provided by the invention; 3) the result detection shows that the reaction product is blue-green under natural illumination, and is judged to be positive when the reaction product is in a white turbid precipitate state under the irradiation of ultraviolet light with the wavelength of 365 nm. The invention overcomes the defects of long detection period, low sensitivity, low specificity, complex operation and high requirements on instruments and equipment in the prior art, and can detect the Monilinia fructicola quickly, conveniently, efficiently, with high specificity and high sensitivity and at low cost.
Description
Technical Field
The invention belongs to the technical field of crop disease detection, identification and prevention, and particularly relates to an LAMP (loop-mediated isothermal amplification) detection primer for Monilinia fructicola and application thereof.
Background
From the genus Streptomyces fungus (Moniliniaspp.) is one of the important fungal diseases which seriously affect the yield and quality of peach trees, and the diseases are widely distributed in the world, are particularly serious in moist and humid areas, mainly damage mature fruits and cause a great amount of rotten fruits. It has been reported that there are 3 main pathogenic fungi causing brown rot of peach, i.e., Monilinia fructicola (Australia type) (A. meyeri)Monilinia fructicola) Brown rot of stone fruit (A), (B), (C)Monilinia laxa) And Monilinia fructicola (Kernel fruit)Monilinia fructigena). Although all 3 pathogens can infect on peach to cause brown rot, there is a certain difference in the distribution and host preference of 3 pathogens, of which brown rot fungus of kernel fruit type (Monilinia fructicola (A) (A))Monilinia fructicola) The wide distribution range in China is the dominant flora of Monilinia fructicola, and the pathogenic bacteria can infect all Prunus (A), (B) and (C)Prunus) In cultivated crops, besides peach fruits, stone fruit trees such as plums, apricots and cherries and kernel fruit trees such as apples and pears are also damaged, flowers, stems and fruits of fruit trees are infected, flowers, fruits are rotted, branches are withered, and in severe disease areas, the loss of the picked fruits is even up to 90%, so that serious economic loss is caused. Therefore, the method has important significance for strengthening the prevention and treatment of the brown rot of the peach, saving the economic loss, improving the income of fruit growers, promoting the healthy development of the peach industry and the like.
The accurate identification and early detection of pathogenic bacteria are the basis of effective prevention and control of diseases, the traditional identification and detection method of the pathogenic bacteria separates the pathogenic bacteria from pathogenic tissues, the morphological characteristics are adopted to carry out preliminary identification on the pathogenic bacteria, and then the pathogenic bacteria are finally determined by the Koehz's rule. The traditional identification method is time-consuming and labor-consuming, the experience of identification personnel can influence the accuracy of results, and obviously, the requirements of large-scale and accurate identification of diseases cannot be met. With the continuous development of scientific technology, the PCR series technology is widely applied to the identification, diagnosis and rapid detection of plant diseases by virtue of the advantages of rapidness, accuracy, strong specificity and the like, but the method also has some defects, such as the need of depending on precise and expensive instruments and equipment such as a PCR amplification instrument, a gel imaging system and the like, more complicated operation steps, the need of certain molecular biology technology for operators and the like, and the defects cause the use and popularization of the technology in economically undeveloped areas and basic agricultural departments to be greatly limited.
The latest technology currently applied to rapid and accurate detection of plant pathogenic microorganisms is Loop-mediated Isothermal Amplification (LAMP). The technology has strong specificity, because the designed detection primers are directed at 6 specific regions of the target gene, in addition, the LAMP method has low requirement on equipment, only a simple water bath or other heating equipment is needed, meanwhile, the method has short reaction time and high efficiency, the whole reaction is completed within 1 hour, and the judgment result can be observed by naked eyes. Until now, no report related to LAMP technology for Monilinia fructicola detection is available. The invention aims to design LAMP detection primers of Monilinia fructicola, and establish an LAMP detection system of Monilinia fructicola on the basis of the designed primers.
Disclosure of Invention
The invention provides a LAMP detection primer for brown rot of peach and application thereof, aiming at solving the problems that the detection and identification of brown rot of peach in the prior art are mainly based on morphological characteristics, the method is long in time consumption, complicated in procedure, strong in experience, low in accuracy and difficult to timely monitor the occurrence of diseases and control the propagation and prevalence of pathogenic bacteria, the existing PCR molecular detection needs expensive instruments such as an amplification instrument and the like, the detection time is long and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
1. designing LAMP detection specific primers for Monilinia fructicola:
by assaying Monilinia fructicola (Macau de.) Hance)Monilinia fructicola) (iii) Internal Transcribed Spacer (ITS) sequences from other pathogenic bacteria, and the ITS sequences were analyzed by alignment using on-line LAMP Primer design software Primer software Explorer V5 (https://primerexplorer.jp/lampv5/ index.html; Eiken Chemical Co., Japan ) Designing a Monilinia fructicola-specific LAMP primer group, which comprises F3, B3, FIP and BIP4 primers, wherein the primer sequences are as follows:
F3：5’-CTTTTTATTAATGTCGTCTGAGTAC-3’，
B3：5’-TACCAAGCTG TGCTTGAG-3’，
FIP：5’-ATTTCGCTGCGTTCTTCATCG-TAGTTAAAACTTTCA ACAACGGATC-3’，
BIP：5’-TGCAGAATTCAGTGAATCATCGAAT-AAATGACG CTCGAACAGG-3’。
2. the establishment of the LAMP detection method for Monilinia fructicola comprises the following steps:
(1) extracting the genome DNA of a sample to be detected:
when the method is used for detecting pure pathogenic bacteria culture, a CTAB method is adopted to extract genome DNA, and the specific method comprises the following steps: a small amount of hyphal powder was taken into a 1.5mL centrifuge tube (hyphal powder covered a semi-circular bottom properly), 900 μ L of 2% CTAB (cetyltrimethylammonium bromide) extract (2% CTAB; 100 mmol/L Tris-HCl, pH 8.0; 20 mmol/L EDTA, pH 8.0; 1.4 mol/L NaCl) and 90 μ L SDS (sodium dodecyl benzene sulfonate) (Note: CTAB, SDS requiring preheating at 60 deg.C, shaking and mixing well with oscillator, water bath at 60 deg.C for 1h (DNA released into buffer), 12000 r.min-1Centrifuging for 15 min; taking 700 mu L of supernatant, adding equal volume of phenol, chloroform and isoamylol (25: 24: 1), gently shaking and uniformly mixing, and 12000 r.min-1Centrifuging for 9 min; taking 500 mu L of supernatant, adding equal volume of chloroform for re-extraction once, 12000 r-min-1Centrifuging for 5 min; taking 350 mu L of supernatant, adding 1/10 volume 3 mol. L-1NaAc and 2 times volume of absolute ethyl alcohol are precipitated for 30 min at-20 ℃ and 12000 r.min-1Centrifuging for 5 min; discarding the supernatant, adding 700 mu L of ice 70% ethanol for washing (slightly centrifuging; dumping the supernatant), airing on an ultra-clean workbench without alcohol smell, adding 30-60 mu L of TE (10 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0) solution for dissolving to obtain DNA solution, detecting the DNA concentration by using an ultraviolet spectrophotometer, and diluting to 100 ng/mu L for later use;
when the method is used for detecting whether Monilinia fructicola exists in plant leaf tissues, the method adopts a NaOH rapid cracking method to extract DNA, and the specific process is as follows: adding 10 muL of 0.5 mol/L NaOH into each mg of plant tissue, fully grinding the tissue into paste in a mortar, transferring the tissue into a 1.5mL centrifuge tube, centrifuging the tissue at 12,000 rpm for 6 min, taking 5 muL of supernatant, adding 495 muL of 0.1 mol/L Tris-HCl (pH = 8.0), uniformly mixing, and taking 1.0 muL as a PCR template for amplification;
(2) establishment of LAMP reaction system: performing LAMP amplification by using the DNA extracted in the step (1) as a template and using an outer primer F3/B3 and an inner primer FIP/BIP, wherein the LAMP detection reaction system is 25 mu L and comprises 1.0 mu L of each of 4 mu M primers F3 and B3 and 1.0 mu L of each of 32 mu M primers FIP and BIP, and an LAMP reaction mixed solution (40 mM Tris-HCl, 20mM (NH)4)2SO4，20 mM KCl，16 mM MgSO41.6mol/L Betaine (Betaine), 2.0mM dNTPs, 0.2wt.% Trion X-100) 12.5. mu.L, 8UBstPolymerase 1.0 μ L, DNA template 1.0 μ L, make up to 25 μ L with sterilized ultrapure water;
(3) LAMP reaction conditions: incubating at 64.5 deg.C for 60 min;
(4) determination of reaction results: by adopting a fluorescent dye visual observation method, after the LAMP reaction is finished, 1.0 microliter of a color-developing agent SYBR green I is added into an amplification product of the LAMP reaction, the reaction product is blue-green under natural illumination, and the judgment that the amplification product is in a white turbid precipitation state under the irradiation of the ultraviolet light with the wavelength of 365nm is positive, namely the existence of the peach brown rot germs, the reaction product is orange or orange under the natural illumination, and the judgment that the amplification product is in a colorless transparent state under the irradiation of the ultraviolet light with the wavelength of 365nm is negative, namely the existence of the peach brown rot germs.
The invention has the beneficial effects that: the invention establishes the LAMP detection technology system with the advantages of rapidness, convenience, strong specificity and high sensitivity for the Monilinia fructicola, can be used for detecting the Monilinia fructicola, or can be used for detecting the early stage and the initial stage of the Monilinia fructicola, and has very important significance for determining the optimal stage of disease control.
Compared with the prior art, the invention has the following technical advantages and positive effects:
1. the specificity is strong: according to the invention, 4 specific primers are designed aiming at 6 regions of the ITS sequence of the monilinia persicae, and any region in the 6 regions can not be matched with the primers for nucleic acid amplification, so that the specificity is extremely high;
2. the sensitivity is high: the detection sensitivity of the kit to the Monilinia fructicola can reach 10 fg/mu L on the DNA level, and the kit has high sensitivity;
3. the detection is efficient and quick: by applying the detection method, the detection result can be obtained within 1-1.5 h, while the detection result can be obtained only within 4-6 h in the conventional PCR or nested PCR detection, so that the method greatly shortens the operation time, and is convenient and quick;
4. the applicability is strong: according to the invention, the LMAP amplification reaction is amplified at one temperature, an expensive PCR amplification instrument is not needed, only a simple water bath kettle or heating equipment is needed, the detection result can be observed by naked eyes, the operation is simple, and the popularization and the use of a basic level are facilitated;
5. the cost is low: the method does not need expensive and precise test instruments such as a PCR instrument and the like, only needs a constant-temperature water bath kettle or other constant-temperature equipment, adopts conventional reagents in the experimental process, and has low cost.
Drawings
FIG. 1 shows the locus of the LAMP specific primer of Monilinia fructicola in the Internal Transformed Spacer (ITS) gene sequence.
FIG. 2 shows the LAMP specificity detection result of Monilinia fructicola of the present invention. FIG. 2a shows the result of detection under natural light, wherein 1-3 are blue-green; FIG. 2b shows the result of detection under 365nm UV light, wherein 1-3 is white turbid precipitate. In the figure, 1-3 is Monilinia fructicola (C.fructicola)Monilinia fructicola) 4-7 peach anthracnose bacteria (respectively)Colletotirchum gloeosporioides) Peach anthracnose bacterium (A. sup. (B. sup.))Colletotrichum acutatum）、Peach bacterial fruit of the species Perforax bacteria (Xanthomonas campestris pv. pruni (Smith) Dye.）、Botrytis cinerea (Botrytis cinerea: (C)Botrytis cinerea) And 8 is a negative control.
FIG. 3 shows LAMP detection sensitivity of Monilinia fructicola in the present invention. FIG. 3a shows the result of the detection under natural light, wherein 1-4 are blue-green; FIG. 3b shows the result of detection under 365nm UV light, wherein 1-4 is a white turbid precipitate. In the figure, the concentrations of template DNAs 1 to 7 were 10 pg, 1 pg, 100 fg, 10fg, 1 fg, 100ag, 10ag, and 8 were negative controls, respectively.
FIG. 4 shows the detection of Monilinia fructicola in diseased fruits by the detection method of the present invention. FIG. 4a shows the result of detection under natural light, wherein 4-5 is blue-green; FIG. 4b is the result of ultraviolet irradiation at 365nm wavelength, wherein 4-5 is in the form of white turbid precipitate. In the figure, 1 is a positive control, 2, 6-8 are healthy peach fruits which are not attacked, 3-4 are peach fruits which are attacked by the peach brown rot, and 5 is a negative control.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The following examples are conducted according to conventional experimental conditions, or according to the protocols reported in the relevant literature, or according to the experimental conditions recommended by the manufacturers.
Example 1: design of specific primers for loop-mediated isothermal amplification (LAMP) detection of Monilinia fructicola (POP) and primer specificity verification
1. Extraction of genomic DNA of test strains
The CTAB method is adopted to extract the genome DNA of the test strain (table 1), and the specific method is as follows: a small amount of hyphal powder was taken into a 1.5mL centrifuge tube (hyphal powder covered a semi-circular bottom properly), 900 μ L of 2% CTAB (cetyltrimethylammonium bromide) extract (2% CTAB; 100 mmol/L Tris-HCl, pH 8.0; 20 mmol/L EDTA, pH 8.0; 1.4 mol/L NaCl) and 90 μ L SDS (sodium dodecyl benzene sulfonate) (Note: CTAB, SDS requiring preheating at 60 deg.C, shaking and mixing well with oscillator, water bath at 60 deg.C for 1h (DNA released into buffer), 12000 r.min-1Centrifuging for 15 min; taking 700 mu L of supernatant, adding phenol, chloroform and isoamylol (25: 24: 1) with equal volume, gently shaking and uniformly mixing, and 12000 r-min-1Centrifuging for 9 min; taking 500 mu L of supernatant, adding equal volume of chloroform for re-extraction once, 12000 r-min-1Centrifuging for 5 min; taking 350 mu L of supernatant, adding 1/10 volume 3 mol. L-1NaAc and 2 times volume of absolute ethyl alcohol are precipitated for 30 min at-20 ℃ and 12000 r.min-1Centrifuging for 5 min; the supernatant was discarded, and 700. mu.L of ice 70% ethanol was added for washing (slight centrifugation; the supernatant was decantedLiquid), airing on an ultra-clean workbench without alcohol smell, adding 30-60 muL TE (10 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0) solution for dissolution to obtain DNA solution, detecting the DNA concentration by using an ultraviolet spectrophotometer and diluting to 100 ng/muL for later use.
TABLE 1 test strains
2. Design of loop-mediated isothermal amplification (LAMP) specific primers for brown rot fungi of peach trees
By assaying Monilinia fructicola (American Australia type)Monilinia fructicola) (iii) Internal Transcribed Spacer (ITS) sequences from other pathogenic bacteria, and the ITS sequences were analyzed by alignment using on-line LAMP Primer design software Primer software Explorer V5 (https://primerexplorer.jp/lampv5/ index.htmlEiken Chemical Co., Japan) designed a Monilinia fructicola-specific LAMP primer set. The primer group consists of 4 primers, including 2 outer primers (F3, B3) and 2 inner primers (FIP, BIP). The primer sequences and their sites in the ITS gene sequences are detailed in Table 2 and FIG. 1.
TABLE 2 used forMonilinia fructicolaLAMP detection primer
3. Establishment of Monilinia fructicola LAMP detection method and primer specificity verification
Using DNA of test strains in Table 1 as a template, LAMP amplification was performed using F3, B3, FIP and BIP, and the LAMP detection reaction system was 25. mu.L, including 4. mu.M primers F3 and B3 each 1.0. mu.L, 32. mu.M primers FIP and BIP each 1.0. mu.L, LAMP reaction mixture [ 40mM Tris-HCl, 20mM (NH) ("40 mM Tris-HCl ])4)2SO4，20 mM KCl，16 mM MgSO41.6mol/L Betaine (Betaine), 2.0mM dNTPs, 0.2% Trion X-100. mu.L, 8UBstPolymerase 1.0 μ L, DNA template 1.0 μ L, make up to 25 μ L with sterilized ultrapure water; LAMP reaction conditions: incubating at 64.5 deg.C for 60 min; determination of reaction results: the measurement was carried out by visual observation using a fluorescent dye. After the LAMP reaction is finished, 1.0 mu L of color-developing agent SYBR green I is added into an amplification product of the LAMP reaction, the reaction product is blue-green under natural illumination, and is judged to be positive if being in a white turbid precipitation state under the irradiation of ultraviolet light with the wavelength of 365nm, namely, the Monilinia fructicola exists, the reaction product is orange or orange under the natural illumination, and is judged to be negative if being in a colorless transparent state under the irradiation of the ultraviolet light with the wavelength of 365nm, namely, the Monilinia fructicola does not exist.
4. Verification result of primer specificity
The LAMP amplification result shows that only the reaction products of the Monilinia fructicola in the tested strains are blue-green under natural illumination and are white turbid precipitates under the irradiation of ultraviolet light with the wavelength of 365nm, and the reaction products of the other tested strains are orange under the natural illumination and are transparent under the irradiation of ultraviolet light with the wavelength of 365nm (shown in figure 2), so that the designed Monilinia fructicola primer F3/B3 and primer FIP/BIP can distinguish the Monilinia fructicola from other pathogenic bacteria, have species specificity and can be used for the rapid and reliable detection and identification of the Monilinia fructicola.
Example 2: loop-mediated isothermal amplification (LAMP) detection sensitivity determination of peach brown rot fungi
1. Preparation of genomic DNA at various concentrations
Diluting the genome DNA of the peach brown rot fungi with sterile ultrapure water to prepare a series of concentrations of 10 times of magnitude for later use;
2. LAMP detection method sensitivity determination and result observation
Performing LAMP amplification by using genomic DNA of the Monilinia fructicola (Takayama) with different concentrations as templates and using a primer F3/B3 and a primer FIP/BIP, wherein the LAMP detection reaction system is 25 mu L and comprises 1.0 mu L of each of 4 mu M primers F3 and B3 and 1.0 mu L of each of 32 mu M primers FIP and BIP, and an LAMP reaction mixed solution [ 40mM Tris-HCl, 20mM (NH)4)2SO4，20 mM KCl，16 mM MgSO41.6mol/L Betaine (Betaine), 2.0mM dNTPs, 0.2% Trion X-100. mu.L, 8UBstPolymerase 1.0 μ L, DNA template 1.0 μ L of different concentration, and sterilized ultrapure water to 25 μ L; LAMP reaction conditions: incubating at 64.5 deg.C for 60 min; determination of reaction results: and (2) measuring by adopting a fluorescent dye visual observation method, adding 1.0 mu L of color-developing agent SYBR green I into an amplification product of LAMP reaction after the LAMP reaction is finished, wherein the reaction product is blue-green under natural illumination, and is judged to be positive when being in a white turbid precipitation state under the irradiation of ultraviolet light with the wavelength of 365nm, the reaction product is orange or orange under the natural illumination, and is judged to be negative when being in a colorless transparent state under the irradiation of the ultraviolet light with the wavelength of 365 nm.
3. LAMP amplification sensitivity detection result
The LAMP amplification sensitivity detection result shows that the detection sensitivity of the established LAMP detection system for the genomic DNA of the Monilinia fructicola can reach 10 fg/muL (figure 3), the reaction products of the genomic DNA of the Monilinia fructicola with the concentrations of 10 pg, 1 pg, 100 fg and 10 fg/muL are blue-green color under natural illumination and are white turbid precipitates under the irradiation of ultraviolet light with the wavelength of 365nm, and the reaction products with the rest concentrations and negative controls are orange under natural illumination and are colorless transparent under the irradiation of ultraviolet light with the wavelength of 365 nm.
Example 3: LAMP detection of Monilinia fructicola in diseased fruits
Collecting samples: collecting fruits typical to the onset symptoms of the peach brown rot and healthy fruits from Fujian Gutian, Fuan and Minqing and bringing the fruits back to a laboratory for later use;
extraction of plant tissue DNA: the method for extracting DNA by adopting NaOH rapid cracking comprises the following specific steps: adding 10 muL of 0.5 mol/L NaOH into each milligram of plant tissue, fully grinding the tissue into paste in a mortar, transferring the paste into a 1.5mL centrifuge tube, centrifuging at 12,000 rpm for 6 min, taking 5 muL of supernatant, adding 495 muL of 0.1 mol/L Tris-HCl (pH = 8.0), uniformly mixing, and taking 1.0 muL as a PCR template for amplification.
Amplification detection and observation: using the extracted DNA as a template and primersF3, B3, FIP and BIP for LAMP amplification, wherein the LAMP detection reaction system is 25 mu L and comprises 1.0 mu L of each of 4 mu M primers F3 and B3 and 1.0 mu L of each of 32 mu M primers FIP and BIP, and the LAMP reaction mixture [ 40mM Tris-HCl, 20mM (NH)4)2SO4，20 mM KCl，16 mM MgSO41.6mol/L Betaine (Betaine), 2.0mM dNTPs, 0.2% Trion X-100. mu.L, 8UBstPolymerase 1.0 μ L, DNA template 1.0 μ L, make up to 25 μ L with sterilized ultrapure water; LAMP reaction conditions: incubating at 64.5 deg.C for 60 min; determination of reaction results: the detection is carried out by adopting a fluorescent dye visual observation method, 1.0 mu L of color-developing agent SYBR green I is added into an amplification product of LAMP reaction after the LAMP reaction is finished, the reaction product is blue-green under natural illumination, and the judgment that the amplification product is white turbid precipitate under the irradiation of the ultraviolet light with the wavelength of 365nm is positive, namely the existence of the peach brown rot germs, the reaction product is orange or orange under the natural illumination, and the judgment that the amplification product is colorless transparent under the irradiation of the ultraviolet light with the wavelength of 365nm is negative, namely the existence of the peach brown rot germs is avoided.
And (3) detection results: the detection result (figure 4) shows that the fruits with the peach brown rot disease are amplified by LAMP, the reaction product is blue-green under natural illumination and is white turbid sediment under the irradiation of ultraviolet light with the wavelength of 365nm, which indicates that the peach brown rot disease exists, while the healthy fruits and the negative control are amplified by LAMP, the reaction product is orange under natural illumination and is colorless transparent under the irradiation of the ultraviolet light with the wavelength of 365nm, which indicates that the peach brown rot disease does not exist, and the technology can be used for the rapid molecular detection of the peach brown rot disease in plant tissues.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture occupational technology college
LAMP (loop-mediated isothermal amplification) detection primer for monilinia persica and application of LAMP detection primer
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> Artificial sequence
<400> 1
ctttttatta atgtcgtctg agtac 25
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence
<400> 2
taccaagctg tgcttgag 18
<210> 3
<211> 46
<212> DNA
<213> Artificial sequence
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atttcgctgc gttcttcatc gtagttaaaa ctttcaacaa cggatc 46
<210> 4
<211> 43
<212> DNA
<213> Artificial sequence
<400> 4
tgcagaattc agtgaatcat cgaataaatg acgctcgaac agg 43
Claims (4)
1. The LAMP detection primer for Monilinia fructicola is characterized in that the nucleotide sequence is as follows:
F3：5’-CTTTTTATTAATGTCGTCTGAGTAC-3’；
B3：5’-TACCAAGCTGTGCTTGAG-3’；
FIP：5’-ATTTCGCTGCGTTCTTCATCG-TAGTTAAAACTTTCAACAACGGATC-3’；
BIP：5’-TGCAGAATTCAGTGAATCATCGAAT-AAATGACGCTCGAACAGG-3’。
2. a LAMP detection method for Monilinia fructicola by using the primers as claimed in claim 1, which comprises the following specific steps:
1) extracting genome DNA of a sample to be detected;
2) establishment of LAMP reaction system: the LAMP detection reaction system is 25 mu L, the DNA extracted in the step 1) is used as a template, the reaction system comprises 1.0 mu L of each of 4 mu M primers F3 and B3, 1.0 mu L of each of 32 mu M primers FIP and BIP, and the LAMP reaction mixed solution is 12.5 mu L and 8UBstPolymerase 1.0 μ L, DNA template 1.0 μ L, make up to 25 μ L with sterilized ultrapure water;
3) LAMP reaction conditions: incubating at 64.5 deg.C for 60 min;
4) determination of reaction results: measuring by adopting a fluorescent dye visual observation method, after the LAMP reaction is finished, mixing 1.0 mu L of color developing agent SYBR green I into an amplification product of the LAMP reaction, wherein the reaction product is blue-green under natural illumination, and the judgment that the reaction product is white turbid precipitate under the irradiation of ultraviolet light with the wavelength of 365nm is positive, namely the existence of the Monilinia fructicola is detected, the reaction product is orange or orange under the natural illumination, and the judgment that the reaction product is colorless transparent under the irradiation of the ultraviolet light with the wavelength of 365nm is negative, namely the existence of the Monilinia fructicola is not detected;
the LAMP reaction mixed solution consists of the following components: 40mM Tris-HCl, 20mM (NH)4)2SO420mM KCl, 16mM MgSO4, 1.6mol/L betaine, 2.0mM dNTPs, 0.2% TrionX-100.
3. The application of the LAMP detection primer for Monilinia fructicola of claim 1 in early diagnosis of Monilinia fructicola and monitoring and identification of germs.
4. The LAMP detection method of Monilinia fructicola according to claim 2, which is applied to early diagnosis of Monilinia fructicola and monitoring and identification of germs.
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