CN114703310B - LAMP (loop-mediated isothermal amplification) detection primer for brown rot of peach and application thereof - Google Patents
LAMP (loop-mediated isothermal amplification) detection primer for brown rot of peach and application thereof Download PDFInfo
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Abstract
The invention discloses a LAMP detection primer for brown rot of peach, which takes ITS genes of brown rot of peach as detection targets, designs and screens a set of specific detection primer groups, and 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) The primer composition provided by the invention is used for carrying out loop-mediated isothermal amplification on the DNA of the sample to be detected; 3) As a result, it was found that the reaction product was blue-green under natural light and that it was judged as positive as white cloudy precipitate under irradiation of ultraviolet light having a wavelength of 365 nm. The invention solves 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 rapidly, conveniently, efficiently, highly specifically and highly sensitively detect the brown rot of peach at low cost.
Description
Technical Field
The invention belongs to the technical field of crop disease detection, identification and prevention and control, and in particular relates to an LAMP detection primer for brown rot of peach and application thereof, which are used for rapid, sensitive and specific molecular detection of brown rot of peach, and can be applied to early diagnosis of brown rot of peach and monitoring and identification of the germs.
Background
Is prepared from the fungus of the genus SclerotiniaMoniliniaspp.) is one of the important fungal diseases that seriously affect the yield and quality of peach trees, and the diseases are widely distributed worldwide, particularly serious in moist areas, and mainly harm mature fruits, resulting in a large number of rotted fruits. It has been reported that there are mainly 3 pathogenic fungi causing brown rot of peach, namely, brown rot of American Australian stone fruitMonilinia fructicola) Brown rot of stone fruitMonilinia laxa) Herenguo brown rot fungiMonilinia fructigena). Although 3 pathogenic bacteria can be infected on peach to cause brown rot, the distribution of the 3 pathogenic bacteria and the preference of a host have certain difference, wherein the brown rot of the American and Australian stone fruit is characterized in thatMonilinia fructicola) The distribution range in China is the widest, and the bacterial strain is dominant bacterial group of brown rot of peach, and the pathogenic bacteria can infect all plumsPrunus) The cultivated crops not only harm peach fruits, but also harm stone fruit like plum, apricot, cherry and kernel fruit like apple and pear, and the like, and the stone fruit like apple flowers, stems and fruits are infected, so that the flowers and the fruits are rotted and the branches are dead, the loss of the picked fruits is even up to 90% in severe areas, and serious economic loss is caused. Therefore, the method has important significance in strengthening the prevention and treatment of brown rot of peach, recovering economic loss, improving income of fruit farmers, promoting healthy development of 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 pathogenic bacteria are separated from pathogenic tissues by the traditional identification and detection method of the pathogenic bacteria, the pathogenic bacteria are primarily identified by adopting morphological characteristics, and the pathogenic bacteria are finally determined by the method of the Koch law. The traditional identification method is time-consuming and labor-consuming, and the experience of the identification personnel also affects the accuracy of the result, so that the requirement of mass and accurate identification of diseases is obviously not met. Along 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 high speed, accuracy, high specificity and the like, but the method also has the defects that the operation steps are complex because of the dependence on precise and precious PCR amplification instruments, gel imaging systems and other instruments and equipment, and operators need to have certain molecular biological technology and the like, so that the technology is greatly limited in use and popularization in underdeveloped areas and basic agricultural departments.
The latest technology applied to the rapid and accurate detection of plant pathogenic microorganisms is Loop-mediated isothermal amplification (Loop-mediated Isothermal Amplification, LAMP) technology. The technology has strong specificity, because the designed detection primer is aimed at 6 specific areas of the target gene, in addition, the LAMP method has low requirements on equipment, only a simple water bath kettle 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. Up to now, related reports of LAMP technology for detecting brown rot of peach are not seen. The invention aims to design LAMP detection primers of brown rot of peach, and establish an LAMP detection system of brown rot of peach based on the designed primers.
Disclosure of Invention
The invention provides an LAMP detection primer for brown rot of peach and application thereof, which are used for 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, complex in procedure, strong in experience and low in accuracy, the occurrence of diseases is difficult to monitor and control the spread and popularity of pathogenic bacteria in time, the existing PCR molecular detection is required to depend on expensive instruments such as an amplification instrument, the detection time is long and the like.
In order to achieve the above purpose, the invention adopts the following technical scheme:
1. design of LAMP detection specific primers for brown rot of peach bacteria:
by measuring brown rot of peach (Meinao stone fruit brown rot)Monilinia fructicola) And Internal Transcribed Spacer (ITS) sequences of other pathogenic bacteria, comparing and analyzing the ITS sequences, and designing software Primer software Explorer V < 5 > by using an online LAMP primerhttps://primerexplorer.jp/lampv5/ index.htmlEiken Chemical co., japan) designed a brown rot peach specific LAMP primer set comprising F3, B3, FIP and BIP4 primers with the following sequences:
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 the brown rot of peach includes the following steps:
(1) Extracting genome DNA of a sample to be detected:
when the method is used for detecting the pure culture of pathogenic bacteria, the CTAB method is adopted to extract the genome DNA, and the specific method is as follows: a small amount of mycelium powder was placed in a 1.5mL centrifuge tube (preferably mycelium powder just covered by a semi-circular bottom), 900. Mu.L of 2% CTAB (cetyltrimethylammonium bromide) extract (2% CTAB;100 mmol/L Tris-HCl, pH8.0; 20 mmol/L EDTA, pH8.0;1.4 mol/L NaCl) and 90. Mu.L SDS (sodium dodecyl benzene sulfonate) were added [ note: CTAB, SDS was preheated at 60℃and mixed with shaking by an oscillator, water-bath at 60℃for 1h (DNA was released into buffer), 12000 r. Min -1 Centrifuging for 15 min; taking 700 mu L of supernatant, adding equal volumes of phenol, chloroform and isoamyl alcohol (25:24:1), gently shaking and uniformly mixing, 12000 r.min -1 Centrifuging for 9 min; taking 500 mu L of supernatant, adding an equal volume of chloroform, extracting once again, 12000 r min -1 Centrifuging for 5 min; taking 350 mu L of supernatant, adding 1/10 volume of 3mol.L -1 NaAc and 2 times volume of absolute ethanol, precipitation at-20deg.C for 30 min,12000 r.min -1 Centrifuging for 5 min; removing supernatant, adding 700 mu L of ice and 70% ethanol for washing (slightly centrifuging; pouring out the supernatant), airing the solution 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 concentration of the DNA by an ultraviolet spectrophotometer and diluting the DNA solution to 100 ng/mu L for later use;
when detecting whether the brown rot of peach 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 [ mu ] L of 0.5 mol/L NaOH into each milligram of plant tissue, fully grinding the tissue into paste in a mortar, transferring into a 1.5mL centrifuge tube, centrifuging at 12,000 rpm for 6 min, taking 5 [ mu ] L of supernatant, adding 495 [ mu ] L of 0.1 mol/L Tris-HCl (pH=8.0), uniformly mixing, and taking 1.0 [ mu ] L as a PCR template for amplification;
(2) Establishing an LAMP reaction system: LAMP amplification was performed using the DNA extracted in step (1) as a template and using the outer primer F3/B3 and the inner primer FIP/BIP, 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, and the LAMP reaction mixture (40 mM Tris-HCl,20mM (NH) 4 ) 2 SO 4 ，20 mM KCl，16 mM MgSO 4 1.6mol/L Betaine (2.0 mM dNTPs,0.2wt% Trion X-100) 12.5. Mu.L, 8UBst1.0. Mu.L of polymerase, 1.0. Mu.L of DNA template, and 25. Mu.L of sterilized ultrapure water;
(3) LAMP reaction conditions: 64.5 Incubating at 60 min;
(4) Determination of the reaction results: after the LAMP reaction is finished, adding 1.0 mu L of a color developing agent SYBR green I into an amplification product of the LAMP reaction, judging that the reaction product is blue-green under natural illumination and is white turbid and precipitative under the irradiation of ultraviolet light with the wavelength of 365nm, namely, the presence of brown rot of peach, and judging that the reaction product is orange or orange yellow under the natural illumination and is colorless and transparent under the irradiation of ultraviolet light with the wavelength of 365nm, namely, the absence of brown rot of peach.
The invention has the beneficial effects that: the LAMP detection technology system with high speed, simplicity, convenience, high specificity and high sensitivity is established, can be used for detecting the brown rot of the peach, or for early detection and early detection of the brown rot of the peach, and has great significance for determining the optimal period 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 for 6 regions of the ITS sequence of the brown rot fungus of peach, and nucleic acid amplification cannot be carried out in any region of the 6 regions which is not matched with the primers, so that the specificity is extremely high;
2. the sensitivity is high: the detection sensitivity of the invention to brown rot of peach can reach 10 fg/mu L on DNA level, and has very high sensitivity;
3. high efficiency and high speed in detection: by applying the detection method, the detection result can be obtained in 1-1.5 h, and the detection result can be obtained only in 4-6 h in the conventional PCR or nested PCR detection, so that the operation time is greatly shortened, and the method is convenient and quick;
4. the applicability is strong: the LMAP amplification reaction of the invention is amplified at a temperature, does not need an expensive PCR amplification instrument, only needs a simple water bath kettle or heating equipment, can observe the detection result by naked eyes, has simple operation, and is convenient for popularization and use of a base layer;
5. the cost is low: the method does not need expensive and precise test instruments such as a PCR instrument, only needs a constant-temperature water bath kettle or other constant-temperature equipment, and has the advantages of low cost due to the fact that the conventional reagents are used in the experimental process.
Drawings
FIG. 1 shows the locus of LAMP specific primers of brown rot peach bacteria in Internal Transcribed Spacer (ITS) gene sequence.
FIG. 2 shows the LAMP specificity detection result of brown rot of peach. FIG. 2a shows the detection result under natural illumination, wherein 1-3 is blue-green; FIG. 2b shows the detection result under irradiation of ultraviolet light with a wavelength of 365nm, wherein 1-3 is in the form of white turbid precipitate. In the figure, 1-3 is brown rot of peachMonilinia fructicola) 4-7 respectively peach anthracnose pathogenColletotirchum gloeosporioides) Peach anthracnose pathogenColletotrichum acutatum）、Bacterial perfora persicaXanthomonas campestrispv.pruni(Smith) Dye.）、Tomato gray mold fungusBotrytis cinerea) And 8 is a negative control.
FIG. 3 shows LAMP detection sensitivity of brown rot of peach. FIG. 3a shows the detection result under natural illumination, wherein 1-4 is blue-green; FIG. 3b shows the detection result under irradiation of ultraviolet light with a wavelength of 365nm, wherein 1-4 is in the form of white turbid precipitate. In the figures, the concentration of 1-7 template DNA is 10 pg, 1 pg, 100 fg, 10fg, 1 fg, 100ag, 10ag and 8 respectively are negative controls.
FIG. 4 shows the detection of brown rot of peach in diseased fruits by the detection method of the present invention. FIG. 4a shows the detection result under natural light, wherein 4-5 is blue-green; FIG. 4b shows the detection result under irradiation of ultraviolet light with a wavelength of 365nm, wherein 4-5 is in the form of a white turbid precipitate. In the figure, 1 is positive control, 2, 6-8 are healthy peach fruits without disease, 3-4 are brown rot disease peach fruits, and 5 is negative control.
Detailed Description
The invention is further illustrated below in connection with specific examples, which are not intended to limit the scope of the invention. The following examples were conducted under conventional experimental conditions, or the procedures described in published related literature, or according to the experimental conditions suggested by the manufacturer.
Example 1: design of loop-mediated isothermal amplification (LAMP) detection specific primers and primer specificity verification of brown rot pathogen
1. Extraction of genomic DNA from test strains
The genomic DNA of the test strain (Table 1) was extracted by CTAB method as follows: a small amount of mycelium powder was placed in a 1.5mL centrifuge tube (preferably mycelium powder just covered by a semi-circular bottom), 900. Mu.L of 2% CTAB (cetyltrimethylammonium bromide) extract (2% CTAB;100 mmol/L Tris-HCl, pH8.0; 20 mmol/L EDTA, pH8.0;1.4 mol/L NaCl) and 90. Mu.L SDS (sodium dodecyl benzene sulfonate) were added [ note: CTAB, SDS was preheated at 60℃and mixed with shaking by an oscillator, water-bath at 60℃for 1h (DNA was released into buffer), 12000 r. Min -1 Centrifuging for 15 min; taking 700 mu L of supernatant, adding equal volumes of phenol, chloroform and isoamyl alcohol (25:24:1), gently shaking and uniformly mixing, 12000 r.min -1 Centrifuging for 9 min; taking 500 mu L of supernatant, adding an equal volume of chloroform, extracting once again, 12000 r min -1 Centrifuging for 5 min; taking 350 mu L of supernatant, adding 1/10 volume of 3mol.L -1 NaAc and 2 times volume of absolute ethanol, precipitation at-20deg.C for 30 min,12000 r.min -1 Centrifuge 5 min; removing supernatant, adding 700 mu L of ice and 70% ethanol for washing (slightly centrifuging; pouring out the supernatant), airing the solution 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 concentration of the DNA by an ultraviolet spectrophotometer and diluting the DNA solution to 100 ng/mu L for later use.
TABLE 1 test strains
2. Design of loop-mediated isothermal amplification (LAMP) specific primer of brown rot of peach
By measuring brown rot of peach (Meinao stone fruit brown rot)Monilinia fructicola) And Internal Transcribed Spacer (ITS) sequences of other pathogenic bacteria, comparing and analyzing the ITS sequences, and designing software Primer software Explorer V < 5 > by using an online LAMP primerhttps://primerexplorer.jp/lampv5/ index.htmlEiken Chemical co., japan) designed a specific LAMP primer set for brown rot peach bacteria. The primer set consists of 4 primers, including 2 outer primers (F3, B3) and 2 inner primers (FIP, BIP). The primer sequences and the sites in ITS gene sequences are shown in Table 2 and FIG. 1.
Table 2 forMonilinia fructicolaPrimers for LAMP detection
3. Establishment of LAMP detection method for brown rot of peach and primer specificity verification
LAMP amplification was performed using the DNA of the test strain of Table 1 as a template and 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) 4 ) 2 SO 4 ，20 mM KCl，16 mM MgSO 4 1.6mol/L Betaine, 2.0mM dNTPs,0.2% Trion X-100, 12.5. Mu.L, 8UBst1.0. Mu.L of polymerase, 1.0. Mu.L of DNA template, and 25. Mu.L of sterilized ultrapure water; LAMP reaction conditions: incubating at 64.5 ℃ for 60 min; determination of the reaction results: the measurement was carried out by visual observation with a fluorescent dye. After the LAMP reaction is finished, adding 1.0 mu L of a color developing agent SYBR green I into an amplification product of the LAMP reaction, judging that the reaction product is blue-green under natural illumination, and the reaction product is white turbid and precipitated under the irradiation of ultraviolet light with the wavelength of 365nm, namely, the presence of brown rot of peach, and judging that the reaction product is orange or orange yellow under the natural illumination, and the reaction product is colorless and transparent under the irradiation of ultraviolet light with the wavelength of 365nm, namely, the absence of brown rot of peach is negative.
4. Primer specificity verification results
LAMP amplification results show that only the reaction products of the brown rot of peach in the strain to be tested are blue-green under natural illumination, and are white turbid precipitate under the irradiation of ultraviolet light with the wavelength of 365nm, and the reaction products of other strains to be tested are orange under the natural illumination and are transparent under the irradiation of ultraviolet light with the wavelength of 365nm (figure 2), so that the designed primers F3/B3 and FIP/BIP of the brown rot of peach can distinguish the brown rot of peach from other pathogenic bacteria, and have the specificity of species, and can be used for rapid and reliable detection and identification of the brown rot of peach.
Example 2: loop-mediated isothermal amplification (LAMP) detection sensitivity determination of brown rot of peach
1. Preparation of genomic DNA at different concentrations
Diluting genomic DNA of brown rot of peach with sterile ultrapure water to prepare a series of concentrations of 10 times of magnitude for later use;
2. LAMP detection method sensitivity measurement and result observation
LAMP amplification is carried out by taking genomic DNA of brown rot of peach with different concentrations as a template and utilizing a primer F3/B3 and a primer FIP/BIP, wherein the LAMP detection reaction system is 25 mu L and comprises 4 mu M primers F3 and B3 of 1.0 mu L and 32 mu M primersFIP and BIP 1.0. Mu.L each, LAMP reaction mixture [ 40mM Tris-HCl,20mM (NH) 4 ) 2 SO 4 ，20 mM KCl，16 mM MgSO 4 1.6mol/L Betaine, 2.0mM dNTPs,0.2% Trion X-100, 12.5. Mu.L, 8UBst1.0 mu L of polymerase and 1.0 mu L of DNA template with different concentrations, and supplementing the template with sterilized ultrapure water to 25 mu L; LAMP reaction conditions: 64.5 Incubating at 60 min; determination of the reaction results: after the LAMP reaction is finished, adding 1.0 mu L of a color developing agent SYBR green I into an amplification product of the LAMP reaction, judging that the reaction product is blue-green under natural light, and is in a white turbidity precipitation state under the irradiation of ultraviolet light with the wavelength of 365nm, and judging that the reaction product is orange or orange yellow under the natural light, and is in a colorless transparent state under the irradiation of ultraviolet light with the wavelength of 365 nm.
3. LAMP amplification sensitivity detection results
The LAMP amplification sensitivity detection results show that the genomic DNA reaction products of the brown rot peach bacteria with the concentrations of 10 pg, 1 pg, 100 fg and 10 fg/. Mu.L are blue-green under natural illumination and are precipitated in a white turbidity state under the irradiation of ultraviolet light with the wavelength of 365nm, and the other concentrations and the negative control reaction products are orange under the natural illumination and are colorless and transparent under the irradiation of ultraviolet light with the wavelength of 365nm, so that the detection sensitivity of the established LAMP detection system of the brown rot peach bacteria on the genomic DNA of the brown rot peach bacteria can reach 10 fg/. Mu.L (shown in figure 3).
Example 3: LAMP detection of brown rot of peach in diseased fruits
Sample collection: collecting typical fruits and healthy fruits of brown rot disease symptoms of peach from Fujian old field, fuan and Minqing, and taking the fruits back to a laboratory for later use;
extraction of plant tissue DNA: the DNA is extracted by adopting a NaOH rapid cracking method, and the specific process is as follows: adding 10 [ mu ] L of 0.5 mol/L NaOH into every milligram of plant tissue, fully grinding the tissue into paste in a mortar, transferring into a 1.5mL centrifuge tube, centrifuging at 12,000 rpm for 6 min, taking 5 [ mu ] L of supernatant, adding 495 [ mu ] L of 0.1 mol/L Tris-HCl (pH=8.0), uniformly mixing, and taking 1.0 [ mu ] L as a PCR template for amplification.
Amplification detection and observation: LAMP amplification was performed using the above-mentioned extracted DNA as a template and primers F3, B3, FIP and BIP, and the LAMP detection reaction system was 25. Mu.L, including 4. Mu.M each of primers F3 and B3, 1.0. Mu.L each of 32. Mu.M each of primers FIP and BIP, LAMP reaction mixture [ 40mM Tris-HCl,20mM (NH) 4 ) 2 SO 4 ，20 mM KCl，16 mM MgSO 4 1.6mol/L Betaine, 2.0mM dNTPs,0.2% Trion X-100, 12.5. Mu.L, 8UBst1.0. Mu.L of polymerase, 1.0. Mu.L of DNA template, and 25. Mu.L of sterilized ultrapure water; LAMP reaction conditions: incubating at 64.5 ℃ for 60 min; determination of the reaction results: after the LAMP reaction is finished, adding 1.0 mu L of a color developing agent SYBR green I into an amplification product of the LAMP reaction, judging that the reaction product is blue-green under natural light irradiation and is in a white turbid precipitate state under the irradiation of ultraviolet light with the wavelength of 365nm to be positive, namely, the presence of brown rot of peach, and judging that the reaction product is orange or orange yellow under the natural light irradiation and is in a colorless transparent state under the irradiation of ultraviolet light with the wavelength of 365nm to be negative, namely, the absence of brown rot of peach.
Detection result: the detection result (figure 4) shows that the disease fruit of the brown rot of the peach is amplified by LAMP, the reaction product is blue-green under natural illumination and is in white turbidity precipitation under the irradiation of ultraviolet light with the wavelength of 365nm, which indicates that the brown rot of the peach exists, while the healthy fruit and the negative control are amplified by LAMP, the reaction product is orange under the natural illumination and is in colorless transparent state under the irradiation of ultraviolet light with the wavelength of 365nm, which indicates that the brown rot of the peach does not exist, and the set of technology can be used for rapid molecular detection of the brown rot of the peach in plant tissues.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> Fujian agricultural occupational technology college
<120> LAMP detection primer for brown rot of peach and application thereof
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> artificial sequence
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ctttttatta atgtcgtctg agtac 25
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<212> DNA
<213> artificial sequence
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taccaagctg tgcttgag 18
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<212> DNA
<213> artificial sequence
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atttcgctgc gttcttcatc gtagttaaaa ctttcaacaa cggatc 46
<210> 4
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<212> DNA
<213> artificial sequence
<400> 4
tgcagaattc agtgaatcat cgaataaatg acgctcgaac agg 43
Claims (4)
1. The LAMP detection primer for brown rot of peach is characterized by comprising the following nucleotide sequences:
F3：5’-CTTTTTATTAATGTCGTCTGAGTAC-3’；
B3：5’-TACCAAGCTGTGCTTGAG-3’；
FIP：5’-ATTTCGCTGCGTTCTTCATCG-TAGTTAAAACTTTCAACAACGGATC-3’；
BIP：5’-TGCAGAATTCAGTGAATCATCGAAT-AAATGACGCTCGAACAGG-3’。
2. a method for carrying out LAMP detection of brown rot of peach using the primer of claim 1, comprising the following specific steps:
1) Extracting genome DNA of a sample to be detected;
2) Establishing an LAMP reaction system: the LAMP detection reaction system was 25. Mu.L, and the DNA extracted in step 1) was used as a template, and the reaction system included 1.0. Mu.L each of the 4. Mu.M primers F3 and B3, 1.0. Mu.L each of the 32. Mu.M primers FIP and BIP, 12.5. Mu.L each of the LAMP reaction mixture, 8UBst1.0. Mu.L of polymerase, 1.0. Mu.L of DNA template, and 25. Mu.L of sterilized ultrapure water;
3) LAMP reaction conditions: incubating at 64.5 ℃ for 60 min;
4) Determination of the reaction results: after the LAMP reaction is finished, 1.0 mu L of a color developing agent SYBR green I is mixed into an amplification product of the LAMP reaction, the reaction product is blue-green under natural illumination, and the reaction product is judged to be positive in a white turbidity precipitation state under the irradiation of ultraviolet light with the wavelength of 365nm, namely, brown rot of peach exists, the reaction product is orange or orange yellow under the natural illumination, and the reaction product is judged to be negative in a colorless transparent state under the irradiation of ultraviolet light with the wavelength of 365nm, namely, brown rot of peach does not exist;
wherein, the LAMP reaction mixed solution consists of the following components: 40mM Tris-HCl,20mM (NH) 4 ) 2 SO 4 ，20mM KCl，16mM MgSO 4 1.6mol/L betaine, 2.0mM dNTPs,0.2% TrionX-100.
3. The use of the LAMP detection primer for brown rot of peach as set forth in claim 1 for early diagnosis of brown rot of peach and monitoring and identification of germs.
4. The application of the LAMP detection method of the brown rot of peach as claimed in claim 2 in early diagnosis of the brown rot of peach and monitoring and identification of germs.
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