CN112852995A

CN112852995A - Primer probe composition, kit and detection method for detecting fusarium graminearum

Info

Publication number: CN112852995A
Application number: CN202110282060.7A
Authority: CN
Inventors: 郭维; 梁晓艳; 张君
Original assignee: Institute of Food Science and Technology
Current assignee: Institute of Food Science and Technology
Priority date: 2021-03-16
Filing date: 2021-03-16
Publication date: 2021-05-28

Abstract

The invention relates to a primer-probe composition, a kit and a detection method for detecting Fusarium graminearum. The primer-probe composition includes a forward primer, a reverse primer and a probe for detecting the 1-500 bp sequence of the galactose oxidase precursor gene of Fusarium graminearum. The primer-probe composition has high specificity and sensitivity, can achieve specific amplification of the galactose oxidase gene of Fusarium graminearum, and has high sensitivity; the detection method has both high specificity And sensitivity, and easy to operate, can well meet the requirements of on-site detection of Fusarium graminearum.

Description

Primer probe composition, kit and detection method for detecting fusarium graminearum

Technical Field

The invention belongs to the technical field of biology, and relates to a primer probe composition, a kit and a detection method for detecting fusarium graminearum.

Background

Corn is the first crop in China in the world, the corn yield in China in 2019 is 26077 ten thousand tons, and accounts for 39.28 percent of the total grain yield. Maize ear rot (maize ear rot) caused by Fusarium graminearum is a major ear disease of maize, not only reducing maize yield, but also affecting seed quality. In addition, Fusarium graminearum can produce a variety of toxic secondary metabolites (trichothecene compounds), such as Deoxynivalenol (DON) and the like. DON can cause animal vomiting and has certain harm to human body, and is recognized as three carcinogens by the international cancer research organization. Therefore, the detection of fusarium graminearum in the corn for feed is of great importance to the safety of human and livestock.

The traditional classification and identification of fusarium is mainly based on morphological characteristics, and is distinguished and identified according to colony morphology, conidia, conidiophores and the like, which plays an important role in fusarium graminearum detection, however, the method is time-consuming and labor-consuming, and requires operators to have professional fusarium morphological identification knowledge and rich practical experience; with the development of molecular technology, Polymerase Chain Reaction (PCR) and real-time fluorescent quantitative PCR (real-time PCR) have been successfully applied to the detection of Fusarium graminearum, but the detection period is long, and the method depends on precise instruments such as a PCR instrument and cannot meet the requirement of rapid detection.

The loop-mediated isothermal amplification (LAMP) method can realize the rapid detection of fusarium graminearum.

CN104313128A discloses a method for detecting Fusarium graminearum based on loop-mediated isothermal amplification technology and a primer composition, wherein the primer composition is applied to detecting the Fusarium graminearum, a detection system can detect the Fusarium graminearum efficiently, specifically and sensitively at the isothermal condition of 62 ℃, but the reaction needs to be carried out at high temperature and needs a water bath kettle or other equipment for assistance.

In conclusion, the establishment of the detection method with lower reaction temperature and more simplified instrument requirements has important significance in the field of method development for rapidly detecting fusarium graminearum.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a primer probe composition, a kit and a detection method for detecting fusarium graminearum, wherein the detection method has high specificity and sensitivity, is simple, convenient and quick to operate, meets the requirement of field detection on the fusarium graminearum, and can be used for early diagnosis and disease monitoring of the field fusarium graminearum.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a primer probe composition for detecting fusarium graminearum, which comprises a forward primer, a reverse primer and a probe for detecting a 1-500 bp sequence of a galactose oxidase precursor gene of fusarium graminearum.

The primer and the probe have high specificity and sensitivity, can realize the specific amplification of galactose oxidase precursor gene of fusarium graminearum, and have high sensitivity.

Preferably, the forward primer comprises the nucleotide sequence shown as SEQ ID No. 1.

Preferably, the reverse primer comprises the nucleotide sequence shown in SEQ ID No. 2.

Preferably, the probe comprises the nucleotide sequence shown in SEQ ID No. 3.

Preferably, the 5' end of the reverse primer is modified with Biotin (Biotin).

Preferably, the 5' end of the probe is modified with a fluorescent group.

Preferably, the fluorophore comprises FAM.

Preferably, the 3' end of the probe is modified with an intermediate arm.

Preferably, the intermediate arm comprises C3-spacer.

Preferably, Tetrahydrofuran (THF) is modified at a position 31-36 bp (for example, 32bp, 33bp, 34bp or 35bp) away from the 5' end in the sequence of the probe.

SEQ ID No.1：

CGGAGGAACTGGAAGTCCTGAAGGGAGTCTT。

SEQ ID No.2：

Biotin-TTTGTGCCATCTGAGCTTAGATAAACCTCA。

SEQ ID No.3：

FAM-CACATTCTATGGGGCCAATGGAGATCCAAAGCC-THF-CCTCAC ACATACAC-C3spacer。

In the invention, the primer and the probe can be used for carrying out Recombinase Polymerase Amplification (RPA), the 5 ' end of the probe is provided with a carboxyfluorescein (FAM) label, the 3 ' end is provided with C3-spacer, the 5 ' end of the reverse primer is provided with Biotin, endonuclease IV (Nfo enzyme) in an RPA reaction system can specifically recognize and cut a THF molecule in a probe sequence, and the cut probe and the downstream primer can be amplified to obtain a double-labeled amplicon with both FAM label and Biotin.

In a second aspect, the present invention provides a kit for detecting fusarium graminearum, wherein the kit comprises the primer probe composition for detecting fusarium graminearum of the first aspect.

Preferably, the kit further comprises a recombinase polymerase amplification enzyme composition.

Preferably, the recombinase polymerase amplification enzyme composition comprises recA recombinase, single-stranded DNA binding protein, strand-displacing DNA polymerase and endonuclease IV (Nfo enzyme).

Preferably, the kit further comprises any one of a buffer, Nuclease-Free Water (Nuclease-Free Water), or magnesium acetate, or a combination of at least two of them.

Preferably, the Buffer comprises a Rehydration Buffer.

Preferably, the kit further comprises a lateral flow chromatography test strip.

In the invention, the primer and the probe can be used for carrying out high-specificity RPA reaction to obtain the dual-labeled amplicon of galactose oxidase precursor gene of the fusarium graminearum, wherein the dual-labeled amplicon carries both FAM (fatty acid amide) labels and Biotin, and the amplicon can be combined with a Biotin ligand on a detection line of a lateral flow chromatography test strip, so that the detection line is developed, and the purpose of detecting the fusarium graminearum is realized.

In a third aspect, the invention provides a primer probe composition for detecting fusarium graminearum in the first aspect and/or an application of the kit for detecting fusarium graminearum in the second aspect in detecting fusarium graminearum.

In a fourth aspect, the present invention provides a method of detecting fusarium graminearum, said method comprising the steps of:

(1) extracting the genome of a sample to be detected as a reaction template, and carrying out recombinase polymerase amplification by using the primer probe composition for detecting fusarium graminearum in the first aspect to obtain an amplification product;

(2) and detecting the amplification product by using a lateral flow chromatography test strip, and judging the detection result.

In the invention, the primer and the probe can be used for carrying out high-specificity RPA reaction to obtain the double-labeled amplicon of galactose oxidase precursor gene of the fusarium graminearum, wherein the double-labeled amplicon is provided with both FAM (fatty acid amide) label and Biotin, and the amplicon can be combined with a Biotin ligand on a detection line of a lateral flow chromatography test strip, so that the detection line is colored, and the purpose of detecting the fusarium graminearum is realized.

Preferably, the reaction system for recombinase polymerase amplification in step (1) comprises 100 fg/. mu.L-100 ng/. mu.L reaction template (e.g., 1 pg/. mu.L, 10 pg/. mu.L, 100 pg/. mu.L, 1 ng/. mu.L, 10 ng/. mu.L, 40 ng/. mu.L, 70 ng/. mu.L or 90 ng/. mu.L), 0.4-0.45. mu.M forward primer (e.g., 0.41. mu.M, 0.42. mu.M, 0.43. mu.M or 0.44. mu.M), 0.4-0.45. mu.M reverse primer (e.g., 0.41. mu.M, 0.42. mu.M, 0.43. mu.M or 0.44. mu.M), 0.05-0.15. mu.M probe (e.06. mu.M, 0.07. mu.M, 0.08. mu.M, 0.09. mu.12. mu.61. mu.M or 0.14. mu.61. M), 59-59% volume buffer (e.59.60.59%, 2.59% buffer, 2.59%, 2.60%, 60.4%, 60.5% or 60.8%), 24% to 26% by volume nuclease-free water (which may be, for example, 24.2%, 24.6%, 24.8%, 25%, 25.2%, 25.6% or 25.8%), 11 to 17mM magnesium acetate (which may be, for example, 12mM, 13mM, 14mM, 15mM or 16mM), and 0.1 to 0.3 mg/. mu.L of the recombinase polymerase amplification enzyme composition.

Preferably, the reaction system for recombinase polymerase amplification in step (1) comprises 1. mu.L of 100 ng/. mu.L reaction template, 2.1. mu.L of 10. mu.M forward primer, 2.1. mu.L of 10. mu.M reverse primer, 0.6. mu.L of 10. mu.M probe, 29.5. mu.L of regeneration Buffer, 12.2. mu.L of Nuclear-Free Water, 2.5. mu.L of 280mM magnesium acetate, and 5mg of recombinase polymerase amplification enzyme composition.

Preferably, the recombinase polymerase amplification temperature in step (1) is 37-42 ℃, including but not limited to 38 ℃, 39 ℃, 40 ℃ or 41 ℃.

Preferably, the recombinase polymerase amplification time in step (1) is 20-30 min, including but not limited to 21min, 22min, 24min, 25min, 26min, 27min or 28 min.

Preferably, the detecting in step (2) comprises mixing the amplification product with a hybridization detection buffer (HybriDetect assay buffer) to obtain a mixture, and placing the sample end of the lateral flow chromatography strip in the mixture.

Preferably, the volume ratio of the amplification product to the HybriDetect assay buffer is (0.03-0.07): 1, including but not limited to 0.04:1, 0.05:1 or 0.06: 1.

Preferably, the standing time is 3-5 min.

As a preferred technical scheme, the method for detecting the fusarium graminearum comprises the following steps of:

(1) extracting a genome of a sample to be detected as a reaction template, and preparing a reaction system: carrying out recombinase polymerase amplification for 20-30 min at 37-42 ℃ by using 0.1-0.3 mu M reaction template, 0.4-0.45 mu M forward primer, 0.4-0.45 mu M reverse primer, 0.5-0.15 mu M probe, 59-61% by volume of buffer solution, 24-26% by volume of nuclease-free water, 11-17 mM magnesium acetate and 0.1-0.3 mg/mu L recombinase polymerase amplification enzyme composition to obtain a reacted material;

(2) and mixing the amplification product with a HybriDetect assay buffer according to the volume ratio of (0.03-0.07): 1 to obtain a mixed solution, vertically placing the sample end of the lateral flow chromatography test strip in the mixed solution for 3-5 min, and judging the detection result.

Compared with the prior art, the invention has the following beneficial effects:

(1) the primer and the probe have higher specificity and sensitivity, can realize the specific amplification of galactose oxidase precursor gene of fusarium graminearum, and have high sensitivity;

(2) the detection method of the invention can carry out high specificity RPA reaction by utilizing the primer and the probe to obtain the galactose oxidase precursor gene of the fusarium graminearum, wherein the galactose oxidase precursor gene of the fusarium graminearum is provided with a FAM marker and a double-labeled amplicon of Biotin, and in addition, the RPA reaction can rapidly amplify nucleic acid to a detectable level at 37-42 ℃, so the detection method of the invention has high specificity and sensitivity, is convenient to operate and can well meet the requirement of field detection on the fusarium graminearum;

(3) the detection method can specifically detect the fusarium graminearum, has high sensitivity, can detect the genome template with the lowest concentration of 100 fg/mu L, and can effectively detect the fusarium graminearum in the corn.

Drawings

FIG. 1 shows the result of screening the RPA primer of Fusarium graminearum, wherein M is DNAmarker, 1-4 are the results of amplification of primer composition 1 against Fusarium graminearum, Fusarium verticillium, Fusarium sporogenes and Fusarium oxysporum, 5-8 are the results of amplification of primer composition 2 against Fusarium graminearum, Fusarium verticillium, Fusarium sporogenes and Fusarium oxysporum, and 9-12 are the results of amplification of primer composition 3 against Fusarium graminearum, Fusarium verticillium, Fusarium sporogenes and Fusarium oxysporum;

FIG. 2 shows the specific detection results of Fusarium graminearum, in which 1 is a positive control strain F.graminearum PH-1; 2 and 3 are f.graminearum isolated from inner mongolia municipalities; 4 is F.graminearum isolated from Liaoning province; 5 and 6 are f.graminearum isolated from shandong province; 7 and 8 are f.graminearum isolated from he province of henna; 9 and 10 are f.graminearum isolated from shanghai city, 11 is f.graminearum isolated from yunnan province; 12 are f.vertetillioides; 13 is f.oxysporum; 14 is f.proliferatum; f.fujikuroi at 15; 16 is Nigrospora sp; 17 is Rhizomucor variabilis; 18 is Phoma sp;

FIG. 3 shows the result of the sensitivity test of Fusarium graminearum, which amplifies the genomic DNA of Fusarium graminearum with different concentrations, from left to right: 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, and 10 fg/. mu.L;

fig. 4 shows symptoms after 2 days of corn kernel inoculation;

FIG. 5 shows the symptoms after 5 days of inoculation of corn stalks;

FIG. 6 shows the results of testing artificially inoculated corn kernels and stalks, wherein 1 is the test result (negative control) using water as a template; 2 is the detection result (positive control) using 100 ng/. mu.LF.graminearum PH-1 genome as the template; 3 is a detection result by taking the corn kernel genome inoculated with water as a template; 4, a detection result by taking corn seeds inoculated with F.graminearum PH-1 as a template; 5 is a detection result by taking the corn stalk genome inoculated with water as a template; and 6, a detection result by taking a corn stalk genome inoculated with F.graminearum PH-1 as a template.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

In the examples of the present invention, the hydration Buffer, magnesium acetate and enzyme dry powder composition were purchased from TwistDx, nucleic-Free Water from ThermoFisher Scientific, and HybriDetect assaybuffer and lateral flow strip from Milenia Biotec GmbH.

Example 1

Based on a bioinformatics analysis tool, a galactose oxidase gene (gaoA) of Fusarium graminearum (F.graminearum) is used as a detection target, gaoA sequences of the Fusarium graminearum and the rest 4 Fusarium graminearum are downloaded from an NCBI database, a specific primer composition is designed in a region with obvious sequence difference between the Fusarium graminearum and the other Fusarium by multi-sequence alignment analysis, 3 groups of primer compositions are preliminarily selected from the designed primer compositions, and the primer composition 1: f1(SEQ ID No. 1: CGGAGGAACTGGAAGTCCTGAAGGGAGTCTT), R1(SEQ ID No. 2: TTTGTGCCATCTGAGCTTAGATAAACCTCA); and (3) primer combination 2: f2(SEQ ID No. 4: CTCTGTTGTTCTTCCAGACGGAAGCACGTTTATTA), R2(SEQ ID No. 5: CTGGTCAGTATTAACCGTGTGTGTCGCTGTACCAT), primer combination 3: f3(SEQ ID No. 6: AGATGGCACAAACTGGGGCAGCCCTGTTGCGTCA), R3(SEQ ID No. 7: AGCGGCCAAGGCCAGGCTGAGGGGCTGTGTAA).

A primer composition with strong specificity is screened through common PCR amplification, fig. 1 shows that only the primer composition 1 can realize the specific amplification of fusarium graminearum and meet the requirement of an RPA experiment on 100-300 bp of an amplification product, the primer composition 1 is adopted for subsequent recombinase polymerase amplification of the fusarium graminearum, F1 is a forward primer, R1 is a reverse primer, and a probe (SEQ ID No. 3: FAM-CACATTCTATGGGGCCAATGGAGATCCAAAGCC-THF-CCTCACACATACAC-C3spacer) is designed according to a probe design principle.

Example 2

The specificity was evaluated by examining f.graminearum PH-1 and f.graminearum isolated from inner mongolian autonomous region, jeanning province, shandong province, henna province, shanghai city, yunnan province, and using other 4 closely related or complex infested strains (f.verteticiloides, f.proliferum, f.oxysporum, f.fujikuroi) and non-fusarium (Phoma sp., Rhizomucor variabilis, Nigrospora sp.) as control strains.

The kit is prepared by adopting a forward primer (SEQ ID No.1), a reverse primer (SEQ ID No.2) and a probe (SEQ ID No.3), and further comprises a regeneration Buffer, Nuclease-Free Water (Nuclease-Free Water), magnesium acetate, a recombinase polymerase amplification enzyme composition (containing recA recombinase, single-stranded DNA binding protein, strand displacement DNA polymerase and endonuclease IV) and a lateral flow chromatography test strip, wherein the concentrations of the forward primer, the reverse primer and the probe are all 10 mu M, and the concentration of the magnesium acetate is 280 mM.

Extracting the genome of the strain to be detected, taking 1 mu L of the genome as a reaction template, carrying out RPA reaction, carrying out two parallel tests, wherein the reaction system is shown in Table 1, and the reaction program is as follows: performing reaction amplification at 40 ℃ for 25min, mixing 5 μ L of the amplification product with 95 μ L of HybriDetect assaybuffer, vertically immersing the sample end of the lateral flow chromatography test strip into the mixed solution, standing at room temperature for 3min, and determining the detection result, as shown in FIG. 2.

TABLE 1

Reagent	Dosage of
		RehydrationBuffer	29.5μL
Nuclease-FreeWater	12.2μL
		Forward primer	2.1μL
Reverse primer	2.1μL
		Probe needle	0.6μL
Genome	1μL
		Recombinase polymerase amplification enzyme compositions	5mg
Magnesium acetate	2.5μL

As can be seen from fig. 2, 1 is a positive control strain f.graminearum PH-1; 2 and 3 are f.graminearum isolated from inner mongolia municipalities; 4 is F.graminearum isolated from Liaoning province; 5 and 6 are f.graminearum isolated from shandong province; 7 and 8 are f.graminearum isolated from he province of henna; 9 and 10 are f.graminearum isolated from shanghai city, 11 is f.graminearum isolated from yunnan province; 12 are f.vertetillioides; 13 is f.oxysporum; 14 is f.proliferatum; f.fujikuroi at 15; 16 is Nigrospora sp; 17 is Rhizomucor variabilis; 18 is Phoma sp, wherein only the detection lines and the control lines of the fusarium graminearum (1-11) have indicator bands to show a positive detection result, and the detection lines of other pathogenic fungi have no indicator bands to show a negative detection result, which indicates that the primer probe composition provided by the invention has higher specificity on the fusarium graminearum.

Example 3

The genome of F.graminearum PH-1 was subjected to 10-fold gradient dilution after the concentration was measured with a spectrophotometer, the DNA concentration was set in the range of 10 ng/. mu.L to 100 ng/. mu.L (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 and 10 fg/. mu.L), the RPA reaction and detection were carried out in the reaction system and under the conditions described in example 2 using 1. mu.L of each diluted concentration of DNA as a template, and the detection results were judged from the change in color of the detection line indicator band, and are shown in FIG. 3.

As can be seen from FIG. 3, the test strip detection line with the DNA concentration of 100 fg/. mu.L-100 ng/. mu.L can be observed to have a band, and the test strip detection line with the DNA concentration of 10 fg/. mu.L has no band, which indicates that the detection requirement can be satisfied when the Fusarium DNA concentration is 100 fg/. mu.L, indicating that the detection method of the present invention has high sensitivity.

Example 4

In this embodiment, corn is taken as an example to detect fusarium graminearum in corn.

Samples to be detected are B73 corn grains and B73 corn stalks, F.graminearum PH-1 is respectively and manually inoculated into the samples, an equal amount of water is inoculated as a control, the symptom after the corn grains are inoculated for 2 days is shown in figure 4, the symptom after the corn stalks are inoculated for 5 days is shown in figure 5, a novel plant genome DNA extraction kit (Tiangen) is adopted to extract genome according to instructions, RPA reaction and detection are carried out according to the method described in example 2, meanwhile, the pure water and the F.graminearum PH-1 genome are used as templates to carry out detection as a control, and the result is shown in figure 6.

As can be seen from FIG. 6, in which 1 is the detection result using water as a template (negative control); 2 is the detection result (positive control) using 100 ng/. mu.L F.graminearum PH-1 genome as the template; 3 is a detection result by taking the corn kernel genome inoculated with water as a template; 4, a detection result by taking corn seeds inoculated with F.graminearum PH-1 as a template; 5 is a detection result by taking the corn stalk genome inoculated with water as a template; and 6, a detection result by taking a corn stalk genome inoculated with F.graminearum PH-1 as a template. The result shows that the corn seeds and stems inoculated with the fusarium graminearum are positive, and the corn seeds and stems not inoculated with the fusarium graminearum are negative, so that the method provided by the invention can quickly and accurately detect the fusarium graminearum in the corn.

In conclusion, the detection method has the characteristics of strong specificity, high accuracy, simple operation and no need of expensive equipment such as a PCR instrument and the like, can realize the on-site rapid detection of whether the corn carries the pathogenic fusarium graminearum, and is easy to popularize and apply in a large scale.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> institute for agricultural product processing of Chinese academy of agricultural sciences

<120> primer probe composition, kit and detection method for detecting fusarium graminearum

<130> 20210312

<160> 7

<170> PatentIn version 3.3

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Claims

1. a primer-probe composition for detecting Fusarium graminearum, it is characterized in that, described primer-probe composition comprises the forward direction of the galactose oxidase precursor gene 1～500bp sequence that detects Fusarium graminearum Primers, reverse primers and probes.

2. primer-probe composition according to claim 1, is characterized in that, described forward primer comprises the nucleotide sequence shown in SEQ IDNo.1;

Preferably, the reverse primer comprises the nucleotide sequence shown in SEQ ID No.2;

Preferably, the probe comprises the nucleotide sequence shown in SEQ ID No.3;

Preferably, the 5' end of the reverse primer is modified with biotin;

Preferably, the 5' end of the probe is modified with a fluorescent group;

Preferably, the fluorescent group includes FAM;

Preferably, the 3' end of the probe is modified with a spacer arm;

Preferably, the spacer arm comprises a C3-spacer;

Preferably, in the sequence of the probe, 31-36 bp from the 5' end is modified with tetrahydrofuran.

3. A test kit for detecting Fusarium graminearum, wherein the test kit comprises the primer-probe composition for detecting Fusarium graminearum according to claim 1 or 2;

Preferably, the kit further comprises a recombinase polymerase amplifying enzyme composition;

Preferably, the recombinase polymerase amplifying enzyme composition comprises recA recombinase, single-stranded DNA binding protein, strand displacement DNA polymerase and endonuclease IV;

Preferably, the kit further comprises any one or a combination of at least two of buffer, nuclease-free water or magnesium acetate;

Preferably, the buffer comprises rehydration buffer;

Preferably, the kit further comprises lateral flow chromatography test strips.

4. the primer probe composition of detecting Fusarium graminearum as claimed in claim 1 or 2 and/or the test kit of detecting Fusarium graminearum according to claim 3 is in the detection of Fusarium graminearum application.

5. a method for detecting Fusarium graminearum, is characterized in that, described method comprises the following steps:

(1) extracting the genome of the sample to be detected as a reaction template, using the primer-probe composition for detecting Fusarium graminearum according to claim 1 or 2 to carry out recombinase polymerase amplification to obtain an amplification product;

(2) Use lateral flow chromatography test strips to detect the amplification product, and determine the detection result.

6 . The method according to claim 5 , wherein the reaction system amplified by the recombinase polymerase in step (1) comprises 100 fg/μL～100ng/μL reaction template, 0.4～0.45 μM forward primer, 0.4 μM ～0.45μM reverse primer, 0.05～0.15μM probe, 59%～61% by volume buffer, 24%～26% by volume nuclease-free water, 11～17mM magnesium acetate and 0.1～0.3mg /μL Recombinase Polymerase Amplification Enzyme Composition.

The method according to claim 5 or 6, wherein the temperature of the recombinase polymerase amplification in step (1) is 37-42°C;

Preferably, the amplification time of the recombinase polymerase in step (1) is 20-30 min.

8. The method according to any one of claims 5-7, wherein the detection in step (2) comprises mixing the amplification product with a hybridization detection buffer to obtain a mixed solution, and the lateral flow chromatography The sample end of the test strip is placed in the mixture.

9. The method according to claim 8, wherein the volume ratio of the amplification product to the hybridization detection buffer is (0.03～0.07):1;

Preferably, the placing time is 3-5 min.

10. The method according to any one of claims 6-9, wherein the method comprises the steps of:

(1) Extract the genome of the sample to be detected as a reaction template, and prepare a reaction system: 0.1-0.3 μM reaction template, 0.4-0.45 μM forward primer, 0.4-0.45 μM reverse primer, 0.5-0.15 μM probe, the volume percentage is 59%-61% buffer, 24%-26% nuclease-free water by volume, 11-17mM magnesium acetate and 0.1mg/μL recombinase polymerase amplifying enzyme composition, reconstituted at 37-42°C Enzyme polymerase amplification for 20-30 minutes to obtain the reacted material;

(2) Mix the amplification product with the hybridization detection buffer in a volume ratio of (0.03-0.07):1 to obtain a mixed solution, and place the sample end of the lateral flow chromatography test strip vertically in the mixed solution 3 ~ 5min, judge the test result.