CN110982922A

CN110982922A - Primer composition and method for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP

Info

Publication number: CN110982922A
Application number: CN201911327198.3A
Authority: CN
Inventors: 张敬泽; 江胡彪; 鲍一丹
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2020-04-10
Anticipated expiration: 2039-12-20
Also published as: CN110982922B

Abstract

The application discloses a primer composition for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP, which comprises the following components: forward inner primer FIP (SEQ ID NO:1), reverse inner primer BIP (SEQ ID NO:2), forward outer primer F3(SEQ ID NO:3), reverse outer primer B3(SEQ ID NO:4), forward loop primer LF (SEQ ID NO:5) and reverse loop primer LB (SEQ ID NO: 6); the application also discloses a kit for rapidly detecting the rice bakanae disease pathogenic bacterium fusarium granatum based on LAMP, which comprises the primer composition. The application also discloses a method for rapidly detecting the rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP, which comprises the following steps: and extracting the genome DNA of the sample to be detected, and carrying out LAMP reaction by using the extracted genome DNA as a DNA template and the primer composition as a primer. The LAMP detection primer and the LAMP detection method provided by the application can be used for detecting diseased seeds and early diagnosing the rice bakanae disease caused by fusarium granatum.

Description

Primer composition and method for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP

Technical Field

The application relates to the technical field of fusarium granatum molecular detection, in particular to a primer composition and a method for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP.

Background

The bakanae disease of rice is one of the important seed-borne diseases of rice. Since its first discovery in japan, the disease has spread to asia, africa, and north america. Typical symptoms of bakanae disease are overgrowth, yellowing of leaves, chlorosis, dwarfing, root and crown rot, and even death. The occurrence of diseases in the seedling stage can cause the yield of rice to be reduced by 10-30%, and the yield loss can reach more than 50% seriously, thereby forming a serious threat to the production of rice crops. Some studies have shown that, although 4 species of fusarium [ fusarium lutescens (fusarium. fujikuroi), fusarium proliferatum (f. verticillium), fusarium verticillioides (f. verticillioides), and f. andiyazi ] can cause varying degrees of rice bakanae symptoms, only fusarium lutescens is the most prominent strong pathogen causing typical symptoms. Current studies demonstrate that only gibberellins GA1, GA3, GA4, and GA7 are biologically active in plants. Fusarium canescens is the only species producing active gibberellins, while others do not have gibberellin products and gene expression, possibly due to gene mutations, or promoter damage. Gibberellins are plant hormones that promote the overgrowth of seedlings. Therefore, the symptom of overgrowth of bakanae disease is closely related to the secretion of gibberellin by fungi.

According to the results of the inventor on the separation of fusarium species from main rice cultivars in Zhejiang, Anhui and Jiangxi, it is shown that 414 segregating lines are obtained from 24 varieties. Morphological and molecular morphological identification shows that the bacillus subtilis is divided into 6 species which are respectively fusarium granatum and account for 81.4 percent; fusarium proliferatum (F. proliferatum), 8.44%; equiseti, 6.03%; asiticum, 0.5%, f incarnatum, 2.66%, f andiayazi, 0.97%. It is shown that Fusarium canopi is the most prominent pathogen causing rice bakanae disease in rice cultivation areas of 3 provinces in south.

With the development of molecular biology technology, more and more molecular biology technologies are applied to the detection of plant pathogenic diseases, for example, ordinary PCR and real-time fluorescence quantitative PCR technologies are widely applied to pathogen detection, but these technologies require a precise and expensive temperature cycling device, the reaction reagents are expensive, special personnel are required to operate, and the detection process is complex, so that the requirement of rapid detection cannot be met. In order to solve this problem, japanese scholars have invented a so-called loop-mediated isothermal amplification (LAMP) technique, which requires designing 3 primer pairs capable of recognizing 6 specific sites of conserved sequences, followed by isothermal amplification with DNA polymerase (Bst DNA polymerase) and direct observation of color change in the tube to determine the presence of pathogenic bacteria. The color change in the tube is due to the addition of highly sensitive DNA fluorescent dye into the reaction tube prior to the reaction. And judging the existence of pathogenic bacteria in the sample according to the characteristics of the dye. The technology has the characteristics of high sensitivity, strong specificity, simple amplification condition, visualized product and the like, and is widely applied to the detection of phytophthora sojae, rice blast bacteria and verticillium grisea. However, the key to LAMP technology is the specificity and sensitivity of the primers. Genomic analysis found that the polyketide synthase gene (polyketide synthase) and non-ribosomal peptide synthase gene (NRPS 31) involved in mycotoxin metabolism are unique among Fusarium canum. Recently, LAMP detection technology was developed based on NRPS31 gene, and LAMP detection technology was developed based on ribosome-specific fragment. However, these are not related to gibberellin metabolic pathways.

Through genome analysis, analysis shows that the promoter region of desaturase gene converting gibberellin GA4 into GA7 (GA 3 is synthesized subsequently) in gibberellin metabolic pathway has great difference with homologous sequences of other species, so that LAMP primers are designed and an LAMP detection technology system is established, which not only has strong specificity and can rapidly and accurately identify the existence of pathogenic bacteria, but also can well predict the generation of gibberellin GA4, GA7 and GA3 generated by the bacteria.

Disclosure of Invention

The application provides a primer composition, a kit and a method for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP.

A primer composition for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP comprises: a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF and a reverse loop primer LB; the base sequence is as follows:

FIP:5′-CTACCAAGGTAAGGTATCTGCTAAGTTGTTTGTCGGCTGATC-3′(SEQ ID NO:1)；

BIP:5′-GCTTCACTGGCCTTGGAGTCATAAGGTTTATGGAGACGCAC-3′(SEQ ID NO:2)；

F3:5′-TTGACAAAGTTCGGTGCC-3′(SEQ ID NO:3)；

B3:5′-TCTGACTTGATTCACAGATG-3′(SEQ ID NO:4)；

LF:5′-GGCGTTCATACCACGACCT-3′(SEQ ID NO:5)；

LB:5′-GTACGGTATCACCGTTGCATT-3′(SEQ ID NO:6)。

the application also provides a kit for rapidly detecting the rice bakanae disease pathogenic bacterium fusarium granatum based on LAMP, and the kit comprises the primer composition.

Optionally, the method includes: 3.2mM forward inner primer FIP and 3.2mM reverse inner primer BIP; 0.2mM forward outer primer F3 and 0.2mM reverse outer primer B3; 0.4mM forward loop primer LF and 0.4mM reverse loop primer LB.

Optionally, the method further includes: 10 × ThermoPol buffer, 1.4mM dNTPs, 0.8M betaine, Bst DNA polymerase and 10000 × dye.

Optionally, the reaction system of the kit is 25 μ l, and comprises: 3.0. mu.l of 10 XTMOL buffer, 3.5. mu.l of 10mM dNTPs, 4.0. mu.l of 5M betaine, LAMP detection primer, 2. mu.l of DNA template, 1.0. mu.l of Bst DNA polymerase and 0.25. mu.l of 10000 Xdye, and ultra-pure water for volume fixing;

wherein the LAMP detection primer comprises: 2.0. mu.l of 40mM forward inner primer FIP, 2.0. mu.l of 40mM reverse inner primer BIP; 0.5. mu.l of 10mM forward outer primer F3 and 0.5. mu.l of 10mM reverse outer primer B3; 1.0. mu.l of 10mM forward loop primer LF and 1.0. mu.l of 10mM reverse loop primer LB.

The application also provides a method for rapidly detecting the rice bakanae disease pathogenic bacterium fusarium granatum based on LAMP, which comprises the following steps:

and extracting the genome DNA of the sample to be detected, and carrying out LAMP reaction by using the extracted genome DNA as a DNA template and the primer composition as a primer.

Several alternatives are provided below, but not as an additional limitation to the above general solution, but merely as a further addition or preference, each alternative being combinable individually for the above general solution or among several alternatives without technical or logical contradictions.

Optionally, the LAMP reaction system is 25 μ l; the method comprises the following steps: 3.0. mu.l of 10 XThermoPol buffer, 3.5. mu.l of 10mM dNTPs, 4.0. mu.l of 5M betaine, LAMP detection primer, 2. mu.l of DNA template, 1.0. mu.l of Bst DNA polymerase and 0.25. mu.l of 10000 Xdye, and ultra-pure water for volume fixing;

Optionally, the LAMP reaction procedure is as follows: reacting for 30-75 min at 60-67 ℃. Alternatively, 65 ℃ for 55 min.

Optionally, the method further includes: and judging the detection result according to the color change of the reaction liquid. SYBR Green I is taken as a dye, and Green represents a positive result; orange indicates a negative result.

Optionally, the sample to be detected is a rice seed or a rice seedling.

Compared with the prior art, the application has at least the following beneficial effects:

(1) the LAMP detection primer and the LAMP detection method provided by the application have strong specificity, can be easily distinguished from fusarium species and other fungi, and are particularly good in repeatability.

(2) The LAMP detection primer and the LAMP detection method have high sensitivity which is 10000 times higher than that of common PCR detection.

(3) The LAMP detection primer and the LAMP detection method can be used for detecting diseased seeds, and the test steps comprise seed grinding, genome DNA extraction, LAMP detection and color change reaction in a detection system, and whether the seeds carry pathogenic bacteria or not is judged.

(4) The LAMP detection technology provided by the application can be used for early diagnosis of the rice bakanae disease caused by fusarium granatum. Collecting asymptomatic rice seedlings (possibly carrying pathogenic bacteria), extracting genome DNA by using a plant DNA extraction Kit (2 XT 5 direct PCR Kit), and carrying out field detection on the pathogenic bacteria by LAMP detection.

Drawings

FIG. 1 shows the position of LAMP detection primer on the partial sequence of desaturase gene promoter.

FIG. 2 is a comparison of the result of detection of Fusarium granatum based on LAMP with the result of electrophoresis of a general PCR amplification product (arrows in the figure are indicated as green).

FIG. 3 shows the results of the sensitivity assay for LAMP detection of Fusarium granatum (arrows in the figure are indicated as green).

FIG. 4 shows the results of detection of the seed of the rice harboring bacteria by LAMP and the ordinary PCR method (arrows in the figure are marked in green).

FIG. 5 shows the results of the LAMP-based detection of artificially inoculated diseased tissues and the corresponding general PCR (arrows in the figure are indicated in green).

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

Example 1 Fusarium granatum LAMP detection primer design and specificity evaluation

(1) LAMP detection primer design

By consulting fusarium fungus classification and metabolic pathway documents and combining genome comparative analysis, particularly gibberellin-related genome sequences are compared, homologous sequences are obtained from desaturase gene promoter regions, and LAMP detection primers are designed according to interspecies base sequence differences (figure 1).

The primer sequence comprises: a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF and a reverse loop primer LB; the specific sequence is as follows:

FIP:5′-GCTACCAAGGTAAGGTATCTGCTAAGTTGTTTGTCGGCTGATC-3′

BIP:5′-GCTTCACTGGCCTTGGAGTCATAAGGTTTATGGAGACGCAC-3′

F3:5′-TTGACAAAGTTCGGTGCC-3′

B3:5′-TCTGACTTGATTCACAGATG-3′

LF:5′-GGCGTTCATACCACGACCT-3′

LB:5′-GTACGGTATCACCGTTGCATT-3′。

(2) LAMP detection system constitution

The LAMP detection system is composed of: the total reaction was 25. mu.l, including 3.0. mu.l 10 × ThermoPol buffer (200mM Tris-HCl (pH 8.8), 100mM KCl, 100mM (NH)₄)₂SO₄、20mM MgSO₄And 1% Triton X-100), 3.5. mu.l of 10mM dNTPs, 4.0. mu.l of 5M betaine, LAMP detection primer (2.0. mu.l of 40mM forward inner primer FIP, 2.0. mu.l of 40mM reverse inner primer BIP; 0.5. mu.l of 10mM forward outer primer F3 and 0.5. mu.l of 10mM reverse outer primer B3; 1.0. mu.l 10mM forward loop primer LF and 1.0. mu.l 10mM reverse loop primer LB), DNA template, 1.0. mu.l Bst DNA polymerase (8U/. mu.l) and 0.25. mu.l 10000 Xdye (SYBR Green I), and finally made up with ultra pure water.

The LAMP reaction program is as follows: 60-67 ℃ for 30-75 min.

The LAMP reaction solution color change is used as a detection result mark: yellow-green indicates a positive result; orange-yellow indicates a negative result.

(3) Strains tested

To compare the specificity of the primers, Fusarium gambogic ZJ01 was chosen as a positive control, 13 other species of the pathogenic bacteria Fusarium and Fusarium isolated from rice were selected, and 10 strains of the pathogenic bacteria and other groups of rice (see Table 1)

TABLE 1 comparison of selected strains and amplification by LAMP and PCR

(4) Extraction of DNA

The 23 strains were inoculated on PDA plates, cultured in the dark at 25 ℃ for 3 days, the cake (0.5 mm directly) was excised from the edge of the colony and transferred to PDB medium, cultured with shaking at 25 ℃ for 3 to 5 days (150rpm), the mycelia were collected by filtration, then ground into powder in liquid nitrogen, and the genomic DNA in each sample was extracted using a fungal crude extract kit (Bio Inc.) (see the description for the specific steps). The obtained DNA was dissolved in TE buffer, and the DNA concentration was measured.

(5) Analysis of LAMP specificity

In the LAMP detection system established above, 2. mu.l of the DNA template of the above 23 test lines and a control (water) were added, respectively, before addition of Bst DNA polymerase. The LAMP reaction program is as follows: 65 ℃ for 55 min.

The assay results are shown in FIG. 2, A, tube No. 1 is green (positive) and is Fusarium gambieri ZJ 01; tube 2 is orange (negative) as a control with water; the reference pathogenic bacteria 3-24 are orange (negative), and the reference pathogenic bacteria 3-24 are respectively: fusarium proliferatum F.proliferatum, Fusarium equiseti F.equiseti No. 4, Fusarium oxysporum F.oxysporum No. 5, Fusarium avenaceum F.avenaceum, Fusarium equiseti F.asiticum No. 7, Fusarium graminearum F.graminearum, Fusarium solani F.solani, Fusarium verticillioides F.verticillioides 10, Fusarium incarnatum F.verticillioides 11, F.commune No. 12, F.amyguium No. 13, F.andiaiyazi, F.boothiai No. 14, Utilia nigra No. 15, Usula utilis J26, Utilia nigra U.escitala J6, Verticillium Verticillium Verticillium 086, Verticillium dahlia 18, Verticillum grandiflorum V. 2, Utilia 19, Utilia oryzae No. 20, Utiliella graminearia solani P23, Utiliella solani P.

The LAMP detection result shows (FIG. 2), the detection result of only Fusarium fujikuroi (Fusarium fujikuroi) of the primer composition of the present application is green (positive), and the detection result of the rest is orange (negative), which indicates that the primer composition has strong specificity.

Further using common PCR to verify, wherein the reaction system is as follows: a25. mu.l reaction system included a 25. mu.l system comprising 12.5. mu.l of a 2X Taq Master mix (including Taq DNA Polymerase, MgCl₂dATP, dCTP, dGTP, dTTP) (Shanghai Huihing Biotech Co., Ltd.), 1.0. mu.l of 10mM reverse outer primer B3 and 1.0. mu.l of 10mM reverse outer primer B3, 2.0. mu.l of genomic DNA of the test strain, and 8.5. mu.l of ultrapure water.

The PCR procedure was: pre-denaturation at 94 ℃ for 3 min; then, the mixture enters circulation, denaturation is carried out for 1min at 94 ℃, annealing is carried out for 1min at 54 ℃, and extension is carried out for 1min at 72 ℃ for 35 cycles; finally, the temperature is prolonged by 10min at 72 ℃.

After the reaction, 3. mu.L of the amplified product was electrophoresed in 1.0% agarose gel and photographed, and the result is shown in B in FIG. 2, lane 1 is a Fusarium granatum PCR product band (234 bp); lane 2 is a water control, no band; and lanes 3-24 are test reference pathogens (supra). The PCR amplification result (B in FIG. 2) shows that only the band amplified from the sample containing the genome of Fusarium granatum is completely consistent with the LAMP detection result.

Example 2 detection of LAMP reaction sensitivity

In order to determine the sensitivity of the LAMP detection method, sensitivity comparison analysis is carried out on the LAMP detection method and the LAMP detection method compared with the sensitivity of common PCR amplification products. The genomic DNA extracted from the above Fusarium granatum ZJ01 was used as a template, and the concentration was determined spectrophotometrically to be 94.0 ng/. mu.l, which was then serially diluted to 9.4ng, 0.94ng, 94pg, 9.4pg, 0.94pg, 94fg, 9.4fg and 0.94 fg. Mu.l of serially diluted DNA was aspirated separately and added to the LAMP detection system. The LAMP reaction program is as follows: 65 ℃ for 55 min. CK is a negative control (no DNA).

The measurement results are shown in fig. 3, where a: the concentrations of the genomic template DNA in tubes Nos. 1 to 9 were 94ng, 9.4ng, 0.94ng, 94pg, 9.4pg, 0.94pg, 94fg, 9.4fg, and 0.94fg, respectively. CK is a negative control (no DNA). Among them, tubes 1 to 7 showed green and positive, 8, 9, and CK showed orange and negative. Template concentrations above 94fg can be detected.

In FIG. 3, B is an electrophoretogram of the product after LAMP reaction, and template concentration above 0.94pg can be detected; c is an electrophoretogram of a PCR amplification product with the same concentration as the LAMP detection template. Template concentrations above 0.94ng could be detected.

The measurement result shows that LAMP detection can reach the template concentration of over 94fg, while ordinary PCR amplification can detect the template concentration of over 0.94 ng. The LAMP detection sensitivity is 10000 times higher than that of the common PCR.

Example 3 detection of Rice seed for carrying bacteria

Seeds on plants with typical symptoms of rice bakanae disease and seeds on corresponding plants without symptoms are collected from 3 fields and dried in the sun for storage according to the harvesting procedure of the seeds. For detecting the seed-carrying bacteria, 10 seeds of the diseased and healthy plants in each field were taken, each sample was ground into powder in liquid nitrogen, and the genomic DNA was extracted using an Ezup column type fungal genomic DNA extraction kit (Shanghai Bioengineering Co., Ltd.) (the specific steps are described in the specification) and dissolved in TE buffer.

In the LAMP detection system established in example 1, 2. mu.l of the DNA template of each test sample was added before addition of Bst DNA polymerase. In each field sample, the diseased seed sample was repeated 6 times, and the control (healthy seed sample) was repeated 4 times. The LAMP reaction program is as follows: 65 ℃ for 55 min.

The results of the measurement are shown in FIG. 4, in which tube 1 is a positive control (DNA from a colony) and shows green color; tube 2 is a negative control (no DNA) showing orange color; 4, 6-9 and 12 tubes of samples from the field 1 are from diseased plant seed samples, and the samples show green and are positive; 3.5, 10, 11 tubes, from healthy plant sample seeds, orange, negative; of the samples from

field

2, 13, 15, 18, 20, 21-22 tubes were from the diseased plant seed samples, which showed green and positive; 14. 16, 17 and 19 tubes are from the healthy plant seed sample, are orange yellow and are negative; of the samples from field 1, 26-27, 29-32 tubes were from the diseased seed sample and positive, 23-25,28 tubes were from the healthy seed sample and negative, showing orange yellow.

The results of the measurement (A in FIG. 4) showed that the reaction tube was green and positive only in the diseased seed sample, and negative in the healthy seed sample.

Using the method of example 2, further ordinary PCR amplification was performed, and the results of electrophoresis of the PCR products showed (B in FIG. 4) that only a band was amplified in the sample containing the genome of Fusarium granatum, which was consistent with the LAMP detection results.

Example 4 detection of Rice seedling carrying bacteria

To detect rice plants infected with bakanae disease, rice seeds (Jinzao 47 variety) were used at a concentration of 10⁶The individual/ml conidia suspension of Fusarium granatum was artificially soaked for 36 hours. The inoculated seeds were sown in plastic pots of 10X 10cm and placed in an illumination incubator at 28 ℃ for cultivation. Seeds were treated by soaking in sterile water for 36 hours as a control. After 15 days of sowing, the stalks of the seedlings which have not been treated with different symptoms are cut by a 0.2-2mm puncher. For on-site detection, genomic DNA was extracted using the Kit 2 XT 5 Direct PCR Kit (Plant) (Shanghai Biotech engineering Co., Ltd.). The stalk sample is put into a centrifuge tube and added with 30ml Lysis Buffer A, and heated for 10min at 95 ℃. After standing, 2. mu.l of supernatant was taken as a template of the LAMP reaction system. Sterilized Water treatment control genomic DNA was obtained in the same manner. The LAMP reaction program is as follows: 65 ℃ for 55 min.

The results of the assay are shown in FIG. 5, where tube 1 is a positive control (DNA from colonies) and appears green; tube 2 is a negative control (inoculated with sterile water) and appears orange; the DNA templates in tubes 3-5, 8-10, 12-15, 17, 20 and 23 were derived from plant tissue inoculated with Fusarium granatum and were yellow-green and positive. Tubes 6-7, 11, 16, 18, 19, 21-22 were from healthy control tissue, shown orange, and negative.

The results of the assay (a in fig. 5) showed that all of the seedling samples inoculated with pathogenic bacteria showed positive, while the seedling samples treated with sterilized water showed negative.

Using the method of example 1, the method of ordinary PCR amplification was carried out, and B in FIG. 5 is the result of ordinary PCR detection, and the results of the measurement showed that all the seedling samples of the seeds inoculated with pathogenic bacteria showed positive, while those treated with sterilized water showed negative, consistent with the LAMP detection result.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Zhejiang university

<120> primer composition and method for rapidly detecting rice bakanae disease pathogenic bacterium fusarium granatum based on LAMP

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Claims

1. The primer composition for rapidly detecting the rice bakanae disease pathogenic bacterium fusarium granatum based on LAMP is characterized by comprising the following components in parts by weight: a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF and a reverse loop primer LB; the base sequence is as follows:

FIP:5′-CTACCAAGGTAAGGTATCTGCTAAGTTGTTTGTCGGCTGATC-3′；

BIP:5′-GCTTCACTGGCCTTGGAGTCATAAGGTTTATGGAGACGCAC-3′；

F3:5′-TTGACAAAGTTCGGTGCC-3′；

B3:5′-TCTGACTTGATTCACAGATG-3′；

LF:5′-GGCGTTCATACCACGACCT-3′；

LB:5′-GTACGGTATCACCGTTGCATT-3′。

2. a kit for rapidly detecting Fusarium celastrum, a pathogenic bacterium of rice bakanae disease based on LAMP, which is characterized by comprising the primer composition according to claim 1.

3. The kit of claim 2, comprising: 3.2mM forward inner primer FIP and 3.2mM reverse inner primer BIP; 0.2mM forward outer primer F3 and 0.2mM reverse outer primer B3; 0.4mM forward loop primer LF and 0.4mM reverse loop primer LB.

4. The kit of claim 2, further comprising: 10 × ThermoPol buffer, 1.4mM dNTPs, 0.8M betaine, Bst DNA polymerase and 10000 × dye.

5. The kit according to claim 2, wherein the reaction system of the kit is 25 μ l, comprising: 3.0. mu.l of 10 XTMOL buffer, 3.5. mu.l of 10mM dNTPs, 4.0. mu.l of 5M betaine, LAMP detection primer, 2. mu.l of DNA template, 1.0. mu.l of Bst DNA polymerase and 0.25. mu.l of 10000 Xdye, and ultra-pure water for volume fixing;

6. A method for rapidly detecting rice bakanae disease pathogenic bacteria Fusarium granatum based on LAMP is characterized by comprising the following steps:

extracting the genomic DNA of the sample to be tested, and carrying out LAMP reaction by using the extracted genomic DNA as a DNA template and the primer composition as claimed in claim 1 as a primer.

7. The method of claim 6, wherein the LAMP reaction is carried out in a volume of 25. mu.l; the method comprises the following steps: 3.0. mu.l 10 XThermoPol buffer, 3.5. mu.l 10mM dNTPs, 4.0. mu.l 5M betaine, LAMP detection primer, 2. mu.l DNA template, 1.0. mu.l Bst DNA polymerase and 0.25. mu.l 10000 Xdye, and ultra-pure water to constant volume;

8. The method of claim 6, wherein the LAMP reaction is programmed as follows: reacting for 30-75 min at 60-67 ℃.

9. The method of claim 6, further comprising: and judging the detection result according to the color change of the reaction liquid.

10. The method of claim 6, wherein the sample to be tested is young rice plants or rice seeds.