CN113186333A - Rapid LAMP (loop-mediated isothermal amplification) detection method for fusarium oxysporum f.sp.cubense - Google Patents
Rapid LAMP (loop-mediated isothermal amplification) detection method for fusarium oxysporum f.sp.cubense Download PDFInfo
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Abstract
The invention discloses a rapid LAMP detection method for watermelon fusarium oxysporum. The method takes the specific DNA fragment of the fusarium oxysporum watermelon specialization type as a target, designs a set of sensitive and specific LAMP primers, and establishes a rapid LAMP detection system of the fusarium oxysporum by combining a DNA rapid extraction method. The LAMP system has the lower limit of detection on FON-1 genome DNA of 10 pg/mu L and the lower limit of detection on the bacteria carrying capacity of plants of 5 multiplied by 102Spores/g. The system only needs about 90min from DNA extraction to detection result acquisition, and pathogenic bacteria hypha and plant tissues can be used as detection materials. The reaction result can be visually identifiedThe method has the characteristics of simple operation, strong specificity, high sensitivity, strong stability and wide applicability, and can provide technical support for rapid diagnosis of field watermelon wilt pathogens.
Description
Technical Field
The invention belongs to the technical field of molecular detection of watermelon fusarium oxysporum, and particularly relates to a rapid LAMP (loop-mediated isothermal amplification) detection method of the watermelon fusarium oxysporum.
Background
Watermelon wilt is an important disease of watermelon (Citrullus lanatus) and occurs all over the world. Pathogenic bacteria of the watermelon fusarium wilt are soil-borne fungi fusarium oxysporum watermelon specialization type, and the pathogenic bacteria invade plants from the root wounds or cells at the top ends of root hairs of the plants, gradually occupy and block the vascular bundles of the plants, influence water delivery, cause water deficiency and wilting of the plants and finally die. The pathogenicity of different fusarium oxysporum watermelon specialization strains to different watermelon varieties is different, and physiological microspecific differentiation exists. Currently, 4 types of fusarium oxysporum watermelon specialization type physiological races are reported, namely No. 0, No.1, No.2 and No.3 physiological races (namely FON-0, FON-1, FON-2 and FON-3) (No. 3 physiological races are not available in nature in China). The race causing watermelon fusarium wilt in China is mainly No.1 physiological race.
The loop-mediated isothermal amplification (LAMP) technology can be completed after the reaction is carried out at 65 ℃ for 1h, and a PCR instrument is not needed, so that the method has a good application prospect. At present, the LAMP detection technology of the fusarium oxysporum has not been reported.
Disclosure of Invention
The invention aims to provide a rapid LAMP detection method for fusarium oxysporum f.sp.cubense for the first time. The invention designs a set of LAMP primer groups by taking the Fusarium oxysporum watermelon specialization type DNA fragments as targets on the basis of the analysis results of the Fusarium oxysporum watermelon specialization type DNA fragments, optimizes a method for extracting the Fusarium oxysporum watermelon specialization type DNA fragments, and establishes a detection system for rapidly detecting the Fusarium oxysporum watermelon Fusarium oxysporum infection. The primer specificity of the system is detected, the system is utilized to detect the roots and stems of watermelon plants which are artificially inoculated with FON-1 and show symptoms and do not show symptoms, and a germ tissue separation method is adopted to detect the result, so as to establish a quick, accurate and sensitive molecular detection method for the fusarium oxysporum f.sp.citrullus, which is suitable for field detection.
The invention relates to a rapid LAMP detection method of fusarium oxysporum f.sp.cubense, wherein the primer is designed according to the gene terminal and part of non-expression sequence region of the fusarium oxysporum f.sp.cubense anchoring protein 3.
The sequence region is a fusarium oxysporum watermelon specialization type specific fragment, and the primer specificity and sensitivity designed according to the fragment have obvious advantages.
In the detection method, the sequences of the gene terminal and part of the non-expression sequence region of the anchoring protein 3 are as follows:
GTTGCTTACGGTTCTAACTGTGCTACCTGTCGTCAGCGCGCCTAATGCTCGTAAGTCAGGTTCATCGTTTCGAGAGCAACTCCTGCTCATCTGGGGCCACATTCGGTGCGCTAGTTAAGCACCTCTTCGAATCAGTCCTGGATAGTTTGGTGTTCGCTTACTGTTTCGTACTTACACAATCTATCTATCTGGCATGAAATCGCTGACTCCAACTTGTTGAAGCCCACAATTCCTTCCAAGTACCC are provided. See SEQ ID NO. 1.
Further, the primer sequence 5 '→ 3' employed:
f3: GTTGCTTACGGTTCTAACTG, see SEQ ID NO. 2.
B3: GGGTACTTGGAAGGAATTGTG, see SEQ ID NO. 3.
FIP: GATTCGAAGAGGTGCTTAACGTTTCGAGAGCAACTCCTGC, see SEQ ID NO. 4.
And (3) BIP: AGTCCTGGATAGTTTGGTGCATGCCAGATAGATAGATTG, see SEQ ID NO. 5.
The detection method is characterized in that the watermelon fusarium wilt bacteria are as follows: fusarium oxysporum watermelon specialization type No. 0, No.1, No.2 physiological races.
The primer can be used for specific detection of Fusarium oxysporum watermelon specialization types 0, 1 and 2, and can completely distinguish Fusarium oxysporum bitter gourd specialization types (F.f.sp.moldicae), Fusarium oxysporum melon specialization types (F.f.sp.melonis), Fusarium oxysporum cucumber specialization types (F.f.sp.cumerinum), Fusarium moniliforme and Fusarium oxysporum slime colony specialization types (F.f.sp.conglutinans) and other Fusarium oxysporum specialization types, as well as Trichoderma harzianum T2-16 (T2-16) and other pathogenic bacteria.
The detection method adopts a DNA simple method to extract DNA during detection.
Compared with a CTAB method with complicated steps, the invention finds that the steps can be simplified and the time and the cost can be saved by extracting the hypha or the DNA of the watermelon plant to be detected by adopting a simple DNA simple extraction method, and the LAMP detection result is not influenced at all.
Further, the DNA extraction method comprises the following steps: adding a proper amount of sample to be detected into a centrifuge tube of 30-70 mul NaOH solution with the concentration of 40-60mmol/L, and carrying out boiling water bath for 5-10 min; adding 2-8 μ L of Tris-HCl buffer solution with pH value of 7-8 for neutralization; centrifuging at the rotation speed of 8000-; taking the supernatant to a new centrifuge tube, and storing at-20 ℃ for later use.
Further, the step of DNA extraction by the DNA simple method is preferably selected, a proper amount of sample to be detected is added into a centrifuge tube of 50 mu L of 50mmol/L NaOH solution, and boiling water bath is carried out for 10 min; then adding 5 mu L of Tris-HCl buffer solution with the pH value of 7.5 for neutralization; centrifuging at 12000r/min for 5 min; taking the supernatant to a new centrifuge tube, and storing at-20 ℃ for later use.
Further, when detecting watermelon wilt pathogen, directly taking a proper amount of hypha of the strain to be detected for extracting DNA, when detecting the infection of watermelon wilt pathogen of watermelon plant, repeatedly washing the tissue of watermelon plant to be detected to be clean with distilled water in an ultra-clean workbench, sucking water by using sterilizing filter paper, cutting off 0.05-0.2g of watermelon plant tissue, and grinding into paste for extracting DNA.
According to the detection method, the LAMP reaction system is 25 mu L: 12.5. mu.L of 2 × LAMP reaction buffer, 0.4. mu.L each of 100. mu. mol/L of the outer primers FIP and BIP, 0.5. mu.L each of 10. mu. mol/L of the inner primers F3 and B3, 6.7. mu.L of deionized water, 1.0. mu.L of Bst DNA polymerase, 2.0. mu.L of template DNA, and 1.0. mu.L of fluorescent color developing agent.
According to the detection method, the reagents are uniformly mixed and then react for 1h at 65 ℃ in a constant-temperature water bath, the color of the product is observed after the reaction is finished, the product with yellow-green color is marked as positive, and the product with orange-yellow color is marked as negative.
The invention designs a set of primers by utilizing a fusarium oxysporum watermelon specialization type specific DNA fragment based on a loop-mediated isothermal technology (LAMP) and establishes an LAMP visual rapid detection system for fusarium oxysporum by combining a DNA rapid extraction method. The system comprises about 90min for DNA extraction and LAMP detection result acquisition. The detection result of the specificity of the primers shows that the primers have higher specificity, and the detection result of the sensitivity shows that the LAMP system is used for FThe lower limit of detection of ON-1 genomic DNA was 10pg/μ L and the lower limit of detection of the amount of bacteria carried by plants was 5X 102Spores/g. Pot experiments show that the LAMP system has good repeatability and stability, and can make correct judgment on whether watermelon seedlings are infected by blight bacteria. Therefore, the established rapid LAMP detection method and system for the watermelon fusarium oxysporum have the characteristics of simple and rapid operation and stable and reliable result, and can meet the detection requirement of the basic level on the watermelon fusarium oxysporum.
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FIG. 1 shows the detection of primer specificity in the detection method of the present invention
A, performing color reaction on LAMP products; yellowish green is positive (tube No. 1-3), orange yellow is negative (tube No. 4-10);
performing gel electrophoresis on the LAMP product; wherein, No.1, No.2 and No.3 are target bands: 1-10: fusarium oxysporum watermelon specialization type No. 0 physiological race (FON-0), Fusarium oxysporum watermelon specialization type No.1 physiological race (FON-1), Fusarium oxysporum watermelon specialization type No.2 physiological race (FON-2), ddH2O, fusarium oxysporum bitter gourd specialization type, fusarium oxysporum melon specialization type, fusarium oxysporum cucumber specialization type, fusarium moniliforme, fusarium oxysporum conglobation type and trichoderma harzianum T2-16; m:
DNA Marker。
FIG. 2 shows the detection of the sensitivity of the LAMP system to FON-1
1-9: negative control (ddH)2O), 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 100 pg/. mu.L, 10 pg/. mu.L, 1 pg/. mu.L, 100 fg/. mu.L, 10 fg/. mu.L FON-1 genomic DNA.
FIG. 3 shows the comparison of LAMP detection of DNA of Fusarium oxysporum F.sp.citrulli extracted by two methods
Performing color reaction on the LAMP product; yellowish green is positive, orange yellow is negative; the simple method is marked as K, and the CTAB method is marked as C;
0: fusarium oxysporum watermelon specialization type No. 0 physiological race, 1: fusarium oxysporum watermelon specialization type 1 physiological race, 2: fusarium oxysporum watermelon specialization type No.2 physiological race and negative control: ddH2O。
FIG. 4 is the LAMP detection of 24 watermelon seedling samples FON-1
6 plants without obvious blight symptom are numbered as A1、A2、A3、A4、A5And A 66 plants with obvious blight symptom are numbered as B1、B2、B3、B4、B5And B6(ii) a The roots are marked as R, the stems are marked as S, the positive control is FON-1, and the negative control is ddH2And O. Rck for root negative control samples and Sck for stem negative control samples.
FIG. 5 shows the result of tissue isolation of a part of a plant
A2And A3Is a plant without obvious blight symptom; b is1Is a plant with obvious blight symptom; the roots are designated R and the stems are designated S.
FIG. 6 shows the sensitivity detection of LAMP system for the root germs of seedlings to be detected
Yang: FON-1, 1-6: 104、5×103、103、5×102、102、101Spore/g, yin: ddH2O。
Detailed Description
The following examples are intended to further illustrate the invention without limiting it.
Example (b):
1 materials and methods
1.1 materials
Test strains: the fusarium oxysporum f.sp.cucumerinum, the fusarium moniliforme f.moniliforme, the fusarium oxysporum f.sp.melo specialization f.f.melo, the fusarium oxysporum f.sp.cucumerinum, the fusarium moniliforme f.moniliforme, the fusarium oxysporum f.sp.conglutination (f.sp.condutitanes) and the Trichoderma harzianum T2-16(Trichoderma harzianum, T2-16) are all deposited by the institute of agricultural biotechnology of agricultural science and institute in the south of the lake province. All test strains are observed morphologically, and the ITS sequences are compared and identified for later use. Zaojia '8424' watermelon seeds (Sinkiang agronomy seed science and technology, Limited liability company).
Reagent: 50mmol/L NaOH (Chemicals group, Ltd.), pH 7.5Tris-HCl (Beijing Solebao science and technology Co., Ltd.), 2 × LAMP reaction buffer, Bst DNA polymerase, ddH2O, a loop-mediated isothermal amplification FDR fluorescence detection reagent (Beijing blue Spectrum Biotechnology Co., Ltd.).
The instrument comprises the following steps: DK-8D model electrothermal constant temperature water tank (Shanghai-constant technology, Inc.), Tanon1600 gel imaging System (Shanghai Tian Neng.), Centrifuge 5424R Small high speed refrigerated Centrifuge (Germany).
1.2 methods
1.2.1 primer design and specificity detection
According to the genome sequence of the watermelon fusarium wilt pathogen, the sequence homologies of related species and other different pathogenic bacteria are compared, and the specific segment of the watermelon fusarium wilt pathogen is found by a genome BLAST method. A set of LAMP reaction primer group (inner primer B3/F3, outer primer BIP/FIP) primer sequences are designed by taking the specific fragment as a target, and are shown in Table 1. The genomic DNAs of Fusarium oxysporum watermelon specialization types 0, 1 and 2, Fusarium oxysporum bitter gourd specialization types, Fusarium oxysporum melon specialization types, Fusarium oxysporum cucumber specialization types, Fusarium moniliforme, Fusarium oxysporum slime lump specialization types and Trichoderma harzianum T2-16 are extracted by a Cetyl Trimethyl Ammonium Bromide (CTAB) method (Liu et al, 2015) and LAMP detection is carried out by using the designed primers. The LAMP reaction system is (25 μ L): 12.5. mu.L of 2 × LAMP reaction buffer, 0.4. mu.L each of the outer primers FIP and BIP (100. mu. mol/L), 0.5. mu.L each of the inner primers F3 and B3 (10. mu. mol/L), 6.7. mu.L of deionized water, 1.0. mu.L of Bst DNA polymerase, 2.0. mu.L of template DNA, and 1.0. mu.L of fluorescent color developer. The operation of loading the sample on ice requires a step of mixing the liquids, and mixing by pipetting is carried out in order to avoid the generation of bubbles. And (3) uniformly mixing the reagents, reacting for 1h at 65 ℃ in a constant-temperature water bath kettle, observing the color of the product after the reaction is finished, and marking the product as positive if the color of the product is yellow-green and marking the product as negative if the color of the product is orange-yellow. And 4.5 mu L of each LAMP reaction product is taken for agarose gel electrophoresis analysis, and the experiment is repeated for 3 times.
TABLE 1 LAMP reaction primers
1.2.2 sensitivity of LAMP for FON-1 detection
The genomic DNA of FON-1 strain was extracted by CTAB method and its concentration was measured by Nanodrop 2000 spectrophotometry. The measured DNA concentrations were diluted in the order of 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, followed by LAMP detection, and the lower limit of detection of the present line was determined based on the color of the LAMP product.
1.2.3 comparison of the effects of pathogen DNA extraction for LAMP detection with CTAB method
Because the step of extracting the DNA by the CTAB method is too complex and the time consumption is too long, the effect of the DNA extracted by the DNA simple method and the DNA extracted by the conventional CTAB method for LAMP detection is compared to determine whether the method is suitable for LAMP detection. The steps of the DNA simplification method are as follows: taking a proper amount of hyphae of the strain to be detected, placing the hyphae into a numbered 1.5mL centrifuge tube, adding 50 mu L (50mmol/L) NaOH solution in advance, and carrying out boiling water bath for 10 min. Then, 5. mu.L (pH 7.5) of Tris-HCl buffer was added for neutralization. Centrifuging at 12000r/min for 10 min. Taking the supernatant to a new centrifuge tube, and storing at-20 ℃ for later use.
The CTAB method refers to the procedure of Liu et al (2015).
1.2.4 detection of artificially inoculated diseased seedlings
In order to determine whether the DNA simple method is suitable for the extraction of plant tissue pathogen DNA and LAMP detection, the method is adopted to detect the roots and stems of watermelon seedlings which are artificially inoculated and have obvious symptoms and do not have obvious symptoms. The operation process is as follows: transferring the separated and purified FON-1 onto PDA plate, activating at 28 deg.C for 7 days, and culturing in 50mL potato glucose broth (PDB) culture medium at 28 deg.C and 120r/min for 7 days. Filtering with four layers of gauze to remove mycelium to obtain spore suspension, detecting spore concentration of spore suspension, mixing appropriate amount of spore suspension with sterilized sandy soil, and making into spore with spore content of 5 × 103Spore/g sand with bacteriaAnd (4) soil. Sowing the watermelon seeds (Zaojia '8424') which are subjected to germination accelerating and white exposure in sandy soil with bacteria, and culturing at 24-28 ℃ in the daytime and 18 ℃ at night. After 7 days of cultivation, 6 plants without blight symptom are selected from the plants, and the number of the plants is A1、A2、A3、A4、A5And A66 plants with obvious blight symptom are numbered as B1、B2、B3、B4、B5And B6Performing LAMP detection, wherein the specific operation method comprises the following steps: repeatedly washing each plant tissue to be clean with distilled water in an ultra-clean workbench, sucking water by using sterilized filter paper, and cutting off 2 parts of root and stem tissues of each numbered plant by using a sterile knife, wherein the weight is 0.1 g. One part is used for LAMP detection by extracting DNA by a simple method, and the other part is used for separating germs by a tissue isolation method. The tissue isolation method comprises the specific steps of putting a sample into a 0.05% sodium hypochlorite solution for treatment for 2min, then transferring the sample into sterile water for cleaning for 1min, finally putting the sample into a PDA culture medium for cultivation at 28 ℃, continuously observing for 4 days, and verifying the result according to whether the pathogenic bacteria of the watermelon fusarium wilt are separated.
In order to determine the lower limit of the LAMP system on the detection of the bacterial load of the plants, 6 parts of plant root tissues with the weight of 0.1g are taken, and 1ul of plant root tissues with the concentration of 10 respectively are added6、5×105、104、5×103、103、102Spore/ml suspension prepared to have bacteria content of 104、5×103、103、5×102、102、101Spores/g of the plant root-bearing bacterial sample. And extracting DNA by using the system, carrying out LAMP amplification and color development reaction, and determining the lower limit of the detection of the system on the plant bacteria carrying amount according to the color development condition. The experiment was repeated 2 times.
In order to further determine the pathogen detection rate of the watermelon wilt pathogen LAMP system on watermelon seedlings, the method is adopted to detect roots and stems of symptomatic and undisplayed watermelon seedlings in an artificial inoculation seedling raising pot, and statistics and analysis are carried out on detection results. The specific operation process is as follows: adding appropriate amount of spore with spore content of 5 × 10 into three seedling-raising pots respectively3Spore/g sand soil with bacteria, planting 30 early-good '8424' watermelon seeds with germination accelerating and white exposing in each pot, and culturingThe culturing method is the same as the above, when about half of seedlings show symptoms, the seedlings are classified according to whether the symptoms appear, the DNA of root and stem tissues of each seedling is extracted by a simple method, LAMP detection is carried out, a tissue isolation method is used for verification, and the results are statistically analyzed.
2 results and analysis
2.1 primer specificity test
By ddH2And taking O as a template as a negative control, and verifying the specificity of the LAMP primer in the system by using genome DNAs of Fusarium oxysporum watermelon specialization types 0, 1 and 2 and other six fungi. The results show that the related LAMP primers can specifically detect Fusarium oxysporum watermelon specialization types 0, 1 and 2 physiological races, and does not detect Fusarium oxysporum bitter gourd specialization type, Fusarium oxysporum melon specialization type, Fusarium oxysporum cucumber specialization type, Fusarium moniliforme, Fusarium oxysporum slime lump specialization type and Trichoderma harzianum T2-16 (figure 1). Therefore, the set of primers is suitable for LAMP detection of fusarium oxysporum f.sp.citrulli.
2.2 sensitivity of LAMP for detecting FON-1
After the genomic DNA of the FON-1 strain is subjected to gradient dilution, LAMP detection is respectively carried out. The lower limit of the LAMP system on the detection of the FON-1 strain genome DNA is judged to be 10 pg/mu L according to the color change of the LAMP product, the result is shown in figure 2, the tubes with the length of 100 ng/mu L-10 pg/mu L are all yellow green, the tube with the length of 1 pg/mu L is lower, and the negative control is orange yellow.
2.3 comparison of the effects of the DNA extraction method and the CTAB method for the LAMP detection
DNA of fusarium oxysporum watermelon specialization No. 0, No.1 and No.2 physiological races is extracted by a simple DNA extraction method and a traditional CTAB method respectively for LAMP detection. The LAMP detection result shows that the pathogen DNA extracted by using the DNA extraction method can be used for LAMP detection, has the effect equivalent to that of the traditional CTAB method, and is yellow green except for negative control (figure 3). Therefore, the DNA simplification method is suitable for the LAMP system.
2.4 detection of artificially inoculated diseased seedlings
The DNA simple method is suitable for detecting the pathogenic bacteria of the plant tissues. As shown in fig. 4: FON-1 is detected from the root and stem parts of 6 plants with obvious blight symptoms. The negative control Rck and the root negative control Sck were both orange yellow, and FON-1 was detected in the roots of 6 plants without significant blight symptoms, wherein FON-1 was not detected in the stems of two plants (i.e., a2 and a6 of S were orange yellow), possibly due to FON-1 not invading the stem bundle tubes. The separation result of the tissue separation method is consistent with LAMP detection (Table 2, figure 5), which shows that the DNA simplification method can carry out LAMP detection by taking plant tissues as materials, and the detection result is reliable.
TABLE 2 tissue isolation results for watermelon seedling samples
+: separating to obtain FON-1; -: not separated into FON-1
The detection results of the plant standard samples with different bacterial contents show that the detection lower limit of the LAMP system to the bacterial carrying quantity of the plants is 5 multiplied by 102Spores/g, see fig. 6, yang: FON-1, 1-6: 104、5×103、103、5×102The spores/g of the tubes were yellow-green, and the remaining tubes and negative control were orange-yellow.
The pathogen detection rate of the watermelon wilt pathogen LAMP system is shown in Table 3, and the LAMP detection result is consistent with the detection result of the tissue isolation method except for the detection result of the stem of an undisplayed plant. As for the detection result of the stem of the plant without manifestation, the LAMP detection rate is slightly higher than that of the tissue isolation method, which is probably caused by that the number of germs at the stem of the plant without manifestation is small, and a small amount of pathogenic bacteria are killed and can not be isolated and cultured in the disinfection process of the tissue isolation method. Experimental results show that the LAMP system has good repeatability and stability, and the reliability of detecting whether the plant is infected by the fusarium oxysporum is higher by using a plant root sample.
LAMP detection and tissue isolation result of roots and stems of table 390 watermelon plants
Watermelon fusarium wilt is a soil-borne disease, germs penetrate root tips or infect wounds at the roots of plants to cause the plants to be infected with diseases, the plants do not show symptoms immediately when infected initially, and no effective medicament is used for treating the plants with symptoms. Therefore, the diagnosis technology before the occurrence of the blight of the plants is particularly important for preventing and treating the blight. The invention designs fusarium oxysporum watermelon specialization type specific LAMP primers and combines a DNA rapid extraction method to establish a rapid LAMP detection system of fusarium oxysporum.
The LAMP technology can generate a large amount of copies in the amplification process and is easy to generate aerosol, so that cross contamination among samples is easy to cause, and the detection result is influenced. The fluorescent color developing agent used in the LAMP system is calcein, and is added into the whole system before LAMP reaction, and a detection dye does not need to be added after the LAMP reaction is finished, so that the possibility of cross contamination is greatly reduced.
The invention finds out the specific segment design of the watermelon fusarium wilt pathogen and screens to obtain the primer by comparing the genomic sequences of the watermelon fusarium wilt pathogen and the related species thereof and other different pathogenic bacteria. Through analysis, the specific segment amplified by the primer is positioned at the end of the gene of which the predicted expression product is Ankyrin 3(Ankyrin 3) and a part of non-expression sequence region. There is currently a lack of relevant studies on the function of this protein in fungi. Ankyrin sequences are mainly present in eukaryotic proteins and have the functions of cell cycle regulation, mitochondrial enzymes, cytoskeletal interactions, signal transduction, stress resistance and the like. The dockerin has important significance in fungus obligate parasitism and functional evolution, and the dockerin 3 with specific DNA motif positioned by the primer of the invention is probably related to obligate parasitism of fusarium oxysporum. The lower limit of detection of the primer on the FON-1 genomic DNA is 10 pg/mu L. Whether the plants are infected by germs can be judged when the plants do not show symptoms, so that the potential pathogenic plants of the watermelon fusarium wilt can be prevented and treated, the generation and the spread of the fusarium wilt can be controlled, and the economic loss can be prevented.
The selected DNA simple extraction method can extract the watermelon fusarium wilt pathogen genome DNA under the boiling water bath condition only by using Tris-HCl and NaOH, and pathogenic bacteria hypha and root and stem tissues of plants to be detected can be used as detection materials. Compared with the common traditional CTAB method and DNA extraction kit method, the DNA simple method is more economic, efficient and convenient.
Lanjianglin et al (2007) adopt a dilution plate tissue isolation method to detect the number of fusarium oxysporum f.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp3And when cfu/g, the blight symptom is not shown, and when the critical threshold value is reached, the blight symptom can be shown initially by the plant. The lower limit of the LAMP system for detecting the bacterial load of the plants is 5 multiplied by 102Spore/g, below the critical value for manifestation, can be used to diagnose whether the plants not infected with disease are infected with fusarium oxysporum. The detection result of the artificial inoculation morbidity experiment shows that for the root sample of the bacteria-carrying plant, the detection rate of the LAMP detection system is 100% consistent with the result of the tissue isolation method, the result is stable and reliable, meanwhile, the system has low requirement on the environment, the result is immediately recognizable, and the LAMP detection system can be applied to judging whether the seedlings carry bacteria or not and detecting field blight bacteria when the seedlings are raised in a factory.
Sequence listing
<110> institute of agricultural biotechnology in Hunan province
<120> quick LAMP detection method for fusarium oxysporum f.sp.cubense
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 245
<212> DNA
<213> Fusarium oxysporum watermelon specialization type (Fusarium oxysporum f. sp. niveum)
<400> 1
gttgcttacg gttctaactg tgctacctgt cgtcagcgcg cctaatgctc gtaagtcagg 60
ttcatcgttt cgagagcaac tcctgctcat ctggggccac attcggtgcg ctagttaagc 120
acctcttcga atcagtcctg gatagtttgg tgttcgctta ctgtttcgta cttacacaat 180
ctatctatct ggcatgaaat cgctgactcc aacttgttga agcccacaat tccttccaag 240
taccc 245
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gggtacttgg aaggaattgt g 21
<210> 4
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gattcgaaga ggtgcttaac gtttcgagag caactcctgc 40
<210> 5
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
agtcctggat agtttggtgc atgccagata gatagattg 39
Claims (9)
1. The rapid LAMP detection method for the fusarium oxysporum f.sp.citrulli is characterized in that the primers are designed according to the gene terminal and part of non-expression sequence region of the fusarium oxysporum f.citrulli anchoring protein 3, and the DNA sequence is specific to the fusarium oxysporum f.citrulli.
2. The method according to claim 1, wherein the sequences of the end of the gene of the anchoring protein 3 of Fusarium oxysporum and a part of the non-expressed sequence region are as follows:
GTTGCTTACGGTTCTAACTGTGCTACCTGTCGTCAGCGCGCCTAATGCTCGTAAGTCAGGTTCATCGTTTCGAGAGCAACTCCTGCTCATCTGGGGCCACATTCGGTGCGCTAGTTAAGCACCTCTTCGAATCAGTCCTGGATAGTTTGGTGTTCGCTTACTGTTTCGTACTTACACAATCTATCTATCTGGCATGAAATCGCTGACTCCAACTTGTTGAAGCCCACAATTCCTTCCAAGTACCC。
3. the detection method according to claim 1, wherein the primer sequence 5 '→ 3':
F3：GTTGCTTACGGTTCTAACTG
B3：GGGTACTTGGAAGGAATTGTG
FIP：GATTCGAAGAGGTGCTTAACGTTTCGAGAGCAACTCCTGC
BIP：AGTCCTGGATAGTTTGGTGCATGCCAGATAGATAGATTG。
4. the detection method according to claim 1, wherein the Fusarium oxysporum F.sp.citrulli is: fusarium oxysporum watermelon specialization type No. 0, No.1, No.2 physiological races.
5. The method according to any one of claims 1 to 4, wherein the detection is carried out by extracting DNA by a DNA extraction method.
6. The detection method according to claim 5, wherein the DNA extraction step comprises: adding a proper amount of sample to be detected into a centrifuge tube of 30-70 mul NaOH solution with the concentration of 40-60mmol/L, and carrying out boiling water bath for 5-10 min; adding 2-8 μ L of Tris-HCl buffer solution with pH value of 7-8 for neutralization; centrifuging at the rotation speed of 8000-; taking the supernatant to a new centrifuge tube, and storing at-20 ℃ for later use.
7. The detection method according to claim 6,
taking a proper amount of a sample to be detected, adding the sample to be detected into a centrifuge tube of 50 mu L of 50mmol/L NaOH solution, and carrying out boiling water bath for 10 min; then adding 5 mu L of Tris-HCl buffer solution with the pH value of 7.5 for neutralization; centrifuging at 12000r/min for 5 min; taking the supernatant to a new centrifuge tube, and storing at-20 ℃ for later use.
8. The detection method according to claim 6, wherein an appropriate amount of hyphae of the strain to be detected is directly taken for extracting DNA when detecting the blight of watermelon, the tissue of the watermelon plant to be detected is repeatedly washed clean with distilled water in an ultra-clean bench when detecting the blight infection of watermelon plant, the water is sucked up by a sterilized filter paper, 0.05-0.2g of the tissue of the watermelon plant is cut, and the cut tissue is ground into paste for extracting DNA.
9. The detection method according to claim 1, wherein the LAMP reaction system is 25 μ L: 12.5. mu.L of 2 × LAMP reaction buffer, 0.4. mu.L each of 100. mu. mol/L of outer primers FIP and BIP, 0.5. mu.L each of 10. mu. mol/L of inner primers F3 and B3, 6.7. mu.L of deionized water, 1.0. mu.L of Bst DNA polymerase, 2.0. mu.L of template DNA, 1.0. mu.L of fluorescent color-developing agent;
and (3) uniformly mixing the reagents, reacting for 1h at 65 ℃ in a constant-temperature water bath kettle, observing the color of the product after the reaction is finished, and marking the product as positive if the color of the product is yellow-green and marking the product as negative if the color of the product is orange-yellow.
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