Method and primer composition for detecting plant anthracnose

Abstract

To provide a method for detecting plant anthracnose and a primer composition.SOLUTION: Disclosed are a method for detecting plant anthracnose and a primer composition. The method for detecting plant anthracnose uses the primer composition to perform a loop-mediated isothermal nucleic acid amplification reaction. The primer composition includes a first primer pair, a second primer pair, and a third primer pair, where the first primer pair, the second primer pair, and the third primer pair each include a nucleotide sequence with a specific sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method and primer composition for detecting plant anthracnose, in particular for strawberry anthracnose, which utilizes loop-mediated isothermal nucleic acid amplification in combination with a lateral flow test. [Background technology]

[0002] Colletotrichum spp. is a common and important plant pathogenic fungus that infects plants or ornamental plants via conidia, such as orchids (Orchidaceae), apples (Malus pumila), peppers (Capsicum spp.), coffee plants (Coffea spp.), grapes (Vitis vinifera), longans (Dimocarpus longan), mangoes (Mangifera indica), kale (Canarium album), bitter orange (Citrus spp.), pears (Pyrus spp.), peaches (Prunus persica), strawberries (Fragaria ananassa), watermelons (Citrullus lanatus), and tea plants (Camellia spp.). When the environment is favorable for the disease to develop or the host plant's immunity is weak, the disease enters the host plant through the water pores on the leaf margins and other natural openings, causing the leaf blades of the host plant to brown or dry out, and then spreads to other parts or surrounding plants, causing the death of many plants, severely affecting plant yields and causing losses to farmers.

[0003] There are 248 known species in the genus Bacillus, and species can be classified and identified by analyzing and comparing sequences from different gene loci. Commonly used gene sequences include those for the internal transcribed spacer (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), histone H3 (HIS3), β-tubulin-2 (TUB2), and glutamine synthetase (GS). Currently, when detecting whether a plant is infected with anthracnose, the gene is often detected using conventional polymerase chain reaction (PCR). However, PCR requires relevant equipment and laboratory staff familiar with molecular biology techniques, and the detection steps are complicated, making it difficult for ordinary users to perform real-time detection outdoors. Therefore, how to detect whether a plant is infected with anthracnose in a simple, rapid, and effective manner is an issue that needs to be resolved immediately. Summary of the Invention

[0004] The present invention comprises the steps of: 1. obtaining a nucleic acid sample isolated from a plant; Step 2: mixing the nucleic acid sample with a primer composition to prepare a reaction solution, and performing a nucleic acid amplification reaction on the reaction solution, wherein the primer composition comprises a first primer pair, a second primer pair, and a third primer pair, wherein the first primer pair comprises the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:3, the second primer pair comprises the nucleotide sequences of SEQ ID NO:4 and SEQ ID NO:5, and the third primer pair comprises the nucleotide sequences of SEQ ID NO:6 and SEQ ID NO:7; and step 3 of detecting whether or not the reaction solution after the nucleic acid amplification reaction contains a nucleic acid amplification product, and determining that the plant is infected with anthrax bacteria if the nucleic acid amplification product is present, and determining that the plant is not infected with anthrax bacteria if the nucleic acid amplification product is not present.

[0005] The present invention also provides a primer composition for detecting plant anthracnose, comprising a first primer pair comprising the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:3, a second primer pair comprising the nucleotide sequences of SEQ ID NO:4 and SEQ ID NO:5, and a third primer pair comprising the nucleotide sequences of SEQ ID NO:6 and SEQ ID NO:7. [Effects of the Invention]

[0006] Therefore, the primer composition provided by the present invention can be used in loop-mediated isothermal nucleic acid amplification reactions, and since there is no need to significantly change the temperature, the overall reaction time can be shortened, experimental equipment can be simplified, and it is useful for quickly detecting whether plants are infected with Bacillus anthracis. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows the results of electrophoresis analysis after loop-mediated isothermal nucleic acid amplification reaction on the genome of Bacillus anthracis using the primer composition disclosed in the present invention. [Figure 2] FIG. 2 shows the results of electrophoresis analysis after loop-mediated isothermal nucleic acid amplification reactions on the genomes of Bacillus anthracis and Trichoderma using the primer composition disclosed in the present invention. [Figure 3] FIG. 3 shows the results of electrophoresis analysis after loop-mediated isothermal nucleic acid amplification reactions were performed on the genomes of Bacillus anthracnose, leaf tissue of healthy strawberry plants, and Bidens frondosa leaf tissue using the primer composition disclosed in the present invention. [Figure 4]FIG. 4 shows the results of electrophoresis analysis after loop-mediated isothermal nucleic acid amplification reactions were performed on plastids containing some genes of Bacillus anthracnose, the genome of Bacillus anthracnose, leaf tissue of a strawberry plant with anthracnose symptoms, and leaf tissue of a healthy strawberry plant using the primer composition disclosed in the present invention. [Figure 5A] FIG. 5A shows the results of electrophoresis analysis after loop-mediated isothermal nucleic acid amplification reactions on leaf tissue from symptom-free strawberry plants and the genome of Bacillus anthracnose using the primer composition disclosed in the present invention. [Figure 5B] FIG. 5B shows the results of a lateral flow test after loop-mediated isothermal nucleic acid amplification reaction was performed on leaf tissue of symptom-free strawberry plants and the genome of Bacillus anthracnose using the primer composition disclosed in the present invention. [Figure 6A] FIG. 6A shows the results of the color change of hydroxynaphthol blue in the reaction tube after 30 minutes of loop-mediated isothermal nucleic acid amplification reaction on different concentrations of Bacillus anthracis genome using the primer composition disclosed in the present invention. [Figure 6B] FIG. 6B shows the results of the color change of hydroxynaphthol blue in the reaction tube after 45 minutes of loop-mediated isothermal nucleic acid amplification reaction on different concentrations of Bacillus anthracis genome using the primer composition disclosed in the present invention. [Figure 6C] FIG. 6C shows the results of the color change of hydroxynaphthol blue in the reaction tube after 60 minutes of loop-mediated isothermal nucleic acid amplification reaction on different concentrations of Bacillus anthracis genome using the primer composition disclosed in the present invention. [Figure 6D] FIG. 6D shows the results of electrophoresis analysis after 30 minutes of loop-mediated isothermal nucleic acid amplification reaction on different concentrations of Bacillus anthracis genome using the primer composition disclosed in the present invention. [Figure 6E] FIG. 6E shows the results of electrophoresis analysis after 60 minutes of loop-mediated isothermal nucleic acid amplification reaction on different concentrations of Bacillus anthracis genome using the primer composition disclosed in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0008] The term "nucleic acid amplification reaction" as used herein means any biochemical technique that increases the copy number of a target nucleotide sequence, and the copies of the target nucleotide sequence increased by the nucleic acid amplification reaction are "nucleic acid amplification products."

[0009] As used herein, the term "primer" refers to a single-stranded oligonucleotide that can bind to a target nucleotide sequence and serve as the initiation point for a nucleic acid amplification reaction under appropriate reaction conditions, and the term "template" refers to a nucleic acid molecule for conducting a nucleic acid amplification reaction, and includes the target nucleotide sequence to be amplified.

[0010] The method for detecting plant anthracnose disclosed in the present invention particularly involves using a primer composition to perform a nucleic acid amplification reaction on the genome of Bacillus anthracis, and detecting whether a nucleic acid amplification product is produced, thereby determining whether the plant is infected with Bacillus anthracis.

[0011] The method disclosed in the present invention includes the following steps: Step 1: Obtain a nucleic acid sample isolated from a plant. In step 2, the nucleic acid sample and a primer composition are mixed to form a reaction solution, and a nucleic acid amplification reaction is carried out on the reaction solution, with the nucleic acid sample serving as a template for the nucleic acid amplification reaction, wherein the primer composition comprises a first primer pair, a second primer pair, and a third primer pair, wherein the first primer pair comprises the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:3, the second primer pair comprises the nucleotide sequences of SEQ ID NO:4 and SEQ ID NO:5, and the third primer pair comprises the nucleotide sequences of SEQ ID NO:6 and SEQ ID NO:7. In step 3, the reaction solution after the nucleic acid amplification reaction is detected to determine whether it contains a nucleic acid amplification product. If the nucleic acid amplification product is present, the plant is determined to be infected with anthrax bacteria, and if the nucleic acid amplification product is not present, the plant is determined to be not infected with anthrax bacteria.

[0012] In step 1 of the method, a nucleic acid sample isolated from a plant is obtained, the nucleic acid sample comprising a sequence of a portion of the β-tubulin (β-tubulin-2, TUB2) genome of Bacillus anthracis, which comprises the nucleotide sequence of SEQ ID NO: 1 or a derived sequence thereof, wherein the derived sequence of SEQ ID NO: 1 has greater than 97% identity to the nucleotide sequence of SEQ ID NO: 1.

[0013] In some embodiments, the nucleic acid sample is obtained from a plant suspected of being infected with anthracnose, the plant being selected from the group consisting of strawberry (Fragaria ananassa), grape (Vitis vinifera), avocado (Avocado), onion (Allium cepa), mango (Mangifera indica), apple (Malus pumila), papaya (Carica papaya), chili pepper (Capsicum annuum), sweet ginger (Dalbergia odorifera), cranberry (Cranberry), bitter orange (Citrus spp.), tea plant (Camellia spp.), garlic (Garlic), poplar (Poplar), guava (Psidium guajava), fig (Ficus carica), and camu camu (Myrciaria dubia, blueberry, Xanthorrhoea, banana, Coffea spp., Chinese chestnut, American ginseng, vegetable soybean, wheat, pomegranate, Eucalyptus, pear, Pyrus spp., Cunninghamia lanceolata, and sweet persimmon, or a combination thereof.

[0014] In some embodiments, the nucleotide sequence of SEQ ID NO: 1 is the Bacillus anthracis species C. siamense isolated from strawberry, and derived sequences having 97% or greater identity to the nucleotide sequence of SEQ ID NO: 1 are the Bacillus anthracis species C. aenigma, C. fructicola, C. alienum, C. gloeosporioides, C. noveboracense, C. viniferum, C. queenslandicum, C. perseae, C. endophyticum, C. makassarense, C. tainan ense, C.hebeiense, C.conoides, C.gigasporum, C.salsolae, C.melanocaulon, C.dianesei, C.hystricis, C.camelliae, C.pandanicola, C. fioriniae, C.changpingense, C.ignotum, C.karstii, C.populi, C.tropicale, C.caricae, C.asianum, C.aeschynomenes, C.chrysophilum, C .australianum, C.parvisporum, C.nupharicola, C.xanthorrhoeae, C.musae, C.endophytica, C.acaciae, C.yuanjiangense, C.nanhuaense , C.dimorphum, C.henanense, C.subhenanense, C.sojae, C.yulongense, C.hymenocallidis, C.gloeosporioides, C.boninense, C.fructicol a, C. cobbittiense, C. ti, C. proteae, C. clidemiae, C. kahawae, C. cigarro, C. jiangxiense, C. wuxiense, C. nullisetosum, C. cangyuanense, C. helleniense, C. fructivorum, C. rhexiae, C. crassipes, C. temperatum, C. analogum, C. horii, or a combination thereof, but are not limited to these.

[0015] In some embodiments, in step 1, the nucleic acid sample may be extracted from tissues of the plant, such as leaf blades, flesh, seeds, stems, or a combination thereof. The method for extracting the nucleic acid sample includes dissolving the plant's cell walls and purifying the intracellular nucleic acids. Methods for dissolving the cell walls include, but are not limited to, ultrasonic shaking, physical polishing, and organic solvent decomposition. Methods for purifying the intracellular nucleic acids include, but are not limited to, separating the nucleic acids from the cytoplasm with an organic solvent, such as purification by adding ethanol or isopropanol. Alternatively, the nucleic acid sample may be obtained from the plant tissue using a commercially available kit. The extraction method may be performed using steps and conditions well known in the art.

[0016] In step 2 of the method, the nucleic acid sample and a primer composition are mixed to form a reaction solution, and a nucleic acid amplification reaction is carried out on the reaction solution, in which the nucleic acid sample serves as a template for the nucleic acid amplification reaction. The primer composition comprises a first primer pair, a second primer pair, and a third primer pair. The first primer pair comprises the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:3, the second primer pair comprises the nucleotide sequences of SEQ ID NO:4 and SEQ ID NO:5, and the third primer pair comprises the nucleotide sequences of SEQ ID NO:6 and SEQ ID NO:7. Table 1 lists the nucleotide sequences of these primer pairs. [Table 1]

[0017] In one embodiment, at least one primer in the primer composition is bound to a detectable marker, which is subsequently used to detect whether a nucleic acid amplification product has been generated or to determine the amount of the nucleic acid amplification product. The detectable marker may be an antigen marker, a fluorescent marker (e.g., ethidium bromide (EtBr), SYBR Green I / II, etc.), a chemiluminescent dye (e.g., horseradish peroxidase (HRP), hydroxynaphthol blue (HNB)), a radioactive marker (e.g., 2 H, 13 C. 15 In one embodiment, a fluorescent marker, 6-carboxyfluorescein (6-FAM), is bound to the nucleotide sequence of SEQ ID NO: 6 of the third primer pair, and an antigen marker, biotin, is bound to the nucleotide sequence of SEQ ID NO: 7.

[0018] In one embodiment, the reaction solution further comprises a DNA polymerase for synthesizing a new nucleotide sequence complementary to the template in a nucleic acid amplification reaction. In one example, the reaction solution further comprises, but is not limited to, deoxyribonucleoside triphosphates (dNTPs), a buffer, a surfactant (e.g., sodium dodecyl sulfate (SDS), polysorbate (Tween)), and a salt (magnesium chloride). The reaction solution may be a commercially available reaction reagent mixture containing the DNA polymerase, deoxyribonucleoside triphosphates, a buffer, and / or a surfactant.

[0019] In one aspect, the nucleic acid amplification reaction may be, but is not limited to, a polymerase chain reaction (PCR), a quantitative polymerase chain reaction (qPCR), a reverse transcription polymerase chain reaction (RT-PCR), a real-time polymerase chain reaction (real-time PCR), a nested polymerase chain reaction (nested PCR), or an isothermal nucleic acid amplification reaction (e.g., recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP)), and the nucleic acid amplification reaction may be performed using steps and conditions well known in the art.

[0020] In one embodiment, a loop-mediated isothermal nucleic acid amplification (LAMP) reaction is used, which can perform nucleic acid amplification reactions under isothermal conditions, and the isothermal conditions are 50 to 75°C. In a preferred embodiment, the isothermal conditions are 60 to 70°C. In a more preferred embodiment, the isothermal conditions are 65°C. Compared to other nucleic acid amplification reactions, the loop-mediated isothermal nucleic acid amplification reaction does not require significant temperature changes, which shortens the overall reaction time and simplifies experimental equipment. In addition, specificity can be improved by using specially designed primer compositions.

[0021] In step 3 of the method, the presence of a nucleic acid amplification product in the reaction solution after the nucleic acid amplification reaction is detected. If the nucleic acid amplification product is present, the plant is determined to be infected with Bacillus anthracis; if the nucleic acid amplification product is absent, the plant is determined to be not infected with Bacillus anthracis. Methods for detecting the presence of a nucleic acid amplification product include, but are not limited to, turbidity measurement, fluorescence detection, bioluminescence detection, gel electrophoresis, colorimetric detection, immunoenzymatic detection, electrochemical detection, and color change in a test strip. These detection methods may be performed using steps and conditions well known in the art. In one embodiment, the nucleic acid amplification reaction is a loop-mediated isothermal nucleic acid amplification reaction. After the loop-mediated isothermal nucleic acid amplification reaction, a large amount of magnesium pyrophosphate is produced as a white precipitate. Therefore, the production of the precipitate can be observed using turbidity measurement. In another embodiment, the reaction mixture can be subjected to electrophoretic analysis using agarose gel electrophoresis and nucleic acid fluorescent staining to determine whether a target nucleic acid amplified fragment of a specific nucleic acid size has been produced.

[0022] In one embodiment, a primer or a nucleic acid amplification product containing a detection marker is applied to a test strip, and the color of the test strip determines whether a nucleic acid amplification product has been produced. The principles used to change the color of the test strip include, but are not limited to, immunological binding, molecular hybridization, colloidal gold labeling technology, or a lateral flow test.

[0023] In another embodiment, a metal indicator, hydroxynaphthol blue (HNB), is added to the reaction solution. After the loop-mediated isothermal nucleic acid amplification reaction, a large amount of magnesium pyrophosphate is produced. Hydroxynaphthol blue changes color depending on the concentration of magnesium ions, and the production of nucleic acid amplification products can be determined from the color change of the reaction solution.

[0024] The present invention further discloses a primer composition for detecting plant anthracnose, comprising a first primer pair comprising the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:3, a second primer pair comprising the nucleotide sequences of SEQ ID NO:4 and SEQ ID NO:5, and a third primer pair comprising the nucleotide sequences of SEQ ID NO:6 and SEQ ID NO:7.

[0025] In one embodiment, the primer composition is used to perform a loop-mediated isothermal nucleic acid amplification reaction, and the loop-mediated isothermal nucleic acid amplification reaction may be performed as a nucleic acid amplification reaction under isothermal conditions, and the isothermal conditions are 50 to 75° C. In a preferred embodiment, the isothermal conditions are 60 to 70° C. In a more preferred embodiment, the isothermal conditions are 65° C.

[0026] In one embodiment, at least one primer in the primer composition is conjugated to a detectable marker, which is subsequently used to detect whether a nucleic acid amplification product has been produced or to determine the amount of the nucleic acid amplification product. In one example, a fluorescent marker, 6-carboxyfluorescein, is conjugated to the nucleotide sequence of SEQ ID NO: 6 of the third primer pair, and an antigen marker, biotin, is conjugated to the nucleotide sequence of SEQ ID NO: 7.

[0027] In the present invention, the following experimental examples demonstrate that the method and the primer composition can detect the gene of Bacillus anthracis. The following examples are provided for illustrative purposes only, and the scope of the present invention is not limited by the examples. Those skilled in the art can obtain other specific embodiments, configurations, and modifications using the disclosure and teachings of the present invention without conducting new experiments.

[0028] (Experimental Example 1) In this example, the method and primer composition disclosed in the present invention were used to perform a loop-mediated isothermal nucleic acid amplification (LAMP) reaction on the genome of Bacillus anthracis.

[0029] First, a nucleic acid sample containing the Bacillus anthracnose genome was obtained by extracting DNA from Bacillus anthracnose isolated from a strawberry anthracnose strain using a plant genome extraction kit (The Genomic DNA Isolation Kit (Plant), NA025-0100, GeneDireX, Inc.). In this example, the Bacillus anthracnose species isolated from the strawberry anthracnose strain was Colletotrichum siamense (isolate number ML133). The nucleic acid sample was then mixed with the primer composition disclosed herein, followed by the addition of a LAMP reaction reagent (WarmStart™ LAMP Kit (DNA & RNA), E1700S, NEB, Inc.) containing Bst 2.0 WarmStart DNA polymerase. In this example, the reaction mixture contained 10 μL of 2× LAMP reaction reagent, 2 μL of 10× primer composition, and 8 μL of the nucleic acid sample.

[0030] The reaction mixture was incubated at 65°C for 60 minutes for nucleic acid amplification, and then terminated at 80°C for 10 minutes. The resulting reaction mixture was then mixed with a nucleic acid fluorescent dye and loaded onto an agarose gel for electrophoresis. After electrophoresis, the gel was illuminated with blue light and the results were recorded. As shown in Figure 1 , "M" was used to mark the reference position where nucleic acid fragments of different sizes appeared as fluorescent bands on the agarose gel. Numerals 1 and 2 represent the results of electrophoresis of the reaction mixture containing the B. anthracis genome, while numerals 3 and 4 represent the results of electrophoresis of a no-template control containing no genome.

[0031] Referring to the electrophoresis results in Figure 1, multiple ladder-like fluorescent bands were observed at positions 1 and 2, indicating that the primer composition effectively amplified the Bacillus anthracis genome, producing multiple nucleic acid amplification products with different fragment sizes. There were no ladder-like fluorescent bands at positions 3 and 4, indicating the absence of nucleic acid amplification products. These experimental results demonstrate that the primer composition disclosed in the present invention can detect whether strawberries are infected with anthracnose.

[0032] (Experimental Example 2) In this experimental example, the primer composition disclosed in the present invention was used to perform a LAMP reaction on the genome of Bacillus anthracis and the genome of Trichoderma sp., to confirm whether the primer composition can specifically amplify the genome of Bacillus anthracis.

[0033] First, DNA was extracted from Bacillus anthracis and Trichoderma using the plant genome extraction kit to obtain nucleic acid samples containing the Bacillus anthracis genome and Trichoderma genome, respectively. In this example, Trichoderma isolates ML001 and ML1425 were used. The nucleic acid samples were then subjected to LAMP reaction and electrophoresis, and the results of the electrophoresis analysis were recorded. The conditions and steps used were the same as in Experimental Example 1. As shown in Figure 2, number 1 indicates the electrophoresis results of the positive control, which is a plastid containing a portion of the Bacillus anthracis gene; number 2 indicates the electrophoresis results of the reaction solution containing the Bacillus anthracis genome; numbers 3 and 4 indicate the electrophoresis results of the reaction solution containing the Trichoderma genome (isolate number ML001); numbers 5 and 6 indicate the electrophoresis results of the reaction solution containing the Trichoderma genome (isolate number ML1425); and numbers 7 and 8 indicate the electrophoresis results of the no-template control group containing no genome at all.

[0034] As shown in Figure 2, multiple ladder-like fluorescent bands were observed at positions 1 and 2, but no ladder-like fluorescent bands were observed at positions 3 to 8. This experimental result indicates that the primer composition disclosed in the present invention amplifies only nucleic acid samples containing the genome of Bacillus anthracis, but does not amplify nucleic acid samples containing the genome of Trichoderma, i.e., the primer composition of the present invention has the specificity to detect Bacillus anthracis genes from genes of different bacterial genera.

[0035] (Experimental Example 3) In this experimental example, the primer composition disclosed in the present invention was used to perform LAMP reactions on the genome of Bacillus anthracis, the leaf tissue of a healthy strawberry plant, and the leaf tissue of Bidens pilosa L. var. radiata Sch., to confirm whether the primer composition specifically amplifies the genome of Bacillus anthracis.

[0036] First, using the plant genome extraction kit, DNA was extracted from anthracnose bacteria, leaf tissue of healthy strawberry plants, and leaf tissue of Bidens frondosa to obtain nucleic acid samples.

[0037] In this experimental example, the LAMP reaction conditions were as follows: the reaction solution containing the nucleic acid sample was placed in an environment at 65°C for 30 minutes to allow nucleic acid amplification, and the nucleic acid amplification was terminated by reaction conditions at 80°C for 10 minutes. Subsequently, the reaction solution after the nucleic acid amplification reaction was mixed with a nucleic acid fluorescent dye and loaded onto an agarose gel for electrophoresis. After electrophoresis was completed, the electrophoresis analysis results were recorded under blue light irradiation. As shown in Figure 3, number 1 indicates the electrophoresis results of the reaction solution containing the genome of Bacillus anthracis; number 2 indicates the genome extracted from strawberry plants using an external device; number 3 indicates the electrophoresis results of the reaction solution containing the genome of healthy strawberry plant leaf tissue; number 4 indicates the electrophoresis results of the reaction solution containing the genome of Bidens frondosa leaf tissue; and number 5 indicates the electrophoresis results of the no-template control group containing no genome.

[0038] As shown in Figure 3, multiple ladder-like fluorescent bands were observed at position number 1, but no ladder-like fluorescent bands were observed at positions numbered 2 to 5. This experimental result indicates that the primer composition disclosed in the present invention amplifies only nucleic acid samples of anthrax genes, but does not amplify nucleic acid samples of healthy plant leaf tissues. In other words, the primer composition of the present invention has the specificity to detect anthrax genes from genes in plant leaf tissues.

[0039] (Experimental Example 4) In this experimental example, the primer composition disclosed in the present invention was used to carry out a LAMP reaction against a gene from a strawberry plant showing symptoms of anthracnose.

[0040] First, using the plant genome extraction kit, DNA was extracted from anthracnose bacteria, leaves of strawberry plants with anthracnose symptoms, and leaf tissue of healthy strawberry plants to obtain nucleic acid samples. Subsequently, a LAMP reaction was performed on the reaction solution containing the nucleic acid sample. In this experimental example, the reaction solution was placed in an environment of 65°C for 30 minutes to allow nucleic acid amplification, and the nucleic acid amplification reaction was terminated by reaction conditions of 80°C for 10 minutes. The reaction solution after the nucleic acid amplification reaction was then mixed with a nucleic acid fluorescent dye and added to an agarose gel for electrophoresis. After electrophoresis was completed, the gel was irradiated with blue light and the electrophoresis analysis results were recorded. As shown in Figure 4, number 1 shows the electrophoresis results of the positive control group, which was a plastid containing some of the genes of Bacillus anthracis; number 2 shows the electrophoresis results of the reaction solution containing the genome of Bacillus anthracis; number 3 shows the electrophoresis results of the reaction solution containing the genome of leaf tissue from a strawberry plant with anthracnose symptoms; number 4 shows the electrophoresis results of the reaction solution containing the genome of leaf tissue from a healthy strawberry plant; and number 5 shows the electrophoresis results of the no-template control group, which did not contain any genome.

[0041] As shown in Figure 4, multiple ladder-like fluorescent bands were observed at positions numbered 1 to 3, but no ladder-like fluorescent bands were observed at positions numbered 4 and 5. This experimental result indicates that the primer composition disclosed in the present invention can perform an amplification reaction not only on nucleic acid samples of anthracnose genes, but also on nucleic acid samples of leaf tissue from strawberry plants with anthracnose symptoms, i.e., the primer composition of the present invention can distinguish strawberry plants with anthracnose symptoms from healthy strawberry plants.

[0042] (Experimental Example 5) In this experimental example, a fluorescent marker, 6-carboxyfluorescein, is bound to the nucleotide sequence of SEQ ID NO: 6 of the primer composition disclosed in the present invention, and an antigen marker, biotin, is bound to the nucleotide sequence of SEQ ID NO: 7, thereby enabling the detection of whether an amplification product has been produced using a lateral flow test.

[0043] First, using the plant genome extraction kit, DNA was extracted from leaf tissue of seven groups of strawberry plants without anthracnose symptoms, the genomes of Bacillus anthracnose (C. siamense) isolated from a strawberry anthracnose strain, another Bacillus anthracnose strain (C. fructicola), and anthracnose (B. anthracnose) isolated from an onion anthracnose strain, and nucleic acid samples were obtained. Subsequently, a LAMP reaction and electrophoresis were performed, and the results of the electrophoresis analysis were recorded. The conditions and steps used were the same as in Experiment 1. Furthermore, in this experiment, a lateral flow test strip was used to detect the reaction solution after the LAMP reaction, and a detection test strip capable of binding to 6-carboxyfluorescein and biotin (Milenia Genline HybriDetect, Milenia Biotec, Inc.) was added, and the color change of the test strip was observed.

[0044] Figure 5A shows the results of electrophoresis analysis of the experimental example, with numbers 1 to 7 indicating the results of electrophoresis of the reaction solution containing the genome of leaf tissue from strawberry plants without anthracnose symptoms, number 8 indicating the results of electrophoresis of the reaction solution containing the genome of anthracnose Bacillus anthracnose (C. siamense) isolated from a strawberry anthracnose strain, number 9 indicating the results of electrophoresis of the reaction solution containing the genome of anthracnose Bacillus anthracnose (C. fructicola) strain, number 10 indicating the results of electrophoresis of the reaction solution containing the genome of anthracnose Bacillus anthracnose isolated from an onion anthracnose strain, and number 11 indicating the results of electrophoresis of a no-template control group containing no genome at all.

[0045] FIG. 5B shows the results of the lateral flow test of this experimental example, and the numbers 1 to 11 of these test strips correspond to the electrophoresis results of the detection of the numbers 1 to 11 in FIG. 5A, respectively.

[0046] As shown in Figures 5A and 5B, the lateral flow test results were consistent with those of conventional electrophoresis analysis. The electrophoresis results for numbers 1-7 (corresponding to the test strips numbered 1-7) all indicate that the reaction solution does not contain the amplification product of the anthrax gene, while the electrophoresis results for numbers 8-10 (corresponding to the test strips numbered 8-10) all indicate that the reaction solution contains the amplification product of the anthrax gene. These experimental results demonstrate that the method disclosed in the present invention can use the lateral flow test to detect whether the reaction solution contains the amplification product of the anthrax gene after the nucleic acid amplification reaction. Compared to electrophoresis analysis, the lateral flow test can be performed without complex equipment such as an electrophoresis tank, gel imager, or micropipette, and is inexpensive and easy to operate. This allows users to quickly detect whether plants are infected with anthrax even outdoors.

[0047] (Experimental Example 6) In this experimental example, the metal indicator hydroxynaphthol blue (HNB) was added to the reaction solution for the LAMP reaction, and the generation of nucleic acid amplification products was detected by utilizing the property of hydroxynaphthol blue that it changes color depending on the change in the concentration of magnesium pyrophosphate generated during the LAMP reaction.

[0048] First, DNA was extracted from an anthracnose strain of Bacillus anthracnose isolated from a strawberry anthracnose strain using the plant genome extraction kit to obtain a nucleic acid sample. The nucleic acid sample was then diluted to 2 ng, 0.2 ng, 0.02 ng, 0.002 ng, and 0.0002 ng (corresponding to numbers 1 to 5, respectively) to obtain the template amount for the nucleic acid amplification reaction. Each diluted nucleic acid sample was mixed with the primer composition disclosed in the present invention and hydroxynaphthol blue to prepare a reaction solution.

[0049] Subsequently, these reaction solutions were subjected to LAMP reaction and electrophoresis, and the results of the electrophoresis analysis were recorded, using the same conditions and steps as in Experimental Example 1. In addition, in this Experimental Example, the color change of hydroxynaphthol blue in the reaction tube was observed by photography after the LAMP reaction had been running for 30, 45, and 60 minutes.

[0050] Figures 6A, 6B, and 6C show the color change of hydroxynaphthol blue in the reaction tubes after the LAMP reaction had continued for 30, 45, and 60 minutes, respectively. In Figure 6A, the reaction solution in reaction tubes numbered 1 to 5 and numbered N was purple; in Figure 6B, the reaction solution in reaction tubes numbered 1 to 3 was blue, and the reaction solution in reaction tubes numbered 4, 5, and numbered N was purple; and in Figure 6C, the reaction solution in reaction tubes numbered 1 to 3 was blue, and the reaction solution in reaction tubes numbered 4, 5, and numbered N was purple. Figures 6D and 6E show the results of electrophoresis analysis after the LAMP reaction had been running for 30 and 60 minutes, respectively. In Figure 6D, clear ladder-like fluorescent bands were observed at positions 1 to 3, but no ladder-like fluorescent bands were observed at positions 4, 5, and N. In Figure 6E, clear ladder-like fluorescent bands were observed at positions 1 to 3, but less clear ladder-like fluorescent bands were observed at positions 4 and 5, but no ladder-like fluorescent bands were observed at position N.

[0051] As shown in Figures 6A, 6B, and 6D, after 30 minutes of LAMP reaction, nucleic acid amplification products were already produced in the reaction tubes using 2 ng, 0.2 ng, and 0.02 ng nucleic acid samples (numbers 1 to 3) as templates. However, a color change in hydroxynaphthol blue was not observed until 45 minutes after the LAMP reaction. After 60 minutes of LAMP reaction, hydroxynaphthol blue, which was inconsistent with the electrophoresis results, appeared in the reaction tubes using 0.002 ng and 0.0002 ng nucleic acid samples (numbers 4 and 5), indicating that the detection limit of the LAMP reaction had been reached. These experimental results indicate that the preferred conditions for implementing the method disclosed herein are to use 2 ng, 0.2 ng, and 0.02 ng nucleic acid samples as templates and to perform the LAMP reaction for 45 to 60 minutes.

Claims

1. Step 1: obtaining a nucleic acid sample isolated from a plant; Step 2, in which the nucleic acid sample and a primer composition are mixed to prepare a reaction solution, and the reaction solution is subjected to a nucleic acid amplification reaction, wherein the primer composition comprises a first primer pair, a second primer pair, and a third primer pair targeting a TUB2 gene region, the first primer pair comprising the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:3, respectively, the second primer pair comprising the nucleotide sequences of SEQ ID NO:4 and SEQ ID NO:5, respectively, and the third primer pair comprising the nucleotide sequences of SEQ ID NO:6 and SEQ ID NO:7, respectively; a step 3 of detecting whether or not the reaction solution after the nucleic acid amplification reaction contains a nucleic acid amplification product, and determining that the plant is infected with Bacillus anthracis if the nucleic acid amplification product is present, and determining that the plant is not infected with Bacillus anthracis if the nucleic acid amplification product is not present; The nucleic acid amplification reaction is a loop-mediated isothermal nucleic acid amplification reaction. Methods for detecting plant anthracnose.

2. 2. The method for detecting plant anthracnose of claim 1, wherein the nucleic acid sample comprises a nucleic acid having the nucleotide sequence of SEQ ID NO:

1.

3. 2. The method for detecting plant anthracnose according to claim 1, wherein at least one primer in the primer composition is bound to a detectable marker, and the detectable marker is a fluorescent marker, an antigen marker, or a combination thereof.

4. 4. The method for detecting plant anthracnose according to claim 3, wherein step 3 is performed by detecting whether the reaction solution after the nucleic acid amplification reaction contains a nucleic acid amplification product using a lateral flow test.

5. a first primer pair consisting of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3; a second primer pair consisting of the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 5; a third primer pair consisting of the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 7 for use in a loop-mediated isothermal nucleic acid amplification reaction; A primer composition for detecting plant anthracnose.

6. The primer composition according to claim 5 , wherein a detectable marker is bound to at least one primer in the primer composition, and the detectable marker is a fluorescent marker, an antigen marker, or a combination thereof.

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