Method for detecting plant anthracnose and primer composition
The primer composition for loop-mediated isothermal nucleic acid amplification with lateral flow testing simplifies and accelerates plant anthracnose detection, addressing the complexity of conventional PCR methods by allowing rapid and user-friendly detection.
Patent Information
- Application Number
- JP2023111516
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2023-07-06
- Publication Date
- 2025-01-20
- Estimated Expiration
- 2043-07-06
AI Technical Summary
Current methods for detecting plant anthracnose, such as those using conventional PCR, are complex and require specialized laboratory equipment and knowledge, making it difficult for ordinary users to perform real-time detection outside the lab.
A primer composition for loop-mediated isothermal nucleic acid amplification combined with lateral flow testing, utilizing specific primer pairs (SEQ ID NO: 2-7) to amplify and detect anthracnose-specific nucleic acid sequences without temperature changes, allowing for simplified and rapid detection.
The method significantly reduces reaction time and simplifies equipment requirements, enabling quick and effective detection of plant anthracnose without the need for advanced laboratory facilities.
Smart Images
Figure 2025008906000001_ABST
Abstract
Description
[Technical field]
[0001] The present invention relates to a method and primer composition for detecting plant anthracnose, in particular for strawberry anthracnose utilizing 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 genus that infects plants or ornamental plants such as orchids (Orchidaceae), apples (Malus pumila), peppers (Capsicum spp.), coffee plants (Coffea spp.), grapes (Vitis vinifera), longans (Dimocarpus longan), mangoes (Mangifera indica), canarium album, bitter orange (Citrus spp.), pears (Pyrus spp.), peaches (Prunus persica), strawberries (Fragaria ananassa), watermelons (Citrullus lanatus), and tea plants (Camellia spp.) via conidia. When the environment is favorable for the development of the disease or when the immunity of the host plant is weak, the disease enters the host plant through the water pores and other natural openings on the leaf margins, 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, seriously affecting the plant yield 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 of different gene loci. Commonly used gene sequences include 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) genes. Currently, when detecting whether a plant is infected with anthracnose, the gene is often detected using conventional polymerase chain reaction (PCR), but PCR requires related equipment and laboratory staff familiar with molecular biology techniques, and the detection steps are complicated, making it difficult for general users to detect in real time outdoors. Therefore, how to detect whether a plant is infected with anthracnose in a simplified, 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: obtaining a nucleic acid sample isolated from a plant; step 2 of mixing the nucleic acid sample with a primer composition to prepare a reaction solution and carrying out a nucleic acid amplification reaction on the reaction solution, the primer composition comprising a first primer pair, a second primer pair and a third primer pair, the first primer pair comprising nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:3, the second primer pair comprising nucleotide sequences of SEQ ID NO:4 and SEQ ID NO:5, and the third primer pair comprising 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, determining that the plant is infected with Bacillus anthracnose if the nucleic acid amplification product is present, and determining that the plant is not infected with Bacillus anthracnose 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. Effect 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 increase or decrease the temperature, the overall reaction time can be shortened, the experimental equipment can be simplified, and it is useful for quickly detecting whether a plant is infected with Bacillus anthracis. [Brief description of the drawings]
[0007] [Figure 1] FIG. 1 shows the results of electrophoretic analysis after loop-mediated isothermal nucleic acid amplification reaction on the genome of Bacillus anthracis using the primer composition disclosed in the present invention. [Diagram 2] FIG. 2 shows the results of electrophoretic 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. [Diagram 3] FIG. 3 shows the results of electrophoretic analysis after loop-mediated isothermal nucleic acid amplification reactions 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 electrophoretic analysis after loop-mediated isothermal nucleic acid amplification reactions were performed on plastids containing some genes of Bacillus anthracnose, the genomes 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 electrophoretic analysis following loop-mediated isothermal nucleic acid amplification reactions on leaf tissue of strawberry plants without anthracnose symptoms 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 on leaf tissue of strawberry plants without anthracnose symptoms and the genome of Bacillus anthracnose using the primer composition disclosed in the present invention. [Figure 6A] FIG. 6A shows the results of hydroxynaphthol blue color change 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 hydroxynaphthol blue color change 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 hydroxynaphthol blue color change 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 electrophoretic analysis after 30 minutes of loop-mediated isothermal nucleic acid amplification reaction on different concentrations of B. anthracis genome using the primer composition disclosed in the present invention. [Figure 6E] FIG. 6E shows the results of electrophoretic analysis after 60 minutes of loop-mediated isothermal nucleic acid amplification reaction on different concentrations of B. anthracis genome using the primer composition disclosed in the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0008] The term "nucleic acid amplification reaction" as used herein means any biochemical technique that increases the number of copies 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, in particular, involves using a primer composition to perform a nucleic acid amplification reaction on the genome of Bacillus anthracnose, and determining whether or not the plant is infected with Bacillus anthracnose by detecting whether or not a nucleic acid amplification product is produced.
[0011] The method disclosed in the present invention includes the following steps. Step 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, using the nucleic acid sample 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, it is detected whether the reaction solution after the nucleic acid amplification reaction contains a nucleic acid amplification product. If the nucleic acid amplification product is present, it is determined that the plant is infected with Bacillus anthracis, and if the nucleic acid amplification product is not present, it is determined that the plant is not infected with Bacillus anthracis.
[0012] Step 1 of the method involves obtaining a nucleic acid sample isolated from a plant, the nucleic acid sample comprising a sequence of a portion of the beta-tubulin (β-tubulin-2, TUB2) genome of Bacillus anthracis, the sequence comprising 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 pepper (Dalbergia odorifera), cranberry (Cranberry), bitter orange (Citrus spp.), tea plant (Camellia spp.), garlic (Garlic), poplar (Poplar), guava (Psidium guajava), fig (Ficus carica), camu camu (Myrciaria dubia, Blueberry, Xanthorrhoea, Banana, Coffea spp., Chinese chestnut, American ginseng, vegetable soybean, wheat, Punica granatum, Eucalyptus, Pyrus spp., Cunninghamia lanceolata, Sweet Persimmon, or a combination thereof.
[0014] In some embodiments, the nucleotide sequence of SEQ ID NO:1 is a 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 a Bacillus anthracis species C. aenigma, C. fructicola, C. alienum, C. gleosporioides, 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. inotum, 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 cell wall of the plant and purifying the nucleic acid in the cell. The method for dissolving the cell wall includes, but is not limited to, ultrasonic shaking, physical polishing, organic solvent dissolution, etc., and the method for purifying the nucleic acid in the cell includes, but is not limited to, separating the nucleic acid 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 tissue of the plant 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 the reaction solution is subjected to a nucleic acid amplification reaction, in which the nucleic acid sample serves as a template for the nucleic acid amplification reaction. The primer composition includes a first primer pair, a second primer pair, and a third primer pair. The first primer pair includes the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:3, the second primer pair includes the nucleotide sequences of SEQ ID NO:4 and SEQ ID NO:5, and the third primer pair includes 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 detection marker, which is subsequently used to detect whether a nucleic acid amplification product has been produced or to determine the amount of a nucleic acid amplification product. The detection 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, the nucleotide sequence of SEQ ID NO:6 of the third primer pair is bound to a fluorescent marker, which is 6-carboxyfluorescein (6-FAM), and the nucleotide sequence of SEQ ID NO:7 is bound to an antigen marker, which is biotin.
[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 embodiment, the reaction solution further comprises, but is not limited to, deoxyribonucleoside triphosphate (dNTP), 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 in which the DNA polymerase, deoxyribonucleoside triphosphate, buffer, and / or surfactant are mixed.
[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), 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 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. Compared with other nucleic acid amplification reactions, the loop-mediated isothermal nucleic acid amplification reaction does not require a large increase or decrease in temperature, which shortens the overall reaction time and simplifies the experimental equipment, and also improves specificity through a specially designed primer composition.
[0021] In step 3 of the method, whether the reaction solution after the nucleic acid amplification reaction contains a nucleic acid amplification product is detected. If the nucleic acid amplification product is present, the plant is determined to be infected with Bacillus anthracis, and if the nucleic acid amplification product is not present, the plant is determined to be not infected with Bacillus anthracis. Methods for detecting whether the nucleic acid amplification product is present include, but are not limited to, turbidity measurement, fluorescence detection, bioluminescence detection, gel electrophoresis, colorimetric detection, immunoenzyme detection, electrochemical detection, color change of a test paper, etc., and these detection methods may be performed using steps and conditions well known in the art. In one embodiment, a loop-mediated isothermal nucleic acid amplification reaction is used as the nucleic acid amplification reaction, and since magnesium pyrophosphate is generated in large amounts as a white precipitate after the loop-mediated isothermal nucleic acid amplification reaction, whether the precipitate is generated can be observed by turbidity measurement. In another embodiment, the reaction product can be subjected to electrophoretic analysis by agarose gel electrophoresis and nucleic acid fluorescent staining to determine whether a target nucleic acid amplification fragment of a specific nucleic acid size is generated.
[0022] In one embodiment, a primer or a nucleic acid amplification product containing a detection marker is applied to a test paper, and the color of the test paper determines whether a nucleic acid amplification product has been produced, where the principle used to change the color of the test paper includes, but is 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 change in magnesium ion concentration, and it is possible to determine whether a nucleic acid amplification product has been produced from the change in color of the reaction solution.
[0024] The present invention further discloses a primer composition for detecting plant anthracnose, comprising 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.
[0025] In one embodiment, the primer composition is utilized to perform a loop-mediated isothermal nucleic acid amplification reaction, which may be performed as a nucleic acid amplification reaction under isothermal conditions, the isothermal conditions being 50-75° C. In a preferred embodiment, the isothermal conditions are 60-70° 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 with a detection marker that 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 embodiment, a fluorescent marker that is 6-carboxyfluorescein is conjugated to the nucleotide sequence of SEQ ID NO:6 of the third primer pair, and an antigen marker that is biotin is conjugated to the nucleotide sequence of SEQ ID NO:7.
[0027] In the present invention, the following experimental examples are used to demonstrate that the method and the primer composition can detect the gene of Bacillus anthracis. The following examples are provided to illustrate the present invention, and the scope of the present invention is not limited by the examples. Those skilled in the art can utilize the disclosure and teachings of the present invention to obtain other specific embodiments, forms and modifications without new experiments.
[0028] (Experimental Example 1) In the experimental 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 plant genome extraction kit (The Genomic DNA Isolation Kit (Plant), NA025-0100, GeneDireX, Inc.) was used to extract DNA from anthracnose bacteria isolated from a strawberry anthracnose strain, thereby obtaining a nucleic acid sample containing the genome of anthracnose bacteria. In this example, the anthracnose species isolated from the strawberry anthracnose strain was Colletotrichum siamense (isolation number ML133). Furthermore, the nucleic acid sample was mixed with the primer composition disclosed in the present invention, and a LAMP reaction reagent (WarmStart (trademark) LAMP Kit (DNA & RNA), E1700S, NEB, Inc.) was added to prepare a reaction solution, and the LAMP reaction reagent contained Bst 2.0 WarmStart DNA polymerase. In this experimental example, the reaction solution contained 10 μL of 2 × LAMP reaction reagent, 2 μL of 10 × the primer composition, and 8 μL of the nucleic acid sample.
[0030] The reaction solution was placed in an environment of 65°C to carry out the nucleic acid amplification reaction for 60 minutes, and the nucleic acid amplification reaction was terminated by reaction conditions of 80°C for 10 minutes. Subsequently, the reaction solution after the nucleic acid amplification reaction was mixed with a nucleic acid fluorescent dye and added to an agarose gel for electrophoresis. After the electrophoresis was completed, blue light was irradiated and the results of the electrophoretic analysis were recorded. As shown in Figure 1, "M" was the reference position where nucleic acid fragments of different sizes were displayed as fluorescent bands in the agarose gel, which was marked using a standard product with a molecular weight of 100 bp (DNA ladder, DM003-R500, GeneDireX, Inc.). Numbers 1 and 2 show the results of electrophoresis of the reaction solution containing the genome of Bacillus anthracis, and numbers 3 and 4 show the results of electrophoresis of the no-template control group that did not contain any genome.
[0031] Referring to the electrophoresis results in FIG. 1, multiple ladder-like fluorescent bands were observed at positions 1 and 2, indicating that the primer composition effectively amplified the genome of Bacillus anthracis, generating multiple nucleic acid amplification products with different fragment sizes. There were no ladder-like fluorescent bands at positions 3 and 4, indicating that there were no nucleic acid amplification products. The experimental results show 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 carry out 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, the plant genome extraction kit was used to extract DNA from Bacillus anthracis and Trichoderma to obtain a nucleic acid sample containing the genome of Bacillus anthracis and a nucleic acid sample containing the genome of Trichoderma, respectively. In this embodiment, Trichoderma with isolation numbers ML001 and ML1425 were used. Then, the nucleic acid sample was subjected to LAMP reaction and electrophoresis, and the results of the electrophoretic analysis were recorded, and the conditions and steps used were the same as those in Experimental Example 1. As shown in FIG. 2, number 1 shows the results of electrophoresis of the positive control group, which is a plastid containing a part of the gene of Bacillus anthracis, number 2 shows the results of electrophoresis of the reaction solution containing the genome of Bacillus anthracis, numbers 3 and 4 show the results of electrophoresis of the reaction solution containing the genome of Trichoderma (isolation number ML001), numbers 5 and 6 show the results of electrophoresis of the reaction solution containing the genome of Trichoderma (isolation number ML1425), and numbers 7 and 8 show the results of electrophoresis of the no-template control group that does not contain any genome.
[0034] As shown in Figure 2, multiple ladder-like fluorescent bands were observed at positions numbered 1 and 2, but no ladder-like fluorescent bands were observed at positions numbered 3 to 8. This experimental result indicates that the primer composition disclosed in the present invention only amplifies 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 a LAMP reaction 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, the plant genome extraction kit was used to extract DNA from anthracnose bacteria, leaf tissue from healthy strawberry plants, and leaf tissue from Bidens frondosa to obtain nucleic acid samples.
[0037] In this experimental example, the conditions used in the LAMP reaction were to place the reaction solution containing the nucleic acid sample in an environment of 65°C to carry out the nucleic acid amplification reaction for 30 minutes, and then to terminate the nucleic acid amplification reaction at 80°C for 10 minutes. Then, the reaction solution after the nucleic acid amplification reaction was mixed with a nucleic acid fluorescent dye and added to an agarose gel for electrophoresis, and after the electrophoresis was completed, blue light was irradiated and the results of the electrophoresis analysis were recorded. As shown in Figure 3, number 1 shows the electrophoresis result of the reaction solution containing the genome of Bacillus anthracis, number 2 shows the genome extracted from strawberry plants by an external device, number 3 shows the electrophoresis result of the reaction solution containing the genome of healthy strawberry plant leaf tissue, number 4 shows the electrophoresis result of the reaction solution containing the genome of Bidens pilosa leaf tissue, and number 5 shows the electrophoresis result of the no-template control group that does not contain any 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 performs an amplification reaction only on a nucleic acid sample of anthrax genes, but does not amplify a nucleic acid sample of healthy plant leaf tissue, i.e., the primer composition of the present invention has the specificity to detect anthrax genes from genes in plant leaf tissue.
[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 of a strawberry plant exhibiting anthracnose symptoms.
[0040] First, the plant genome extraction kit was used to extract DNA from anthracnose bacteria, leaves of strawberry plants with symptoms of anthracnose, and leaf tissues of healthy strawberry plants to obtain nucleic acid samples. Then, a LAMP reaction was performed on the reaction solution containing the nucleic acid sample. In this experimental example, the conditions used in the LAMP reaction were to place the reaction solution in an environment of 65°C to carry out a nucleic acid amplification reaction for 30 minutes, and to terminate the nucleic acid amplification reaction under reaction conditions of 80°C and 10 minutes. Then, the reaction solution after the nucleic acid amplification reaction was mixed with a nucleic acid fluorescent dye, added to an agarose gel, electrophoresed, and after completion of electrophoresis, irradiated with blue light to record the results of the electrophoretic analysis. As shown in Figure 4, number 1 shows the electrophoresis result of the positive control group, which was a plastid containing some of the genes of Bacillus anthracis, number 2 shows the electrophoresis result of the reaction solution containing the genome of Bacillus anthracis, number 3 shows the electrophoresis result of the reaction solution containing the genome of leaf tissue of a strawberry plant with anthracnose symptoms, number 4 shows the electrophoresis result of the reaction solution containing the genome of leaf tissue of a healthy strawberry plant, and number 5 is the electrophoresis result of the no-template control group containing no genome at all.
[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 a nucleic acid sample of anthracnose genes, but also on a nucleic acid sample of leaf tissue of a strawberry plant with anthracnose symptoms, i.e., the primer composition of the present invention can distinguish between strawberry plants with anthracnose symptoms and 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 a lateral flow test to be used to detect whether an amplification product has been produced.
[0043] First, the plant genome extraction kit was used to extract DNA from the leaf tissues of seven groups of strawberry plants without symptoms of anthracnose, the genomes of anthracnose bacteria (C. siamense) isolated from a strawberry anthracnose strain, another anthracnose strain (C. fructicola), and anthracnose bacteria isolated from an onion anthracnose strain, and nucleic acid samples were obtained. Then, the LAMP reaction and electrophoresis were performed, and the results of the electrophoresis analysis were recorded, and the conditions and steps used were the same as those in Experimental Example 1. In addition, in this experimental example, a lateral flow test paper was used to detect the reaction solution after the LAMP reaction, and a detection test paper (Milenia Genline HybriDetect, milenia biotec, Inc.) that can bind to 6-carboxyfluorescein and biotin was added to observe the color change of the test paper.
[0044] Figure 5A shows the results of electrophoresis analysis of the experimental example, where numbers 1 to 7 respectively show the results of electrophoresis of the reaction solution of the genome of leaf tissue of strawberry plants without anthracnose symptoms, number 8 shows the result of electrophoresis of the reaction solution containing the genome of anthracnose Bacillus anthracnose strain (C. siamense) isolated from a strawberry anthracnose strain, number 9 shows the result of electrophoresis of the reaction solution containing the genome of anthracnose Bacillus anthracnose strain (C. fructicola), number 10 shows the result of electrophoresis of the reaction solution containing the genome of anthracnose Bacillus anthracnose strain isolated from an onion anthracnose strain, and number 11 is the result of electrophoresis of the 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 numbers 1 to 11 of these test strips correspond to the electrophoretic results of detection of numbers 1 to 11 in FIG. 5A, respectively.
[0046] As shown in Figures 5A and 5B, the lateral flow test is consistent with the experimental results of conventional electrophoretic analysis, and the electrophoretic results of numbers 1 to 7 (corresponding to the test strips of numbers 1 to 7) all indicate that the reaction solution does not contain the amplification product of the anthrax gene, and the electrophoretic results of numbers 8 to 10 (corresponding to the test strips of numbers 8 to 10) all indicate that the reaction solution contains the nucleic acid amplification product of the anthrax gene. The experimental results show that the method disclosed in the present invention can use the lateral flow test to detect whether the reaction solution after the nucleic acid amplification reaction contains the nucleic acid amplification product of the anthrax gene. Compared with electrophoretic analysis, the lateral flow test can be performed without complex equipment such as an electrophoretic tank, a gel imager, and a micropipette, and is low-cost and easy to operate, so that users can operate it themselves even outdoors to quickly detect whether plants are infected with anthrax.
[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 production of nucleic acid amplification products was detected by utilizing the property of hydroxynaphthol blue that it changes color depending on the change in concentration of magnesium pyrophosphate produced during the LAMP reaction.
[0048] First, the plant genome extraction kit was used to extract DNA from the anthracnose bacteria isolated from the strawberry anthracnose strain to obtain a nucleic acid sample. The nucleic acid sample was then diluted to 2ng, 0.2ng, 0.02ng, 0.002ng, and 0.0002ng (corresponding to numbers 1 to 5, respectively) to obtain the template amount for the nucleic acid amplification reaction. Each of the diluted nucleic acid samples was mixed with the primer composition disclosed in the present invention and hydroxynaphthol blue to prepare a reaction solution.
[0049] Next, the reaction solutions were subjected to LAMP reaction and electrophoresis, and the results of the electrophoretic 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 allowed to proceed for 30 minutes, 45 minutes, and 60 minutes.
[0050] Figures 6A, 6B, and 6C show the color change of hydroxynaphthol blue in the reaction tubes after 30, 45, and 60 minutes of LAMP reaction, respectively, where the reaction solution in reaction tubes numbered 1 to 5 and N in Figure 6A was purple, the reaction solution in reaction tubes numbered 1 to 3 in Figure 6B was blue, and the reaction solution in reaction tubes numbered 4, 5, and N was purple, and the reaction solution in reaction tubes numbered 1 to 3 in Figure 6C was blue, and the reaction solution in reaction tubes numbered 4, 5, and N was purple. Figures 6D and 6E show the results of electrophoretic analysis after the LAMP reaction had run 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 band was observed at position N.
[0051] As shown in the experimental results of Figures 6A, 6B and 6D, when the LAMP reaction had been running for 30 minutes, the reaction tubes using 2ng, 0.2ng and 0.02ng of nucleic acid samples (numbers 1 to 3) as template already produced nucleic acid amplification products, but the color change of hydroxynaphthol blue was observed after 45 minutes of LAMP reaction. After 60 minutes of LAMP reaction, the reaction tubes using 0.002ng and 0.0002ng of nucleic acid samples (numbers 4 and 5) as template showed hydroxynaphthol blue that did not match the results of electrophoresis, which should have reached the detection limit of the LAMP reaction. The experimental results show that the preferred implementation conditions of the method disclosed in the present invention are to use 2ng, 0.2ng and 0.02ng of nucleic acid samples as template and to carry out the LAMP reaction for 45 to 60 minutes.
Claims
1. Step 1: obtaining a nucleic acid sample isolated from a plant; Step 2, which comprises mixing the nucleic acid sample with a primer composition to prepare a reaction solution and subjecting the reaction solution to a nucleic acid amplification reaction, wherein the primer composition comprises a first primer pair, a second primer pair, and a third primer pair, the first primer pair comprising nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:3, the second primer pair comprising nucleotide sequences of SEQ ID NO:4 and SEQ ID NO:5, and the third primer pair comprising nucleotide sequences of SEQ ID NO:6 and SEQ ID NO:7; and step 3 of detecting whether the reaction solution after the nucleic acid amplification reaction contains a nucleic acid amplification product, 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.
2. 2. The method for detecting plant anthracnose as described in claim 1, wherein the nucleic acid sample comprises the nucleotide sequence of SEQ ID NO:
1.
3. 2. The method for detecting plant anthracnose according to claim 1, wherein the nucleic acid amplification reaction is a loop-mediated isothermal nucleic acid amplification reaction.
4. 4. The method for detecting plant anthracnose according to claim 3, wherein at least one nucleotide sequence in the primer composition is linked to a detectable marker, the detectable marker being a fluorescent marker, an antigen marker or a combination thereof.
5. 5. The method for detecting plant anthracnose according to claim 4, wherein step 3 is to detect whether the reaction solution after the nucleic acid amplification reaction contains a nucleic acid amplification product by a lateral flow test.
6. 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; A primer composition for detecting plant anthracnose, comprising: a primer pair comprising the nucleotide sequences of SEQ ID NO:6 and SEQ ID NO:
7.
7. The primer composition according to claim 6, which is used in a loop-mediated isothermal nucleic acid amplification reaction.
8. The primer composition according to claim 7, wherein a detectable marker is bound to at least one nucleotide sequence in the primer composition, and the detectable marker is a fluorescent marker, an antigen marker, or a combination thereof.