Alternania alternania RPA lateral flow test strip detection kit and application thereof in Bo-chrysanthemum disease detection

Abstract

The invention discloses an Alternania alternana RPA (recombinase polymerase amplification) lateral flow test strip detection kit and application thereof in Bo-chrysanthemum disease detection, and belongs to the field of rapid detection of plant pathogenic fungi. The detection kit provided by the invention comprises a reagent for detecting Alt a 1, the reagent comprises a first RPA primer, and the nucleotide sequences of the first RPA primer are respectively SEQ ID NO.1 and SEQ ID NO.2; the reagent for detecting TEF1 alpha comprises an RPA primer II, and the nucleotide sequences of the RPA primer II are respectively SEQ ID NO.4 and SEQ ID NO.5; and an RPA reaction component. Compared with the prior art, the kit disclosed by the invention has the advantages that through mutual verification of double targets, the detection specificity and accuracy are improved, false negative caused by gene variation of a single target is avoided, and the identification capability on a complex sample is improved.

Description

Technical Field

[0001] This invention relates to the field of rapid detection of plant pathogenic fungi, specifically to... Alternaria alternata RPA lateral flow test strip test kit and its application in the detection of diseases in Bozhou chrysanthemum. Background Technology

[0002] Bo chrysanthemum is the dried capitulum of the chrysanthemum plant (Chrysanthemum morifolium), a member of the Asteraceae family. The flowers are large and white, and it is mainly produced in Bozhou City, Anhui Province. Bo chrysanthemum is a traditional Chinese medicine with the character "Bo" in its name, and it possesses functions such as dispelling wind and clearing heat, calming the liver and improving eyesight, and clearing heat and detoxifying.

[0003] Alternaria alternata *Alternaria alternata* is a common plant pathogenic fungus that can cause leaf spot, fruit rot, and other diseases in various crops. Traditional detection methods include microscopic morphological identification and molecular detection techniques: morphological identification relies on microscopic observation of colony morphology and conidial characteristics, but... Alternaria Because species are morphologically similar, identification results are easily influenced by subjective factors. In recent years, isothermal amplification technologies (such as LAMP and RPA) have been introduced into the field of plant pathogen detection. For example, Chinese patent CN106987653B discloses a method for detecting plant fungi based on LAMP technology, but this method has a complex detection process, long reaction time, and often requires a fluorescence detector, making it inconvenient for field use.

[0004] The ITS area serves as a universal fungal barcode. A. alternata and A. tenuissima , A. arborescens The ITS regions of closely related species are highly similar, and conventional PCR methods are prone to cross-reactivity, which reduces the accuracy of detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes an Alternaria alternata RPA lateral flow test strip detection kit and its application in the detection of diseases in Bozhou chrysanthemum.

[0006] The objective of this invention can be achieved through the following technical solutions: A first aspect of the present invention relates to a detection kit, comprising: Used for detection Above 1 The reagents include RPA primer 1, with nucleotide sequences SEQ ID NO.1 and SEQ ID NO.2, respectively; Used for detection TEF1α The reagents include RPA primer 2, with nucleotide sequences SEQ ID NO.4 and SEQ ID NO.5, respectively; And RPA reaction components.

[0007] Optionally, the RPA reaction components include: recombinase, single-strand binding protein, strand displacement DNA polymerase, and buffer.

[0008] Optionally, the method for detection Above 1 The reagents also include probe one, whose nucleotide sequence is: SEQ ID NO. 3; Optionally, the reagent for detecting TEF1α further includes probe II, with the nucleotide sequence: SEQ ID NO.6.

[0009] Optionally, the 5' end of probe one and / or probe two is marked with FAM.

[0010] Optionally, the middle section of probe one and / or probe two contains a dSpacer cutting site, and the 3′ end has a C3Spacer blocking extension.

[0011] Optionally, it also includes a method for detection IT Reagents for the region.

[0012] A second aspect of the present invention relates to detection Above 1 The reagents or the kits mentioned above are used in the detection Alternaria alternate Applications in [the context of the text].

[0013] Optionally, the detection Alternaria alternata Including distinction Alternaria alternata and A. very thin , A. arborescens .

[0014] The beneficial effects of this invention are: 1. Significantly improved detection specificity pass Above 1 Gene+ TEF1-α Gene dual-target combination detection effectively distinguishes A. alternata and A. tenuissima, A. arborescens Avoid closely related species. IT Cross-reactivity issues that are prone to occur in area testing.

[0015] 2. The dual targets mutually verify each other, reducing false positives and false negatives and improving the reliability of test results.

[0016] 3. High detection sensitivity and short reaction time Using optimized RPA-specific primers and probes, amplification can be completed in 20 minutes at low temperature (37–42℃). Detection sensitivity reaches the picomolar (pM) level, enabling the detection of low-copy-level pathogen DNA, making it suitable for early disease detection.

[0017] 4. Multi-channel visual inspection The designed multi-channel lateral flow test strip can simultaneously detect multiple gene loci and includes a control line (C line), enabling parallel detection of multiple targets in a single reaction. Detection results can be directly interpreted via colorimetric analysis, eliminating the need for expensive laboratory testing equipment.

[0018] 5. Strong on-site portable testing capability The test reagents are in the form of lyophilized RPA reaction components, which can be stored for a long time at room temperature (≥6 months), and are convenient to transport and store. Attached Figure Description

[0019] The invention will now be further described with reference to the accompanying drawings.

[0020] Figure 1 This is a schematic diagram showing the sensitivity test results of the detection kit of this application; 10 0 ~10 2 Significant fluorescence signals were obtained at all template DNA concentrations, with the concentration decreasing to 10. -4 ~10 -1 The fluorescence intensity decreased significantly, but remained observable. Figure 2 This is a schematic diagram of the detection results of the test kit of this application for samples with dilution ratios of 50 to 5000 times; in the figure, the control line (C line) and the test line (T line) of the test strip are clearly colored, showing a positive result, indicating that the kit can stably produce positive detection results under various labeled conditions, reflecting the robustness of the detection method; Figure 3 This diagram illustrates the detection results of the test kit of this application for samples diluted 10 to 1000 times. At low dilutions (20 times, 10 times), the test line (T line) is dark, indicating a strong positive result. As the dilution increases to 100, the T line becomes lighter, but is still observable. When diluted to 1000 times, the T line essentially disappears, and the result tends to be negative. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] In some embodiments of the present invention, a method for detecting Alternaria alternata The reagent kit.

[0023] In some examples of the present invention, the reagent kit is selected Alt a1 Genes, as the core detection target, are species-specific and can accurately distinguish between species. A. alternata and A. tenuissima , A. arborescens Closely related species.

[0024] In some examples of the present invention, the reagent kit is selected from... TEF1-α Genes serve as secondary barcodes to further enhance species identification capabilities. ITS regions are selected as universal detection targets for fungi or as positive internal controls.

[0025] In some examples of this invention, a pair of RPA primers were designed for each target gene (the forward primer has a Biotin label at the 5′ end, and the reverse primer is unlabeled), with a primer length of 30–35 bp and an amplified fragment length of 100–300 bp.

[0026] For each target gene, an RPA probe (with a FAM marker at the 5′ end, a dSpacer (THF) cleavage site in the middle, and a C3 Spacer to block extension at the 3′ end) was designed. The probe was 46–52 bp in length, located in the middle of the amplification product, and did not overlap with the primers.

[0027] In some examples of this invention, the primer and probe sequences used in the kit are shown in the table below: In some embodiments of the present invention, RPA refers to Recombinase Polymerase Amplification; the RPA primers may be oligonucleotide fragments designed from the specific gene sequences of the target fungus.

[0028] In some examples, the components required for the RPA reaction may include recombinase, single-stranded DNA binding protein, strand-displacing DNA polymerase, etc. The recombinase is used to guide the primer to a homologous sequence of the template DNA. The single-stranded DNA binding protein (SSB) binds to and stabilizes the single strand of DNA uncracked by the recombinase-primer complex, creating conditions for polymerase binding and elongation. The strand-displacing DNA polymerase is used to synthesize new DNA strands and achieve continuous amplification.

[0029] In some examples, the target fungal-specific gene sequence includes, but is not limited to, those described above. ITS、TEF1α、Alt a1One or more of the following. The detection method for the target fungal-specific gene sequence can be PCR amplification and sequencing.

[0030] In some examples, the FAM label refers to the Carboxyfluorescein fluorescent label.

[0031] In some examples, the dSpacer (THF) cleavage site refers to a structure artificially inserted into the DNA strand that can be recognized and cleaved by a specific enzyme. C3 Spacer extension blocking can be a non-nucleotide spacer consisting of a three-carbon chain (propylene glycol group) used to prevent DNA polymerase from extending the DNA.

[0032] The kit may also include Mg²⁺ starter solution and buffer, positive control DNA and negative control (without template), and multichannel lateral flow test strips (each detection channel corresponds to a gene target, with a detection line T and a control line C).

[0033] In other embodiments of the present invention, a method for detecting using the kit described in the above embodiments is disclosed. Alternaria alternata The method may include the following steps: Sample preparation: Take samples of diseased tissue from plant leaves and fruits, and perform rapid DNA extraction or crude lysis.

[0034] Reaction preparation: Add template DNA, lyophilized RPA components, corresponding primers, probes, and initiation solution to the reaction tube and mix well.

[0035] Isothermal amplification: React at 37–42 ℃ for 20 minutes, gently mixing once during the process.

[0036] Lateral flow detection: Add 5–10 μL of amplification product to the sample well of the lateral flow test strip, add buffer, let stand for 3–5 minutes, and observe the color development results.

[0037] Result interpretation rules: C-line color development: The testing process is effective.

[0038] T-line staining: positive for the corresponding gene target.

[0039] Multi-channel interpretation: Above 1 Positive and TEF1-α Positive result is A. alternata ; IT A positive result indicates a positive fungal test result within the internal control.

[0040] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0041] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A detection kit, comprising: Reagents for detecting Alt a 1 RPA primers, nucleotide sequences of which are SEQ ID NO. 1 and SEQ ID NO. 2, respectively; Reagents for detecting TEF1 a RPA primer two, nucleotide sequences of which are SEQ ID NO. 4 and SEQ ID NO. 5, respectively; and RPA reaction components.

2. The test kit according to claim 1, characterized in that, The RPA reaction components comprise: recombinase, single-strand binding protein, strand displacement DNA polymerase and buffer.

3. The test kit according to claim 1, characterized in that, The reagent for detecting Alt a 1 The reagent for detecting TEF1 a The reagent for detecting 4. The test kit according to claim 1, characterized in that, The 5' end of SEQ ID NO. 1 of RPA primer one and / or SEQ ID NO. 4 of RPA primer two is labeled with biotin.

5. The test kit according to claim 3, characterized in that, The 5' end of the probe one and / or probe two is labeled with FAM.

6. The test kit according to claim 3, characterized in that, The middle section of the probe one and / or probe two contains a dSpacer cleavage site, and the 3' end is blocked with C3 Spacer.

7. The test kit according to claim 1, characterized in that, Also included are reagents for detecting the ITS region.

8. The test kit according to claim 7, characterized in that The reagents for detecting the ITS region include PCR amplification and sequencing related reagents.

9. Use of a reagent for detecting Alt a 1 or a kit according to any one of claims 1 to 8 for detecting Alternaria alternata ​ 10. The assay of claim 9 Alt a 1 or the kit of any one of claims 1 to 8 for use in the detection Alternaria alternata wherein the assay is a lateral flow assay. The detection Alternaria alternata including distinguishing between diseases in chrysanthemum Alternaria alternata Alternaria alternata with A. tenuissima, A. arborescens.

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