Method for detecting grapefruit botryosphaeria hemsleyana based on combination of RPA and MIRA-LFD

Abstract

The invention discloses an RPA (recombinase polymerase amplification)-combined MIRA-LFD (multiple-input multiple-output lateral flow dipstick) detection method for grapevine leptosphaeria The invention belongs to the technical field of biology, and particularly relates to a grapevine botryosphaeria hemsleyana detection method based on combination of RPA and MIRA-LFD. The composition for identifying or assisting in identifying the grapefruit botryosphaeria hemsleyana is a composition A or a composition B, wherein the composition A consists of RPA-F1 and RPA-R1; the composition B is composed of RPA-F1, RPA-R2 and a specific fluorescent probe sequence LFD; the RPA-F1 is DNA (deoxyribonucleic acid) with a nucleotide sequence shown as SEQ ID No. 1; the RPA-R1 is DNA (deoxyribonucleic acid) of which the nucleotide sequence is SEQ ID No.2; the RPA-R2 is DNA (deoxyribonucleic acid) of which the nucleotide sequence is SEQ ID No.3; the sequence LFD of the probe is DNA with the nucleotide sequence of SEQ ID No.4.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for detecting grape winged leafroller based on RPA combined with MIRA-LFD. Background Technology

[0002] Grape flower winged moth ( Lobesia botrana The grape leafroller (Lobesia variegata) belongs to the order Lepidoptera, family Tortricidae, and genus Lobesia. It was officially listed in the "List of Quarantine Pests of Plants Entering China" in 2007 and is considered an important global quarantine economic pest. Initially discovered in southern Italy, it is now distributed throughout Europe, North and West Africa, the Middle East and Far East in Asia, and parts of the United States. This insect feeds on approximately 40 different plant species; however, its feeding on grapes makes the grape leafroller a significant economic pest in grape production. It can directly feed on inflorescences and fruits, or cause severe damage to grapes by promoting infections with pathogenic fungi such as gray mold and black mold. It can also promote attacks by secondary pests, including fruit flies such as the black-bellied fruit fly and the spotted-winged fruit fly. Therefore, the grape leafroller is a very serious pest in vineyards, requiring long-term monitoring and control. In actual port quarantine, intercepted insects are often incomplete stages, with indistinct external morphological characteristics in the egg, pupal, or larval stages. They must be reared to adulthood for further morphological observation or dissection of the external genitalia of male and female adults for identification, a time-consuming process that makes accurate and rapid differentiation of these pests and their similar species challenging. Therefore, rapid and efficient molecular identification techniques play a crucial role in early monitoring and protecting agricultural and forestry safety from these invasive pests.

[0003] Currently, new molecular identification technologies are still being developed and applied. For example, the combination of recombinase polymerase amplification (RPA) technology with multi-enzyme isothermal rapid amplification and lateral flow chromatography strips (MIRA-LFD) provides a new approach for the molecular identification of invasive insects. Summary of the Invention

[0004] The main problem this invention aims to solve is how to quickly identify the grape flower leaf roller moth.

[0005] To address the aforementioned problems, the present invention provides a composition for identifying or assisting in the identification of the grape vine leafroller moth.

[0006] This invention provides a composition for identifying or assisting in the identification of the grape bud roller moth, the composition being composition A or composition B, wherein composition A consists of an upstream primer named RPA-F1 and a downstream primer named RPA-R1; The composition B is composed of an upstream primer with the name of RPA-F1, a downstream primer with the name of RPA-R2, and a fluorescent probe sequence LFD specific to the grape flower winged leaf roller to be detected; The RPA-F1 is a DNA with the nucleotide sequence of SEQ ID No. 1 in the sequence listing; The RPA-R1 is a DNA with the nucleotide sequence of SEQ ID No. 2 in the sequence listing; The RPA-R2 is a DNA with the nucleotide sequence of SEQ ID No. 3 in the sequence listing; The probe sequence LFD is a DNA with the nucleotide sequence of SEQ ID No. 4 in the sequence listing.

[0007] The present application provides the use of the composition described above in any one of the following: a1) preparing a product for identifying or discriminating grape flower winged leaf rollers; a2) identifying or discriminating grape flower winged leaf rollers; a3) preparing a product for detecting whether grape flower winged leaf rollers are contained in a sample to be detected; a4) detecting whether grape flower winged leaf rollers are contained in a sample to be detected.

[0008] The present application also provides a kit for identifying or assisting in identifying grape flower winged leaf rollers, which contains the composition described above and M or N or a lateral flow test strip. The M is MgOAC, Primer Free Rehydration Buffer, enzyme dry powder, and ddH2O. The N is A Buffer, B Buffer, a small tube containing enzyme dry powder, and ddH2O.

[0009] In the above-mentioned kit, the kit is an isothermal amplification kit.

[0010] The M can be a TwistAmp@Basic Kit (TwistDx, TABAS03KIT, UK) kit.

[0011] The N can be a DNA isothermal rapid amplification kit (colloidal gold type) (Anpu Future, product number: WLN8203KIT).

[0012] The lateral flow test strip can be a nucleic acid detection test strip (Anpu Future, WLFS8204).

[0013] The application also provides a preparation method of the kit, comprising the steps of separately packaging the upstream primer of RPA-F1, the downstream primer with the name of RPA-R1, the downstream primer with the name of RPA-R2 and the probe sequence LFD specific to the grape Adoxophyes orana.

[0014] The application also provides the use of the kit in any one of the following: a1) preparing a product for identifying or discriminating Adoxophyes orana; a2) identifying or discriminating Adoxophyes orana; a3) preparing a product for detecting whether a sample to be tested contains Adoxophyes orana; a4) detecting whether a sample to be tested contains Adoxophyes orana.

[0015] The application also provides a method for detecting Adoxophyes orana, comprising detecting whether a sample to be tested contains the nucleic acid of Adoxophyes orana by using the composition.

[0016] The application also provides a method for identifying or assisting in identifying Adoxophyes orana, comprising the following steps: S1: extracting the genomic DNA of a suspected sample of Adoxophyes orana; S2: using the genomic DNA of the sample as the template DNA, performing the multi-enzyme constant-temperature rapid amplification by using the kit to obtain an amplification product, and then performing a color development reaction by using a test strip, so as to determine whether the sample to be tested is Adoxophyes orana according to the color of the color development reaction; The specific determination method is as follows: If the detection line of the test strip presents red, the sample to be tested is Adoxophyes orana; if the detection line of the test strip does not present red, the sample to be tested is not Adoxophyes orana.

[0017] In the above method, the reaction condition of the multi-enzyme constant-temperature rapid amplification is 37 ℃ for 6 min.

[0018] In a specific embodiment, the MIRA-LFD reaction system is (50 μL): 29 μL A buffer, 2 μL RPA-F1 (10 μM), 2 μL RPA-R2 (10 μM), 2.5 μL B buffer, 9 μL ddH2O, 1 μL LFD-LB-Probe (10 μM) and 5 μL template DNA.

[0019] The template DNA can be the DNA of the larvae, pupae or adult of Adoxophyes orana.

[0020] The present application is based on the establishment of the grape moth detection method of RPA combined with MIRA-LFD, and the detection process can catalyze the reaction at room temperature, so that the identification is not limited in the laboratory, and without the help of PCR instrument and other high-cost, inconvenient equipment, not only can achieve the purpose of simple, rapid, sensitive and specific detection, but also can judge the detection result by naked eye, and the technical requirement of personnel is not high, and better visual service can be provided for on-site and field rapid identification of important invasive pests, and has wide popularization and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a MIRA-LFD visual identification flowchart. Wherein C is a quality control line, and T is a detection line.

[0022] Figure 2 It is a specific gel electrophoresis result map of grape moth based on RPA detection. Wherein M: D2000Marker; 1: grape moth; 2: larch moth (Zhouzhi County, Shaanxi Province); 3: larch moth (Ningqiang County, Shaanxi Province); 4: mulberry moth; 5: giant moth; N: ddH2O.

[0023] Figure 3 It is a specific verification of MIRA-LFD detection of different geographical populations of grape moth. Wherein 1: grape moth; 2: larch moth (Zhouzhi County, Shaanxi Province); 3: larch moth (Ningqiang County, Shaanxi Province); 4: mulberry moth; 5: giant moth; N: ddH2O.

[0024] Figure 4 It is a result map of MIRA-LFD detection of grape moth visual reaction temperature and time optimization. Wherein (A) 1-5: the reaction temperature is 33, 35, 37, 39, 41 ℃; (B) 1-5: the reaction time is 2, 4, 6, 8, 10 min.

[0025] Figure 5 It is the sensitivity of MIRA-LFD detection of grape moth. Wherein 1-7: 100.0 ng / μL, 10.0 ng / μL, 1.0 ng / μL, 1.0×10 -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 ng / μL; N: ddH2O.

[0026] Figure 6 It is a result map of MIRA-LFD detection of different instar crude extracts of grape moth. Wherein 1: larva; 2: pupa; 3: adult; N: ddH2O. DETAILED DESCRIPTION

[0027] The present application will be further described in conjunction with the preferred embodiments thereof, given only by way of illustration of the present application, and not to be construed as a limitation of the present application. The following examples provided as a guide for those skilled in the art to make further improvements, and do not constitute any limitation of the present application in any way.

[0028] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0029] The quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged, unless otherwise specified.

[0030] The grape flower winged small-rolled moth in the following examples is donated by John Vontas, which has been recorded in: Albaz E, Katsavou E, Cagatay N S, et al. Analysis of insecticide resistance and de novo transcriptome assembly of resistance associated genes in the European grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae) [J]. Bulletin of Entomological Research, 2024, 114(1): 88-98. The biological material can be obtained from the applicant, and can only be used for repeating the experiments of the present application, and cannot be used for other purposes.

[0031] The detection operation flowchart of the RPA-based MIRA-LFD for detecting grape flower winged small-rolled moth provided by the present application is as shown in Figure 1 : First, the suspected sample is collected and DNA is extracted, then the MIRA-LFD reaction is carried out, and finally the result is determined by the test strip.

[0032] Example 1, design of RPA primer and MIRA-LFD probe The design of the RPA specific primer is based on the DNA barcode sequence of cytochrome C oxidase No. 1 (COI) gene (COI gene nucleotide sequence is SEQ ID No. 5) of grape flower winged small-rolled moth (different geographical populations) and other 3 similar species (different geographical populations).

[0033] The RPA primer design principles referenced the primer design requirements in the TwistDx manual (https: / / www.twistdx.co.uk / wp-content / uploads / 2021 / 04 / twistamp-assay-design-manual-v2-5.pdf). Primer length: 30-36 bp, amplification length: 100-300 bp, maximum 500 bp, dG < -4 kcal, CG content = 20%-70%, Tm = 50-100℃, maximum allowed single-base repeats: 5), and NCBI-Primer Blast was used for design. All used sequences were aligned using Clustal W in MEGA X, and highly variable sites were manually selected and recorded in BioEdit. Based on the Primer Blast output, the primer sequences were manually adjusted to ensure primer specificity and amplified fragment synthesis.

[0034] The design of MIRA-LFD primers and probes also referenced the primer design requirements in the TwistDx operating instructions. It should be noted that the downstream primer of MIRA-LFD needs to be biotin-tagged at the 5' end. Probe design principle: Design a probe of 46-52 nucleotides in length, complementary to the target fragment, and located between the upstream and downstream primers. The probe sequence should not overlap with the recognition site of the specific primer. Avoid the presence of palindromic sequences, internal secondary structures, and continuous repeating bases in the probe sequence. The probe has three modification sites: (1) Modify the 5' end with an antigen marker (FAM). (2) Introduce dSpacer (tetrahydrofuran, THF) at about 30 nucleotides from the 5' end as the recognition site for nfo. (3) Place THF at about 15 nucleotides from the 3' end and modify the 3' end with a modifying group such as C3-spacer. The primers were synthesized by Qingke Biotechnology (Beijing) and stored in a freezer at -20℃. The sequences are shown in Table 1.

[0035] Table 1. Primer sequence information of the present invention

[0036] Note: The C at position 1 of the LFD-LB-Probe can be labeled FAM, the T at position 33 can be replaced with tetrahydrofuran (THF), and the 3' end can be labeled C3-spacer.

[0037] Example 2: Study on RPA-based MIRA-LFD visualization molecular identification technology for the grape flowering leafroller moth 1. RPA-specific primer screening DNA from different geographical populations of the grape budworm was used as the target species, along with two other fruit borers and ddH2O as negative controls. RPA reactions were performed using the TwistAmp@Basic Kit (TwistDx, TABAS03KIT, UK). The reaction mixture consisted of 50 μL of the following: 2 μL (10 μM) forward primer RPA-F1, 2 μL (10 μM) reverse primer RPA-R1, 29.5 μL Primer-Free Rehydration Buffer, 9 μL ddH2O, and 5 μL template DNA. After mixing, the mixture was quickly transferred to a reaction tube containing enzyme powder. The enzyme powder was dissolved, and finally 2.5 μL of MgOAc was added. The mixture was then inverted and mixed thoroughly.

[0038] The RPA amplification conditions were: amplification at 39 ℃ for 20 min, followed by stopping the reaction at 95 ℃ for 5 min. To purify the RPA product, 25 μL of a phenol / chloroform / isoamyl alcohol mixture (25:24:1) was added and centrifuged at 12000 rpm for 10 min. 5 μL of the amplified product was subjected to 1% agarose gel electrophoresis at 220 V for 30 min. The optimal primers were selected by observing the amplification results under UV light using a Gel Logic 212 PRO (Carestream Kodak, Canada). Figure 2 If the amplified product is 278 bp in size, the sample is Grape Flowerwing Moth; if there is no amplified product of 278 bp in size, the sample is not Grape Flowerwing Moth.

[0039] Figure 2 The results showed that if the grape flower winged moth showed a clear and bright band at the corresponding fragment size (278 bp), while other negative controls did not show obvious bands, it indicated that the primers were specific and could be used for subsequent experiments.

[0040] 2. Visualized Specificity Detection of Grape Flower Leaf Roller using MIRA-LFD DNA was extracted from the grape vine leafroller using a blood / cell / tissue genomic DNA extraction kit (centrifuge column type) (Tiangen, DP304).

[0041] The overall MIRA-LFD reaction system (50 μL) using the DNA Isothermal Rapid Amplification Kit (Colloidal Gold Form) (AMP Future, Catalog No.: WLN8203KIT) was as follows: 29 μL A buffer, 2 μL RPA-F1 (10 μM), 2 μL reverse primer RPA-R2 (10 μM), 2.5 μL B buffer, 9 μL ddH2O, 1 μL LFD-LB-Probe (10 μM) and 5 μL template DNA.

[0042] After the reaction, the amplification product was diluted 10-fold with ddH2O and mixed thoroughly. The diluted mixture was then applied to the sample wells of colloidal gold test strips to observe the color change. Results could be obtained within 5 minutes by observing the presence or absence of the control and detection lines.

[0043] The specificity of the MIRA-LFD identification method for the grapevine leafroller was visualized. Results showed that only the test line on the grapevine leafroller test strip was red, indicating a positive reaction. Conversely, only the control line in the other negative controls was blue, demonstrating that the MIRA-LFD detection also has high specificity. Figure 3 ).

[0044] 3. Optimization of MIRA-LFD Visualized Response Parameters for Grape Flower Leaf Roller Five reaction time gradients were set for MIRA-LFD (33℃, 35℃, 37℃, 39℃, and 41℃), and five reaction time gradients were also set for each gradient (2 min, 4 min, 6 min, 8 min, and 10 min). After the reaction, the amplification product was diluted 10-fold with ddH2O and thoroughly mixed. The diluted mixture was then applied to the sample wells of colloidal gold test strips to observe the color change. Results could be obtained within 5 minutes by observing the presence or absence of the control line and the test line.

[0045] The only difference between step 3 and step 2 in the MIRA-LFD reaction is the temperature and time. The results show that the optimal reaction time and temperature combination for the MIRA-LFD identification method of the grape budworm is 6 min - 37℃. Figure 4 (A) and (B)).

[0046] 4. Visual Sensitivity Detection of Grape Flower Leaf Roller using MIRA-LFD The template DNA from the grapevine flower-winged leafroller was diluted with ddH2O in six serial concentrations: 100.0 ng / μL, 10.0 ng / μL, 1.0 ng / μL, and 1.0 × 10⁻⁶ ng / μL. -1 ng / μL, 1.0 × 10 -2ng / μL, 1.0 × 10 -3 ng / μL. After the reaction, the amplification product was diluted 10-fold with ddH2O and mixed thoroughly. Then, the diluted mixture was applied to the sample wells of colloidal gold test strips to observe the color change. Results could be obtained within 5 minutes by observing the presence or absence of the control line and the test line.

[0047] The only difference between step 4 and step 2 in the MIRA-LFD reaction is the concentration of the grape vine leafroller template DNA used in the MIRA-LFD reaction. The results show that the detection sensitivity can reach 1.0 × 10⁻⁶. -2 ng / μL, exhibiting high sensitivity ( Figure 5 ).

[0048] 5. Rapid on-site MIRA-LFD visualization detection of grape flowering leafrollers in different developmental stages based on crude extraction. Rapid DNA extraction was performed on different developmental stages (larva, pupa, and adult) of the grape vine leafroller, and MIRA-LFD detection was conducted after extraction.

[0049] The RPA-based MIRA-LFD detection method is as follows: Using a sterilized pipette tip, tissue samples from *Gnaphalium affine* at different developmental stages are ground or aspirated into 0.2 mL centrifuge tubes. 20-30 μL of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) is added, and the 0.2 mL tubes are incubated in a 95°C water bath for 5 minutes. The supernatant contains crudely extracted DNA, which serves as the reaction template.

[0050] The overall MIRA-LFD reaction system, performed using the DNA Isothermal Rapid Amplification Kit (Colloidal Gold Form) (AMP Future, China), consisted of 50 μL: 29 μL A buffer, 2 μL RPA-F1 (10 μM), 2 μL RPA-R2 (10 μM), 2.5 μL B buffer, 9 μL ddH2O, 1 μL LFD-LB-Probe (10 μM), and 5 μL template DNA.

[0051] After the reaction, the amplification product was diluted 10-fold with ddH2O and mixed thoroughly. The diluted mixture was then applied to the sample wells of colloidal gold test paper to observe the color change. Results were obtained within 5 minutes by observing the presence or absence of the control and detection lines. The optimal reaction time and temperature combination for the MIRA-LFD identification method for the grapevine leafroller was determined to be 6 min–37℃.

[0052] Figure 6The results showed that the reaction system containing DNA from the larvae, pupae, and adults of the grape vine leafroller could produce clear control and detection lines on the test strips simultaneously, while the reaction system containing the blank control could only produce control lines on the test strips. This indicates that template DNA from the grape vine leafroller can be detected in all three developmental stages based on crude DNA extraction.

[0053] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A composition for identifying or aiding in identifying grape moth, said composition is composition A or composition B, said composition A consists of an upstream primer with the name of RPA-F1, a downstream primer with the name of RPA-R1; Said composition B consists of an upstream primer with the name of RPA-F1, a downstream primer with the name of RPA-R2, a fluorescent probe sequence LFD specific to grape moth to be detected; Said RPA-F1 is a DNA with the nucleotide sequence of SEQ ID No. 1 in the sequence listing; Said RPA-R1 is a DNA with the nucleotide sequence of SEQ ID No. 2 in the sequence listing; Said RPA-R2 is a DNA with the nucleotide sequence of SEQ ID No. 3 in the sequence listing; Said probe sequence LFD is a DNA with the nucleotide sequence of SEQ ID No. 4 in the sequence listing.

2. The use of the composition of claim 1 in any one of the following: a1) preparing a product for identifying or identifying grape moth; a2) identifying or identifying grape moth; a3) preparing a product for detecting whether the sample to be detected contains grape moth; a4) detecting whether the sample to be detected contains grape moth.

3. A kit for identifying or aiding in the identification of grape leaffolder, characterized in that, The kit contains the composition of claim 1 and M or N or lateral flow test strip; Said M is MgOAC, Primer Free Rehydration Buffer, a small tube containing enzyme dry powder, ddH2O; Said N is A Buffer, B Buffer, a small tube containing enzyme dry powder, ddH2O; Said lateral flow test strip is a nucleic acid detection test strip.

4. The kit of claim 3, wherein: The kit is an isothermal amplification kit.

5. The preparation method of the kit of claim 3 or 4, comprising the step of separately packaging the upstream primer of RPA-F1, the downstream primer with the name of RPA-R2, and the probe sequence LFD specific to grape moth to be detected.

6. The use of the kit of claim 3 or 4 in any one of the following: a1) preparing a product for identifying or identifying grape moth; a2) identifying or identifying grape moth; a3) preparing a product for detecting whether the sample to be detected contains grape moth; a4) detecting whether the sample to be detected contains grape moth.

7. A method for detecting grape moth, comprising using the composition of claim 1 to detect whether the sample to be detected contains the nucleic acid of grape moth.

8. A method for identifying or aiding in identifying grape moth, comprising the following steps: S1: extracting the genomic DNA of the suspected sample of grape moth; S2: using the genomic DNA of the sample as template DNA, using the kit of claim 3 or 4 to perform multi-enzyme isothermal rapid amplification to obtain amplification products, and then using test strips to perform color development reaction, and determining whether the sample to be detected is grape moth according to the color of the color development reaction; The specific determination method is as follows: If the detection line of the test strip presents red color, the sample to be measured is grape flower winged small-roller moth; if the detection line of the test strip does not present color, the sample to be measured is not grape flower winged small-roller moth.

9. The method of claim 8, wherein, The reaction condition of the isothermal amplification is 37 DEG C for 6 min.

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