Method for detecting pine wood nematodes

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for detecting bursaphelenchus xylophilus in water extract. The method comprises the following steps: firstly, enriching aqueous solutions containing different concentrations of pine wood nematode eDNA by adopting filter membranes made of different materials, and screening out a filter membrane material suitable for enriching eDNA in wood pine wood nematode; furthermore, a method for rapidly, simply and conveniently detecting pine wood nematodes is established based on the material. RPA detection is carried out after sample nucleic acid is enriched through the filter membrane material screened in the invention, the defect of low eDNA abundance in a wood water immersion liquid is overcome, large-sample-size detection can be carried out, and compared with a traditional means, the filter membrane material is quicker, simpler and more convenient.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for detecting pine wood nematodes in aqueous solutions. Background Technology

[0002] Pine wilt disease (PWD), also known as pine blight, is caused by the pine wilt nematode Bursaphelenchus xylophilus (FUTAI K. Pinewood nematode, Bursaphelenchus xylophilus [J]. Annual Review of Phytopathology, 2013, 51(1):61-83.). Numerous studies have shown that pine wilt nematodes originated in North America (IWAHORIH, KANZAKIN, FUTAIK. Bursaphelenchus xylophilus and B. mucronatus in Japan: Where are they from? [C] / / Fourth International Congress of Nematology, 2002. Nematology Monographs and Perspectives 2, 2004: 793-803.). In 1982, pine wilt nematodes were first isolated from black pines at the Sun Yat-sen Mausoleum in Nanjing, China (Sun Yongchun. Pine wilt nematodes discovered at the Sun Yat-sen Mausoleum in Nanjing [J]. Jiangsu Forestry Science and Technology, 1982: 47.). Since then, pine wilt disease has spread rapidly in my country, seriously threatening the country's ecological security. Timely and accurate monitoring of pine wilt nematodes and clarifying their distribution are the primary steps in effectively controlling the spread of this disease. In the early stages of pine wilt disease, the nematode is unevenly distributed within the pine host tree, necessitating the collection of large quantities of wood samples for isolation and identification to effectively prevent missed detection. However, identifying pine wilt nematodes from large quantities of wood typically requires the use of a modified Behman funnel method, which is cumbersome and time-consuming. Directly using molecular detection methods such as PCR or real-time fluorescence PCR to detect wood samples only allows for the extraction of small amounts of wood nucleic acid for each test, easily leading to misjudgments, and the experimental process is relatively time-consuming (Li Min, Ye Jianren, Chen Fengmao. Research progress on pine wilt nematode detection technology [J]. Chinese Journal of Forest Diseases and Insect Pests, 2022, 41(3):52-58.). Therefore, there is an urgent need to develop a rapid and simple method for detecting and identifying pine wilt nematodes from large quantities of wood.

[0003] Environmental DNA (eDNA) is a technology developed in the 1990s. It refers to DNA extracted from environmental samples such as water and sediments, including intracellular DNA and extracellular DNA, and can be used to monitor the species richness of aquatic organisms (REESHC, MADDISONBC, MIDDLEDITCHDJ, et al. The detection of aquatic animal species using environmental DNA - a review of eDNA as a survey tool in ecology[J]. Journal of Applied Ecology, 2014, 51(5):1450-1490.). Unlike traditional biomonitoring methods, eDNA does not require manipulation of the species themselves but only of the environmental samples. It has advantages such as low investment, simple sampling, and high sensitivity, and is gradually being applied to biological species monitoring, biomass estimation, etc. (Xing Yingchun, Gao Wanru, Bai Jie, et al. Application of environmental DNA in lake biodiversity research[J]. Acta Hydrobiologica Sinica, 2022, 46(1):137-148.). Pine wood nematode primarily parasitizes the water-conducting tissues of the vascular bundles in pine trees. Therefore, monitoring for pine wood nematode by collecting eDNA from a large number of pine wood samples in aqueous extracts is feasible. However, the abundance of eDNA in large quantities of wood extracts is low, so an efficient enrichment method needs to be established before detection.

[0004] Currently, commonly used filter membranes for enriching eDNA in aquatic environments include mixed cellulose membranes, glass fiber membranes, polycarbonate membranes, nitrocellulose membranes, and nylon membranes. The enrichment mechanism of these membranes is based on physical retention due to membrane pore size and adsorption retention based on the membrane's chemical properties and electrostatic attraction. Factors affecting the detection efficiency of filter membranes are related to the material of the membrane. Related research results show that filter membranes of different materials have different enrichment effects for different environments and research subjects. Liang and Keeley studied the effects of different filter membrane types and pore sizes on the recovery rate of microbial DNA in surface water samples. The results showed that MCE had the best DNA recovery effect in environmental water samples (Liang Z, Keeley A. Filtration Recovery of Extracellular DNA from Environmental Water Samples[J]. Environmental Science & Technology, 2013, 47(16): 9324-9331.). Li Hongting et al. compared the enrichment effects of nitrocellulose membranes, cellulose acetate membranes, glass fiber membranes, and polycarbonate membranes on environmental DNA in water bodies to study aquatic biodiversity. The results showed that cellulose acetate membranes obtained the highest concentration of eDNA (Li Hongting, Zhang Shuai, Zou Keshu, et al. Establishment and optimization of environmental DNA extraction method in Pearl River Estuary [J]. Southern Fisheries Science, 2022, 18(3): 8.). Li Jiaqiao et al. compared the enrichment effects of different filter membranes such as nitrocellulose membranes, polycarbonate membranes, and glass fiber membranes on eDNA in seawater and concluded that nitrocellulose membranes were the best. Mixed cellulose membranes are composed of a mixture of cellulose acetate and cellulose nitrate membranes (Li Jiaqiao, Xie Yanhui, Zhang Na, et al. Preliminary study on the enrichment of Vibrio parahaemolyticus of acute hepatopancreatic necrosis disease in water bodies based on eDNA technology [J]. China Port Science and Technology, 2023, 5(4): 60-68.). Summary of the Invention

[0005] This invention first enriches aqueous solutions containing different concentrations of pine wood nematode eDNA using filter membranes of different materials, screening out suitable filter membrane materials for enriching eDNA from pine wood nematodes. Furthermore, based on this material, a rapid and simple method for detecting pine wood nematodes is established. Thus, this invention is complete.

[0006] In a first aspect, the present invention provides an RPA primer for detecting pine wood nematodes in an aqueous extract, the primer comprising an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2, the RPA primer being used to amplify pine wood nematode DNA.

[0007] Secondly, the present invention provides an RPA-nfo probe for detecting pine wood nematodes in a water immersion solution. The nucleotide sequence of the probe is shown in SEQ ID NO: 3. The probe generates a visible signal on the test strip by specifically recognizing or binding to pine wood nematode DNA.

[0008] Thirdly, the present invention provides a kit for detecting pine wood nematode, the kit comprising an RPA primer pair and an RPA-nfo probe, the primers comprising an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2, and the RPA-nfo probe nucleotide sequence as shown in SEQ ID NO: 3.

[0009] Furthermore, the RPA primers can be used to amplify pine wood nematode DNA.

[0010] Furthermore, the RPA-nfo probe generates a visible signal on the test strip by specifically recognizing or binding to pine wood nematode DNA.

[0011] Fourthly, the present invention provides a method for detecting pine wood nematodes, the method comprising the following steps:

[0012] S1. Collect samples and prepare sample aqueous extract;

[0013] S2. Total nucleic acids are enriched by filtering the aqueous extract prepared in S1 through a filter membrane;

[0014] S3. Extract the total nucleic acids enriched in S2 according to the DNA extraction kit instructions;

[0015] S4. Amplify the total nucleic acid extracted in S3 using RPA primers;

[0016] S5. Detect the amplified product from S4 and interpret the results.

[0017] Furthermore, in step S1, when the water extract is a water extract of pine wood nematode tissue, the water extract is obtained by repeatedly freezing and thawing the nematode and then grinding it.

[0018] Furthermore, in step S1, when the water extract is a water extract containing pine wood nematodes, the water extract is obtained by soaking the wood containing pine wood nematodes in water.

[0019] Furthermore, in step S2, the filter membrane is a cellulose membrane.

[0020] Furthermore, the cellulose membrane is selected from one or more of nitrocellulose membranes, polycarbonate membranes, glass fiber membranes, and / or mixed cellulose membranes.

[0021] Preferably, the cellulose membrane is a mixed cellulose membrane.

[0022] Furthermore, in step S4, the RPA primers include an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2.

[0023] Furthermore, the RPA amplification reaction time is 10-40 min.

[0024] Furthermore, the RPA amplification reaction time is 15-35 min.

[0025] Preferably, the RPA amplification reaction time is 20-30 min.

[0026] More preferably, the RPA amplification reaction time is 30 min.

[0027] Furthermore, the RPA amplification reaction temperature is 36-42℃.

[0028] Furthermore, the RPA amplification temperature is 37-41℃.

[0029] Preferably, the RPA amplification temperature is 38-40℃.

[0030] More preferably, the RPA amplification temperature is 39°C.

[0031] Furthermore, in step S5, the result interpretation method includes, but is not limited to, electrophoresis and / or test strip method.

[0032] Furthermore, the test strip method utilizes the RPA primers described in the first aspect and the RPA-nfo probes described in the second aspect to specifically recognize or bind to pine wood nematode DNA, thereby generating a visible signal on the test strip.

[0033] Furthermore, when using test strips for detection, if both the control line and the test line of the test strip show bands, then the sample to be tested is or contains pine wood nematode; if the control line of the test strip shows a band but the test line does not show a band, then the sample to be tested is not or does not contain pine wood nematode.

[0034] Beneficial effects

[0035] 1. This invention creatively proposes a method for detecting pine wood nematode through wood. This method can directly filter the sample and then enrich and extract DNA. Compared with the traditional method of extracting DNA by grinding wood, this method reduces the wood grinding step and can be used for enrichment and extraction detection on a large number of pieces of wood (pine wood nematode is unevenly distributed and few in number in the early stage of disease). This method reduces the chance of missed detection and the test procedure is also simpler.

[0036] 2. This invention focuses on the enrichment of pine wood nematode eDNA in aqueous solutions using commonly used filter membranes for enriching eDNA in aquatic environments. A filter membrane material that can efficiently enrich large amounts of eDNA in wood water extracts is selected, with a detection limit of 4 μL.

[0037] 3. This invention utilizes RPA technology to obtain specific primers and probes based on the genomic DNA sequence of *Pinus wood nematode*, enabling the detection of the target nematode. Based on the designed primer pairs and probes, this invention establishes a method for detecting *Pinus wood nematode* from a large volume of wood extracts. This method monitors *Pinus wood nematode* using eDNA from a large number of pine wood samples, exhibiting high specificity and sensitivity. However, this method can only specifically detect *Pinus wood nematode*, and does not show specific band fragments for other nematodes; the sensitivity of the RPA method is 3.69 × 10⁻⁶. -3 ng / μL.

[0038] 4. The filter membrane material selected in this application enriches the nucleic acid of the sample before RPA detection, which not only overcomes the defect of low eDNA abundance in wood water extract, but also enables large sample volume detection, which is faster and simpler than traditional methods. Attached Figure Description

[0039] Figure 1 This is a standard curve for real-time fluorescent PCR of pine wood nematode nucleic acid.

[0040] Figure 2 A comparison of the recovery rates of eDNA enriched by filter membranes at different concentrations.

[0041] Figure 3 The graphs show the MCE fluorescence curves at different concentrations.

[0042] Figure 4 For the selection of primer pairs.

[0043] Figure 5 Optimize RPA reaction time. (Note: M: DL2000 Marker; 1-7: amplification time is 10, 15, 20, 25, 30, 35, 40 min; 8: NC).

[0044] Figure 6 Optimization of RPA reaction temperature. (Note: M: DL2000 Marker; 1-7: 36, 37, 38, 39, 40, 41, 42℃; 8: NC).

[0045] Figure 7 For RPA-specific detection. (Note: M: DL2000 Marker; 1-6: Pine Wood Nematode; 7: Pine Wood Nematode; 8: Bean-shell Nematode; 9: Conical-tailed Nematode; 10: False-shell Nematode; 11: NC).

[0046] Figure 8 For specific detection of colloidal gold test strips.

[0047] Figure 9 For RPA sensitivity detection. (M: DL2000 Marker; 1-7: concentrations of 36.9, 36.9×10⁻¹~36.9×10⁻⁶ ng / μL, 8: NC).

[0048] Figure 10 Sensitivity testing of the test strip detection system. (Note: Concentrations from left to right are 78 ng / μL - 78 × 10⁻⁷ ng / μL). Detailed Implementation

[0049] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0050] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0051] Example 1: Filter membrane screening for pine wood nematode eDNA enrichment

[0052] 1. Materials and Methods

[0053] 1.1 Test Materials

[0054] Pine wood nematode (No. B00160) and diseased pine wood were preserved in the Nematode Laboratory of the Institute of Plant Inspection and Quarantine, Chinese Academy of Inspection and Quarantine Sciences; mixed cellulose membrane MCE (50 mm, 0.45 μm), polyethersulfone membrane PES (50 mm, 0.45 μm), and cellulose acetate membrane CA (50 mm, 0.45 μm).

[0055] 1.2 Preparation of Standard Curve

[0056] One hundred pine wood nematodes were selected and placed in centrifuge tubes. After adding sterile water, the tissues were repeatedly frozen and thawed in liquid nitrogen. The filtrate was then collected and diluted with sterile water to a final volume. This mixture was used as the initial nematode tissue mixture (equivalent to a concentration of 1 nematode / mL).

[0057] Take the above initial nematode mixture and dilute it at a ratio of 1:10 to four concentration gradients. Dilute the lowest concentration (1:10) to four different concentrations. -4 The concentration of nematodes / mL is specified as 1, therefore the concentration gradient of the diluted standard solution is 10. 4 10 3 102 10.1. DNA was extracted using the DNAsecure novel plant genomic DNA extraction kit and then detected by real-time fluorescent PCR.

[0058] A standard curve was constructed with the logarithm of the nematode mixture concentration as the Y-axis and the number of real-time fluorescence PCR reaction cycles as the X-axis. The filter membrane recovery rate was calculated as the sample recovery concentration / the actual concentration added.

[0059] 1.3 Sample Preparation

[0060] Aspirate from the initial nematode tissue mixture into a graduated cylinder and bring the volume to a final volume with sterile water to obtain concentration A. After thoroughly mixing concentration A, dilute it serially at a ratio of 1:10 to obtain concentrations B and C. Bring each treatment to a final volume and set aside. Repeat each experiment three times.

[0061] 1.4 Nucleic acid enrichment of samples

[0062] Place the three types of filter membranes (MCE, PES, and CA) on the filter respectively, pour the prepared nematode tissue mixtures of different concentrations into the filter cups respectively, turn on the diaphragm vacuum pump, and wait for all the liquid to pass through the filter membrane; remove the filter membrane, fold it and place it in a centrifuge tube, label it, and store it until DNA extraction is performed.

[0063] 1.5 Nucleic acid extraction from samples

[0064] After thawing the filter membrane, cut it into thin strips and place them in centrifuge tubes. Extract DNA according to the instructions of the DNAsecure Plant Genomic DNA Extraction Kit. Store the extracted DNA in a refrigerator for later use.

[0065] 1.6 Real-time fluorescent PCR detection of pine wood nematode

[0066] use wait

[17] ( C, Castagnone C, Boonham N, et al. Satellite DNA as a target for TaqMan real-time PCR detection of the pine wood nematode, Bursaphelenchus xylophilus. [J] Molecular Plant Pathology. 2007, 8, 803–809.) established a real-time fluorescent PCR detection method for pine wood nematode, and performed real-time fluorescent PCR detection on the standard curve nucleic acid and sample nucleic acid prepared in 1.2.

[0067] Table 1. Specific primers and probes for real-time fluorescent PCR detection of pine wood nematode.

[0068]

[0069] 1.7 Statistical Analysis

[0070] Data were statistically analyzed using Excel and SPSS 26 software. One-way ANOVA was used to compare the significance of differences in the concentration of different nematode tissue mixtures and the enrichment efficiency of different filter membrane materials, with the error controlled within the 95% confidence interval.

[0071] 2. Results and Analysis

[0072] 2.1 Drawing the standard curve

[0073] A standard curve was established using real-time fluorescent PCR amplification, with the logarithmic values ​​of the concentrations of the serially diluted nematode mixture as the Y-axis and the number of real-time fluorescent PCR reaction cycles (CT value) as the X-axis. Figure 1 The concentration gradient of the nematode standard mixture is 10. 4 10 3 10 2 The concentration logarithms of 1, 10, and 1 were 13.28, 9.96, 6.64, 3.32, and 0, respectively, and the correlation coefficient R of the fitted curve was 0. 2 =0.9961, indicating that the eDNA concentration and CT value have a good linear relationship, and the established standard curve can accurately reflect the amplification of pine wood nematode DNA.

[0074] 2.2 eNDA recovery rate after membrane enrichment

[0075] eDNA from three different concentrations of nematode tissue mixtures was enriched and filtered using a mixed cellulose membrane (MCE), a polyethersulfone membrane (PES), and a cellulose acetate membrane (CA). The filtered membranes were then used to extract the eDNA using a nucleic acid extraction kit for real-time fluorescence PCR detection. The results (Table 2) showed that, under concentration A, the recoveries of MCE, PES, and CA were 55.70%, 59.79%, and 28.96%, respectively; under concentration B, the recoveries were 35.86%, 31.94%, and 15.29%, respectively; and under concentration C, the recoveries were 25.46%, 20.30%, and 11.30%, respectively. Analysis of variance showed significant differences in enrichment efficiency of *Pinus fibrocarpa* eDNA among different concentrations (F = 0.16.922, P = 0.000) and among different filter membrane types (F = 9.795, P = 0.001), but there was no interaction between concentration and filter membrane type (F = 0.788, P = 0.548).

[0076] Table 23 Recovery Rates of Filter Membranes

[0077]

[0078] Note: Different lowercase letters after the data in the same column in the table indicate significant differences between different treatments (P<0.05), while the same lowercase letter indicates no significant differences between different treatments (P>0.05).

[0079] 2.3 Comparison of Filter Membrane Recovery Rates at Different eDNA Concentrations

[0080] Under three different eDNA concentrations, the recovery rates of all three filter membranes decreased as the eDNA concentration decreased. Figure 2 Pairwise comparisons show that under concentration A, the membrane recovery rate is significantly better than that under concentrations B and C (P = 0.001, P = 0.000); while the recovery rates under concentrations B and C decrease significantly, and the difference in membrane recovery rates between the two is not significant (P = 0.109).

[0081] The recovery rates of the three filter membranes were compared pairwise, and the results showed that ( Figure 2 The difference between MCE and PES was not significant (P = 0.750), but both were significantly better than CA (P = 0.001; P = 0.002). Among them, PES had the highest recovery rate under concentration A, but MCE had a higher recovery rate than PES under concentrations B and C.

[0082] 3. Conclusion

[0083] This study compared and analyzed the enrichment effects of three types of filter membranes—mixed cellulose membrane (MCE), polyethersulfone membrane (PES), and cellulose acetate membrane (CA)—on nematode tissue mixtures, and selected MCE as the optimal filter membrane with a pore size of 0.45 μm.

[0084] Example 2: Detection of pine wood nematode based on MCE-enriched wood eDNA

[0085] Diseased pine wood was collected and cut into small pieces using an electric cutter. The pieces were then placed in sterile distilled water and stirred. 10 μL, 8 μL, 6 μL, 4 μL, and 2 μL of the supernatant from the soaked diseased pine wood were respectively transferred to graduated cylinders and diluted to volume with sterile water. Samples of different concentrations were then poured into filter cups equipped with MCE membranes and filtered for enrichment using the same method as in 1.4. DNA was extracted for later use. Each experiment was repeated three times, with sterile water serving as a blank control.

[0086] Using the mixed cellulose membrane (MCE) screened in previous studies as the experimental material, nucleic acids from pine wood nematodes in the mixed sap of diseased pine wood were enriched. After extracting the nucleic acids from the membrane, CT values ​​were measured by qPCR. Figure 3 ).

[0087] The limits of detection (LOCs) for MCE were determined by taking 10 μL, 8 μL, 6 μL, 4 μL, and 2 μL of the mixed solution of diseased pine wood, respectively (Table 3). The CT value was <35 for samples of 10 μL and 8 μL, and >35 for samples smaller than 6 μL. The CT value was 37.24 at a concentration of 4 μL, and no CT value was found at lower concentrations. Therefore, the LOC for MCE in diseased pine wood is 4 μL.

[0088] Table 3 Comparison of CT values ​​for MCE membrane enrichment detection at different concentrations

[0089]

[0090] Example 3: Establishment of the RPA method for pine wood nematode

[0091] 1. Experimental materials

[0092] The tested nematodes were: 103, 105, 116, 130, 142, 160, 205, 302, 601, and 701. Specific information is shown in Table 4.

[0093] Table 4. Number and source of the tested nematodes

[0094] Serial Number Collection Number scientific name Place of origin 1 B00103 Bursaphelenchusxylophilus Canada 2 B00105 Bursaphelenchusxylophilus USA 3 B00116 Bursaphelenchusxylophilus Spain 4 B00130 Bursaphelenchusxylophilus South Korea 5 B00142 Bursaphelenchusxylophilus Canada 6 B00106 Bursaphelenchusxylophilus Liaoning 7 B00205 Bursaphelenchusmucronatus Greece 8 B00302 Bursaphelenchusdoui South Korea 9 B00601 Bursaphelenchusconicaudatus Ningbo 10 B00701 Bursaphelenchusfraudulentus Ningbo

[0095] 2. RPA primer and probe design

[0096] Based on the rDNA-ITS gene sequence of Pine Wood Nematode published in NCBIGenBank, and referring to the RPA primer and nfo probe design recommendations provided by www.twistdx.co.uk, five pairs of RPA primers were designed by comparing the gene differences between Pine Wood Nematode and other nematodes of the genus *Scleroderma* using BioEdit software. The specificity of the five primer pairs was verified by PCR.

[0097] like Figure 4 As shown, three pairs (pairs 2, 4, and 5) exhibited non-specific amplification, while one pair (pair 3) showed weak amplification bands. Pair 1 was ultimately selected for subsequent experiments, and probes were designed. The primer and probe sequences are shown in Table 5.

[0098] Table 5 Primer and probe design

[0099]

[0100] 3. Sample preparation

[0101] Take the above-mentioned pine wood nematodes into centrifuge tubes, add sterile water, and repeatedly freeze and thaw the tissues with liquid nitrogen. Collect all the grinding liquid and add sterile water to make up the volume.

[0102] 4. Nucleic acid enrichment of samples

[0103] Place the MCE membrane on the filter, pour the prepared nematode tissue mixture into the filter cup, turn on the diaphragm vacuum pump, and wait for all the liquid to pass through the membrane. Remove the membrane, fold it, place it in a centrifuge tube, label it, and store it until DNA extraction is performed.

[0104] 5. Extraction of nematode DNA

[0105] Extract DNA according to the instructions of the DNAsecure Novel Plant Genomic DNA Extraction Kit and store it for later use.

[0106] 6. Optimization of the test strip reaction system

[0107] Using the extracted nematode DNA as a template, seven different reaction times and seven different reaction temperatures were evaluated. The reaction systems are shown in Table 6.

[0108] (1) Add Abuffer to each dry powder reaction tube;

[0109] (2) Add the upstream primer, downstream primer and probe to each reaction tube respectively;

[0110] (3) Add nucleic acid template and ddH2O to the reaction tube in sequence;

[0111] (4) Finally, add Bbuffer to the reaction tube and mix thoroughly;

[0112] (5) After mixing, shake (or centrifuge quickly) the reaction solution to the bottom of the tube, and then immediately place the reaction tube into a constant temperature device for incubation.

[0113] (6) After the reaction is complete, dilute and mix the amplification product with ddH2O, then perform color development on the test strip, and observe the results of the control line and the detection line within 5 minutes.

[0114] Judgment criteria: If both the control line and the test line of the test strip show bands, then the sample to be tested is or contains pine wood nematode; if the control line of the test strip shows a band but the test line does not show a band, then the sample to be tested is not or does not contain pine wood nematode.

[0115] Table 6 System Configuration

[0116] Components Volume (μL) Abuffer 29.4 Upstream primer (10 μM) 2 Downstream primer (10 μM) 2 Probe (10μM) 0.6 <![CDATA[ddH2O]]> 13.5 Nucleic acid template 2.5 Bbuffer 50

[0117] 6.1 Optimization of different amplification times

[0118] The amplification time was set to 10, 15, 20, 25, 30, 35, and 40 minutes respectively before detection.

[0119] Test results as follows Figure 5As shown, when the RPA reaction time is 15 min, a band can be amplified, but the band color is relatively light. As the reaction time increases, the band color gradually becomes clearer and brighter, but there is little difference when the RPA reaction time is 30 min or more. Considering all factors, an RPA reaction time of 30 min was chosen for subsequent experiments.

[0120] 6.2 Optimization of different reaction temperatures

[0121] The reaction temperatures were set to 36, 37, 38, 39, 40, 41 and 42 °C respectively, and the results were measured.

[0122] The results are as follows Figure 6 As shown, bands were successfully amplified at reaction temperatures ranging from 36℃ to 42℃. The bands amplified at reaction temperatures of 39℃ were not significantly different from those at 40℃, 41℃, and 42℃. Considering all factors, 39℃ was chosen for subsequent experiments.

[0123] 7. Specificity of the RPA electrophoresis detection system for pine wood nematode

[0124] Using the pine wood nematode and other nematode species listed in Table 4 as templates, the above-screened system configuration was used for the test strip method. Each sample was tested three times. The specificity of the detection method was determined by analyzing the test strip results using electrophoresis and test strips.

[0125] Electrophoresis results as follows Figure 7 As shown, electrophoresis results 1-6 show obvious bands, while results 7-11 show no bands; therefore, the RPA primers for pine wood nematode designed in Table 3-2 have good specificity.

[0126] Test strip results as follows Figure 8 As shown, clear bands appeared on the detection lines for all six different types of pine wood nematodes. Other nematode species and water only showed bands on the control line and not on the detection line, indicating that the established test strip method can specifically detect pine wood nematodes.

[0127] 8. Sensitivity detection of the RPA electrophoresis system for pine wood nematode

[0128] The extracted DNA from the pine wood nematode (sample B00130) was serially diluted 10-fold to an initial concentration of 78 ng / μL. Eight concentrations were then obtained, and the DNA was used as a template for detection by electrophoresis and test strip methods. Each sample was repeated three times.

[0129] Electrophoresis results as follows Figure 9 As shown, the RPA product of *Pinus fibrous tissue* at a concentration of 36.9 ng / μL exhibited clear and bright bands. The band brightness gradually decreased with decreasing concentration, reaching a maximum at 36.9 × 10⁻⁶ ng / μL. -4The presence of only a weak band at ng / μL of RPA product indicates that the RPA method has a sensitivity of 36.9 × 10⁻⁶. -4 ng / μL.

[0130] Results of the test strip method are as follows Figure 10 As shown, a band of pine wood nematode RPA product at a concentration of 78 ng / μL appeared on the test strip. The band color became lighter as the DNA concentration decreased, with the highest concentration at 78 × 10⁻⁶ ng / μL. -4 The presence of only a faint red band at ng / μL indicates that the detection limit of the colloidal gold test strip method is 78 × 10⁻⁶. -4 ng / μL.

Claims

1. An RPA primer for detecting pine wood nematode in an aqueous extract, said primer comprising an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2, said RPA primer being used to amplify pine wood nematode DNA.

2. An RPA-nfo probe for detecting pine wood nematodes in an aqueous extract, the nucleotide sequence of which is shown in SEQ ID NO: 3, wherein the probe generates a visible signal on a test strip by specifically recognizing or binding to pine wood nematode DNA.

3. A kit for detecting pine wood nematode, the kit comprising an RPA primer pair and an RPA-nfo probe, the primers comprising an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2, and the RPA-nfo probe having a nucleotide sequence as shown in SEQ ID NO:

3.

4. A method for detecting pine wood nematode, the method comprising the following steps: S1. Collect samples and prepare sample aqueous extract; S2. Total nucleic acids are enriched by filtering the aqueous extract prepared in S1 through a filter membrane; S3. Extract the total nucleic acids enriched in S2 according to the DNA extraction kit instructions; S4. Amplify the total nucleic acid extracted in S3 using RPA primers; S5. Detect the amplified product from S4 and interpret the results.

5. The method as described in claim 4, in step S1, when the water extract is a water extract of pine wood nematode tissue, the water extract is obtained by repeatedly freezing and thawing the nematode and then grinding it; when the water extract is a water extract of wood containing pine wood nematodes, the water extract is obtained by soaking the wood containing pine wood nematodes in water.

6. The method according to any one of claims 4 or 5, wherein in step S2, the filter membrane is a cellulose membrane, the cellulose membrane being selected from one or more of nitrocellulose membranes, polycarbonate membranes, glass fiber membranes, and / or mixed cellulose membranes.

7. The method according to any one of claims 4-6, wherein in step S4, the RPA primer comprises an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO:

2.

8. The method according to any one of claims 4-7, wherein the RPA amplification reaction time is 10-40 min; and the RPA amplification reaction temperature is 36-42℃.

9. The method according to any one of claims 4-8, wherein in step S5, the result interpretation method includes, but is not limited to, electrophoresis and / or test strip method.

10. The method of claim 9, wherein the test strip method utilizes the RPA primers of claim 1 and the RPA-nfo probe of claim 2 to specifically recognize or bind to pine wood nematode DNA, thereby generating a visible signal on the test strip.

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