Method for extracting DNA (deoxyribonucleic acid) of pine wood nematode in wood sample

Abstract

The invention discloses a method for extracting pine wood nematode DNA in a wood sample, and pine wood nematode diseases are pine tree diseases caused by pine wood nematode, have high destructiveness and severely harm forest resources. Therefore, the detection and prevention and control of the pine wood nematode disease are of great significance. However, an existing pine wood nematode disease detection method still has various limitations, and particularly, the process of extracting pine wood nematode DNA from a wood sample is complex in operation, long in time and high in cost. According to the scheme, extraction conditions are screened, and it is determined that an ultrasonic cell crusher is used for carrying out bursaphelenchus xylophilus cell crushing on a wood sample; by sampling different positions of the cross section of the tree, the distribution of the pine wood nematodes in the inner ring of the wood is determined; through screening by a sampling method, tree sampling by using the growth cone is determined. Finally, the method for rapidly extracting the DNA of the pine wood nematode in the wood sample is established, and the method can make the diagnosis of the pine wood nematode disease faster, simpler and more economical, and lays a foundation for the prevention and treatment of the pine wood nematode disease.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and wood detection technology, and in particular to a method for extracting pine wood nematode DNA from wood samples. Background Art

[0002] Pine wilt disease, also known as pine wilt disease, is caused by pine wood nematodes, a common and devastating epidemic in pine forests. It has a strong pathogenicity, a long incubation period, a fast onset, a fast host death rate, multiple transmission routes, and hidden sites of disease. Once it occurs, it is difficult to control. At present, there are few technical reports on the rapid and efficient prevention and control of pine wood nematode disease. Therefore, pine wood nematode disease is also called the "cancer" of pine trees. How to effectively prevent and control pine wood nematode disease is an important task that requires continuous in-depth research. Therefore, the rapid and accurate identification of pine wood nematode pathogens is of great significance for the prevention and control of pine wood nematode diseases.

[0003] So far, rapid diagnostic tools for detecting pine wood nematodes are still very limited, so they still need to be continuously studied and improved. The main detection methods for pine wood nematode disease are biological detection, remote sensing monitoring and molecular biological detection. Biological detection is to detect pine wood nematode disease by observing whether pine trees have symptoms, but sometimes the symptoms are not obvious, which can easily lead to missed detection and misdetection. Or directly observe the nematodes under a microscope to complete the judgment, but it takes a long time to separate the nematodes and is difficult to identify. Remote sensing monitoring can capture discolored diseased wood of pine trees infected with pine wood nematodes, but it is affected by factors such as climate and topography, and it is still a manual visual interpretation method, which is highly subjective and easy to miss the best treatment period. Molecular biological detection uses PCR amplification technology to quickly and accurately detect the presence of pine wood nematodes in a short time, but it is necessary to extract pine wood nematode DNA from wood samples for detection. This process requires special equipment and procedures and is complicated to operate. In past studies, a variety of DNA extraction methods have been developed, such as CTAB method and column chromatography. These methods have high extraction efficiency, but the biggest problem is that currently obtaining pine wood nematodes from wood samples requires the Baermann funnel method to separate the nematodes, which generally takes 24 hours and requires felling trees for inspection, causing irreversible damage to the trees. There are disadvantages such as complex operation, long time, and high cost. How to directly extract nematode DNA from wood samples and conduct PCR testing has become a key issue limiting the efficiency of nematode detection.

[0004] In view of these limitations, this study established a method for rapid extraction of pine wilt DNA from wood samples without felling trees. Combined with PCR amplification technology, pine wilt disease can be detected quickly and accurately. This study can provide a theoretical basis and technical support for the scientific and effective prevention and control of pine wilt disease. Breakthroughs in the diagnosis of pine wilt disease can allow us to control the pathogen at the source, control the spread of the disease, protect the ecological environment, and reduce economic losses, which has great social, economic and ecological significance. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for extracting pine wood nematode DNA from wood samples, which is reliable in implementation, flexible in application and has good reference results.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0007] A method for extracting pine wood nematode DNA from a wood sample, comprising:

[0008] S01. Remove wood strip samples from the pine wood to be tested according to preset requirements;

[0009] S02, immersing the wood stick sample in a lysis solution, and then subjecting it to cell disruption treatment using a cell disruption device under a low temperature environment;

[0010] S03, taking out the wood strip sample treated by S02, and then soaking it in a cleaning solution to remove impurities;

[0011] S04. Immerse the wood sample treated by S03 in ddH2O so that the pine wood nematode DNA is eluted into the solution, thereby completing the extraction of pine wood nematode DNA from the wood sample.

[0012] As a possible implementation, further, in the present solution S01, the sample is removed from the inner ring portion between the core and the outer ring of the pine wood to be tested by the growth cone.

[0013] As a possible implementation mode, further, in the present solution S02, the pH value of the lysate is 8, and it includes the following components at the final concentration:

[0014] Tris 20 mM;

[0015] NaCl 25 mM;

[0016] EDTA 2.5 mM;

[0017] The lysate also includes SDS with a mass fraction content of 0.05%.

[0018] As a possible implementation manner, further, in the present solution S02, after the sample is immersed in the lysis solution, it is placed on ice to create a low-temperature environment, and the cells are disrupted for 1 minute using a tissue grinder or an ultrasonic cell disruptor.

[0019] As a possible implementation manner, further, in the present solution S03, the pH value of the cleaning solution is 8, and the cleaning solution includes Tris with a final concentration of 10 mM and also includes Tween-20 with a mass fraction content of 0.1%.

[0020] As a possible implementation manner, further, in the present solution S01, the pine wood to be detected is a felled pine wood segment or an unfelled pine wood trunk.

[0021] Based on the above, the present invention also provides a wood sample inspection method, which includes the above-mentioned method for extracting pine wood nematode DNA from wood samples.

[0022] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0023] 1. This scheme directly uses the cellulose in the wood strips (sampling strips) of the pine wood to be tested to assist in the adsorption of pine wood nematode DNA, overcoming the problem of poor adsorption effect of pine wood chips. It does not need to use other carriers for auxiliary adsorption of pine wood nematode DNA as in the traditional scheme. After the wood strips obtained after sampling in this scheme are soaked in lysis solution, washed with cleaning solution and eluted, an eluate containing pine wood nematode DNA can be obtained, which can be directly used as a template for PCR amplification, real-time fluorescence quantitative PCR and loop-mediated isothermal amplification (LAMP) for detection.

[0024] 2. This method can take samples from the sampled wood, and then break the cells of the pine wood nematodes in the sampled wood strips, eliminating the complex process of separating nematodes using the Baermann funnel method and manually identifying nematodes in the traditional method. This method is not only easy to operate, but also short in time and has good detection effect. The traditional method of extracting pine wood nematode DNA from wood requires the use of the Baermann funnel method to separate nematodes, which generally takes 24 hours.

[0025] 3. This scheme uses growth cone sampling to quickly extract pine wood nematode DNA from wood samples and complete the test, which can obtain accurate and effective results and avoid the damage to trees caused by cutting down trees to obtain wood samples.

[0026] 4. This scheme has high detection sensitivity. As long as there is one pine wood nematode in the sample, the result can be detected and a specific band can be obtained, which proves that the detection sensitivity of this method is very high. As long as there is pine wood nematode, it can be detected using this method. It overcomes the problems of large errors, strong subjectivity and inaccurate results of traditional biological detection methods and remote sensing detection.

[0027] 5. The primers designed in this scheme are specific and can amplify the specific bands of pine wood nematodes, while no bands are amplified from the DNA of pine wood nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a comparison of the test results of different sample processing methods in this scheme, where M: DNA Marker; 1: Detection of crude DNA extract of pine wood nematode obtained by tissue grinder; 3: Detection of crude DNA extract of pine wood nematode obtained by ultrasonic cell crusher; 5: Detection of crude DNA extract of pine wood nematode obtained by ultrasonic cleaning machine;

[0030] Figure 2 This is the comparison of the results of the specific experiment of this scheme, where M: DNA Marker; 1, 3: using pine wood nematode DNA as template; 2, 4: using pine wood nematode DNA as template;

[0031] Figure 3 This is a sensitivity experiment of different concentrations of pine wood nematodes in this scheme, where M: DNA Marker; 1: 300 pine wood nematodes; 2: 200 pine wood nematodes; 3: 100 pine wood nematodes; 4: 50 pine wood nematodes; 5: 25 pine wood nematodes; 6: 1 pine wood nematode;

[0032] Figure 4 This is a comparison of the results of the simulated diseased wood detection in this scheme, where M: DNA Marker; 1: 300 pine wood nematodes were quickly determined using cellulose paper; 3: 300 pine wood nematodes were dropped into sawdust for rapid determination;

[0033] Figure 5 This is a comparison of the effects of NaCl on the binding rate of DNA to cellulose in this scheme, where M: DNA Marker; 1: The wood sample was soaked in 150 Mm NaCl solution in advance; 2: The wood sample was not soaked in 150 Mm NaCl solution;

[0034] Figure 6 This is a schematic diagram of the wood pile sampling and wood strips in this scheme;

[0035] Figure 7This is the comparison of the results of the three groups of pine samples tested at different sampling locations. M: DNA Marker; 1-17: correspond to Figure 6 17 sampling sites;

[0036] Figure 8 The figure is a comparison of the effects of different sampling methods on the test results of this scheme, where M: DNA Marker; 1, 2, 3: sampling using growth cones; 4, 5, 6: sampling using an electric drill. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that the following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Similarly, the following examples are only partial embodiments of the present invention rather than all embodiments, and all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0038] This embodiment provides a method for extracting pine wood nematode DNA from a wood sample, which comprises:

[0039] S01, removing a wood strip sample from the inner circle between the core and the outer circle of the pine wood to be tested through a growth cone, wherein the pine wood to be tested is a felled pine wood section or an unfelled pine wood trunk;

[0040] S02, immersing the wood stick sample in a lysis solution, and then subjecting it to cell disruption treatment using a cell disruption device under a low temperature environment;

[0041] S03, taking out the wood strip sample treated by S02, and then soaking it in a cleaning solution to remove impurities;

[0042] S04. Immerse the wood sample treated by S03 in ddH2O so that the pine wood nematode DNA is eluted into the solution, thereby completing the extraction of pine wood nematode DNA from the wood sample.

[0043] Wherein, in the present scheme S02, the pH value of the lysate is 8, and it includes the following components with final concentrations:

[0044] Tris 20 mM;

[0045] NaCl 25 mM;

[0046] EDTA 2.5 mM;

[0047] The lysate also includes SDS with a mass fraction content of 0.05%.

[0048] As a possible implementation, further, in this solution S02, after the sample is immersed in the lysis solution, it is placed on ice to create a low temperature environment, and the cells are disrupted for 1 minute by a tissue grinder or an ultrasonic cell disruptor. The purpose of the disruption on ice is that the ultrasonic cell disruptor has a better effect on the disruption of pine wood nematode cells, and the low temperature during the disruption on ice inhibits the endogenous DNase of the pine wood nematode tissue from degrading DNA, so as to obtain accurate results.

[0049] As a possible implementation manner, further, in the present solution S03, the pH value of the cleaning solution is 8, and the cleaning solution includes Tris with a final concentration of 10 mM and also includes Tween-20 with a mass fraction content of 0.1%.

[0050] In step S03 of this scheme, when the wood strip sample is soaked in the cleaning solution, the cellulose in the wood strip is used to adsorb the large molecular weight pine wood nematode DNA and is retained during the cleaning process, while other smaller compounds, including some DNA amplification inhibitors, either do not bind to the cellulose or are quickly released into the washing solution.

[0051] Based on the above, the present invention also provides a wood sample inspection method, which includes the above-mentioned method for extracting pine wood nematode DNA from wood samples.

[0052] In order to facilitate the detailed description of each step of the present embodiment, the present implementation scheme is described in comparison with some prior art and the single variable method as follows.

[0053] 1. Experimental Materials

[0054] (1) Biological materials: Pine wood nematodes and Pine wood nematodes. Pine wood nematodes and Pine wood nematodes were isolated from infected wood of Pinus massoniana in Lianjiang County, Fujian Province. Larvae after artificial breeding for multiple generations in the laboratory were used for the test. Pine wood nematodes were raised in PDA culture medium and placed in a constant temperature and humidity chamber (humidity 80%, temperature 30°C). After 6-7 days of breeding, the larval suspension was separated by the Baermann funnel method for subsequent tests.

[0055] (2) Samples and chemical materials: cellulose paper, wood samples collected from Putian, Fujian, Masson pine sawdust, cleaning solution (10 mM Tris [pH 8.0], 0.1% Tween-20), lysis solution (20 mM Tris [pH 8.0], 25 mM NaCl, 2.5 mM EDTA, 0.05% SDS), cellulose paper, 150 mM NaCl solution.

[0056] The above experimental materials are the materials used in the subsequent steps of this embodiment.

[0057] 2. Primer design

[0058] According to the pine wood nematode gene sequence published in GenBank, specific primers were designed using the differences in the gene sequences of pine wood nematode and its non-pathogenic pine wood nematode (B. mucronatus), as shown in Table 1. Among them, the primers were commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0059] Table 1 Primer sequences

[0060]

[0061] 3. Extraction condition screening

[0062] Three portions of 300 pine wood nematodes were selected and placed in 10 μL of clean water, 90 μL of lysis solution was added, and the mixture was placed on ice. Different sample treatment methods were used for the treatments, as follows:

[0063] The first part was cell disruption using a tissue grinder for 1 min;

[0064] The second sample was subjected to cell disruption using an ultrasonic cell disruptor for 1 minute (on-ice disruption instrument (Xinzhi JY92-IIDN), parameters: power 45%, disruption for 10 seconds, stop for 5 seconds, duration 1 minute)

[0065] The third portion was subjected to cell disruption for 1 minute using an ultrasonic cleaning machine to obtain a crude DNA extract of pine wood nematodes. (KH-300DE CNC ultrasonic cleaning machine, parameters: power 100%, temperature 1, disruption 1 min)

[0066] 4. Extraction of pine wood nematode DNA

[0067] Based on the ability of cellulose paper to capture or adsorb DNA under specific conditions, the cellulose paper strip is soaked in the crude DNA extract of pine wood nematode (three portions prepared in step 3) to bind nucleic acid. Then the cellulose paper strip is soaked in 100 μL of cleaning solution to remove impurities. Finally, the cellulose paper strip is soaked in the amplification reaction solution to elute the nucleic acid. The eluate at this time can be directly used as a template for PCR amplification.

[0068] The method for making the cellulose paper strip is as follows: first, cut the cellulose filter paper into filter paper strips of 44mm×20cm in size, and draw a shallow horizontal line with a pencil at 40mm from the long side of the filter paper, and then neatly immerse the long side of the cellulose filter paper in paraffin. Due to capillary action, the paraffin will diffuse upward along the filter paper. When the paraffin is about to diffuse to the marked position, pull out the filter paper and place it flat on a clean table to let it stand. The paraffin will continue to diffuse upward until it reaches the marked position. Subsequently, a 40mm long hydrophobic area wrapped by paraffin will be formed on the filter paper (as a handheld area), and the 4mm filter paper area not wrapped by paraffin will serve as a DNA binding area. Finally, the filter paper is cut to obtain a cellulose paper strip of 2×44mm in size.

[0069] 5. Establishment of PCR system

[0070] The DNA of pine wood nematodes in wood samples was used as a template for PCR amplification. The reaction system of 10 μL is shown in Table 2. The reaction procedure was: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 55.4°C for 15 seconds, extension at 72°C for 15 seconds, 29 cycles; and finally extension at 72°C for 5 minutes. 10 μL of PCR product was subjected to 1.5% agarose gel electrophoresis.

[0071] Table 2 PCR reaction system

[0072]

[0073] 6. Specificity experiment of detection method

[0074] Using the DNA of pine wood nematode and pine wood nematode as template, PCR amplification was performed using the primers designed in step 2, and then the PCR products were characterized by electrophoresis. The results are as follows: Figure 1 As shown, Figure 1 In the experiment, group numbers 1 and 3 used pine wood nematode DNA as templates, and group numbers 2 and 4 used pine wood nematode DNA as templates. It can be seen that group 1 and group 3 both have obvious specific bands, while group 2 and group 4 have no obvious specific bands. The experimental results show that the DNA of pine wood nematode can amplify specific bands, while the DNA of pine wood nematode does not amplify any bands. The primers designed in the experiment are specific and can amplify the specific bands of pine wood nematode.

[0075] 7. Comparative experiment on extraction conditions

[0076] DNA was extracted from the three pine wood nematode DNA crude extracts prepared in step 3 by step 4, and then the eluates were directly used as templates for PCR amplification, and PCR amplification was performed, and then the PCR products were characterized by electrophoresis. The results are as follows: Figure 2As shown, it can be known that using different cell disruption instruments to disrupt pine wood nematodes in wood samples has different effects.

[0077] Figure 2 In the test, group number 1 is the test group corresponding to the crude DNA extract of pine wood nematode obtained after cell disruption using a tissue grinder; group number 3 is the test group corresponding to the crude DNA extract of pine wood nematode obtained after cell disruption using an ultrasonic cell crusher; group number 5 is the test group corresponding to the crude DNA extract of pine wood nematode obtained after cell disruption using an ultrasonic cleaning machine; it can be seen that Figure 1 Groups 1 and 3 shown in the figure obtained obvious specific bands with almost the same concentrations, while group 5 did not obtain obvious specific bands. The results showed that the tissue grinder and ultrasonic cell disruptor had a good effect on the cell disruption of pine wood nematodes, and the effects of the two on the cell disruption of pine wood nematodes were similar; the ultrasonic cleaning machine group did not obtain specific bands and had almost no effect on the cell disruption of pine wood nematodes. Tissue grinders and ultrasonic cell disruptors can be used in the cell disruption of pine wood nematodes, but ultrasonic cleaning machines are not used.

[0078] 8. Sensitivity test of detection method

[0079] Referring to the extraction conditions in step 3, the concentration of pine nematodes was diluted from 300 to 1 (cell disruption was performed using an ultrasonic cell disruptor), and then the pine nematode DNA was extracted according to the extraction method in step 4. Then, PCR amplification was performed on the DNA at different concentration gradients in combination with step 5, and the amplified products were detected by electrophoresis using 1.5% agarose gel. The results are as follows: Figure 3 As shown, Figure 3 The number of pine wood nematodes used in each group is: group 1 is 300 pine wood nematodes, group 2 is 200 pine wood nematodes, group 3 is 100 pine wood nematodes, group 4 is 50 pine wood nematodes, group 5 is 25 pine wood nematodes, and group 6 is 1 pine wood nematode.

[0080] Depend on Figure 3 The results of the sensitivity experiment showed that the brightness of the amplified product band became darker as the concentration of pine wood nematodes decreased, and a faint specific band could still be amplified when the template amount was 1 pine wood nematode, indicating that the sensitivity of the PCR detection method was 1 pine wood nematode.

[0081] 9. Testing of simulated diseased wood samples

[0082] Compared with the existing technology, one of the technical cores of this scheme is to use the cellulose contained in pine wood to assist in the adsorption of pine wood nematode DNA. At the same time, cellulose can effectively bind to DNA and will not inhibit the amplification reaction. In order to verify it, this scheme uses 0.2g of Masson pine sawdust to simulate samples drilled from diseased trees, adds 300 pine wood nematodes to the sawdust, mixes it thoroughly to simulate the infected wood sample, and uses the cellulose in the sawdust instead of the cellulose paper of the traditional scheme to adsorb, clean and release the pine wood nematode DNA. By adding a lysis solution that can cover the sawdust, mixing thoroughly, pine wood nematode cells were broken (ultrasonic cell crusher was used for cell breaking), centrifuged for 1 minute at 12000rpm, the supernatant was discarded, and a cleaning solution that can cover the sawdust was added. After mixing thoroughly, centrifuged for 1 minute at 12000rpm, the supernatant was discarded, 30μL ddH2O was added, mixed thoroughly, centrifuged for 1 minute at 12000rpm, and the supernatant was taken to obtain purified pine wood nematode DNA, which was used as a template for PCR amplification. PCR detection was performed according to method 5. The results are shown in FIG. Figure 4 shown.

[0083] Figure 4 The results of using sawdust to add pine wood nematode simulated diseased wood, using cellulose in sawdust instead of cellulose paper to absorb pine wood nematode DNA, and using the traditional cellulose paper adsorption method as a control group, and then testing the results, wherein group number 1 is the result of 300 pine wood nematodes using cellulose paper to measure DNA, and group number 3 is the result of 300 pine wood nematodes dripped into sawdust and using the scheme of this embodiment to measure DNA. It can be seen that the test results of group 1 and group 3 both have obvious specific bands, and the band of group 1 is deeper than that of group 3. The experiment shows that the sample simulating diseased wood can detect the results, but the cellulose in sawdust is slightly less effective than the cellulose in cellulose paper in adsorbing, washing, and eluting pine wood nematode DNA.

[0084] 10. Sample preprocessing optimization

[0085] Some studies have shown that salt can increase the degree of binding between DNA and cellulose paper by counteracting electrostatic repulsion. Therefore, this embodiment scheme uses 150mM NaCl solution to verify whether cellulose in wood has an effect on the adsorption capacity of pine wood nematode DNA.

[0086] Before testing the wood samples, the samples were soaked in 150 mM NaCl to increase the degree of binding between pine wood nematode DNA and wood. The pine wood nematode DNA in the wood samples was extracted by referring to the methods of steps 3 and 4, and PCR testing was performed according to step 5. The results are as follows: Figure 5 shown.

[0087] Figure 5The comparison of the detection of using 150 Mm NaCl to increase the adsorption capacity of pine wood nematode DNA to cellulose in wood strips is shown. Group No. 1 is the experimental group in which the wood strip samples are pre-immersed in 150 Mm NaCl solution, and Group No. 2 is the experimental group in which the wood strip samples are not immersed in 150 Mm NaCl solution. It can be seen that the color of the strips in Group 2 is darker than that of the strips in Group 1. The experiment shows that NaCl will reduce the adsorption capacity of pine wood nematode DNA to cellulose in wood strips. Therefore, when using the detection method of this embodiment, the pine wood samples are not treated with NaCl.

[0088] 11. Comparison of detection rates of wood sample sampling locations

[0089] refer to Figure 6 The pine wood pile samples were equally divided into 8 areas, 8 samples were taken from the inner and outer circles of each of the 8 areas, and 1 sample was taken from the heart of the tree. A total of 17 samples were cut into small wooden strips, and the cellulose in the wooden strips was used to absorb the pine wood nematode DNA (i.e., the wooden strips were used instead of cellulose paper to absorb the pine wood nematode DNA), and then the pine wood nematode DNA was washed and eluted; the specific processing steps include:

[0090] S01, remove wood strip samples from each area of ​​the pine wood pile sample through the growth cone;

[0091] S02, immersing the wood stick sample in the lysis solution, then placing it on ice, and subjecting it to cell disruption by an ultrasonic cell disruptor;

[0092] S03, taking out the wood strip sample treated by S02, and then soaking it in a cleaning solution to remove impurities;

[0093] S04. Soak the wood sample treated with S03 in 30 μL ddH2O so that the pine wood nematode DNA is eluted into the solution, thereby completing the extraction of pine wood nematode DNA from the wood sample.

[0094] The eluate at this time is the purified pine wood nematode DNA, which can be directly used as a template for PCR amplification. Use the PCR system in step 5 for detection, and the results are as follows: Figure 7 shown.

[0095] Figure 7 Shows the Figure 6 The regional division shown in the figure shows the results of sampling and extracting DNA from 17 parts of three different pine samples and PCR amplification, and then testing. It can be seen that the specific bands obtained in groups 1 to 8 and 17 are fewer and lighter, and the specific bands obtained in groups 9 to 16 are more and darker. The experiment shows that the detection efficiency of the outer circle and the core is low, and the detection efficiency of the inner circle is high. For the pine wood to be tested, the inner samples should be selected as much as possible for testing (i.e. the inner circle between the core and the outer circle).

[0096] 12. Screening of wood sample sampling methods

[0097] Use a growth cone to take out small wood strips from the pine wood sample or use an electric drill to drill wood chips as samples for pine wood nematode disease detection. The wood strip detection method refers to the method used in the comparison of detection rates of wood sample sampling locations in 11, and the wood chip detection method refers to the method used in the detection of simulated diseased wood in 9. The results are as follows Figure 8 shown.

[0098] Figure 8 In the experiment, groups 1, 2, and 3 were sampled using growth cones, and groups 4, 5, and 6 were sampled using electric drills. The inner circle of the pine samples was sampled using growth cones or electric drills. The test results showed that specific bands were detected in groups 1 to 3, but not in groups 4 to 6. The experiment showed that both growth cone sampling and rapid testing on pine samples yielded test results, while both electric drill sampling and rapid testing did not yield results. In practical applications, it is easier to detect results using growth cone sampling.

[0099] The above descriptions are only some embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any equivalent device or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for extracting pine wood nematode DNA from wood samples. It is characterized in that It includes: S01. Remove wood strip samples from the pine wood to be tested according to preset requirements; S02, immersing the wood stick sample in a lysis solution, and then subjecting it to cell disruption treatment using a cell disruption device under a low temperature environment; S03, taking out the wood strip sample treated by S02, and then soaking it in a cleaning solution to remove impurities; S04: Soak the wood strip samples treated with S03 in ddH 2 O, so that the pine wood nematode DNA is eluted into the solution, completing the extraction of pine wood nematode DNA from the wood sample.

2. The method for extracting pine wood nematode DNA from wood samples according to claim 1, It is characterized in that In S01, the sample is removed from the inner circle between the core and the outer circle of the pine wood to be tested through the growth cone.

3. The method for extracting pine wood nematode DNA from wood samples according to claim 1, It is characterized in that In S02, the pH value of the lysate is 8, and it includes the following components at final concentrations: Tris 20 mM; NaCl 25 mM; EDTA 2.5 mM; The lysate also includes SDS with a mass fraction content of 0.05%.

4. The method for extracting pine wood nematode DNA from wood samples according to claim 1, It is characterized in that In S02, the sample is immersed in the lysis solution and then placed on ice to create a low-temperature environment, and the cells are disrupted by a tissue grinder or an ultrasonic cell disruptor for 1 minute.

5. The method for extracting pine wood nematode DNA from wood samples according to claim 1, It is characterized in that In S03, the pH value of the cleaning solution is 8, and the cleaning solution includes Tris with a final concentration of 10 mM and Tween-20 with a mass fraction content of 0.1%.

6. The method for extracting pine wood nematode DNA from wood samples according to claim 1, It is characterized in that In S01, the pine wood to be detected is a felled pine wood segment or an unfelled pine wood trunk.

7. A method for testing a wood sample, It is characterized in that The method comprises the method for extracting pine wood nematode DNA from a wood sample according to any one of claims 1 to 6.

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