A specific primer for rapid identification of northern root-knot nematodes in soil and its application

Abstract

The present invention provides a specific primer for quickly identifying northern root-knot nematodes in soil and its application, and relates to the separation, single nematode DNA extraction and identification of northern root-knot nematodes in soil. The specific primer is an upstream primer Mhs-F: TGTCTGGCTTTGGGCTAC; a downstream primer Mhs-R: GGTGACCGAAAGAGCATT. The present invention overcomes the shortcomings of the prior art, and the primer can be used to quickly and accurately identify northern root-knot nematodes, laying a foundation for the prevention and control of northern root-knot nematodes.

Description

Technical Field

[0001] The invention relates to the technical field of root-knot nematode identification, and in particular to a specific primer for rapidly identifying northern root-knot nematodes in soil and an application thereof. Background Art

[0002] Root-knot nematodes are a type of obligate plant parasitic nematodes that are widely distributed in agricultural production areas around the world. Due to their wide range of hosts, root-knot nematodes can infect up to more than 5,500 plant species, including vegetables, beans, grains, medicinal plants, fruit trees and woody ornamental plants, etc., causing losses of more than 100 billion US dollars to agricultural production each year. In 2013, they were listed as the top ten plant parasitic nematodes by the scientific community. There are more than 100 species of root-knot nematodes, among which the southern root-knot nematode, the northern root-knot nematode, the peanut root-knot nematode and the Java root-knot nematode are the four most common ones that harm medicinal plants.

[0003] Yunnan Province is the main production area of ​​Chinese medicinal materials in China, and many varieties of Chinese medicinal materials have been widely cultivated. In the process of cultivating Chinese medicinal materials, the inventors found in their preliminary investigations that many medicinal plants such as Panax notoginseng, Yunnan Angelica, Yunnan Aucklandia, Gentiana macrophylla, Aconite, and Aconitum carmichaelii were infected by root-knot nematodes, which were identified as northern root-knot nematodes by morphological and molecular biological identification. The infection of northern root-knot nematodes has led to a decrease in the yield and quality of medicinal materials, posing a serious threat to the sustainable and healthy development of the Chinese medicinal materials industry. The prevention and control of northern root-knot nematode diseases has become an urgent problem to be solved in the cultivation of Chinese medicinal materials in Yunnan.

[0004] Root-knot nematodes live in the soil at a depth of 5 to 20 cm, and complete their life cycle from eggs to larvae to adults by absorbing nutrients from the root system of host plants. The population density of root-knot nematodes in the soil is closely related to the degree of damage to the plant host, and there are significant differences between different root-knot nematode species. Therefore, rapid and accurate identification of root-knot nematode species in the soil is the premise and basis for the prevention and control of plant root-knot nematode diseases. Detection of root-knot nematode DNA by PCR is currently the most important method for identifying root-knot nematodes, which has the advantages of being fast, accurate, easy to operate, and not affected by the environment and host. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a specific primer for quickly identifying northern root-knot nematodes in soil and its application. The primer can be used to quickly and accurately identify northern root-knot nematodes, laying the foundation for preventing and controlling northern root-knot nematodes.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0007] A specific primer for rapid identification of northern root-knot nematodes in soil, the sequence of the specific primer is as follows:

[0008] Upstream primer Mhs-F: TGTCTGGCTTTGGGCTAC;

[0009] Downstream primer Mhs-R: GGTGACCGAAAGAGCATT.

[0010] The method for applying the above-mentioned specific primers to quickly identify northern root-knot nematodes in soil comprises the following steps:

[0011] S1, Isolation of nematodes in soil;

[0012] S2, quickly extracting DNA from a single nematode to obtain a DNA extract;

[0013] S3, DNA amplification using nematode-specific primers, specifically comprising the following steps:

[0014] S3-1. Set up the PCR system as follows: 12.5 μL of PCR Master, 1 μL each of upstream primer Mhs-F and downstream primer Mhs-R, 2 μL of DNA extraction solution, and add sterile double distilled water to 25 μL;

[0015] S3-2, PCR program: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 30 s, 61 °C annealing for 30 s, 72 °C extension for 1 min, 35 cycles; 72 °C extension for 5 min, 12 °C storage;

[0016] S3-3. Detect the PCR product by agarose gel electrophoresis and observe the bands to determine whether it is the northern root-knot nematode.

[0017] Preferably, the specific method of step S1 comprises the following steps:

[0018] S1-1. After mixing the collected soil evenly, take 100 g of soil and put it into a 2 L beaker, add 1 L of water, stir with a glass rod for 3 min, let it stand for 1 min, filter with an 80-mesh sieve and collect the filtrate;

[0019] S1-2, add 1 L of water to the mud at the bottom, stir, let stand, filter, and repeat the operation twice;

[0020] S1-3. Combine the collected filtrates, pass through a 500-mesh sieve, and rinse with clean water until the water leaking from the filter is clear. Place the remaining nematode residues on the sieve on a funnel covered with paper towels, and ensure that there is water to submerge the residues. Leave it for more than 12 hours until the water level that submerges the residues is below the residues.

[0021] S1-4. Release the water at the bottom of the funnel and observe the living nematodes collected in the soil under a stereo microscope.

[0022] Preferably, the specific method in step S2 comprises the following steps:

[0023] Preferably, a separated nematode is picked out with an acupuncture needle, placed in a PCR tube, 9 μL 2×TaqBuffer, 1 μL 1μg / μL proteinase K are added, low-speed centrifugation is performed for 30 s, liquid nitrogen is quick-frozen for 3 min, and then placed in a PCR instrument. The program is set to 65°C, 90 min, and 95°C, 15 min to obtain a DNA extract, which is stored at -20°C for later use.

[0024] A kit for detecting northern root-knot nematodes, wherein the sequences of the specific primers in the kit are as follows:

[0025] Upstream primer Mhs-F: TGTCTGGCTTTGGGCTAC;

[0026] Downstream primer Mhs-R: GGTGACCGAAAGAGCATT.

[0027] The present invention provides a specific primer for quickly identifying northern root-knot nematodes in soil and its application, which has the advantages of:

[0028] Through the technical scheme provided by the present invention, nematode DNA is amplified using specific primers, and the measured band is single and bright, with strong specificity and high sensitivity, and finally a specific primer for quickly identifying northern root-knot nematodes in soil is provided, laying a foundation for preventing and controlling northern root-knot nematodes; and the identification process includes the separation of northern root-knot nematodes in soil, the rapid extraction of single nematode DNA and the DNA amplification of northern root-knot nematode specific primers, the method has a high nematode yield and the nematode surface is relatively clean; the extraction of single nematode DNA is fast and simple, and the demand for nematode samples is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the soil suspension in Example 1 of the present invention;

[0030] Figure 2 This is a schematic diagram of the soil suspension filtrate passing through a 500-mesh screen in Example 1 of the present invention;

[0031] Figure 3 This is a schematic diagram of separating root-knot nematodes in Example 1 of the present invention;

[0032] Figure 4 This is a schematic diagram of the root-knot nematodes separated from the soil in Example 1 of the present invention;

[0033] Figure 5 This is a gel image of the DNA amplification effect of northern root-knot nematode DNA and elephant ear bean root-knot nematode DNA using specific primers in the present invention;

[0034] Figure 6 This is a gel image of the amplification effect of the nematode DNA extraction template obtained in Example 2 and the template extracted by the conventional method. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Embodiment 1:

[0036] Isolation of root-knot nematodes in soil:

[0037] The root-knot nematodes in soil were separated by combining the Kappa sieving method and the modified Behmann funnel method:

[0038] After mixing the collected soil evenly, take 100 g of soil and put it into a 2 L beaker, add 1 L of water, stir with a glass rod for 3 min, let it stand for 1 min, filter with an 80-mesh sieve and collect the filtrate.

[0039] Add 1 L of water to the mud at the bottom, stir, let stand, filter, and repeat the operation twice. Combine the collected 3 L of filtrate, pass through a 500-mesh sieve, and rinse with clean water until the water leaking from the filter is clear. Place the remaining nematode residue on the sieve on a funnel covered with paper towels, and ensure that there is water to submerge the residue. Leave it for more than 12 hours until the water level of the residue is below the residue.

[0040] Drain the water at the bottom of the funnel, and the living root-knot nematodes collected in the soil can be observed under a stereo microscope. The separation process is as follows: Figure 1-4 shown. Embodiment 2:

[0041] Rapid extraction of single nematode DNA:

[0042] Use an acupuncture needle to pick out a root-knot nematode isolated from the soil from the water, put it into a PCR tube, add 9 μL 2× Taq Buffer and 1 μL 1μg / μL proteinase K, centrifuge at low speed for 30 s to prevent the nematode from sticking to the tube wall, freeze it in liquid nitrogen for 3 minutes, place it in a PCR instrument, set the program to 65 ℃ 90 min, 95 ℃ 15 min, obtain DNA extract, and store it at -20 ℃ for later use. Embodiment 3:

[0043] Design of primers specific for northern root-knot nematode:

[0044] Based on the northern root-knot nematode-specific protein sequence:

[0045] MKTLSFSIFIFLICILKESMAGRGGGNLGPYGQGQSDQRRGSLITNNDHQMSGFGLPYPMKGGDTPTIQQKIDFLIKTKMNKQKKKYKSDKEQQLVDFLNGIKKEIIDAGYNNLNLRKYCTNYIITSKLVIINQLYKHFFDMSQAQPFSSQNVNSNDQVNATYRDGNRLDLGCFFTNYWGFH;

[0046] And its corresponding coding gene sequence (northern root-knot nematode-specific gene):

[0047]

[0048] Using TBtools-Ⅱ, the gene sequence was compared with the genomes of common southern root-knot nematodes, Javan root-knot nematodes, peanut root-knot nematodes, and elephant-ear bean root-knot nematodes. The results showed that the gene only existed in northern root-knot nematodes, and did not exist in the other four root-knot nematodes. The comparison results showed that the gene was a specific gene of northern root-knot nematodes.

[0049] Based on the northern root-knot nematode-specific gene, specific primers were designed:

[0050] Mhs-F:TGTCTGGCTTTGGGCTAC;

[0051] Mhs-R: GGTGACCGAAAGAGCATT.

[0052] Detection:

[0053] Detection 1. DNA amplification with primers specific for northern root-knot nematodes

[0054] (1) The PCR system is as follows: 12.5 μL of PCR Master Mix, 1 μL each of Mhs-F and Mhs-R, 2 μL of template (i.e., DNA extract), and sterile double-distilled water to make up to 25 μL.

[0055] (2) The PCR program was as follows: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 61 °C for 30 s, extension at 72 °C for 1 min, 35 cycles; extension at 72 °C for 5 min, and storage at 12 °C.

[0056] (3) The PCR product was sequenced and verified to be consistent with the gene sequence in the genome of northern root-knot nematode.

[0057] Detection 2: Specificity verification:

[0058] The root-knot nematode of Elephant bean was selected as a specific control, and the following two primers were designed for amplification of the DNA extract of the root-knot nematode of Elephant bean (the amplification conditions are the same as those in 1 above):

[0059] (1) Mhs-F and Mhs-R were used as primers to amplify the DNA of the root-knot nematode of the elephant ear bean;

[0060] (2) Use Me-F and Me-R (specific primers for R. elegans) as primers to amplify R. elegans DNA, where:

[0061] Me-F: AACTTTTGTGAAAGTGCCCTG;

[0062] Me-R:TCAGTTCAGGCAGGATCAACC.

[0063] The PCR products of the above-mentioned test 1 and test 2 were respectively tested by agarose gel electrophoresis. The specific results are as follows Figure 5 As shown, the DNA templates in lanes 1-3 are northern root-knot nematode DNA, and the primers are northern root-knot nematode specific primers Mhs-F and Mhs-R; the DNA templates in lanes 4-6 are elephant bean root-knot nematode DNA, and the primers are northern root-knot nematode specific primers Mhs-F and Mhs-R; the DNA templates in lanes 7-9 are elephant bean root-knot nematode DNA, and the primers are elephant bean root-knot nematode specific primers Me-F and Me-R. As can be seen from the figure, the PCR product with northern root-knot nematode DNA as the template is detected by agarose gel electrophoresis, and the bands are bright and single; the DNA of elephant bean root-knot nematode is amplified with Mhs-F and Mhs-R primers, and no bands are observed, but the DNA of elephant bean root-knot nematode is amplified with elephant bean root-knot nematode specific primers, and the corresponding bands are obtained, indicating that the elephant bean root-knot nematode DNA template used is normal. In summary, the results show that the northern root-knot nematode primers are specific.

[0064] Test 3: Verify that the method in Example 2 is feasible for obtaining DNA extract:

[0065] A conventional method was used to extract the DNA of northern root-knot nematodes as a control, specifically using Dzup genomic DNA extraction reagent (purchased from Sangon Biotech) as a control. Dzup uses guanidine salt and other chaotropic agents and denaturants under high pH conditions to lyse tissues and hydrolyze RNA, and then precipitate with ethanol to obtain genomic DNA. Using this method to extract DNA, 50-100 mg of root-knot nematodes are required, and they need to be ground with liquid nitrogen. The DNA obtained by this method and the DNA obtained in Example 2 are amplified by PCR according to the above-mentioned detection method 1;

[0066] The results are as follows Figure 6 As shown, lanes 1-3 are DNA templates extracted by conventional methods, and lanes 4-6 are DNA templates obtained in Example 2. The PCR product bands of the two methods are single and bright, with no difference, indicating that the concentration and quality of DNA extracted by the method of the present invention are guaranteed and feasible. In addition, in Example 2, single nematode DNA is quickly extracted, and only one nematode is required, and the overall extraction is fast and the loss is small.

[0067] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A specific primer for rapid identification of northern root-knot nematodes in soil, characterized in that: The sequences of the specific primers are as follows: Upstream primer Mhs-F: TGTCTGGCTTTGGGCTAC; Downstream primer Mhs-R: GGTGACCGAAAGAGCATT.

2. Use of the specific primers as claimed in claim 1 in rapid identification of northern root-knot nematodes in soil.

3. The use according to claim 2, characterized in that: The method for applying the specific primers to quickly identify northern root-knot nematodes in soil comprises the following steps: S1, Isolation of nematodes in soil; S2, quickly extracting DNA from a single nematode to obtain a DNA extract; S3, DNA amplification using nematode-specific primers, specifically comprising the following steps: S3-1. Set up the PCR system as follows: 12.5 μL of PCR Master, 1 μL each of upstream primer Mhs-F and downstream primer Mhs-R, 2 μL of DNA extraction solution, and add sterile double distilled water to 25 μL; S3-2, PCR program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 61℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ extension for 5 min, 12℃ storage; S3-3. Detect the PCR product by agarose gel electrophoresis and observe the bands to determine whether it is the northern root-knot nematode.

4. The use according to claim 3, characterized in that: The specific method of step S1 comprises the following steps: S1-1. After mixing the collected soil evenly, take 100 g of soil and put it into a 2 L beaker, add 1 L of water, stir with a glass rod for 3 min, let it stand for 1 min, filter with an 80-mesh sieve and collect the filtrate and bottom mud; S1-2, add 1 L of water to the mud at the bottom, stir, let stand, filter, and repeat the operation twice; S1-3. Combine the collected filtrates, pass through a 500-mesh sieve, and rinse with clean water until the water leaking from the filter is clear. Place the remaining nematode residues on the sieve on a funnel covered with paper towels, and ensure that there is water to submerge the residues. Leave it for more than 12 hours until the water level that submerges the residues is below the residues. S1-4. Release the water at the bottom of the funnel and observe the living nematodes collected in the soil under a stereo microscope.

5. The use according to claim 3, characterized in that: The specific method in step S2 comprises the following steps: Use an acupuncture needle to pick out a separated nematode, put it into a PCR tube, add 9μL 2×Taq Buffer, 1μL 1μg / μL proteinase K, centrifuge at low speed for 30 s, quick freeze in liquid nitrogen for 3 min, place in a PCR instrument, set the program to 65℃, 90 min, 95℃, 15 min, obtain DNA extract, and store at -20℃ for later use.

6. A kit for detecting northern root-knot nematodes, characterized in that: The sequences of the specific primers in the kit are as follows: Upstream primer Mhs-F: TGTCTGGCTTTGGGCTAC; Downstream primer Mhs-R: GGTGACCGAAAGAGCATT.

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