Primer group for detecting plasmodiophora brassicae in rape seeds as well as detection method and application of primer group

Abstract

The invention discloses a primer group for plasmodiophora brassicae as well as a detection method and application of the primer group. The invention provides a primer group. The primer group comprises at least one of a Pb06745-R / F primer group, a Pb08600-R / F primer group and a Pb03781-R / F primer group. The invention provides three qPCR primer groups for quantitative detection of plasmodiophora brassicae of rape seeds, can specifically detect 10 resting spores of plasmodiophora brassicae, has the advantages of high sensitivity and high specificity, can detect different physiological race types of plasmodiophora brassicae, and has the advantage of high stability. A safe and reliable detection technology is provided for oilseed rape production, whether oilseed rape seeds are polluted by plasmodiophora brassicae or not and the bacterium carrying amount can be rapidly and accurately judged, rapid spreading of oilseed rape clubroot is relieved, clubroot is prevented and controlled, and meanwhile the method has guiding significance in inspection and quarantine of entry and exit related plants and products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganism detection, in particular to a primer set for detecting Plasmodiophora brassicae in rapeseed and a detection method and application thereof. BACKGROUND

[0002] Rape is the first source of domestic plant edible oil in China, and it plays a decisive role in the domestic edible oil market. Rapeseed root rot is a major destructive soil-borne disease caused by the protozoan Plasmodiophora brassicae. In recent years, it has occurred and spread on a large scale in China, causing root swelling, rotting, and large-scale death of rapeseed plants, resulting in significant yield loss and seriously threatening the safety and healthy development of China's rapeseed industry. At present, the rapid spread of rapeseed root rot has not been effectively controlled. Since root rot only harms the roots of rapeseed, it is not clear whether rapeseed seeds can carry Plasmodiophora brassicae and spread root rot.

[0003] Plasmodiophora brassicae is a typical biotrophic obligate parasite with complex infection cycles and diverse life forms, which cannot be cultured in vitro, making pathogen identification difficult. In soil, the main life form of Plasmodiophora brassicae is dormant spores, which have a thick cell wall on the outside, making it difficult to extract and quantify Plasmodiophora brassicae genomic DNA. There have been reports on the identification and detection of Plasmodiophora brassicae in soil and diseased roots. The main methods are based on PCR principles, including conventional PCR, quantitative PCR (qPCR), droplet digital PCR, RNase H2-dependent PCR, loop-mediated isothermal amplification (LAMP), etc. Among them, qPCR is a technology for selective amplification and real-time quantitative detection of DNA (or cDNA, RNA), which combines PCR amplification and fluorescence detection technology to monitor the change of fluorescence signal in real time during PCR, thereby realizing the quantitative analysis of target DNA. Due to its high sensitivity, high specificity, and fast detection speed, qPCR is widely used in the detection of plant pathogens.

[0004] Currently, the detection methods of Plasmodiophora brassicae have certain controversy in sensitivity and reliability, and there is a lack of detection technology for Plasmodiophora brassicae carried on the surface of seeds. Establishing a precise and efficient quantitative detection method for Plasmodiophora brassicae carried on seeds can provide key technical support for early warning and prevention and control of root rot. SUMMARY

[0005] The purpose of the present application is to overcome the above-mentioned deficiencies of the prior art, and to provide a primer set for detecting Plasmodiophora brassicae in rapeseed and a detection method and application thereof.

[0006] The application detects Plasmodiophora brassicae by three groups of stable and efficient specific detection primers, has high sensitivity and specificity, provides a safe and reliable detection technology for rape production, can quickly and accurately judge whether the rape seed is contaminated by Plasmodiophora brassicae and the size of the bacterial load, is beneficial to slow down the rapid spread of Plasmodiophora brassicae and prevent and control the Plasmodiophora brassicae, and has guiding significance for inspection and quarantine of relevant plants and products entering and leaving the country.

[0007] The first object of the application is to provide a primer group.

[0008] The second object of the application is to provide the application of the primer group in detecting Plasmodiophora brassicae.

[0009] The third object of the application is to provide a kit for detecting Plasmodiophora brassicae.

[0010] The fourth object of the application is to provide a method for detecting Plasmodiophora brassicae.

[0011] In order to achieve the above objects, the application is realized by the following scheme:

[0012] A primer group, the primer group comprises at least one group of Pb06745-R / F, Pb08600-R / F and Pb03781-R / F primer groups;

[0013] The nucleotide sequences of the Pb06745-R / F primer group are shown as SEQ ID NO: 1 and 2;

[0014] The nucleotide sequences of the Pb08600-R / F primer group are shown as SEQ ID NO: 3 and 4;

[0015] The nucleotide sequences of the Pb03781-R / F primer group are shown as SEQ ID NO: 5 and 6.

[0016] The application of the primer group in detecting Plasmodiophora brassicae.

[0017] Further, the Plasmodiophora brassicae includes P2, P3, P4 and P8 physiological races identified by the Williams identification system, and 16 / 15 / 31 physiological races identified by the ECD identification system.

[0018] Further, the Plasmodiophora brassicae includes Plasmodiophora brassicae and / or Plasmodiophora brassicae spores in rape and / or soil.

[0019] Further, the Plasmodiophora brassicae includes Plasmodiophora brassicae and / or Plasmodiophora brassicae spores in rape seeds or roots.

[0020] A kit for detecting Plasmodiophora brassicae, the kit contains the primer group.

[0021] A method for detecting root knotweed bacteria, using the primer set for detection.

[0022] Furthermore, DNA of the sample to be tested is extracted, and PCR amplification or qPCR amplification is performed using the primer set with the DNA of the sample to be tested as a template.

[0023] Furthermore, the sample to be tested is mixed with steel beads, fully broken up, and DNA of the sample to be tested is extracted.

[0024] Preferably, the method for extracting DNA from the sample to be tested includes the CTAB method.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention provides three sets of qPCR primer sets that can be used for the quantitative detection of root-knot fungi in rapeseed seeds. The primer sets can specifically detect as few as 10 dormant spores of root-knot fungi, have the advantages of high sensitivity and high specificity, can detect different physiological subspecies types of root-knot fungi, and have the advantage of high stability.

[0027] (2) The present invention can extract dormant spores of root knot fungi from nodules infected with clubroot, remove the host tissue, and then extract DNA. High-purity root knot fungi DNA can be directly obtained without connecting the DNA to a vector to obtain a recombinant plasmid. qPCR quantification can be directly performed, which has the advantages of simple operation and short time.

[0028] (3) The present invention fully breaks the cell walls of dormant spores of root knotweed fungi by using steel balls, thereby improving the release efficiency of DNA and thus improving the efficiency of DNA extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of primer specificity detection are shown in Figure 1. A is the PCR electrophoresis result, M: DNA marker; lanes 1-3: soil DNA from non-diseased areas; lane 4: DNA from healthy rapeseed leaves; lane 5: DNA from dormant spores of root knotweed fungus; lane 6: soil DNA from a diseased area; lane 7: blank control; and B is the melting peak diagram of the qPCR amplification.

[0030] Figure 2 These are the amplified sequence sites for the primer sets Pb06745-R / F, Pb08600-R / F, and Pb03781-R / F.

[0031] Figure 3The qPCR amplification results of 30 rapeseed samples. Among them, A is the qPCR amplification curve of Pb08600-R / F primer group; B is the qPCR amplification curve of Pb06745-R / F primer group; C is the qPCR amplification curve of Pb03781-R / F primer group; D is the qPCR amplification melting peak graph of Pb08600-R / F primer group; E is the qPCR amplification melting peak graph of Pb06745-R / F primer group; F is the qPCR amplification melting peak graph of Pb03781-R / F primer group.

[0032] Figure 4 The detection sensitivity and qPCR standard curve. Among them, A is the DNA amount extracted from different numbers of dormant spores; B is the PCR amplification result of different numbers of dormant spores; C is the qPCR amplification curve of different numbers of dormant spores; D is the standard curve of qPCR amplification. DETAILED DESCRIPTION

[0033] The present application will be further described in conjunction with the drawings and specific embodiments of the present application, which are used to explain the present application and are not used to limit the scope of the present application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0034] Example 1 primer design

[0035] 1. Materials: 3 portions of non-gall nematode field soil, 1 portion of gall nematode field soil (physiological race type P2); rape leaf; gall nematode dormant spores (physiological race type P4).

[0036] 2. Primer design: based on the whole genome sequence of the gall nematode (NCBI accession number PRJNA1080080), gene sequence alignment was performed in the whole species gene database, other species genes and GC abnormal sequences were excluded, and 12 genes were randomly selected from the final sequence to design optimal primer pairs as detection primers (Table 1). In order to adapt to the requirements of qPCR, the expected product size after amplification is between 100-250bp.

[0037] Table 1

[0038]

[0039]

[0040] 2. DNA Extraction: 0.01 g of each material was added to two steel balls and cooled in liquid nitrogen for 30 seconds. The sample was then ground in a grinder three times for 1 minute each time. Between each grinding step, the sample was cooled in liquid nitrogen and then ground again. After thorough disruption, 10 μL of 20 mg / mL proteinase K was added to prepare a pretreated sample. DNA from the pretreated sample was extracted using the CTAB (cetyltrimethylammonium bromide) method and used as a template for qPCR amplification.

[0041] The steps of the CTAB method are as follows: (1) Add 1 mL of 2% (w / v) CTAB preheated at 60°C to the pretreated sample as soon as possible, mix well, and then keep it at 60°C for 45 minutes. During the keeping period, turn it upside down and mix it three times; (2) Add 1 mL of chloroform to the sample treated in step (1), vortex mix for 1 minute, and centrifuge at 12000 rpm for 15 minutes at room temperature; repeat this step until there is almost no brown substance in the middle layer; (3) Transfer the supernatant treated in step (2) to a 2 mL centrifuge tube, add an equal volume of isopropanol precooled at -20°C to the supernatant, and place it at -20°C for precipitation for more than 1 hour; (4) Centrifuge the sample treated in step (3) at 12000 rpm for 15 minutes, discard the supernatant, and collect the precipitate; (5) Add 1 mL of 75% (v / v) ethanol, centrifuge at 12000 rpm for 8 min, discard the supernatant, and observe the color change of the precipitate; repeat this step until the precipitate is almost white; (6) place the precipitate obtained in step (5) at 37°C for 1 h to dry, and add 100 μL ddH2O to dissolve the DNA.

[0042] 3. PCR amplification: PCR amplification was performed using each of the 12 primer sets listed in Table 1. The total PCR amplification system was 30 μL, containing 20 μL of 2× ES Taq MasterMix, 7 μL of double-distilled water, 1 μL of forward primer, 1 μL of reverse primer, and 1 μL of DNA extracted in step 2. The amplification program was as follows: 95°C, 3 min; 95°C, 30 s, 58°C, 30 s, 72°C, 11 s, 30 cycles; 72°C, 11 s.

[0043] 4. qPCR amplification: qPCR amplification was performed using each of the 12 primer sets listed in Table 1. The total qPCR amplification system (30 μL) contained 20 μL of SYBR Green qPCR Master Mix, 7 μL of double-distilled water, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, and 1 μL of DNA extracted in step 2. The amplification program was as follows: 95°C for 3 min; 40 cycles of 95°C for 30 s and 58°C for 30 s; and 72°C for 11 s.

[0044] 2. Experimental Results

[0045] The nucleotide sequence of the product amplified by the primer set Pb06888-R / F is shown in SEQ ID NO: 25, with a length of 169 bp; the nucleotide sequence of the product amplified by the primer set Pb02115-R / F is shown in SEQ ID NO: 26, with a length of 196 bp; the nucleotide sequence of the product amplified by the primer set Pb07172-R / F is shown in SEQ ID NO: 27, with a length of 156 bp; the nucleotide sequence of the product amplified by the primer set Pb06938-R / F is shown in SEQ ID NO: 28, with a length of 146 bp; the nucleotide sequence of the product amplified by the primer set Pb03088-R / F is shown in SEQ ID NO: 29, with a length of 152 bp; the nucleotide sequence of the product amplified by the primer set Pb10053-R / F is shown in SEQ ID NO: 30, with a length of 100 bp; the nucleotide sequence of the product amplified by the primer set Pb03906-R / F is shown in SEQ ID NO: NO: 31, with a length of 189 bp; the nucleotide sequence of the product amplified by the primer set Pb02219-R / F is shown in SEQ ID NO: 32, with a length of 259 bp; the nucleotide sequence of the product amplified by the primer set Pb04953-R / F is shown in SEQ ID NO: 33, with a length of 169 bp; the nucleotide sequence of the product amplified by the primer set Pb06745-R / F is shown in SEQ ID NO: 34, with a length of 137 bp; the nucleotide sequence of the product amplified by the primer set Pb08600-R / F is shown in SEQ ID NO: 35, with a length of 179 bp; and the nucleotide sequence of the product amplified by the primer set Pb03781-R / F is shown in SEQ ID NO: 36, with a length of 243 bp.

[0046] like Figure 1 As shown in A, among the 12 primer sets tested, the primer sets Pb06938-R / F, Pb03906-R / F, Pb03088-R / F, Pb07172-R / F, Pb10053-R / F, Pb02219-R / F, Pb06888-R / F, and Pb02115-R / F were able to amplify bands in soil (lanes 1-3) or rapeseed leaves (lane 4) without clubroot fungi, indicating that these primer sets had poor specificity for clubroot fungi; Figure 1As shown in Figure B, after PCR amplification by Pb06745-R / F, Pb08600-R / F, Pb04953-R / F and Pb03781-R / F primer groups, only lanes 5 (plasmodiophora resting spores) and lanes 6 (sick field soil) containing plasmodiophora amplified bands, and lanes without plasmodiophora had no bands, indicating that the four primer groups can specifically amplify plasmodiophora DNA; Pb06745-R / F, Pb08600-R / F and Pb03781-R / F primer groups were amplified by qPCR, and the variation range of the melting peak map was smaller and the peak shape was sharper, indicating that the specificity of the three primer groups was optimal. Figure 2 The sequence sites corresponding to the Pb06745-R / F, Pb08600-R / F and Pb03781-R / F primer groups.

[0047] Example 2 qPCR quantitative detection of plasmodiophora in 30 parts of rapeseed harvested from plasmodiophora fields

[0048] I. Experimental method

[0049] 1. Materials: 30 parts of rapeseed harvested from plasmodiophora disease fields in 7 provinces nationwide, and the plasmodiophora physiological race types are P2, P3, P4, P8 (Williams identification system) and other unknown types (not identified).

[0050] 2. Isolation and enrichment of plasmodiophora on the surface of rapeseed: take rapeseed, add appropriate amount of water to cover the seeds, stir well, elute the surface attachments, centrifuge to obtain the precipitate, dry and weigh to obtain the surface attachments of the seeds.

[0051] 3. DNA extraction: weigh 0.01 g of seed surface attachments, add 2 steel balls, cool in liquid nitrogen for 30 s, then put into a grinding instrument and grind 3 times to break it up. After the last grinding, extract DNA by CTAB method, the method is the same as in Example 1, which is used as a qPCR amplification template.

[0052] 4. qPCR amplification: use Pb06745-R / F, Pb08600-R / F or Pb03781-R / F primer groups in Table 1 for qPCR amplification, the total amplification system is 30 μL, containing qPCR SYBR Green Master Mix 20 μL, double distilled water 7 μL, concentration 10 μM of forward primer 1 μL, concentration 10 μM of reverse primer 1 μL, template DNA 1 μL; the amplification program is: 95℃, 3 min; 95℃, 30 s, 58℃, 30 s, cycle 40 times; 72℃, 11 s.

[0053] 5. According to the Ct value (based on the average Ct value of the three primer groups) and the qPCR standard curve regression equation, the seed bacterial load is calculated.

[0054] II. Experimental results

[0055] The results are shown in Figure 3 A-F. The Ct value of the water control after qPCR amplification was greater than 35, indicating that the detection process was reliable. The Ct value of the 30 oilseed rape samples was between 25.01 and 33.03. After calculation, the amount of Plasmodiophora on the surface of these seeds was 0.06 x 10 5 ~ 84.84 x 10 5 spores per 500 g of seeds.

[0056] Example 3: Detection sensitivity and qPCR standard curve preparation

[0057] I. Experimental methods

[0058] 1. Materials: diseased oilseed rape roots collected from the field, and the physiological race of Plasmodiophora was P2 (Williams identification system) and 16 / 15 / 31 (ECD identification system).

[0059] 2. Preparation of different concentrations of Plasmodiophora resting spore solution: (1) Take the diseased oilseed rape swollen roots and put them in a juicer to crush them with sterile water to obtain the crushed tissue of the diseased oilseed rape swollen roots; (2) Filter the crushed tissue of the diseased oilseed rape swollen roots with six layers of gauze, collect the filtrate, and centrifuge at 300 rpm for 8 min; take the supernatant and centrifuge at 4500 rpm for 8 min, and retain the precipitate; (3) Add 8 mL of 50% (w / v) sucrose solution to the precipitate in step (2) and centrifuge at 4500 rpm for 8 min, and take the supernatant; (4) Add 30 mL of sterile water to the supernatant in step (3) and centrifuge at 4500 rpm for 8 min, discard the supernatant, and retain the precipitate; (5) Resuspend the precipitate in step (4) in 30 mL of sterile water, centrifuge at 4500 rpm for 8 min, discard the supernatant, and retain the precipitate, which is the purified Plasmodiophora resting spores.

[0060] Resuspend the purified Plasmodiophora resting spores in 30 mL of sterile water, and under a light microscope, use a hemocytometer to count the concentration of the resting spores, adjust the spore concentration to 1 x 10 8 / mL, and serially dilute with sterile water by a factor of 10 to 1 x 10 3 / mL.

[0061] 3. DNA extraction: Take 1 mL of each concentration of resting spore suspension, centrifuge at 4500 rpm for 8 min to collect the resting spores, add 2 steel balls, cool in liquid nitrogen for 30 s, and then grind with a grinder for 3 times, 1 min each time. After thorough crushing, extract the Plasmodiophora resting spore DNA using the CTAB method, which is the same as in Example 1. Dissolve the DNA with 100 μL of ddH2O, and measure the DNA concentration with 1 μL of DNA using a NanoDrop microspectrophotometer.

[0062] 4. PCR amplification: PCR amplification was performed using the primer sets Pb08600-R / F, Pb06745-R / F, and Pb03781-R / F in Table 1, respectively. The total PCR amplification system was 30 μL, containing 20 μL of 2× ES Taq MasterMix, 7 μL of double-distilled water, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, and 1 μL of DNA (equivalent to a 100-fold dilution of the original dormant spore solution, corresponding to 10 to 10 dormant spores). 6 The amplification program was as follows: 95°C, 3 min; 95°C, 30 s, 58°C, 30 s, 72°C, 11 s, 30 cycles; 72°C, 11 s.

[0063] 5. qPCR amplification: qPCR amplification was performed using primer sets Pb08600-R / F, Pb06745-R / F, and Pb03781-R / F, respectively. The total qPCR amplification system was 30 μL, containing 20 μL qPCR SYBR Green Master Mix, 7 μL double-distilled water, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, and 1 μL DNA (corresponding to 10 to 10 dormant spores). 6 The amplification program was as follows: 95°C, 3 min; 95°C, 30 s, 58°C, 30 s, 40 cycles; 72°C, 11 s.

[0064] 6. Construct a standard curve: Use the common logarithmic value of the number of dormant spores as the X-axis and the cycle number Ct value as the Y-axis to draw a standard curve, and calculate the regression equation.

[0065] 2. Experimental Results

[0066] 1. If Figure 4 As shown in A, the DNA concentration was measured using a NanoDrop micro-spectrophotometer and found to be 1×10 6 , 1×10 5 and 1×10 4 The DNA content of dormant spores was 555.67 ng, 61.33 ng and 6.97 ng, decreasing exponentially, while 1×10 3 The DNA amount of dormant spores or less cannot be effectively measured using the NanoDrop micro-spectrophotometer.

[0067] 2. If Figure 4 As shown in B, in the PCR reaction, 100 dormant spores can be detected, but 10 dormant spores cannot be detected. Figure 4 As shown in Figure C, the qPCR reaction can detect the DNA of 10 dormant spores of Rhizoctonia solani. The sensitivity of the qPCR detection in the present invention is 10 times that of ordinary PCR.

[0068] 3. If Figure 4 As shown in D, a standard curve was drawn with the common logarithmic value of the number of dormant spores as the X-axis and the cycle number Ct value as the Y-axis, and the regression equation was calculated. The results showed that there was a significant linear negative correlation between the logarithmic value of the number of dormant spores (x) and the cycle number Ct value (y). The regression equation was: x = -0.2920y + 11.0068, and the correlation coefficient R 2 =0.9986.

[0069] Example 4 A kit for detecting root knot fungi

[0070] 1. Composition

[0071] Pb06745-R / F primer set, the nucleotide sequences are shown in SEQ ID NOs: 1 and 2;

[0072] Pb08600-R / F primer set, the nucleotide sequences are shown in SEQ ID NOs: 3 and 4;

[0073] Pb03781-R / F primer set, the nucleotide sequences are shown in SEQ ID NOs: 5 and 6;

[0074] Double-distilled water (can be used as a negative control);

[0075] The dormant spore solution of root knotweed (used to establish the standard curve and regression equation, and can also be used as a positive control) can be 1×10 8 spores / mL, 1×10 7 spores / mL, 1×10 6 spores / mL, 1×10 5 spores / mL, 1×10 4 spores / mL and 1×10 3 spores / mL.

[0076] 2. Usage

[0077] 1. DNA extraction

[0078] The sample to be tested is rapeseed seeds: the seed surface attachments are obtained according to the method of Example 2, and DNA is extracted using the CTAB method to serve as a template.

[0079] The sample to be tested is rapeseed root: dormant spores of Plasmodium fumigatus were extracted and purified from rapeseed root according to the method of Example 2, and DNA of the dormant spores of Plasmodium fumigatus was extracted using the CTAB method and used as a template.

[0080] The sample to be tested is soil: the soil sample to be tested is treated according to the method of Example 1, and the DNA of dormant spores of root knotweed fungus is extracted by the CTAB method and used as a template.

[0081] 2. qPCR amplification: qPCR amplification was performed using the Pb08600-R / F, Pb06745-R / F, and Pb03781-R / F primer sets, respectively. The total qPCR amplification system was 30 μL, containing 20 μL of SYBR Green qPCR Master Mix, 7 μL of double-distilled water, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, and 1 μL of template DNA. The amplification program was as follows: 95°C for 3 min; 95°C for 30 s, 58°C for 30 s, 40 cycles; 72°C for 11 s.

[0082] 3. Determination method

[0083] A standard curve and regression equation were constructed using the common logarithm of the number of spores in the dormant spore solution of R. truncatum as the horizontal axis and the corresponding qPCR cycle threshold (Ct) as the vertical axis. The Ct value was the average Ct value of the three primer sets. The bacterial count of the test sample was calculated based on the Ct value of the amplified sample (based on the average Ct value of the three primer sets) and the qPCR standard curve regression equation.

[0084] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A primer set, characterized in that: The primer set includes at least one of the primer sets Pb06745-R / F, Pb08600-R / F and Pb03781-R / F; The nucleotide sequences of the Pb06745-R / F primer set are shown in SEQ ID NOs: 1 and 2; The nucleotide sequences of the Pb08600-R / F primer set are shown in SEQ ID NOs: 3 and 4; The nucleotide sequences of the Pb03781-R / F primer set are shown in SEQ ID NOs: 5 and 6.

2. Use of the primer set according to claim 1 in detecting root knot fungi.

3. The use according to claim 2, characterized in that The root knot fungus includes physiological races P2, P3, P4, and P8 identified by the Williams identification system, or physiological races 16 / 15 / 31 identified by the ECD identification system.

4. The use according to claim 2, characterized in that The root-knot bacteria include root-knot bacteria and / or root-knot bacteria spores in rapeseed and / or soil.

5. The use according to claim 4, characterized in that The root-knot fungi include root-knot fungi and / or root-knot fungi spores in rapeseed seeds or roots.

6. A kit for detecting root knot fungus, characterized in that, The kit contains the primer set according to claim 1.

7. A method for detecting root knot fungus, characterized in that, Detection is performed using the primer set described in claim 1.

8. The method according to claim 7, characterized in that Extract DNA from the sample to be tested, use the DNA from the sample to be tested as a template, and perform PCR amplification or qPCR amplification using the primer set of claim 1.

9. The method according to claim 8, characterized in that The sample to be tested is mixed with steel beads, fully crushed, and the DNA of the sample to be tested is extracted.

10. The method according to claim 9, characterized in that Methods for extracting DNA from test samples include the CTAB method.

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