CN108300791B - Synchronous detection and identification method for three main pathogenic nematodes in tobacco root knot nematode disease - Google Patents
Synchronous detection and identification method for three main pathogenic nematodes in tobacco root knot nematode disease Download PDFInfo
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Abstract
The invention discloses a synchronous detection and identification method for three main pathogenic nematodes in tobacco root knot nematode disease. Comprises the steps of PCR primer design, DNA template preparation, PCR amplification and product detection. The invention designs the PCR primers containing 6 for identifying the species of the 3 kinds of root-knot nematodes according to the species specific region sequences of the meloidogyne incognita, the meloidogyne arachidis and the meloidogyne javanica, and the identification stability and the accuracy are good; the designed 6 primers are combined for use, so that 1 time of PCR reaction can be realized to complete the synchronous detection of the pathogenic nematodes of the 3 tobacco root-knot nematode diseases, and the identification is convenient and time-saving; a common PCR instrument and a conventional reagent are adopted, so that the detection cost is low, and the popularization and the application are facilitated; the invention comprises a DNA preparation method aiming at diseased root samples and soil samples, can identify the species of root-knot nematodes in various development stages including eggs, larvae and adults, and has wide application range.
Description
Technical Field
The invention belongs to the technical field of plant disease pathogen detection, and particularly relates to a synchronous detection and identification method for three main pathogenic nematodes in tobacco root-knot nematode disease.
Background
Root knot nematode (A), (B) and (C)Meloidogynespp.) is the most diverse, widespread and most harmful class of plant pathogenic nematodes. The nematodes are mainly in a soil-spreading form, harm the root systems of plants and enable the roots of the damaged plants to form tumor-shaped root knots, so that the root knot nematode disease seriously influencing the normal growth and development of the plants occurs, known plants damaged by the root knot nematode disease mainly comprise more than 3000 species of solanaceae, leguminosae, cucurbitaceae, gramineae, umbelliferae and the like, and the plant damage in temperate regions, subtropical regions and tropical regions is particularly serious. Particularly, the tobacco is most seriously damaged by root-knot nematodes, and according to the estimation of the food and agriculture organization of the United nations, the tobacco loss caused by the root-knot nematodes in developed countries is 4% per year on average, the average loss in the world is estimated to be 10%, and the loss in developing countries exceeds 25%. Since the middle and later stages of the 80 th century in China, tobacco root knot nematode disease areas are continuously enlarged and serious, the tobacco root knot nematode disease areas are common in tobacco areas such as Henan, Shandong, Yunnan, Guizhou, Sichuan and Hubei, the tobacco roots knot nematode disease areas are serious in harm, the disease incidence rate of the disease areas is generally 70% -100%, the serious disease is more than 90%, the yield is reduced by 30% -70%, and the tobacco root knot nematode disease areas become main diseases in tobacco production in China.
Root-knot nematodes belong to soil inhabitation organisms, and are difficult to act on the root-knot nematodes due to physical and chemical factors externally applied under the restriction of the soil environment, and most of chemical nematicides for preventing and controlling root-knot nematodes have the disadvantages of high toxicity and high residue and are generally forbidden worldwide in recent years. Thus miningThe safe and efficient root knot nematode disease prevention and control method becomes a great trend. In the process of preventing and controlling the tobacco root knot nematode disease, selecting and adopting a proper disease-resistant tobacco variety is considered as an economic, efficient and green measure. However, the root-knot nematodes have various varieties, different root-knot nematodes have different pathogenic abilities on host plants, the resistance range of disease-resistant varieties to the root-knot nematodes is limited, and the root-knot nematodes which are widely distributed in tobacco and most seriously harmful are mainly peanut root-knot nematodes (A)Meloidogyne arenaria) Meloidogyne incognita (C.), (M. incognita) And root-knot nematode of Java: (M. javanica) The three kinds of root-knot nematodes have very similar morphological and molecular biological characteristics, but have larger pathogenicity difference for different tobacco varieties. Therefore, accurate and rapid identification of the three root-knot nematodes is a precondition for breeding and utilizing disease-resistant tobacco varieties.
The most common method for identifying the species of the root-knot nematodes is a morphological method, which is to determine the species of the root-knot nematodes by cutting the cuticle membrane of the perineal region of female root-knot nematodes through a micromanipulation method and observing the differences of patterns in the perineal region under a microscope. The method has the advantages of simplicity, convenience and intuition, and has the following disadvantages: 1. the variety of the root-knot nematodes is multiple, and the characteristics of many varieties of perineal patterns are easy to be confused, so that the identification accuracy completely depends on the experience of an operator, and the misjudgment probability is high; 2. the perineal patterns of the same type of root-knot nematodes have individual differences, and the female adults with more than 20 heads at least need to be observed to confirm the diagnosis in actual operation, which is labor-consuming and time-consuming; 3. the morphological identification is only applicable to adults and is not suitable for larvae, and the root-knot nematodes separated from the soil exist in the form of second-instar larvae, so that the method is limited in practical application. In addition, the isoenzyme electrophoresis method is also a commonly used method in the identification of the root-knot nematode, the method carries out polyacrylamide electrophoresis on a crude protein extract of female adult root-knot nematode, and carries out species judgment according to the spectrum type of an enzyme band after the isoenzyme of esterase and malate dehydrogenase is dyed.
With the development of molecular biology technology in recent years, rapid and accurate identification of root-knot nematodes by using PCR technology has become a trend. Compared with morphological and isoenzyme electrophoresis identification methods, the PCR identification method has wider application range, and eggs, second instar larvae and male and female adults of the root-knot nematodes can be used as identification objects. Early PCR identification included RAPD-PCR technology using random primer amplification or PCR-RFLP technology using nematode universal primer amplification followed by restriction of the amplified product, which have the common disadvantage of low stability and accuracy due to the use of specific primers not specific for root-knot nematodes. Therefore, the domestic and foreign scholars have started to use the species-specific PCR primer amplification to identify the species of the root-knot nematodes in recent years. Xujianhua et al, a primer for rapid molecular detection of root-knot nematodes and a use method thereof (patent number: ZL 03131763.4), and a method for identifying root-knot nematodes in tobacco (patent number: ZL201610585565. X), provide a novel method for PCR identification of pathogenic nematodes of tobacco root-knot nematode, and the method adopts three pairs of specific primers of species of root-knot nematodes to perform PCR detection on the root-knot nematodes separated from diseased roots of tobacco and soil respectively, so that the identification is convenient, the accuracy is high, and the diagnosis efficiency of the pathogenic nematodes of tobacco root-knot nematode is greatly improved. However, because a step-by-step PCR detection method is adopted, at least more than three PCR reactions are required to complete the identification of one sample, so that the identification process is complicated to operate and takes long time. Therefore, it is necessary to develop a detection and identification method that can solve the above-mentioned problems.
Disclosure of Invention
The invention aims to provide a synchronous detection and identification method for three main pathogenic nematodes in tobacco root knot nematode disease.
The invention aims to realize the method, comprises the steps of PCR primer design, DNA template preparation, PCR amplification and product detection, and specifically comprises the following steps:
A. designing a PCR primer: 6 PCR primers are designed according to species-specific sequences of Meloidogyne incognita, Meloidogyne arachidis and Meloidogyne javanica, namely TR1, TR2, TR3, TR4, TR5 and TR6 are used for PCR identification of the 3 Meloidogyne incognita, and the sequences are respectively:
TR1：5'-AAGCAAAAGACGAAGCACCAAAA-3'
TR2：5'-GAGGATTCAGCTCCCCAGCAC-3'
TR3：5'-GAGTACGGATTTGAAATACAGGG-3'
TR4：5'-TGAATGCTATGCCATCAGGAG-3'
TR5：5'-CGATTGAACTGAGCCCAGACT-3'
TR6：5'-TTTATTCGCAAGACAACACCC-3'；
B. preparing a DNA template:
when detecting tobacco diseased root samples: removing attachments on the surface of a diseased root sample, selecting a root-knot nematode egg mass or a root-knot tissue, placing the root-knot nematode egg mass or the root-knot tissue in a sterilized mortar, adding liquid nitrogen, grinding the root-knot nematode egg mass or the root-knot tissue into powder, transferring the powder into a centrifuge tube, adding a DNA extraction buffer solution, placing the centrifuge tube in a water bath at 50-60 ℃, keeping the temperature for 50-70 min, cooling to room temperature, sequentially extracting with a phenol-chloroform-isoamylol mixed solvent and a chloroform-isoamylol mixed solvent which have the same volume, taking supernate, adding isopropanol which has the same volume to precipitate, washing the precipitate with an ethanol solution with the volume percentage concentration of 60-80%, drying, dissolving the precipitate in sterilized purified water, and adjusting the concentration value of DNA to be 50 ng/mu l to obtain a DNA template preparation solution for PCR detection;
when the detection sample is tobacco rhizosphere soil: separating soil nematodes from a soil sample by a conventional method, placing the obtained nematode suspension under a dissecting mirror for observation, picking 2-3 to 10 mu l of 100 ng/mu proteinase K solution of the nematodes suspected to be root-knot nematodes, namely second-instar larvae or male adults, cutting the nematodes by using a dissecting needle, heating at 95 ℃ for 10min after 1h in water bath at 60 ℃, and cooling to room temperature to obtain a DNA template preparation solution for PCR detection;
C. and (3) PCR amplification: carrying out PCR amplification by using DNA template preparation solution for PCR detection to obtain a PCR amplification product;
D. and (3) product detection: and (3) carrying out 1.5% agarose gel electrophoresis on 5-10 mu l of PCR amplification product, dyeing by ethidium bromide after finishing the electrophoresis, and observing under an ultraviolet transmission instrument, wherein if an electrophoresis band with the molecular weight of 700bp is detected, the detected sample contains meloidogyne incognita, if an electrophoresis band with the molecular weight of 260bp is detected, the detected sample contains meloidogyne arachidis hypogaeae, and if an electrophoresis band with the molecular weight of 570bp is detected, the detected sample contains meloidogyne javanica.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the invention designs 6 PCR primers for identifying the species of the 3 kinds of root-knot nematodes according to the species specific region sequences of the meloidogyne incognita, the meloidogyne arachidis and the meloidogyne javanica, and the identification stability and the identification accuracy are good.
2. The 6 primers designed by the invention are used in combination, so that 1 time of PCR reaction can be realized to complete the synchronous detection of the pathogenic nematodes of 3 tobacco root knot nematode diseases, and the identification is convenient and time-saving.
3. The invention adopts a common PCR instrument and a conventional reagent, has detection cost and is beneficial to popularization and application.
4. The invention comprises a DNA preparation method aiming at diseased root samples and soil samples, can identify the species of root-knot nematodes in various development stages including eggs, larvae and adults, and has wide application range.
Drawings
FIG. 1 is a schematic diagram showing the PCR amplification result of a tobacco nematodosis diseased root sample according to the present invention;
wherein M is a DNA molecular weight standard (DL 2000), and 1-12 are tobacco root knot nematode disease root samples collected from Yunan province, position, Zymuan county, Shanbei county, teacher county, Gunn county, Mongolian city, Screen side county, Binchuan county, inkstone county, Jianshui county and Maitreya city in sequence;
FIG. 2 is a schematic diagram showing the PCR amplification result of the rhizosphere soil sample of tobacco root knot nematode disease according to the present invention;
wherein M is DNA molecular weight standard (DL 2000), and 1-2 are tobacco root knot nematode rhizosphere soil samples collected from the provinces of Yunnan province, the provinces of Shanbei and Jiangchuan.
Detailed Description
The present invention is further illustrated by the following examples and the accompanying drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
The synchronous detection and identification method of three main pathogenic nematodes in tobacco root knot nematode disease comprises the steps of PCR primer design, DNA template preparation, PCR amplification and product detection, and specifically comprises the following steps:
A. designing a PCR primer: 6 PCR primers are designed according to species-specific sequences of Meloidogyne incognita, Meloidogyne arachidis and Meloidogyne javanica, namely TR1, TR2, TR3, TR4, TR5 and TR6 are used for PCR identification of the 3 Meloidogyne incognita, and the sequences are respectively:
TR1：5'-AAGCAAAAGACGAAGCACCAAAA-3'
TR2：5'-GAGGATTCAGCTCCCCAGCAC-3'
TR3：5'-GAGTACGGATTTGAAATACAGGG-3'
TR4：5'-TGAATGCTATGCCATCAGGAG-3'
TR5：5'-CGATTGAACTGAGCCCAGACT-3'
TR6：5'-TTTATTCGCAAGACAACACCC-3'；
B. preparing a DNA template:
when detecting tobacco diseased root samples: removing attachments on the surface of a diseased root sample, selecting a root-knot nematode egg mass or a root-knot tissue, placing the root-knot nematode egg mass or the root-knot tissue in a sterilized mortar, adding liquid nitrogen, grinding the root-knot nematode egg mass or the root-knot tissue into powder, transferring the powder into a centrifuge tube, adding a DNA extraction buffer solution, placing the centrifuge tube in a water bath at 50-60 ℃, keeping the temperature for 50-70 min, cooling to room temperature, sequentially extracting with a phenol-chloroform-isoamylol mixed solvent and a chloroform-isoamylol mixed solvent which have the same volume, taking supernate, adding isopropanol which has the same volume to precipitate, washing the precipitate with an ethanol solution with the volume percentage concentration of 60-80%, drying, dissolving the precipitate in sterilized purified water, and adjusting the concentration value of DNA to be 50 ng/mu l to obtain a DNA template preparation solution for PCR detection;
when the detection sample is tobacco rhizosphere soil: separating soil nematodes from a soil sample by a conventional method, placing the obtained nematode suspension under a dissecting mirror for observation, picking 2-3 to 10 mu l of 100 ng/mu proteinase K solution of the nematodes suspected to be root-knot nematodes, namely second-instar larvae or male adults, cutting the nematodes by using a dissecting needle, heating at 95 ℃ for 10min after 1h in water bath at 60 ℃, and cooling to room temperature to obtain a DNA template preparation solution for PCR detection;
C. and (3) PCR amplification: carrying out PCR amplification by using DNA template preparation solution for PCR detection to obtain a PCR amplification product;
D. and (3) product detection: and (3) carrying out 1.5% agarose gel electrophoresis on 5-10 mu l of PCR amplification product, dyeing by ethidium bromide after finishing the electrophoresis, and observing under an ultraviolet transmission instrument, wherein if an electrophoresis band with the molecular weight of 700bp is detected, the detected sample contains meloidogyne incognita, if an electrophoresis band with the molecular weight of 260bp is detected, the detected sample contains meloidogyne arachidis hypogaeae, and if an electrophoresis band with the molecular weight of 570bp is detected, the detected sample contains meloidogyne javanica.
The DNA extraction buffer described in step B consisted of 200mmol/L Tris-HCl pH 8.5, 250mmol/L NaCl, 25mmol/L EDTA, 0.5% SDS.
And the volume ratio of the phenol to the chloroform to the isoamylol in the phenol-chloroform-isoamylol mixed solvent in the step B is 25:24: 1.
And the volume ratio of chloroform to isoamylol in the chloroform-isoamylol mixed solvent in the step B is 24: 1.
The PCR reaction system in step C is a 25. mu.l PCR reaction system with 0.4. mu.M each of 1 XPCR buffer, 1U Taq enzyme, 0.2mM dNTPs, and primers TR1, TR2, TR3, TR4, TR5 and TR 6.
The PCR reaction condition in the step C is pre-denaturation at 94 ℃ for 2 min; 30 cycles, each cycle comprising: 45s at 94 ℃, 60s at 52 ℃ and 90s at 72 ℃; finally, the extension is carried out for 10min at 72 ℃.
The synchronous detection and identification method for three main pathogenic nematodes in tobacco root knot nematode disease specifically comprises the following operations:
(1) 6 PCR primers are designed according to species-specific sequences of Meloidogyne incognita, Meloidogyne arachidis and Meloidogyne javanica, namely TR1, TR2, TR3, TR4, TR5 and TR6 are used for PCR identification of the 3 Meloidogyne incognita, and the sequences are respectively:
TR1：5'-AAGCAAAAGACGAAGCACCAAAA-3'
TR2：5'-GAGGATTCAGCTCCCCAGCAC-3'
TR3：5'-GAGTACGGATTTGAAATACAGGG-3'
TR4：5'-TGAATGCTATGCCATCAGGAG-3'
TR5：5'-CGATTGAACTGAGCCCAGACT-3'
TR6：5'-TTTATTCGCAAGACAACACCC-3'
(2) the preparation of the DNA template for the PCR detection of the pathogenic nematodes of the tobacco root knot nematode disease comprises the following steps:
when detecting a tobacco diseased root sample, impurities such as soil attached to the surface of the diseased root sample are washed clean by tap water, surface moisture is absorbed by filter paper, the meloidogyne egg masses are picked by a needle picker of a dissector, or the meloidogyne egg masses are directly cut off by scissors, 0.2g of the egg masses or the meloidogyne tissues are weighed and placed in a mortar which is sterilized by high pressure, liquid nitrogen is added, the mixture is ground into powder and then transferred into a centrifugal tube of 1.5ml, 0.5ml of DNA extraction buffer solution (200 mmol/L Tris-HCl pH 8.5, 250mmol/L NaCl, 25mmol/L EDTA and 0.5% SDS) is added, and the mixture is placed in a water bath at 55 ℃ for heat preservation for 1 h. After cooling to room temperature, the reaction mixture was cooled with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and chloroform: sequentially extracting isoamyl alcohol (24: 1), and adding equal volume of isopropanol into supernate for precipitation. And washing and drying the precipitate by 70% ethanol, dissolving the precipitate in sterilized purified water, and adjusting the DNA concentration to 50 ng/mu l to obtain a DNA template preparation solution for PCR detection.
When the detection sample is tobacco rhizosphere soil, firstly, 200g of soil sample is taken to carry out soil nematode separation by a conventional method, the obtained nematode suspension is placed in a dissecting environment for observation, 2-3 to 10 mu l of 100 ng/mu proteinase K solution of suspected root-knot nematode second-age larvae or male adults is picked, the nematodes are cut by a dissecting needle, the solution is heated for 10min at 95 ℃ after being subjected to water bath at 60 ℃, and the DNA template preparation solution for PCR detection is obtained after the solution is cooled to room temperature.
(3) And (3) PCR amplification: adding 2 μ l of the DNA template preparation solution obtained in step (2) into 25 μ l of PCR reaction system containing 0.4 μ M each of 1 XPCR buffer, 1U Taq enzyme, 0.2mM dNTPs, and primers TR1, TR2, TR3, TR4, TR5 and TR 6; placing the mixture in a PCR instrument for amplification, wherein the optimized amplification conditions are as follows: pre-denaturation at 94 ℃ for 2 min; 30 cycles, each cycle comprising: 45s at 94 ℃, 60s at 52 ℃ and 90s at 72 ℃; finally, the extension is carried out for 10min at 72 ℃.
(4) And (3) detecting a PCR product: and (3) carrying out 1.5% agarose gel electrophoresis on 5-10 μ l of the PCR amplification product obtained in the step (3), observing the agarose gel under an ultraviolet transilluminator after the agarose gel is subjected to ethidium bromide staining after the electrophoresis is finished, wherein the detected sample contains the meloidogyne incognita if an electrophoresis band with the molecular weight of 700bp is detected, the detected sample contains the meloidogyne incognita if an electrophoresis band with the molecular weight of 260bp is detected, and the detected sample contains the meloidogyne javanica if an electrophoresis band with the molecular weight of 570bp is detected.
The invention is further illustrated by the following specific examples:
example 1
1. The design of specific primers for identifying three pathogenic nematodes of tobacco root knot nematode disease: according to the genome sequence information of the meloidogyne incognita, the meloidogyne arachidis and the meloidogyne javanica published by the National Center for Biotechnology Information (NCBI), DNASTAR 7.0 software is used for analysis to find out the species-specific sequences of three meloidogyne incognita, and primer Premier 5.0 software is used for designing 6 PCR primers for identifying the species of the three meloidogyne incognita:
TR1：5'-AAGCAAAAGACGAAGCACCAAAA-3'
TR2：5'-GAGGATTCAGCTCCCCAGCAC-3'
TR3：5'-GAGTACGGATTTGAAATACAGGG-3'
TR4：5'-TGAATGCTATGCCATCAGGAG-3'
TR5：5'-CGATTGAACTGAGCCCAGACT-3'
TR6：5'-TTTATTCGCAAGACAACACCC-3'
the 6 oligonucleotide primers were synthesized by a DNA synthesizer.
2. Preparing a DNA template for PCR identification of pathogenic nematodes of tobacco root knot nematode: respectively collecting tobacco root knot nematode disease root samples from Yunan province, Tu Yi county, Jiang Chuan county, Hibei county, teacher county, Gunn county, Mong city, Wen Bing county, Bin Chuan county, inkstone county, Jianshui county and Maitreya city, washing impurities such as soil attached to the surface of the disease root with tap water, draining off surface water with filter paper, picking up root knot nematode egg masses with a needle pick-up or directly cutting off root knot parts with scissors, weighing 0.2g of egg masses or root knot tissues in a high-pressure sterilized mortar, adding liquid nitrogen, grinding into powder, transferring into a 1.5ml centrifuge tube, adding 0.5ml of DNA extraction buffer (200 mmol/L Tris-HCl pH 8.5, 250mmol/L NaCl, 25mmol/L EDTA, 0.5% SDS), and placing in 55 ℃ water bath for 1 h. After cooling to room temperature, the reaction mixture was cooled with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and chloroform: sequentially extracting isoamyl alcohol (24: 1), and adding equal volume of isopropanol into supernate for precipitation. And washing and drying the precipitate by 70% ethanol, dissolving the precipitate in sterilized purified water, and adjusting the DNA concentration to 50 ng/mu l to obtain a diseased root DNA template preparation solution for PCR detection.
3. PCR specific amplification of pathogenic nematodes: adding 2 μ l of diseased root DNA template preparation solution into 25 μ l of PCR reaction system containing 0.4 μ M each of 1 XPCR buffer, 1U Taq enzyme, 0.2mM dNTPs, and primers TR1, TR2, TR3, TR4, TR5 and TR 6; placing the mixture in a PCR instrument for amplification, wherein the optimized amplification conditions are as follows: pre-denaturation at 94 ℃ for 2 min; 30 cycles, each cycle comprising: 45s at 94 ℃, 60s at 52 ℃ and 90s at 72 ℃; finally, the extension is carried out for 10min at 72 ℃.
4. And (3) detecting a PCR product: adding 5 μ l of PCR amplification product into 2 μ l of 6 × electrophoresis sample buffer solution, and performing 1.5% agarose gel electrophoresis; after the electrophoresis is finished, the gel block is stained by ethidium bromide and observed under an ultraviolet transilluminator, and the result shows that a strip with the molecular weight of 570bp is amplified in the samples in the Luzhou county and the Yiyi county, which indicates that the detected sample contains the javanica; amplifying a band with the molecular weight of 700bp in samples in the Shanbei county, the teacher county, the gunn county, the Mongolian city, the screen county and the Bingchuan county, and indicating that the detected sample contains the southern root knot nematode; a band with the molecular weight of 260bp is amplified from samples of Jianchuan, inkstone county, Jianshui county and Maitreya city, and the detected samples contain the peanut root-knot nematode (figure 1).
Example 2
1. The design of specific primers for identifying three pathogenic nematodes of tobacco root knot nematode disease: according to the genome sequence information of the meloidogyne incognita, the meloidogyne arachidis and the meloidogyne javanica published by the National Center for Biotechnology Information (NCBI), DNASTAR 7.0 software is used for analysis to find out the species-specific sequences of three meloidogyne incognita, and primer Premier 5.0 software is used for designing 6 PCR primers for identifying the species of the three meloidogyne incognita:
TR1：5'-AAGCAAAAGACGAAGCACCAAAA-3'
TR2：5'-GAGGATTCAGCTCCCCAGCAC-3'
TR3：5'-GAGTACGGATTTGAAATACAGGG-3'
TR4：5'-TGAATGCTATGCCATCAGGAG-3'
TR5：5'-CGATTGAACTGAGCCCAGACT-3'
TR6：5'-TTTATTCGCAAGACAACACCC-3'
the 6 oligonucleotide primers were synthesized by a DNA synthesizer.
2. Preparing a DNA template for PCR identification of pathogenic nematodes of tobacco root knot nematode: collecting rhizosphere soil samples of tobacco root knot nematode diseases in the northern county and the Jianshui county of Yunnan province respectively, taking 200g of soil for each sample, separating soil nematodes by a conventional method, placing the obtained nematode suspension in an anatomical environment for observation, picking 2-3 suspected root knot nematode second-instar larvae or male adults in the soil into 10 mu l of 100 ng/mu proteinase K solution, cutting the nematodes by using a dissecting needle, heating at 95 ℃ for 10min after water bathing at 60 ℃ for 1h, and cooling to room temperature to obtain the soil DNA template preparation solution for PCR detection.
3. PCR specific amplification of pathogenic nematodes: adding 2 μ l of soil DNA template preparation solution into 25 μ l of PCR reaction system containing 0.4 μ M each of 1 XPCR buffer, 1U Taq enzyme, 0.2mM dNTPs, and primers TR1, TR2, TR3, TR4, TR5 and TR 6; placing the mixture in a PCR instrument for amplification, wherein the optimized amplification conditions are as follows: pre-denaturation at 94 ℃ for 2 min; 30 cycles, each cycle comprising: 45s at 94 ℃, 60s at 52 ℃ and 90s at 72 ℃; finally, the extension is carried out for 10min at 72 ℃.
4. And (3) detecting a PCR product: adding 5 μ l of PCR amplification product into 2 μ l of 6 × electrophoresis sample buffer solution, and performing 1.5% agarose gel electrophoresis; after the electrophoresis is finished, the gel block is stained by ethidium bromide and observed under an ultraviolet transilluminator, and the result shows that a band with the molecular weight of 700bp is amplified in a sample in the northern prefecture, which indicates that the detected sample contains the meloidogyne incognita, and a band with the molecular weight of 260bp is amplified in a sample in the Jiangchang, which indicates that the detected sample contains the meloidogyne incognita (figure 2).
SEQUENCE LISTING
<110> research institute of tobacco agricultural science in Yunnan province
<120> a synchronous detection and identification method for three main pathogenic nematodes in tobacco root knot nematode disease
<130> 2018
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> TR1
<400> 1
aagcaaaaga cgaagcacca aaa 23
<210> 2
<211> 21
<212> DNA
<213> TR2
<400> 2
gaggattcag ctccccagca c 21
<210> 3
<211> 23
<212> DNA
<213> TR3
<400> 3
gagtacggat ttgaaataca ggg 23
<210> 4
<211> 21
<212> DNA
<213> TR4
<400> 4
tgaatgctat gccatcagga g 21
<210> 5
<211> 21
<212> DNA
<213> TR5
<400> 5
cgattgaact gagcccagac t 21
<210> 6
<211> 21
<212> DNA
<213> TR6
<400> 6
tttattcgca agacaacacc c 21
Claims (4)
1. A synchronous detection and identification method for three main pathogenic nematodes in tobacco root knot nematode disease is characterized by comprising the steps of PCR primer design, DNA template preparation, PCR amplification and product detection, and specifically comprises the following steps:
A. designing a PCR primer: 6 PCR primers are designed according to species-specific sequences of Meloidogyne incognita, Meloidogyne arachidis and Meloidogyne javanica, namely TR1, TR2, TR3, TR4, TR5 and TR6 are used for PCR identification of the 3 Meloidogyne incognita, and the sequences are respectively:
TR1：5'-AAGCAAAAGACGAAGCACCAAAA-3'
TR2：5'-GAGGATTCAGCTCCCCAGCAC-3'
TR3：5'-GAGTACGGATTTGAAATACAGGG-3'
TR4：5'-TGAATGCTATGCCATCAGGAG-3'
TR5：5'-CGATTGAACTGAGCCCAGACT-3'
TR6：5'-TTTATTCGCAAGACAACACCC-3'；
B. preparing a DNA template:
when detecting tobacco diseased root samples: removing attachments on the surface of a diseased root sample, selecting a root-knot nematode egg mass or a root-knot tissue, placing the root-knot nematode egg mass or the root-knot tissue in a sterilized mortar, adding liquid nitrogen, grinding the root-knot nematode egg mass or the root-knot tissue into powder, transferring the powder into a centrifuge tube, adding a DNA extraction buffer solution, placing the centrifuge tube in a water bath at 50-60 ℃, keeping the temperature for 50-70 min, cooling to room temperature, sequentially extracting with a phenol-chloroform-isoamylol mixed solvent and a chloroform-isoamylol mixed solvent which have the same volume, taking supernate, adding isopropanol which has the same volume to precipitate, washing the precipitate with an ethanol solution with the volume percentage concentration of 60-80%, drying, dissolving the precipitate in sterilized purified water, and adjusting the concentration value of DNA to be 50 ng/mu l to obtain a DNA template preparation solution for PCR detection;
when the detection sample is tobacco rhizosphere soil: separating soil nematodes from a soil sample by a conventional method, placing the obtained nematode suspension under a dissecting mirror for observation, picking 2-3 to 10 mu l of 100 ng/mu proteinase K solution of the nematodes suspected to be root-knot nematodes, namely second-instar larvae or male adults, cutting the nematodes by using a dissecting needle, heating at 95 ℃ for 10min after 1h in water bath at 60 ℃, and cooling to room temperature to obtain a DNA template preparation solution for PCR detection;
C. and (3) PCR amplification: carrying out PCR amplification by using DNA template preparation solution for PCR detection to obtain a PCR amplification product; the PCR reaction system is a 25 mu l PCR reaction system with 1 × PCR buffer, 1U Taq enzyme, 0.2mM dNTPs and 0.4 mu M of each of primers TR1, TR2, TR3, TR4, TR5 and TR6, and the PCR reaction condition is pre-denaturation at 94 ℃ for 2 min; 30 cycles, each cycle comprising: 45s at 94 ℃, 60s at 52 ℃ and 90s at 72 ℃; finally, extending for 10min at 72 ℃;
D. and (3) product detection: and (3) carrying out 1.5% agarose gel electrophoresis on 5-10 mu l of PCR amplification product, dyeing by ethidium bromide after finishing the electrophoresis, and observing under an ultraviolet transmission instrument, wherein if an electrophoresis band with the molecular weight of 700bp is detected, the detected sample contains meloidogyne incognita, if an electrophoresis band with the molecular weight of 260bp is detected, the detected sample contains meloidogyne arachidis hypogaeae, and if an electrophoresis band with the molecular weight of 570bp is detected, the detected sample contains meloidogyne javanica.
2. The method for synchronously detecting and identifying three main pathogenic nematodes in tobacco Meloidogyne incognita according to claim 1, wherein the DNA extraction buffer in step B consists of 200mmol/L Tris-HCl pH 8.5, 250mmol/L NaCl, 25mmol/L EDTA, and 0.5% SDS.
3. The method for synchronously detecting and identifying three main pathogenic nematodes in tobacco meloidogyne disease according to claim 1, wherein the volume ratio of phenol, chloroform and isoamyl alcohol in the phenol-chloroform-isoamyl alcohol mixed solvent in the step B is 25:24: 1.
4. The method for synchronously detecting and identifying three main pathogenic nematodes in tobacco Meloidogyne incognita disease according to claim 1, wherein the volume ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol mixed solvent in the step B is 24: 1.
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