CN108300791A - A kind of synchronization Testing and appraisal method of three kinds of main pathogen nematodes in tobacco root-knot - Google Patents
A kind of synchronization Testing and appraisal method of three kinds of main pathogen nematodes in tobacco root-knot Download PDFInfo
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Abstract
The invention discloses a kind of synchronization Testing and appraisal methods of three kinds of main pathogen nematodes in tobacco root-knot.Including PCR primer design, DNA profiling preparation, PCR amplification, product detection step.The present invention devises the Identification of Species that above-mentioned 3 kinds of root-knot nematodes are used for comprising 6 PCR primers according to the special region sequence of type of Meloidogyne incognita, peanut root-knot nematode and javanese root knot nematode, and the stability of identification is good, accuracy is high；6 designed primers are applied in combination, and 1 PCR reaction may be implemented and complete the 3 synchronous detections for growing tobacco Pathogen of Root-knot Disease nematode, identification is convenient, time saving；Using regular-PCR instrument and conventional reagent, testing cost is conducive to promote and apply；The present invention includes the DNA preparation methods devised for old complaint sample and pedotheque, can carry out Identification of Species by object of the root-knot nematode of each stage of development including ovum, larva and adult, have extensive use scope.
Description
Technical field
The invention belongs to plant disease technical field of detection of pathogeny, and in particular to three kinds of masters in a kind of tobacco root-knot
Want the synchronization Testing and appraisal method of pathogenic nematode.
Background technology
Root-knot nematode（MeloidogyneSpp.）It is that most species in plant pathogeny line insect, distribution are most wide and harm is most tight
A kind of nematode of weight.This kind of nematode mainly in the form of soil-borne, endangers the root system of plant, and the root of aggrieved plant is made to be formed
The root knot of tumour shape, to seriously affect the root knot nematode disease of plant normal growth development, it is known that be critically ill by root-knot nematode
Harmful plant includes mainly more than 3000 kinds of Solanaceae, pulse family, Curcurbitaceae, grass family, Carrot family etc., in temperate zone, subtropical zone and heat
Hazard of plant with area is especially serious.It is wherein especially the most serious by root-knot nematode harm with tobacco, according to FAO (Food and Agriculture Organization of the United Nation)
Estimation, developed country's tobacco loss caused by root-knot nematode is every year on average 4%, and global average loss is estimated as 10%,
The loss of developing country is more than 25%.In China since middle and later periods the 1980s, tobacco root-knot lesion is continuous
Expand, harm aggravates, and more universal, heavier, the lesion of harm occurs in cigarette districts such as Henan, Shandong, Yunnan, Guizhou, Sichuan, Hubei
Incidence is generally 70%-100%, and severe one becomes the main disease on China's tobacco leaf production up to 90% or more, underproduction 30%-70%
Evil.
Root-knot nematode belongs to soil habitat biology, due to being limited physics, the chemistry that outside applies by soil environment
Factor is difficult to work to root-knot nematode, and especially in recent years, most chemical nematicides for root knot nematode disease prevention are deposited
High poison, the high residue the drawbacks of, worldwide generally disabled.Therefore safe and efficient root knot nematode disease prevention and control are taken
Method has become trend of the times.During the prevention and control of tobacco root-knot, selection is using suitable disease-resistant tobacco bred quilt
It is considered an economy, measure efficiently, green.However, root-knot nematode species are various, different root-knot nematodes are to host plant
Pathogenecity is different, and disease-resistant variety is also limited to the resistant range of root-knot nematode, is distributed most extensive harm on current country tobacco
The root-knot nematode of most serious is mainly peanut root-knot nematode（Meloidogyne arenaria）, Meloidogyne incognita（M. incognita）And javanese root knot nematode（M. javanica）, the form of these three root-knot nematodes and molecular biological characteristics pole
To be similar, but there are larger Difference in Pathogenicity to different tobacco breds.Therefore accurate, the above-mentioned three kinds of root-knot nematodes of Rapid identification
It is selection and breeding and the precondition using disease-resistant tobacco bred.
Identify that the most common method of root-knot nematode species is morphological method, this method cuts root by micromanipulation method
Tie lines worm female adult pest perineal region cutin membrane observes the difference of perineal region decorative pattern to determine the type of root-knot nematode under the microscope.
The advantages of this method be it is easy, intuitive, there are the drawbacks of be：1. root-knot nematode species are more, the perineal pattern of numerous species is special
Point is easier to obscure so that the accuracy of identification fully relies on the experience of operator, and the probability of erroneous judgement is higher；2. same
There are individual differences for the perineal pattern of the root-knot nematode of class, at least need to carry out 20 or more female adult pests in practical operation
Observation can just make a definite diagnosis, and take a lot of work, is time-consuming；3. Morphological Identification is only applicable to adult, it is not suitable for larva, and be separated in soil
Root-knot nematode is existed in the form of second instar larvae, and this method made is restricted in practical applications.In addition, isodynamic enzyme is electric
Swimming method is also common method in root-knot nematode identification, and this method carries out poly- third with the protein crude extract administration of root-knot nematode female adult pest
Acrylamide electrophoresis should by carrying out type judgement according to the spectral pattern of enzyme band after esterase and Chances of Malic Dehydrogenase Isoenzyme dyeing
The shortcomings that method part overcomes Morphological Identification can more fast and accurately judge root-knot nematode species, but still can only
It is used as identification object with female adult pest.
Recently as the development of Protocols in Molecular Biology, quick, precise Identification is carried out to root-knot nematode using round pcr
Have become trend.Compared with morphology and isozyme electrophoresis identification method, the PCR identification method scope of applications are more wide, root knot line
Ovum, second instar larvae and the female male imago of worm all can be used as the object of identification.The PCR identifications of early stage include being expanded using random primer
RAPD-PCR technologies or using the PCR-RFLP of restricted digestion is carried out to amplified production again after the amplification of nematode universal primer
Technology, their common drawback are to cause the stability of identification and accurate due to the use of the dedicated special primer of non-root-knot nematode
Property is not high.Therefore, domestic and foreign scholars are started then are expanded using root-knot nematode species Specific PCR primers to carry out root in recent years
The Identification of Species of tie lines worm.The inventions such as Xu Jianhua《A kind of root-knot nematode rapid molecular detection primer and usage thereof》（The patent No.：
ZL03131763.4）, once built quick equal inventions《One identification method for growing tobacco middle root-knot nematode》（The patent No.：
ZL201610585565.X）The new method of tobacco root-knot pathogenic nematode PCR identifications is provided, this method uses three pairs of roots
Knot line insect types special primer carries out PCR detections respectively to the root-knot nematode for being isolated from tobacco old complaint and soil, and identification is convenient, accurate
True rate is high, substantially increases the diagnosis efficiency of tobacco root-knot cause of disease.But as a result of substep PCR detection methods, at least
The identification of a sample could be completed by needing to carry out the PCR reactions above three times so that qualification process is cumbersome, time-consuming.
Therefore, a kind of Testing and appraisal method that can solve above-mentioned technical problem of exploitation is very important.
Invention content
The purpose of the present invention is to provide a kind of synchronous detection mirror of three kinds of main pathogen nematodes in tobacco root-knot
Determine method.
The object of the present invention is achieved like this, including PCR primer design, DNA profiling preparation, PCR amplification, product detection
Step specifically includes：
A, PCR primer designs：According to Meloidogyne incognita, peanut root-knot nematode and the species specificity sequence for grabbing root-knot nematode
6 PCR primers are designed, i.e. TR1, TR2, TR3, TR4, TR5 and TR6 is identified for above-mentioned 3 kinds of root-knot nematodes PCR, sequence point
It is not：
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, prepared by DNA profiling：
When detecting tobacco old complaint sample：The attachment of old complaint sample surfaces is removed, picking root-knot nematode pieces of an egg or root knot group
It knits in the mortar after being placed in sterilizing, liquid nitrogen grinding is added and is transferred in centrifuge tube at powder, DNA Extraction buffers are added, it is placed in 50 ~
60 DEG C of water-baths keep the temperature 50 ~ 70min, are cooled to room temperature, with isometric phenol-chloroform-isoamyl alcohol mixed solvent and chloroform-isoamyl alcohol
Mixed solvent extracts successively, and supernatant is taken to add isometric isopropanol precipitating, precipitates the ethyl alcohol through concentration expressed in percentage by volume 60 ~ 80%
It after solution is cleaned and dried, is dissolved in sterile purified water, adjusts DNA concentration value 50ng/ μ l, obtain the DNA moulds detected for PCR
Plate preparation solution；
When it is tobacco rhizosphere soil to detect sample：It takes pedotheque to carry out soil nematodes separation using conventional method, will obtain
Nematode suspension be placed in dissection under the microscope, the nematode 2 ~ 3 of the wherein doubtful root-knot nematode second instar larvae of picking or male imago
Into the Proteinase K Solution of the 100ng/ μ of 10 μ l, nematode is cut off with dissecting needle, 95 DEG C of heating 10min after 60 DEG C of water-bath 1h,
It is cooled to room temperature up to the DNA profiling preparation solution for PCR detections；
C, PCR amplification：Pcr amplification product is obtained for the DNA profiling preparation solution progress PCR amplification of PCR detections；
D, product detection：The pcr amplification product of 5 ~ 10 μ l is taken to carry out 1.5% agarose gel electrophoresis electrophoresis, through bromination after terminating
It is observed under ultraviolet transilluminator again after the dyeing of second ingot, such as detects that molecular weight is that the electrophoretic band of 700bp shows to contain in by sample product
There is Meloidogyne incognita, such as detects that molecular weight is that the electrophoretic band of 260bp shows to be contained peanut root-knot nematode by sample product, such as
Detect that molecular weight is that the electrophoretic band of 570bp shows to contain javanese root knot nematode in by sample product.
Compared with prior art, the present invention its advantage and good effect are shown：
1, the present invention is designed according to the special region sequence of type of Meloidogyne incognita, peanut root-knot nematode and javanese root knot nematode
It is used for the Identification of Species of above-mentioned 3 kinds of root-knot nematodes comprising 6 PCR primers, the stability of identification is good, accuracy is high.
2,6 primers designed by the present invention are applied in combination, and 1 PCR reaction may be implemented complete 3 to grow tobacco root-knot nematode
The synchronous detection of sick pathogenic nematode, identification are convenient, time saving.
3, the present invention uses regular-PCR instrument and conventional reagent, testing cost to be conducive to promote and apply.
4, the present invention includes the DNA preparation methods devised for old complaint sample and pedotheque, can be to include ovum, children
The root-knot nematode of each stage of development including worm and adult is that object carries out Identification of Species, has extensive use scope.
Description of the drawings
Fig. 1 is the PCR amplification result schematic diagram of tobacco Root-knot old complaint sample of the present invention；
Wherein, M is DNA molecular amount standard（DL2000）, 1-12, which is followed successively by, picks up from the counties Lu Quan of Yunnan Province, Zhanyi County, Jiangchuan County, mound
Bei Xian, Shizong County, the counties Geng Ma, Mengzi city, the counties Ping Bian, Binchuan County, Yanshan County, Jianshui County and Muller city tobacco root-knot
Old complaint sample；
Fig. 2 is the PCR amplification result schematic diagram of tobacco root-knot rhizosphere soil sample of the present invention；
Wherein, M is DNA molecular amount standard（DL2000）, 1-2 is respectively the tobacco root knot for picking up from Yunnan Province Qiubei County and Jiangchuan County
Nematodiasis Rhizosphere Soil sample.
Specific implementation mode
With reference to embodiment and attached drawing, the present invention is further illustrated, but is not subject in any way to the present invention
Limitation, based on present invention teach that made by it is any transform or replace, all belong to the scope of protection of the present invention.
The synchronization Testing and appraisal method of three kinds of main pathogen nematodes in tobacco root-knot of the present invention, including
PCR primer design, DNA profiling preparation, PCR amplification, product detection step, specifically include：
A, PCR primer designs：According to Meloidogyne incognita, peanut root-knot nematode and the species specificity sequence for grabbing root-knot nematode
6 PCR primers are designed, i.e. TR1, TR2, TR3, TR4, TR5 and TR6 is identified for above-mentioned 3 kinds of root-knot nematodes PCR, sequence point
It is not：
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, prepared by DNA profiling：
When detecting tobacco old complaint sample：The attachment of old complaint sample surfaces is removed, picking root-knot nematode pieces of an egg or root knot group
It knits in the mortar after being placed in sterilizing, liquid nitrogen grinding is added and is transferred in centrifuge tube at powder, DNA Extraction buffers are added, it is placed in 50 ~
60 DEG C of water-baths keep the temperature 50 ~ 70min, are cooled to room temperature, with isometric phenol-chloroform-isoamyl alcohol mixed solvent and chloroform-isoamyl alcohol
Mixed solvent extracts successively, and supernatant is taken to add isometric isopropanol precipitating, precipitates the ethyl alcohol through concentration expressed in percentage by volume 60 ~ 80%
It after solution is cleaned and dried, is dissolved in sterile purified water, adjusts DNA concentration value 50ng/ μ l, obtain the DNA moulds detected for PCR
Plate preparation solution；
When it is tobacco rhizosphere soil to detect sample：It takes pedotheque to carry out soil nematodes separation using conventional method, will obtain
Nematode suspension be placed in dissection under the microscope, the nematode 2 ~ 3 of the wherein doubtful root-knot nematode second instar larvae of picking or male imago
Into the Proteinase K Solution of the 100ng/ μ of 10 μ l, nematode is cut off with dissecting needle, 95 DEG C of heating 10min after 60 DEG C of water-bath 1h,
It is cooled to room temperature up to the DNA profiling preparation solution for PCR detections；
C, PCR amplification：Pcr amplification product is obtained for the DNA profiling preparation solution progress PCR amplification of PCR detections；
D, product detection：The pcr amplification product of 5 ~ 10 μ l is taken to carry out 1.5% agarose gel electrophoresis electrophoresis, through bromination after terminating
It is observed under ultraviolet transilluminator again after the dyeing of second ingot, such as detects that molecular weight is that the electrophoretic band of 700bp shows to contain in by sample product
There is Meloidogyne incognita, such as detects that molecular weight is that the electrophoretic band of 260bp shows to be contained peanut root-knot nematode by sample product, such as
Detect that molecular weight is that the electrophoretic band of 570bp shows to contain javanese root knot nematode in by sample product.
DNA Extraction buffers described in step B are 200mmol/L Tris-HCl pH=8.5,250mmol/L
NaCl, 25mmol/L EDTA, 0.5%SDS composition.
The volume proportion of phenol-chloroform-isoamyl alcohol in the mixed solvent phenol, chloroform and isoamyl alcohol described in step B is 25:
24:1。
The volume proportion of chloroform-isoamyl alcohol in the mixed solvent chloroform and isoamyl alcohol described in step B is 24:1.
PCR reaction systems are 1 × PCR buffer, 1U Taq enzymes in step C, 0.2mM dNTPs and primer TR1,
The 25 μ l PCR reaction systems of each 0.4 μM of TR2, TR3, TR4, TR5 and TR6.
PCR reaction conditions are 94 DEG C of 2min of pre-degeneration in step C；30 cycles, each cycle include：94 DEG C of 45s, 52
DEG C 60s, 72 DEG C of 90s；Last 72 DEG C of extension 10min again.
The synchronization Testing and appraisal method of three kinds of main pathogen nematodes is specifically grasped in tobacco root-knot of the present invention
Make as follows：
（1）According to 6 PCR of species specificity sequence design of Meloidogyne incognita, peanut root-knot nematode and javanese root knot nematode
Primer, i.e. TR1, TR2, TR3, TR4, TR5 and TR6 identify that sequence is respectively for above-mentioned 3 kinds of root-knot nematodes PCR：
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）It is prepared by the DNA profiling for the PCR detections of tobacco root-knot pathogenic nematode, including：
When detecting tobacco old complaint sample, the impurity such as the soil of the attachment of old complaint sample surfaces are rinsed well with tap water, and
Surface moisture is blotted with filter paper, and anatomical lens is with choosing needle picking root-knot nematode pieces of an egg, or is directly cut root knot part with scissors, claims
It takes the pieces of an egg of 0.2g or root knot tissue to be placed in and is transferred to 1.5ml's at powdered through liquid nitrogen grinding in autoclaved mortar, is added
In centrifuge tube, 0.5ml DNA Extraction buffers are added（200mmol/L Tris-HCl pH=8.5,250mmol/L NaCl,
25mmol/L EDTA, 0.5%SDS）, it is placed in 55 DEG C of water-baths and keeps the temperature 1h.After being cooled to room temperature, with isometric phenol：Chloroform：
Isoamyl alcohol（25:24:1）And chloroform：Isoamyl alcohol（24:1）It extracts successively, supernatant is taken to add isometric isopropanol precipitating.Precipitation warp
It after 70% ethyl alcohol is cleaned and dried, is dissolved in sterile purified water, adjusts DNA concentration to 50ng/ μ l, obtain detecting for PCR
DNA profiling preparation solution.
When it is tobacco rhizosphere soil to detect sample, 200g pedotheques is first taken to carry out soil nematodes point using conventional method
From, obtained nematode suspension is placed under dissection border and is observed, the wherein doubtful root-knot nematode second instar larvae of picking or male imago
In nematode 2-3 items to the Proteinase K Solution of the 100ng/ μ of 10 μ l, nematode is cut off with dissecting needle, is added for 95 DEG C after 60 DEG C of water-bath 1h
Hot 10min is cooled to room temperature up to the DNA profiling preparation solution for PCR detections.
（3）PCR amplification：Take 2 μ l in step（2）The buffer containing 1 × PCR, 1U is added in obtained DNA profiling preparation solution
The 25 μ l PCR reaction systems of Taq enzyme, 0.2mM dNTPs and each 0.4 μM of primer TR1, TR2, TR3, TR4, TR5 and TR6
In；It is placed in PCR instrument and is expanded, optimized amplification condition is：94 DEG C of 2min of pre-degeneration；30 cycles, each cycle packet
It includes：94 DEG C of 45s, 52 DEG C of 60s, 72 DEG C of 90s；Last 72 DEG C of extension 10min again.
（4）PCR product detects：Take 5~10 μ l steps（3）Obtained pcr amplification product carries out 1.5% Ago-Gel
Electrophoresis, Ago-Gel terminate to observe under ultraviolet transilluminator after ethidium bromide staining in electrophoresis, such as detect that molecular weight is
The electrophoretic band of 700bp shows to contain Meloidogyne incognita in by sample product, such as detects that molecular weight is the electrophoretic band of 260bp
Show to be contained peanut root-knot nematode by sample product, such as detects that molecular weight is that the electrophoretic band of 570bp shows to contain in by sample product
There is javanese root knot nematode.
Case is embodied, the present invention will be further described below：
Embodiment 1
1. for three grow tobacco Pathogen of Root-knot Disease nematode analysis special primer design：According to American National biology skill
Art information centre（NCBI）Meloidogyne incognita, peanut root-knot nematode and the javanese root knot nematode Genomic sequence information of announcement,
It is analyzed with 7.0 softwares of DNASTAR, finds out the species specificity sequence of three kinds of root-knot nematodes, utilize primer Premier 5.0
PCR primer of the Software for Design 6 for the identification of three kinds of root-knot nematode species：
TR1：5'-AAGCAAAAGACGAAGCACCAAAA-3'
TR2：5'-GAGGATTCAGCTCCCCAGCAC-3'
TR3：5'-GAGTACGGATTTGAAATACAGGG-3'
TR4：5'-TGAATGCTATGCCATCAGGAG-3'
TR5：5'-CGATTGAACTGAGCCCAGACT-3'
TR6：5'-TTTATTCGCAAGACAACACCC-3'
DNA synthesizer synthesizes above-mentioned 6 oligonucleotide primers.
2. prepared by the DNA profiling for the PCR identifications of tobacco root-knot pathogenic nematode：Yunnan Province's Lu is picked up from respectively to advise
County, Zhanyi County, Jiangchuan County, Qiubei County, Shizong County, the counties Geng Ma, Mengzi city, the counties Ping Bian, Binchuan County, Yanshan County, Jianshui County and more
The tobacco root-knot old complaint sample for strangling city, the impurity such as the soil of the attachment on old complaint surface are rinsed well with tap water, and
Surface moisture is blotted with filter paper, and anatomical lens is with choosing needle picking root-knot nematode pieces of an egg, or is directly cut root knot part with scissors, claims
It takes the pieces of an egg of 0.2g or root knot tissue to be placed in and is transferred to 1.5ml's at powdered through liquid nitrogen grinding in autoclaved mortar, is added
In centrifuge tube, 0.5ml DNA Extraction buffers are added（200mmol/L Tris-HCl pH=8.5,250mmol/L NaCl,
25mmol/L EDTA, 0.5%SDS）, it is placed in 55 DEG C of water-baths and keeps the temperature 1h.After being cooled to room temperature, with isometric phenol：Chloroform：
Isoamyl alcohol（25:24:1）And chloroform：Isoamyl alcohol（24:1）It extracts successively, supernatant is taken to add isometric isopropanol precipitating.Precipitation warp
It after 70% ethyl alcohol is cleaned and dried, is dissolved in sterile purified water, adjusts DNA concentration to 50ng/ μ l, obtain the disease detected for PCR
Root DNA profiling preparation solution.
3. the PCR specific amplifieds of pathogenic nematode：Take 2 μ l old complaint DNA profiling preparation solutions that the buffer containing 1 × PCR is added,
The 25 μ l PCR reactants of 1U Taq enzymes, 0.2mM dNTPs and each 0.4 μM of primer TR1, TR2, TR3, TR4, TR5 and TR6
In system；It is placed in PCR instrument and is expanded, optimized amplification condition is：94 DEG C of 2min of pre-degeneration；30 cycles, it is each to recycle
Including：94 DEG C of 45s, 52 DEG C of 60s, 72 DEG C of 90s；Last 72 DEG C of extension 10min again.
4. PCR product detects：It takes the pcr amplification product of 5 μ l that 2 μ l 6 × electrophoresis sample-loading buffers are added, carries out 1.5%
Agarose gel electrophoresis；It is observed under ultraviolet transilluminator after gel piece ethidium bromide staining after electrophoresis, as a result, it has been found that Lu
The band for amplifying that a molecular weight is 570bp in the sample of county, Zhanyi County is advised, shows the line of root knot containing Java in examined samples
Worm；Qiubei County, Shizong County, the counties Geng Ma, Mengzi city, the counties Ping Bian and Binchuan County sample in amplify a molecular weight be 700bp
Band, show to contain Meloidogyne incognita in examined samples；It is expanded in river and mountain, Yanshan County, Jianshui County and the sample in Muller city
Go out the band that a molecular weight is 260bp, shows to contain peanut root-knot nematode (Fig. 1) in by sample product.
Embodiment 2
1. for three grow tobacco Pathogen of Root-knot Disease nematode analysis special primer design：According to American National biology skill
Art information centre（NCBI）Meloidogyne incognita, peanut root-knot nematode and the javanese root knot nematode Genomic sequence information of announcement,
It is analyzed with 7.0 softwares of DNASTAR, finds out the species specificity sequence of three kinds of root-knot nematodes, utilize primer Premier 5.0
PCR primer of the Software for Design 6 for the identification of three kinds of root-knot nematode species：
TR1：5'-AAGCAAAAGACGAAGCACCAAAA-3'
TR2：5'-GAGGATTCAGCTCCCCAGCAC-3'
TR3：5'-GAGTACGGATTTGAAATACAGGG-3'
TR4：5'-TGAATGCTATGCCATCAGGAG-3'
TR5：5'-CGATTGAACTGAGCCCAGACT-3'
TR6：5'-TTTATTCGCAAGACAACACCC-3'
DNA synthesizer synthesizes above-mentioned 6 oligonucleotide primers.
2. prepared by the DNA profiling for the PCR identifications of tobacco root-knot pathogenic nematode：Yunnan Province Qiubei is acquired respectively
The rhizosphere soil sample of the tobacco root-knot in county and Jianshui County, fetch earth earth 200g per sample, is carried out using conventional method
Soil nematodes detaches, and obtained nematode suspension is placed under dissection border and is observed, the wherein doubtful root-knot nematode second instar larvae of picking
Or in the nematode 2-3 items of male imago to the Proteinase K Solution of the 100ng/ μ of 10 μ l, nematode is cut off with dissecting needle, 60 DEG C of water-baths
95 DEG C of heating 10min, are cooled to room temperature up to the soil DNA template preparation solution for PCR detections after 1h.
3. the PCR specific amplifieds of pathogenic nematode：Take 2 μ l soil DNA template preparation solutions that the buffer containing 1 × PCR is added,
The 25 μ l PCR reactants of 1U Taq enzymes, 0.2mM dNTPs and each 0.4 μM of primer TR1, TR2, TR3, TR4, TR5 and TR6
In system；It is placed in PCR instrument and is expanded, optimized amplification condition is：94 DEG C of 2min of pre-degeneration；30 cycles, it is each to recycle
Including：94 DEG C of 45s, 52 DEG C of 60s, 72 DEG C of 90s；Last 72 DEG C of extension 10min again.
4. PCR product detects：It takes the pcr amplification product of 5 μ l that 2 μ l 6 × electrophoresis sample-loading buffers are added, carries out 1.5%
Agarose gel electrophoresis；It is observed under ultraviolet transilluminator after gel piece ethidium bromide staining after electrophoresis, as a result, it has been found that mound
The band that a molecular weight is 700bp is amplified in the sample of Bei Xian, shows to contain Meloidogyne incognita in examined samples, river and mountain
Sample in amplify a molecular weight be 260bp band, show in by sample product contain peanut root-knot nematode (Fig. 2).
SEQUENCE LISTING
<110>Yunnan Academy of Tobacco Agricultural Science
<120>A kind of synchronization Testing and appraisal method of three kinds of main pathogen nematodes in tobacco root-knot
<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 (6)
1. a kind of synchronization Testing and appraisal method of three kinds of main pathogen nematodes in tobacco root-knot, it is characterised in that including
PCR primer design, DNA profiling preparation, PCR amplification, product detection step, specifically include：
A, PCR primer designs：According to Meloidogyne incognita, peanut root-knot nematode and the species specificity sequence for grabbing root-knot nematode
6 PCR primers are designed, i.e. TR1, TR2, TR3, TR4, TR5 and TR6 is identified for above-mentioned 3 kinds of root-knot nematodes PCR, sequence point
It is not：
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, prepared by DNA profiling：
When detecting tobacco old complaint sample：The attachment of old complaint sample surfaces is removed, picking root-knot nematode pieces of an egg or root knot group
It knits in the mortar after being placed in sterilizing, liquid nitrogen grinding is added and is transferred in centrifuge tube at powder, DNA Extraction buffers are added, it is placed in 50 ~
60 DEG C of water-baths keep the temperature 50 ~ 70min, are cooled to room temperature, with isometric phenol-chloroform-isoamyl alcohol mixed solvent and chloroform-isoamyl alcohol
Mixed solvent extracts successively, and supernatant is taken to add isometric isopropanol precipitating, precipitates the ethyl alcohol through concentration expressed in percentage by volume 60 ~ 80%
It after solution is cleaned and dried, is dissolved in sterile purified water, adjusts DNA concentration value 50ng/ μ l, obtain the DNA moulds detected for PCR
Plate preparation solution；
When it is tobacco rhizosphere soil to detect sample：It takes pedotheque to carry out soil nematodes separation using conventional method, will obtain
Nematode suspension be placed in dissection under the microscope, the nematode 2 ~ 3 of the wherein doubtful root-knot nematode second instar larvae of picking or male imago
Into the Proteinase K Solution of the 100ng/ μ of 10 μ l, nematode is cut off with dissecting needle, 95 DEG C of heating 10min after 60 DEG C of water-bath 1h,
It is cooled to room temperature up to the DNA profiling preparation solution for PCR detections；
C, PCR amplification：Pcr amplification product is obtained for the DNA profiling preparation solution progress PCR amplification of PCR detections；
D, product detection：The pcr amplification product of 5 ~ 10 μ l is taken to carry out 1.5% agarose gel electrophoresis electrophoresis, through bromination after terminating
It is observed under ultraviolet transilluminator again after the dyeing of second ingot, such as detects that molecular weight is that the electrophoretic band of 700bp shows to contain in by sample product
There is Meloidogyne incognita, such as detects that molecular weight is that the electrophoretic band of 260bp shows to be contained peanut root-knot nematode by sample product, such as
Detect that molecular weight is that the electrophoretic band of 570bp shows to contain javanese root knot nematode in by sample product.
2. the synchronization Testing and appraisal method of three kinds of main pathogen nematodes in tobacco root-knot according to claim 1,
It is characterized in that the DNA Extraction buffers described in step B are 200mmol/L Tris-HCl pH=8.5,250mmol/L
NaCl, 25mmol/L EDTA, 0.5%SDS composition.
3. the synchronization Testing and appraisal method of three kinds of main pathogen nematodes in tobacco root-knot according to claim 1,
It is characterized in that the volume proportion of phenol-chloroform-isoamyl alcohol in the mixed solvent phenol, chloroform and isoamyl alcohol described in step B is 25:
24:1。
4. the synchronization Testing and appraisal method of three kinds of main pathogen nematodes in tobacco root-knot according to claim 1,
It is characterized in that the volume proportion of the chloroform-isoamyl alcohol in the mixed solvent chloroform and isoamyl alcohol described in step B is 24:1.
5. the synchronization Testing and appraisal method of three kinds of main pathogen nematodes in tobacco root-knot according to claim 1,
It is characterized in that PCR reaction systems are 1 × PCR buffer, 1U Taq enzymes in step C, 0.2mM dNTPs and primer TR1,
The 25 μ l PCR reaction systems of each 0.4 μM of TR2, TR3, TR4, TR5 and TR6.
6. the synchronization Testing and appraisal method of three kinds of main pathogen nematodes in tobacco root-knot according to claim 1,
It is characterized in that PCR reaction conditions are 94 DEG C of 2min of pre-degeneration in step C；30 cycles, each cycle include：94 DEG C of 45s,
52 DEG C of 60s, 72 DEG C of 90s；Last 72 DEG C of extension 10min again.
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| AU3081500A (en) * | 1998-12-30 | 2000-07-24 | Stichting Dienst Landbouwkundig Onderzoek | Dna oligonucleotide which is specific for meloidogyne species, dna vector and host cell containing the oligonucleotide, use and kit |
| CN1482254A (en) * | 2003-07-28 | 2004-03-17 | 南京农业大学 | Primer and method for rapid molecular detection of eelworm |
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