CN102102100B - Method for assisting in identifying root-knot nematodes and special primer pair thereof - Google Patents
Method for assisting in identifying root-knot nematodes and special primer pair thereof Download PDFInfo
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- CN102102100B CN102102100B CN 201010175658 CN201010175658A CN102102100B CN 102102100 B CN102102100 B CN 102102100B CN 201010175658 CN201010175658 CN 201010175658 CN 201010175658 A CN201010175658 A CN 201010175658A CN 102102100 B CN102102100 B CN 102102100B
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Abstract
The invention discloses a method for assisting in identifying root-knot nematodes and a special primer pair thereof. The invention provides a special primer consisting of a primer pair A and a primer pair B, wherein the primer pair A consists of DNA (Deoxyribose Nucleic Acid) shown as a sequence 1 in a sequence table and DNA shown as a sequence 2 in the sequence table; the primer pair B consists of DNA shown as a sequence 3 in the sequence table and DNA shown as a sequence 4 in the sequence table; and the special primer can be used for assisting in identifying the root-knot nematodes. The method provided by the invention can be used for correctly distinguishing four common root-knot nematodes, has high sensitivity, and can be used for identifying the types of root-knot nematodes by using a single egg capsule or a single second stage juvenile. The root-knot nematode can be directly separated from diseased tissues without purification culturing, the purification culturing time of the nematodes can be greatly shortened, and the detection efficiency can be improved.
Description
Technical field
The method and the primer special thereof that the present invention relates to a kind of auxiliary discriminating root knot nematode are right.
Background technology
The plant nematode disease is the Plant diseases that generally takes place in a kind of world wide, endangers 3000 various plants.The annual loss that causes because of nematode in the whole world is up to more than 1,000 hundred million dollars.The plant root-knot nematode disease takes place more extensive, especially more serious at the southern area hazard ratio in China, like banana root knot nematode, watermelon root knot nematode, capsicum root knot nematode.Owing to have significant pathogenic difference between the root knot nematode kind; The application-dependent of success such as crop resistance and Plant Quarantine is in the root knot nematode kind is identified fast and accurately; Thereby the authentication method of taking to classify reliably is the basis of plant nematode diseases control.
At present; Chinese scholars often utilizes methods such as mtDNA-RFLP, rDNA-ITS and SCAR to identify the kind of root knot nematodes, however the purpose that can not reach fast, accurately identify because of its separately limitation (mtDNA-RFLP consuming time long, the rDNA-ITS tolerance range is not high, SCAR sensitivity is not strong).
Summary of the invention
The method and the primer special thereof that the purpose of this invention is to provide a kind of auxiliary discriminating root knot nematode are right.
The present invention protects the primer pair B that DNA forms shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table.Said primer pair B can be used for the auxiliary Meloidogyne incognita of differentiating.
The present invention also protects a kind of method of auxiliary discriminating Meloidogyne incognita, comprises the steps:
(1) genomic dna of extraction root knot nematode to be measured;
(2) be template with the genomic dna, carry out pcr amplification with said primer pair B; If obtain pcr amplification product, root knot nematode to be measured is candidate's a Meloidogyne incognita; If do not obtain pcr amplification product, root knot nematode to be measured is candidate's a non-Meloidogyne incognita.Said pcr amplification product specifically can be 400bp-600bp.
The present invention also protects the primer special of primer to first and primer pair B composition; Said primer is made up of DNA shown in the sequence 2 of DNA shown in the sequence 1 of sequence table and sequence table first; Said primer pair B is made up of DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table.Said primer special can be used for the auxiliary root knot nematode of differentiating.
The present invention also protects the method for a kind of auxiliary discriminating root knot nematode, comprises the steps:
(1) genomic dna of extraction root knot nematode to be measured;
(2) be template with the genomic dna, carry out pcr amplification with said primer special;
When carrying out pcr amplification with said primer pair B: if obtain pcr amplification product, root knot nematode to be measured is candidate's a Meloidogyne incognita; If do not obtain pcr amplification product, root knot nematode to be measured is candidate's a non-Meloidogyne incognita;
When with said primer first being carried out pcr amplification: if obtain the pcr amplification product of 1.6kb-1.8kb, root knot nematode to be measured is candidate's Meloidogyne incognita or a javanese root knot nematode; If obtain the pcr amplification product of 1.0kb-1.2kb, root knot nematode to be measured is candidate's a peanut root-knot nematode; If obtain the pcr amplification product of 0.4kb-0.6kb, root knot nematode to be measured is candidate's a northern root knot nematode.
Carry out the pcr amplification product that pcr amplification obtains with said primer pair B and specifically can be 400bp-600bp.
The present invention also protects DNA forms shown in the sequence 2 of DNA shown in the sequence 1 of sequence table and sequence table primer to first.Said primer can be used for the auxiliary root knot nematode of differentiating to first.
The invention provides the another kind of auxiliary method of differentiating root knot nematode, comprise the steps:
(1) genomic dna of extraction root knot nematode to be measured;
(2) be template with the genomic dna, first carried out pcr amplification with said primer; If obtain the pcr amplification product of 1.6kb-1.8kb, root knot nematode to be measured is candidate's Meloidogyne incognita or a javanese root knot nematode; If obtain the pcr amplification product of 1.0kb-1.2kb, root knot nematode to be measured is candidate's a peanut root-knot nematode; If obtain the pcr amplification product of 0.4kb-0.6kb, root knot nematode to be measured is candidate's a northern root knot nematode.
More than in the method for arbitrary said auxiliary discriminating root knot nematode:
Said root knot nematode to be measured is Meloidogyne incognita, javanese root knot nematode, peanut root-knot nematode or northern root knot nematode.
Said genomic dna is taken from adult, larva or egg capsule.
Take from the used primer of adult or larva, said PCR to for said primer during to first when genomic dna, the response procedures of said PCR is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 1min, 48 ℃ of annealing 1min, 72 ℃ of extension 2min carry out 40 circulations; Last 72 ℃ of insulation 5min.Take from the used primer of egg capsule, said PCR to for said primer during to first when genomic dna, the response procedures of said PCR is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 1min, 48 ℃ of annealing 1min, 72 ℃ of extension 2min carry out 35 circulations; Last 72 ℃ of insulation 5min.
Take from the used primer of adult or larva, said PCR to for said primer pair B the time when genomic dna, the response procedures of said PCR is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 45S, 58 ℃ of annealing 45S, 72 ℃ of extension 45S carry out 40 circulations; Last 72 ℃ of insulation 5min.Take from the used primer of egg capsule, said PCR to for said primer pair B the time when genomic dna, the response procedures of said PCR is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 45S, 58 ℃ of annealing 45S, 72 ℃ of extension 45S carry out 35 circulations; Last 72 ℃ of insulation 5min;
More than arbitrary said method all can be used for the assistant identification root knot nematode plant that catches an illness.
When using primer of the present invention to assistant identification, genomic dna both can have been taken from nematode adult, nematode larval, nematode egg capsule, also can take from the root knot (root nodule) of the plant that infects root knot nematode.
Can not identify the deficiency of root knot nematode quickly and accurately in order to overcome existing method, this patent provides a kind of method of identifying root knot nematode, and this PCR method can not only accurately be distinguished four kinds of common root knot nematodes; And it is highly sensitive; Single egg capsule capable of using or wall scroll second instar larvae are identified the root knot nematode kind, can directly from diseased tissues, separate to obtain, and need not purifying and cultivate; Can shorten nematode purifying incubation time greatly, improve detection efficiency.
Description of drawings
Fig. 1 adopts the electrophorogram of primer to the pcr amplification product of first among the embodiment 2.
Fig. 2 is the electrophorogram that adopts the pcr amplification product of primer pair B among the embodiment 2.
Fig. 3 adopts the electrophorogram of primer to the pcr amplification product of first among the embodiment 5.
Fig. 4 is the electrophorogram that adopts the pcr amplification product of primer pair B among the embodiment 5.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment like no specified otherwise, is the quality percentage composition.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
8 root knot nematode populations: 5 Meloidogyne incognita populations (separation) from 5 infected plants; 1 javanese root knot nematode population; 1 peanut root-knot nematode population; 1 northern root knot nematode population.
The preparation of embodiment 1, primer
Following four primers of synthetic:
#C2F3:5 '-GGTCAA TGTCAGAAA TTTGTGG-3 '; Sequence 1;
#1108:5 '-TACCTTTGACCAA TCACGCT-3 '; Sequence 2;
MI-F:5 '-GGGCAAGTAAGGATGCTCTGAC-3 '; Sequence 3;
MI-R (5 '-CTTTCATAGCCACGTCGCGATC-3 '; Sequence 4.
The primer that #C2F3 and #1108 form to as primer to first, the primer of MI-F and MI-R composition is to as primer pair B.Primer to first to zone between CO II in the Mitochondrial DNA (mtDNA) and LrRNA.Primer pair B is to the esophageal gland protein gene.
One, with primer first is assisted the discriminating root knot nematode
1, extracts the genomic dna of wall scroll root knot nematode (adult).
2, be that template is carried out the PCR reaction with the genomic dna, obtain pcr amplification product.
PCR reaction system (25 μ L): 17.1 μ l ddH20; 2.5 μ l 10 * buffer; 2 μ l dNTP (2.5mM); 1 μ l primer #C2F3 (20 μ M); 1 μ l primer #1108 (20 μ M); 0.4 μ l Taq enzyme (5U/ μ l); 1 μ l template.
PCR response procedures: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 1min, 48 ℃ of annealing 1min, 72 ℃ of extension 2min carry out 40 circulations; Last 72 ℃ of insulation 5min.
3, pcr amplification product carries out 1% (containing 0.5 μ g/mL EB) agarose gel electrophoresis, and electrophoretic buffer is 0.5 * TBE, and voltage is 80V, the about 30min of electrophoresis.In gel images system (Tanon 4500, day can scientific and technological (Shanghai) Co., Ltd.), take pictures and preserve, and carry software (day ability GIS gel images treatment system with system; Edition 4 .00, software registration number: 0003952) stripe size is analyzed.
The result sees Fig. 1.Among Fig. 1: M: standard molecular weight; 1-5: Meloidogyne incognita; 6: javanese root knot nematode; 7: peanut root-knot nematode; 8: northern root knot nematode; 9:CK (not adding template).The stripe size analytical results: the pcr amplification product of Meloidogyne incognita and javanese root knot nematode is about 1.7kb (between the 1.6kb-1.8kb); The pcr amplification product of peanut root-knot nematode is about 1.1kb (between the 1.0kb-1.2kb), and the pcr amplification product of northern root knot nematode is about 0.5kb (between the 0.4kb-0.6kb).
Two, with the auxiliary discriminating of primer pair B root knot nematode adult
1, extracts the genomic dna of wall scroll root knot nematode (adult).
2, be that template is carried out the PCR reaction with the genomic dna, obtain pcr amplification product.
PCR reaction system (25 μ L): 17.1 μ l ddH20; 2.5 μ l 10 * buffer; 2 μ l dNTP (2.5mM); 1 μ l primer MI-F (20 μ M); 1 μ l primer MI-R (20 μ M); 0.4 μ l Taq enzyme (5U/ μ l); 1 μ l template.
PCR response procedures: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 45S, 58 ℃ of annealing 45S, 72 ℃ of extension 45S carry out 40 circulations; Last 72 ℃ of insulation 5min.
3, pcr amplification product carries out 1% (containing 0.5 μ g/mL EB) agarose gel electrophoresis, and electrophoretic buffer is 0.5 * TBE, and voltage is 80V, the about 30min of electrophoresis.In gel images system (Tanon 4500, day can scientific and technological (Shanghai) Co., Ltd.), take pictures and preserve, and carry software (day ability GIS gel images treatment system with system; Edition 4 .00, software registration number: 0003952) stripe size is analyzed.
The result sees Fig. 2.Among Fig. 2: M: standard molecular weight; 1-5: Meloidogyne incognita; 6: javanese root knot nematode; 7: peanut root-knot nematode; 8: northern root knot nematode; 9:CK (not adding template).Except Meloidogyne incognita, other three kinds of root knot nematodes all do not obtain pcr amplification product.The stripe size analytical results: the pcr amplification product of Meloidogyne incognita is about 500bp (between the 400b-600b).
One, with primer first is assisted the discriminating root knot nematode
1, extracts the genomic dna of wall scroll root knot nematode (2 instar larvae).
2, be that template is carried out the PCR reaction with the genomic dna, obtain pcr amplification product.
PCR reaction system (25 μ L): 17.1 μ l ddH20; 2.5 μ l 10 * buffer; 2 μ l dNTP (2.5mM); 1 μ l primer #C2F3 (20 μ M); 1 μ l primer #1108 (20 μ M); 0.4 μ l Taq enzyme (5U/ μ l); 1 μ l template.
PCR response procedures: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 1min, 48 ℃ of annealing 1min, 72 ℃ of extension 2min carry out 40 circulations; Last 72 ℃ of insulation 5min.
3, pcr amplification product carries out 1% (containing 0.5 μ g/mL EB) agarose gel electrophoresis, and electrophoretic buffer is 0.5 * TBE, and voltage is 80V, the about 30min of electrophoresis.In gel images system (Tanon 4500, day can scientific and technological (Shanghai) Co., Ltd.), take pictures and preserve, and carry software (day ability GIS gel images treatment system with system; Edition 4 .00, software registration number: 0003952) stripe size is analyzed.
The stripe size analytical results: the pcr amplification product of Meloidogyne incognita and javanese root knot nematode is about 1.7kb (between the 1.6kb-1.8kb); The pcr amplification product of peanut root-knot nematode is about 1.1kb (between the 1.0kb-1.2kb), and the pcr amplification product of northern root knot nematode is about 0.5kb (between the 0.4kb-0.6kb).
Two, with the auxiliary discriminating of primer pair B root knot nematode adult
1, extracts the genomic dna of wall scroll root knot nematode (2 instar larvae).
2, be that template is carried out the PCR reaction with the genomic dna, obtain pcr amplification product.
PCR reaction system (25 μ L): 17.1 μ l ddH20; 2.5 μ l 10 * buffer; 2 μ l dNTP (2.5mM); 1 μ l primer MI-F (20 μ M); 1 μ l primer MI-R (20 μ M); 0.4 μ l Taq enzyme (5U/ μ l); 1 μ l template.
PCR response procedures: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 45S, 58 ℃ of annealing 45S, 72 ℃ of extension 45S carry out 40 circulations; Last 72 ℃ of insulation 5min.
3, pcr amplification product carries out 1% (containing 0.5 μ g/mL EB) agarose gel electrophoresis, and electrophoretic buffer is 0.5 * TBE, and voltage is 80V, the about 30min of electrophoresis.In gel images system (Tanon 4500, day can scientific and technological (Shanghai) Co., Ltd.), take pictures and preserve, and carry software (day ability GIS gel images treatment system with system; Edition 4 .00, software registration number: 0003952) stripe size is analyzed.
Except Meloidogyne incognita, other three kinds of root knot nematodes all do not obtain pcr amplification product.The stripe size analytical results: the pcr amplification product of Meloidogyne incognita is about 500bp (between the 400b-600b).
One, with primer first is assisted the discriminating root knot nematode
1, extracts the genomic dna of root knot nematode egg capsule.
2, be that template is carried out the PCR reaction with the genomic dna, obtain pcr amplification product.
PCR reaction system (25 μ L): 17.1 μ l ddH20; 2.5 μ l 10 * buffer; 2 μ l dNTP (2.5mM); 1 μ l primer #C2F3 (20 μ M); 1 μ l primer #1108 (20 μ M); 0.4 μ l Taq enzyme (5U/ μ l); 1 μ l template.
PCR response procedures: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 1min, 48 ℃ of annealing 1min, 72 ℃ of extension 2min carry out 35 circulations; Last 72 ℃ of insulation 5min.
3, pcr amplification product carries out 1% (containing 0.5 μ g/mL EB) agarose gel electrophoresis, and electrophoretic buffer is 0.5 * TBE, and voltage is 80V, the about 30min of electrophoresis.In gel images system (Tanon 4500, day can scientific and technological (Shanghai) Co., Ltd.), take pictures and preserve, and carry software (day ability GIS gel images treatment system with system; Edition 4 .00, software registration number: 0003952) stripe size is analyzed.
The stripe size analytical results: the pcr amplification product of Meloidogyne incognita and javanese root knot nematode is about 1.7kb (between the 1.6kb-1.8kb); The pcr amplification product of peanut root-knot nematode is about 1.1kb (between the 1.0kb-1.2kb), and the pcr amplification product of northern root knot nematode is about 0.5kb (between the 0.4kb-0.6kb).
Two, with the auxiliary discriminating of primer pair B root knot nematode adult
1, extracts the genomic dna of root knot nematode egg capsule.
2, be that template is carried out the PCR reaction with the genomic dna, obtain pcr amplification product.
PCR reaction system (25 μ L): 17.1 μ l ddH20; 2.5 μ l 10 * buffer; 2 μ l dNTP (2.5mM); 1 μ l primer MI-F (20 μ M); 1 μ l primer MI-R (20 μ M); 0.4 μ l Taq enzyme (5U/ μ l); 1 μ l template.
PCR response procedures: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 45S, 58 ℃ of annealing 45S, 72 ℃ of extension 45S carry out 35 circulations; Last 72 ℃ of insulation 5min.
3, pcr amplification product carries out 1% (containing 0.5 μ g/mL EB) agarose gel electrophoresis, and electrophoretic buffer is 0.5 * TBE, and voltage is 80V, the about 30min of electrophoresis.In gel images system (Tanon 4500, day can scientific and technological (Shanghai) Co., Ltd.), take pictures and preserve, and carry software (day ability GIS gel images treatment system with system; Edition 4 .00, software registration number: 0003952) stripe size is analyzed.
Except Meloidogyne incognita, other three kinds of root knot nematodes all do not obtain pcr amplification product.The stripe size analytical results: the pcr amplification product of Meloidogyne incognita is about 500bp (between the 400b-600b).
The plant sample of catching an illness of taking from Hainan is following: banana, tomato, capsicum, muskmelon, celery, cucumber.
One, with primer first is detected the plant that catches an illness
1, gets the root knot (root nodule) of the plant that catches an illness, extract total DNA.
2, be that template is carried out the PCR reaction with total DNA, obtain pcr amplification product.
PCR reaction system (25 μ L): 17.1 μ l ddH20; 2.5 μ l 10 * buffer; 2 μ l dNTP (2.5mM); 1 μ l primer #C2F3 (20 μ M); 1 μ l primer #1108 (20 μ M); 0.4 μ l Taq enzyme (5U/ μ l); 1 μ l template.
PCR response procedures: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 1min, 48 ℃ of annealing 1min, 72 ℃ of extension 2min carry out 35 circulations; Last 72 ℃ of insulation 5min.
3, pcr amplification product carries out 1% (containing 0.5 μ g/mL EB) agarose gel electrophoresis, and electrophoretic buffer is 0.5 * TBE, and voltage is 80V, the about 30min of electrophoresis.In gel images system (Tanon 4500, day can scientific and technological (Shanghai) Co., Ltd.), take pictures and preserve, and carry software (day ability GIS gel images treatment system with system; Edition 4 .00, software registration number: 0003952) stripe size is analyzed.
The result sees Fig. 3.Among Fig. 3: M: standard molecular weight; 1: banana; 2: tomato; 3: capsicum; 4: muskmelon; 5: celery; 6: cucumber.6 kinds of plants that catch an illness all obtain being about the pcr amplification product of 1.7kb (between the 1.6kb-1.8kb).Explain that 6 kind of plant are Meloidogyne incognita or javanese root knot nematode infects.
Two, detect the plant that catches an illness with primer pair B
1, gets the root knot (root nodule) of the plant that catches an illness, extract total DNA.
2, be that template is carried out the PCR reaction with total DNA, obtain pcr amplification product.
PCR reaction system (25 μ L): 17.1 μ l ddH20; 2.5 μ l 10 * buffer; 2 μ l dNTP (2.5mM); 1 μ l primer MI-F (20 μ M); 1 μ l primer MI-R (20 μ M); 0.4 μ l Taq enzyme (5U/ μ l); 1 μ l template.
PCR response procedures: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 45S, 58 ℃ of annealing 45S, 72 ℃ of extension 45S carry out 40 circulations; Last 72 ℃ of insulation 5min.
3, pcr amplification product carries out 1% (containing 0.5 μ g/mL EB) agarose gel electrophoresis, and electrophoretic buffer is 0.5 * TBE, and voltage is 80V, the about 30min of electrophoresis.In gel images system (Tanon 4500, day can scientific and technological (Shanghai) Co., Ltd.), take pictures and preserve, and carry software (day ability GIS gel images treatment system with system; Edition 4 .00, software registration number: 0003952) stripe size is analyzed.
The result sees Fig. 4.Among Fig. 4: M: standard molecular weight; 1: banana; 2: tomato; 3: capsicum; 4: muskmelon; 5: celery; 6: cucumber.Banana, tomato, capsicum, muskmelon, celery all obtain being about the pcr amplification product of 500bp (between the 400b-600b), and cucumber does not obtain pcr amplification product.
The result of integrating step one, reach a conclusion as follows: banana, tomato, capsicum, muskmelon, celery are Meloidogyne incognita and infect.Cucumber is that javanese root knot nematode infects.
Sequence table
< 110>Research Institute of Environment and Plant Protection, Chinese Academy of Tropi
< 120>method and the primer special thereof of auxiliary discriminating root knot nematode are right
<130>CGGNARY102310
<160>4
<210>1
<211>22
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>1
ggtcaatgtc?agaaatttgt?gg 22
<210>2
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>2
tacctttgac?caatcacgct 20
<210>3
<211>22
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>3
gggcaagtaa?ggatgctctg?ac 22
<210>4
<211>22
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>4
ctttcatagc?cacgtcgcga?tc 22
Claims (4)
1. the primer pair B formed of DNA shown in the sequence 4 of DNA and sequence table shown in the sequence 3 of sequence table.
2. the said primer pair B of claim 1 is in the auxiliary application of differentiating in the Meloidogyne incognita.
3. an auxiliary method of differentiating Meloidogyne incognita comprises the steps:
(1) genomic dna of extraction root knot nematode to be measured;
(2) with the genomic dna be template, carry out pcr amplification with the said primer pair B of claim 1; If obtain pcr amplification product, root knot nematode to be measured is candidate's a Meloidogyne incognita; If do not obtain pcr amplification product, root knot nematode to be measured is candidate's a non-Meloidogyne incognita;
Said genomic dna is taken from adult, larva or egg capsule;
Take from the used primer of adult or larva, said PCR to for said primer pair B the time when genomic dna, the response procedures of said PCR is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 45S, 58 ℃ of annealing 45S, 72 ℃ of extension 45S carry out 40 circulations; Last 72 ℃ of insulation 5min;
Take from the used primer of egg capsule, said PCR to for said primer pair B the time when genomic dna, the response procedures of said PCR is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 45S, 58 ℃ of annealing 45S, 72 ℃ of extension 45S carry out 35 circulations; Last 72 ℃ of insulation 5min;
Use said primer pair B to carry out the pcr amplification product that pcr amplification obtains and be 400bp-600bp.
4. the application of the said method of claim 3 in the assistant identification Meloidogyne incognita is caught an illness plant.
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| CN102268482B (en) * | 2011-08-10 | 2013-01-23 | 中国农业大学 | Meloidogyne enterolobii detection kit based on loop-mediated isothermal amplification and application thereof |
| CN102367482B (en) * | 2011-11-07 | 2013-05-01 | 中国农业大学 | Specific primer pair for assisting booklice identification and application thereof |
| CN102367484B (en) * | 2011-11-08 | 2013-01-30 | 华南农业大学 | Method for detecting single root knot of enterolobium cyclocarpum meloidogyne and application thereof |
| CN102766687B (en) * | 2012-07-05 | 2013-12-25 | 华南农业大学 | Primers and method for directly and simultaneously detecting Meloidogyne and Rotylenchulus reniformis in soil |
| CN103343169B (en) * | 2013-07-30 | 2015-07-01 | 中国农业科学院烟草研究所 | Meloidogyne molecular identification primer set, and preparation method and application thereof |
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| 杨佩文等.三七病原根结线虫核糖体基因ITS区段的克隆测序及其在检测中的应用.《江西农业大学学报》.2008,第30卷(第1期),全文. * |
| 王天阳.南方根结线虫Mi-flp-16基因的克隆及原位杂交分析.《中国蔬菜》.2009,(第4期),全文. * |
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