CN107815490A - A kind of tobacco root-knot pathogeny detecting reagent box and its application method based on loop-mediated isothermal amplification technique - Google Patents
A kind of tobacco root-knot pathogeny detecting reagent box and its application method based on loop-mediated isothermal amplification technique Download PDFInfo
- Publication number
- CN107815490A CN107815490A CN201711124726.6A CN201711124726A CN107815490A CN 107815490 A CN107815490 A CN 107815490A CN 201711124726 A CN201711124726 A CN 201711124726A CN 107815490 A CN107815490 A CN 107815490A
- Authority
- CN
- China
- Prior art keywords
- root
- knot
- mmol
- reaction solutions
- isothermal amplification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
Abstract
The invention discloses a kind of tobacco root-knot pathogeny detecting reagent box and its application method based on loop-mediated isothermal amplification technique.The described tobacco root-knot pathogeny detecting reagent box based on loop-mediated isothermal amplification technique includes being used for the I reaction solutions for detecting Meloidogyne incognita, detect the A reaction solutions of peanut root-knot nematode, detect the J reaction solutions of javanese root knot nematode, Bst archaeal dna polymerases large fragment and 100 × SYBR Green I dyeing liquors；The present invention is based on loop-mediated isothermal amplification technique, and the requirement to testing conditions is low, only needs water-bath or insulating box to can be achieved with detecting, beneficial to popularization and application.The kit and method that the present invention includes are due to that need not carry out gel electrophoresis, naked eyes can directly observe testing result, it is thus time-consuming short, 1.5h can complete to detect, kit of the present invention contains the primer for the detection LAMP detections of Meloidogyne incognita, peanut root-knot nematode and javanese root knot nematode, synchronously three kind cause of disease root-knot nematodes common to tobacco root-knot can detect.
Description
Technical field
The invention belongs to plant disease technical field of detection of pathogeny, and in particular to one kind is based on loop-mediated isothermal amplification technique
Tobacco root-knot pathogeny detecting reagent box and its application method.
Background technology
Tobacco root-knot is a kind of soil-borne disease for having a strong impact on tobacco leaf production.In recent years, for root-knot nematode
Be present the drawbacks of high poison, high residue in most chemical nematicides of disease preventing and treating, worldwide generally disabled.Therefore adopt
Safe and efficient root knot nematode disease preventing control method is taken to turn into trend of the times.During the prevention and control of tobacco root-knot, choosing
It is considered as an economy, measure efficiently, green to select using suitable disease-resistant tobacco bred.However, root-knot nematode species are numerous
More, different root-knot nematodes are different to the pathogenecity of host plant, and disease-resistant variety is also limited to the resistant range of root-knot nematode, when
It is mainly peanut root-knot nematode that the most extensive root-knot nematode for endangering most serious is distributed on preceding domestic tobacco（Meloidogyne arenaria）, Meloidogyne incognita（M. incognita）And javanese root knot nematode（M. javanica）, these three root knot lines
The form and molecular biological characteristics of worm are very much like, but larger Difference in Pathogenicity to different tobacco breds be present.Therefore it is accurate
Really, the above-mentioned three kinds of root-knot nematodes of Rapid identification are seed selection and the precondition using disease-resistant tobacco bred.
Identify that the most frequently used 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, the difference for observing perineal region decorative pattern under the microscope determine the species of root-knot nematode.
The advantages of this method is easy, directly perceived, and the drawbacks of existing is：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；It is 2. same
There is individual difference in the perineal pattern of the root-knot nematode of class, the female adult pest progress to more than 20 is at least needed in practical operation
Observation can just make a definite diagnosis, and take a lot of work, be 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 present in the form of second instar larvae, and this method made is restricted in actual applications.In addition, isodynamic enzyme is electric
Swimming method is also the method commonly used 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 species judgement according to the spectral pattern of enzyme band after esterase and Chances of Malic Dehydrogenase Isoenzyme dyeing
It 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
Turn into trend.Compared with morphology and isozyme electrophoresis authentication 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 as the objects of identification.The PCR identifications of early stage include expanding using random primer
RAPD-PCR technologies or using the PCR-RFLP for carrying out restricted digestion after the amplification of nematode universal primer to amplified production again
Technology, their common drawback are due to cause the stability of identification and accurate using the special special primer of non-root-knot nematode
Property is not high.Therefore, domestic and foreign scholars are started then 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 the invention such as quick《One authentication 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 enters performing PCR detection to the root-knot nematode for being isolated from tobacco old complaint and soil respectively, 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 Standard PCR detection method so that
Qualification process is cumbersome, time-consuming, and needs instrument and equipment costly.
The content of the invention
The first object of the present invention is to provide a kind of tobacco root-knot disease based on loop-mediated isothermal amplification technique
Former detection kit；Second purpose is to provide the tobacco root-knot cause of disease based on loop-mediated isothermal amplification technique
The application method of detection kit.
The first object of the present invention is achieved in that the described Tobacco Root tie lines based on loop-mediated isothermal amplification technique
Parasitosis pathogeny detecting reagent box includes being used for the I reaction solutions for detecting Meloidogyne incognita, detects the A reactions of peanut root-knot nematode
Liquid, detect the J reaction solutions of javanese root knot nematode, Bst archaeal dna polymerases large fragment and 100 × SYBR Green I dyeing liquors；
Wherein, the solute of I reaction solutions and its concentration are：Each 0.2 μm of olL of primer MiF3 and MiB3-1, primer MiFIP
1.6 μm olL each with MiBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl（20 mmol·L-1, pH
8.8）, KCl（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2 SO4 （10 mmol·L-1）,
0.1% Triton x-100；
MiF3：5'-GTGCTTCGTCTTTTGCTT-3'；
MiB3：5'-ACTTTCCTTGGAATTGGAACA-3'；
MiFIP：
5'-AGGAAGGTATACTATCCAAGACCCA-TTTAAAATCTGTTTCGGCACAC-3'；
MiBIP：
5'-TTCACAAAAAACCCAATATGTCAGC-CGATATCTAGGGGTGTTTGA-3'；
The solute and its concentration of A reaction solutions be：Each 0.2 μm of olL of primer MaF3 and MaB3-1, primer MaFIP and
Each 1.6 μm of olL of MaBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl（20 mmol·L-1, pH 8.8）, KCl
（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2 SO4 （10 mmol·L-1）, 0.1% Triton
x-100；
MaF3：5'-TGCTTTCAACGCGGTATG-3'；
MaB3：5'-ATTTCTCTCCACGAGTTTCT-3'；
MaFIP：
5'-CCACTTTGCCTCAGCGAAATTT-GTCGTAATCAATGGGTTGTC-3'；
MaBIP：
5'-GTGGCTTTTCGATGTTCGCT-TAGCCCAATTTGAGTTTTCC-3'；
J reaction solute and its concentration be：Each 0.2 μm of olL of primer MjF3 and MjB3-1, primer MjFIP and
Each 1.6 μm of olL of MjBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl（20 mmol·L-1, pH 8.8）, KCl
（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2 SO4 （10 mmol·L-1）, 0.1% Triton
x-100；
MjF3：5'-TCGGAAATGACGAAGGTG-3'；
MjB3：5'- TTATAAACCCAGCTAGGGAC-3'；
MjFIP：
5'-TTAGGCTGATTTCCGATTTCCGAC-TCGGAAATGGGAACTGTC-3'；
MjBIP：
5'-ATGTCGGAAATCGGAATTCCAG-CCCCATTTATTCGCAAGAC-3'。
The second object of the present invention, which is achieved in that, carries the DNA containing tobacco root-knot original nematode to be measured
Liquid is taken, is separately added into I reaction solutions, A reaction solutions and J that the kit of tobacco root-knot pathogenic nematode LAMP detections is included
In reaction solution, ring mediated isothermal amplification is carried out in the presence of Bst DNA polymerase Large fragments, then carries out SYBR Green I
Dyeing, it is seen that observed under light or ultraviolet light, if I reaction solutions are presented green and represent that testing sample contains Meloidogyne incognita, be in
Existing Chinese red represents that testing sample is free of Meloidogyne incognita；If A reaction solutions are presented green and represent that testing sample contains peanut
Root-knot nematode, Chinese red is presented and represents that testing sample is free of peanut root-knot nematode；If J reaction solutions are presented green and represent to treat test sample
Product contain javanese root knot nematode, and Chinese red is presented and represents that testing sample is free of javanese root knot nematode.
Ring mediated isothermal amplification (loop-mediatedisothermal amplification, LAMP), it is a kind of new
Nucleic acid amplification method, be characterized in for target gene 6 regions design 4 species-specific primers, in strand displacement archaeal dna polymerase
In the presence of (Bst DNA polymerase), 60--65 DEG C of constant-temperature amplification, or so 15-60 minutes can be achieved 109~1010Times
Nucleic acid amplification, there is simple to operate, high specificity.Low is required to instrument and equipment, a water-bath or insulating box are just
It can realize and react, detection as a result is also very simple, it is not necessary to gel electrophoresis, loop-mediated isothermal amplification are carried out as PCR
Result observe by the naked eye the generation of white opacity or green fluorescence to judge, it is simple and efficient, be adapted to basic unit's quick diagnosis.Mesh
The fields such as the preceding qualitative and quantitative detection for being successfully applied to the diagnosing of clinical disease, popular bacterium or virus, but there is no application
In the report of tobacco root-knot Pathogen identification.
Compared with prior art, its advantage and good effect are shown the present invention：
It is 1. simple to operate：The present invention be based on loop-mediated isothermal amplification technique, the requirement to testing conditions is low, only need water-bath or
Insulating box can be achieved with detecting, beneficial to popularization and application.
2. detection efficiency is high：The kit and method that the present invention includes, visually can be with due to that need not carry out gel electrophoresis
Testing result is directly observed, thus it is time-consuming short, and 1.5h can complete to detect.
3. have a wide range of application：Kit of the present invention is contained for Meloidogyne incognita, peanut root-knot nematode and Java root
The primer of tie lines worm detection LAMP detections, synchronously three kind cause of disease root-knot nematodes common to tobacco root-knot can be examined
Survey.
Embodiment
With reference to embodiment, the present invention is further illustrated, but the present invention is not any limitation as in any way,
Based on present invention teach that any conversion or replacement made, belong to protection scope of the present invention.
Tobacco root-knot pathogeny detecting reagent box of the present invention based on loop-mediated isothermal amplification technique includes
For detecting the I reaction solutions of Meloidogyne incognita, the A reaction solutions of peanut root-knot nematode are detected, the J for detecting javanese root knot nematode is anti-
Answer liquid, Bst archaeal dna polymerases large fragment and 100 × SYBR Green I dyeing liquors；
Wherein, the solute of I reaction solutions and its concentration are：Each 0.2 μm of olL of primer MiF3 and MiB3-1, primer MiFIP
1.6 μm olL each with MiBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl（20 mmol·L-1, pH
8.8）, KCl（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2 SO4 （10 mmol·L-1）,
0.1% Triton x-100；
MiF3：5'-GTGCTTCGTCTTTTGCTT-3'；
MiB3：5'-ACTTTCCTTGGAATTGGAACA-3'；
MiFIP：
5'-AGGAAGGTATACTATCCAAGACCCA-TTTAAAATCTGTTTCGGCACAC-3'；
MiBIP：
5'-TTCACAAAAAACCCAATATGTCAGC-CGATATCTAGGGGTGTTTGA-3'；
The solute and its concentration of A reaction solutions be：Each 0.2 μm of olL of primer MaF3 and MaB3-1, primer MaFIP and
Each 1.6 μm of olL of MaBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl（20 mmol·L-1, pH 8.8）, 10
mmol·L -1 KCl, MgSO4（6.0 mmol·L-1）, 10 mmolL-1 (NH4) 2 SO4 , 0.1% Triton x-
100；
MaF3：5'-TGCTTTCAACGCGGTATG-3'；
MaB3：5'-ATTTCTCTCCACGAGTTTCT-3'；
MaFIP：
5'-CCACTTTGCCTCAGCGAAATTT-GTCGTAATCAATGGGTTGTC-3'；
MaBIP：
5'-GTGGCTTTTCGATGTTCGCT-TAGCCCAATTTGAGTTTTCC-3'；
J reaction solute and its concentration be：Each 0.2 μm of olL of primer MjF3 and MjB3-1, primer MjFIP and
Each 1.6 μm of olL of MjBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl（20 mmol·L-1, pH 8.8）, KCl
（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2 SO4 （10 mmol·L-1）, 0.1% Triton
x-100；
MjF3：5'-TCGGAAATGACGAAGGTG-3'；
MjB3：5'- TTATAAACCCAGCTAGGGAC-3'；
MjFIP：
5'-TTAGGCTGATTTCCGATTTCCGAC-TCGGAAATGGGAACTGTC-3'；
MjBIP：
5'-ATGTCGGAAATCGGAATTCCAG-CCCCATTTATTCGCAAGAC-3'。
The concentration of Bst DNA polymerase Large fragments is 8U/ μ l.
Tobacco root-knot pathogeny detecting reagent box of the present invention based on loop-mediated isothermal amplification technique makes
With method, it is by the DNA extract solutions containing tobacco root-knot original nematode to be measured, is separately added into tobacco root-knot cause of disease
In I reaction solutions, A reaction solutions and J reaction solutions that the kit of nematode LAMP detections is included, in Bst DNA polymerase Large fragments
In the presence of carry out ring mediated isothermal amplification, then carry out SYBR Green I and dye, it is seen that observed under light or ultraviolet light, if I
Reaction solution is presented green and represents that testing sample contains Meloidogyne incognita, and Chinese red is presented and represents that testing sample is free of Root Knot
Nematode；If A reaction solutions are presented green and represent that testing sample contains peanut root-knot nematode, Chinese red is presented and represents testing sample not
Containing peanut root-knot nematode；If J reaction solutions are presented green and represent that testing sample contains javanese root knot nematode, Chinese red is presented and represents
Testing sample is free of javanese root knot nematode.
So that case is embodied, the present invention will be further described below：
Embodiment 1
The preparation of tobacco root-knot pathogenic nematode LAMP detection kit
（1) be used for three grow tobacco Pathogen of Root-knot Disease nematode analysis LAMP 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,
Using Primer Explorer V4 software LAMP primers.Devise the LAMP primer for the identification of above-mentioned three kinds of root-knot nematodes
Each one group, it is respectively：
Meloidogyne incognita LAMP primer：
MiF3：5'-GTGCTTCGTCTTTTGCTT-3'
MiB3：5'-ACTTTCCTTGGAATTGGAACA-3'
MiFIP：
5'-AGGAAGGTATACTATCCAAGACCCA-TTTAAAATCTGTTTCGGCACAC-3'
MiBIP：
5'-TTCACAAAAAACCCAATATGTCAGC-CGATATCTAGGGGTGTTTGA-3'
Peanut root-knot nematode LAMP primer：
MaF3：5'-TGCTTTCAACGCGGTATG-3'
MaB3：5'-ATTTCTCTCCACGAGTTTCT-3'
MaFIP：
5'-CCACTTTGCCTCAGCGAAATTT-GTCGTAATCAATGGGTTGTC-3'
MaBIP：5'-GTGGCTTTTCGATGTTCGCT-TAGCCCAATTTGAGTTTTCC-3'
Javanese root knot nematode LAMP primer：
MjF3：5'-TCGGAAATGACGAAGGTG-3'
MjB3：5'- TTATAAACCCAGCTAGGGAC-3'
MjFIP：
5'-TTAGGCTGATTTCCGATTTCCGAC-TCGGAAATGGGAACTGTC-3'
MjBIP：
5'-ATGTCGGAAATCGGAATTCCAG-CCCCATTTATTCGCAAGAC-3'
（2）The preparation of I reaction solutions：The solute and its concentration of described I reaction solutions be：Each 0.2 μ of primer MiF3 and MiB3
mol·L -1, each 1.6 μm of olL of primer MiFIP and MiBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl
（20 mmol·L-1, pH 8.8）, KCl（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2 SO4
（10 mmol·L-1）, 0.1% Triton x-100.
（3）The preparation of A reaction solutions：The solute and its concentration of described A reaction solutions be：Primer MaF3 and MaB3 is each
0.2μmol·L -1, each 1.6 μm of olL of primer MaFIP and MaBIP-1, dNTPs（2.4 mmol·L -1）, Tris-
HCl（20 mmol·L-1, pH 8.8）, KCl（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2
SO4 （10 mmol·L-1）, 0.1% Triton x-100.
(4）The preparation of J reaction solutions：The solute and its concentration of described J reactions be：Primer MjF3 and MjB3 each 0.2
μmol·L -1, each 1.6 μm of olL of primer MjFIP and MjBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl
（20 mmol·L-1, pH 8.8）, KCl（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2 SO4
（10 mmol·L-1）, 0.1% Triton x-100.
（5）The composition of tobacco root-knot pathogenic nematode LAMP detection kit：Described kit is by detecting south
The I reaction solutions of root-knot nematode, the A reaction solutions of peanut root-knot nematode are detected, detect the J reaction solutions of javanese root knot nematode, concentration is
8U/ μ l Bst DNA polymerase Large fragments, the dyeing liquors of 100 × SYBR Green I composition, each component are individually packed.
Embodiment 2
Tobacco root-knot sample DNA extracts
It is placed in from clip root knot 10-20 on the old complaint sample of clean tobacco root-knot through in autoclaved mortar, adding
Enter liquid nitrogen grinding to be transferred in advance added with 100 μ l 100ng μ l into powder-1Proteinase K Solution 1.5ml centrifuge tube in, 60
95 DEG C of heating 10min, 12000rmin after DEG C water-bath 1h-11min is centrifuged, the supernatant of gained is the DNA for being used for PCR detections
Extract solution.
Embodiment 3
Detected using tobacco root-knot pathogenic nematode LAMP detection kit
3 0.2ml through aseptic process PCR pipe is taken, is separately added into and adds tobacco root-knot pathogenic nematode LAMP detections
Kit the I reaction solutions, A reaction solutions and the J reaction solutions that are included respectively take 22 μ l；Root knot nematode in tobacco is sequentially added in each pipe
The sick μ l of 2 μ l and Bst DNA polymerase Large fragments of sample DNA extract solution 1；After well mixed, PCR pipe is placed in 60-65 DEG C of perseverance
45 min, 82 DEG C of 5 min of insulation are incubated in tepidarium.After insulation terminates, 2 100 × SYBR of uL Green are often added in pipe
I, observe color change under ultraviolet irradiation after mixing；If I reaction solutions are presented green and represent that testing sample contains Root Knot line
Worm, Chinese red is presented and represents that testing sample is free of Meloidogyne incognita；If A reaction solutions are presented green and represent that testing sample contains
Peanut root-knot nematode, Chinese red is presented and represents that testing sample is free of peanut root-knot nematode；If J reaction solutions are presented green and represent to treat
Test sample product contain javanese root knot nematode, and Chinese red is presented and represents that testing sample is free of javanese root knot nematode.
SEQUENCE LISTING
<110>Yunnan Academy of Tobacco Agricultural Science
<120>A kind of tobacco root-knot pathogeny detecting reagent box and its user based on loop-mediated isothermal amplification technique
Method
<130> 2017
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> MiF3
<400> 1
gtgcttcgtc ttttgctt 18
<210> 2
<211> 21
<212> DNA
<213> MiB3
<400> 2
actttccttg gaattggaac a 21
<210> 3
<211> 47
<212> DNA
<213> MiFIP
<400> 3
aggaaggtat actatccaag acccatttaa aatctgtttc ggcacac 47
<210> 4
<211> 45
<212> DNA
<213> MiBIP
<400> 4
ttcacaaaaa acccaatatg tcagccgata tctaggggtg tttga 45
<210> 5
<211> 18
<212> DNA
<213> MaF3
<400> 5
tgctttcaac gcggtatg 18
<210> 6
<211> 20
<212> DNA
<213> MaB3
<400> 6
atttctctcc acgagtttct 20
<210> 7
<211> 42
<212> DNA
<213> MaFIP
<400> 7
ccactttgcc tcagcgaaat ttgtcgtaat caatgggttg tc 42
<210> 8
<211> 40
<212> DNA
<213> MaBIP
<400> 8
gtggcttttc gatgttcgct tagcccaatt tgagttttcc 40
<210> 9
<211> 18
<212> DNA
<213> MjF3
<400> 9
tcggaaatga cgaaggtg 18
<210> 10
<211> 20
<212> DNA
<213> MjB3
<400> 10
ttataaaccc agctagggac 20
<210> 11
<211> 42
<212> DNA
<213> MjFIP
<400> 11
ttaggctgat ttccgatttc cgactcggaa atgggaactg tc 42
<210> 12
<211> 41
<212> DNA
<213> MjBIP
<400> 12
atgtcggaaa tcggaattcc agccccattt attcgcaaga c 41
Claims (3)
1. a kind of tobacco root-knot pathogeny detecting reagent box based on loop-mediated isothermal amplification technique, it is characterised in that described
The tobacco root-knot pathogeny detecting reagent box based on loop-mediated isothermal amplification technique include be used for detect Root Knot line
The I reaction solutions of worm, the A reaction solutions of peanut root-knot nematode are detected, detect the J reaction solutions of javanese root knot nematode, Bst archaeal dna polymerases
Large fragment and 100 × SYBR Green I dyeing liquors；
Wherein, the solute of I reaction solutions and its concentration are：Each 0.2 μm of olL of primer MiF3 and MiB3-1, primer MiFIP and
Each 1.6 μm of olL of MiBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl（20 mmol·L-1, pH 8.8）, KCl
（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2 SO4 （10 mmol·L-1）, 0.1% Triton
x-100；
MiF3：5'-GTGCTTCGTCTTTTGCTT-3'；
MiB3：5'-ACTTTCCTTGGAATTGGAACA-3'；
MiFIP：
5'-AGGAAGGTATACTATCCAAGACCCA-TTTAAAATCTGTTTCGGCACAC-3'；
MiBIP：
5'-TTCACAAAAAACCCAATATGTCAGC-CGATATCTAGGGGTGTTTGA-3'；
The solute and its concentration of A reaction solutions be：Each 0.2 μm of olL of primer MaF3 and MaB3-1, primer MaFIP and
Each 1.6 μm of olL of MaBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl（20 mmol·L-1, pH 8.8）,
KCl（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2 SO4 （10 mmol·L-1）, 0.1%
Triton x-100；
MaF3：5'-TGCTTTCAACGCGGTATG-3'；
MaB3：5'-ATTTCTCTCCACGAGTTTCT-3'；
MaFIP：
5'-CCACTTTGCCTCAGCGAAATTT-GTCGTAATCAATGGGTTGTC-3'；
MaBIP：
5'-GTGGCTTTTCGATGTTCGCT-TAGCCCAATTTGAGTTTTCC-3'；
J reaction solute and its concentration be：Each 0.2 μm of olL of primer MjF3 and MjB3-1, primer MjFIP and
Each 1.6 μm of olL of MjBIP-1, dNTPs（2.4 mmol·L -1）, Tris-HCl（20 mmol·L-1, pH 8.8）,
KCl（10 mmol·L-1）, MgSO4（6.0 mmol·L-1）, (NH4) 2 SO4 （10 mmol·L-1）, 0.1%
Triton x-100；
MjF3：5'-TCGGAAATGACGAAGGTG-3'；
MjB3：5'- TTATAAACCCAGCTAGGGAC-3'；
MjFIP：
5'-TTAGGCTGATTTCCGATTTCCGAC-TCGGAAATGGGAACTGTC-3'；
MjBIP：
5'-ATGTCGGAAATCGGAATTCCAG-CCCCATTTATTCGCAAGAC-3'。
2. the tobacco root-knot Pathogen test reagent according to claim 1 based on loop-mediated isothermal amplification technique
Box, it is characterised in that the concentration of Bst DNA polymerase Large fragments is 8U/ μ l.
A kind of 3. examination of the tobacco root-knot Pathogen test based on loop-mediated isothermal amplification technique described in claim 1 or 2
The application method of agent box, it is characterised in that be by the DNA extract solutions containing tobacco root-knot original nematode to be measured, be separately added into
In I reaction solutions, A reaction solutions and J reaction solutions that the kit of tobacco root-knot pathogenic nematode LAMP detections is included,
Ring mediated isothermal amplification is carried out in the presence of Bst DNA polymerase Large fragments, SYBR Green I is then carried out and dyes, it is seen that light
Or observed under ultraviolet light, if I reaction solutions are presented green and represent that testing sample contains Meloidogyne incognita, Chinese red is presented and represents
Testing sample is free of Meloidogyne incognita；If A reaction solutions are presented green and represent that testing sample contains peanut root-knot nematode, present
Chinese red represents that testing sample is free of peanut root-knot nematode；If J reaction solutions are presented green and represent that testing sample contains Java root
Tie lines worm, Chinese red is presented and represents that testing sample is free of javanese root knot nematode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711124726.6A CN107815490A (en) | 2017-11-14 | 2017-11-14 | A kind of tobacco root-knot pathogeny detecting reagent box and its application method based on loop-mediated isothermal amplification technique |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711124726.6A CN107815490A (en) | 2017-11-14 | 2017-11-14 | A kind of tobacco root-knot pathogeny detecting reagent box and its application method based on loop-mediated isothermal amplification technique |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107815490A true CN107815490A (en) | 2018-03-20 |
Family
ID=61608987
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711124726.6A Pending CN107815490A (en) | 2017-11-14 | 2017-11-14 | A kind of tobacco root-knot pathogeny detecting reagent box and its application method based on loop-mediated isothermal amplification technique |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107815490A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110423748A (en) * | 2019-09-12 | 2019-11-08 | 中国农业科学院麻类研究所 | A kind of LAMP primer and its application, detection method of quick detection javanese root knot nematode |
| CN110499357A (en) * | 2018-05-20 | 2019-11-26 | 安徽省农业科学院植物保护与农产品质量安全研究所 | A kind of primer and probe using RPA technology detection Meloidogyne incognita |
| CN110499370A (en) * | 2018-05-20 | 2019-11-26 | 安徽省农业科学院植物保护与农产品质量安全研究所 | A kind of primer and probe based on RPA technology detection javanese root knot nematode |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011246A (en) * | 2016-06-01 | 2016-10-12 | 宁波大学 | Isothermal amplification detection primer sequences for five meloidogyne spp. based on micro-fluidic chip technology and application of primer sequences |
-
2017
- 2017-11-14 CN CN201711124726.6A patent/CN107815490A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011246A (en) * | 2016-06-01 | 2016-10-12 | 宁波大学 | Isothermal amplification detection primer sequences for five meloidogyne spp. based on micro-fluidic chip technology and application of primer sequences |
Non-Patent Citations (1)
| Title |
|---|
| T O POWERS: "A polymerase chain reaction method for identification of five major meloidogyne species", 《J NEMATOL》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110499357A (en) * | 2018-05-20 | 2019-11-26 | 安徽省农业科学院植物保护与农产品质量安全研究所 | A kind of primer and probe using RPA technology detection Meloidogyne incognita |
| CN110499370A (en) * | 2018-05-20 | 2019-11-26 | 安徽省农业科学院植物保护与农产品质量安全研究所 | A kind of primer and probe based on RPA technology detection javanese root knot nematode |
| CN110423748A (en) * | 2019-09-12 | 2019-11-08 | 中国农业科学院麻类研究所 | A kind of LAMP primer and its application, detection method of quick detection javanese root knot nematode |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Böhm et al. | Real‐time quantitative PCR: DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants | |
| CN108060257B (en) | Primer composition for detecting pythium androsaceus based on loop-mediated isothermal amplification technology and detection method thereof | |
| CN102586461A (en) | Loop mediated isothermal amplification (LAMP) detection method for meloidogyne hapla and application of method | |
| CN108220474A (en) | A kind of LAMP detection primer of Fusarium graminearum and its application | |
| CN107815490A (en) | A kind of tobacco root-knot pathogeny detecting reagent box and its application method based on loop-mediated isothermal amplification technique | |
| CN102321747A (en) | Kit for detecting meloidogyne incognita based on loop-mediated isothermal amplification, and application thereof | |
| CN110982922B (en) | Primer composition and method for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP | |
| CN102010910A (en) | Loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method | |
| CN106222298A (en) | The LAMP detection kit of a kind of RNA viruses, detection method and application | |
| CN107828905A (en) | Tobacco smoke pollution LAMP detection primer and detection method | |
| CN107828912A (en) | A kind of dosporium cucumerinumand LAMP detection primer and detection method | |
| CN107881254A (en) | Loop-mediated isothermal amplification method detects the primer and its detection method of ramie mould bacterium | |
| CN101586168B (en) | Primer set for detecting J subgroup fowl leukemia virus gene, detection method and rapid detection kit | |
| CN105219879B (en) | The application of Primer composition and its application, the kit and kit that are made from it for detecting citrus Tylenchulus Semipenetrans | |
| CN107937617A (en) | Detect the RT LAMP primer compositions thing and its kit and method of Sai Neijia paddy viruses | |
| KR20140086234A (en) | Primer composition for loop-mediated isothermal amplification reaction for detecting Cucumber Mosaic Virus, and use thereof | |
| CN110878371A (en) | Xinjiang isolate LAMP (loop-mediated isothermal amplification) rapid detection method for apricot chlorotic leafroll phytoplasma | |
| KR101937390B1 (en) | Primer set for detecting Peronospora destructor Berk., Diagnostic kit, and Detecting method using the same | |
| CN104962659A (en) | Pseudomonas syringae pv.cucumber cucumber strain Padlock probe and application thereof | |
| CN108754021A (en) | A kind of ring mediated isothermal amplification detection reagent of duck hepatitis B virus | |
| CN104513853B (en) | The Fields detection test kit of Fructus Persicae brown rot fungus anti-DMI series bactericidal agent | |
| CN105087558B (en) | The detection kit and detection method of genetically engineered soybean | |
| CN107142329A (en) | A kind of upland rice cyst roundworm LAMP quick determination methods | |
| KR20140002907A (en) | Composition for detecting magnaporthe oryzae and method for quantitative analysis of magnaporthe oryzae using the same | |
| KR102647443B1 (en) | LAMP primer set for detecting Cicadella viridis and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180320 |
|
| RJ01 | Rejection of invention patent application after publication |