CN108315392A - A kind of nest type PCR detection reagent and its application in the detection of smoke tree wilt - Google Patents
A kind of nest type PCR detection reagent and its application in the detection of smoke tree wilt Download PDFInfo
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Abstract
The present invention provides a kind of nest type PCR detection reagent, the kit includes：Primer P1 and primer P2 for first round PCR amplification；With the primer PN1 and primer PN2 for the second wheel PCR amplification；Described primer P1, P2, PN1 and PN2 are respectively provided with and the same or analogous amplification functions of SEQ ID NO.1 to 4 in pcr amplification reaction.The present invention also provides application of the kit in detection smoke tree wilt (Verticillium dahliae).The present invention also provides a kind of methods detecting smoke tree wilt using the kit.The present invention can highly sensitive, high accuracy rapidly detect smoke tree wilt.
Description
Technical field
The invention belongs to Ornamental Diseases detection, identification and Prevention Technique fields, and in particular to one kind is for detecting Huang
The detection kit and its application method of smoke tree wilt.
Background technology
Smoke tree (Cotinus coggygria) also known as ecliptic smoke tree or red autumnal leaves, Anacardiaceae fuste；Machaka or
Dungarunga, 3 meters to 5 meters of plant height, autumn leaf color bright red after frost is the seeds of great ornamental values and the economic values, as north
The main red-leaf tree species of capital Fragrant Hill Park, attract a large amount of domestic and foreign tourists, have in the succession of red autumnal leaves culture irreplaceable
Effect.
But since the eighties in last century, because infection verticillium dahliae (Verticillium dahliae), Beijing,
There is the withered phenomenon of large area in smoke tree on the ground such as Shandong, and wherein Xiangshan park smoke tree woods is withered because large area occurs in droop
Dead phenomenon, annual withered smoke tree were once reaching thousands of strains, and smoke tree droop incidence is up to 46.2% in garden, seriously affects
The red autumnal leaves landscape in Fragrance Hill.Smoke tree droop has become a kind of destructive disease for smoke tree of causing harm at present, is the life of Beijing city gardens
One of letter problem to be solved in state system Construction.
Beijing Forestry University and Fragrant Hill Park are to the pathogenic bacteria of smoke tree droop, factors affecting the disease, chemical control side for many years
Method etc. has made intensive studies, but still generation (Song Lizhou, Du Wanguang, the Li Weiwei .2011. of control smoke tree droop not yet in effect
Fragrant Hill Park smoke tree droop Study of Prevention Technology Landscape Architecture in Beijing, 27 (2):51-56；Ge Yuxuan, Zhou Xiaohong, Liu Yang, etc.
.2014. smoke tree category germ plasm resource, Lycopersicum esculentum, chemical composition, leaf color study on regulation progress gardening journals, 41 (9):1833-
1845；Thunder increases the research Beijing Forestry University journal of the general Beijing areas .1993. Verticillium Causing Cotinus coggygria Wilt, 15 (3):88-
93) it is more than 200 kinds that, one of the main reasons, which is the known host plant of verticillium dahliae, and many ornamental plants and hayashishita weeds are all
It is its asymptomatic carrier, although these plants contain verticillium dahliae in vivo, itself does not show any symptom, becomes yellow
Important mediator (Inderbitzin, P., the and Subbarao, K.V.2014.Verticillium that smoke tree droop is propagated
systematics and evolution:How confusion impedes Verticillium wilt management
And how to resolve it.Phytopathology, 104:564-574；King's just sweet smell verticillium dahliaes
The host types identification Northwest Agricultural Universities journal of (Verticillium dahliae Kleb.), 1987,15 (1)：35-
48).Traditional pathogen detaches, identification technology is time-consuming and laborious, it is difficult to is used for quickly detecting to a large amount of nursery stocks.Therefore, it has sought
Effect, the detection technique of quick smoke tree wilt (Verticillium dahliae) have weight to the prevention of smoke tree droop
The production practices meaning wanted.
The development of round pcr has pushed plant disease diagnosis and pathogen identification work, Zhu to have bravely equal (1999) according to cotton
The alkali yl coding sequence of flower verticillium wilt pathogen (Verticillium dahlia) ribosomal gene ITS sections, devises a pair of of 26bp
PCR specificity amplification primers be used for the Molecular Identification and molecular monitoring of verticillium dahliae, but cannot distinguish between different pathogenic classes
Type.Encarnacion etc. (2000) selects 17 plants of verticillium dahliaes and 5 plants of olive verticillium wilt pathogen (Verticillium
Dahlia) defoliation bacterial strain and 18 plants of verticillium dahliaes and the non-defoliation bacterial strain of 7 plants of olive verticillium wilt pathogens utilize
RAPD-PCR methods screen the signature band of defoliation verticillium wilt pathogen and non-defoliation verticillium wilt pathogen respectively, to the specificity
Band is sequenced, and carries out Blast comparisons, no similarity segments report.But in produce reality, due to soil physical chemistry
The complexity of property and the ecosystem, the verticillium dahliae proportion in soil is very low, by conventional PCR method it is difficult to examine
Measure its presence.In addition, due to the difference of plant genome sequences and the quick variation of pathogen, existing detection technique is not suitable for
Carry out the quick detection of verticillium dahliae.
Therefore, the emergency administered for the above insufficient and smoke tree droop, there is an urgent need to one kind being capable of quick, spirit
Method that is quick, accurately detecting smoke tree wilt.
Invention content
(1) technical problems to be solved
It is an object of the invention to overcome the shortcomings of that verticillium dahliae Testing and appraisal method quick, sensitive etc., carries
For a kind of nest type PCR detection reagent and its application method, realize to the fast of the smoke tree wilt in plant tissue and soil
Fast qualitative detection, and can be in the form of kit in the diagnosis and treatment field large-scale promotion of smoke tree droop.
(2) technical solution
In order to reach the above-mentioned purpose of the present invention, the above-mentioned technical problem of the present invention is solved, first aspect present invention provides
A kind of nest type PCR detection reagent, wherein the kit includes：(1) it is used for the primer P1 of first round PCR amplification and draws
Object P2；(2) the primer PN1 and primer PN2 of the second wheel PCR amplification are used for；Wherein, with smoke tree wilt
(Verticillium dahliae) genomic DNA as template carry out PCR amplification when, the primer P1 with the primer P2
Pairing as primer when with the identical function as primer of primer sequence as shown in SEQ ID NO.1；With smoke tree
Wilt (Verticillium dahliae) genomic DNA as template carry out PCR amplification when, the primer P2 with institute
When stating primer P1 pairing as primer with the identical function as primer of primer sequence as shown in SEQ ID NO.2；
And it is described when carrying out PCR amplification using smoke tree wilt (Verticillium dahliae) genomic DNA as template
Primer PN1 has the primer sequence as shown in SEQ ID NO.3 when carrying out PCR amplification as primer with primer PN2 pairings
Arrange the identical function as primer, the primer PN2 have when carrying out PCR amplification as primer with primer PN1 pairings with
The identical function as primer of primer sequence as shown in SEQ ID NO.4.
Second aspect of the present invention provides the kit described in first aspect present invention in detection smoke tree wilt
Application in (Verticillium dahliae).
Third aspect present invention provides a kind of method of detection smoke tree wilt (Verticillium dahliae),
Wherein, the method is detected using the kit described in first aspect.
(3) advantageous effect
The present invention unexpectedly may be used by the discovery that studies for a long period of time using two sets of primers of P1/P2 and PN1/PN2 as nest-type PRC primer
To detect the genomic DNA of 3fg/ μ L, sensitivity exceedes 1000 times than the raising of general PCR detection techniques.Utilize the present invention's
Kit can be accurate, quick, sensitive and specifically withered to the smoke tree in soil, plant tissue such as plant leaf blade (kind seedling leaf)
The germ that withers carries out specific molecular detection, is particularly useful for the early diagnosis of smoke tree droop and detection and the mirror of pathogen
It is fixed.
Description of the drawings
Fig. 1 is the result expanded using the smoke tree Fusarium oxysporum in P1/P2 primer pair soil；In figure, M：Nucleic acid molecules
Measure D2000；1-3：Normal pure forest smoke tree rhizosphere soil；4-6：Morbidity pure forest smoke tree rhizosphere soil；7-9：Smoke tree rhizosphere under arbor-vitae
Soil；10-12：Spiraea trilobata rhizosphere soil；13-15：Soybean rhizosphere；16-18：Sabina vulgaris Ant rhizosphere soil；19：It is negative
It compares (distilled water)；20：Positive control (smoke tree wilt genomic DNA).
Fig. 2 is the result expanded using the smoke tree Fusarium oxysporum in PN1/PN2 primer pair soil；In figure, M：Nucleic acid point
Son amount D2000；1-3：Normal pure forest smoke tree rhizosphere soil；4-6：Morbidity pure forest smoke tree rhizosphere soil；7-9：Pubescent smoketree root under arbor-vitae
Border soil；10-12：Spiraea trilobata rhizosphere soil；13-15：Soybean rhizosphere；16-18：Sabina vulgaris Ant rhizosphere soil；19：Sun
Property control (smoke tree wilt genomic DNA)；20：Negative control (distilled water).
Fig. 3 is the result expanded using the smoke tree Fusarium oxysporum in P1/P2 primer pair plant leaf blades；In figure, M：Nucleic acid
Molecular weight D2000；1-3：Three-bristle cudrania leaf piece；4-6：Leptopus Chinensis (Bunge) Pojarkova blade；7-9：Soybean leaves；10-12：Normal smoke tree blade；13-
16：With the smoke tree blade of Sabina vulgaris Ant companion planting；17：Negative control (distilled water)；18：Positive control (smoke tree wilt genome
DNA)。
Fig. 4 is the result expanded using the smoke tree Fusarium oxysporum in PN1/PN2 primer pair plant leaf blades；In figure, M：Core
Acid molecule amount D2000；1-3：Three-bristle cudrania leaf piece；4-6：Leptopus Chinensis (Bunge) Pojarkova blade；7-9：Soybean leaves；10-12：Normal smoke tree blade；
13-16：With the smoke tree blade of Sabina vulgaris Ant companion planting；17：Negative control (distilled water)；18：Positive control (smoke tree wilt gene
Group DNA).
Specific implementation mode
As described above, first aspect present invention provides a kind of nest type PCR detection reagent, wherein the kit packet
It includes：(1) the primer P1 and primer P2 of first round PCR amplification are used for；(2) the primer PN1 and primer of the second wheel PCR amplification are used for
PN2；
Wherein, PCR expansions are being carried out using smoke tree wilt (Verticillium dahliae) genomic DNA as template
When increasing, the primer P1 has and the primer sequence as shown in SEQ ID NO.1 when with primer P2 pairings as primer
The identical function as primer；Using smoke tree wilt (Verticillium dahliae) genomic DNA as template
Carry out PCR amplification when, the primer P2 with the primer P1 pairing as primer when have with as shown in SEQID NO.2
The identical function as primer of primer sequence；And
When carrying out PCR amplification using smoke tree wilt (Verticillium dahliae) genomic DNA as template,
The primer PN1 has and draws as shown in SEQ ID NO.3 when carrying out PCR amplification as primer with primer PN2 pairings
The identical function as primer of object sequence, primer PN2 tools when carrying out PCR amplification as primer with primer PN1 pairings
Have and the identical function as primer of primer sequence as shown in SEQ ID NO.4.
In some embodiments, the primer P1：(1) include the sequence as shown in SEQ ID NO.1；(2) by SEQ
Sequence shown in ID NO.1 forms；Or (3) are that can be matched by template of smoke tree wilt genomic DNA with primer P2
Go out first round amplified production as primer amplification；Primer P2：(1) include the sequence as shown in SEQ ID NO.2；(2) by SEQ
Sequence shown in ID NO.2 forms；Or (3) are that can be matched by template of smoke tree wilt genomic DNA with primer P1
Amplify the first round amplified production；Wherein, the first round amplified production is sequence and SEQ shown in SEQ ID NO.1
The sequence that the pairing of sequence shown in ID NO.2 is obtained as template by PCR amplification as primer and using smoke tree wilt genomic DNA
Row, such as sequence shown in SEQ ID NO.5；Also, primer PN1：(1) include the sequence as shown in SEQ ID NO.3；(2)
Sequence forms shown in SEQ ID NO.3；Or (3) be can be with the first round amplified production (or smoke tree wilt
Genomic DNA) it is that template goes out the second wheel amplified production with primer PN2 pairings as primer amplification；Primer PN2：(1) include such as
Sequence shown in SEQ ID NO.4；(2) sequence shown in SEQ ID NO.4 forms；Or (3) be can be with described first
Wheel amplified production is that template amplifies the second wheel amplified production with primer PN1 pairings；Wherein, the second wheel amplified production
Production is expanded for the pairing of sequence shown in the sequence shown in SEQ ID NO.3 and SEQ ID NO.4 as primer and with the first round
Object (or smoke tree wilt genomic DNA) is the sequence that template is obtained by PCR amplification, such as shown in SEQ ID NO.6
Sequence.
In some embodiments, the kit further includes：(3) PCR reaction buffers；(4)dNTPs；(5) Taq enzyme；
And/or (6) sterile water.It is further preferred that the kit further includes：(3) PCR reaction buffers；(4)dNTPs；(5)Taq
Enzyme；(6) sterile water.
In some preferred embodiments, the PCR amplification that the PCR reaction buffers are 10 to 50 times preferably 10 times
With concentration (with PCR amplification densimeter)；It is further preferred that the PCR reaction buffers contain Tris-HCl, MgCl2And KCl.
It is further preferred that the composition of the PCR reaction buffers is as follows：Tris-HCl buffer solution, 15mMs of the pH of 100mM for 8.3
MgCl2With 500mM KCl.
In some embodiments, the dNTPs (i.e. dATP, dCTP, dGTP and dTTP equimolar mixture) is concentration
DNTPs mixed liquors (being, for example, 2.5 to 20mmol/L) not less than 2.5mmol/L；The Taq enzyme is that concentration is not less than 5U/ μ L
Taq enzyme solution (be, for example, 5 to 50U/ μ L)；Described primer P1, P2, PN1 and PN2 are each independently concentration and are not less than 10 μ
The primer solution of mol/ (being, for example, 10 to 50mmol/L).
In certain embodiments, the kit includes：(1) 10 × PCR reaction buffers, described 10 ×
The composition of PCR reaction buffers is as follows：100mM Tris-HCl buffer solutions (pH8.3), 15mM MgCl2、500mM KCl；(2)
2.5mmol/L dNTPs mixed liquors；(3) 5U/ μ L Taq enzymes；First round primer, that is, primer the P1 and primer P2 of (4) 10 μm of ol/L,
Wherein the sequence of P1 is the sequence as shown in SEQ ID NO.1, and the sequence of P2 is the sequence as shown in SEQ ID NO.2；(5)10μ
Second wheel primer, that is, primer PN1 and primer PN2 of mol/L, the wherein sequence of primer PN1 is the sequence as shown in SEQ ID NO.3,
The sequence of PN2 is the sequence as shown in SEQ ID NO.4；(6) sterile water, such as sterile purified water (such as sterilizing distilled water).
Certainly, as long as the concentration and pH that can be diluted to needed for amplification system, these reagents and Tris- in kit
The pH of HCl buffer solutions can also be other concentration or pH.
Second aspect of the present invention provides the kit described in first aspect present invention in detection smoke tree wilt
Application in (Verticillium dahliae).
Third aspect present invention provides a kind of method of detection smoke tree wilt (Verticillium dahliae),
Wherein, the method is detected using the kit described in first aspect.
In some embodiments, described method includes following steps：(1) DNA is extracted from measuring samples, obtains sample
DNA；(2) using the sample DNA as template, using in the kit the primer P1 and primer P2 carry out the as primer
One wheel PCR amplification, obtains first round amplified production；(3) using the first round amplified production as template, in the kit
The primer PN1 and primer PN2 as primer carry out second wheel PCR amplification, obtain the second wheel amplified production；(4) institute is detected
It states and whether occurs target stripe (sometimes referred to as target product or target amplification product) in the second wheel amplified production, and according to mesh
The presence or absence of mark band judges to whether there is smoke tree wilt in the sample.
In certain embodiments, described method includes following steps：
(1) extraction of measuring samples DNA：Measuring samples about 0.25g is taken, liquid nitrogen grinding is added, then extracts DNA, obtains
Sample DNA；
(2) first round PCR amplification：PCR expansions are carried out using the sample DNA as template DNA using primer P1 and primer P2
Increase, PCR reaction systems are：React 25 μ L of total volume：1 μ L of template DNA, 0.2 μ L of 5U/ μ L Taq enzymes, 10 × PCR reaction bufferings
Each 1 μ L of primer P1 and primer P2 of 2.5 μ L of liquid, 2.0 μ L of 2.5mmol/L d NTPs mixed liquors, 10 μm of ol/L, aseptic double-distilled water
Complement to 25 μ L；By reaction mixture in being expanded in PCR instrument, response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation
30s, 56 DEG C of annealing 40s, 72 DEG C of extension 60s, 30 recycle；72 DEG C of extension 10min, obtain first round amplified production；
(3) second wheel PCR amplifications：Take first round amplified production described in 1 μ L as the second wheel template DNA, with primer PN1 and
Primer PN2 is the second wheel PCR amplification primer, and PCR reaction systems are：React 25 μ L of total volume：1 μ L of template DNA, 5U/ μ L Taq
The primer PN1 of 0.2 μ L of enzyme, 10 × PCR reaction buffers, 2.5 μ L, 2.0 μ L of 2.5mmol/L d NTPs mixed liquors, 10 μm of ol/L
With each 1 μ L of primer PN2, aseptic double-distilled water complements to 25 μ L；By reaction mixture in being expanded in PCR instrument, response procedures are：
94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 56 DEG C of annealing 40s, 72 DEG C of extension 60s, 30 recycle；72 DEG C of extension 10min, obtain
To the second wheel amplified production；Take 5 μ L second wheel amplified production electrophoresis 20min, electrophoretic voltage in 1.0% Ago-Gel be
150V is imaged using gel imaging system, and the DNA bands observed in gel then judge institute if there is the DNA bands of 176bp
It detects and contains smoke tree wilt in sample, it is on the contrary then be not present.
In the methods of the invention, measuring samples are not particularly limited, as long as DNA can be extracted therefrom.The sample
Product are, for example, pedotheque or Plant tissue samples, such as the root of the seed of plant, plant seedlings, stem and leaf；Adult plant
Organs or its hetero-organization such as root, stem, leaf, flower, fruit.
DNA extraction method may be used known method those skilled in the art and can select to use according to different samples
Method appropriate, such as in the case where measuring samples are pedotheque, such as Tiangeng soil extracting genome DNA may be used
Kit (DP336) is extracted according to specification therein.It, can in the case where measuring samples are plant tissue such as blade
To be extracted according to specification therein using high-efficiency plant genome DNA extracting reagent kit (DP350).
Embodiment
Hereafter by by the form of embodiment, invention is further explained.But these embodiments are only for example
Illustration purpose.Protection scope of the present invention is not limited to these examples.
Embodiment 1：The nest-type PRC detection of smoke tree wilt in plant rhizosphere soil
Smoke tree Fusarium oxysporum in soil is detected.Pedotheque includes：(1) normal pure forest smoke tree rhizosphere soil；(2)
Morbidity pure forest smoke tree rhizosphere soil；(3) smoke tree rhizosphere soil under arbor-vitae；(3) Spiraea trilobata rhizosphere soil；(4) soybean rhizosphere
Soil；(5) Sabina vulgaris Ant rhizosphere soil.Each pedotheque detects three parts.It is as follows：
The extraction of plant rhizosphere soil DNA
It takes pedotheque about 0.25g, after liquid nitrogen grinding, is said according to Tiangeng soil genome DNA extracting reagent kit (DP336)
The operating procedure of bright book extracts DNA, obtains DNA sample.
First round PCR amplification
Using primer P1 and primer P2 (particular sequence see the table below) to DNA sample (as template DNA) for extracting in (1) into
Row amplification, PCR reaction systems are：React 25 μ L of total volume：1 μ L of template DNA, 0.2 μ L of 5U/ μ L Taq enzymes, 10 × PCR are anti-
Answer buffer solution (comprising 100mM pH be 8.3 Tris-HCl buffer solutions, 15mM MgCl2 and 500mM KCl) 2.5 μ L,
Each 1 μ L of primer P1 and P2 of 2.0 μ L of 2.5mmol/L d NTPs mixed liquors, 10 μm of ol/L, aseptic double-distilled water complement to 25 μ L.It will
Reaction mixture is expanded on BIOMETRA T-Gradient grads PCR instrument, and response procedures are：94 DEG C of pre-degeneration 5min；
94 DEG C of denaturation 30s, 56 DEG C of annealing 40s, 72 DEG C of extension 60s, 30 recycle；72 DEG C of extension 10min；Thus first round product is obtained
(particular sequence see the table below).
Second wheel PCR amplification
Take 1 μ L first round product as the template DNA of the second wheel PCR amplification, with primer PN1 and primer PN2 (particular sequences
See the table below) it is the second primer for taking turns PCR amplification, PCR reaction systems are：React 25 μ L of total volume：1 μ L of template DNA, 5U/ μ L
0.2 μ L of Taq enzyme, 10 × PCR reaction buffers (comprising 100mM pH be 8.3 Tris-HCl buffer solutions, 15mM MgCl2 and
500mM KCl) 2.5 μ L, 2.0 μ L of 2.5mmol/L d NTPs mixed liquors, 10 μm of ol/L primer PN1 and PN2 each 1 μ L, it is sterile
Distilled water complements to 25 μ L.Reaction mixture is expanded on BIOMETRA T-Gradient grads PCR instrument, reaction interval
Sequence is：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 56 DEG C of annealing 40s, 72 DEG C of extension 60s, 30 recycle；72 DEG C of extensions
10min；Thus obtain the second wheel product (particular sequence see the table below).
The sequence of the sequence and first round product AP1 of the primer that 1 two-wheeled PCR amplification of table uses and the second wheel product
AP2。
Detected through gel electrophoresis
The first round product of 5 μ L and 5 μ L second is taken to take turns product in 1.0% Ago-Gel (nucleic acid containing Goldview respectively
Dyestuff) in electrophoresis 20min (150V), gel imaging system imaging, observe band, if there are the mesh of 324bp for first round product
Mark band or the second wheel product then judge to contain smoke tree wilt in detected sample there are the target stripe of 176bp, on the contrary
If the target stripe of 324bp is not present in first round product, and also there is no judge if the target stripe of 176bp for the second wheel product
There is no the bacterium.
The electrophoresis result of first round product is as shown in Figure 1.In figure, M：Nucleic acid molecular weight D2000；1-3：Normal pure forest smoke tree
Rhizosphere soil；4-6：Morbidity pure forest smoke tree rhizosphere soil；7-9：Smoke tree rhizosphere soil under arbor-vitae；10-12：Spiraea trilobata rhizosphere
Soil；13-15：Soybean rhizosphere；16-18：Sabina vulgaris Ant rhizosphere soil；19：Negative control (distilled water)；20：Positive control
(smoke tree wilt genomic DNA).As seen from Figure 1, only there is the target stripe of 324bp in positive control.
The electrophoresis result of second wheel product is as shown in Figure 2.In figure, M：Nucleic acid molecular weight D2000；1-3：Normal pure forest smoke tree
Rhizosphere soil；4-6：Morbidity pure forest smoke tree rhizosphere soil；7-9：Smoke tree rhizosphere soil under arbor-vitae；10-12：Spiraea trilobata rhizosphere
Soil；13-15：Soybean rhizosphere；16-18：Sabina vulgaris Ant rhizosphere soil；19：Positive control (smoke tree wilt genome
DNA)；20：Negative control (distilled water).As seen from Figure 2, occur in swimming lane 3,7,8,10 to 13 and 15,17 and 18
The target stripe of 176bp shows that there are smoke tree wilts for the corresponding sample of these swimming lanes.
Embodiment 2：The nest-type PRC detection of smoke tree wilt in plant leaf blade
The extraction of plant leaf blade DNA
Plant leaf blade sample about 0.25g is taken, after liquid nitrogen grinding, according to high-efficiency plant genome DNA extracting reagent kit
(DP350) operating procedure of specification extracts DNA, obtains DNA sample.
First round PCR amplification
PCR amplification, PCR reactants are carried out to the DNA sample of extraction using primer P1 and primer P2 (as template DNA)
System is：React 25 μ L of total volume：1 μ L of template DNA, 0.2 μ L of 5U/ μ L Taq enzymes, 10 × PCR reaction buffers (include 100mM
PH be 8.3 Tris-HCl buffer solutions, 15mM MgCl2 and 500mM KCl) 2.5 μ L, 2.5mmol/L d NTPs mixed liquors
Each 1 μ L of primer P1 and P2 of 2.0 μ L, 10 μm of ol/L, aseptic double-distilled water complement to 25 μ L.By reaction mixture in BIOMETRA
It is expanded on T-Gradient grads PCR instrument, response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 56 DEG C of annealing
40s, 72 DEG C of extension 60s, 30 cycles；72 DEG C of extension 10min；Obtain first round product.
Second wheel PCR amplification
It takes 1 μ L first round product as the second wheel template, is the second wheel primer, PCR reaction systems with primer PN1/PN2
For：React 25 μ L of total volume：1 μ L of template DNA, 0.2 μ L of 5U/ μ LTaq enzymes, 10 × PCR reaction buffers (include the pH of 100mM
Tris-HCl buffer solutions, 15mM MgCl2 and 500mM KCl for 8.3) 2.5 μ L, 2.0 μ of 2.5mmol/L d NTPs mixed liquors
L, each 1 μ L of primer PN1 and PN2 of 10 μm of ol/L, aseptic double-distilled water complement to 25 μ L.By reaction mixture in BIOMETRA T-
It is expanded on Gradient grads PCR instrument, response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 56 DEG C of annealing
40s, 72 DEG C of extension 60s, 30 cycles；72 DEG C of extension 10min；Obtain the second wheel product.
Detected through gel electrophoresis
The first round product and 5 μ L second wheel amplified productions for taking 5 μ L respectively (contain Goldview in 1.0% Ago-Gel
Nucleic acid dye) in electrophoresis 20min (150V), gel imaging system imaging, observe band, if there are 324bp for first round product
Target stripe or the second wheel product there are the target stripes of 176bp then to judge in detect sample containing smoke tree wilt,
If instead the target stripe of 324bp is not present in first round product, and also the target stripe of 176bp is not present then in the second wheel product
Judge that the bacterium is not present.
The electrophoresis result of first round product is as shown in Figure 3.In figure, M：Nucleic acid molecular weight D2000；1-3：Three-bristle cudrania leaf piece；4-
6：Leptopus Chinensis (Bunge) Pojarkova blade；7-9：Soybean leaves；10-12：Normal smoke tree blade；13-16：With the smoke tree blade of Sabina vulgaris Ant companion planting；
17：Negative control (distilled water)；18：Positive control (smoke tree wilt genomic DNA).As seen from Figure 3, only positive
There is the band of 324bp in control.
The electrophoresis result of second wheel product is as shown in Figure 4.In figure, M：Nucleic acid molecular weight D2000；1-3：Three-bristle cudrania leaf piece；4-
6：Leptopus Chinensis (Bunge) Pojarkova blade；7-9：Soybean leaves；10-12：Normal smoke tree blade；13-16：With the smoke tree blade of Sabina vulgaris Ant companion planting；
17：Negative control (distilled water)；18：Positive control (smoke tree wilt genomic DNA).As seen from Figure 4, in swimming lane 1
The target stripe for 176bp occur to 13 shows that there are smoke tree wilts for the corresponding sample of these swimming lanes.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, it will be understood by those of ordinary skill in the art that：It still may be used
With technical scheme described in the above embodiments is modified or equivalent replacement of some of the technical features；
And these modifications or replacements, various embodiments of the present invention technical solution that it does not separate the essence of the corresponding technical solution spirit and
Range.
Sequence table
<110>Ornamental Plants of Beijing District research institute
<120>A kind of nest type PCR detection reagent and its application in the detection of smoke tree wilt
<130> GY17100352
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>It is artificial synthesized
<400> 1
catcagtctc tctgtttata ccaac 25
<210> 2
<211> 26
<212> DNA
<213>It is artificial synthesized
<400> 2
cgatgcgagc tgtaactact acgcaa 26
<210> 3
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 3
ccactgcttt taagggccta 20
<210> 4
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 4
cagcgaaacg cgatatgtag 20
<210> 5
<211> 324
<212> DNA
<213>Smoke tree wilt (Verticillium dahliae)
<400> 5
cgatgcgagc tgtaactact acgcaaggaa gggccacgcg ggtccgccac tgcttttaag 60
ggcctacaga cgtagatccc caacaccggg ccactggggc tcgagggttg aaacgacgct 120
cggacaggca tgcctcccag gatactggaa ggcgccatgt gcgttcaaag attcgatgat 180
tcactgaatt ctgcaattca cactacatat cgcgtttcgc tgcgttcttc atcgatgcta 240
gagccaagag atccgttgtt aaaagtttta atagttcgct aagaacactc agaagtatcg 300
ttggtataaa cagagagact gatg 324
<210> 6
<211> 176
<212> DNA
<213>Smoke tree wilt (Verticillium dahliae)
<400> 6
ccactgcttt taagggccta cagacgtaga tccccaacac cgggccactg gggctcgagg 60
gttgaaacga cgctcggaca ggcatgcctc ccaggatact ggaaggcgcc atgtgcgttc 120
aaagattcga tgattcactg aattctgcaa ttcacactac atatcgcgtt tcgctg 176
Claims (9)
1. a kind of nest type PCR detection reagent, which is characterized in that the kit includes：
(1) the primer P1 and primer P2 of first round PCR amplification are used for；
(2) the primer PN1 and primer PN2 of the second wheel PCR amplification are used for；
Wherein, PCR amplification is being carried out using smoke tree wilt (Verticillium dahliae) genomic DNA as template
When, the primer P1 has and the primer sequence phase as shown in SEQ ID NO.1 when with primer P2 pairings as primer
The same function as primer；Using smoke tree wilt (Verticillium dahliae) genomic DNA as template into
When row PCR amplification, the primer P2 has when with primer P1 pairings as primer to be drawn with as shown in SEQ ID NO.2
The identical function as primer of object sequence；And
It is described when carrying out PCR amplification using smoke tree wilt (Verticillium dahliae) genomic DNA as template
Primer PN1 has and the primer as shown in SEQ ID NO.3 when carrying out PCR amplification as primer with primer PN2 pairings
The identical function as primer of sequence, the primer PN2 have when carrying out PCR amplification as primer with primer PN1 pairings
With the identical function as primer of primer sequence as shown in SEQ ID NO.4.
2. kit according to claim 1, it is characterised in that：
The primer P1 is that can be matched by template of the smoke tree wilt genomic DNA as primer with the primer P2
Go out first round amplified production by PCR amplification, and the primer P2 is that can be with the smoke tree wilt genomic DNA
Template is matched with the primer P1 goes out the first round amplified production by PCR amplification；Wherein, the first round amplified production is
The pairing of sequence shown in sequence and SEQ ID NO.2 shown in SEQ ID NO.1 is as primer and with the smoke tree wilt base
Because group DNA is the sequence that template is obtained by PCR amplification；The primer PN1 can be using the first round amplified production as template
It is matched as primer with the primer PN2 and the second wheel amplified production is gone out by PCR amplification, and the primer PN2 can be with institute
It is that template goes out the second wheel amplified production with primer PN1 pairings by PCR amplification to state first round amplified production；Wherein,
The second wheel amplified production is that sequence pairing shown in sequence and SEQ ID NO.4 shown in SEQ ID NO.3 is used as primer simultaneously
The sequence obtained by PCR amplification as template using the first round amplified production；
Preferably, the primer P1 includes the sequence as shown in SEQ ID NO.1, more preferably shown in SEQ ID NO.1
Sequence composition；And/or the primer P2 includes the sequence as shown in SEQ ID NO.2, more preferably by SEQ ID NO.2 institutes
The sequence composition shown；And/or the primer PN1 includes the sequence as shown in SEQ ID NO.3, more preferably by SEQ ID
Sequence shown in NO.3 forms；And/or the primer PN2 includes the sequence as shown in SEQ ID NO.4, more preferably by SEQ
Sequence shown in ID NO.4 forms.
3. kit according to claim 1, which is characterized in that the kit further includes：
(3) PCR reaction buffers；
(4)dNTPs；
(5) Taq enzyme；
(6) sterile water.
4. according to the method described in claim 2, it is characterized in that：
The PCR reaction buffers are 10 times of PCR amplification concentration；It is further preferred that the PCR reaction buffers contain
Tris-HCl、MgCl2And KCl；Preferably, the composition of the PCR reaction buffers is as follows：The pH of 100mM is 8.3
Tris-HCl buffer solutions, 15mM MgCl2With 500mM KCl.
5. kit according to claim 3, it is characterised in that：
The dNTPs is the dNTPs mixed liquors that concentration is not less than 2.5mmol/L；
The Taq enzyme is the Taq enzyme solution that concentration is not less than 5U/ μ L；
Described primer P1, P2, PN1 and PN2 are each independently the primer solution that concentration is not less than 10 μm of ol/.
6. the kit described in any one of claim 1 to 5 is in detection smoke tree wilt (Verticillium
Dahliae the application in).
7. a kind of method of detection smoke tree wilt, which is characterized in that the method is using any one of claim 1 to 5
The kit is detected.
8. according to the method described in claim 8, it is characterized in that, described method includes following steps：
(1) DNA is extracted from measuring samples, obtains sample DNA；
(2) using the sample DNA as template, using in the kit the primer P1 and primer P2 carry out the as primer
One wheel PCR amplification, obtains first round amplified production；
(3) using the first round amplified production as template, using in the kit the primer PN1 and primer PN2 as
Primer carries out the second wheel PCR amplification, obtains the second wheel amplified production；
(4) it detects and whether occurs target stripe in the second wheel amplified production, and according to described in the judgement of the presence or absence of target stripe
It whether there is smoke tree wilt in sample.
9. the method according to the description of claim 7 is characterized in that described method includes following steps：
(1) extraction of measuring samples DNA
Measuring samples about 0.25g is taken, liquid nitrogen grinding is added, then extracts DNA, obtains sample DNA, wherein the measuring samples are
Pedotheque or plant leaf blade sample；
(2) first round PCR amplification
PCR amplification is carried out using the sample DNA as template DNA using primer P1 and primer P2, PCR reaction systems are：Reaction
25 μ L of total volume：1 μ L of template DNA, 0.2 μ L of 5U/ μ L Taq enzymes, 10 × PCR reaction buffers, 2.5 μ L, 2.5mmol/L
Each 1 μ L of primer P1 and primer P2 of 2.0 μ L of dNTPs mixed liquors, 10 μm of ol/L, aseptic double-distilled water complement to 25 μ L；Reaction is mixed
Liquid is closed in being expanded in PCR instrument, response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C denaturation 30s, 56 DEG C annealing 40s, 72 DEG C
Extend 60s, 30 cycles；72 DEG C of extension 10min, obtain first round amplified production；
(3) second wheel PCR amplifications
It takes first round amplified production described in 1 μ L as the second wheel template DNA, is that the second wheel PCR expands with primer PN1 and primer PN2
Increase primer, PCR reaction systems are：React 25 μ L of total volume：1 μ L of template DNA, 0.2 μ L of 5U/ μ L Taq enzymes, 10 × PCR reactions
Primer PN1 and primer PN2 each 1 μ L of 2.5 μ L of buffer solution, 2.0 μ L of 2.5mmol/L dNTPs mixed liquors, 10 μm of ol/L, it is sterile double
It steams water and complements to 25 μ L；By reaction mixture in being expanded in PCR instrument, response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C of changes
Property 30s, 56 DEG C annealing 40s, 72 DEG C extension 60s, 30 cycle；72 DEG C of extension 10min, obtain the second wheel amplified production；
(4) detected through gel electrophoresis
Take 5 μ L second take turns amplified production the electrophoresis 20min in 1.0% Ago-Gel, electrophoretic voltage 150V, using gel at
As system imaging, the DNA bands observed in gel then judge to contain in detected sample if there is the DNA bands of 176bp
Smoke tree wilt, it is on the contrary then be not present.
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| CN101698882A (en) * | 2009-11-12 | 2010-04-28 | 黑龙江省科学院微生物研究所 | Method for checking cucumber fusarium axysporum molecules |
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| CN108018214A (en) * | 2017-12-06 | 2018-05-11 | 中国农业科学院棉花研究所 | The method for separating verticillium dahliae in soil |
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2018
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101698882A (en) * | 2009-11-12 | 2010-04-28 | 黑龙江省科学院微生物研究所 | Method for checking cucumber fusarium axysporum molecules |
| CN104630371A (en) * | 2015-02-13 | 2015-05-20 | 南京林业大学 | Molecular detection method for elm blight pathogen |
| CN108018214A (en) * | 2017-12-06 | 2018-05-11 | 中国农业科学院棉花研究所 | The method for separating verticillium dahliae in soil |
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| Title |
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| J. MERCADO-BLANCO等: "Detection of the nondefoliating pathotype of Verticillium dahliae in infected olive plants by nested PCR", 《PLANT PATHOLOGY》 * |
| PAULINA KUCHTA等: "THE SUITABILITY OF PCR-BASED TECHNIQUES FOR DETECTING Verticillium dahliae IN STRAWBERRY PLANTS AND SOIL", 《JOURNAL OF FRUIT AND ORNAMENTAL PLANT RESEARCH》 * |
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Application publication date: 20180724 |