CN106350598A - PCR primer pair, kit and method for early warning of Citrus Huanglongbing - Google Patents
PCR primer pair, kit and method for early warning of Citrus Huanglongbing Download PDFInfo
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- 241000207199 Citrus Species 0.000 title claims abstract description 62
- 235000020971 citrus fruits Nutrition 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 38
- 241001478315 Candidatus Liberibacter asiaticus Species 0.000 title claims abstract description 37
- 238000001514 detection method Methods 0.000 claims abstract description 35
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 6
- 244000052616 bacterial pathogen Species 0.000 claims description 29
- 238000012408 PCR amplification Methods 0.000 claims description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 14
- 239000000463 material Substances 0.000 claims description 13
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 238000001962 electrophoresis Methods 0.000 claims description 9
- 230000004087 circulation Effects 0.000 claims description 8
- 238000013399 early diagnosis Methods 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 6
- 238000012856 packing Methods 0.000 claims description 6
- 230000009182 swimming Effects 0.000 claims description 6
- 238000000137 annealing Methods 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 230000007850 degeneration Effects 0.000 claims description 4
- 238000001502 gel electrophoresis Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000007400 DNA extraction Methods 0.000 claims description 2
- 210000001367 artery Anatomy 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 20
- 241000196324 Embryophyta Species 0.000 abstract description 9
- 238000003745 diagnosis Methods 0.000 abstract description 7
- 230000002265 prevention Effects 0.000 abstract description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract 1
- 239000003085 diluting agent Substances 0.000 description 12
- 201000010099 disease Diseases 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 238000007689 inspection Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 241001672694 Citrus reticulata Species 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
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- 238000013461 design Methods 0.000 description 3
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- 239000012634 fragment Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
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- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 238000002887 multiple sequence alignment Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002420 orchard Substances 0.000 description 2
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- -1 /or Species 0.000 description 1
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- 239000008272 agar Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
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- 102000036639 antigens Human genes 0.000 description 1
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- 238000009371 citriculture Methods 0.000 description 1
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- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
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- 238000011068 loading method Methods 0.000 description 1
- LLLILZLFKGJCCV-UHFFFAOYSA-M n-methyl-n-[(1-methylpyridin-1-ium-4-yl)methylideneamino]aniline;methyl sulfate Chemical compound COS([O-])(=O)=O.C=1C=CC=CC=1N(C)\N=C\C1=CC=[N+](C)C=C1 LLLILZLFKGJCCV-UHFFFAOYSA-M 0.000 description 1
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- 239000011550 stock solution Substances 0.000 description 1
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- 238000012795 verification Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
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Abstract
The invention discloses a PCR primer pair, a kit and a method for early warning of Citrus Huanglongbing, and belongs to plant molecule detection techniques. The nucleotide sequences of the primer pair are as follows: an upstream primer HLBF468: 5'-GCGATTAAGTTAGAGGTGA-3'; and a downstream primer HLBR877: 5'-TACCATCTCTGATATCGTCCTATA-3'. Ultra-high sensitivity can be achieved by using a common PCR method with the primers; and based on the primer pair, the invention further provides a kit and a detection method which are simple and feasible to operate, good in specificity, ultra-high in sensitivity, greatly improved in detection diagnosis efficiency and applicable to early warning and prevention of the Citrus Huanglongbing.
Description
Technical field
The invention belongs to plant molecular detection technique is and in particular to be used for the pcr primer of Citrus Huanglongbing pathogen early warning, examination
Agent box and method.
Background technology
Citrus Huanglongbing pathogen (citrus huanglongbing, hlb) is the most destructive citrus disease of world wide, gives
Orange Producing causes huge loss, is the great plant quarantine disease of domestic and international Citrus main production country.Citrus HUANGLONG in 1994
The cause of disease of disease is confirmed as " candidatusliberibacter spp. ", is that one kind is limited to sieve tissue endoparasitism, by Citrus chachiensis Hort.
The louse-borne gram negative bacteria of Fructus Citri tangerinae wood.Citrus Huanglongbing pathogen occurs in most of Citrus producing region of China, seriously restricts China
The existence of Citrus Industry and sound development.According to incompletely statistics, China is every year because the yellow twig underproduction is more than 750,000 tons, direct economy
Loss is more than 1,500,000,000 yuan.In 19 citriculture provinces of China, area, existing 11 endangered by yellow twig, and injured area exceedes Citrus chachiensis Hort.
The 80% of the gross area cultivated by Fructus Citri tangerinae.Meanwhile, developing rapidly with international trade, tourism and traffic, HUANGLONG pathogenic bacteria is from external epidemic-stricken area
The danger of invasion China also increasingly increases, and after invasion, easily outburst is caused disaster and led to huge economic loss, its quarantine detection
Particularly efficiently the demand of quick detection is more urgent.
At present, the method that predominantly detects of Citrus Huanglongbing pathogen bacterium candidatusliberibacter spp. has symptomatic diagnosises
Method, electron microscopic observation method, serological method and pcr method.Because Citrus Huanglongbing pathogen symptom is complex, with various plants nutritional deficiency disease phase
Seemingly, according to the diagnostic method of symptom, there is in the early detection of yellow twig significant limitation, be unfavorable for the early stage of yellow twig
Preventing and treating.And electron microscopic observation method is due to being affected to make greatly recall rate relatively low by objective factors such as sample collecting and process, only
60%～70%.Serological method is numerous and diverse due to antibody preparation process, and obtained antibody can only be with immunizing antigen disease locality
Former react, easily cause false negative, thus be not suitable for not widely using.Pcr detection method be a kind of with a high credibility, can batch
Carry out, qualitative and quantitative analysis can not only be carried out to the cause of disease of the plant tissue of aobvious disease moreover it is possible to not showing the vegetable material of disease, Jie
Pathogen in body insecticide is detected, has become one of reliable method of Citrus Huanglongbing pathogen diagnosis at present.Currently reported
Yellow twig pcr detection technique mainly include conventional pcr method, nido pcr method and real time fluorescent quantitative pcr method.
The conventional pcr detection method of HUANGLONG pathogenic bacteria has simple and quick, testing cost than other pcr detection methods
Low feature, generally can complete the qualitative detection to yellow twig, be applied to the quick of HUANGLONG pathogenic bacteria nearly ten years
Detection, becomes the common method of test in laboratory HUANGLONG pathogenic bacteria.From the point of view of currently reported, overwhelming majority routine pcr detection
Method establishes method for quick just for HUANGLONG pathogenic bacteria, or but lacking data explanation in terms of sensitivity, or reaching
Insufficient sensitivity high, for example Hu Hao in 2006 etc. " Scientia Agricultura Sinica " deliver " the conventional pcr of citrus yellow shoot disease and
Fluorescent quantitation pcr detects " middle report, its detection sensitivity arrival 0.439pg/ μ l.
For the infected plant not showing disease, because yellow twig pathogenic bacteria is Citrus plant in-vivo content is relatively low and skewness
Even, therefore in pcr template, the dna content of yellow twig pathogenic bacteria is extremely low, and this frequently results in detection false negative, the standard of impact early warning
Really property, is unfavorable for the early prevention and treatment of HUANGLONG pathogenic bacteria, therefore, the sensitivity of pcr method is more high more is conducive to the accurate of early warning
Property.
Content of the invention
The above-mentioned HUANGLONG pathogenic bacteria detection accuracy being existed based on above-mentioned field this area is low, false negative, missing inspection easily, no
The problems such as beneficial to Citrus Huanglongbing pathogen early prevention and treatment, the present invention is provided to the pcr primer of Citrus Huanglongbing pathogen early warning, test kit
And method, even if only with conventional pcr method and apparatus, also enabling the sensitivity of superelevation, being very suitable for early warning
Diagnosis.
Technical scheme is as follows:
For the pcr primer pair of Citrus Huanglongbing pathogen early warning, its nucleotide sequence is as follows:
Forward primer hlbf468:5`-gcgattaagttagaggtga-3 '；
Downstream primer hlbr877:5`-taccatctctgatatcgtcctata-3 '.
Test kit for Citrus Huanglongbing pathogen early warning is it is characterised in that include described pcr primer pair.
Further, also include carrying out the required conventional reagent of pcr reaction, and/or the conventional reagent needed for electrophoresis detection.
A kind of pcr method for Citrus Huanglongbing pathogen early warning is it is characterised in that comprise the steps:
(1) dna extraction is carried out to the Citrus material taking from area to be measured, obtain containing Citrus material dna and there may be
HUANGLONG pathogenic bacteria dna template to be measured；
(2) pcr amplification is carried out to described template to be measured using the primer pair described in claim 1；
(3) detected through gel electrophoresis pcr amplified production；If the target stripe of 433bp in swimming lane, the corresponding Citrus chachiensis Hort. of this swimming lane
It is set to HUANGLONG pathogenic bacteria at the beginning of Fructus Citri tangerinae material to carry or infected material.
Further, described pcr amplification reaction system is:
2 × taq pcr mastermix15 μ l, 10 μm of each 1 μ l of upstream and downstream primer hlbf468 and hlbr877, to be measured
Template 2 μ l, ddh2o 11 μ l.
The reaction condition of described pcr amplification is: 95 DEG C of denaturations 3min；With 95 DEG C of degeneration 30s, 55 DEG C annealing extend 30s,
72 DEG C extend 30s is 1 circulation, totally 40 circulations；72 DEG C of extension 5min.
Described Citrus material is Citrus leaf middle arteries.
A kind of method of detection HUANGLONG pathogenic bacteria (candidatusliberibacter spp.) it is characterised in that include as
Lower step:
(1) extract the dna of pathogenic bacteria to be measured as template to be measured；
(2) pcr amplification is carried out to described template to be measured using described primer pair；
(3) detected through gel electrophoresis pcr amplified production；If swimming lane appears as the target stripe of 433bp, pathogenic bacteria to be measured is yellow
Imperial pathogenic bacteria or contain HUANGLONG pathogenic bacteria.
Described pcr amplification reaction system is:
2 × taq pcr mastermix 15 μ l, 10 μm of each 1 μ l of upstream and downstream primer hlbf468 and hlbr877, to be measured
Template 2 μ l, ddh2O 11 μ l, and/or
The reaction condition of described pcr amplification is: 95 DEG C of denaturations 3min；With 95 DEG C of degeneration 30s, 55 DEG C annealing extend 30s,
72 DEG C extend 30s is 1 circulation, totally 40 circulations；72 DEG C of extension 5min.
The preparation method of mentioned reagent box is it is characterised in that in the packing box indicating Citrus Huanglongbing pathogen early diagnosiss purposes
Pcr primer pair described in inner packing claim 1.
Inventor retrieves HUANGLONG pathogenic bacteria candidatusliberibacterspp. in genbank data base and its belongs to together
Other strain sequence lengths are more than the 16s rdna DNA homolog sequence of 1000bp, 16s rdna gene a plurality of to 40 being obtained
Homologous sequence carries out Multiple Sequence Alignment with dnaman software, finds and select HUANGLONG pathogenic bacteria
Candidatusliberibacterspp. conserved positions, selected conserved positions can specifically be distinguished again and belong to other bacterium together
Kind, go out some candidate drugs in selected conserved positions engineer, using oligo software, these candidate drugs are commented
Estimate, and the primer higher to assessment fraction verify, selection can specifically be distinguished and belong to other strains together, dna organized by Citrus,
And the good primer of sensitivity.
In selected primer, the accidentally middle sensitivity finding one pair of which primer surpasss the expectation several times inventor, compiles
Number be hlbf468/hlbr877, respectively as shown in seq id no.1 and seq id no.2.As described in embodiment 2, this primer
To can to HUANGLONG pathogenic bacteria dna concentration be only 1.17 × 10-9The template amplification of ng/ μ l goes out detectable band, is existing report
3000 times of the sensitivity 0.439pg/ μ l of Hu Hao in 2006 et al. report, i.e. sensitivity improves more than 3000 times, this primer
Beyond high sensitivity unprecedented expected from this area, this high sensitivity is for detection Citrus Huanglongbing pathogen bacterium and Citrus
Significant for the early diagnosiss of yellow twig, can overcome because yellow twig pathogenic bacteria Citrus plant in-vivo content relatively low and be distributed
The uneven false negative causing and cause missing inspection, diagnostic error the problems such as, thus the early stage realizing Citrus Huanglongbing pathogen accurately examine
Disconnected, according to this early diagnosis, reject the seedling of bacteria infection early, in order to avoid causing further large area sense after plant adult onset
Dye, thus avoid causing large area to lose.
Based on described pcr primer pair hlbf468/hlbr877, present invention also offers a kind of yellow for efficient detection Citrus
The test kit of imperial pathogenic bacteria or early diagnosiss Citrus Huanglongbing pathogen and pcr method.
For improving the availability of test kit of the present invention, preferably further include the required conventional reagent of pcr amplification,
And/or the conventional reagent needed for electrophoresis.
On the other hand, the present invention by design of primers that pcr is detected and screening, optimize conventional pcr reaction system with anti-
Answer program, establish a kind of pcr detection method of efficient detection Citrus Huanglongbing pathogen bacterium, i.e. using above-mentioned pcr primer pair or reagent
Box, carries out pcr amplification and electrophoresis detection using conventional pcr detection meanss to Citrus genome dna.The pcr inspection of the present invention
Survey method can improve the sensitivity of conventional pcr detection method, improves than the domestic at present conventional pcr detection method having been reported that
More than 3000 times, it can be avoided that detection false negative situation, meet the needs of the early stage Accurate Diagnosis to Citrus Huanglongbing pathogen.
The present invention is also claimed the preparation production method of mentioned reagent box, the reagent based on commercial object of any scale
The production behavior of box both falls within protection scope of the present invention.Described production behavior includes, and uses indicating the detection of Citrus Huanglongbing pathogen bacterium
The pcr primer pair described in claim 1 is loaded in the packing box on way.Further, described production behavior is included in described packing box
The required conventional reagent of interior loading pcr amplification, and/or the conventional reagent needed for electrophoresis.
In sum, carry out identification and the diagnosis of Citrus Huanglongbing pathogen using the pcr primer pair that the present invention provides, simple to operate
Easy, high specificity, sensitivity superelevation, checkout and diagnosis efficiency can be greatly improved, more targetedly to cultivate healthy Citrus,
Generational loss is down to minimum.The present invention establishes with high sensitivity and blanket Citrus Huanglongbing pathogen Rapid identification simultaneously
Technical system, is HUANGLONG disease early diagnosis and prevention and control, protection China Citrus Industry develop in a healthy way and provide strong technical support.
Brief description
Fig. 1 is Citrus Huanglongbing pathogen specific primer the result figure.
Wherein, m:d2000marker；1-7: the newly infection yellow twig sample in meeting orchard；8: detoxic seedling sample；9: negative right
According to (ddh20).
Fig. 2 is Citrus Huanglongbing pathogen specific primer detection sensitivity result figure.
Wherein, m:d2000marker；1:pcr purified product stock solution (11.7ng/ μ l)；2:10 times of diluent；3:102Times
Diluent；4:103Times diluent；5:104Times diluent；6:105Times diluent；7:106Times diluent；8:107Times diluent；9:
108Times diluent；10:109Times diluent；11:1010Times diluent；12: negative control (ddh20).
Specific embodiment
Further describe product of the present invention with reference to specific embodiment, but the present invention is not limited with this
Scope.If no special instructions, following adopted reagent are all commercially available with material, and the method being adopted is conventional behaviour
Make.
Biomaterial
Positive is newly infection yellow twig in orchard by Guangdong Kang Shi biotech inc Jiangmen city
Mandarin tree leaf tissue；
Negative sample is the Citrus detoxic seedling leaf tissue being provided by Guangzhou Qi Mei plant protection company limited.
Reagent consumptive material
Plant Genome dna extracts kit, a large amount of dna Product Purification Kit, 2 × taq pcr mastermix is equal
Purchased from TIANGEN Biotech (Beijing) Co., Ltd.；
Embodiment 1, the design of Citrus Huanglongbing pathogen specific primer
Retrieve HUANGLONG pathogenic bacteria candidatusliberibacter spp. and its belong to other together in genbank data base
Strain sequence length is more than 16s rdna DNA homolog sequence (40) of 1000 bp, to all 16s rdna bases being obtained
Because sequence carries out Multiple Sequence Alignment with dnaman software, find the guarantor of HUANGLONG pathogenic bacteria candidatusliberibacter spp.
Keep site, this conserved positions can specifically be different from again and belong to other strains together simultaneously, the site engineer selecting at this
Go out a series of, some primer pair is estimated to primer using oligo software, and carry out experimental verification one by one, final determine
Go out a pair available primer pair, as shown in table 1 below:
The specific primer of Citrus Huanglongbing pathogen identified by table 1
Above-mentioned primer pair upstream and downstream primer sequence corresponds to seq id no.1 and seq id no.2 respectively.
Above-mentioned target fragment length 433bp, sequence is as shown in seq id no.3.
Embodiment 2, test kit of the present invention
The present embodiment provides the test kit of a kind of efficient detection Citrus Huanglongbing pathogen bacterium and/or early diagnosiss Citrus Huanglongbing pathogen,
It includes the pcr primer pair described in embodiment 1, and the conventional reagent that pcr amplification is required, and/or the conventional examination needed for electrophoresis
Agent.
The required conventional reagent of described pcr amplification includes: pcr buffer, dntp, taq dna polymerase, te buffer,
Distilled water etc., or all kinds of pcr reaction mix mixed liquors containing taq polymerase of commercialization.
Conventional reagent needed for described electrophoresis includes: agar powder, distilled water, eb (ethidium bromide) etc..
Embodiment 3, the foundation of Citrus Huanglongbing pathogen Rapid identification system
(1) dna extracts:
Using " Plant Genome dna extracts kit " (TIANGEN Biotech (Beijing) Co., Ltd.), plant sample is entered
Row genome dna extracts, and concrete operations flow process, with reference to description, is verified for follow-up primer.
(2) primer specificity checking:
With above extracted dna as template, carry out pcr amplification using designed primer, anti-by continuous adjustment pcr
Answer reagent dosage and annealing temperature, time etc., determine final reaction system.
After amplified reaction terminates, the amplified production taking 5 μ l pcr special carries out electrophoresis inspection on 2.0% agarose gel
Survey, eb dyes 15 minutes, observed result under uviol lamp.Result is illustrated in fig. 1 shown below.
Result shows hlbf468/hlbr877 primer pair under the pcr reaction system and reaction condition of standard, to and only right
There is positive reaction in the Citrus tissue of all infection yellow twigs, produce specificity purpose band, be organized as the moon to healthy mandarin tree
Property reaction (no specific band generation), result such as Fig. 1.
(3) sensitivity technique:
Specificity bar will be obtained using " a large amount of dna Product Purification Kit " (TIANGEN Biotech (Beijing) Co., Ltd.)
, with carrying out product purification, concrete operations flow process is with reference to description for the pcr reactant liquor of band.
UsingNd-1000 spectrophotometer carries out the dna Concentration Testing of pcr product after purification, inspection
Survey result is 11.7 ng/ μ l, for follow-up sensitivity technique.
The pcr reaction purification product of concentration 11.7 ng/ μ l is serially diluted using te buffer, obtain 11 kinds dilute
Release liquid；Purpose fragment dna concentration in 11 kinds of diluents be not: 11.7 ng/ μ l, 1.17 ng/ μ l, 0.117 ng/ μ l,
1.17×10-2ng/μl、1.17×10-3ng/μl、1.17×10-4ng/μl、1.17×10-5ng/μl、1.17×10-6ng/μl、
1.17×10-7ng/μl、1.17×10-8Ng/ μ l and 1.17 × 10-9ng/μl.
Respectively with every kind of diluent as template (water is as negative control), with primer pair hlbf468/hlbr877 respectively to not
Template dna with concentration carries out pcr amplification.
Pcr reaction system and amplification condition are with (2).
After amplified reaction terminates, take 5ul pcr special amplified production that electrophoresis inspection is carried out on 2.0% agarose gel
Survey, eb dyes 15 minutes, observed result under uviol lamp.Result is illustrated in fig. 2 shown below.
Result shows that detectable template concentrations reach 1.17 × 10-9Ng/ μ l, than prior art (Hu Hao, 2006 " Citrus
The conventional pcr of yellow twig and fluorescent quantitation pcr detection ") the conventional pcr detection sensitivity 4.39pg/ μ l of report improves
More than 3000 times.
sequence listing
<110>Planning Design Inst., Ministry of Agricultry
<120>it is used for pcr primer, test kit and the method for Citrus Huanglongbing pathogen early warning
<130> p160618/nyb
<160> 3
<170> patentin version 3.5
<210> 1
<211> 19
<212> dna
<213> artificial sequence
<220>
<223>forward primer hlbf468
<400> 1
gcgattaagt tagaggtga 19
<210> 2
<211> 24
<212> dna
<213> artificial sequence
<220>
<223>downstream primer hlbr877
<400> 2
taccatctct gatatcgtcc tata 24
<210> 3
<211> 433
<212> dna
<213> artificial sequence
<220>
<223>target fragment
<400> 3
gcgattaagt tagaggtgaa atcccagggc tcaaccttgg aactgccttt aatactggtt 60
gtctagagtt taggagaggt gagtggaatt ccgagtgtag aggtgaaatt cgtagatatt 120
cggaggaaca ccggtggcga aggcggctca ctggcctgat actgacgctg aggcgcgaaa 180
gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 240
gctgttgggt ggtttaccat tcagtggcgc agctaacgca ttaagcactc cgcctgggga 300
gtacggtcgc aagattaaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 360
tgtggtttaa ttcgatgcaa cgcgcagaac cttaccagcc cttgacatgt ataggacgat 420
atcagagatg gta 433
Claims (10)
1. it is used for the pcr primer pair of Citrus Huanglongbing pathogen early warning, its nucleotide sequence is as follows:
Forward primer hlbf468:5`-gcgattaagttagaggtga-3 '；
Downstream primer hlbr877:5`-taccatctctgatatcgtcctata-3 '.
2. it is used for the test kit of Citrus Huanglongbing pathogen early warning it is characterised in that including the pcr primer pair described in claim 1.
3. test kit according to claim 2 it is characterised in that also include carries out the required conventional reagent of pcr reaction,
And/or the conventional reagent needed for electrophoresis detection.
4. a kind of pcr method for Citrus Huanglongbing pathogen early warning is it is characterised in that comprise the steps:
(1) dna extraction is carried out to the Citrus material taking from area to be measured, obtain and contain Citrus material dna and Huang that may be present
The template to be measured of imperial pathogenic bacteria dna；
(2) pcr amplification is carried out to described template to be measured using the primer pair described in claim 1；
(3) detected through gel electrophoresis pcr amplified production；If the target stripe of 433bp in swimming lane, this swimming lane corresponding Citrus material
Material is just set to HUANGLONG pathogenic bacteria and carries or infected material.
5. method according to claim 4 is it is characterised in that described pcr amplification reaction system is:
2 × taq pcr mastermix 15 μ l, 10 μm of each 1 μ l of upstream and downstream primer hlbf468 and hlbr877, template to be measured
2 μ l, ddh2o 11μl.
6. the method according to claim 4 or 5 is it is characterised in that the reaction condition of described pcr amplification is: 95 DEG C of pre- changes
Property 3min；Extend 30s, 72 DEG C of extension 30s with 95 DEG C of degeneration 30s, 55 DEG C of annealing for 1 circulation, totally 40 circulations；72 DEG C of extensions
5min.
7. method according to claim 4 is it is characterised in that described Citrus material is Citrus leaf middle arteries.
8. a kind of method of detection HUANGLONG pathogenic bacteria (candidatusliberibacter spp.) is it is characterised in that include as follows
Step:
(1) extract the dna of pathogenic bacteria to be measured as template to be measured；
(2) pcr amplification is carried out to described template to be measured using the primer pair described in claim 1；
(3) detected through gel electrophoresis pcr amplified production；If swimming lane appears as the target stripe of 433bp, pathogenic bacteria to be measured is yellow twig
Bacterium or contain HUANGLONG pathogenic bacteria.
9. method according to claim 8 is it is characterised in that described pcr amplification reaction system is:
2 × taq pcr mastermix 15 μ l, 10 μm of each 1 μ l of upstream and downstream primer hlbf468 and hlbr877, template to be measured
2 μ l, ddh2O 11 μ l, and/or
The reaction condition of described pcr amplification is: 95 DEG C of denaturations 3min；With 95 DEG C of degeneration 30s, 55 DEG C annealing extend 30s, 72 DEG C
Extending 30s is 1 circulation, totally 40 circulations；72 DEG C of extension 5min.
10. the preparation method of the test kit described in claim 2 is it is characterised in that use indicating Citrus Huanglongbing pathogen early diagnosiss
The pcr primer pair described in packing box inner packing claim 1 on way.
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Cited By (3)
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| CN109828019A (en) * | 2019-02-21 | 2019-05-31 | 南昌大学 | The method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen |
| CN112391493A (en) * | 2020-12-10 | 2021-02-23 | 浙江省检验检疫科学技术研究院 | RAA fluorescence detection method, primer probe and kit for citrus greening disease Asian species |
| CN115015462A (en) * | 2022-07-01 | 2022-09-06 | 广西特色作物研究院 | Citrus huanglongbing detection method based on LC-MS (liquid chromatography-mass spectrometry) non-targeted analysis |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109828019A (en) * | 2019-02-21 | 2019-05-31 | 南昌大学 | The method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen |
| CN112391493A (en) * | 2020-12-10 | 2021-02-23 | 浙江省检验检疫科学技术研究院 | RAA fluorescence detection method, primer probe and kit for citrus greening disease Asian species |
| CN115015462A (en) * | 2022-07-01 | 2022-09-06 | 广西特色作物研究院 | Citrus huanglongbing detection method based on LC-MS (liquid chromatography-mass spectrometry) non-targeted analysis |
| CN115015462B (en) * | 2022-07-01 | 2023-06-23 | 广西特色作物研究院 | Citrus yellow dragon disease detection method based on liquid chromatography-mass spectrometry non-targeted analysis |
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