CN109762912A - Identify the primer pair and its application of bactrocera tsuneonis and the big trypetid of tangerine - Google Patents
Identify the primer pair and its application of bactrocera tsuneonis and the big trypetid of tangerine Download PDFInfo
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Abstract
The invention discloses a kind of primer pairs and its application for identifying bactrocera tsuneonis and the big trypetid of tangerine.The present invention devises the primer pair of identification bactrocera tsuneonis and the big trypetid of tangerine, and develops the Molecular Identification technology based on Standard PCR.Proved by test: the Identification of Species to bactrocera tsuneonis and the big trypetid of tangerine may be implemented in the Molecular Identification technology of the invention based on Standard PCR, has the advantages that high specificity, high sensitivity, at low cost, easy to operate, time-consuming is short.The Identification of Species problem of non-adult form trypetid and trypetid adult residuum is not only solved, also the epidemic monitoring for bactrocera tsuneonis and the big trypetid of tangerine, the distribution in China citrus main producing region and invasion status and its subsequent plant quarantine and field, which are administered, provides practical technique and theoretical foundation.To effectively preventing its further invasion and diffusion, protection agricultural production and ecological safety, economic trade development in China's promoted to be of great significance.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to identification bactrocera tsuneonis and the big trypetid of tangerine primer pair and
It is applied.
Background technique
Anastrepha Diptera Tephritidae, it is widely distributed in the whole world, there are about 500 belong to 4,500 kinds, wherein have economic significance
Trypetid have 223 kinds of 26 category.The host range of trypetid is extensive, while causing harm the industrial crops such as water fruits and vegetables, also serious shadow
The foreign trade for ringing fruits and vegetables, causes trade barrier, leads to huge economic loss.Bactrocera tsuneonis Bactrocera
The tsuneonis and big trypetid B.minax of tangerine is 2 kinds of oligophagies of the big trypetid subgenus Tetradacus of Bactrocera Bactrocera
Trypetid, the sweet orange of the Rutaceae that only causes harm Citrus Citrus, bitter orange, Ponkan, citrus unshiu Marcovitch, lemon, citron, fingered citron, Fortunella
The cumquat of Fortunella, the citrus crop such as trifoliate orange of Poncirus Ponciru.Larva is the main worm state of citrus of causing harm, larva
Feeding pulp after hatching, leads to the part or all of fruit rot of citrus and at paste, it is made to have completely lost edible value and warp
Ji value；Or it causes the underdone first Huang of fruit and falls off.The moth fruit of the larva rate that causes harm is generally 5-20%, and some areas may be up to
50% or more, or even total crop failure.
The history of life of the big trypetid of tangerine and bactrocera tsuneonis is 1 year 1 generation, there are diapause phenomenon, not yet realization laboratory after
Generation raising is to obtain stable worm sources, therefore also constrain its biology, Physiology and biochemistry, science of heredity and control strategy etc.
Research.The big trypetid of tangerine is mainly distributed on part province (Chongqing, Guangxi, Guizhou, Hubei, Hunan, the Shan of Bhutan, India and China
West, Sichuan and Yunnan), and bactrocera tsuneonis only limits to a small number of provinces (Guangxi, Hunan, Sichuan, the cloud for being distributed in Japan and China
South), and it is listed in China's agricultural quarantine harmful organisms.It is mutually tied based on the place CLIMEX comparison model and ArcGIS interpolation analysis
The method of conjunction shows that all citrus planting areas in China are the big trypetid of tangerine and honey to the prediction result of its potential geographical distribution
The middle height normal region of the big trypetid of mandarin orange.Once big trypetid intrudes into new citrus planting area, disaster will be caused to Citrus Industry
The consequence of property.With climate change, the increase of the adjustment of pattern of farming and international trade, harm is gradually expanded, epidemic situation be in by
Aggravate trend year, bactrocera tsuneonis is arrived in trapping in 2016 in Guangdong Province Huaiji County, it was confirmed that it, which has, further invades diffusion
Trend.
Since the trypetid of intercept and capture is mostly extremely approximate non-adult worm state in form, generally need to be through raising in 1-2 weeks
Could be by precise Identification after adult, and bactrocera tsuneonis and the big trypetid of tangerine are even more to need 5-6 through aging pupate to sprouting wings of larva
The time of the moon, greatly reduce the timeliness of quarantine.Traditional trypetid identification work relies primarily on morphological feature, and 2
The formalness feature of kind big trypetid is quite similar, is only capable of that (the big trypetid of tangerine has 1 pair of scapulet by the hair on the neck sequence of adult mesonotum
Hair on the neck, preceding supraalar bristle lack such as；Bactrocera tsuneonis has 2 pairs of scapular bristles and 1-2 to preceding supraalar bristle) and the oopod feature of female adult (tangerine is big
Trypetid female adult oopod base pitch is longer, and long close etc. with the sum of 2-5 abdomen backboard length, end is slightly sharp；The oviposition of bactrocera tsuneonis female adult
Device base pitch is shorter, is about the sum of the 4th and the 5th abdomen backboard length, end tri-lobed) it is used as appraisal basis, this gives the shape of 2 kinds big trypetid
State identification causes extreme difficulties.With the development of international fruits and vegetables trade, the risk that Fruit fly is passed to China significantly increases
Add, the demand of quarantine identification especially Rapid identification is more urgent.
Whether Molecular Identification by trypetid sample ontogeny state and morphological feature due to not influenced completely, it is considered to be
Effective compensation process of Morphological Identification.The concept of DNA bar code is proposed by Hebert etc., it is believed that can be based on a segment standard
The short sequence of DNA effectively distinguishes different plant species, to realize the accurate Rapid identification of species, i.e. DNA bar code technology
(DNA barcoding)；And it was found that the cytochrome C oxidase subunit base I on mitochondrial genomes (mtDNA)
(cytochrome c oxidase subunit I, COI) gene the preceding paragraph length be 658bp sequence can be used for effectively into
The segment, is defined as the DNA bar code of animal by the identification of action object.In recent years, based on DNA bar code sequence design specificity
Primer, probe and the Standard PCR technology that develops, real-time fluorescent PCR technology, loop-mediated isothermal amplification technique LAMP are also widely applied
In the Molecular Identification of trypetid.Wherein polymerase chain reaction (Polymerase Chain Reaction, PCR) by Mullis in
It invents within 1985, is a kind of Protocols in Molecular Biology for selective amplification in vitro specific DNA fragments.One typical PCR
Process includes multiple " denaturation-annealing-extension " circulation, reaction system generally from DNA profiling, positive/negative (contain to primer, buffer
Mg2+Deng), dNTP and archaeal dna polymerase etc., i.e., it is single-stranded for being denaturalized at high temperature using DNA, primer and single stranded DNA mould when low temperature
Plate is combined by the principle of base pair complementarity, then is warming up to the optimal reactive temperature of archaeal dna polymerase, using dNTP as raw material, by alkali
The principle of base complementary pairing and semi-conservative replication extends the new DNA chain complementary with template DNA of synthesis along 5 ' -3 ' directions.It is based on
Standard PCR (species-specific PCR, SS-PCR) technology of species-specific primers, fully considers primer in design of primers
Species specificity, unknown species template DNA is expanded with species-specific primer, pass through the presence or absence of electrophoresis detection target fragment, identify
Target species and other species.Standard PCR technology is due to operating fast and convenient, high specificity, high sensitivity, saving the advantages that cost
It is widely applied in the Molecular Identification of trypetid.
Summary of the invention
The first purpose of the invention is to provide for identifying the primer set pair of tangerine big trypetid and bactrocera tsuneonis.
Primer set provided by the invention is formed to by primer pair first and primer pair B；
The primer pair first is made of Bm-F and Bm-R；
The primer pair B is made of Bt-F and Bt-R；
The Bm-F is following a1) or a2):
A1) single strand dna shown in sequence 1 in sequence table；
A2) there is phase by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1
The single strand dna of congenerous；
The Bm-R is following a3) or a4):
A3) single strand dna shown in sequence 2 in sequence table；
A4) there is phase by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The single strand dna of congenerous；
The Bt-F is following b1) or b2):
B1) single strand dna shown in sequence 3 in sequence table；
B2) there is phase by sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3
The single strand dna of congenerous；
The Bt-R is following b3) or b4):
B3) single strand dna shown in sequence 4 in sequence table；
B4) there is phase by sequence 4 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4
The single strand dna of congenerous.
Further, the primer pair first and the molar ratio of two single strand dnas in the primer pair B are 1:
1.Two equal independent packagings of single strand dna in the primer pair first and the primer pair B.
A second object of the present invention is to provide a kind of for identifying the kit of tangerine big trypetid and bactrocera tsuneonis.
Kit provided by the invention includes above-mentioned primer set pair.
Further, the kit may also include the reagent for carrying out standard PCR amplification, such as 2 × Taq PCR
MasterMix and ddH2O etc..
Third object of the present invention is to provide above-mentioned primer set pair or the new applications of mentioned reagent box.
The present invention provides above-mentioned primer sets pair or mentioned reagent box in following c1)-c8) in it is any in application:
C1) the product of preparation identification tangerine big trypetid and bactrocera tsuneonis；
C2) the big trypetid of identification tangerine and bactrocera tsuneonis；
C3) preparation identifies that trypetid to be measured is the product of the big trypetid of tangerine or bactrocera tsuneonis；
C4) identify that trypetid to be measured is the big trypetid of tangerine or bactrocera tsuneonis；
C5 the product of the epidemic monitoring of the big trypetid of tangerine and bactrocera tsuneonis) is prepared；
C6) the epidemic monitoring of tangerine big trypetid and bactrocera tsuneonis；
C7) the product of invasion and/or the diffusion of the big trypetid of preparation prevention tangerine and bactrocera tsuneonis；
C8) prevent the invasion and/or diffusion of tangerine big trypetid and bactrocera tsuneonis.
Fourth object of the present invention, which is to provide, a kind of identifies or assists to identify that trypetid to be measured is the big trypetid of tangerine or mandarin orange
The method of big trypetid.
It is provided by the invention to identify or assist to identify that trypetid to be measured includes for the method for the big trypetid of tangerine or bactrocera tsuneonis
Following steps: using the genomic DNA of trypetid to be measured as template, above-mentioned primer pair first is respectively adopted and primer pair B carries out PCR expansion
Increase；
If primer pair first PCR amplification obtains the target fragment that size is 422bp, trypetid to be measured is or candidate is the big reality of tangerine
Fly；
If primer pair B PCR amplification obtains the target fragment that size is 456bp, trypetid to be measured is or candidate is that mandarin orange is big
Trypetid.
Above-mentioned primer pair first is in following d1)-d8) in it is any in application also belong to protection scope of the present invention:
D1) the product of the preparation identification big trypetid of tangerine；
D2 the big trypetid of tangerine) is identified；
D3) preparation identify trypetid to be measured whether be the big trypetid of tangerine product；
D4) identify whether trypetid to be measured is the big trypetid of tangerine；
D5 the product of the epidemic monitoring of the big trypetid of tangerine) is prepared；
D6) the epidemic monitoring of the big trypetid of tangerine；
D7) the product of the invasion and/or diffusion of the preparation prevention big trypetid of tangerine；
D8) prevent the invasion and/or diffusion of the big trypetid of tangerine.
Above-mentioned primer pair B is in following e1)-e8) in it is any in application also belong to protection scope of the present invention:
E1) the product of preparation identification bactrocera tsuneonis；
E2 bactrocera tsuneonis) is identified；
E3) preparation identify trypetid to be measured whether be bactrocera tsuneonis product；
E4) identify whether trypetid to be measured is bactrocera tsuneonis；
E5 the product of the epidemic monitoring of bactrocera tsuneonis) is prepared；
E6) the epidemic monitoring of bactrocera tsuneonis；
E7) the product of invasion and/or the diffusion of preparation prevention bactrocera tsuneonis；
E8) prevent the invasion and/or diffusion of bactrocera tsuneonis.
Fifth object of the present invention is to provide it is a kind of identify or assist to identify trypetid to be measured whether be the big trypetid of tangerine side
Method.
It is provided by the invention identify or assist to identify trypetid to be measured whether be the big trypetid of tangerine method include the following steps: with
The genomic DNA of trypetid to be measured is template, carries out PCR amplification using above-mentioned primer pair first；
If PCR amplification obtains the target fragment that size is 422bp, trypetid to be measured is or candidate is the big trypetid of tangerine；
If PCR amplification does not obtain the target fragment that size is 422bp, trypetid to be measured is not or candidate is not the big reality of tangerine
Fly.
It identifies or assists to identify whether trypetid to be measured is bactrocera tsuneonis sixth object of the present invention is to provide a kind of
Method.
It is provided by the invention to identify or assist to identify whether trypetid to be measured is that the method for bactrocera tsuneonis includes the following steps:
Using the genomic DNA of trypetid to be measured as template, PCR amplification is carried out using above-mentioned primer pair B；
If PCR amplification obtains the target fragment that size is 456bp, trypetid to be measured is or candidate is bactrocera tsuneonis；
If PCR amplification does not obtain the target fragment that size is 456bp, trypetid to be measured is not or candidate is not the big reality of mandarin orange
Fly.
In any of the above-described the method, the target fragment that the size is 422bp is in the big trypetid mtDNA COI gene of tangerine
DNA bar code sequence 134-555 shown in DNA fragmentation；DNA bar shaped in the big trypetid mtDNA COI gene of tangerine
Code sequence is to carry out PCR amplification using the big trypetid genomic DNA of tangerine as template using universal primer LCO1490/HCO2198 and obtain
PCR product sequence.
The target fragment that the size is 456bp is the DNA bar code sequence in bactrocera tsuneonis mtDNA COI gene
DNA fragmentation shown in 140-595；DNA bar code sequence in the bactrocera tsuneonis mtDNA COI gene is with the big reality of tangerine
Fly genomic DNA is template, carries out the PCR product sequence that PCR amplification obtains using universal primer LCO1490/HCO2198.
In any of the above-described the method, the PCR reaction system by 3 μ l of DNA profiling, forward primer (100 μM) 1.5 μ l,
Reverse primer (100 μM) 1.5 μ l, 2 × Taq PCR MasterMix 25 μ l and ddH219 μ l of O composition.The forward primer and
The reverse primer is Bm-F and Bm-R in the primer pair first or Bt-F and Bt-R in the primer pair B.The Bm-
F, the final concentration of the Bm-R, the Bt-F and the Bt-R in the PCR reaction system is 0.3 μM.
The PCR reaction condition is as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 1min of 30 circulations, 65 DEG C of annealing are prolonged
Stretch 1min；65 DEG C of extension 10min.
In any of the above-described the method, the trypetid can be bactrocera tsuneonis and/or the big trypetid of tangerine.The trypetid specifically may be used
For the ovum of the trypetid, larva, pupa and/or adult.
7th purpose of the invention is to provide the DNA bar code sequence in the big trypetid mtDNA COI gene of above-mentioned tangerine
DNA fragmentation shown in 134-555 or the DNA bar code sequence 140-595 in above-mentioned bactrocera tsuneonis mtDNA COI gene
DNA fragmentation shown in position.
DNA fragmentation shown in DNA bar code sequence 134-555 in the above-mentioned big trypetid mtDNA COI gene of tangerine and/
Or DNA fragmentation shown in DNA bar code sequence 140-595 in bactrocera tsuneonis mtDNA COI gene is in following f1)-
F16 in) it is any in application also belong to protection scope of the present invention:
F1) the product of preparation identification tangerine big trypetid and bactrocera tsuneonis；
F2) the big trypetid of identification tangerine and bactrocera tsuneonis；
F3) preparation identifies that trypetid to be measured is the product of the big trypetid of tangerine or bactrocera tsuneonis；
F4) identify that trypetid to be measured is the big trypetid of tangerine or bactrocera tsuneonis；
F5) the product of the preparation identification big trypetid of tangerine；
F6 the big trypetid of tangerine) is identified；
F7) preparation identify trypetid to be measured whether be the big trypetid of tangerine product；
F8) identify whether trypetid to be measured is the big trypetid of tangerine；
F9) the product of preparation identification bactrocera tsuneonis；
F10 bactrocera tsuneonis) is identified；
F11) preparation identify trypetid to be measured whether be bactrocera tsuneonis product；
F12) identify whether trypetid to be measured is bactrocera tsuneonis；
F13 the product of the epidemic monitoring of the big trypetid of tangerine and/or bactrocera tsuneonis) is prepared；
F14) the epidemic monitoring of the big trypetid of tangerine and/or bactrocera tsuneonis；
F15) the product of invasion and/or the diffusion of the big trypetid of preparation prevention tangerine and/or bactrocera tsuneonis；
F16) the invasion and/or diffusion of the big trypetid of prevention tangerine and/or bactrocera tsuneonis.
Passing through test proves: the Molecular Identification technology of the invention based on Standard PCR may be implemented to bactrocera tsuneonis and
The Identification of Species of the big trypetid of tangerine has the advantages that high specificity, high sensitivity, at low cost, easy to operate, time-consuming is short, not only solves
The Identification of Species problem for non-adult form trypetid and the trypetid adult residuum of having determined also is the epidemic situation of bactrocera tsuneonis and the big trypetid of tangerine prison
Survey, the distribution of China citrus main producing region and invasion status and its subsequent plant quarantine and field administer provide it is practical
Technology and theoretical foundation.To effectively preventing its further invasion and diffusion, protection agricultural production and ecological safety, promote China
Economic trade development is of great significance.
Detailed description of the invention
Fig. 1 is the big trypetid primer specificity testing result of tangerine.
Fig. 2 is bactrocera tsuneonis primer specificity testing result.
Fig. 3 is the big trypetid primer specificity testing result of former tangerine.
Fig. 4 is former bactrocera tsuneonis primer specificity testing result.
Fig. 5 is that the big trypetid special primer of tangerine expands the big trypetid sample electrophoresis figure of Shaanxi Province's tangerine.
Fig. 6 is that the big trypetid special primer of tangerine expands the big trypetid sample electrophoresis figure of Hunan Province's tangerine.
Fig. 7 is that the big trypetid special primer of tangerine expands the big trypetid sample electrophoresis figure of Guizhou Province's tangerine.
Fig. 8 is that the big trypetid special primer of tangerine expands the big trypetid sample electrophoresis figure of Chongqing City's tangerine.
Fig. 9 is that the big trypetid special primer of tangerine expands the big trypetid sample electrophoresis figure of Yunnan Province's tangerine.
Figure 10 is that the big trypetid special primer of tangerine expands the big trypetid sample electrophoresis figure of Hubei Province's tangerine.
Figure 11 is that bactrocera tsuneonis special primer expands Sichuan Province's bactrocera tsuneonis sample electrophoresis figure.
Figure 12 is that bactrocera tsuneonis special primer expands Yunnan Province's bactrocera tsuneonis sample electrophoresis figure.
Figure 13 is that bactrocera tsuneonis special primer expands Hunan Province's bactrocera tsuneonis sample electrophoresis figure.
Figure 14 is that bactrocera tsuneonis special primer expands Guangxi province bactrocera tsuneonis sample electrophoresis figure.
Figure 15 is that bactrocera tsuneonis special primer expands Guizhou Province's bactrocera tsuneonis sample electrophoresis figure.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.In following embodiments, unless otherwise specified, the 1st of each nucleotide sequence is the 5 ' ends of corresponding DNA in sequence table
Nucleotide, last bit are the 3 ' terminal nucleotides of corresponding DNA.
The big trypetid of tangerine and bactrocera tsuneonis primer in following embodiments are synthesized by giving birth to work biology (Shanghai) Co., Ltd..
The DNA bar code sequence in trypetid mtDNA COI gene in following embodiments is using trypetid genomic DNA as mould
Plate carries out the PCR product sequence that PCR amplification obtains using universal primer LCO1490/HCO2198.
The acquisition of the primer pair of embodiment 1, the big trypetid of identification tangerine and bactrocera tsuneonis
One, the acquisition of trypetid mtDNA COI gene order
1, for trying trypetid sample
As shown in table 1 for examination trypetid sample, sample is placed in dehydrated alcohol, and -20 DEG C save backup.
Table 1, for examination trypetid sample type and locality information
2, PCR amplification
Using " blood/cell/tissue genome DNA extracting reagent kit " (TIANGEN Biotech (Beijing) Co., Ltd.
DP304 the genomic DNA of each trypetid sample in table 1) is extracted respectively, and concrete operations process is respectively obtained referring to specification
The genomic DNA of each trypetid sample in table 1.
Respectively using the genomic DNA of each trypetid sample in table 1 as template, universal primer LCO1490/ is used
HCO2198 carries out PCR amplification, respectively obtains the pcr amplification product of each trypetid sample in table 1.
PCR amplification total system (25 μ l): 2 × Taq PCR MasterMix (TIANGEN Biotech (Beijing) Co., Ltd.,
KT201) 12.5 μ l, each 1 μ l of upstream and downstream primer (10 μM, Sangon Biotech's synthesis), ddH2O
9.5 μ l, 1 μ l of DNA profiling.
PCR amplification condition: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s of 35 circulations, 50 DEG C of annealing 30s, 72 DEG C extend
30s；72 DEG C of extension 10min.
3, PCR product detection and sequencing
It takes 5 μ l pcr amplification products to carry out electrophoresis detection respectively, will test qualified pcr amplification product and directly transfer to Hua Da
Gene Co., Ltd carries out purifying and bidirectional sequencing.Sequencing peak figure is checked using Chromas software, with DNAMAN software to sequencing
As a result spliced.
The big trypetid of published tangerine and bactrocera tsuneonis sequence in GenBank are downloaded, and filters out the standard of length 658bp
DNA bar code sequence in mtDNA COI gene.1 reality for belonging to 2 kinds 1,041 is obtained altogether by testing and downloading in GenBank
DNA bar code sequence in fly mtDNA COI gene.
Two, the design of tangerine big trypetid and bactrocera tsuneonis specific primer pair
By comparing the DNA bar code sequence in the 2 kinds 1,041 trypetid mtDNA COI genes that above-mentioned steps one obtain
Column find the site SNPs, design primer pair, finally obtain 2 pairs of primers, and 2 pairs of primer information are as shown in table 2, by raw work biology
's synthesis.Primer pair Bm-F/Bm-R is used for the big trypetid of specificity identification tangerine；Primer pair Bt-F/Bt-R is used for
Specificity identification bactrocera tsuneonis.
The big trypetid of table 2, tangerine and bactrocera tsuneonis special primer information
Three, the verifying of the primer pair of the big trypetid of identification tangerine and bactrocera tsuneonis
1, for trying trypetid sample
As shown in table 1 for examination trypetid sample, sample is placed in dehydrated alcohol, and -20 DEG C save backup.
2, PCR amplification
Respectively using the genomic DNA of each trypetid sample in table 1 as template, respectively using primer pair Bm-F/Bm-R, draw
Object carries out PCR amplification to Bt-F/Bt-R, respectively obtains the pcr amplification product of each trypetid sample in table 1.
PCR amplification system (50 μ l) is as shown in table 3.
Table 3, PCR reaction system
| template DNA | 3μl |
| Forward Primer(100μM) | 1.5μl |
| Reverse Primer(100μM) | 1.5μl |
| 2×Taq PCR MasterMix | 25μl |
| ddH2O | 19μl |
| Total volume | 25.0μl |
PCR amplification condition: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 1min of 30 circulations, 65 DEG C of annealing extend 1min；65
DEG C extend 10min.
3, PCR product detects
It takes 5 μ l pcr amplification products to carry out electrophoresis detection respectively, will test qualified pcr amplification product and directly transfer to Hua Da
Gene Co., Ltd carries out purifying and bidirectional sequencing.
The result shows that: amplification obtains size as 422bp to primer pair Bm-F/Bm-R in the big trypetid DNA sample of all tangerines
Aim sequence, as shown in the DNA bar code sequence (658bp) 134-555 in the big trypetid mtDNA COI gene of tangerine
DNA fragmentation, and without amplified production in all bactrocera tsuneonis DNA samples.Primer pair Bt-F/Bt-R is in the big reality of all mandarin oranges
Amplification obtains the aim sequence that size is 456bp, the as DNA in bactrocera tsuneonis mtDNA COI gene in fly DNA sample
DNA fragmentation shown in bar code sequence (658bp) 140-595, and produced without amplification in the big trypetid DNA sample of all tangerines
Object.
The specific detection of the primer pair of embodiment 2, the big trypetid of identification tangerine and bactrocera tsuneonis
(1) the method for the present invention
One, the method for detecting specificity of the primer pair of the big trypetid of identification tangerine and bactrocera tsuneonis
1, for trying trypetid sample
As shown in table 4 for examination trypetid sample, sample is placed in dehydrated alcohol, and -20 DEG C save backup.
The corresponding trypetid species of table 4, routine PCR reaction
2, PCR amplification
Using " blood/cell/tissue genome DNA extracting reagent kit " (TIANGEN Biotech (Beijing) Co., Ltd.
DP304 the genomic DNA of each trypetid sample in table 4) is extracted respectively, and concrete operations process is respectively obtained referring to specification
The genomic DNA of each trypetid sample in table 4.
Respectively using the genomic DNA of each trypetid sample in table 4 as template, primer Bm-F/Bm-R, primer are used respectively
Bt-F/Bt-R carries out PCR amplification, respectively obtains the pcr amplification product of each trypetid sample in table 4.
PCR reaction system and PCR amplification condition are the same as 2 in the step of embodiment 1 three.
3, PCR product detects
Standard PCR amplification result is detected by agarose gel electrophoresis, using D2000marker, takes each 5 μ of amplified production
L, 1.5% agarose gel electrophoresis 25 minutes in 1 × TAE buffer, electrophoresis result are obtained by ultraviolet imager.
Two, the specific detection result of the primer pair of the big trypetid of identification tangerine and bactrocera tsuneonis
The principle of specificity verification is the primer pair of every kind of trypetid only in the DNA mould of corresponding target trypetid
Aim sequence is amplified in plate, occurs the single band that becomes clear of corresponding length in electrophoresis result, and without mesh in other DNA profilings
Sequence be amplified, do not occur band in electrophoresis result.I.e. the primer pair is big in the big trypetid of tangerine established and mandarin orange
Under trypetid specific primer PCR identification technology system, target trypetid species can be specifically identified.
If occurring the single band that becomes clear of corresponding length in the corresponding DNA cloning electrophoresis result of certain primer pair, to
Survey trypetid is or candidate is the corresponding target trypetid of the primer pair；
If not occurring band in the corresponding DNA cloning electrophoresis result of certain primer pair, trypetid to be measured is not or candidate
Target trypetid not corresponding for the special primer.
The corresponding target trypetid of primer pair Bm-F/Bm-R is the big trypetid of tangerine；The size of its respective strap is 422bp.
The corresponding target trypetid of primer pair Bt-F/Bt-R is bactrocera tsuneonis；The size of its respective strap is 456bp.
Specific detection result is as shown in Figure 1, 2: in 46 trypetid samples, the big trypetid primer pair Bm-F/Bm-R of tangerine
Successful amplification goes out aim sequence (target fragment that size is 422bp) in serial number the 1-29 big trypetid DNA sample of totally 29 tangerines,
Occur the single band that becomes clear in electrophoresis result, and do not amplify aim sequence in the big trypetid DNA sample of non-tangerine, in electrophoresis
As a result without band in；Bactrocera tsuneonis primer pair Bt-F/Bt-R is in serial number 30-38 totally 9 bactrocera tsuneonis DNA samples
Middle Successful amplification goes out aim sequence the target fragment of 456bp (size be), occurs the single band that becomes clear in electrophoresis result, and
Aim sequence is not amplified in non-bactrocera tsuneonis DNA sample, without band in electrophoresis result.
The above results show: the primer pair of the big trypetid of identification tangerine and bactrocera tsuneonis of the invention has good spy
The opposite sex, the big trypetid of tangerine through the invention and bactrocera tsuneonis primer pair can realize the quick mirror of this 2 kinds of quarantine fruit flies
It is fixed.
(2) it compares
Using the genomic DNA of each trypetid sample in table 4 as template, using document " Jiang F, Li Z H, Wu J
J,et al.A rapid diagnostic tool for two species of Tetradacus(Diptera:
Tephritidae:Bactrocera)based on species-specific PCR[J].Journal of Applied
Entomology, 2014,138 (6): primer pair and the big trypetid of method identification tangerine and bactrocera tsuneonis in 418-422. ".
As a result as shown in Figure 3 and Figure 4.The result shows that: document " Jiang F, Li Z H, Wu J J, et al.A rapid
diagnostic tool for two species of Tetradacus(Diptera:Tephritidae:Bactrocera)
based on species-specific PCR[J].Journal of Applied Entomology,2014,138(6):
In 418-422. " based on Guizhou, Sichuan, the big trypetid of tangerine of 3 geographical population in Hunan and 2 Guizhou, Sichuan geographical population honey
The big trypetid DNA bar code sequence of mandarin orange, the special primer of the tangerine of design big trypetid and bactrocera tsuneonis, when the model for expanding geographical population
Occurs non-specific band after enclosing, in electrophoresis result.The big trypetid special primer of its Central Plains tangerine goes out in 30,31,36 sample lane of number
There is non-specific band in 2,8,20,29 sample lane of number in existing non-specific band, former bactrocera tsuneonis special primer.
The sensitivity technique of the primer pair of embodiment 3, the big trypetid of identification tangerine and bactrocera tsuneonis
One, the sensitivity detection method of the primer pair of the big trypetid of identification tangerine and bactrocera tsuneonis
Selection derives from 1 representative population DNA sample of each province and municipality directly under the Central Government in the big trypetid DNA sample of tangerine, simultaneously
Also 1 representative population DNA sample of the selection from each province and municipality directly under the Central Government in bactrocera tsuneonis DNA sample.6 have been selected altogether
A big trypetid DNA sample of tangerine, respectively from Shaanxi, Hubei, Hunan, Guizhou, Chongqing, Yunnan；5 bactrocera tsuneonis DNA samples,
Respectively from Sichuan, Yunnan, Guizhou, Hunan, Guangxi.Specifying information is as shown in table 5.
The big trypetid of table 5, tangerine and bactrocera tsuneonis sample source ground information
Selected DNA sample is serially diluted, obtain 100ng/ μ l, 10ng/ μ l, 1ng/ μ l, 0.1ng/ μ l,
The DNA sample of 0.01ng/ μ l, 0.001ng/ μ l concentration, respectively using the genomic DNA of the trypetid sample after diluting as template, point
It Shi Yong not primer Bm-F/Bm-R, Bt-F/Bt-R progress PCR amplification.PCR reaction system and PCR amplification condition are the same as embodiment 1
2 in step 3.
Two, the sensitivity technique result of the primer pair of the big trypetid of identification tangerine and bactrocera tsuneonis
The sensitivity technique result of the primer pair of the big trypetid of tangerine is as shown in Figure 5-10, the special primer of bactrocera tsuneonis
Pair sensitivity technique result as shown in figs. 11 and 15.As seen from the figure, two kinds of primer pairs can be only 0.1ng/ μ in DNA concentration
Obvious band is gone out by standard PCR amplification under the conditions of l, it is seen that the big trypetid of identification tangerine and bactrocera tsuneonis of the invention is specifically drawn
The sensitivity of object pair is higher.
Sequence table
<110>China Agricultural University
<120>primer pair and its application of bactrocera tsuneonis and the big trypetid of tangerine are identified
<160>4
<170>PatentIn version 3.5
<210>1
<211>25
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>1
aatttataac gtaatcgtta cagcc 25
<210>2
<211>25
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>2
aagtattgtg atagctccgg ctagg 25
<210>3
<211>25
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>3
taatgtaatc gttactgctc acgcc 25
<210>4
<211>25
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>4
ctgggtcaaa gaaggatgta tttag 25
Claims (10)
1. being made of for identifying the primer set pair of tangerine big trypetid and bactrocera tsuneonis primer pair first and primer pair B；
The primer pair first is made of Bm-F and Bm-R；
The primer pair B is made of Bt-F and Bt-R；
The Bm-F is following a1) or a2):
A1) single strand dna shown in sequence 1 in sequence table；
A2 sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 1
The single strand dna of energy；
The Bm-R is following a3) or a4):
A3) single strand dna shown in sequence 2 in sequence table；
A4 sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 2
The single strand dna of energy；
The Bt-F is following b1) or b2):
B1) single strand dna shown in sequence 3 in sequence table；
B2 sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 3
The single strand dna of energy；
The Bt-R is following b3) or b4):
B3) single strand dna shown in sequence 4 in sequence table；
B4 sequence 4 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 4
The single strand dna of energy.
2. a kind of for identifying the kit of tangerine big trypetid and bactrocera tsuneonis comprising primer set described in claim 1
It is right.
3. primer set pair described in claim 1 or kit as claimed in claim 2 are in following c1)-c8) in it is any in
Application:
C1) the product of preparation identification tangerine big trypetid and bactrocera tsuneonis；
C2) the big trypetid of identification tangerine and bactrocera tsuneonis；
C3) preparation identifies that trypetid to be measured is the product of the big trypetid of tangerine or bactrocera tsuneonis；
C4) identify that trypetid to be measured is the big trypetid of tangerine or bactrocera tsuneonis；
C5 the product of the epidemic monitoring of the big trypetid of tangerine and bactrocera tsuneonis) is prepared；
C6) the epidemic monitoring of tangerine big trypetid and bactrocera tsuneonis；
C7) the product of invasion and/or the diffusion of the big trypetid of preparation prevention tangerine and bactrocera tsuneonis；
C8) prevent the invasion and/or diffusion of tangerine big trypetid and bactrocera tsuneonis.
4. a kind of method for identifying or assisting to identify that trypetid to be measured is the big trypetid of tangerine or bactrocera tsuneonis, includes the following steps:
Using the genomic DNA of trypetid to be measured as template, primer pair first described in claim 1 is respectively adopted and primer pair B carries out
PCR amplification；
If primer pair first PCR amplification obtains the target fragment that size is 422bp, trypetid to be measured is or candidate is the big trypetid of tangerine；
If primer pair B PCR amplification obtains the target fragment that size is 456bp, trypetid to be measured is or candidate is the big reality of mandarin orange
Fly.
5. the primer pair first in claim 1 is in following d1)-d8) in it is any in application:
D1) the product of the preparation identification big trypetid of tangerine；
D2 the big trypetid of tangerine) is identified；
D3) preparation identify trypetid to be measured whether be the big trypetid of tangerine product；
D4) identify whether trypetid to be measured is the big trypetid of tangerine；
D5 the product of the epidemic monitoring of the big trypetid of tangerine) is prepared；
D6) the epidemic monitoring of the big trypetid of tangerine；
D7) the product of the invasion and/or diffusion of the preparation prevention big trypetid of tangerine；
D8) prevent the invasion and/or diffusion of the big trypetid of tangerine.
6. the primer pair B in claim 1 is in following e1)-e8) in it is any in application:
E1) the product of preparation identification bactrocera tsuneonis；
E2 bactrocera tsuneonis) is identified；
E3) preparation identify trypetid to be measured whether be bactrocera tsuneonis product；
E4) identify whether trypetid to be measured is bactrocera tsuneonis；
E5 the product of the epidemic monitoring of bactrocera tsuneonis) is prepared；
E6) the epidemic monitoring of bactrocera tsuneonis；
E7) the product of invasion and/or the diffusion of preparation prevention bactrocera tsuneonis；
E8) prevent the invasion and/or diffusion of bactrocera tsuneonis.
7. it is a kind of identify or assist to identify trypetid to be measured whether be the big trypetid of tangerine method, include the following steps: with trypetid to be measured
Genomic DNA be template, using described in claim 1 primer pair first carry out PCR amplification；
If PCR amplification obtains the target fragment that size is 422bp, trypetid to be measured is or candidate is the big trypetid of tangerine；
If PCR amplification does not obtain the target fragment that size is 422bp, trypetid to be measured is not or candidate is not the big trypetid of tangerine.
8. it is a kind of identify or assist to identify trypetid to be measured whether be bactrocera tsuneonis method, include the following steps: with reality to be measured
The genomic DNA of fly is template, carries out PCR amplification using primer pair B as stated in claim 2；
If PCR amplification obtains the target fragment that size is 456bp, trypetid to be measured is or candidate is bactrocera tsuneonis；
If PCR amplification does not obtain the target fragment that size is 456bp, trypetid to be measured is not or candidate is not bactrocera tsuneonis.
9. DNA fragmentation shown in DNA bar code sequence 134-555 in the big trypetid mtDNA COI gene of tangerine or mandarin orange are big
DNA fragmentation shown in DNA bar code sequence 140-595 in trypetid mtDNA COI gene.
10. DNA fragmentation and/or honey shown in DNA bar code sequence 134-555 in the big trypetid mtDNA COI gene of tangerine
DNA fragmentation shown in DNA bar code sequence 140-595 in the big trypetid mtDNA COI gene of mandarin orange is in following f1)-f16)
In it is any in application:
F1) the product of preparation identification tangerine big trypetid and bactrocera tsuneonis；
F2) the big trypetid of identification tangerine and bactrocera tsuneonis；
F3) preparation identifies that trypetid to be measured is the product of the big trypetid of tangerine or bactrocera tsuneonis；
F4) identify that trypetid to be measured is the big trypetid of tangerine or bactrocera tsuneonis；
F5) the product of the preparation identification big trypetid of tangerine；
F6 the big trypetid of tangerine) is identified；
F7) preparation identify trypetid to be measured whether be the big trypetid of tangerine product；
F8) identify whether trypetid to be measured is the big trypetid of tangerine；
F9) the product of preparation identification bactrocera tsuneonis；
F10 bactrocera tsuneonis) is identified；
F11) preparation identify trypetid to be measured whether be bactrocera tsuneonis product；
F12) identify whether trypetid to be measured is bactrocera tsuneonis；
F13 the product of the epidemic monitoring of the big trypetid of tangerine and/or bactrocera tsuneonis) is prepared；
F14) the epidemic monitoring of the big trypetid of tangerine and/or bactrocera tsuneonis；
F15) the product of invasion and/or the diffusion of the big trypetid of preparation prevention tangerine and/or bactrocera tsuneonis；
F16) the invasion and/or diffusion of the big trypetid of prevention tangerine and/or bactrocera tsuneonis.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110117666A (en) * | 2019-06-06 | 2019-08-13 | 中国农业大学 | Identify the primer pair and its application of tyrophagus putrescentiae and ellipse dispersion |
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| CN102517388A (en) * | 2011-12-20 | 2012-06-27 | 中国农业大学 | Kit for identifying fruit flies and special primers therefor |
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| CN102517388A (en) * | 2011-12-20 | 2012-06-27 | 中国农业大学 | Kit for identifying fruit flies and special primers therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110117666A (en) * | 2019-06-06 | 2019-08-13 | 中国农业大学 | Identify the primer pair and its application of tyrophagus putrescentiae and ellipse dispersion |
| CN110117666B (en) * | 2019-06-06 | 2022-04-26 | 中国农业大学 | Specific primer pair for identifying Tyrophagus putrescentiae and Tyrophagus ovatus and application thereof |
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