CN110117666A - Identify the primer pair and its application of tyrophagus putrescentiae and ellipse dispersion - Google Patents
Identify the primer pair and its application of tyrophagus putrescentiae and ellipse dispersion Download PDFInfo
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- CN110117666A CN110117666A CN201910490713.3A CN201910490713A CN110117666A CN 110117666 A CN110117666 A CN 110117666A CN 201910490713 A CN201910490713 A CN 201910490713A CN 110117666 A CN110117666 A CN 110117666A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention discloses the primer pairs and its application of identification tyrophagus putrescentiae and ellipse dispersion.The present invention is based on the Molecular Identification technologies of Standard PCR to realize the Identification of Species to tyrophagus putrescentiae and ellipse dispersion, the primer pair of identification tyrophagus putrescentiae and ellipse dispersion that the present invention designs 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 mite and mite adult residuum is not only solved, also the epidemic monitoring for tyrophagus putrescentiae and ellipse dispersion, the distribution in China and invasion status and its subsequent improvement provide 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 tyrophagus putrescentiae and ellipse dispersion primer pair and
It is applied.
Background technique
Mite is under the jurisdiction of Arachnoidea Arachinida, Acari Acari, Acariformes Acariformes, and individual is small, at
The long general 0.1-0.2mm of mite body, is visually difficult to observe.Storage harmful mite can largely multiply in stored goods, eat the battalion of stored goods
Support part, cause the loss of storage amount of substance sum number amount, meanwhile, the activity for harmful mite of storing in a warehouse will increase heat, and its excreta with
And the microorganism carried with it can quickly cause stored goods under heat catalysis and pollute, go mouldy on a large scale.Importantly,
Most of kinds in storage harmful mite can all cause the anaphylactias such as asthma, endanger the health of people.
Discovery is investigated from stored goods in recent years, tyrophagus putrescentiae is the sociales in storage harmful mite, recall rate highest, part
Area reaches 49.3%, is the strongest storage harmful mite of current adaptability.During harmful mite is stored in a warehouse in raising, the discovery overwhelming majority
The type of storage harmful mite change, through detecting, all polluted by ellipse dispersion, and in population based on ellipse dispersion.
It can be seen that the competitiveness of ellipse dispersion is stronger.But since tyrophagus putrescentiae and ellipse dispersion individual are small, when Morphological Identification
Need to make slide sample, and identification relies primarily on the features such as position and the length of bristle, this gives the Morphological Identification of 2 kinds of mites
Cause extreme difficulties.Therefore how it is fast and accurately identified, is especially non-adult form mite and mite adult residuum
Identification of Species is a problem to be solved.
DNA bar code technology (DNA barcoding) is to be carried out fastly using one section of conservative fragments in organism DNA to species
The emerging technology of fast precise Identification.In recent years, based on DNA bar code sequence design specific primer, probe and the routine developed
Round pcr, real-time fluorescent PCR technology, loop-mediated isothermal amplification technique LAMP are widely used to the Molecular Identification of species.Its
In, Standard PCR technology has been reflected in the molecule of mite due to operating fast and convenient, high specificity, high sensitivity, saving the advantages that cost
It is widely applied in fixed.
Summary of the invention
The first purpose of the invention is to provide for identifying the primer pair of tyrophagus putrescentiae and ellipse dispersion.
The primer pair first is made of Tp-F and Tp-R；
The primer pair B is made of Ao-F and Ao-R；
The Tp-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 Tp-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 Ao-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 Ao-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 tyrophagus putrescentiae and ellipse dispersion.
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 tyrophagus putrescentiae and ellipse dispersion；
C2 tyrophagus putrescentiae and ellipse dispersion) are identified；
C3) preparation identifies that mite to be measured is the product of tyrophagus putrescentiae or ellipse dispersion；
C4) identify that mite to be measured is tyrophagus putrescentiae or ellipse dispersion；
C5 the product of the epidemic monitoring of tyrophagus putrescentiae and/or ellipse dispersion) is prepared；
C6) the epidemic monitoring of tyrophagus putrescentiae and/or ellipse dispersion；
C7) the product of invasion and/or the diffusion of preparation prevention tyrophagus putrescentiae and/or ellipse dispersion；
C8) prevent the invasion and/or diffusion of tyrophagus putrescentiae and/or ellipse dispersion.
Fourth object of the present invention, which is to provide, a kind of identifies or assists to identify that mite to be measured is tyrophagus putrescentiae or oval food
The method of flour mite.
It is provided by the invention to identify or assist to identify that mite to be measured is the method for tyrophagus putrescentiae or ellipse dispersion including such as
Lower step: using the genomic DNA of mite to be measured as template, above-mentioned primer pair first is respectively adopted and primer pair B carries out PCR amplification；
If primer pair first PCR amplification obtains the target fragment that size is 484bp, mite to be measured is or candidate is saprophage junket
Mite；
If primer pair B PCR amplification obtains the target fragment that size is 639bp, mite to be measured is or candidate is oval baking soda
Mite.
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 preparation identification tyrophagus putrescentiae；
D2 tyrophagus putrescentiae) is identified；
D3) preparation identify mite to be measured whether be tyrophagus putrescentiae product；
D4) identify whether mite to be measured is tyrophagus putrescentiae；
D5 the product of the epidemic monitoring of tyrophagus putrescentiae) is prepared；
D6) the epidemic monitoring of tyrophagus putrescentiae；
D7) the product of invasion and/or the diffusion of preparation prevention tyrophagus putrescentiae；
D8) prevent the invasion and/or diffusion of tyrophagus putrescentiae.
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 ellipse dispersion；
E2 ellipse dispersion) is identified；
E3) preparation identify mite to be measured whether be ellipse dispersion product；
E4) identify whether mite to be measured is ellipse dispersion；
E5 the product of the epidemic monitoring of ellipse dispersion) is prepared；
E6) the epidemic monitoring of ellipse dispersion；
E7) the product of invasion and/or the diffusion of preparation prevention ellipse dispersion；
E8) prevent the invasion and/or diffusion of ellipse dispersion.
Fifth object of the present invention is to provide it is a kind of identify or assist to identify mite to be measured whether be tyrophagus putrescentiae method.
It is provided by the invention identify or assist to identify mite to be measured whether be tyrophagus putrescentiae method include the following steps: with to
The genomic DNA for surveying mite is template, carries out PCR amplification using above-mentioned primer pair first；
If PCR amplification obtains the target fragment that size is 484bp, mite to be measured is or candidate is tyrophagus putrescentiae；
If PCR amplification does not obtain the target fragment that size is 484bp, mite to be measured is not or candidate is not tyrophagus putrescentiae.
Sixth object of the present invention is to provide it is a kind of identify or assist to identify mite to be measured whether be ellipse dispersion side
Method.
It is provided by the invention identify or assist to identify mite to be measured whether be ellipse dispersion method include the following steps: with
The genomic DNA of mite to be measured is template, carries out PCR amplification using above-mentioned primer pair B；
If PCR amplification obtains the target fragment that size is 639bp, mite to be measured is or candidate is ellipse dispersion；
If PCR amplification does not obtain the target fragment that size is 639bp, mite to be measured is not or candidate is not oval baking soda
Mite.
In the above method, the target fragment that the size is 484bp is NAD4 gene in tyrophagus putrescentiae mitochondrial genomes
DNA fragmentation shown in (sequence 5) 405-889.The target fragment that the size is 639bp is ellipse dispersion mitochondria base
Because of DNA fragmentation shown in NAD2 gene (sequence 6) 201-840 in group.
In any of the above-described the method, the PCR reaction system is by 1.5 μ l of DNA profiling, forward primer (10 μM) 1 μ l, anti-
To primer (10 μM) 1 μ l, 2 × Taq PCR MasterMix 12.5 μ l and ddH29 μ l of O composition.
The forward primer and the reverse primer are Tp-F in the primer pair first and Tp-R or the primer pair B
In Ao-F and Ao-R.The end of the Tp-F, the Tp-R, the Ao-F and the Ao-R in the PCR reaction system is dense
Degree is 0.6 μM.
The PCR reaction condition is as follows:
Tyrophagus putrescentiae: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 30s of 35 circulations, 62 DEG C of annealing 30s, 72 DEG C extend
30s；72 DEG C of extension 5min.
Ellipse dispersion: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 30s of 35 circulations, 60 DEG C of annealing extend 1min；60℃
Extend 5min.
In any of the above-described the method, the mite concretely ovum of the mite, larva, pupa and/or adult.
Following f1)-f16) in it is any in application also belong to protection scope of the present invention:
F1) the product of preparation identification tyrophagus putrescentiae and ellipse dispersion；
F2 tyrophagus putrescentiae and ellipse dispersion) are identified；
F3) preparation identifies that mite to be measured is the product of tyrophagus putrescentiae or ellipse dispersion；
F4) identify that mite to be measured is tyrophagus putrescentiae or ellipse dispersion；
F5) the product of preparation identification tyrophagus putrescentiae；
F6 tyrophagus putrescentiae) is identified；
F7) preparation identify mite to be measured whether be tyrophagus putrescentiae product；
F8) identify whether mite to be measured is tyrophagus putrescentiae；
F9) the product of preparation identification ellipse dispersion；
F10 ellipse dispersion) is identified；
F11) preparation identify mite to be measured whether be ellipse dispersion product；
F12) identify whether mite to be measured is ellipse dispersion；
F13 the product of the epidemic monitoring of tyrophagus putrescentiae and/or ellipse dispersion) is prepared；
F14) the epidemic monitoring of tyrophagus putrescentiae and/or ellipse dispersion；
F15) the product of invasion and/or the diffusion of preparation prevention tyrophagus putrescentiae and/or ellipse dispersion；
F16) prevent the invasion and/or diffusion of tyrophagus putrescentiae and/or ellipse dispersion.
The present invention is based on the Molecular Identification technologies of Standard PCR to realize the Identification of Species to tyrophagus putrescentiae and ellipse dispersion,
The specific primer of identification tyrophagus putrescentiae and ellipse dispersion that the present invention designs is to high specificity, high sensitivity, cost
Low, easy to operate, time-consuming short advantage, not only solves the Identification of Species problem of non-adult form mite and mite adult residuum, also for
The epidemic monitoring of ellipse dispersion and tyrophagus putrescentiae is provided in the distribution in China and invasion status and its subsequent improvement
Practical technique and theoretical foundation.To effectively preventing its further invasion and diffusion, protection agricultural production and ecological safety, promotion
Economic trade development in China's is of great significance.
Detailed description of the invention
Fig. 1 is tyrophagus putrescentiae primer specificity testing result.1: tyrophagus putrescentiae；2: sweet tea mite eats in family；3: ellipse dispersion；4:
The thermophilic junket mite of line；5: Acarus siro；6: Cheyletusmoorei；7: Cheyletus eruditus.
Fig. 2 is ellipse dispersion primer specificity testing result.1: ellipse dispersion；2: tyrophagus putrescentiae；3: sweet tea mite eats in family；
4: the thermophilic junket mite of line；5: Acarus siro；6: Cheyletusmoorei；7: Cheyletus eruditus.
Fig. 3 is tyrophagus putrescentiae special primer sensitivity technique result.1:26.62ng/ul；2:13.31ng/ul；3:
6.66ng/ul；4:3.33ng/ul；5:1.66ng/ul.
Fig. 4 is ellipse dispersion special primer sensitivity technique result.1:15ng/ul；2:5ng/ul；3:1.67ng/ul；
4:0.56ng/ul；5:0.18ng/ul.
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.Nucleic acid sequence in following embodiments, unless otherwise specified, the 1st of each nucleotide sequence are the 5 ' ends of corresponding DNA
Terminal nucleotide, last bit are the 3 ' terminal nucleotides of corresponding DNA.Tyrophagus putrescentiae and ellipse dispersion primer are equal in following embodiments
It is synthesized by raw work biology (Shanghai) Co., Ltd..
NAD4 gene order such as 5 institute of sequence in sequence table in tyrophagus putrescentiae mitochondrial genomes in following embodiments
Show；NAD2 gene order is as shown in the sequence 6 in sequence table in ellipse dispersion mitochondrial genomes in following embodiments.
The acquisition of embodiment 1, the primer pair of identification tyrophagus putrescentiae and ellipse dispersion
One, mite mitochondrial genomes obtain
1, for trying mite sample
It is collected in Czech for examination ellipse dispersion, is collected in BeiJing, China for examination tyrophagus putrescentiae, sample is soaked in anhydrous second
In alcohol, -20 DEG C are saved backup.
2, mitochondrial genomes sequence
Using Tiangeng company minim DNA extracts kit extract for try mite sample DNA, minim DNA extracts kit at
Divide as shown in table 1.
Table 1, minim DNA extracts kit ingredient
| Kit forms (50 times) | The amount of each component |
| Buffer GA | 15ml |
| Buffer GB | 15ml |
| Buffer GD | 13ml |
| Rinsing liquid PW | 15ml |
| Elution buffer TB | 15ml |
| Proteinase K | 1ml |
| Carrier RNA | 310μg |
| RNase-free dd H2O | 1ml |
| Adsorption column CR2 | 50 |
| Collecting pipe (2ml) | 50 |
Steps are as follows for specific experiment:
1) it by dissection sem observation and the adult mite of picking 50 purifying raisings, is placed in the centrifuge tube of 1.5ml, with anhydrous second
Alcohol is cleaned, naturally dry.
2) the GA buffer of 180ul and the protease of 20ul is added, shakes up, 56 DEG C of water-baths are stayed overnight after brief centrifugation.
Centrifuge tube is taken out, surface water droplet, brief centrifugation are wiped.
3) the GB solution of 200ul and the Carrier RNA solution of 1ul is added, sufficiently shake up (there is precipitating to generate at this time,
Precipitating need to be made to disperse) 70 DEG C of water-bath 10min afterwards, limpid no precipitating in pipe at this time.Taking-up is dried, brief centrifugation.
4) dehydrated alcohol (dehydrated alcohol is put into 4 DEG C of refrigerators when being higher than 26 DEG C and is pre-chilled by room temperature) of 200ul is added, up and down
It is reverse to shake up, 5min is stood after brief centrifugation, and then the liquid in centrifuge tube is transferred completely on collecting pipe using liquid-transfering gun
Adsorption tube in, room temperature 12000rpm be centrifuged 1min.It takes out, outwells waste liquid.
5) the GD solution of 500ul is added, stands 5min, then 12000rpm is centrifuged 1min.It takes out, outwells waste liquid.
6) the PW solution of 600ul is added, stands 5min, then 12000rpm is centrifuged 1min, takes out, outwells waste liquid.Weight
Duplicate step is twice.
7) blank pipe 12000rpm is centrifuged 2min, takes out, outwells waste liquid, adsorption tube is placed on clean blotting paper and is dried in the air naturally
It is placed on clean centrifuge tube after dry (about 5min).
8) ddH of 30ul is added2O stands 5min, and 12000rpm is centrifuged 2min, by the liquid liquid-transfering gun in centrifuge tube
Draw, after rejoin in adsorption tube, repeat the above steps.
9) with the ddH of 1ul2O is the solubility of measurement of comparison DNA solution, and reaching 2ug is then success, takes suitable DNA solution
For I gene sequencing of CO, remaining send to company carries out the sequencing of two generations.
10) amplification of I gene order of CO is carried out with the PCR system of 25ul, obtained PCR product is placed in 4 DEG C of refrigerators and protects
It deposits.Then PCR product is sent to company and is sequenced, obtain I sequence of CO.
11) after obtaining company's two generations sequencing result, using I sequence of CO as anchor series, Geneious software splicing line is used
Mitochondrial genes group sequence obtains complete mitochondrial genomes sequence.
12) by the mitochondrial genomes sequence obtained send to MITOS line upper mounting plate carry out each gene location and length just
Step determines.
13) with initiation codon, terminator codon, close kind of blood relationship of same gene length and position are reference, are determined
The length of each gene and position.Because the tRNA degradation of mite is serious, software can not mostly be identified, need to according to the secondary structure of tRNA and
Anticodon is determined and draws manually.
14) the mitochondrial genomes ring figure comprising 37 gene locations is obtained using Genieous software.
Two, the design of the primer pair of tyrophagus putrescentiae and ellipse dispersion
It one determines mitochondrial genomes sequence through the above steps, finds specific site, design primer pair, finally obtain
2 primer pairs are obtained, as shown in table 2, are synthesized by raw work biology (Shanghai) Co., Ltd..Primer pair Tp-F/Tp-R is for spy
Opposite sex identification tyrophagus putrescentiae, primer pair Ao-F/Ao-R are used for specificity identification ellipse dispersion.
Table 2, tyrophagus putrescentiae and ellipse dispersion special primer information
Three, the verifying of the primer pair of tyrophagus putrescentiae and ellipse dispersion is identified
1, for trying mite sample
It is consistent with mitochondrial genomes sequencing sample used for examination mite sample.
2, PCR amplification
Tyrophagus putrescentiae carries out PCR amplification using Tp-F/Tp-R primer pair.PCR amplification system is 2 × Taq PCR
MasterMix 12.5ul, upstream and downstream primer (10 μM) each 1ul, DNA profiling 1.5ul, ddH2O 9ul.PCR amplification condition such as table
Shown in 3.
Table 3, tyrophagus putrescentiae PCR amplification condition
Ellipse dispersion carries out PCR amplification using Ao-F/Ao-R primer.PCR amplification system is 2 × Taq PCR
MasterMix 12.5ul, upstream and downstream primer (10 μM) each 1ul, DNA profiling 1.5ul, ddH2O 9ul.PCR amplification condition such as table
Shown in 4.
Table 4, ellipse dispersion PCR amplification condition
3, PCR product detects
It takes 8ul pcr amplification product to carry out electrophoresis detection respectively, will test qualified pcr amplification product and directly meet at Hua Da
Gene Co., Ltd carries out purifying and bidirectional sequencing.
The result shows that: primer pair Tp-F/Tp-R is expanded in tyrophagus putrescentiae sample and is obtained the aim sequence of 484bp, as
DNA fragmentation shown in NAD4 gene (sequence 5) 405-889 in tyrophagus putrescentiae mitochondrial genomes, and in ellipse dispersion
In all without product.Primer pair Ao-F/Ao-R is expanded in ellipse dispersion sample and is obtained the aim sequence of 639bp, as oval
DNA fragmentation shown in NAD2 gene (sequence 6) 201-840 in baking soda mite mitochondrial genomes, and in tyrophagus putrescentiae all
Without product.
The specific detection of embodiment 2, the primer pair of identification tyrophagus putrescentiae and ellipse dispersion
One, the method for detecting specificity of the primer pair of tyrophagus putrescentiae and ellipse dispersion is identified
1, for trying mite sample
As shown in table 5 for examination mite, sample mite is placed in dehydrated alcohol, and -20 DEG C save backup.
Mite sample used in table 5, identification special primer specific PCR
| Chinese name | Section | Latin literary fame |
| Ellipse dispersion | Tyroglyphidae | Aleuroglyphus ovatus |
| Acarus siro | Tyroglyphidae | Acarus siro |
| Tyrophagus putrescentiae | Tyroglyphidae | Tyrophagus putrescentiae |
| The thermophilic junket mite of line | Tyroglyphidae | Tyroborus lini |
| Family food sweet tea mite | Shi Tian mite section | Glycyphagus domesticus |
| Cheyletus eruditus | Cheyletidae | Cheyletus eruditus |
| Cheyletusmoorei | Cheyletidae | Cheyletus malaccensis |
1, DNA is extracted
The DNA of the various mites in table 5 is extracted using Tiangeng " minim DNA extracts kit ", specific extraction process is as follows:
1) it by dissection sem observation and the adult mite of picking 50 purifying raisings, is placed in the centrifuge tube of 1.5ml, with anhydrous second
Alcohol is cleaned, naturally dry.
2) the GA buffer of 180ul and the protease of 20ul is added, shakes up, 56 DEG C of water-baths are stayed overnight after brief centrifugation.
Centrifuge tube is taken out, surface water droplet, brief centrifugation are wiped.
3) the GB solution of 200ul and the Carrier RNA solution of 1ul is added, sufficiently shake up (there is precipitating to generate at this time,
Precipitating need to be made to disperse) 70 DEG C of water-bath 10min afterwards, limpid no precipitating in pipe at this time.Taking-up is dried, brief centrifugation.
4) dehydrated alcohol (dehydrated alcohol is put into 4 DEG C of refrigerators when being higher than 26 DEG C and is pre-chilled by room temperature) of 200ul is added, up and down
It is reverse to shake up, 5min is stood after brief centrifugation, and then the liquid in centrifuge tube is transferred completely on collecting pipe using liquid-transfering gun
Adsorption tube in, room temperature 12000rpm be centrifuged 1min.It takes out, outwells waste liquid.
5) the GD solution of 500ul is added, stands 5min, then 12000rpm is centrifuged 1min.It takes out, outwells waste liquid.
6) the PW solution of 600ul is added, stands 5min, then 12000rpm is centrifuged 1min, takes out, outwells waste liquid.Weight
Duplicate step is twice.
7) blank pipe 12000rpm is centrifuged 2min, takes out, outwells waste liquid, adsorption tube is placed on clean blotting paper and is dried in the air naturally
It is placed on clean centrifuge tube after dry (about 5min).
8) ddH of 30ul is added2O stands 5min, and 12000rpm is centrifuged 2min, by the liquid liquid-transfering gun in centrifuge tube
Draw, after rejoin in adsorption tube, repeat the above steps.
2, PCR amplification
Respectively using the DNA of mite sample each in table 5 as template, primer pair Tp-F/Tp-R and primer pair Ao-F/ is used respectively
Ao-R carries out PCR amplification, respectively obtains the pcr amplification product of each mite sample in table 5.
PCR reaction system and PCR amplification condition are the same as the 2 of 1 step 3 of embodiment.
3, PCR product detects
Standard PCR amplification result is detected by agarose gel electrophoresis, using D2000marker, takes each amplified production
8ul, the 1.5% agarose gel electrophoresis 30min in 1 × TAE buffer, electrophoresis result are obtained by ultraviolet imager.
Two, the specific detection result of the primer pair of tyrophagus putrescentiae and ellipse dispersion is identified
The principle of specific detection is the primer pair of every kind of mite only in the DNA profiling of corresponding target mite
Aim sequence is amplified, occurs the single band that becomes clear of corresponding length in electrophoresis result, and without purpose sequence in other DNA profilings
Column are amplified, and do not occur band in electrophoresis result, i.e., the primer pair is in the tyrophagus putrescentiae and ellipse dispersion established
Under specific primer PCR identification technology system, target mite kind 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 mite is or candidate is the corresponding target mite of the primer pair.
If not occurring band in the corresponding DNA cloning electrophoresis result of certain primer pair, mite to be measured be not or it is candidate not
For the corresponding target mite of the primer pair.
The corresponding target mite of primer pair Tp-F/Tp-R is tyrophagus putrescentiae；The size of its respective strap is 484bp.
The corresponding target mite of primer pair Ao-F/Ao-R is ellipse dispersion；Its respective strap size is 639bp.
Tyrophagus putrescentiae specific detection result is as shown in Figure 1.The result shows that: in 7 mite samples, tyrophagus putrescentiae special primer
To Tp-F/Tp-R, Successful amplification goes out aim sequence (target fragment that size is 484bp) in the tyrophagus putrescentiae sample of serial number 1,
Occur bright single band in electrophoresis result, and nothing amplifies target sequence in the DNA sample of non-tyrophagus putrescentiae, electricity
Without band in result of swimming.
Ellipse dispersion specific detection result is as shown in Figure 2.The result shows that: in 7 mite samples, ellipse dispersion is special
Primer pair Ao-F/Ao-R Successful amplification in the ellipse dispersion sample of serial number 1 goes out aim sequence, and (size is the target of 639bp
Segment), occur bright single band in electrophoresis result, and nothing amplifies mesh in the DNA sample of non-ellipse dispersion
Sequence is marked, without band in electrophoresis result.
The above results show: the primer pair of identification tyrophagus putrescentiae and ellipse dispersion of the invention has good spy
It is anisotropic, it can be achieved that quick, precise Identification to tyrophagus putrescentiae and ellipse dispersion.
The sensitivity technique of embodiment 3, the primer pair of identification tyrophagus putrescentiae and ellipse dispersion
One, the sensitivity detection method of the primer pair of tyrophagus putrescentiae and ellipse dispersion is identified
Tyrophagus putrescentiae: it is in the same manner as in Example 1 using mite sample, the DNA sample of tyrophagus putrescentiae is diluted, is obtained dense
Degree is respectively the DNA sample of 26.62ng/ul, 13.31ng/ul, 6.66ng/ul, 3.33ng/ul and 1.66ng/ul, respectively
It using the DNA sample being diluted as template, is expanded using primer pair Tp-F/Tp-R, PCR amplification system and amplification condition and reality
Apply 1 step 3 of example 2 are identical.
Ellipse dispersion: it is in the same manner as in Example 1 using mite sample, the DNA sample of ellipse dispersion is diluted, point
The DNA sample that concentration is 15ng/ul, 5ng/ul, 1.67ng/ul, 0.56ng/ul and 0.18ng/ul is not obtained, respectively with quilt
Diluted DNA sample is template, is expanded using primer pair Ao-F/Ao-R, PCR amplification system and amplification condition and embodiment
The 2 of 1 step 3 are identical.
Two, the sensitivity technique result of tyrophagus putrescentiae and ellipse dispersion primer pair is identified
The sensitivity technique result of the primer pair of tyrophagus putrescentiae as shown in figure 3, ellipse dispersion primer pair
Sensitivity technique result it is as shown in Figure 4.Identifying tyrophagus putrescentiae primer pair as seen from the figure can be in DNA template concentration
Purpose band is gone out by standard PCR amplification under the conditions of 6.66ng/ul, identification ellipse dispersion primer pair can be in DNA mould
Plate concentration by standard PCR amplification goes out purpose band under the conditions of being 1.67ng/ul, it is seen that identification tyrophagus putrescentiae of the invention and ellipse
The sensitivity of circle baking soda mite primer pair is good.
The present invention is had been described in detail above.To those skilled in the art, do not depart from spirit of the invention and
Range, and without carrying out under unnecessary experimental conditions, can synchronization parameters, concentration and under the conditions of, it is real in a wider range
Apply the present invention.Although The present invention gives particular embodiments, it is understood that, the present invention can be improved further.
In short, pressing the principle of the present invention, the application is intended to include any change, purposes or improvement of the present invention, including departing from this Shen
Please in the open scope, and the change carried out with routine techniques known in the art.By the range of following attached claims,
It can carry out the application of some essential characteristics.
Sequence table
<110>China Agricultural University
<120>primer pair and its application of tyrophagus putrescentiae and ellipse dispersion are identified
<160>6
<170>PatentIn version 3.5
<210>1
<211>21
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>1
gtgttttcct tgccttttct c 21
<210>2
<211>21
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>2
ataagtagag ccccataaac c 21
<210>3
<211>21
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>3
tcttccacag aacaaaatat g 21
<210>4
<211>21
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>4
cggttctttc gttagttact g 21
<210>5
<211>1301
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>5
ataaaaatga taatgagagt cttttttttg ttgtctgttt tttatgtttt tcttgatttt 60
ttctttttta attgagtttt cttatttatg gtttttttta tttttaattt ttacgggggc 120
tttacggatg gattttttta cagagattat ttttctcttc ttttggtttt tgttactttt 180
tgggtttttt tattttcttt tctttctatg aagttttcta atcctaattt tgttgtgctt 240
tgagttataa tattttttct tttttttagt ttttttactg ttaattattt atttttctat 300
gttttttttg agtttgtttt tgttataata tttatttttc ttttaggttg aggtaaaact 360
ctggagcgtt tacaggcttc tttttatatg tttttttaca ctatagtgtt ttccttgcct 420
tttctcgttt ttttgatcta tctaaatttt tcaatttcag attctttttc ttcattaact 480
ttttctcaat atgatgataa tcttatctat aatgattttt tttgagtttt tataatctta 540
gtttttgtag ttaagcttcc cctttttggg tttcatcttt gattacctaa ggcccatgtg 600
gaggctcctg ttgctggttc tataattttg gctggggttc tactaaaatt agggggttat 660
ggtatttttc gttttttctc ttctgtgggt tgtctcaatt tttcttatag agtgttattt 720
tcctatattt tttatgtctc tctttacggg gctgtgtttg taagcttgat ttgtattcgt 780
caaatagacc ttaagatact cattgcttac tcttctgttg ttcacataag agttataatc 840
ttaggtattt taagtttttc tatttggggg gtttatgggg ctctacttat gataattgct 900
catggtttta tttcccctat gatattttat ctcataactt acctttatga aaatcaccat 960
tctcgtagaa ttatagtact taagggggtg ttgatctgta accctttatt ttgtctttta 1020
tggtttctat gttgttcttt aaatttaagg gctcctcctt ttatgtcttt ttattcagag 1080
gttattattt ttggttcttt aggttcttta ggtttacttg agtgactaat aattacttta 1140
agttgttttt ttactggggt ttactgtatc tatagttatg tggtggtgag acacggttct 1200
tctctttcta gattgtttta tttcttagat tataaaattg tcttagtttc agtttctcac 1260
tttatttttg ttatttttta ccctattatc ttttttttgt a 1301
<210>6
<211>969
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>6
atggtactca ctttaatagt aactaccatt tcaggaatag catgctcaag atgattaata 60
gtatgaatac tattagaagc aaacaccctg gctatgtgtt ttttagtatc ccatgaatca 120
aaaacaagaa taaaaagaga aaaagcaacc atgacatatt tcatagtaca aatcttagca 180
tcaatcctta ttcttctagc ttcttccaca gaacaaaata tgttatcttc aactctgatc 240
ataggaggca ttctcgccaa aataggagtc tgaccagccc acctatggta cataaaaata 300
attaaccttc ttcaaataaa acaaaattcc ttactaatct taataacatg gcaaaaaatc 360
ctaccagctt tcctagcaat aagaataaga aaaaatatag aaagagaaac ccctttaata 420
atagtagcct tagtatccct agtaacccct cttaccatat taaaatcaaa cttaactaca 480
aaaagaatta tagccttatc ctcattaaac aacaacgcat ggctagtaat agcaatctcc 540
ttatccacaa aatcattcac agcattctta gctttatact cttccacact tataatactt 600
ctaaaaacaa taaactgact aacaaaaaaa tcagaaagaa caggaaaaag attctgatac 660
tcaatagtaa tattcggtaa tctgggaggc ctaccacctt taacaatatt ttgaataaaa 720
gtaataattt taaaaacctt aatggagagc aacacaagaa cagaaatctg cggaattcta 780
atattatcag catgtattat actttaccac tacctatgaa cagtaactaa cgaaagaacc 840
gcctcaccag taaaaaggca aaaccaagcc atcttaaaca aaaaagaaag aaaaacctat 900
atcaccataa ttttacccat aagtttcgtg agagcgtacc taacaataat tatattaggc 960
ttaacctaa 969
Claims (10)
1. being made of for identifying the primer set pair of tyrophagus putrescentiae and ellipse dispersion primer pair first and primer pair B；
The primer pair first is made of Tp-F and Tp-R；
The primer pair B is made of Ao-F and Ao-R；
The Tp-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 Tp-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 Ao-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 Ao-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 tyrophagus putrescentiae and ellipse dispersion 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 tyrophagus putrescentiae and ellipse dispersion；
C2 tyrophagus putrescentiae and ellipse dispersion) are identified；
C3) preparation identifies that mite to be measured is the product of tyrophagus putrescentiae or ellipse dispersion；
C4) identify that mite to be measured is tyrophagus putrescentiae or ellipse dispersion；
C5 the product of the epidemic monitoring of tyrophagus putrescentiae and/or ellipse dispersion) is prepared；
C6) the epidemic monitoring of tyrophagus putrescentiae and/or ellipse dispersion；
C7) the product of invasion and/or the diffusion of preparation prevention tyrophagus putrescentiae and/or ellipse dispersion；
C8) prevent the invasion and/or diffusion of tyrophagus putrescentiae and/or ellipse dispersion.
4. a kind of identify or assist to identify that mite to be measured is the method for tyrophagus putrescentiae or ellipse dispersion, include the following steps: with
The genomic DNA of mite to be measured is template, and primer pair first described in claim 1 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 484bp, mite to be measured is or candidate is tyrophagus putrescentiae；
If primer pair B PCR amplification obtains the target fragment that size is 639bp, mite to be measured is or candidate is ellipse dispersion.
5. the primer pair first in claim 1 is in following d1)-d8) in it is any in application:
D1) the product of preparation identification tyrophagus putrescentiae；
D2 tyrophagus putrescentiae) is identified；
D3) preparation identify mite to be measured whether be tyrophagus putrescentiae product；
D4) identify whether mite to be measured is tyrophagus putrescentiae；
D5 the product of the epidemic monitoring of tyrophagus putrescentiae) is prepared；
D6) the epidemic monitoring of tyrophagus putrescentiae；
D7) the product of invasion and/or the diffusion of preparation prevention tyrophagus putrescentiae；
D8) prevent the invasion and/or diffusion of tyrophagus putrescentiae.
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 ellipse dispersion；
E2 ellipse dispersion) is identified；
E3) preparation identify mite to be measured whether be ellipse dispersion product；
E4) identify whether mite to be measured is ellipse dispersion；
E5 the product of the epidemic monitoring of ellipse dispersion) is prepared；
E6) the epidemic monitoring of ellipse dispersion；
E7) the product of invasion and/or the diffusion of preparation prevention ellipse dispersion；
E8) prevent the invasion and/or diffusion of ellipse dispersion.
7. it is a kind of identify or assist to identify mite to be measured whether be tyrophagus putrescentiae method, include the following steps: with the base of mite to be measured
Because group DNA is template, PCR amplification is carried out using the primer pair first described in claim 1；
If PCR amplification obtains the target fragment that size is 484bp, mite to be measured is or candidate is tyrophagus putrescentiae；
If PCR amplification does not obtain the target fragment that size is 484bp, mite to be measured is not or candidate is not tyrophagus putrescentiae.
8. it is a kind of identify or assist to identify mite to be measured whether be ellipse dispersion method, include the following steps: with mite to be measured
Genomic DNA 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 639bp, mite to be measured is or candidate is ellipse dispersion；
If PCR amplification does not obtain the target fragment that size is 639bp, mite to be measured is not or candidate is not ellipse dispersion.
9. DNA fragmentation shown in NAD4 gene 405-889 or ellipse dispersion line grain in tyrophagus putrescentiae mitochondrial genomes
DNA fragmentation shown in NAD2 gene 201-840 in body genome.
10. DNA fragmentation and/or ellipse dispersion shown in NAD4 gene 405-889 in tyrophagus putrescentiae mitochondrial genomes
DNA fragmentation shown in NAD2 gene 201-840 is in following f1 in mitochondrial genomes)-f16) in it is any in application:
F1) the product of preparation identification tyrophagus putrescentiae and ellipse dispersion；
F2 tyrophagus putrescentiae and ellipse dispersion) are identified；
F3) preparation identifies that mite to be measured is the product of tyrophagus putrescentiae or ellipse dispersion；
F4) identify that mite to be measured is tyrophagus putrescentiae or ellipse dispersion；
F5) the product of preparation identification tyrophagus putrescentiae；
F6 tyrophagus putrescentiae) is identified；
F7) preparation identify mite to be measured whether be tyrophagus putrescentiae product；
F8) identify whether mite to be measured is tyrophagus putrescentiae；
F9) the product of preparation identification ellipse dispersion；
F10 ellipse dispersion) is identified；
F11) preparation identify mite to be measured whether be ellipse dispersion product；
F12) identify whether mite to be measured is ellipse dispersion；
F13 the product of the epidemic monitoring of tyrophagus putrescentiae and/or ellipse dispersion) is prepared；
F14) the epidemic monitoring of tyrophagus putrescentiae and/or ellipse dispersion；
F15) the product of invasion and/or the diffusion of preparation prevention tyrophagus putrescentiae and/or ellipse dispersion；
F16) prevent the invasion and/or diffusion of tyrophagus putrescentiae and/or ellipse dispersion.
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| CN201910490713.3A CN110117666B (en) | 2019-06-06 | 2019-06-06 | Specific primer pair for identifying Tyrophagus putrescentiae and Tyrophagus ovatus and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350522A (en) * | 2016-11-02 | 2017-01-25 | 崔玉宝 | Gene cloning and expression purification for recently found allergen of tyrophagus putrescentiae and application thereof |
| CN109055573A (en) * | 2018-09-17 | 2018-12-21 | 皖南医学院 | A method of based on the common reserve flour mite of multiple PCR technique Rapid identification |
| CN109762912A (en) * | 2019-03-18 | 2019-05-17 | 中国农业大学 | Identify the primer pair and its application of bactrocera tsuneonis and the big trypetid of tangerine |
-
2019
- 2019-06-06 CN CN201910490713.3A patent/CN110117666B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350522A (en) * | 2016-11-02 | 2017-01-25 | 崔玉宝 | Gene cloning and expression purification for recently found allergen of tyrophagus putrescentiae and application thereof |
| CN109055573A (en) * | 2018-09-17 | 2018-12-21 | 皖南医学院 | A method of based on the common reserve flour mite of multiple PCR technique Rapid identification |
| CN109762912A (en) * | 2019-03-18 | 2019-05-17 | 中国农业大学 | Identify the primer pair and its application of bactrocera tsuneonis and the big trypetid of tangerine |
Non-Patent Citations (4)
| Title |
|---|
| BIN YANG ET AL.: "Identification of astigmatid mites using ITS2 and COI regions", 《PARASITOL RES》 * |
| NCBI: "KJ571488.1", 《GENBANK》 * |
| 吴志刚 等: "中国储粮害虫DNA条形码鉴定系统研究", 《中国农业大学学报》 * |
| 郑凌霄 等: "基于DNA条形码技术的粉螨种类鉴定研究", 《中国媒介生物学及控制杂志》 * |
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