CN105586339B - It is a kind of identification Dysmicoccus neobrevipes Beardsley primer pair and its application - Google Patents
It is a kind of identification Dysmicoccus neobrevipes Beardsley primer pair and its application Download PDFInfo
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Abstract
The invention discloses a kind of primer pair of identification Dysmicoccus neobrevipes Beardsley and its applications.The primer pair of the present invention is made of primer D.neo 28SF and primer D.neo 28SR, respectively as shown in sequence table 1,2.The present invention also protects the primer pair identifying or assisting to identify whether insect to be measured be in application in Dysmicoccus neobrevipes Beardsley, detection biological sample to be measured containing the application in Dysmicoccus neobrevipes Beardsley.The present invention also protect it is a kind of identify insect to be measured whether be in the method for Dysmicoccus neobrevipes Beardsley, a kind of detection biological sample to be measured whether the method containing Dysmicoccus neobrevipes Beardsley.Whether the detection method that the present invention establishes can judgement sample be rapidly and accurately Dysmicoccus neobrevipes Beardsley, quickly detection provides foundation with accurately identifying to the Dysmicoccus neobrevipes Beardsley that is established as of this method, for effectively stop Dysmicoccus neobrevipes Beardsley China diffusion is propagated further and harm provides technical guarantee, provide guarantee for imports and exports safety.
Description
Technical field
The invention belongs to technical field of plant quarantine, and in particular to it is a kind of identification Dysmicoccus neobrevipes Beardsley primer pair and its answer
With.
Background technology
Dysmicoccus neobrevipes Beardsley (Dysmicoccus neobrevipes Beardsley) belongs to Semiptera (Hemiptera), a red-spotted lizard
Superfamily (Coccoidea), Pseudococcidae (Pseudococcidae).
Dysmicoccus neobrevipes Beardsley host range is very extensive, endangers the agricultural economy such as pineapple, banana, manaca, citrus, grape
Crop is mainly distributed on tropical and subtropical region, is locally distributed note in China only Hainan, Taiwan, Guangdong at present
It carries.Dysmicoccus neobrevipes Beardsley colonizes in host plant surface with ovum, larva and adult, thus is easily propagated with host plant, is arranged
Enter in the inward plant quarantine harmful organism register of the People's Republic of China (PRC), often there are intercepting and capturing in port quarantine.
Dysmicoccus neobrevipes Beardsley category identification is based primarily upon the formalness feature of female adult pest, including body is long, porous gland type,
Quantity and arrangement, and the discriminating for the nymph or residuum that these identification features often intercept and capture port is then helpless, and with passing
Typoiogical classification of uniting carries out coccid qualification process complexity, need to be a series of through fixation, transparent, dyeing, decoloration, shaping, mounting, microscopy etc.
Step, this is quickly tested with Imported Fruits puts, speeds passage through customs to form contradiction.Therefore, traditional morphological method is difficult to meet port
The current demand for quickly identifying and identifying to Dysmicoccus neobrevipes Beardsley during quarantine and the allocation and transportation of fruit and seedling, especially works as intercepting and capturing
Sample be nymph or residuum when, and Dysmicoccus neobrevipes Beardsley quick and precisely identification be effectively to prevent it that diffusion is propagated further
Prerequisite.
Invention content
The object of the present invention is to provide a kind of primer pair of identification Dysmicoccus neobrevipes Beardsley and its applications.
The present invention provides a kind of primer pair, is made of primer D.neo-28SF and primer D.neo-28SR；
The primer D.neo-28SF is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) sequence 1 is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 1
The DNA molecular of identical function；
The primer D.neo-28SR is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) sequence 2 is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 2
The DNA molecular of identical function.
The purposes of the primer pair is following (b1) or (b2) or (b3) or (b4)：
(b1) it identifies or assists to identify whether insect to be measured is Dysmicoccus neobrevipes Beardsley；
(b2) prepare for identify or assist to identify insect to be measured whether be Dysmicoccus neobrevipes Beardsley kit；
(b3) detect in biological sample to be measured whether contain Dysmicoccus neobrevipes Beardsley；
(b4) prepare for detect in biological sample to be measured whether the kit containing Dysmicoccus neobrevipes Beardsley.
The present invention also protects the kit containing the primer pair；The purposes of the kit is following (c1) or (c2)：
(c1) it identifies or assists to identify whether insect to be measured is Dysmicoccus neobrevipes Beardsley；
(c2) detect in biological sample to be measured whether contain Dysmicoccus neobrevipes Beardsley.
The present invention also protects the preparation method of the kit, includes the steps that individually packing each primer.
The present invention also protect it is a kind of identify insect to be measured whether be Dysmicoccus neobrevipes Beardsley method, include the following steps：
(1) genomic DNA of insect to be measured is extracted；
(2) using the genomic DNA of step (1) extraction as template, PCR amplification is carried out using the primer pair, if PCR
The DNA fragmentation containing 230bp-240bp, insect to be measured are in amplified production or candidate is Dysmicoccus neobrevipes Beardsley, if PCR amplification
DNA fragmentation, insect to be measured in product without containing 230bp-240bp are or candidate is non-Dysmicoccus neobrevipes Beardsley.
The DNA fragmentation of the DNA fragmentation of the 230bp-240bp concretely 235bp.
The DNA fragmentation of the 230bp-240bp is concretely following (d1) or (d2)：
(d1) single strand dna shown in the sequence 3 of sequence table；
(d2) there is the DNA molecular of 95% or more homology with the sequence of sequence table 3.
The present invention also protect it is a kind of identify insect to be measured whether be Dysmicoccus neobrevipes Beardsley method, include the following steps：
It detects in the genomic DNA of insect to be measured and whether contains specific DNA fragment, if containing specific DNA fragment, to be measured
Insect is or candidate is Dysmicoccus neobrevipes Beardsley, if not containing specific DNA fragment, insect to be measured is or candidate is non-new pineapple ash
Mealybug；
The specific DNA fragment is the target sequence of the primer pair described in the genome of Dysmicoccus neobrevipes Beardsley.
The specific DNA fragment is concretely following (d1) or (d2)：
(d1) single strand dna shown in the sequence 3 of sequence table；
(d2) there is the DNA molecular of 95% or more homology with the sequence of sequence table 3.
The present invention also protect in a kind of detection biological sample to be measured whether the method containing Dysmicoccus neobrevipes Beardsley, including it is as follows
Step：
(1) total DNA of biological sample to be measured is extracted；
(2) using the total DNA of step (1) extraction as template, PCR amplification is carried out using the primer pair, if PCR amplification
DNA fragmentation containing 230bp-240bp in product contains or doubtful containing Dysmicoccus neobrevipes Beardsley in biological sample to be measured, if
It is not contained in DNA fragmentation, biological sample to be measured in pcr amplification product without containing 230bp-240bp or doubtful without containing new spinach
Trailing plants ash mealybug.
The DNA fragmentation of the DNA fragmentation of the 230bp-240bp concretely 235bp.
The DNA fragmentation of the 230bp-240bp is concretely following (d1) or (d2)：
(d1) single strand dna shown in the sequence 3 of sequence table；
(d2) there is the DNA molecular of 95% or more homology with the sequence of sequence table 3.
The present invention also protect in a kind of detection biological sample to be measured whether the method containing Dysmicoccus neobrevipes Beardsley, including it is as follows
Step：
It detects in the total DNA of biological sample to be measured and whether contains specific DNA fragment, if containing specific DNA fragment, to be measured
Contain in biological sample or doubtful containing Dysmicoccus neobrevipes Beardsley, if without containing being free of in specific DNA fragment, biological sample to be measured
Have or doubtful without containing Dysmicoccus neobrevipes Beardsley；
The specific DNA fragment is the target sequence of the primer pair described in the genome of Dysmicoccus neobrevipes Beardsley.
The present invention also protects a kind of specific DNA fragment, is the target of the primer pair described in the genome of Dysmicoccus neobrevipes Beardsley
Sequence.
The specific DNA fragment is concretely following (d1) or (d2)：
(d1) single strand dna shown in the sequence 3 of sequence table；
(d2) there is the DNA molecular of 95% or more homology with the sequence of sequence table 3.
Any description above insect concretely mealybug.The mealybug concretely Dysmicoccus neobrevipes Beardsley, Jack Bei Ershi
Mealybug, ocean stern line mealybug, tangerine stern line mealybug, glandular spines mealybug, pineapple ash mealybug or Lee's Bi Lishi ash mealybugs.
Dysmicoccus neobrevipes Beardsley in any description above " whether containing Dysmicoccus neobrevipes Beardsley in biological sample to be measured " can be
Adult, nymph or residuum.
In the reaction system of any description above PCR amplification, the working concentration of primer D.neo-28SF is 10 μm of ol/L, is drawn
The working concentration of object D.neo-28SR is 10 μm of ol/L.
The reaction system of any description above PCR amplification is concretely：Easy Taq Buffer (include 10mmol/
LMgCl2) 2.5 μ L, 2.5 μm of each 2 μ L, Easy Taq of ol/L dNTP Mixture 2 μ L, D.neo-28SF and D.neo-28SR
DNA Polymerase (5U/ μ L) 0.25 μ L, template DNA 4 μ L, ddH2O 12.25μL。
The response procedures of any description above PCR amplification are concretely：95 DEG C of pre-degeneration 3min；94 DEG C denaturation 45s, 60 DEG C
Anneal 1min, and 72 DEG C of extension 1min run 35 cycles；Last 72 DEG C of extensions 7min.
The present invention is around the Dysmicoccus neobrevipes Beardsley easily spread in china with the allocation and transportation of plant product and its nursery stock etc., to agricultural
There are grave dangers for industry production, the problem of but being difficult to quick and precisely be differentiated, using the technology and method of specific PCR, from
The angle for detecting monitoring, studies its Fast Detection Technique, meanwhile, with the 6 of other categories kind that Pseudococcidae is often intercepted and captured in port
Kind mealybug etc. is control, carries out species specificity inspection, is cut with different host fruits or same host fruit but different batches fruit
The Dysmicoccus neobrevipes Beardsley obtained carries out application verification.The detection method that the present invention establishes can rapidly and accurately judgement sample whether be
Dysmicoccus neobrevipes Beardsley, quickly detection provides foundation with accurately identifying to the Dysmicoccus neobrevipes Beardsley that is established as of this method, for effectively resistance
Cut Dysmicoccus neobrevipes Beardsley China diffusion is propagated further and harm provides technical guarantee, provide guarantor for imports and exports safety
Card.
Description of the drawings
Fig. 1 is the corresponding electrophoretogram of specific detection in embodiment 3.
Fig. 2 is that versatility detects corresponding electrophoretogram in embodiment 4.
Fig. 3 is that 5 medium sensitivity of embodiment detects corresponding electrophoretogram.
Specific implementation mode
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.
Dysmicoccus neobrevipes Beardsley：Bibliography：Xu Lang, Yu Daojian, Jiao Yi wait the DNA of Dysmicoccus neobrevipes Beardsleys and its allied species
Bar code identifies [J] plant quarantine, 2013,27 (3)：66-69.；The public can examine from Xiamen Entry-Exit Inspection and Quarantine Bureau
Epidemic disease technique center obtains.
Jack's Bei Ershi mealybugs：Bibliography：Xu Lang, Yu Daojian, Jiao Yi wait Dysmicoccus neobrevipes Beardsleys and its allied species
DNA bar code identifies [J] plant quarantine, 2013,27 (3)：66-69.；The public can examine from Xiamen Entry-Exit Inspection and Quarantine Bureau
Test the acquisition of Quarantine Techniques center.
Ocean stern line mealybug：Bibliography：Xu Lang, Yu Daojian, Jiao Yi wait the DNA of Dysmicoccus neobrevipes Beardsleys and its allied species
Bar code identifies [J] plant quarantine, 2013,27 (3)：66-69.；The public can examine from Xiamen Entry-Exit Inspection and Quarantine Bureau
Epidemic disease technique center obtains.
Tangerine stern line mealybug：Bibliography：Ni Xingwu, Gao Quanzhun, Yu Fangping, Zhang Zhenmin, the ten thousand inner Taiwans .2006. of Zheng
The pest plant quarantine that may be carried on fruit, 20 (3)：155~157.；The public can enter and leave the border from Xiamen and examine inspection
Epidemic disease office inspection and quarantine technique center obtains.
Glandular spines mealybug：Bibliography：Ni Xingwu, Gao Quanzhun, Yu Fangping, waiting may carrying on the fruit of the Taiwans
Pest [J] plant quarantine, 2006,20 (3)：155-157.；The public can be from the inspection and quarantine of Xiamen Entry-Exit Inspection and Quarantine Bureau
Technique center obtains.
Pineapple ash mealybug：Bibliography：Xu Lang, Yu Daojian, Jiao Yi wait the DNA items of Dysmicoccus neobrevipes Beardsleys and its allied species
Shape code identifies [J] plant quarantine, 2013,27 (3)：66-69.；The public can be from the inspection and quarantine of Xiamen Entry-Exit Inspection and Quarantine Bureau
Technique center obtains.
Lee's Bi Lishi ash mealybugs：Bibliography：Xu Lang, Yu Daojian, Jiao Yi wait Dysmicoccus neobrevipes Beardsleys and its allied species
DNA bar code identifies [J] plant quarantine, 2013,27 (3)：66-69.；The public can examine from Xiamen Entry-Exit Inspection and Quarantine Bureau
Test the acquisition of Quarantine Techniques center.
Embodiment 1, design of primers
It carries out a large amount of sequence analyses, compare several primers obtained for identifying Dysmicoccus neobrevipes Beardsley.By each primer
Preliminary experiment is carried out, compares the performances such as sensitivity, specificity, finally obtains the pair of primers for identifying Dysmicoccus neobrevipes Beardsley.Such as
Shown in lower：
D.neo-28SF (sequence 1 of sequence table)：5’-CGTGTGCGCTCGACGGGGTT-3’；
D.neo-28SR (sequence 2 of sequence table)：5’-CCGAAGCGAACGCCGCAA-3’.
Embodiment 2, detection method are established
One, the extraction of genomic DNA
With reference to TIANamp Genomic DNA Kit blood/cell/tissue genome DNA extracting reagent kit (centrifugal column
Type), genomic DNA is extracted, is carried out in accordance with the following steps successively：
1, mealybug to be measured is placed in the centrifuge tube of 0.6mL, the pure water of 0.4mL is added, covers tightly lid, in circumferential oscillation device
Upper concussion 15s takes out mealybug and is placed on the filter paper that high-temperature sterilization is crossed, and to suck moisture, drying keeps polypide fully dry.
2,1.5mL centrifuge tubes are taken, polypide is placed in centrifuge tube, is smashed to pieces.
3, into centrifuge tube, power enters the Buffer GA of 200 μ L, then power enters 5 μ L RNaseA and 20 μ L Proteinase K,
Slight concussion.
4, centrifuge tube is placed in 56 DEG C of water bath with thermostatic control shaking tables and places 30min (take out and shaken after 10min), taken out
Wink is to remove the droplet on tube wall afterwards.
5, the Buffer GB of 200 μ L are added into centrifuge tube, overturn mixing, wink is from 70 DEG C of water bath with thermostatic control shaking tables are placed
10min, after taking-up, wink from.
6, be added 200 μ L absolute ethyl alcohols into centrifuge tube, shake 15s, wink from.
7, CB3 adsorption columns are put into collecting pipe, the solution in centrifuge tube are transferred in CB3 adsorption columns, 12000rpm
30s is centrifuged, waste liquid is outwelled, CB3 adsorption columns is put back in collecting pipe.
8, Buffer GD, 12000rpm the centrifugation 30s of 500 μ L are added into CB3 adsorption columns, outwells waste liquid, CB3 is inhaled
Attached column is put back in collecting pipe.
9, Buffer PW, 12000rpm the centrifugation 30s of 600 μ L are added into CB3 adsorption columns, outwells waste liquid, CB3 is inhaled
Attached column is put back in collecting pipe.
10, repetitive operation step 9.
11, CB3 adsorption columns are put back in collecting pipe, 12000rpm centrifuges 2min, outwells waste liquid, adsorption column CB3 is placed in
It is placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
12, the 1.5mL cleaning centrifuge tubes that high pressure sterilization is crossed are taken.CB3 adsorption columns are transferred in centrifuge tube, and to adsorbed film
The elution buffer TE of 50 μ L is vacantly added dropwise in intermediate position, is placed at room temperature for 5min, and then 12000rpm centrifuges 2min, obtains gene
Group DNA solution.
13, the Genomic DNA solution extracted is put in -20 DEG C of preservations, it is spare.
Two, PCR amplification
Using the genomic DNA of step 1 as template, using the primer pair of D.neo-28SF and primer D.neo-28SR compositions
Carry out PCR amplification.
The reaction system of PCR amplification is：Easy Taq Buffer (include 10mmol/L MgCl2) 2.5 μ L, 2.5 μm of ol/
Each 2 μ L, Easy Taq DNA Polymerase of 2 μ L, D.neo-28SF and D.neo-28SR of L dNTP Mixture (5U/ μ L)
0.25 μ L, template DNA 4 μ L, ddH2O12.25μL.In the reaction system of PCR, the concentration of D.neo-28SF and D.neo-28SR
It is 10 μm of ol/L.
The response procedures of PCR amplification are：95 DEG C of pre-degeneration 3min；94 DEG C of denaturation 45s, 60 DEG C of annealing 1min, 72 DEG C extend
1min runs 35 cycles；Last 72 DEG C of extensions 7min.
Pcr amplification product is detected into row agarose gel electrophoresis, if obtaining the amplified production of 230bp-240bp, is waited for
Survey mealybug is Dysmicoccus neobrevipes Beardsley, if the amplified production for not obtaining 230bp-240bp mealybug to be measured is non-new pineapple ashes
A red-spotted lizard.
Embodiment 3, specificity
Sample to be tested is respectively：Dysmicoccus neobrevipes Beardsley, Jack Bei Ershi mealybugs, ocean stern line mealybug, tangerine stern line mealybug, gland
Pierce mealybug, pineapple ash mealybug, Lee's Bi Lishi ash mealybugs.
Each sample to be tested is detected using the method that embodiment 2 is established respectively.
Agarose gel electrophoresis the result is shown in Figure 1.In Fig. 1, M is the DNA Marker of 100bp, and swimming lane 1 is new pineapple ashes
The pcr amplification product of a red-spotted lizard genomic DNA, swimming lane 2 are the pcr amplification product of Jack's Bei Ershi mealybug genomic DNAs, and swimming lane 3 is
The pcr amplification product of ocean stern line mealybug genomic DNA, swimming lane 4 are the pcr amplification product of tangerine stern line mealybug genomic DNA, swimming
Road 5 is the pcr amplification product of glandular spines mealybug genomic DNA, and swimming lane 6 is the pcr amplification product of pineapple ash mealybug genomic DNA,
Swimming lane 7 is the pcr amplification product of Lee's Bi Lishi ash mealybug genomic DNAs.The results show that the PCR amplification production of Dysmicoccus neobrevipes Beardsley
The DNA fragmentation (through sequencing, as shown in the sequence 3 of sequence table) of detection 235bp in object, and Jack Bei Ershi mealybugs, ocean stern line
Mealybug, tangerine stern line mealybug, glandular spines mealybug, pineapple ash mealybug, Lee's Bi Lishi ash mealybugs pcr amplification product in be not detected
The DNA fragmentation of 230bp-240bp shows that the PCR method established has good specificity, can be used for the fast of Dysmicoccus neobrevipes Beardsley
Speed detection.
Embodiment 4, versatility
Sample to be tested is respectively：It is fragrant from Taiwan pineapple, Indonesia's rambutan, Hong Kong rambutan, Taiwan
The Dysmicoccus neobrevipes Beardsley intercepted and captured in any of several broadleaf plants, saba (batch 1), saba (batch 2), saba (batch 3).
Each sample to be tested is detected using the method that embodiment 2 is established respectively.
Agarose gel electrophoresis result is shown in Fig. 2.In Fig. 2, M is the DNA Marker of 100bp, and swimming lane 1 is Taiwan spinach
The pcr amplification product of Dysmicoccus neobrevipes Beardsley genomic DNA on trailing plants, swimming lane 2 are the new pineapple ash on Indonesia's rambutan
The pcr amplification product of mealybug genomic DNA, swimming lane 3 are that the PCR of the Dysmicoccus neobrevipes Beardsley genomic DNA on the rambutan of Hong Kong expands
Increase production object, swimming lane 4 is the pcr amplification product of the Dysmicoccus neobrevipes Beardsley genomic DNA on saba (batch 1), and swimming lane 5 is
The pcr amplification product of Dysmicoccus neobrevipes Beardsley genomic DNA on the banana of Taiwan, swimming lane 6 are on saba (batch 2)
Dysmicoccus neobrevipes Beardsley genomic DNA pcr amplification product, swimming lane 7 be saba 3 on Dysmicoccus neobrevipes Beardsley genome
The pcr amplification product of DNA.The results show that the above sample can amplify the PCR product of 230bp-240bp, illustrate new pineapple ash
Mealybug special primer versatility is good.
Embodiment 5, sensitivity
1, Dysmicoccus neobrevipes Beardsley genomic DNA is extracted using the step of embodiment 2 one method, obtaining DNA concentration is
The DNA solution of 11.5 μ g/ μ L；
2, the DNA solution obtained with 10 times of gradient dilution steps 1 of TE buffer solutions, obtains each dilution；
3, take each dilution that step 2 arrives as template, using primer D.neo-28SF and primer D.neo-28SR compositions
Primer pair according to the step of embodiment 2 two method carry out PCR amplification；
Since the dilution of the dilution of use is different, following different reaction system is formed：
In reaction system 1, Dysmicoccus neobrevipes Beardsley genomic DNA initial content is：46μg；
In reaction system 2, Dysmicoccus neobrevipes Beardsley genomic DNA initial content is：4.6μg；
In reaction system 3, Dysmicoccus neobrevipes Beardsley genomic DNA initial content is：460ng；
In reaction system 4, Dysmicoccus neobrevipes Beardsley genomic DNA initial content is：46ng；
In reaction system 5, Dysmicoccus neobrevipes Beardsley genomic DNA initial content is：4.6ng；
In reaction system 6, Dysmicoccus neobrevipes Beardsley genomic DNA initial content is：460pg；
In reaction system 7, Dysmicoccus neobrevipes Beardsley genomic DNA initial content is：46pg；
In reaction system 8, Dysmicoccus neobrevipes Beardsley genomic DNA initial content is：4.6pg.
Pcr amplification product into row agarose gel electrophoresis and is taken pictures.
Agarose gel electrophoresis result is shown in Fig. 3.In Fig. 3, M is the DNA Marker of 100bp, and swimming lane 1-8, which is corresponding in turn to, to be adopted
With the amplified production of PCR when reaction system 1-8, the results show that Dysmicoccus neobrevipes Beardsley genomic DNA initial content is 4.6ng-46
When μ g, template amplification can amplify the PCR product of 230bp-240bp, and brightness gradually weakens, Dysmicoccus neobrevipes Beardsley genome
DNA initial contents are that the purpose band of the template amplification of 4.6ng is relatively fuzzyyer, and the template of other dilutions does not amplify purpose item
Band.The result shows that the PCR method of foundation has good sensitivity, the quick detection of Dysmicoccus neobrevipes Beardsley is cannot be only used for, and
And the PCR of 230bp-240bp can be also amplified when the content of Dysmicoccus neobrevipes Beardsley genomic DNA in PCR system is down to 4.6ng
Product, sensitivity are high.
Claims (7)
1. primer pair is made of primer D.neo-28SF and primer D.neo-28SR；
The primer D.neo-28SF is single strand dna shown in the sequence 1 of sequence table；
The primer D.neo-28SR is single strand dna shown in the sequence 2 of sequence table.
2. the application of primer pair described in claim 1, for following (b1) or (b2) or (b3) or (b4):
(b1) it identifies or assists to identify whether insect to be measured is Dysmicoccus neobrevipes Beardsley；
(b2) prepare for identify or assist to identify insect to be measured whether be Dysmicoccus neobrevipes Beardsley kit；
(b3) detect in biological sample to be measured whether contain Dysmicoccus neobrevipes Beardsley；
(b4) prepare for detect in biological sample to be measured whether the kit containing Dysmicoccus neobrevipes Beardsley.
3. application as claimed in claim 2, it is characterised in that：In (b1) or (b2), the insect is mealybug.
4. the kit containing primer pair described in claim 1；The purposes of the kit is following (c1) or (c2)：
(c1) it identifies or assists to identify whether insect to be measured is Dysmicoccus neobrevipes Beardsley；
(c2) detect in biological sample to be measured whether contain Dysmicoccus neobrevipes Beardsley.
5. the preparation method of kit described in claim 4 includes the steps that individually packing each primer.
6. it is a kind of identify insect to be measured whether be Dysmicoccus neobrevipes Beardsley method, include the following steps：
(1) genomic DNA of insect to be measured is extracted；
(2) using the genomic DNA of step (1) extraction as template, PCR amplification is carried out using primer pair described in claim 1, such as
The DNA fragmentation containing 230bp-240bp, insect to be measured are Dysmicoccus neobrevipes Beardsley in fruit pcr amplification product, if PCR amplification is produced
DNA fragmentation, insect to be measured in object without containing 230bp-240bp are non-Dysmicoccus neobrevipes Beardsley.
7. in a kind of detection biological sample to be measured whether the method containing Dysmicoccus neobrevipes Beardsley, include the following steps：
(1) total DNA of biological sample to be measured is extracted；
(2) using the total DNA of step (1) extraction as template, PCR amplification is carried out using primer pair described in claim 1, if
DNA fragmentation containing 230bp-240bp in pcr amplification product contains Dysmicoccus neobrevipes Beardsley in biological sample to be measured, if PCR
Dysmicoccus neobrevipes Beardsley is not contained in DNA fragmentation, biological sample to be measured in amplified production without containing 230bp-240bp.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610159773.3A CN105586339B (en) | 2016-03-21 | 2016-03-21 | It is a kind of identification Dysmicoccus neobrevipes Beardsley primer pair and its application |
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| CN201610159773.3A CN105586339B (en) | 2016-03-21 | 2016-03-21 | It is a kind of identification Dysmicoccus neobrevipes Beardsley primer pair and its application |
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| CN109355430A (en) * | 2018-11-19 | 2019-02-19 | 海南省农业科学院热带果树研究所 | A kind of multiple RT-PCR detection method of synchronous detection 3 kinds of pineapples wilting correlated virus and pineapple bacilliform DNA virus |
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| CN108441950A (en) * | 2018-04-28 | 2018-08-24 | 无锡英特派金属制品有限公司 | A kind of preparation method of nano silver wires with high length-diameter ratio |
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| CN109355430A (en) * | 2018-11-19 | 2019-02-19 | 海南省农业科学院热带果树研究所 | A kind of multiple RT-PCR detection method of synchronous detection 3 kinds of pineapples wilting correlated virus and pineapple bacilliform DNA virus |
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