CN105586339A - Primer pair used for identifying dysmicoccus neobrevipes and application thereof - Google Patents
Primer pair used for identifying dysmicoccus neobrevipes and application thereof Download PDFInfo
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Abstract
The invention discloses a primer pair used for identifying dysmicoccus neobrevipes and application thereof. The primer pair is composed of a primer D.neo-28SF and a primer D.neo-28SR which are as shown in a sequence table 1 and a sequence table 2. The invention further provides application of the primer pair in identifying or assisting in identifying whether insects to be detected are dysmicoccus neobrevipes or not and detecting whether biological samples to be detected contain dysmicoccus neobrevipes or not. The invention further discloses a method for identifying whether the insects to be detected are dysmicoccus neobrevipes or not and a method for detecting whether the biological samples to be detected contain dysmicoccus neobrevipes or not. The built detection methods can be used for quickly and accurately judging whether the samples are dysmicoccus neobrevipes or not. Bases are provided for quick detection and accurate recognition of dysmicoccus neobrevipes due to establishment of the methods, a technological guarantee is provided for effectively intercepting further spreading and damage of dysmicoccus neobrevipes in China, and a guarantee is provided for import and export security.
Description
Technical field
The invention belongs to plant quarantine technical field, be specifically related to a kind of primer pair and application thereof of identifying new pineapple ash mealybug.
Background technology
New pineapple ash mealybug (DysmicoccusneobrevipesBeardsley) belongs to Semiptera (Hemiptera), Coccoidea (Coccoidea), Pseudococcidae (Pseudococcidae).
New pineapple ash mealybug host range is very extensive, the agricultural industrial crops such as harm pineapple, banana, manaca, oranges and tangerines, grape, and it is mainly distributed in tropical and subtropical region, and in China, only there is the record of distribution Hainan, Taiwan, part, Guangdong at present. New pineapple ash mealybug colonizes in host plant surface with ovum, larva and adult, thereby very easily propagates with host plant, is put into the People's Republic of China (PRC) and enters the territory in plant quarantine harmful organism register, often has intercepting and capturing in port quarantine.
The mainly formalness feature based on female adult of new pineapple ash mealybug kind identification, comprise body length, porous gland type, quantity and arrangement, the nymph that these recognition features are are often intercepted and captured for port or the discriminating of residual body are helpless, and classify and carry out coccid qualification process complexity with traditional form, need be through series of steps such as fixing, transparent, dyeing, decolouring, shaping, mounting, microscopies, this and Imported Fruits are tested the formation contradiction of putting, speed passage through customs fast. Therefore, traditional morphological method is difficult to meet in port quarantine and fruit and seedling allocation and transportation process to the identification and the current demand of identifying fast of new pineapple ash mealybug, especially when the sample of intercepting and capturing is while being nymph or residual body, and the quick and precisely identification of new pineapple ash mealybug is effectively to stop it further to propagate the prerequisite of diffusion.
Summary of the invention
The object of this invention is to provide a kind of primer pair and application thereof of identifying new pineapple ash mealybug.
The invention provides a kind of primer pair, formed by primer D.neo-28SF and primer D.neo-28SR;
Described 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 had to the DNA molecular of identical function through the replacement of one or several nucleotides and/or disappearance and/or interpolation and with sequence 1;
Described 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 had to the DNA molecular of identical function through the replacement of one or several nucleotides and/or disappearance and/or interpolation and with sequence 2.
The purposes of described primer pair is following (b1) or (b2) or (b3) or (b4):
(b1) identify or whether assistant identification insect to be measured is new pineapple ash mealybug;
(b2) for the preparation of identifying or whether assistant identification insect to be measured is the kit of new pineapple ash mealybug;
(b3) detect in biological specimen to be measured, whether to contain new pineapple ash mealybug;
(b4) for the preparation of detecting the kit that whether contains new pineapple ash mealybug in biological specimen to be measured.
The present invention also protects the kit that contains described primer pair; The purposes of described kit is following (c1) or (c2):
(c1) identify or whether assistant identification insect to be measured is new pineapple ash mealybug;
(c2) detect in biological specimen to be measured, whether to contain new pineapple ash mealybug.
The present invention also protects the preparation method of described kit, comprises each the primer step of packaging separately.
The present invention also protects and a kind ofly identifies that whether insect to be measured is the method for new pineapple ash mealybug, comprises the steps:
(1) extract the genomic DNA of insect to be measured;
(2) genomic DNA extracting taking step (1) is as template, adopt described primer pair to carry out pcr amplification, if in pcr amplification product, contain the DNA fragmentation of 230bp-240bp, insect to be measured for or candidate be new pineapple ash mealybug, if in pcr amplification product, do not contain the DNA fragmentation of 230bp-240bp, insect to be measured for or candidate be non-new pineapple ash mealybug.
The DNA fragmentation of described 230bp-240bp specifically can be the DNA fragmentation of 235bp.
The DNA fragmentation of described 230bp-240bp specifically can be as follows (d1) or (d2):
(d1) single strand dna shown in the sequence 3 of sequence table;
(d2) there is the DNA molecular of 95% above homology with the sequence 3 of sequence table.
The present invention also protects and a kind ofly identifies that whether insect to be measured is the method for new pineapple ash mealybug, comprises the steps:
Detect in the genomic DNA of insect to be measured whether contain specific DNA fragment, if contain specific DNA fragment, insect to be measured for or candidate be new pineapple ash mealybug, if do not contain specific DNA fragment, insect to be measured for or candidate be non-new pineapple ash mealybug;
Described specific DNA fragment is the target sequence of the primer pair described in the genome of new pineapple ash mealybug.
Described specific DNA fragment specifically can be as follows (d1) or (d2):
(d1) single strand dna shown in the sequence 3 of sequence table;
(d2) there is the DNA molecular of 95% above homology with the sequence 3 of sequence table.
The present invention also protects a kind of method that whether contains new pineapple ash mealybug in biological specimen to be measured that detects, and comprises the steps:
(1) extract total DNA of biological specimen to be measured;
(2) the total DNA extracting taking step (1) is as template, adopt described primer pair to carry out pcr amplification, in the DNA fragmentation of 230bp-240bp, biological specimen to be measured, contain or doubtfully contain new pineapple ash mealybug if contained in pcr amplification product, in the DNA fragmentation of 230bp-240bp, biological specimen to be measured, do not contain or doubtfully do not contain new pineapple ash mealybug if do not contained in pcr amplification product.
The DNA fragmentation of described 230bp-240bp specifically can be the DNA fragmentation of 235bp.
The DNA fragmentation of described 230bp-240bp specifically can be as follows (d1) or (d2):
(d1) single strand dna shown in the sequence 3 of sequence table;
(d2) there is the DNA molecular of 95% above homology with the sequence 3 of sequence table.
The present invention also protects a kind of method that whether contains new pineapple ash mealybug in biological specimen to be measured that detects, and comprises the steps:
Detect in total DNA of biological specimen to be measured and whether contain specific DNA fragment, in specific DNA fragment, biological specimen to be measured, contain or doubtfully contain new pineapple ash mealybug if contained, in specific DNA fragment, biological specimen to be measured, do not contain or doubtfully do not contain new pineapple ash mealybug if do not contained;
Described specific DNA fragment is the target sequence of the primer pair described in the genome of new pineapple ash mealybug.
The present invention also protects a kind of specific DNA fragment, is the target sequence of the primer pair described in the genome of new pineapple ash mealybug.
Described specific DNA fragment specifically can be as follows (d1) or (d2):
(d1) single strand dna shown in the sequence 3 of sequence table;
(d2) there is the DNA molecular of 95% above homology with the sequence 3 of sequence table.
Arbitrary described insect specifically can be mealybug above. Described mealybug specifically can be new pineapple ash mealybug, Jack Bei Ershi mealybug, ocean stern line mealybug, tangerine stern line mealybug, glandular spines mealybug, pineapple ash mealybug or Lee Bi Lishi ash mealybug.
New pineapple ash mealybug in arbitrary described " whether containing new pineapple ash mealybug in biological specimen to be measured " can be adult, nymph or residual body above.
In the reaction system of arbitrary described pcr amplification, the working concentration of primer D.neo-28SF is 10 μ mol/L above, and the working concentration of primer D.neo-28SR is 10 μ mol/L.
The reaction system of arbitrary described pcr amplification specifically can be above: EasyTaqBuffer (comprises 10mmol/LMgCl2) 2.5 μ L, 2.5 μ mol/LdNTPMixture2 μ L, the each 2 μ L of D.neo-28SF and D.neo-28SR, EasyTaqDNAPolymerase (5U/ μ L) 0.25 μ L, template DNA 4 μ L, ddH2O12.25μL。
The response procedures of arbitrary described pcr amplification specifically can be above: 95 DEG C of denaturation 3min; 94 DEG C of sex change 45s, 60 DEG C of annealing 1min, 72 DEG C are extended 1min, move 35 circulations; Last 72 DEG C are extended 7min.
The present invention is around the very easily its spread in china with the allocation and transportation of plant product and nursery stock etc. thereof of new pineapple ash mealybug, agriculture and forestry are produced and had grave danger, but be difficult to the problem of quick and precisely differentiating, adopt the technology and method of specific PCR, from detecting the angle of monitoring, study its Fast Detection Technique, simultaneously, 6 kinds of mealybugs that other genus of often intercepting and capturing in port taking Pseudococcidae are planted etc. are contrast, carry out species specificity inspection, with different host fruits or same host fruit but the new pineapple ash mealybug that different batches fruit is intercepted and captured carries out application verification. Whether the detection method that the present invention sets up rapidly and accurately judgement sample is new pineapple ash mealybug, the new pineapple ash mealybug fast detecting that is established as of the method provides foundation with accurate identification, provide technical guarantee for effectively stopping new pineapple ash mealybug in further propagation diffusion and the harm of China, for imports and exports safety provides guarantee.
Brief description of the drawings
Fig. 1 is electrophoretogram corresponding to specific detection in embodiment 3.
Fig. 2 is that in embodiment 4, versatility detects corresponding electrophoretogram.
Fig. 3 is that embodiment 5 medium sensitivities detect corresponding electrophoretogram.
Detailed description of the invention
Following embodiment is convenient to understand better the present invention, but does not limit the present invention. Experimental technique in following embodiment, if no special instructions, is conventional method. Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop. Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
New pineapple ash mealybug: bibliography: Xu Lang, Yu Daojian, Jiao Yi, etc. the DNA bar code qualification [J] of new pineapple ash mealybug and allied species thereof. plant quarantine, 2013,27 (3): 66-69.; The public can obtain from Inspection and Quarantine Center of Xiamen Entry-Exit Inspection and Quarantine Bureau.
Jack Bei Ershi mealybug: bibliography: Xu Lang, Yu Daojian, Jiao Yi, etc. the DNA bar code qualification [J] of new pineapple ash mealybug and allied species thereof. plant quarantine, 2013,27 (3): 66-69.; The public can obtain from Inspection and Quarantine Center of Xiamen Entry-Exit Inspection and Quarantine Bureau.
Ocean stern line mealybug: bibliography: Xu Lang, Yu Daojian, Jiao Yi, etc. the DNA bar code qualification [J] of new pineapple ash mealybug and allied species thereof. plant quarantine, 2013,27 (3): 66-69.; The public can obtain from Inspection and Quarantine Center of Xiamen Entry-Exit Inspection and Quarantine Bureau.
Tangerine stern line mealybug: bibliography: Ni Xingwu, Gao Quanzhun, Yu Fangping, Zhang Zhenmin, the insect that may carry on ten thousand li of .2006. Taiwan fruits of Zheng. plant quarantine, 20 (3): 155～157.; The public can obtain from Inspection and Quarantine Center of Xiamen Entry-Exit Inspection and Quarantine Bureau.
Glandular spines mealybug: bibliography: Ni Xingwu, Gao Quanzhun, Yu Fangping, etc. the insect [J] that may carry on the fruit of Taiwan. plant quarantine, 2006,20 (3): 155-157.; The public can obtain from Inspection and Quarantine Center of Xiamen Entry-Exit Inspection and Quarantine Bureau.
Pineapple ash mealybug: bibliography: Xu Lang, Yu Daojian, Jiao Yi, etc. the DNA bar code qualification [J] of new pineapple ash mealybug and allied species thereof. plant quarantine, 2013,27 (3): 66-69.; The public can obtain from Inspection and Quarantine Center of Xiamen Entry-Exit Inspection and Quarantine Bureau.
Lee Bi Lishi ash mealybug: bibliography: Xu Lang, Yu Daojian, Jiao Yi, etc. the DNA bar code qualification [J] of new pineapple ash mealybug and allied species thereof. plant quarantine, 2013,27 (3): 66-69.; The public can obtain from Inspection and Quarantine Center of Xiamen Entry-Exit Inspection and Quarantine Bureau.
Embodiment 1, design of primers
Carry out a large amount of sequence analyses, compare the some primers that obtained for the identification of new pineapple ash mealybug. Each primer is carried out to preliminary experiment, and the performances such as relatively sensitivity, specificity, finally obtain the pair of primers for the identification of new pineapple ash mealybug. As follows:
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 set up
One, the extraction of genomic DNA
With reference to TIANampGenomicDNAKit blood/cell/tissue genome DNA extracting reagent kit (centrifugal column type), extract genomic DNA, carry out in accordance with the following steps successively:
1, mealybug to be measured is placed in to the centrifuge tube of 0.6mL, adds the pure water of 0.4mL, cover tightly lid, on circumference oscillator, shake 15s, take out mealybug and be placed on the filter paper that high-temperature sterilization crosses, to suck moisture, dry and make polypide fully dry.
2, get 1.5mL centrifuge tube, polypide is inserted in centrifuge tube, smash to pieces.
3, enter the BufferGA of 200 μ L to power in centrifuge tube, then power enters 5 μ LRNaseA and 20 μ LProteinaseK, slightly concussion.
4, centrifuge tube is placed in to 56 DEG C of water bath with thermostatic control shaking tables and places 30min (takes out and shake) after 10min, take out afterwards wink from, to remove the globule on tube wall.
5, to the BufferGB that adds 200 μ L in centrifuge tube, put upside down and mix, wink from, 70 DEG C of water bath with thermostatic control shaking tables are placed 10min, after taking-up, wink from.
6, in centrifuge tube, add 200 μ L absolute ethyl alcohols, concussion 15s, wink from.
7, CB3 adsorption column is put into collecting pipe, the solution in centrifuge tube is transferred in CB3 adsorption column, the centrifugal 30s of 12000rpm, outwells waste liquid, and CB3 adsorption column is put back in collecting pipe.
8, to the BufferGD that adds 500 μ L in CB3 adsorption column, the centrifugal 30s of 12000rpm, outwells waste liquid, and CB3 adsorption column is put back in collecting pipe.
9, to the BufferPW that adds 600 μ L in CB3 adsorption column, the centrifugal 30s of 12000rpm, outwells waste liquid, and CB3 adsorption column is put back in collecting pipe.
10, repeat step 9.
11, CB3 adsorption column is put back in collecting pipe, the centrifugal 2min of 12000rpm, outwells waste liquid, adsorption column CB3 is placed in to room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
12, get the clean centrifuge tube of 1.5mL that autoclaving is crossed. CB3 adsorption column is transferred in centrifuge tube, and to adsorbed film middle part the elution buffer TE of unsettled dropping 50 μ L, room temperature is placed 5min, then the centrifugal 2min of 12000rpm, obtains genomic DNA solution.
13, the genomic DNA solution having extracted is put in to-20 DEG C of preservations, for subsequent use.
Two, pcr amplification
Taking the genomic DNA of step 1 as template, adopt the primer pair of D.neo-28SF and primer D.neo-28SR composition to carry out pcr amplification.
The reaction system of pcr amplification is: EasyTaqBuffer (comprises 10mmol/LMgCl2) 2.5 μ L, 2.5 μ mol/LdNTPMixture2 μ L, the each 2 μ L of D.neo-28SF and D.neo-28SR, EasyTaqDNAPolymerase (5U/ μ L) 0.25 μ L, template DNA 4 μ L, ddH2O12.25 μ L. In the reaction system of PCR, the concentration of D.neo-28SF and D.neo-28SR is 10 μ mol/L.
The response procedures of pcr amplification is: 95 DEG C of denaturation 3min; 94 DEG C of sex change 45s, 60 DEG C of annealing 1min, 72 DEG C are extended 1min, move 35 circulations; Last 72 DEG C are extended 7min.
Pcr amplification product is carried out to agarose gel electrophoresis detection, if obtain the amplified production of 230bp-240bp, mealybug to be measured is new pineapple ash mealybug, is non-new pineapple ash mealybug if do not obtain the amplified production of 230bp-240bp mealybug to be measured.
Embodiment 3, specificity
Sample to be tested is respectively: new pineapple ash mealybug, Jack Bei Ershi mealybug, ocean stern line mealybug, tangerine stern line mealybug, glandular spines mealybug, pineapple ash mealybug, Lee Bi Lishi ash mealybug.
The method that adopts embodiment 2 to set up detects respectively each sample to be tested.
Agarose gel electrophoresis the results are shown in Figure 1. In Fig. 1, M is the DNAMarker of 100bp, swimming lane 1 is the pcr amplification product of new pineapple ash mealybug genomic DNA, swimming lane 2 is the pcr amplification product of Jack Bei Ershi mealybug genomic DNA, swimming lane 3 is the pcr amplification product of ocean stern line mealybug genomic DNA, swimming lane 4 is the pcr amplification product of tangerine stern line mealybug genomic DNA, swimming lane 5 is the pcr amplification product of glandular spines mealybug genomic DNA, swimming lane 6 is the pcr amplification product of pineapple ash mealybug genomic DNA, and swimming lane 7 is the pcr amplification product of Lee Bi Lishi ash mealybug genomic DNA. Result shows, the DNA fragmentation that detects 235bp in the pcr amplification product of new pineapple ash mealybug is (through order-checking, as shown in the sequence 3 of sequence table), and in the pcr amplification product of Jack Bei Ershi mealybug, ocean stern line mealybug, tangerine stern line mealybug, glandular spines mealybug, pineapple ash mealybug, Lee Bi Lishi ash mealybug, all do not detect the DNA fragmentation of 230bp-240bp, show that the PCR method of setting up has good specificity, can be used for the fast detecting of new pineapple ash mealybug.
Embodiment 4, versatility
Sample to be tested is respectively: the new pineapple ash mealybug of intercepting and capturing from Taiwan pineapple, Indonesia's rambutan, Hong Kong rambutan, Taiwan banana, saba (batch 1), saba (batch 2), saba (batch 3).
The method that adopts embodiment 2 to set up detects respectively each sample to be tested.
Agarose gel electrophoresis the results are shown in Figure 2. in Fig. 2, M is the DNAMarker of 100bp, swimming lane 1 is the pcr amplification product of the new pineapple ash mealybug genomic DNA on the pineapple of Taiwan, swimming lane 2 is the pcr amplification product of the new pineapple ash mealybug genomic DNA on Indonesia's rambutan, swimming lane 3 is the pcr amplification product of the new pineapple ash mealybug genomic DNA on the rambutan of Hong Kong, swimming lane 4 is the pcr amplification product of the new pineapple ash mealybug genomic DNA on saba (batch 1), swimming lane 5 is the pcr amplification product of the new pineapple ash mealybug genomic DNA on the banana of Taiwan, swimming lane 6 is the pcr amplification product of the new pineapple ash mealybug genomic DNA on saba (batch 2), swimming lane 7 is the pcr amplification product of the new pineapple ash mealybug genomic DNA on saba 3. result demonstration, above sample all can amplify the PCR product of 230bp-240bp, illustrates that new pineapple ash mealybug special primer versatility is good.
Embodiment 5, sensitivity
1, adopt the method for the step 1 of embodiment 2 to extract new pineapple ash mealybug genomic DNA, obtaining DNA concentration is the DNA solution of 11.5 μ g/ μ L;
2, the DNA solution obtaining by 10 times of gradient dilution steps 1 of TE buffer solution, obtains each dilution;
3, get each dilution that step 2 arrives as template, adopt the primer pair of primer D.neo-28SF and primer D.neo-28SR composition to carry out pcr amplification according to the method for the step 2 of embodiment 2;
Due to the dilution factor difference of the dilution adopting, form following different reaction system:
In reaction system 1, new pineapple ash mealybug genomic DNA initial content is: 46 μ g;
In reaction system 2, new pineapple ash mealybug genomic DNA initial content is: 4.6 μ g;
In reaction system 3, new pineapple ash mealybug genomic DNA initial content is: 460ng;
In reaction system 4, new pineapple ash mealybug genomic DNA initial content is: 46ng;
In reaction system 5, new pineapple ash mealybug genomic DNA initial content is: 4.6ng;
In reaction system 6, new pineapple ash mealybug genomic DNA initial content is: 460pg;
In reaction system 7, new pineapple ash mealybug genomic DNA initial content is: 46pg;
In reaction system 8, new pineapple ash mealybug genomic DNA initial content is: 4.6pg.
Pcr amplification product is carried out to agarose gel electrophoresis and takes pictures.
Agarose gel electrophoresis the results are shown in Figure 3. In Fig. 3, M is the DNAMarker of 100bp, swimming lane 1-8 is the corresponding amplified production of PCR while adopting reaction system 1-8 successively, result shows, when new pineapple ash mealybug genomic DNA initial content is 4.6ng-46 μ g, template amplification all can amplify the PCR product of 230bp-240bp, and brightness weakens gradually, the object band of the template amplification that new pineapple ash mealybug genomic DNA initial content is 4.6ng is fuzzyyer, and other dilution templates do not amplify object band. Result shows, the PCR method of setting up has good sensitivity, not only can be used for the fast detecting of new pineapple ash mealybug, and in the time that the content of new pineapple ash mealybug genomic DNA in PCR system is low to moderate 4.6ng, also can amplify the PCR product of 230bp-240bp, sensitivity is high.
Claims (10)
1. primer pair, is made up of primer D.neo-28SF and primer D.neo-28SR;
Described primer D.neo-28SF is following (a1) or (a2):
(a1) single strand dna shown in the sequence 1 of sequence table;
(a2) by sequence 1 through the replacement of one or several nucleotides and/or disappearance and/or interpolation and with sequence 1 toolThere is the DNA molecular of identical function;
Described primer D.neo-28SR is following (a3) or (a4):
(a3) single strand dna shown in the sequence 2 of sequence table;
(a4) by sequence 2 through the replacement of one or several nucleotides and/or disappearance and/or interpolation and with sequence 2 toolsThere is the DNA molecular of identical function.
2. the application of primer pair claimed in claim 1, for following (b1) or (b2) or (b3) or (b4):
(b1) identify or whether assistant identification insect to be measured is new pineapple ash mealybug;
(b2) for the preparation of identifying or whether assistant identification insect to be measured is the kit of new pineapple ash mealybug;
(b3) detect in biological specimen to be measured, whether to contain new pineapple ash mealybug;
(b4) for the preparation of detecting the kit that whether contains new pineapple ash mealybug in biological specimen to be measured.
3. application as claimed in claim 2, is characterized in that: described (b1) or (b2) in, described insect isMealybug.
4. contain the kit of primer pair described in claim 1; The purposes of described kit is following (c1) or (c2):
(c1) identify or whether assistant identification insect to be measured is new pineapple ash mealybug;
(c2) detect in biological specimen to be measured, whether to contain new pineapple ash mealybug.
5. the preparation method of kit described in claim 4, comprises each the primer step of packaging separately.
6. identify that whether insect to be measured is a method for new pineapple ash mealybug, comprises the steps:
(1) extract the genomic DNA of insect to be measured;
(2) genomic DNA extracting taking step (1), as template, adopts primer pair claimed in claim 1 to carry outPcr amplification, if in pcr amplification product, contain the DNA fragmentation of 230bp-240bp, insect to be measured for or candidate beNew pineapple ash mealybug, if in pcr amplification product, do not contain the DNA fragmentation of 230bp-240bp, insect to be measured for orCandidate is non-new pineapple ash mealybug.
7. identify that whether insect to be measured is a method for new pineapple ash mealybug, comprises the steps:
Detect in the genomic DNA of insect to be measured whether contain specific DNA fragment, if contain specific DNA fragment,Insect to be measured is or candidate is new pineapple ash mealybug, if do not contain specific DNA fragment, insect to be measured for or candidate beNon-new pineapple ash mealybug;
Described specific DNA fragment is the target sequence of primer pair claimed in claim 1 in the genome of new pineapple ash mealybug.
8. detect a method that whether contains new pineapple ash mealybug in biological specimen to be measured, comprise the steps:
(1) extract total DNA of biological specimen to be measured;
(2) the total DNA extracting taking step (1), as template, adopts primer pair claimed in claim 1 to carry out PCRAmplification, contains in the DNA fragmentation of 230bp-240bp, biological specimen to be measured or doubts if contained in pcr amplification productSeemingly contain new pineapple ash mealybug, if do not contain the DNA fragmentation of 230bp-240bp, life to be measured in pcr amplification productIn thing sample, do not contain or the doubtful grey mealybug of new pineapple that do not contain.
9. detect a method that whether contains new pineapple ash mealybug in biological specimen to be measured, comprise the steps:
Detect in total DNA of biological specimen to be measured whether contain specific DNA fragment, if contain specific DNA fragment,In biological specimen to be measured, contain or the doubtful grey mealybug of new pineapple that contains, if do not contain specific DNA fragment, biology to be measuredIn sample, do not contain or the doubtful grey mealybug of new pineapple that do not contain;
Described specific DNA fragment is the target sequence of primer pair claimed in claim 1 in the genome of new pineapple ash mealybug.
10. a specific DNA fragment, for primer pair claimed in claim 1 in the genome of new pineapple ash mealybugTarget sequence.
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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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| Publication number | Priority date | Publication date | Assignee | Title |
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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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