CN102534007A - Specific sequence characterized amplified region (SCAR) marker for Trialeurodes vaporariorum, specific primers, and quick molecular identification method - Google Patents
Specific sequence characterized amplified region (SCAR) marker for Trialeurodes vaporariorum, specific primers, and quick molecular identification method Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, in particular to a specific sequence characterized amplified region (SCAR) marker for Trialeurodes vaporariorum, specific primers, and a quick molecular identification method. According to the specific SCAR marker for the Trialeurodes vaporariorum, the nucleotide sequence of the specific SCAR marker is shown as SEQ ID NO.1. The invention also relates to probes hybridized with the specific SCAR marker for the Trialeurodes vaporariorum, or annealing marker primer pairs for the specific SCAR marker. The quick molecular identification method for the Trialeurodes vaporariorum comprises a step of hybridizing by using the specific SCAR marker probes for the Trialeurodes vaporariorum or performing polymerase chain reaction (PCR) amplification by using the annealing specific primers. The SCAR molecular marker has the advantages of high repeatability, high specificity, high sensitivity and the like, and can quickly and accurately identify the Trialeurodes vaporariorum at low cost. The SCAR molecular marker can quickly and cheaply identify the Trialeurodes vaporariorum in fields quickly, and has important significance for pertinently controlling the Trialeurodes vaporariorum in the fields.
Description
Technical field
The present invention relates to biology field, particularly, relate to Trialeurodes vaporariorum Westwood specific SCAR label, Auele Specific Primer and rapid molecular authentication method.
Background technology
Trialeurodes vaporariorum Westwood (Trialeurodes vaporariorum) is worldwide disastrous insect, and the host plant of having reported surpasses 900 kinds (Kirk etc., 2000).It draws host plant juice with adult and nymph, makes that blade fades, flavescence, wilting, can secrete a large amount of honeydews, pollution fruit and blade.What is more important is propagated the viroses of plant, like cucumber yellow, tomato chlorisis etc., can cause when serious that blade is withered, plant death etc.20 world's the mid-1970s break out in north China, are primary pest (Zhu Guoren etc., China's Vegetable, 2006 (6): 49-51) of north China afterwards always.Particularly in recent decades; Deep variation has taken place in the northern area field of vegetables ecosystem; Particularly heat the developing rapidly of greenhouse and vegetables in vinyl house production; For this kind insect provides the good environment of procreation in winter, and form base, worm source, make the insect that originally can not survive the winter that the possibility of big generation arranged at open country; Greenhouse, booth and outdoor vegetable produce closely linking, interlaced, make the Trialeurodes vaporariorum Westwood population be able to procreation and anniversary generation, and harm increases the weight of, and normal wildness is caused disaster on large-scale attached-greenhouse fruit and vegetable.Trialeurodes vaporariorum Westwood is still the critical limitation factor (Shi Yujuan, Zhang Mingxin, Liu Jiae, Li Xiaoying, 2011, Chinese fruit and vegetable, (2): 26-28) that the northern China vegetables produce at present.
In north China, Trialeurodes vaporariorum Westwood is overlapping with the Bemisia tabaci host, the harm habit is similar, and the life history is close, and same area mixes and take place, appearance similar and be difficult to distinguish.Though there is document to propose its formalness or microscope inspection recognition feature (Zhu Guoren etc., China's Vegetable, 2006 (6): 49-51; Chu Dong etc., insect knowledge, 2008; 45 (1): 154-155); But the quick differentiation on the formalness of these two kinds of insects is still relatively more difficult for non-aleyrodid research professional person, and because the plasticity-of aleyrodid insect is strong, can there be variation in various degree in its morphological specificity because of envrionment conditions, host plant difference etc.; Especially be kept at the aleyrodid insect in the alcohol or preserve incomplete sample etc. for the strange land collection, adopt top two kinds of methods can't differentiate at all.And the Molecular Identification technology is relatively accurate, quick, and simple relatively for the preservation of sample, has nothing to do with the whether complete of sample, therefore, is necessary to develop its rapid molecular authenticate technology.
Along with development of molecular biology; Protocols in Molecular Biology development in recent years rapidly; Utilize molecular marking technique only to need a spot of DNA just can identify caste rapidly and accurately, such technology not only can detect adult, and other each worm attitude is also had similar detection usefulness.There was the investigator to adopt amplification order-checking mtCOI sequence or ITS2 sequence in the past; And make up clustering tree with this gene order of logining on GenBank; Learn that by cluster analysis the sibship between analyzing samples and the known array is far and near; And then infer affiliated kind (Hinomoto etc., 2007 of this gene; Vera etc., 2007; Tselila etc., 2007).But this Technology Need sample checks order after pcr amplification, understands the financial resources and the labour of labor like this.Restriction fragment length polymorphism (Restriction fragment length polymorphism; Be called for short RFLP) and single strand conformation polymorphism (Single-Stranded Confirmation Polymorphisms; Be called for short SSCP) wait technology also to be used to carry out the Molecular Identification of caste usually, but its process complicacy needs enzyme to cut, increase or amplification, polyacrylamide gel electrophoresis combination silver staining method etc.; Need take a long time, waste time and energy.Also have investigator's ITS zone fragment that increases to combine restriction endonuclease sites; After cutting, enzyme judges the difference (Jeong etc. that plant according to segmental length; 2010; Molecular identification of two trichogramma species (Hymenoptera:Trichogrammatidae) in Korea.Journal ofAsia-Pacific Entomology; 13:41-44), but this kind method still needs and wants two operation stepss, and enzyme is cut the result and received multiple factor affecting and be prone to and cut not open or because the phenomenons such as restriction enzyme site change that the amplification mispairing causes.Characteristic sequence amplification region (sequence characterized amplified regions; SCAR) labeling technique is through randomly amplified polymorphic DNA (random amplified polymorphic DNA; RAPD) technology screening goes out the specific fragment of target kind; Then target RAPD specific fragment is cloned and check order, and according to the base sequence design specific primers at these target fragment two ends; With this Auele Specific Primer is carried out pcr amplification once more to genomic DNA fragment then, and then will differentiate out with the corresponding single site of former RAPD fragment; SCAR is labeled as codominant inheritance, and the difference of DNA sample room to be checked can having and not having and show (Li Yu etc., 1999) through amplified production.Compare with above-mentioned other molecule marking method, have advantages such as easy and simple to handle, sensitivity height, can be used for the extensive detection of sample.
The simultaneous Bemisia tabaci biotype of field and Trialeurodes vaporariorum Westwood was merely Type B in the past; Yet at present, variation has taken place in the dominant type that the Bemisia tabaci of harm takes place in the domestic majority area, and succession is Q type Bemisia tabaci (Pan etc.; 2011, Journal ofEconomic Entomology.).But, the molecule marker that can differentiate Type B, Q type Bemisia tabaci and Trialeurodes vaporariorum Westwood is not simultaneously also arranged at present.Therefore, the present invention finds the specific band of Trialeurodes vaporariorum Westwood, and then is converted into stable SCAR mark through the RAPD amplification, carries out the evaluation identification of Trialeurodes vaporariorum Westwood.The quick identification of using the present invention can carry out the field Trialeurodes vaporariorum Westwood fast, is at an easy rate identified, and is significant for the specific aim control in its field.
Summary of the invention
The purpose of this invention is to provide a kind of Trialeurodes vaporariorum Westwood specific SCAR label.
Another object of the present invention provides the probe of partially or completely hybridizing with above-mentioned Trialeurodes vaporariorum Westwood specific SCAR label.
It is right that another object of the present invention provides with above-mentioned Trialeurodes vaporariorum Westwood specific SCAR label annealed Trialeurodes vaporariorum Westwood specific SCAR label primer.
Another object of the present invention provides a kind of Trialeurodes vaporariorum Westwood rapid molecular authentication method.
Another object of the present invention provides the test kit that is used to identify Trialeurodes vaporariorum Westwood.
Another object of the present invention provides the application of above-mentioned Trialeurodes vaporariorum Westwood specific SCAR label in identifying Trialeurodes vaporariorum Westwood.
Another object of the present invention provides above-mentioned probe and primer to the application in identifying Trialeurodes vaporariorum Westwood.
The present invention is a template with the genomic dna to multiple aleyrodid, carries out the RAPD amplification, obtains the specific SCAR label of Trialeurodes vaporariorum Westwood, and the nucleotide sequence of said specific SCAR label is shown in SEQ ID NO.1:
gaccgcttgt?tgacccacag?acagagtagc?caggaattca?tttttctctc?tcttttccca 60
ttcaaacaca?cgtgtgtgaa?aaagacagat?ggggggggga?gggtatcacg?tgactttagc 120
tcctgtatat?ctgtcatttc?ctaacacaga?tgatcaaatg?ggtaatgaga?gagaaagacg 180
aactcctgga?tatagctata?tgccgacagc?tgtcttaaag?tcgttaaggt?gaatccgccg 240
tcgagggaac?agttataggt?gatttcggaa?cagacgttgg?aatcggaatt?gggataacaa 300
tgacccattt?cttttggtca?catctttcgt?ttgcgtagag?atggaggggt?ccctccaaaa 360
ctccacaaga?ctcgatcact?caaatcaatc?gtttgatgaa?gcagatttag?tcagcgattt 420
cgcttcaact?tttatctacc?atcgaagtgc?tgctgatcga?tttacggtta?attactactc 480
agactatatt?cctcagagct?tctccaaaac?aatggaccta?ttgcatgggt?ctgaaaattg 540
aatgaatttg?ttcgaaaaat?gaaagtgctc?agtgagctga?tgccagagat?ggttttttca 600
ttcattttgg?acaagcggtc 620
The nucleotide fragments of the specific SCAR label of this Trialeurodes vaporariorum Westwood is 620bp.
According to above-mentioned Trialeurodes vaporariorum Westwood specific SCAR label sequence, the present invention has designed the probe of partially or completely hybridizing with above-mentioned Trialeurodes vaporariorum Westwood specific SCAR label, to be used for the specific marker Trialeurodes vaporariorum Westwood.
According to above-mentioned Trialeurodes vaporariorum Westwood specific SCAR label sequence, it is right that the present invention has designed Trialeurodes vaporariorum Westwood specific SCAR label primer, is used to the Trialeurodes vaporariorum Westwood specific SCAR label that increases above-mentioned.Wherein, preferred a pair of specific SCAR label primer, its upstream primer nucleotide sequence is shown in SEQ ID NO.2:
Tv-F：5’-tgacccacag?acagagta-3’。
According to above-mentioned preferred Trialeurodes vaporariorum Westwood specific SCAR label primer, the nucleotide sequence of its downstream primer is shown in SEQ ID NO.3:
Tv-R：5’-cgaaatcgct?gactaaat-3’。
Trialeurodes vaporariorum Westwood rapid molecular authentication method of the present invention, wherein, said method comprises to be used above-mentioned probe to hybridize or uses above-mentioned primer to carrying out the step of pcr amplification.
The present invention uses above-mentioned preferred Trialeurodes vaporariorum Westwood specific SCAR label primer right, amplifies 412bp Trialeurodes vaporariorum Westwood specific SCAR label, and its nucleotide sequence is shown in SEQ ID NO.4:
TGACCCACAGACAGAGTAGCCAGGAATTCATTTTTCTCTCTCTTTTCCCATTCAAACACACGTGTGTGAAAAAGACAGATGGGGGGGGGAGGGTATCACGTGACTTTAGCTCCTGTATATCTGTCATTTCCTAACACAGATGATCAAATGGGTAATGAGAGAGAAAGACGAACTCCTGGATATAGCTATATGCCGACAGCTGTCTTAAAGTCGTTAAGGTGAATCCGCCGTCGAGGGAACAGTTATAGGTGATTTCGGAACAGACGTTGGAATCGGAATTGGGATAACAATGACCCATTTCTTTTGGTCACATCTTTCGTTTGCGTAGAGATGGAGGGGTCCCTCCAAAACTCCACAAGACTCGATCACTCAAATCAATCGTTTGATGAAGCAG
ATTTAGTCAGCGATTTCG
The SCAR mark of the 620bp that the present invention obtains is the specific sequence fragment that belongs to Trialeurodes vaporariorum Westwood; It is right to design other Auele Specific Primer in view of the above; Become through mark behind the pcr amplification and to have species specific radioactivity of Trialeurodes vaporariorum Westwood height or nonradioactive probe; Adopt this probe that the genomic dna of Trialeurodes vaporariorum Westwood is hybridized; Trialeurodes vaporariorum Westwood will show stronger hybridization signal, and other species are equal amixia signals then, have or do not have a Trialeurodes vaporariorum Westwood of identifying according to hybridization signal.Perhaps based on the sequence of this SCAR mark; It is right that the synthetic specific TaqMan fluorescent probe of design reaches the Auele Specific Primer supporting with it; Adopt fluorescent quantitative PCR technique then can detect the fluorescent signal of Trialeurodes vaporariorum Westwood; Other kinds are not then possessed detectivity, but the copy number of detection by quantitative target dna fragmentation also simultaneously.
The test kit that is used to identify Trialeurodes vaporariorum Westwood of the present invention, wherein, said test kit comprises that the probe and/or the above-mentioned Trialeurodes vaporariorum Westwood specific SCAR label primer of partially or completely hybridizing with above-mentioned Trialeurodes vaporariorum Westwood specific SCAR label are right.
The present invention also provides the application of above-mentioned Trialeurodes vaporariorum Westwood specific SCAR label in identifying Trialeurodes vaporariorum Westwood.
The present invention also provides above-mentioned probe and primer to the application in identifying Trialeurodes vaporariorum Westwood.
The present invention filters out the specific fragment of Trialeurodes vaporariorum Westwood through the RAPD amplification, reclaims order-checking and is converted into stable SCAR molecule marker afterwards, carries out the evaluation identification of Trialeurodes vaporariorum Westwood.This SCAR molecule marker has advantages such as good reproducibility, high specificity, sensitivity height, can identify Trialeurodes vaporariorum Westwood rapidly and accurately, and cost.The present invention can carry out the quick identification of field Trialeurodes vaporariorum Westwood fast, at an easy rate and identify, and is significant for the specific aim control in its field.
Description of drawings
Fig. 1 primer OPA-17 is to the amplification collection of illustrative plates of different aleyrodid class pests, M:200bp Marke r; 1-5: Trialeurodes vaporariorum Westwood; 6-10:B type Bemisia tabaci; 11-15:Q type Bemisia tabaci; 16: blank.
The checking of Fig. 2 Trialeurodes vaporariorum Westwood SCAR primer specificity, M: swimming lane 1-7: be respectively Trialeurodes vaporariorum Westwood, Type B Bemisia tabaci, Q type Bemisia tabaci, ZH-2 type Bemisia tabaci, Aleurocanthus spiniferus, citrus whitefly, spiral aleyrodid; 8: negative control.
The SCAR primer amplification result of the Trialeurodes vaporariorum Westwood population in Fig. 3 Yunnan, Shanxi, Shandong and Jilin, M: swimming lane 1-3: Kunming, Yunnan Trialeurodes vaporariorum Westwood; 4-6: Yuncheng, Shanxi Trialeurodes vaporariorum Westwood; 7-9: Jinan, Shandong Trialeurodes vaporariorum Westwood; 10-12: Jilin Area Trialeurodes vaporariorum Westwood; 13 swimming lanes: blank; Each geographic Trialeurodes vaporariorum Westwood population is increased 20 respectively, and diagram only shows 3 here.
To the amplification sensitivity detected result of SCAR primer, the M:1-8 swimming lane is respectively single head Trialeurodes vaporariorum Westwood genomic dna stoste, dilution 10 under the different extension rates of Fig. 4 single head Trialeurodes vaporariorum Westwood adult DNA
1, 10
2, 10
3, 10
4, 10
5, 10
6, 10
7Times the time the SCAR amplification.
Embodiment
Test materials and reagent
The examination worm:
Trialeurodes vaporariorum Westwood examination worm is provided by Plant Protection institute, Chinese Academy of Agricultral Sciences, and host plant is a wild cabbage.Type B Bemisia tabaci and Q type Bemisia tabaci are raised in Vegetable & Flower Inst., Chinese Academy of Agriculture Science, and host plant is respectively wild cabbage and cotton, during do not use any agricultural chemicals.Other contrast examination worm samples; Be collected in October, 2011 in the institute of the Chinese Academy of Agricultural Sciences on the chrysanthemum host like the oranges and tangerines aleyrodid, spiral aleyrodid and Aleurocanthus spiniferus are collected in respectively on October 25th, 2011 on the sour tangerine host in piscidia host and Fu Chang village, Haikou Da Po town of Danzhou, Hainan.ZJ-2 type Bemisia tabaci sample is provided by Plant Protection institute, Chinese Academy of Agricultral Sciences.
The confession agent of having a try:
Random primer adopts the RAPD universal primer series of Operon company, adopts OPA, OPB, OPC series, and each series comprises 20 primers.Random primer and Auele Specific Primer are completed bio-engineering corporation by Shanghai English respectively, and synthetic to hold up bio-engineering corporation of section synthetic with Beijing.The sequence order-checking is held up biotechnology ltd of section by Beijing and is carried out.The polysaccharase that uses in the pcr amplification is that 2 * Taq 10MASTER Mix (0.1U TaqE/ μ l) is available from skill Development Co., Ltd of Beijing Olympic Competition Boke.The genome DNA extracting reagent kit of examination worm and clone's test kit are respectively available from hundred Tyke Bioisystech Co., Ltd and Beijing Quanshijin Biotechnology Co., Ltd, Beijing.
Explain: make the experimental methods of molecular biology specify in following examples, all carry out, perhaps carry out according to test kit and product description with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.
The extraction of genomic dna adopts the genome of hundred Tyke Science and Technology Ltd.s to extract test kit; Concrete operations are following: (1) places the single head aleyrodid to drip to be had on the Parafilm film of 10 μ L lysates; Fully grind as homogenizer PCR pipe bottom with 0.2mL; Lysate with other 20 μ L cleans homogenizer 2 times, merges mixing; (2) move in the 1.5mL centrifuge tube with micropipet, add 2 μ L Proteinase Ks, mixing, 55 ℃ of following water-baths were perhaps spent the night in 4 hours; (3) add RNase 2.5 μ L centrifugal a few second, places 37 ℃ of baking oven 15-30min; (4) take out above-mentioned sample, dry in the air, add albumen precipitation liquid 25 μ L, shake 25s on the vortex oscillation device at a high speed to room temperature; (5) ice bath 5min, protein precipitation; Room temperature is centrifugal, 13000r/min, centrifugal 5min; The careful supernatant of drawing (does not suck deposition) in another new pipe; (6) add isopyknic Virahol 50 μ L, put upside down mixing 30 times; (7) the centrifugal 10min of 12000r/min under the room temperature abandons supernatant, adds 1mL 70% ethanol and puts upside down rinsing DNA deposition, and the centrifugal 2min of 12000r/min abandons supernatant under the room temperature; (8) add the 1mL absolute ethyl alcohol and put upside down rinsing DNA deposition, the centrifugal 2min of room temperature 12000r/min abandons supernatant; (9) add 20 μ L DNA lysates, 65 ℃ of following water-bath 60min, dissolving DNA, it is subsequent use to put-20 ℃ of preservations then.
Embodiment 2RAPD amplification
Genomic dna with the confession test agent in the above-mentioned materials and methods is a template, adopts 60 random primers to carry out random amplification respectively, writes down the specific band and the corresponding amplimer that screen, and these bands are carried out mark.The amplification TV is 20 μ L, comprises 10 μ L2 * Mix, dna profiling 1 μ L, RAPD primer (10mM) 3 μ L and 6 μ Ldd H respectively
2O.The PCR response procedures is: at first carries out 94 ℃ of preparatory sex change 4min, carries out 40 circulations afterwards, comprise 94 ℃ of 1min, and 37 ℃ of annealing 50s, 72 ℃ are extended 1min 35s, and last 72 ℃ are extended 10min.Get appearance electrophoresis on the pcr amplification product 6 μ L of each sample respectively.
In the RAPD random amplification; When finding the OPA-17 primer amplification, show the specificity of very strong kind for Trialeurodes vaporariorum Westwood, the DNA band of a 620bp occurs in Trialeurodes vaporariorum Westwood; And this specific band is very bright; And all stable appearance the in 24 individualities that detect, and in other Bemisia tabaci biotypes or other aleyrodid kind, all do not occur, see Fig. 1.
Recovery clone, SCAR design of primers and the pcr amplification of embodiment 3 specific amplification products
To finding in the RAPD amplification that the specific band in the Trialeurodes vaporariorum Westwood sample carries out mark.On agarose gel electrophoresis, reclaim this specific band, adopt DNA purification kit (hundred Tykes, Beijing) to carry out purifying; Be connected to this purifying fragment on the pEASY-T1 carrier (Quan Shijin, Beijing), be transformed on the TOP10 competent cell; Be inverted overnight cultures under 37 ℃ of conditions; Picking white positive colony adds 37 ℃ of overnight shakings cultivations in the LB liquid nutrient medium, adopts the plasmid extraction kit of Beijing hundred Tyke Bioisystech Co., Ltd to extract plasmid at last; Positive plasmid send Beijing friendship to hold up biotechnology ltd of section and checks order after adopting universal primer M13 forward and reverse primer amplification checking.
Sequencing result is shown in SEQ ID NO.1:
gaccgcttgt?
tgacccacag?acagagtagc?caggaattca?tttttctctc?tcttttccca 60
ttcaaacaca?cgtgtgtgaa?aaagacagat?ggggggggga?gggtatcacg?tgactttagc 120
tcctgtatat?ctgtcatttc?ctaacacaga?tgatcaaatg?ggtaatgaga?gagaaagacg 180
aactcctgga?tatagctata?tgccgacagc?tgtcttaaag?tcgttaaggt?gaatccgccg 240
tcgagggaac?agttataggt?gatttcggaa?cagacgttgg?aatcggaatt?gggataacaa 300
tgacccattt?cttttggtca?catctttcgt?ttgcgtagag?atggaggggt?ccctccaaaa 360
ctccacaaga?ctcgatcact?caaatcaatc?gtttgatgaa?gcag
atttag?tcagcgattt 420
cgcttcaact?tttatctacc?atcgaagtgc?tgctgatcga?tttacggtta?attactactc 480
agactatatt?cctcagagct?tctccaaaac?aatggaccta?ttgcatgggt?ctgaaaattg 540
aatgaatttg?ttcgaaaaat?gaaagtgctc?agtgagctga?tgccagagat?ggttttttca 600
ttcattttgg?acaagcggtc 620
The specific amplification fragment of Trialeurodes vaporariorum Westwood is 620bp, and this gene order is carried out the BLAST comparison on the NCBI website, does not find that any and this gene fragment has the sequence of higher similarity, explains that this fragments specific is very strong.
Sequencing result based on specific fragment; Adopt a pair of SCAR Auele Specific Primer of Primer 5 software designs, the upstream and downstream primer sequence is respectively: Tv-F:5 '-tgacccacag acagagta-3 ' and Tv-R:5 '-cgaaatcgctgactaaat-3 ' (underscore part in the sequence), and reaction system is set at 20 μ L; Wherein 2 * ES Mix is 10 μ L; Upstream primer and downstream primer are respectively 1 μ L, and dna profiling is 1 μ L, ddH
2O is 7 μ L.Response procedures is following: 94 ℃ of preparatory sex change 3min, carry out 35 circulations (72 ℃ are extended 1min 50sec for 94 ℃ of sex change 1min, 59 ℃ of annealing 45sec) afterwards, and last 72 ℃ are extended 10min.Expection amplified fragments size is 507bp.
Adopt Type B Bemisia tabaci, Q type Bemisia tabaci, Aleurocanthus spiniferus, spiral aleyrodid, oranges and tangerines aleyrodid and ZJ-2 type Bemisia tabaci to plant, through the specificity of pcr amplification checking Trialeurodes vaporariorum Westwood SCAR primer as contrast.After the extracting genome DNA with single head Trialeurodes vaporariorum Westwood adult, carry out 10 times of gradient dilutions successively, check the susceptibility of this SCAR primer amplification.
The employing specificity SCAR primers is right: and Tv-F (5 '-tgacccacag acagagta-3 ') and Tv-R (5 '-cgaaatcgctgactaaat-3 ') come the Trialeurodes vaporariorum Westwood population is carried out pcr amplification; With other Bemisia tabaci different biotypes or other aleyrodid kind as the contrast population; Find only in Trialeurodes vaporariorum Westwood, to occur the specific band of a 412bp, and this band after reclaiming cloning and sequencing, find with original RAPD specific band in the sequence of homologous segment in full accord.And any band does not all appear in as other aleyrodid kind that contrasts or Bemisia tabaci biotype, and see Fig. 2, explain that this Auele Specific Primer exists the specificity of planting for Trialeurodes vaporariorum Westwood.In addition,, adopt the check of increasing of this SACR primer to gathering after the Trialeurodes vaporariorum Westwood population in Yunnan and field, Shanxi carries out identification of morphology, the result be illustrated in every 412bp that tries all to have occurred in the worm to expect dna fragmentation (SEQ ID.4, Fig. 3).After the genomic dna of single head Trialeurodes vaporariorum Westwood gradient dilution successively, find that dna profiling is diluted to 10
7The Shi Weijian amplified band is seen Fig. 4, shows that this SCAR primer is to having very high amplification sensitivity.
Claims (8)
1. a Trialeurodes vaporariorum Westwood specific SCAR label is characterized in that, the nucleotide sequence of said specific SCAR label is shown in SEQ ID NO.1.
2. the probe of partially or completely hybridizing with the described Trialeurodes vaporariorum Westwood specific SCAR label of claim 1.
3. right with the described Trialeurodes vaporariorum Westwood specific SCAR label of claim 1 annealed Trialeurodes vaporariorum Westwood specific SCAR label primer.
4. Trialeurodes vaporariorum Westwood specific SCAR label primer according to claim 3 is right, it is characterized in that, said primer is to comprising the primer of nucleotide sequence shown in SEQ ID NO.2 and SEQ ID NO.3.
5. a Trialeurodes vaporariorum Westwood rapid molecular authentication method is characterized in that, said method comprise use the said probe of claim 2 hybridize or the said primer of claim 3 to carrying out the step of pcr amplification.
6. a test kit that is used to identify Trialeurodes vaporariorum Westwood is characterized in that, said test kit comprises that the said probe of claim 2 and/or the said primer of claim 3 are right.
7. the application of the said Trialeurodes vaporariorum Westwood specific SCAR label of claim 1 in identifying Trialeurodes vaporariorum Westwood.
8. said probe of claim 2 or the said primer of claim 3 are to the application in identifying Trialeurodes vaporariorum Westwood.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103088147A (en) * | 2013-02-07 | 2013-05-08 | 中国农业科学院蔬菜花卉研究所 | PCR (polymerase chain reaction) method and kit for simultaneously identifying specificities of Q-type bemisia tabaci and B-type bemisia tabaci |
| CN103898225A (en) * | 2014-04-11 | 2014-07-02 | 山东省农业科学院生物技术研究中心 | Primers and method of identifying cryptic specie of bemisia tabaci and trialeurodes vaporariorum |
| CN106305625A (en) * | 2016-08-15 | 2017-01-11 | 青岛农业大学 | Application of trialeurodes vaporarionm westwood to separation of tomato chlorosis viruses (ToCVs) |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088147A (en) * | 2013-02-07 | 2013-05-08 | 中国农业科学院蔬菜花卉研究所 | PCR (polymerase chain reaction) method and kit for simultaneously identifying specificities of Q-type bemisia tabaci and B-type bemisia tabaci |
| CN103088147B (en) * | 2013-02-07 | 2014-08-06 | 中国农业科学院蔬菜花卉研究所 | PCR (polymerase chain reaction) method and kit for simultaneously identifying specificities of Q-type bemisia tabaci and B-type bemisia tabaci |
| CN103898225A (en) * | 2014-04-11 | 2014-07-02 | 山东省农业科学院生物技术研究中心 | Primers and method of identifying cryptic specie of bemisia tabaci and trialeurodes vaporariorum |
| CN103898225B (en) * | 2014-04-11 | 2015-10-21 | 山东省农业科学院生物技术研究中心 | A kind of primer and discrimination method differentiating the hidden kind of Bemisia tabaci and trialeurodes vaporariorum |
| CN106305625A (en) * | 2016-08-15 | 2017-01-11 | 青岛农业大学 | Application of trialeurodes vaporarionm westwood to separation of tomato chlorosis viruses (ToCVs) |
| CN106305625B (en) * | 2016-08-15 | 2019-05-28 | 青岛农业大学 | Method of the Trialeurodes vaporariorum Westwood in separation tomato chlorisis virus |
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|---|---|
| CN102534007B (en) | 2013-10-09 |
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