CN103088147B - PCR (polymerase chain reaction) method and kit for simultaneously identifying specificities of Q-type bemisia tabaci and B-type bemisia tabaci - Google Patents
PCR (polymerase chain reaction) method and kit for simultaneously identifying specificities of Q-type bemisia tabaci and B-type bemisia tabaci Download PDFInfo
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Abstract
The invention discloses a PCR (polymerase chain reaction) method and a kit for simultaneously identifying specificities of Q-type bemisia tabaci and B-type bemisia tabaci, and belongs to the technology of molecular biological detection. The PCR method comprises the steps of carrying out PCR amplification on DNA of the bemisia tabaci to be tested, and detecting PCR amplification product. The PCR method is characterized in that the primer pair adopted for PCR amplification is as follows: an upstream primer Bt-F:5'-TCT CAT CGG TGT CTTATT G-3', and a downstream primer Bt-R:5'-CTT CGG GTC GTT TCT CGT-3'; the detection of the PCR amplification product means that gel electrophoresis is carried out on the PCR amplification product to observe whether characteristic belt type appears; the characteristic belt type of the Q-type bemisia tabaci is a 507bp stripe; and the characteristic belt type of the B-type bemisia tabaci is 507bp and 660pb stripes. A kit is also provided on the basis of the method.
Description
Technical field
The invention belongs to Measurement for Biotechnique, particularly a kind of specific PCR method and test kit of simultaneously differentiating Q type and Type B Bemisia tabaci.
Background technology
Bemisia tabaci Bemisia taabaci belongs to Hemiptera, and Aleyrodidae, is distributed widely in different continents, by direct feeding plant juice, secretion honeydew Pollution Plant and propagation plant virus, agricultural and horticultural production is caused to serious harm.Bemisia tabaci invasion property is extremely strong, past two during the last ten years, Bemisia tabaci intrudes into global multiple countries and regions and has replaced local multiple indigenous Bemisia tabaci, multiple important crops has all over the world been caused to serious harm, inhale water except thorn itself and cause that blade is even plant wither, host plant physiologic derangement, the most important thing is that the sick big area of the louse-borne geminivirus infection disease of tobacco powder-tomato yellow leaf curl virus breaks out cause harm (Brown & Czosnek, 2002; Czosnek & Ghanim, 2011), and spread to the north (Zhang Sui etc., 2006 by south; Wu Yonghan etc., 2007; Zhou Ying etc., 2010).The popular of virus disease brought grave danger to tomato production, and the tomato booth in partial area even has no harvest.Thereby Bemisia tabaci becomes " super insect " (Liu et al., 2007 that the whole world is paid close attention to; Martinez-Carrillo and Brown, 2007; Bethke et al., 2009).Bemisia tabaci is one and comprises many genotypical aggregate species, the biotype of being named has 24 (Dinsdale et al. at least, 2010), by the end of 2011, the subspecies of having reported have 32 at least, at least there are 6 kinds of biotype (B in China at present, Q, ZHJ-1, ZHJ-2, ZHJ-3, FJ-l) (great incomparably etc., 2009), this wherein distributes the most extensively, the most serious biotype of harm is to invade in recent years China and progressively form the Type B and the Q type Bemisia tabaci that spread diffusion tendency.According to the Phylogenetic Analysis document of delivering recently, cross experiment and mating behavior observations show that Bemisia tabaci at least comprises 28 genetic construction difference obviously but hidden kind (Dinsdale et al., 2010 that formalness cannot be distinguished; Xu et al., 2010; De Barro et al., 2011; Hu et al., 2011).But whether the strong evidence and the sorting technique of genetics angle that still lack on morphology reasonable, the nomenclature that makes hidden kind on taxonomy without sufficient foundation, so still continue to use the concept of biotype in the application.In the biotype of all Bemisia tabaci, Type B and Q type Bemisia tabaci surely belong to invasion property strong, in worldwide, extensively distribute, hazardness is the most serious.
Type B Bemisia tabaci originates from area, the Middle East-Asia Minor (De Barro et al., 2011), the middle and later periods nineties, and Type B Bemisia tabaci invasion China, and brought financial loss to agricultural and horticultural production.Within 2005, find first Q type Bemisia tabaci (Chu etc. in Chinese yunnan; 2005); in subsequently several years; find that Q type Bemisia tabaci has replaced Type B Bemisia tabaci gradually; 2009 the end of the year China had at least 21 provinces and cities to find that Q type tobacco powder has replaced the Type B Bemisia tabaci (Pan et al., 2011) originally causing harm; But in some areas, field finds that the two is to mix (Chu Dong etc., 2007 that occur; Chu Dong etc., 2009).
Type B Bemisia tabaci is apparent consistent with Q type Bemisia tabaci; in form, cannot distinguish; but the two exists notable difference on biological characteristics, host plant, physiology and molecular level, for example Q type Bemisia tabaci has stronger host's adaptability and resistance (Muniz, 2000 than Type B Bemisia tabaci; Horowitz et al., 2003), to stronger (pan et al., 2012) etc. of the transmission capacity of virus disease, and between much genotype, there is reproduction isolation.At present adopt exactly mtCOI Auele Specific Primer to carry out specific PCR amplification through the molecular assay method that is usually used in distinguishing its biotype, then pcr amplification product is checked order and sequence alignment, and then determine it is which kind of biotype (Luo Chen etc., 2002).But this method need to be passed through all multi-steps such as pcr amplification, clone, order-checking, sequence alignment, consuming time longer, and need a large amount of funds as support.
And further develop along with molecular biological, investigator attempts searching can obviously identify the method for two biotypes by pcr amplification.Such as Shatters JRG etc. (2009) report is the mtCOI sequence between the two by relatively Type B and Q type Bemisia tabaci, designs respectively Auele Specific Primer separately for specific sequence separately, differentiates this two biotypes by the method for pcr amplification.The technology of more common qualification species also comprises randomly amplified polymorphic DNA (random amplifiedpolymorphic DNA in addition, RAPD) technology is in conjunction with characteristic sequence amplification region (sequence characterized amplified regions, SCAR) labeling technique filters out the specific fragment that need to identify species, then target specific fragment is carried out to Cloning and sequencing, and carry out Auele Specific Primer design with this fragment sequence, then with this, Auele Specific Primer is carried out to pcr amplification again to genomic DNA fragment, and then the site corresponding with former specific fragment identified.SCAR is labeled as codominant inheritance, and the difference between DNA to be checked can be directly by having or not amplified production to show (Li Yu etc., 1999).Carry out species mirror method for distinguishing and restrictive fragment length polymerphism (the restriction fragment lengthpolymorphism conventionally adopting according to the size of specific amplification fragment length like this, RFLP), base sequence is measured, the technology such as quantitative fluorescent PCR are compared, remove process that screening restriction enzyme and base sequence measure or costly from, the unfavourable condition that plant and instrument requirement condition is high, not only easy and simple to handle, cost is low, and result is stable, highly sensitive, reproducible, once and Auele Specific Primer obtains, can be used for the detection analysis of extensive sample, can differentiate target sample fast.But the primer pair that at present Type B Bemisia tabaci and Q type Bemisia tabaci is carried out to adopt in the molecule marker of Biotype Determination only can be identified single biotype (Shatters et al., 2009).
Summary of the invention
The invention provides a kind of method for the identification of Type B Bemisia tabaci and Q type Bemisia tabaci and the test kit based on the method.Technical scheme of the present invention is as follows:
For detection of the method for Type B Bemisia tabaci and Q type Bemisia tabaci, comprise to the DNA of Bemisia tabaci to be measured is carried out pcr amplification and detects pcr amplification product, it is characterized in that:
The primer pair that described pcr amplification adopts is as follows:
Upstream primer Bt-F:5 '-TCT CAT CGG TGT CTTATT G-3 ',
Downstream primer Bt-R:5 '-CTT CGG GTC GTT TCT CGT-3 ';
Described detection pcr amplification product refers to pcr amplification product carry out gel electrophoresis and observe whether occur that feature banding pattern, the feature banding pattern of Q type Bemisia tabaci are a 507bp band; Type B Bemisia tabaci feature banding pattern is 507bp and 660bp two bands.
The system of described pcr amplification is: the concentration of Taq archaeal dna polymerase 0.05U/ μ l, 1 × Taq damping fluid, magnesium chloride 1.5mM, dNTPs 200 μ M, upstream primer and downstream primer is respectively 0.5 μ M, DNA profiling concentration is 0.17 × 10
-5~ 0.2ng/ul, adds ddH
2o to 20 μ l.
The program of described pcr amplification is: 94 DEG C of denaturations 3 minutes; Then carry out 30 circulations: 45 seconds, 72 DEG C extensions of 1 minute, 59 DEG C annealing of 94 DEG C of sex change 110 seconds; Last 72 DEG C are extended 10min.
For detection of the test kit of Type B Bemisia tabaci and Q type Bemisia tabaci, it is characterized in that comprising one couple of PCR primers:
Upstream primer Bt-F:5 '-TCT CAT CGG TGT CTTATT G-3 ',
Downstream primer Bt-R:5 '-CTT CGG GTC GTT TCT CGT-3 ';
Also comprise the reagent detecting for PCR: Taq archaeal dna polymerase, dNTPs, magnesium chloride, Taq DNA polymerase buffer liquid and sterilized water.
Described PCR primer is mixed in PCR premix system in advance, and described PCR premix system is as follows: Taq archaeal dna polymerase 0.05U/ μ l, 1 × Taq damping fluid, 1.5mM magnesium chloride, 200 μ M dNTPs, upstream primer and downstream primer be 0.5 μ M and sterilized water respectively.
The present invention adopts OPA, OPB, OPC series, each series comprise 20 totally 60 RAPD random primers to carrying out pcr amplification for the DNA in examination worm source respectively.In amplification, find that a random primer can amplify the specific band of Q type Bemisia tabaci (nucleotide sequence is as shown in Seq ID No.1), this band does not occur in the amplified production of Type B Bemisia tabaci.By separating and check order this band, and based on this strip design Q type bemisa babaci specificity SCAR primers, in the primer of designing, find that a pair of Auele Specific Primer all amplifies feature band in Type B and Q type Bemisia tabaci.Through the checking of field specificity, this can be stablized and amplify feature banding pattern in Type B and Q type Bemisia tabaci Auele Specific Primer, and the band that can not increase in the Bemisia tabaci of other subgroup.
The present invention is based on this to Auele Specific Primer, be provided for detecting PCR method and the test kit of Type B Bemisia tabaci and Q type Bemisia tabaci, so only carrying out a pcr amplification can be by judges Type B Bemisia tabaci and Q type Bemisia tabaci to the identification of amplification bands of a spectrum simultaneously fast, thereby significantly shorten the decision technology of Bemisia tabaci biotype, also saved cost, that its Molecular Identification technical is improved greatly, rapid detection is carried out in the distribution that this technological improvement is Bemisia tabaci different biotypes and identification provides easier method, is more suitable for the large batch of sample test in field.
Brief description of the drawings
The pcr amplification result of Fig. 1 OPA-17 to three kinds of Bemisia tabaci
(M:200bp ladder; 1-5:Q type Bemisia tabaci; 6-10:B type Bemisia tabaci; 11-15: Trialeurodes vaporariorum Westwood; 16: blank; )
The pcr amplification result of Fig. 2 specificity SCAR primers in different aleyrodid kinds
(M:200bp ladder; 1-2:Q type Bemisia tabaci; 3-4:B type Bemisia tabaci; 5-6: Trialeurodes vaporariorum Westwood; 7-8: the green aleyrodid of tangerine; 9-10: Aleurocanthus spiniferus; 11-12:ZJ-2 type Bemisia tabaci)
Amplification under Fig. 3 SCAR primer pair single head Q type Bemisia tabaci adults DNA and different concns thereof
(M:200bp Marker; 1: single head Q type Bemisia tabaci genomic dna stoste 3.4ng/ μ l; 2:DNA dilution 10
1doubly; 3:DNA dilution 10
2doubly; 4:DNA dilution 10
3doubly; 5:DNA dilution 10
4doubly; 6:DNA dilution 10
5doubly.）
The detected result of Fig. 4 Auele Specific Primer to 9 Bemisia tabacis in amplification field
(M:Marker II; Other swimming lane is followed successively by Shandong, Zhejiang, Shanxi, Henan, Shanghai, Jiangxi, Hainan, Tianjin and Hebei population)
The detected result of Fig. 5 Auele Specific Primer to 9 Bemisia tabacis in amplification field
(M:Marker II; Other swimming lane is followed successively by Guangdong, Anhui, Beijing, Changsha, Guangxi, Liaoning, Xinjiang and Gansu population)
Embodiment
Below by specific experiment process and experimental result explanation the present invention:
Biomaterial
Type B Bemisia tabaci and Q type Bemisia tabaci are raised in Vegetable & Flower Inst., Chinese Academy of Agriculture Science, raise so far from 2009, and host plant is respectively wild cabbage, poinsettia, and population maintains and between feeding period, do not use any agricultural chemicals;
The green aleyrodid of tangerine gathers in institute of the Chinese Academy of Agricultural Sciences, and the known references of record is: Liu Xun, and incomparably great, Zhang Guifen, 2009, insect journal, 52 (8): 895-900.
Trialeurodes vaporariorum Westwood, Aleurocanthus spiniferus and Zhejiang Province's Bemisia tabaci ZJ-2 type are provided by the Zhang Guifen researcher of Plant Protection institute, Chinese Academy of Agricultral Sciences; The known references of recording is: Liu Xun, and incomparably great, Zhang Guifen, 2009, insect journal, 52 (8): 895-900.
Be within 2011, to gather the aleyrodid class pest adult on deifferent regions.China and host for the field population of examination, more than tentatively judge its kind or whether be Bemisia tabaci according to the morphological specificity of adult whitefly for planting experimentally group, first Bemisia tabaci sample wherein adopts mtCOI gene amplification and sequence alignment (Luo Chen etc., 2002) determine its biotype, Partial Species also adopts Type B and the Q type Bemisia tabaci primer pair separately that publish (2009) such as Shatters to identify its biotype simultaneously, afterwards as the checking material in this research.
Biomaterial granting statement:
More than be Bemisia tabaci different biotypes that recorded in current document or known or different sorts for examination insect, those skilled in the art can be by gathering and the acquisition of conventional verification method, also there is preservation in this laboratory, can from the applying date, in 20 years, provide for proof test to the public.
Random primer adopts the RAPD universal primer series of Operon company, adopt OPA, OPB, OPC series, each series comprises 20 primers, and random primer and Auele Specific Primer respectively and Beijing Qing Ke bio-engineering corporation synthetic by Shanghai Ying Jun bio-engineering corporation synthesize.
Sequence order-checking is undertaken by Beijing Qing Ke bio-engineering corporation.
The polysaccharase using in pcr amplification is that (0.1U TaqE/ μ is l) purchased from skill Development Co., Ltd of Olympic Competition Boke for 2 × Taq10MASTER Mix.
The genome DNA extracting reagent kit of aleyrodid insect and clone's test kit are respectively purchased from Beijing hundred Tyke Bioisystech Co., Ltd and Beijing Quanshijin Biotechnology Co., Ltd.
Embodiment 1RAPD amplification screening specificity random primer
2.1 single head aleyrodid insect DNA extraction
The extraction of genomic dna adopts DNA extraction test kit (Beijing hundred Tyke Bioisystech Co., Ltd, solution-type), and method reference reagent box specification sheets carries out.
Concrete operations are as follows: single head Bemisia tabaci adults is placed in to drip to be had on the Parafilm film of cell pyrolysis liquid (test kit provides) of 10 μ l, fully grind as homogenizer bottom using the PCR pipe of 0.2ml, lysate with another 20 cleans homogenizer 2 times, and merging mixes; Then move into 1.5ml centrifuge tube with micropipet; Add Proteinase K, 2 μ l, mix, and 55 DEG C, water-bath 4 hours or spend the night; Centrifugal several seconds, add RNase A2.5 μ l(10 μ g/ μ l), be placed in 37 DEG C of baking oven 15-30min; Take out above-mentioned sample, dry in the air to room temperature, add albumen precipitation liquid 25 μ l, on vortex oscillation device, shake at a high speed 25s; Ice bath 5min, protein precipitation; Room temperature is centrifugal, 13000r/min, centrifugal 5min; The careful supernatant liquor of drawing (does not suck precipitation) in another new pipe; Add isopyknic Virahol 50 μ l, put upside down and mix 30 times; Room temperature is centrifugal, 12000r/min, and centrifugal 10min, abandons supernatant; Add 1ml 70% ethanol to put upside down rinsing DNA precipitation, room temperature is centrifugal, 12000r/min, and centrifugal 2min, abandons supernatant; Add 1ml dehydrated alcohol to put upside down rinsing DNA precipitation, room temperature is centrifugal, 12000r/min, and centrifugal 2min, abandons supernatant; Add 20 μ l DNA lysates, 65 DEG C of dissolving DNAs, water-bath 60min, is put in-20 DEG C and saves backup.
2.2RAPD amplification
Application RAPD amplification technique, taking the DNA of the test sample in above-mentioned materials and methods as template, adopts 60 random primers to carry out respectively pcr amplification, records the specific band and the corresponding amplimer that screen, and these bands are carried out to mark.
Amplification cumulative volume is 20 μ l, comprises respectively 10 μ l2 × Taq10MASTER Mix(comprising Taq DNA Polymerase0.1U/ μ l, 2 × Taq PCR Buffer, 3mM MgCl
2with 400 μ M dNTP mix), RAPD primer 3 μ l and the 6 μ l dd H of DNA profiling 1 μ l, 10mM
2o.
PCR response procedures is: first carries out 94 ° of C denaturation 4min, carries out afterwards 40 circulations, comprise 94 ° of C1min, and 37 ° of C annealing 50s, 72 ° of C extend 1min35s, and last 72 ° of C extend 10min.
2.3 specific amplification products reclaim clone
In RAPD amplification, while finding OPA-17 primer amplification, occur a very bright amplified band in Q type Bemisia tabaci, size is about in 721bp(Fig. 1 shown in arrow), but in Type B Bemisia tabaci, do not see the appearance of this band.In Trialeurodes vaporariorum Westwood kind, also do not occur, see Fig. 1.
On agarose gel electrophoresis, reclaim this specific band, adopt DNA purification kit (hundred Tykes, Beijing) to carry out purifying, afterwards this purifying fragment is connected on pEASY-T carrier (the full formula gold in Beijing), be transformed into TOP10 competent cell, under 37 ° of C conditions, be inverted overnight incubation, picking white positive colony, add 37 ° of C overnight shakings in LB liquid nutrient medium to cultivate, finally adopt the plasmid extraction kit of Beijing hundred Tyke Bioisystech Co., Ltd to extract plasmid, positive plasmid adopts after primer amplified checking, send Beijing biotechnology company limited of Jiao Qing section to check order.
As can be seen from Figure 1, the specific amplification fragment of Q type Bemisia tabaci is 721bp, reclaims rear its sequence of measuring of clone as shown in Seq IDNo.1.
The design of 2.4 specificity SCAR primers and checking
The specific band Seq ID No.1 occurring in RAPD amplification based on Q type Bemisia tabaci, adopts PrimerPremier5.0 software design SCAR Auele Specific Primer, and wherein a pair of Auele Specific Primer is as follows:
Bt-F（5’-TCT CAT CGG TGT CTTATT G-3’）；
Bt-R(5’-CTT CGG GTC GTT TCT CGT-3’)，
Adopt this primer pair Q type Bemisia tabaci to carry out specific PCR amplification checking, with other aleyrodid kind (Type B Bemisia tabaci, Trialeurodes vaporariorum Westwood, Aleurocanthus spiniferus, the green aleyrodid of tangerine and ZJ-2 type Bemisia tabaci) population in contrast.
Pcr amplification condition and the amplification program in the present invention, optimized are as follows:
Reaction system is set as 20 μ l, and wherein 2 × Taq10MASTER Mix(is comprising TaqDNAPolymerase0.1U/ μ l, 2 × Taq PCR Buffer, 3mM MgCl
2with 400 μ M dNTP mix) be 10 μ l, upstream primer and downstream primer (10 μ M) are respectively 1 μ l, and DNA profiling is 1 μ l, ddH
2o is 7 μ l.
The response procedures of optimizing is as follows: 94 DEG C of denaturation 3min, carry out afterwards 30 circulations (72 DEG C are extended 1min50sec for 94 DEG C of sex change 1min, 59 DEG C of annealing 45sec), and last 72 DEG C are extended 10min.After reaction finishes, draw PCR product 5 μ l and carry out agarose gel electrophoresis detection.
Pcr amplification result shows: the specific band (1-2 swimming lane in seeing Fig. 2) that occurs a 507bp in Q type Bemisia tabaci; And in Type B Bemisia tabaci, occur that two bands, size are about 507bp and 660bp(is shown in 3-4 swimming lane in Fig. 2), and all there is not any band in other aleyrodid kinds in contrast, see Fig. 2.
Specificity and the stability checking of 2.5 Auele Specific Primers to field Bemisia tabaci
Specificity and the stability of the different population checking primer pair in field in all parts of the country is picked up from utilization, and the population of collection first adopts mtCOI gene amplification and after sequencing, determines its biotype or kind, and details see the following form.
The Information Monitoring of the field Bemisia tabaci adopting in proof test
Record in the PCR program of specificity checking and system same 2.4.
Amplification is shown in Fig. 4 and Fig. 5.
Result shows, in Q type Bemisia tabaci, there is a very strong single band, and in Type B Bemisia tabaci, there are two bands, to gather from the result of 18 Bemisia tabaci field populations biotypes in field with mtCOI the biotype the result after in conjunction with sequencing analysis consistent, amplification repeatability and stability that this Auele Specific Primer is described are very strong, are suitable for the biotype rapid detection of field Bemisia tabaci.
The sensitivity test of 2.6 Auele Specific Primers to Q type Bemisia tabaci
Because primer pair amplifies a DNA fragmentation in Q type Bemisia tabaci, therefore adopt Q type Bemisia tabaci to check the sensitivity of primer pair amplification.
Be 3.4ng/ μ l by the genomic dna concentration of single head Q type Bemisia tabaci, after gradient dilution, amplification program and the sampling mode of employing 2.4 increase and detect successively, found that DNA profiling dilution 10
5times time have no amplified band, see Fig. 3, show that this SCAR primer pair has very high amplification sensitivity, can reach pg level.
The Auele Specific Primer that to sum up the present invention screens can accurately be differentiated Type B Bemisia tabaci and Q type Bemisia tabaci according to the difference of amplification bands of a spectrum to (Bt-F(5 '-TCT CAT CGG TGT CTTATT G-3 ') and Bt-R (5 '-CTT CGG GTC GTT TCT CGT-3 '), and this SCAR mark is highly sensitive, can in the time that being 3.4ng/ μ l, the genomic dna concentration of single head Q type Bemisia tabaci adults dilute 10000 times of laggard performing PCR amplifications, still can successfully detect very strong, single expection specific band, illustrate that this specificity SCAR primers has very high sensitivity, amplification sensitivity can reach pg rank.Can differentiate primer pair Type B and Q type Bemisia tabaci have realized greatly fast and have saved time and cost simultaneously, belong to the improvement of technology.Type B and Q type Bemisia tabaci in sensing chamber and field all show very strong specificity and suitability, therefore can be directly used in the rapid detection in enormous quantities of field Bemisia tabaci.
Claims (6)
1. for detection of the method for Type B Bemisia tabaci and Q type Bemisia tabaci, comprise to the DNA of Bemisia tabaci to be measured carried out pcr amplification and detects pcr amplification product, it is characterized in that:
The primer pair that described pcr amplification adopts is as follows:
Upstream primer Bt-F:5 '-TCT CAT CGG TGT CTT ATT G-3 ',
Downstream primer Bt-R:5 '-CTT CGG GTC GTT TCT CGT-3 ';
Described detection pcr amplification product refers to pcr amplification product carry out gel electrophoresis and observe whether occur that feature banding pattern, the feature banding pattern of Q type Bemisia tabaci are a 507bp band; Type B Bemisia tabaci feature banding pattern is 507bp and 660bp two bands;
In the system of described pcr amplification, the DNA concentration of Bemisia tabaci to be measured is 0.17 × 10
-4～0.2ng/ul.
2. method according to claim 1, the system of described pcr amplification also comprises: Taq archaeal dna polymerase 0.05U/ μ l, 1 × Taq damping fluid, magnesium chloride 1.5mM, dNTPs200 μ M, concentration be respectively 0.5 μ M upstream primer and downstream primer, add ddH
2o to 20 μ l.
3. method according to claim 1 and 2, the program of described pcr amplification is: 94 DEG C of denaturations 3 minutes; Then carry out 30 circulations: 45 seconds, 72 DEG C extensions of 1 minute, 59 DEG C annealing of 94 DEG C of sex change 110 seconds; Last 72 DEG C are extended 10min.
4. for detection of the test kit of Type B Bemisia tabaci and Q type Bemisia tabaci, it is characterized in that comprising one couple of PCR primers:
Upstream primer Bt-F:5 '-TCT CAT CGG TGT CTT ATT G-3 ',
Downstream primer Bt-R:5 '-CTT CGG GTC GTT TCT CGT-3 '.
5. test kit according to claim 4, also comprises the reagent detecting for PCR: Taq archaeal dna polymerase, dNTPs, magnesium chloride, Taq DNA polymerase buffer liquid and sterilized water.
6. test kit according to claim 4, described PCR primer is mixed in PCR premix system in advance, and described PCR premix system is as follows: Taq archaeal dna polymerase 0.05U/ μ l, 1 × Taq damping fluid, 1.5mM magnesium chloride, 200 μ M dNTPs, upstream primer and downstream primer be 0.5 μ M and sterilized water respectively.
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