CN102719560A - Chip for screening apscaviroid viroid and application of chip - Google Patents
Chip for screening apscaviroid viroid and application of chip Download PDFInfo
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- CN102719560A CN102719560A CN2012101933948A CN201210193394A CN102719560A CN 102719560 A CN102719560 A CN 102719560A CN 2012101933948 A CN2012101933948 A CN 2012101933948A CN 201210193394 A CN201210193394 A CN 201210193394A CN 102719560 A CN102719560 A CN 102719560A
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Abstract
The invention discloses a chip for screening apscaviroid viroid and application of the chip. A probe group identifying or assisting in identifying the apscaviroid viroid consists of 35 probes, and the nucleotide sequences of the 35 probes are the sequences from 1 to 35 in a sequence table respectively. The invention also provides a gene chip for screening the apscaviroid viroid, which is obtained by fixing the probes on the surface of a chip base.. The experiment proves that the nucleotide sequence of the apscaviroid viroid is analyzed by a bioinformatics method, the compatible probes of the group are designed, and the standard viroid sample verification result indicates that a probe effect is good. The chip for screening the apscaviroid viroid disclosed by the invention can be applied to the quarantine identification of the apscaviroid viroid.
Description
Technical field
The present invention relates to information biology and biological quarantine authenticate technology field, relate in particular to chip and the application thereof of examination apple rust fruit Tobamovirus viroid.
Background technology
Apple rust fruit Tobamovirus viroid (Apscaviroid) belongs to pospiviroidae (Pospiviroidae); According to (the International Committee on Taxonomy of Viruses of ICTV; ICTV) the 8th subseries report; This genus comprises apple rust fruit virus (Apple scar skin viroid; ASSVd), the recessed viroid of apple (Apple dimple fruit viroid; ADFVd), the bent leaf viroid of citrus (Citrus bent leaf viroid, CiBLV), Australia grape viroid (Australian grapevine viroid, AGVd), citrus viroid III (Citrus viroid III; CiVd III), the yellow point of grape viroid 1 (Grapevine yellow speckle viroid 1; GYSVd-1), the yellow point of grape viroid 2 (Grapevine yellow speckle viroid 2, GYSVd-2) and pears apple pox viroid (Pear blister canker viroid PBCVd) waits 8 kinds of viroids.The viroid of this genus can be infected multiple important fruit trees such as apple, pears, peach, oranges and tangerines and grape.Its representative species apple rust fruit virus infection apple can cause that apple rough skin disease causes fruit to become flat, paint face, deformity and dehiscent fruit, is one of destructive viroid disease in the apple production; Can pass through seed, grafting and amboceptor entomochory, velocity of propagation is fast in the orchard, can between plant, connect propagation through root, does not still have the generation that effective medicine prevents this disease at present; In a single day apple tree catches an illness, and the state of an illness increases the weight of and become the permanent disease of complete stool year by year, must remove diseased plant and could control spreading of disease; It can make the apple fruit commodity value reduce greatly, and this not only has a strong impact on orchard worker's income, has also restricted the sound development of China's apple industry simultaneously, is the internal quarantine object; In recent years, the expansion of this viroid spreads rapidly, and the technician is trans-regional to stride the orchard grafting, prune operation, has more increased the weight of its propagation and development.The grape Huang is selected viroid and is then caused grape diseased plant blade yellow spotting or yellow class in irregular shape piece, causes a large amount of underproduction of grape.Pears apple pox viroid harm pear tree causes its yield and quality to descend, and can cause when serious that fruit tree sharply fails, and does sth. in advance withered and has no harvest, and brings very big influence to production of fruit trees.This belongs to medium pears apple pox viroid is the quarantine harmful organisms in 2007 issue " the People's Republic of China enter the territory Plant Quarantine property harmful organism register ".
Because this accessory has the possibility of potential New Development quarantine property viroid; Can only belong to known class virus to this and carry out specific detection and be used for method that this genus viroid detects such as plant indicator method, polyacrylamide gel electrophoresis, nucleic acid dot hybridization technology and Protocols in Molecular Biology etc. at present; Viroid monitoring for the unknown and New Development is powerless; So cause dangerous viroid omission easily and propagate diffusion, and then cause enormous economic loss and bad social influence.
Summary of the invention
An object of the present invention is to provide the probe groups of a kind of evaluation or assistant identification apple rust fruit Tobamovirus viroid.
The present invention provides and identifies or the probe groups of assistant identification apple rust fruit Tobamovirus viroid, by probe 1-probe 35 totally 35 probes form, the nucleotide sequence of said probe 1-probe 35 respectively is the sequence 1-35 in the sequence table.
In the above-mentioned probe, said apple rust fruit Tobamovirus viroid is that apple rust fruit virus, the recessed viroid of apple, the bent leaf viroid of citrus, Australia grape viroid, citrus viroid III, grape are yellowly selected viroid 1, the grape Huang is selected viroid 2 or pears apple pox viroid.
Another object of the present invention provides the gene chip of a kind of examination apple rust fruit Tobamovirus viroid.
The gene chip of examination apple rust fruit Tobamovirus viroid provided by the invention is the gene chip that above-mentioned probe stationary is obtained at the sheet primary surface.
In the said gene chip; Said base is the aldehyde radical glass chip; What adopt in an embodiment of the present invention is brilliant core
the microarray substrate of Boao Biological Co., Ltd, catalog number: 420022.
In the said gene chip, said apple rust fruit Tobamovirus viroid is that apple rust fruit virus, the recessed viroid of apple, the bent leaf viroid of citrus, Australia grape viroid, citrus viroid III, grape are yellowly selected viroid 1, the grape Huang is selected viroid 2 or pears apple pox viroid.
The 3rd purpose of the present invention provides the test kit of a kind of examination apple rust fruit Tobamovirus viroid.
Test kit provided by the invention comprises above-mentioned gene chip.
The mentioned reagent box comprises that also the primer of cDNA of the said apple rust fruit Tobamovirus viroid that is used to increase is right, said primer to be specially primer to 1 or primer to 2;
Said primer is formed (apple that is used to increase rust fruit virus) to 1 by the dna molecular shown in the sequence 37 in sequence in the sequence table 36 and the sequence table;
Said primer is formed (citrus virus III is used to increase) to 2 by the dna molecular shown in the sequence 39 in sequence in the sequence table 38 and the sequence table;
Said apple rust fruit Tobamovirus viroid is apple rust fruit virus or citrus virus III.
Above-mentioned probe groups, said gene chip or mentioned reagent box are following 1)-4) in application, also be the scope that the present invention protects:
1) evaluation or assistant identification apple rust fruit Tobamovirus viroid;
2) preparation is identified or assistant identification apple rust fruit Tobamovirus viroid product;
3) evaluation or assistant identification plant infection apple rust to be measured fruit Tobamovirus viroid;
4) preparation is identified or assistant identification plant infection apple rust to be measured fruit Tobamovirus viroid product.
In the above-mentioned application, said apple rust fruit Tobamovirus viroid is that apple rust fruit virus, the recessed viroid of apple, the bent leaf viroid of citrus, Australia grape viroid, citrus viroid III, grape are yellowly selected viroid 1, the grape Huang is selected viroid 2 or pears apple pox viroid; The said measuring plants of treating is apple, citrus, grape or pears.
The 4th purpose of the present invention provides the method for a kind of examination or auxiliary examination plant infection apple rust to be measured fruit Tobamovirus viroid.
Method provided by the invention comprises the steps:
The cDNA that 1) will treat the tissue of measuring plants carries out mark, obtains the mark after product;
2) mark after product that step 1) is obtained and above-mentioned gene chip are hybridized, and obtain hybridizing the back chip;
3) with step 2) chip scanning after the hybridization that obtains,
If the signal absolute value of at least one said probe on the said gene chip be not less than 600 and SNR be not less than 3.0, plant infection then to be measured or candidate infect apple rust fruit Tobamovirus viroid.
In the aforesaid method, in the step 1), saidly be labeled as with said cDNA to be template, to carrying out pcr amplification, to obtain the PCR product, more said PCR product is carried out the Klenow enzyme labelling, obtain the mark after product with the said primer in the above-mentioned test kit;
Step 2) in, the temperature of said hybridization is 42 ℃, and the time of said hybridization is 12h;
In said step 2) also comprise the step that the back chip of said hybridization is washed before back and the step 3);
Said at least one said probe is any in the above-mentioned probe groups;
The said blade that is organized as;
Said apple rust fruit Tobamovirus viroid, said primer to the said measuring plants following 1 of treating) or 2):
1) said apple rust fruit Tobamovirus viroid is an apple rust fruit virus, and said primer is to being that said primer is to 1; The said measuring plants of treating is an apple;
2) said apple rust fruit Tobamovirus viroid is citrus viroid III, and said primer is to being that said primer is to 2; The said measuring plants of treating is a citrus.
Of the present invention experiment showed, the probe in the examination apple provided by the invention rust fruit Tobamovirus viroid chip have on the apple rust fruit Tobamovirus viroid genus level highly compatible with belong in specificity, required sample size is few, generally only needs 0.1g.The analysis of data combines with Computer Image Processing software in addition, and reaching analytical results can visualize, visual.The present invention adopts bioinformatics method that the nucleotide sequence of apple rust fruit Tobamovirus viroid is analyzed, and has designed the compatible probe of this genus, and standard class viral sample checking result proves that probe is respond well.The quarantine that apple rust fruit Tobamovirus viroid chip of the present invention can be used for apple rust fruit Tobamovirus viroid is identified.
Description of drawings
Fig. 1 is apple rust fruit Tobamovirus viroid examination chip probe dot matrix synoptic diagram (10 * 12)
Fig. 2 is for using the result of apple rust fruit viral sample checking apple rust fruit Tobamovirus viroid examination chip
Fig. 3 is for using the result of citrus virus III sample checking apple rust fruit Tobamovirus viroid examination chip
Fig. 4 is an examination chip sensitivity detected result
Fig. 5 is a PCR sensitivity detected result
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The preparation of the chip of embodiment 1, examination apple rust fruit Tobamovirus viroid
1, belongs to the design of level highly compatible oligonucleotide probe
Belong to full genome and nucleotide sequence from the U.S. state-run biotechnology information center (NCBI) and the international virusology classification council (ICTV) DB download viroid; Removing 90% above length and other sequence has the nucleotide sequence of 95% similarity; As at interval, extract the nucleotide sequence of all 40mer with 5 bases continuously; With 40%≤GC content≤60%, single base contents≤50%, repeat base number≤4 continuously; And the hairpin structure that does not have greater than 6 bases is that standard is screened nucleotide sequence, and in ncbi database, carries out homology relatively to guarantee the specificity of institute's acquisition probe.
Designed 35 probes (probe 1-probe 35) of apple rust fruit Tobamovirus viroid according to mentioned above principle, its nucleotide sequence is followed successively by respectively shown in the sequence 1-35 in the sequence table.
Can above-mentioned 35 probes of synthetic.
2, the preparation of chip
Above-mentioned 1 35 probes obtaining are used point sample damping fluid (brilliant core
the chip sampling liquid of Boao Biological Co., Ltd respectively; Catalog number: 440010) dissolving; Concentration is 50 μ M; Every probe laterally repeats 3 and selects at aldehyde radical glass chip chip (brilliant core
the microarray substrate of Boao Biological Co., Ltd; Catalog number: 420022); Every about 0.25nL; The about 180 μ m of spot diameter, dot spacing is 300 μ m, the standard variance of point sample uniformity coefficient is 15%.The chip point is had one side hydration 10s on 65 ℃ of water-baths of probe, chip is 3cm apart from water surface distance, in the air at room temperature seasoning, is carrying out a hydration.The one side that point is had a probe upwards, it is crosslinked to be placed in the UV-crosslinked appearance 250mJ.Chip is placed on 42 ℃ of preheatings, and 0.5%SDS cleans 10min.Chip transferred in 42 ℃ of preheating zero(ppm) water clean 2min.Be placed on chip in the 50mL taper centrifuge tube, the centrifugal 1min of 2000rpm to remove the liquid of chip surface, obtains the chip of examination apple rust fruit Tobamovirus viroid.
The chip of examination apple rust fruit Tobamovirus viroid is as shown in Figure 1; 1-35 is apple rust fruit Tobamovirus viroid examination chip probe 1-35 (corresponding sequence 1-35) among Fig. 1; Hex is a fixedly positive quality control of chip, and PC is the hybridization positive quality control, and NC is the negative Quality Control of hybridization.
The application of the chip of embodiment 2, examination apple rust fruit Tobamovirus viroid
One, examination chip detection sample
1, the total RNA of the sample that is used to detect extracts
1) gets apple blade (the apple rust fruit virus of A pple scar skin viroid that infects apple rust fruit virus; Be documented in: several kinds of process for extracting of apple rust fruit virus and the comparison of RT-PCR detection sensitivity. plant protection; 2006; 32 (2): 95-97., the public can obtain from China Inst. of Quarantine Inspection Sciences.) and infect citrus virus III the oranges and tangerines blade (citrus virus III latin name Citrus viroid III. is documented in: the Molecular Identification and the somatotype of citrus virus. Scientia Agricultura Sinica; 2008; 41 (9): 2670-2677, the public can obtain from China Inst. of Quarantine Inspection Sciences.) each 0.1g sample, powdered with liquid nitrogen grinding, move in the 1.5mL centrifuge tube of sterilization, add the Trizol reagent of 1mL then, concuss shakes up;
2) 4 ℃, the centrifugal 10min of 12000rpm changes supernatant in the one new 1.5mL centrifuge tube over to;
3) add the 0.5mL chloroform, thermal agitation 15s, room temperature is placed 15min; 4 ℃, the centrifugal 15min of 12000rpm;
4) with the upper water phase transition in new 1.5mL centrifuge tube, add the equal-volume Virahol, put upside down mixing, room temperature keeps 15min;
5) 4 ℃, the centrifugal 10min of 12000rpm; Outwell supernatant, add 75% cold washing with alcohol deposition, 4 ℃ then, the centrifugal 5min of 12000rpm abandons ethanol;
7) under the deposition room temperature after the thorough drying, be dissolved in the 40 μ L distilled waters (DEPC processing) ,-20 ℃ of preservations are subsequent use, obtain total RNA.
2, sample mark and hybridization
The above-mentioned total RNA reverse transcription that obtains is obtained cDNA.
Pcr amplification promptly adds cDNA product 2 μ L, upstream primer 0.5 μ L (final concentration is 0.5mmol/L), downstream primer 0.5 μ L (final concentration is 0.5mmoL/L), dNTP Mix (10mmol/L) 0.5 μ L, Taq enzyme (5U/ μ L) 0.5 μ L, PCR damping fluid (10 *) 2 μ L and DEPC-H in the reaction tubes of 0.2mL
2O 14 μ L increase according to the PCR response procedures then.
The upstream and downstream primer by the primer that is respectively applied for amplification apple rust fruit virus or citrus virus III to forming.
The primer of the above-mentioned apple rust fruit virus that is used to increase is to comprising
Upstream primer: 5 '-
GGTAAACACCGTGCGGTCCC-3 ' (sequence 36);
Downstream primer: 5 '-
GGGAAACACCTATTGTGTTT-3 ' (sequence 37);
Its PCR response procedures is: 94 ℃ of 5min; 94 ℃ of 30s, 54 ℃ of 30s, 72 ℃ of 30s; Totally 30 circulations; 72 ℃ are extended 10min.
The primer of the above-mentioned citrus virus III that is used to increase is to comprising
Upstream primer: 5 '-GGCAGCTAAGTTGGTGACGC-3 ' (sequence 38);
Downstream primer: 5 '-TTCGTCGACGACGACAGGTA-3 ' (sequence 39);
Its PCR response procedures is: 95 ℃ of 3min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s; Totally 35 circulations; 72 ℃ are extended 10min.
Klenow enzyme labelling system is 25 μ L, promptly in the PCR of 0.2mL reaction tubes, adds PCR product 5 μ L, 9N random primer (invitrogen, Cat.No.48190-011) (100 μ mol/L) 2 μ L and H
2O 12 μ L, 95 ℃ of sex change 3min, ice bath 5min.In reaction tubes, add 10 * Klenow enzyme buffer liquid, 2.5 μ L then, dNTP (2.5mmol/L) 2 μ L, cy3-dCTP 0.5 μ L (Amersham, Cat.No.PA 53021 final concentrations are 5nmol/L), klenow enzyme (5U/ μ L) 1 μ L.37 ℃ of reaction 1.5h, 70 ℃ of sex change 5min, ice bath 5min obtains the mark sample.
Hybridization: system is 16 μ L; Comprise 2.4 μ L SSC (final concentration 3 *), 0.32 μ L SDS (final concentration 0.2%), 4 μ L methane amides (final concentration 25%), 1.6 μ L Denhardt ' s (Ameresco; Cat.No.E717, final concentration 5 *) and mark sample 7.68 μ L.95 ℃ of sex change 3min, ice bath 5min is instantaneous centrifugal.Hybridization solution is added on the chip, and deckglass is built, and 42 ℃ of water-bath hybridization are spent the night (12h), the chip (citrus virus III) after chip after obtaining respectively hybridizing (apple rust fruit virus) and the hybridization.
3, washing, scanning
Cleaning system and program are following:
Earlier washing lotion I, II are placed on and are preheating to 42 ℃ in the microwave oven, transfer in the cleaning box.After hybridization finishes, the chip after the hybridization is transferred in the cleaning box that holds washing lotion, hybridization surface upwards is placed on the horizontal shaking table and slowly cleans.After chip cleans, be placed in the 50mL taper centrifuge tube, the centrifugal 1min of 2000rpm removes the liquid of chip surface, obtains chip to be detected (apple rust fruit virus), chip to be detected (citrus virus III) respectively.
Place scanner to carry out scanning analysis above-mentioned chip to be detected (apple rust fruit virus), chip to be detected (citrus virus III); PMT is made as 900, obtains data such as each point fluorescence intensity and background intensity.
Use the Bo Ao LuxScan of biotech firm 3.0 chip scanners extraction data from chip, the signal value of probe is the median of the median subtracting background value of probe prospect value.SNR is the ratio of interior all signal value medians of image corresponding points and background value median.
If signal value >=600 and SNR >=3 of at least one said probe are judged to the positive (for apple rust fruit Tobamovirus viroid infects); The signal value of probe＜600 and SNR＜2 are judged to feminine gender (not infecting for apple rust fruit Tobamovirus viroid); All the other situation are judged to suspicious, are judged to suspiciously, need repeated authentication.
The probe of Fig. 2, Fig. 3 is arranged identical with Fig. 1, and apple rust fruit Tobamovirus viroid probe is 1-35.
The result of apple rust fruit virus is as shown in Figure 2, and the signal value of probe 1,4,5,6,9 positions is respectively 6642,48016,14567,1892 and 5453, all greater than 600; SNR is respectively 19,330,41,6 and 16, all greater than 3, explains that the present invention can detect the apple rust fruit virus of apple rust fruit Tobamovirus viroid;
The result of citrus virus III is as shown in Figure 3, and the signal value of probe 2,3 positions is respectively 14093,8598 all greater than 600; SNR is respectively 34,20, all greater than 3, explains that the present invention can detect the citrus virus III of apple rust fruit Tobamovirus viroid;
The fixedly positive quality control of said chip, hybridize positive quality control and hybridize negative Quality Control performance good, the standard model hybridization signal is strong, explains that the chip examination program of designed probe and foundation has the good operation effect.
Two, examination chip and PCR detection sensitivity are relatively
Accurately take by weighing 0.1g and infect the apple tissue of apple rust fruit virus, extract total RNA, be respectively applied for apple rust fruit Tobamovirus viroid examination chip detection and PCR and detect, both used RNA all carry out 10
0, 10
1, 10
2With 10
3Times gradient dilution.
The method of chip detection is with above-mentioned one, and the result is as shown in Figure 4, and the probe of Fig. 4 is arranged identical with Fig. 1, wherein A:10
1Dilution; B:10
2Dilution; C:10
3Dilution; The signal value of A middle probe 1,4,5,6,9 positions is respectively 1152,3155,2692,1058,991, all greater than 600; SNR is respectively 4,9,8,4,4, all greater than 3, so probe 1,4,5,6,9 is judged to the positive.The signal value of B middle probe 6 positions is respectively 723, greater than 600; SNR is 9, can judge that probe 6 is positive.The C no signal.Therefore, can to detect the highly diluted multiple of object to be checked be 10 to apple rust fruit Tobamovirus viroid examination chip
2
The primer that PCR detects does
Upstream primer: 5 '-
GGTAAACACCGTGCGGTCCC-3 ';
Downstream primer: 5 '-
GGGAAACACCTATTGTGTTT-3 ',
Base is for protecting base in the square frame in the above-mentioned primer, and the italic base is a restriction enzyme site BamHI base, and the underscore base is an apple rust fruit virus sequence.
The result that PCR detects is as shown in Figure 5,1:10
0Dilution; 2:10
1Dilution; 3:10
2Dilution; 4:10
3Dilution; 5: blank; M:Marker DL2000 can find out 10
0, 10
1With 10
2The dilution template all obtains the product (the 1-330 position Nucleotide of apple rust fruit virus Genbank HQ840722) of 355bp, and the highly diluted multiple of object to be checked is 10
2
Therefore, apple rust fruit Tobamovirus viroid examination chip detection sensitivity is suitable with PCR method sensitivity.
Claims (10)
1. identify or the probe groups of assistant identification apple rust fruit Tobamovirus viroid for one kind, by probe 1-probe 35 totally 35 probes form, the nucleotide sequence of said probe 1-probe 35 respectively is the sequence 1-35 in the sequence table.
2. probe groups according to claim 1 is characterized in that: said apple rust fruit Tobamovirus viroid is that apple rust fruit virus, the recessed viroid of apple, the bent leaf viroid of citrus, Australia grape viroid, citrus viroid III, grape are yellowly selected viroid 1, the grape Huang is selected viroid 2 or pears apple pox viroid.
3. the gene chip of examination apple rust fruit Tobamovirus viroid is for being fixed on the gene chip that the sheet primary surface obtains with claim 1 or 2 described probe groups.
4. gene chip according to claim 3 is characterized in that: said base is the aldehyde radical glass chip; Said apple rust fruit Tobamovirus viroid is that apple rust fruit virus, the recessed viroid of apple, the bent leaf viroid of citrus, Australia grape viroid, citrus viroid III, grape are yellowly selected viroid 1, the grape Huang is selected viroid 2 or pears apple pox viroid.
5. the test kit of an examination apple rust fruit Tobamovirus viroid comprises claim 3 or 4 described gene chips.
6. test kit according to claim 5 is characterized in that: said test kit comprises that also the primer of cDNA of the said apple rust fruit Tobamovirus viroid that is used to increase is right, said primer to be specially primer to 1 or primer to 2; Said primer is made up of the dna molecular shown in the sequence 37 in sequence in the sequence table 36 and the sequence table 1;
Said primer is made up of the dna molecular shown in the sequence 39 in sequence in the sequence table 38 and the sequence table 2;
Said apple rust fruit Tobamovirus viroid is apple rust fruit virus or citrus virus III.
7. claim 1 or 2 said probe groups, claim 3 or 4 described gene chips, claim 5 or 6 described test kits
Following 1)-4) in application, also be the scope that the present invention protects:
1) evaluation or assistant identification apple rust fruit Tobamovirus viroid;
2) preparation is identified or assistant identification apple rust fruit Tobamovirus viroid product;
3) evaluation or assistant identification plant infection apple rust to be measured fruit Tobamovirus viroid;
4) preparation is identified or assistant identification plant infection apple rust to be measured fruit Tobamovirus viroid product.
8. application according to claim 7 is characterized in that: said apple rust fruit Tobamovirus viroid is that apple rust fruit virus, the recessed viroid of apple, the bent leaf viroid of citrus, Australia grape viroid, citrus viroid III, grape are yellowly selected viroid 1, the grape Huang is selected viroid 2 or pears apple pox viroid; The said measuring plants of treating is apple, citrus, grape or pears.
9. the method for an examination or auxiliary examination plant infection apple rust to be measured fruit Tobamovirus viroid comprises the steps:
The cDNA that 1) will treat the tissue of measuring plants carries out mark, obtains the mark after product;
2) the mark after product that step 1) is obtained is hybridized with claim 3 or 4 described gene chips, obtains hybridizing the back chip;
3) with step 2) chip scanning after the hybridization that obtains,
If the signal absolute value of at least one said probe on the said gene chip be not less than 600 and SNR be not less than 3.0, plant infection then to be measured or candidate infect apple rust fruit Tobamovirus viroid.
10. method according to claim 9 is characterized in that:
In the step 1), saidly be labeled as with said cDNA to be template, to carrying out pcr amplification, to obtain the PCR product, more said PCR product is carried out the Klenow enzyme labelling, obtain the mark after product with the said primer in the described test kit of claim 6;
Step 2) in, the temperature of said hybridization is 42 ℃, and the time of said hybridization is 12h;
In said step 2) also comprise the step that the back chip of said hybridization is washed before back and the step 3);
Said at least one said probe is any in the said probe groups;
The said blade that is organized as;
Said apple rust fruit Tobamovirus viroid, said primer to the said measuring plants following 1 of treating) or 2):
1) said apple rust fruit Tobamovirus viroid is an apple rust fruit virus, and said primer is to being that said primer is to 1; The said measuring plants of treating is an apple;
2) said apple rust fruit Tobamovirus viroid is citrus viroid III, and said primer is to being that said primer is to 2; The said measuring plants of treating is a citrus.
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| CN103382508A (en) * | 2013-07-12 | 2013-11-06 | 西南大学 | Method for synchronous detection of 4 apple viruses and viroid |
| CN104164517A (en) * | 2014-07-21 | 2014-11-26 | 中国农业大学 | Multiple RT-PCR detection method for apple latent viruses and viroid |
| CN105969766A (en) * | 2016-05-19 | 2016-09-28 | 山东出入境检验检疫局检验检疫技术中心 | Pear blister canker viroid (PBCVd) molecular standard sample and preparation method thereof |
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| CN102226195A (en) * | 2011-05-12 | 2011-10-26 | 湖南农业大学 | Method for cloning viroid complete-genome sequence |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103382508A (en) * | 2013-07-12 | 2013-11-06 | 西南大学 | Method for synchronous detection of 4 apple viruses and viroid |
| CN104164517A (en) * | 2014-07-21 | 2014-11-26 | 中国农业大学 | Multiple RT-PCR detection method for apple latent viruses and viroid |
| CN104164517B (en) * | 2014-07-21 | 2016-06-22 | 中国农业大学 | The multiple RT-PCR detection method of apple latent virus and viroid |
| CN105969766A (en) * | 2016-05-19 | 2016-09-28 | 山东出入境检验检疫局检验检疫技术中心 | Pear blister canker viroid (PBCVd) molecular standard sample and preparation method thereof |
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