CN102719561A - Chip for screening pospiviroid viroid and application of chip - Google Patents
Chip for screening pospiviroid viroid and application of chip Download PDFInfo
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- CN102719561A CN102719561A CN2012101934048A CN201210193404A CN102719561A CN 102719561 A CN102719561 A CN 102719561A CN 2012101934048 A CN2012101934048 A CN 2012101934048A CN 201210193404 A CN201210193404 A CN 201210193404A CN 102719561 A CN102719561 A CN 102719561A
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Abstract
The invention discloses a chip for screening pospiviroid viroid and application of the chip. A probe group identifying or assisting in identifying the pospiviroid viroid consists of 19 probes, and the nucleotide sequences of the 19 probes are the sequences from 1 to 19 in a sequence table respectively. The invention also provides a gene chip for screening the pospiviroid viroid, which is obtained by fixing the probe group on the surface of a chip base. The experiment proves that the nucleotide sequence of the pospiviroid 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 pospiviroid viroid disclosed by the invention can be applied to the quarantine identification of the pospiviroid 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 pospiviroid viroid.
Background technology
Pospiviroid viroid (Pospiviroid) belongs to pospiviroidae (Pospiviroidae); According to (the International Committee on Taxonomy of Viruses of ICTV; ICTV) the 8th subseries report; This genus comprises potato spindle tuber viroid (Potato spindle tuber viroid; PSTVd), chrysanthemum stunt viroid (Chrysanthemum stunt viroid; CSVd), oranges and tangerines bark viroid (Citrus exocortis viroid, CEVd), goldfish flower hide viroid (Columnea latent viroid, CLVd), Iresine herbetii viroid 1 (Iresine viroid 1; IrVd-1), Mexico's lobus cardiacus eggplant viroid (Mexican papita viroid; MPVd-1), tomato synthlipsis viroid (Tomato apical stunt viroid, TASVd) and tomato planto macho viroid (Tomato planta macho viroid TPMVd) waits 8 kinds of viroids.The viroid of this genus can be infected multiple important crops such as yam, tomato, capsicum, avocado, chrysanthemum, oranges and tangerines, goldfish flower, Iresine herbetii, lobus cardiacus eggplant.Its representative species potato spindle tuber viroid is the quarantine harmful organisms in 2007 issue " the People's Republic of China enter the territory Plant Quarantine property harmful organism register ", in the potato planting district of the U.S., Canada, the FSU generation is arranged all; In South Africa, this viroid also causes tomato disease.Lentogenic strains is more than heavy strain, and its ratio is about 10: 1.Light-duty diseased plant causes that heavy losses are 15% ~ 25%; Heavy strain causes serious symptoms, causes production loss can reach 65%.In recent years; The host of this cause of disease is expansion trend, and successively on the root of Manchurian Currant of Datura, jasmine eggplant, Herba Herminii, Petunia and the Turkey of the Solanum ornamental plant of Zelanian capsicum, Holland and European countries and Czech and Germany, finding should virus.Expansion along with this virus host scope; Various countries' increasing in various degree to this viral attention degree with to the degree of concern of virus host harmful organism variation tendency; And in time take the quarantine measures of various flexibles, and in the quarantine that enters the territory, adjusted the inspection object off the record.The detection of Duo stem tuber virus is spun to China outlet pepper seed enforcement yam suddenly by Korea S like in February, 2010, and as transporting the treating part goods by moving back.
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 pospiviroid viroid.
The probe groups of evaluation provided by the invention or assistant identification pospiviroid viroid, by probe 1-probe 19 totally 19 probes form, the nucleotide sequence of said probe 1-probe 19 respectively is the sequence 1-19 in the sequence table.
In the above-mentioned probe, said pospiviroid viroid is potato spindle tuber viroid, chrysanthemum stunt viroid, oranges and tangerines bark viroid, goldfish colored hide viroid, Iresine herbetii viroid 1, Mexico lobus cardiacus eggplant viroid, tomato synthlipsis viroid or tomato planto macho viroid.
Another object of the present invention provides the gene chip of a kind of examination pospiviroid viroid.
The gene chip of examination pospiviroid viroid provided by the invention is for being fixed on the gene chip that the sheet primary surface obtains with above-mentioned probe groups.
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 pospiviroid viroid is potato spindle tuber viroid, chrysanthemum stunt viroid, oranges and tangerines bark viroid, goldfish colored hide viroid, Iresine herbetii viroid 1, Mexico lobus cardiacus eggplant viroid, tomato synthlipsis viroid or tomato planto macho viroid.
The 3rd purpose of the present invention provides the test kit of a kind of examination pospiviroid 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 pospiviroid viroid that is used to increase is right, said primer to be specially primer to 1 or primer to 2;
Said primer is formed (being used to the chrysanthemum stunt viroid that increases) to 1 by the dna molecular shown in the sequence 21 in sequence in the sequence table 20 and the sequence table;
Said primer is formed (being used to the tomato planto macho viroid that increases) to 2 by the dna molecular shown in the sequence 23 in sequence in the sequence table 22 and the sequence table;
Said pospiviroid viroid is chrysanthemum stunt viroid or tomato planto macho viroid.
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 pospiviroid viroid;
2) preparation is identified or assistant identification pospiviroid viroid product;
3) evaluation or assistant identification plant infection pospiviroid to be measured viroid;
4) preparation is identified or assistant identification plant infection pospiviroid to be measured viroid product.
In the above-mentioned application, said pospiviroid viroid is potato spindle tuber viroid, chrysanthemum stunt viroid, oranges and tangerines bark viroid, goldfish colored hide viroid, Iresine herbetii viroid 1, Mexico lobus cardiacus eggplant viroid, tomato synthlipsis viroid or tomato planto macho viroid; The said measuring plants of treating is yam, chrysanthemum, oranges and tangerines, goldfish flower, Iresine herbetii, lobus cardiacus eggplant or tomato.
The 4th purpose of the present invention provides the method for a kind of examination or auxiliary examination plant infection pospiviroid to be measured 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's potato-infecting fusiform tubers 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 pospiviroid viroid, said primer to the said measuring plants following 1 of treating) or 2):
1) said pospiviroid viroid is a chrysanthemum stunt viroid, and said primer is to being that said primer is to 1; The said measuring plants of treating is a chrysanthemum;
2) said pospiviroid viroid is a tomato planto macho viroid, and said primer is to being that said primer is to 2; The said measuring plants of treating is a tomato.
Experiment of the present invention proves; Probe in the examination pospiviroid viroid chip provided by the invention has the compatibility and the interior specificity of genus on the pospiviroid viroid genus level; 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 pospiviroid 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.Pospiviroid viroid chip of the present invention can be used for the quarantine of pospiviroid viroid to be identified.
Description of drawings
Fig. 1 is a pospiviroid viroid examination chip probe dot matrix synoptic diagram (6 * 5)
Fig. 2 is for using the result of chrysanthemum stunt viroid sample checking pospiviroid viroid examination chip
Fig. 3 is for using the result of tomato planto macho viroid sample checking pospiviroid 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 pospiviroid 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 19 probes (probe 1-probe 19) of pospiviroid viroid according to mentioned above principle, its nucleotide sequence is followed successively by respectively shown in the sequence 1-19 in the sequence table.
Can above-mentioned 19 probes of synthetic.
2, the preparation of chip
Above-mentioned 1 19 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 5 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 pospiviroid viroid.
The chip of examination pospiviroid viroid is as shown in Figure 1; 1-19 is pospiviroid viroid examination chip probe 1-19 (corresponding sequence 1-19) among Fig. 1; Hex is a fixedly positive quality control of chip; 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 pospiviroid viroid
One, the chip detection sample of examination
1, the total RNA of the sample that is used to detect extracts
1) gets chrysanthemum blade (the chrysanthemum stunt viroid Chrysanthemum stunt viroid that infects chrysanthemum stunt viroid; Be documented in: the foundation and the comparison of two kinds of detection methods of chrysanthemum stunt viroid; Plant protection; 2002,28 (2): 48-50., the public can obtain from China Inst. of Quarantine Inspection Sciences.) and (tomato planto macho viroid: Tomato planta macho viroid. is documented in: Mexican papita viroid and tomato planta macho viroid belong to a single species in the genus Pospiviroid to infect the tomato leaf of tomato planto macho viroid; Archives of Virology; 2011; 156 (8): 1433-1437, 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 chrysanthemum stunt viroid and tomato planto macho viroid to forming.
The primer of the above-mentioned chrysanthemum stunt viroid that is used to increase to by
Upstream primer 5 '-CGGGACTTACTTGTGGTTCC-3 ' (sequence 20) and
Downstream primer 5 '-AGGGAACAAAACTAAGGTTCCAC-3 ' (sequence 21) is formed.
Its PCR response procedures is: 94 ℃ of 5min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; Totally 30 circulations; 72 ℃ are extended 8min.
The primer of the above-mentioned tomato planto macho viroid that is used to increase to by
Upstream primer 5 '-GGATCCCCGGGGAAACCT-3 ' (sequence 22) and
Downstream primer 5 '-CTGAAGCGCTCCTTTGGC-3 ' (sequence 23) is formed.
Its PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s; Totally 30 circulations; 72 ℃ are extended 8min.
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 LSSC (final concentration 3 *), 0.32 μ LSDS (final concentration 0.2%), 4 μ L methane amides (final concentration 25%), 1.6 μ LDenhardt ' 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 (tomato planto macho viroid) after chip (chrysanthemum stunt viroid) after obtaining respectively hybridizing 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 (chrysanthemum stunt viroid), chip to be detected (tomato planto macho viroid) respectively.
Place scanner to carry out scanning analysis above-mentioned chip to be detected (chrysanthemum stunt viroid) and chip to be detected (tomato planto macho viroid); 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 the pospiviroid viroid infects); The signal value of probe＜600 and SNR＜2 are judged to feminine gender (not infecting for the pospiviroid viroid); All the other situation are judged to suspicious, need repeated authentication.
The probe of Fig. 2, Fig. 3 is arranged identical with Fig. 1, and pospiviroid viroid probe is 1-19.
The result of chrysanthemum stunt viroid is as shown in Figure 2, and the signal value of probe 1,3,9 positions is respectively 3823,2008,5285, all greater than 600; SNR is respectively 13,8,19, and all greater than 3, all are judged to the positive with probe 1,3,9, explains that the present invention can detect the chrysanthemum stunt viroid of pospiviroid viroid.
The result of tomato planto macho viroid is as shown in Figure 3, and the signal value of probe 1,3,4,18 positions is respectively 1600,1792,3472, all greater than 600; SNR is respectively 6,7,7,14, all greater than 3, so probe 1,3,4,18 is judged to the positive, explains that the present invention can detect the tomato planto macho viroid of pospiviroid 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, the chip of examination and PCR detection sensitivity are relatively
Accurately take by weighing the chrysanthemum tissue of 0.1g infection chrysanthemum stunt viroid, extract total RNA, be respectively applied for bell potato spindle tubers Tobamovirus 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 1,3,8,9 positions is respectively 9447,5796,1093,16571, all greater than 600; SNR is respectively 27,16,5,50, all greater than 3, so probe 1,3,8,9 is judged to the positive.The signal value of B middle probe 1,3,8,9 positions is respectively 3107,1889,769,5258, all greater than 600; SNR is respectively 29,21,12,75, all greater than 3, so probe 1,3,8,9 is judged to the positive.The signal value of C middle probe 9 positions is 719, greater than 600; SNR is 13; Can judge that probe 9 is positive.Therefore, can to detect the highly diluted multiple of object to be checked be 10 to pospiviroid viroid examination chip
3
The primer that PCR detects does
Upstream primer: 5 '-CGGGACTTACTTGTGGTTCC-3 ';
Downstream primer: 5 '-AGGGAACAAAACTAAGGTTCCAC-3 '.
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
1, 10
2With 10
3The dilution template all obtains the product (the 1-355 position Nucleotide of chrysanthemum stunt viroid Genbank FN646407) of 355bp, and the highly diluted multiple of object to be checked is 10
3
Therefore, pospiviroid viroid examination chip is suitable with PCR method sensitivity.
Claims (10)
1. identify or the probe groups of assistant identification pospiviroid viroid for one kind, by probe 1-probe 19 totally 19 probes form, the nucleotide sequence of said probe 1-probe 19 respectively is the sequence 1-19 in the sequence table.
2. probe groups according to claim 1 is characterized in that: said pospiviroid viroid is potato spindle tuber viroid, chrysanthemum stunt viroid, oranges and tangerines bark viroid, goldfish colored hide viroid, Iresine herbetii viroid 1, Mexico lobus cardiacus eggplant viroid, tomato synthlipsis viroid or tomato planto macho viroid.
3. the gene chip of an examination pospiviroid 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 pospiviroid viroid is potato spindle tuber viroid, chrysanthemum stunt viroid, oranges and tangerines bark viroid, goldfish colored hide viroid, Iresine herbetii viroid 1, Mexico lobus cardiacus eggplant viroid, tomato synthlipsis viroid or tomato planto macho viroid.
5. the test kit of an examination pospiviroid 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 pospiviroid 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 21 in sequence in the sequence table 20 and the sequence table 1;
Said primer is made up of the dna molecular shown in the sequence 23 in sequence in the sequence table 22 and the sequence table 2;
Said pospiviroid viroid is chrysanthemum stunt viroid or tomato planto macho viroid.
7. claim 1 or 2 said probe groups, claim 3 or 4 described gene chips, claim 5 or 6 described test kits are following 1)-4) in application:
1) evaluation or assistant identification pospiviroid viroid;
2) preparation is identified or assistant identification pospiviroid viroid product;
3) evaluation or assistant identification plant infection pospiviroid to be measured viroid;
4) preparation is identified or assistant identification plant infection pospiviroid to be measured viroid product.
8. application according to claim 7 is characterized in that: said pospiviroid viroid is potato spindle tuber viroid, chrysanthemum stunt viroid, oranges and tangerines bark viroid, goldfish colored hide viroid, Iresine herbetii viroid 1, Mexico lobus cardiacus eggplant viroid, tomato synthlipsis viroid or tomato planto macho viroid; The said measuring plants of treating is yam, chrysanthemum, oranges and tangerines, goldfish flower, Iresine herbetii, lobus cardiacus eggplant or tomato.
9. the method for an examination or auxiliary examination plant infection pospiviroid to be measured 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's potato-infecting fusiform tubers Tobamovirus viroid.
10. method according to claim 9 is characterized in that:
In the step 1), said to be labeled as with said cDNA be template, to carrying out pcr amplification, obtains the PCR product with the said primer in the described test kit of claim 6, more said PCR product carried out the Klenow enzyme labelling, obtains the mark after product;
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 pospiviroid viroid, said primer to the said measuring plants following 1 of treating) or 2):
1) said pospiviroid viroid is a chrysanthemum stunt viroid, and said primer is to being that said primer is to 1; The said measuring plants of treating is a chrysanthemum;
2) said pospiviroid viroid is a tomato planto macho viroid, and said primer is to being that said primer is to 2; The said measuring plants of treating is a tomato.
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| CN108330110A (en) * | 2018-02-02 | 2018-07-27 | 中国医学科学院药用植物研究所 | A kind of primer and method for detecting Dwarfing of Chrysanthemum encephalapthy agent |
| CN108330110B (en) * | 2018-02-02 | 2021-02-26 | 中国医学科学院药用植物研究所 | Primer and method for detecting pathogen of chrysanthemum dwarfing disease |
| CN113774053A (en) * | 2021-08-19 | 2021-12-10 | 中国农业科学院植物保护研究所 | Nucleic acid probe and kit for simultaneously detecting multiple tomato viruses and application of nucleic acid probe and kit |
| CN113774053B (en) * | 2021-08-19 | 2023-11-17 | 中国农业科学院植物保护研究所 | Nucleic acid probe and kit for simultaneously detecting various tomato viruses and application of nucleic acid probe and kit |
| CN116042922A (en) * | 2022-12-29 | 2023-05-02 | 武汉海关技术中心 | Tomato male strain virus TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, kit and application |
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