CN102719561B - 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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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 obtainedby 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 is spent viroid (the Columnea latent viroid that hides, CLVd), Iresine herbetii viroid 1(Iresine viroid 1, IrVd-1), Mexico 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 potato, 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 " People's Republic of China (PRC) enter the territory Plant Quarantine harmful organism register ", in the potato planting district of the U.S., Canada, USSR (Union of Soviet Socialist Republics) 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, successively finds this virus in Datura, jasmine eggplant, Herba Herminii, Petunia and the Turkey of the Solanum ornamental plant of Zelanian capsicum, Holland and European countries and Czech and the root of Manchurian Currant of Germany.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 potato suddenly by Korea S as 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 the method for this genuss viroid detection 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 helpless, 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 described probe 1-probe 19 respectively is the sequence 1-19 in the sequence table.
In the above-mentioned probe, described 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, described base is the aldehyde radical glass chip, and what adopt in an embodiment of the present invention is the brilliant core of Boao Biological Co., Ltd
The microarray substrate, catalog number: 420022.
In the said gene chip, described 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.
It is right that the mentioned reagent box also comprises for the primer of cDNA of the described pospiviroid viroid of amplification, described primer to be specially primer to 1 or primer to 2;
Described primer is formed (being used for the amplification chrysanthemum stunt viroid) to 1 by the dna molecular shown in the sequence 21 in sequence in the sequence table 20 and the sequence table;
Described primer is formed (being used for the amplification tomato planto macho viroid) to 2 by the dna molecular shown in the sequence 23 in sequence in the sequence table 22 and the sequence table;
Described 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 of protection of the invention:
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, described 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 described measuring plants for the treatment of is potato, 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:
1) cDNA that 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 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 described probe on the described gene chip be not less than 600 and signal to noise ratio be not less than 3.0, then plant infection to be measured or candidate's potato-infecting fusiform tubers Tobamovirus viroid.
In the aforesaid method, in the step 1), describedly be labeled as with described cDNA to be template, to carrying out pcr amplification, to obtain the PCR product with the described primer in the above-mentioned test kit, more described PCR product is carried out the Klenow enzyme labelling, obtain the mark after product;
Step 2) in, the temperature of described hybridization is 42 ℃, and the time of described hybridization is 12h;
In described step 2) also comprise the step that the back chip of described hybridization is washed before back and the step 3);
Described at least one described probe is any in the above-mentioned probe groups;
The described blade that is organized as;
Described pospiviroid viroid, described primer to the described measuring plants following 1 for the treatment of) or 2):
1) described pospiviroid viroid is chrysanthemum stunt viroid, and described primer is to being that described primer is to 1; The described measuring plants for the treatment of is chrysanthemum;
2) described pospiviroid viroid is tomato planto macho viroid, and described primer is to being that described primer is to 2; The described measuring plants for the treatment of is tomato.
Of the present invention experimental results show that, probe in the examination pospiviroid viroid chip provided by the invention has the compatibility on the pospiviroid viroid genus level and belongs to interior 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 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 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 examination chip sensitivity detected result
Fig. 5 is PCR sensitivity detected result
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels 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) database 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 carries out homology relatively to guarantee the specificity of institute's acquisition probe in ncbi database.
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 that obtain are used point sample damping fluid (the brilliant core of Boao Biological Co., Ltd respectively
The chip sampling liquid, catalog number: 440010) dissolving, concentration is 50 μ M, every probe laterally repeats 5 points at aldehyde radical glass chip chip (the brilliant core of Boao Biological Co., Ltd
The microarray substrate, 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 instrument 250mJ.Chip is placed on 42 ℃ of preheatings, and 0.5%SDS cleans 10min.Chip transferred in 42 ℃ of pre-hot distilled waters clean 2min.Chip is placed in the 50mL taper centrifuge tube, and 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 as shown in Figure 1,1-19 is the corresponding sequence 1-19 of pospiviroid viroid examination chip probe 1-19(among Fig. 1), Hex is 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 for detection of sample extracts
1) gets chrysanthemum blade (the chrysanthemum stunt viroid Chrysanthemum stunt viroid that infects chrysanthemum stunt viroid; be documented in: 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 liquor 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 liquor, add 75% cold washing with alcohol precipitation, 4 ℃ then, the centrifugal 5min of 12000rpm abandons ethanol;
7) under the precipitation room temperature fully after the drying, be dissolved in the 40 μ L distilled waters (DEPC processing) ,-20 ℃ of preservations are standby, obtain total RNA.
2, sample mark and hybridization
The above-mentioned total RNA reverse transcription that obtains is obtained cDNA.
Pcr amplification, namely in the reaction tubes of 0.2mL, add 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
2O 14 μ L increase according to the PCR response procedures then.
The upstream and downstream primer by the primer of be respectively applied to increase chrysanthemum stunt viroid and tomato planto macho viroid to forming.
Above-mentioned for the amplification chrysanthemum stunt viroid primer 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.
Above-mentioned for the amplification tomato planto macho viroid primer 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, namely adds PCR product 5 μ L, 9N random primer (invitrogen, Cat.No.48190-011) (100 μ mol/L) 2 μ L and H in the PCR of 0.2mL reaction tubes
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 concentrations 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 cover glass is built, and 42 ℃ of water-bath hybridization are spent the night (12h), the chip (tomato planto macho viroid) after the chip (chrysanthemum stunt viroid) after obtaining respectively hybridizing and the hybridization.
3, washing, scanning
Cleaning system and program are as follows:
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 to extract data from chip, the signal value of probe is the median of the median subtracting background value of probe prospect value.Signal to noise ratio is the ratio of interior all the signal value medians of image corresponding points and background value median.
If signal value 〉=600 and signal to noise ratio 〉=3 of at least one described probe are judged to the positive (for the pospiviroid viroid infects); The signal value of probe＜600 and signal to noise ratio＜2 are judged to feminine gender (not infecting for the pospiviroid viroid); All the other situations 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 as shown in Figure 2, the signal value of probe 1,3,9 positions is respectively 3823,2008,5285, all greater than 600; Signal to noise ratio is respectively 13,8,19, and all greater than 3, all are judged to the positive with probe 1,3,9, illustrates that the present invention can detect the chrysanthemum stunt viroid of pospiviroid viroid.
The result of tomato planto macho viroid as shown in Figure 3, the signal value of probe 1,3,4,18 positions is respectively 1600,1792,3472, all greater than 600; Signal to noise ratio is respectively 6,7,7,14, all greater than 3, so probe 1,3,4,18 is judged to the positive, illustrates 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, illustrates 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 to 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, the result as shown in Figure 4, 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; Signal to noise ratio 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; Signal to noise ratio 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; Signal to noise ratio 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 is
Upstream primer: 5 '-CGGGACTTACTTGTGGTTCC-3 ';
Downstream primer: 5 '-AGGGAACAAAACTAAGGTTCCAC-3 '.
The result that PCR detects as shown in Figure 5,1:10
0Dilution; 2:10
1Dilution; 3:10
2Dilution; 4:10
3Dilution; 5: blank; M:Marker DL2000, as can be seen, 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 (8)
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 described probe 1-probe 19 respectively is the sequence 1-19 in the sequence table;
Described pospiviroid viroid is chrysanthemum stunt viroid or tomato planto macho viroid.
2. the gene chip of an examination pospiviroid viroid is for being fixed on the gene chip that the sheet primary surface obtains with the described probe groups of claim 1.
3. gene chip according to claim 2, it is characterized in that: described base is the aldehyde radical glass chip; Described pospiviroid viroid is chrysanthemum stunt viroid or tomato planto macho viroid.
4. the test kit of an examination pospiviroid viroid comprises claim 2 or 3 described gene chips.
5. test kit according to claim 4 is characterized in that: it is right that described test kit also comprises for the primer of the cDNA of the described pospiviroid viroid of amplification, described primer to be specially primer to 1 or primer to 2;
Described 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;
Described 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;
Described pospiviroid viroid is chrysanthemum stunt viroid or tomato planto macho viroid.
6. the described probe groups of claim 1, claim 2 or 3 described gene chips, claim 4 or 5 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;
Described pospiviroid viroid is chrysanthemum stunt viroid or tomato planto macho viroid; The described measuring plants for the treatment of is chrysanthemum or tomato.
7. the method for an examination or auxiliary examination plant infection pospiviroid to be measured viroid comprises the steps:
1) cDNA that 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 and claim 2 or 3 described gene chips 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 described probe on the described gene chip be not less than 600 and signal to noise ratio be not less than 3.0, then plant infection to be measured or candidate's potato-infecting fusiform tubers Tobamovirus viroid;
Described pospiviroid viroid, described primer to the described measuring plants for the treatment of be following 1) or 2):
1) described pospiviroid viroid is chrysanthemum stunt viroid, and described primer is to being that described primer is to 1; The described measuring plants for the treatment of is chrysanthemum;
2) described pospiviroid viroid is tomato planto macho viroid, and described primer is to being that described primer is to 2; The described measuring plants for the treatment of is tomato.
8. method according to claim 7 is characterized in that:
In the step 1), described to be labeled as with described cDNA be template, to carrying out pcr amplification, obtains the PCR product with the described primer in the described test kit of claim 5, more described PCR product carried out the Klenow enzyme labelling, obtains the mark after product;
Step 2) in, the temperature of described hybridization is 42 ℃, and the time of described hybridization is 12h;
In described step 2) also comprise the step that the back chip of described hybridization is washed before back and the step 3);
Described at least one described probe is any in the above-mentioned probe groups;
The described blade that is organized as.
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| 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 |
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