CN102732641B - Chip for screening Pelamoviroid and application thereof - Google Patents
Chip for screening Pelamoviroid and application thereof Download PDFInfo
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Abstract
The invention discloses a chip for screening Pelamoviroid and an application thereof. The invention provides probes for identification or assistant identification of Pelamoviroid, comprising nine probes, nucleotide sequences of the nine probs are respectively the sequence 1-9 in the sequence table. The invention also provides a gene chip for screening Pelamoviroid, and the gene chip is obtained by fixing the probes on the surface of a film base. Experiments of the invention prove that by analyzing the nucleotide sequence of Pelamoviroid through using bioinformatics methods, the compatible probes are designed, and the results of verifying standard viroid samples prove that the probes have good effect. The chip for screening Pelamoviroid disclosed herein can be used for quarantine and identification of Pelamoviroid.
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 pelamoviroid viroid.
Background technology
Pelamoviroid viroid (Pelamoviroid) belongs to avocado sunspot viroid section (Avsunviroidae), according to (the International Committee on Taxonomy of Viruses of ICTV, ICTV) the 8th subseries report, this genus comprises peach floral leaf viroid (the Peach latent mosaic viroid that hides, PLMVd) and chrysanthemum chlorotic mottle virus (Chrysanthemum chlorotic mottle viroid, CChMVd).The peach floral leaf viroid that hides is distributed in countries such as South and North America, Australia, Austria, China, Croatia, France, Greece, Japan, Morocco, Romania, Serbia, Spain and Tunisia, it is one of former to be confirmed as systemic infection venereal disease the most dangerous on the fruit tree in many countries, mainly infects tone fruit trees such as peach, Lee, apricot.Infect peach and generally show as floral leaf, retarded germination causes early ageing even death when serious, causes serious economy loss.This viroid can be infected through grafting and black peach aphid, with the asexual propagation material long-distance communications, in peach pierre and seedling allocation and transportation process, is easy to cause the diffusion of this virus to spread.Chrysanthemum chlorotic mottle virus mainly is distributed in Denmark, France, India, Japan, Korea S and China, infects chrysanthemum etc. and makes the plant leaf of being injured present mottling, and the back is chlorisis fully; Growth retardation is downgraded.
Along with the expansion of China's foreign exchanges, international and domestic germplasm exchange is frequent day by day, strengthens quarantine and examination, will play important effect to the propagation of blocking this genus viroid (especially virulent strain department).Setting up effective examination technology is the key that improves viroid quarantine and examination effect, but the method that is used for this genus viroid detection at present in the world mainly contains woody plant indicator biological assay, RT-PCR, LAMP and making nucleic acid molecular hybridization technology, these technology can only belong to known class virus to this and carry out specific detection, 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 pelamoviroid viroid.
The probe groups of evaluation provided by the invention or assistant identification pelamoviroid viroid, by probe 1-probe 9 totally 9 probes form, the nucleotide sequence of described probe 1-probe 9 respectively is the sequence 1-9 in the sequence table.
In the above-mentioned probe, described pelamoviroid viroid is hide floral leaf viroid or chrysanthemum chlorotic mottle virus of peach.
Another object of the present invention provides the gene chip of a kind of examination pelamoviroid viroid.
The gene chip of examination pelamoviroid 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 Boao Biological Co., Ltd
The microarray substrate, catalog number: 420022.
In the said gene chip, described pelamoviroid viroid is hide floral leaf viroid or chrysanthemum chlorotic mottle virus of peach.
The 3rd purpose of the present invention provides the test kit of a kind of examination pelamoviroid viroid.
Test kit provided by the invention comprises above-mentioned gene chip.
The mentioned reagent box also comprise for the amplification described pelamoviroid viroid cDNA primer right, described primer to be specially primer to 1 or primer to 2;
Described primer is formed (be used for amplification peach hide floral leaf viroid) to 1 by the dna molecular shown in the sequence 11 in sequence in the sequence table 10 and the sequence table;
Described primer is formed (being used for the amplification chrysanthemum chlorotic mottle virus) to 2 by the dna molecular shown in the sequence 13 in sequence in the sequence table 12 and the sequence table;
Described pelamoviroid viroid is hide floral leaf viroid or chrysanthemum chlorotic mottle virus of peach.
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 pelamoviroid viroid;
2) preparation is identified or assistant identification pelamoviroid viroid product;
3) evaluation or assistant identification plant infection pelamoviroid to be measured viroid;
4) preparation is identified or assistant identification plant infection pelamoviroid to be measured viroid product.
In the above-mentioned application, described pelamoviroid viroid is specially peach hide floral leaf viroid or chrysanthemum chlorotic mottle virus; The described measuring plants for the treatment of is peach or chrysanthemum.
The 4th purpose of the present invention provides the method for a kind of examination or auxiliary examination plant infection pelamoviroid 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 infect the pelamoviroid 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 pelamoviroid viroid, described primer to the described measuring plants following 1 for the treatment of) or 2):
1) described pelamoviroid viroid is the peach floral leaf viroid that hides, and described primer is to being that described primer is to 1; The described measuring plants for the treatment of is peach;
2) described pelamoviroid viroid is chrysanthemum chlorotic mottle virus, and described primer is to being that described primer is to 2; The described measuring plants for the treatment of is chrysanthemum.
Of the present invention experiment showed, the probe in the examination pelamoviroid viroid chip provided by the invention have the highly compatible on the pelamoviroid viroid genus level and 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 pelamoviroid 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.Genus chip of the present invention can be used for the quarantine of pelamoviroid viroid to be identified.
Description of drawings
Fig. 1 is pelamoviroid viroid examination chip probe dot matrix synoptic diagram (10 * 12)
Fig. 2 is for using the result of chrysanthemum chlorotic mottle virus sample checking pelamoviroid viroid examination chip
Fig. 3 is examination chip sensitivity detected result
Fig. 4 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 pelamoviroid 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 9 probes (probe 1-probe 9) of pelamoviroid viroid according to mentioned above principle, its nucleotide sequence is followed successively by respectively shown in the sequence 1-9 in the sequence table.
Can above-mentioned 9 probes of synthetic.
2, the preparation of chip
Above-mentioned 19 probes that obtain are used point sample damping fluid (Boao Biological Co., Ltd respectively
The chip sampling liquid, catalog number: 440010) dissolving, concentration is 50 μ M, every probe laterally repeats 3 points in aldehyde radical glass chip chip (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 pelamoviroid viroid.
The chip of examination pelamoviroid viroid as shown in Figure 1,1-9 is the corresponding sequence 1-9 of pelamoviroid viroid examination chip probe 1-9(among Fig. 1), Hex is 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 pelamoviroid viroid
One, examination chip detection sample
1, the total RNA of for detection of sample extracts
1) gets and infect hide peach blade (the peach floral leaf viroid latin name Peach latent mosaic viroid that hides of floral leaf viroid of peach, be documented in: peach hide clone and the sequential analysis .2005 of floral leaf viroid Chinese pathogenic strain P3, Plant Pathology 35 (4): 300-304., the public can obtain from China Inst. of Quarantine Inspection Sciences.) and the chrysanthemum blade of chrysanthemum chlorotic mottle virus (chrysanthemum chlorotic mottle virus Chrysanthemum chlorotic mottle viroid. is called for short ATCC available from American type culture collection, PV-120) each 0.1g sample adds 1mL grinding buffer solution [Na
2HPO
412H
2O 3.58g, KH
2PO
40.27g, NaCl 8g, KCl 0.2g, Na
2SO
31.3g, polyvinylpyrrolidone (PVP) MW 24-40 000 20g, NaN
30.2g, chicken egg protein (pow dered egg album in) 2.0g, Tween-20 20.0mL, be dissolved in the 1000mL deionized water, regulating pH value is 7.5], grind evenly in the back immigration 1.5mL centrifuge tube, the centrifugal 5min of 6000r/min gets supernatant;
2) in the PCR pipe with washed with de-ionized water nanometer magnetic bead 2 times, discard the supernatant that adds 100 μ L behind the ionized water, mixing is placed under the room temperature in conjunction with 10min, puts upside down the mixing sample therebetween for several times.Abandon supernatant behind the magnet absorption nanometer magnetic bead, add 200 μ L PBS[NaCl 8.0g, Na
2HPO
4, 1.15g, KH
2PO
40.2g, KCl 0.2g, add the 900mL dissolved in distilled water, regulate pH value to 7.4, adding distil water is settled to 1L] clean nanometer magnetic bead;
3) after the cleaning, add 50 μ L ddH
2O is the suspended nano magnetic bead again, handles 5min for 95 ℃; Draw supernatant liquor behind the magnet absorption nanometer magnetic bead and be RNA ,-20 ℃ of preservations are standby.
2, sample mark and hybridization
The above-mentioned 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 hides the primer of floral leaf viroid or chrysanthemum chlorotic mottle virus to forming by the peach that is respectively applied to increase.
Above-mentionedly hide the primer of floral leaf viroid to comprising for the amplification peach
Upstream primer: 5 '-CCAGGTAACGCCGTAGAAACTG-3 ' (sequence 10);
Downstream primer: 5 '-ATCACACCCTCCTCGGAACCAA-3 ' (sequence 11);
Its PCR response procedures is: 95 ℃ of 3min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; 30 circulations; 72 ℃ of 10min.
Above-mentioned for the amplification chrysanthemum chlorotic mottle virus primer to comprising
Upstream primer: 5 '-GGCACCTGATGTCGGTGT-3 ' (sequence 12);
Downstream primer: 5 '-GACCTCTTGGGGGTTTCAAAC-3 ' (sequence 13);
Its PCR response procedures is: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s; 30 circulations; 72 ℃ of 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 L SSC(final concentrations 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 cover glass is built, and 42 ℃ of water-baths hybridization is spent the night (12h), the chip after obtaining respectively hybridizing (peach hide floral leaf viroid) and hybridize after chip (chrysanthemum chlorotic mottle virus).
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 (peach hide floral leaf viroid), chip to be detected (chrysanthemum chlorotic mottle virus) respectively.
Place scanner to carry out scanning analysis above-mentioned chip to be detected (peach hide floral leaf viroid), chip to be detected (chrysanthemum chlorotic mottle virus); 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 pelamoviroid viroid infects); The signal value of probe＜600 and signal to noise ratio＜2 are judged to feminine gender (not infecting for the pelamoviroid viroid); All the other situations are judged to suspicious, need repeated authentication.
The probe of Fig. 2 is arranged identical with Fig. 1, and positive probe is 1-9.
The checking result of chrysanthemum chlorotic mottle virus as shown in Figure 2, the signal value of probe 7,8,9 positions is respectively 1992,20970,18483, all greater than 600; Signal to noise ratio is respectively 6,52,48, all greater than 3, illustrates that the present invention can detect the chrysanthemum chlorotic mottle virus of pelamoviroid 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, examination chip and PCR detection sensitivity are relatively
Accurately take by weighing 0.1g and infect the hide peach tissue of floral leaf viroid of peach, extract total RNA, be respectively applied to pelamoviroid viroid examination chip detection and PCR detection, both used RNA all carry out 10
1With 10
2Times gradient dilution.
The method of chip detection is with above-mentioned one, the result as shown in Figure 3, the probe of Fig. 3 is arranged identical with Fig. 1, wherein A:10
1Dilution; B:10
2Dilution.The signal value of A middle probe 1,2 positions is respectively 3913,1004, all greater than 600; Signal to noise ratio is 8,3 respectively, all 〉=3; So probe 1,2 is judged to the positive.The signal value of B middle probe 1 position is 1531, greater than 600; Signal to noise ratio is 4; Can judge that probe 1 is positive.Therefore, can to detect the highly diluted multiple of object to be checked be 10 to pelamoviroid viroid examination chip
2
The primer that PCR detects is
Upstream primer: 5 '-CCAGGTAACGCCGTAGAAACTG-3 ';
Downstream primer: 5 '-ATCACACCCTCCTCGGAACCAA-3 '.
The result that PCR detects as shown in Figure 4,1:10
1Dilution; 2:10
2Dilution; 3: blank; M:Marker DL2000, as can be seen, 10
1With 10
2The dilution template all obtains the product (the 1-337 position Nucleotide of Genbank GU290549) of 337bp, can detect object 10 to be checked
2Extension rate.
Therefore, pelamoviroid viroid examination chip sensitivity is suitable with PCR method sensitivity.
Claims (5)
1. the test kit of an examination pelamoviroid viroid comprises the gene chip of examination pelamoviroid viroid; Described gene chip is for probe 1-probe 9 totally 9 gene chips that probe stationary obtains at the sheet primary surface; Described test kit also comprise for the amplification described pelamoviroid viroid cDNA primer right, 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 11 in sequence in the sequence table 10 and the sequence table 1;
Described primer is made up of the dna molecular shown in the sequence 13 in sequence in the sequence table 12 and the sequence table 2.
2. test kit according to claim 1, it is characterized in that: described base is the aldehyde radical glass chip.
3. claim 1 or 2 described test kits are following 1) or 2) in application:
1) evaluation or assistant identification pelamoviroid viroid;
2) preparation is identified or assistant identification pelamoviroid viroid product;
Described pelamoviroid viroid, described primer are to following 1) or 2):
1) described pelamoviroid viroid is the peach floral leaf viroid that hides, and described primer is to being that described primer is to 1;
2) described pelamoviroid viroid is chrysanthemum chlorotic mottle virus, and described primer is to being that described primer is to 2.
4. claim 1 or 2 described test kits are following 3) or 4) in application
3) evaluation or assistant identification plant infection pelamoviroid to be measured viroid;
4) preparation is identified or assistant identification plant infection pelamoviroid to be measured viroid product;
Described pelamoviroid viroid, described primer to the described measuring plants following 1 for the treatment of) or 2):
1) described pelamoviroid viroid is the peach floral leaf viroid that hides, described primer to be described primer to 1, the described measuring plants for the treatment of is peach;
2) described pelamoviroid viroid is chrysanthemum chlorotic mottle virus, described primer to be described primer to 2, the described measuring plants for the treatment of is chrysanthemum.
5. the method for an examination or auxiliary examination plant infection pelamoviroid 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; 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 described test kit of claim 1, more described PCR product is carried out the Klenow enzyme labelling, obtain the mark after product;
2) the mark after product that step 1) is obtained and the gene chip described in the claim 1 are hybridized, and obtain hybridizing the back chip; The temperature of described hybridization is 42 ℃, and the time of described hybridization is 12h;
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 infect the pelamoviroid viroid;
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 pelamoviroid viroid, described primer to the described measuring plants following 1 for the treatment of) or 2):
1) described pelamoviroid viroid is the peach floral leaf viroid that hides, and described primer is to being that described primer is to 1; The described measuring plants for the treatment of is peach;
2) described pelamoviroid viroid is chrysanthemum chlorotic mottle virus, and described primer is to being that described primer is to 2; The described measuring plants for the treatment of is chrysanthemum.
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| CN101451164A (en) * | 2008-12-19 | 2009-06-10 | 北京市农林科学院 | Method for detecting chrysanthemum chlorotic mottle virus |
| CN102242225B (en) * | 2011-07-20 | 2013-05-08 | 南京农业大学 | Double RT-PCR (reverse transcription-polymerase chain reaction) detection method of CVB (chrysanthemum virus B) and CChMVd (chrysanthemum chlorotic mottle viroid) |
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