CN102719563B - Chip for screening coleus scutellarioides viroid and application of chip - Google Patents
Chip for screening coleus scutellarioides viroid and application of chip Download PDFInfo
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- CN102719563B CN102719563B CN201210193802.XA CN201210193802A CN102719563B CN 102719563 B CN102719563 B CN 102719563B CN 201210193802 A CN201210193802 A CN 201210193802A CN 102719563 B CN102719563 B CN 102719563B
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Abstract
The invention discloses a chip for screening coleus scutellarioides viroid and application of the chip. A probe group identifying or assisting in identifying the scutellarioides viroid consists of 6 probes, and the nucleotide sequences of the 6 probes are the sequences from 1 to 6 in a sequence table respectively. The invention also provides a gene chip for screening the scutellarioides viroid, which is obtained by fixing the probes on the surface of a chip base. The experiment proves that the nucleotide sequence of the scutellarioides 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 scutellarioides viroid disclosed by the invention can be applied to the quarantine identification of the scutellarioides viroid.
Description
Technical field
The present invention relates to information biology and biological quarantine identification technical field, particularly relate to chip and application thereof that examination five colours Soviet Union viroid belongs to viroid.
Background technology
Five colours Soviet Union viroid belongs to viroid (Coleviroid) and belongs to pospiviroidae (Pospiviroidae), find that viroid has 6 kinds (Coleus blumei viroid1-6, CbVd-1 ~ CbVd-6) and the five colours reported are revived up to now.Five colours Soviet Union viroid finds in Brazil at first, and rear Germany, Canada, Japan, India, Indonesia and China report in succession.Five colours Soviet Union infects and to there will be yellow or the symptom such as mottled after viroid, and minority has dwarfism to infect.
This genus viroid, by mechanical inoculation, also can pass through seed dispersal.Along with the increase of domestic and international plants and plant product trade, this genus occurs that the frequency of " newly " viroid is more and more higher, this " newly " is mainly manifested in: novel species possible in belonging to, as two coleus blumei viroid 1 novel species (CbVd-5, CbVd-6) that Hou Wanying etc. finds for 2009; Find new host and plant interior possible variation; These 3 kinds of situations all can cause the new viroid occurring in this genus having quarantine risk.These potential new viroids are needed to set up broad spectrum examination technology, to reach the examination of (primary first-order equation can detect all viroids of this genus in same sample), wide spectrum (can not occur undetected because of variation etc.) fast.And existing technology is as low in biological characteristis, forward and reverse polyacrylamide gel electrophoresis and RT-PCR isoflux, a kind of viroid in sample once can only be detected; And belong to specific detection, cannot detect large and that variant sites is many viroid that makes a variation.In order to address these problems, domestic and international researchist has carried out the research of broad spectrum high-throughput examination technology, and result shows that the categorization levels from pathogen is higher carries out examination, effectively can improve the Effect of screening of neopathy pathogenic microorganism; This technology in conjunction with the high-throughput feature of chip, thus can improve the efficiency of examination greatly simultaneously.If adopt this examination chip technology to carry out viroid Quarantine control at seed stage, utilize and obtain healthy plant and carry out vegetative propagation again, just can be the propagation that controls five colours Soviet Union viroid and produce nontoxic coleus and create favorable conditions.
Summary of the invention
An object of the present invention is to provide a kind of qualification or assistant identification five colours Soviet Union viroid belongs to the probe groups of viroid.
Qualification provided by the invention or assistant identification five colours Soviet Union viroid belong to the probe groups of viroid, by probe 1-probe 6 totally 6 probes form, the nucleotide sequence of described probe 1-probe 6 respectively is the sequence 1-6 in sequence table.
In above-mentioned probe, it is CbVd-1, CbVd-2, CbVd-3, CbVd-4, CbVd-5 or CbVd-6 that described five colours Soviet Union viroid belongs to viroid.
Another object of the present invention is to provide the gene chip that a kind of examination five colours Soviet Union viroid belongs to viroid.
Examination five colours Soviet Union provided by the invention viroid belongs to the gene chip of viroid, for above-mentioned probe being fixed on the gene chip that sheet primary surface obtains.
In said gene chip, described base is aldehyde radical glass chip, and what adopt in an embodiment of the present invention is the brilliant core of Boao Biological Co., Ltd
microassay substrate, catalog number: 420022.
In said gene chip, it is CbVd-1, CbVd-2, CbVd-3, CbVd-4, CbVd-5 or CbVd-6 that described five colours Soviet Union viroid belongs to viroid.
3rd object of the present invention is to provide the test kit that a kind of examination five colours Soviet Union viroid belongs to viroid.
Test kit provided by the invention, comprises above-mentioned gene chip.
Mentioned reagent box also comprises the primer pair belonging to the cDNA of viroid for described five colours Soviet Union viroid of increasing, and described primer pair is specifically made up of the DNA molecular shown in sequence 8 in sequence in sequence table 7 and sequence table;
It is CbVd-1 that described five colours Soviet Union viroid belongs to viroid.
Above-mentioned probe groups, said gene chip or mentioned reagent box are following 1)-4) in application, be also the scope of protection of the invention:
1) qualification or assistant identification five colours Soviet Union viroid belong to viroid;
2) characterization or assistant identification five colours Soviet Union viroid belong to viroid product;
3) qualification or the assistant identification plant infection five colours to be measured Soviet Union viroid belong to viroid;
4) characterization or the assistant identification plant infection five colours to be measured Soviet Union viroid belong to viroid product.
In above-mentioned application, described in treat that measuring plants is coleus; It is CbVd-1 that described five colours Soviet Union viroid belongs to viroid.
4th object of the present invention is to provide a kind of method that examination or auxiliary examination plant infection to be measured five colours Soviet Union viroid belong to viroid.
Method provided by the invention, comprises the steps:
1) cDNA of the tissue treating measuring plants is marked, obtain marking after product;
2) mark after product step 1) obtained and above-mentioned gene chip are hybridized, and obtain hybridizing rear chip;
3) by step 2) chip scanning after the hybridization that obtains,
If the signal absolute value of probe described on described gene chip at least one is not less than 600 and signal to noise ratio is not less than 3.0, then plant infection to be measured or candidate infect the five colours Soviet Union viroid belong to viroid.
In aforesaid method, in step 1), described in be labeled as with described cDNA for template, carry out pcr amplification with the described primer pair in above-mentioned test kit, obtain PCR primer, more described PCR primer is carried out Klenow enzyme labelling, obtain mark after product;
Step 2) in, the temperature of described hybridization is 42 DEG C, and the time of described hybridization is 12h;
In described step 2) also comprise the step of being carried out by chip after described hybridization washing afterwards with before step 3);
Probe described in described at least one is any one in above-mentioned probe groups;
Describedly be organized as blade; Describedly treat that measuring plants is coleus;
It is CbVd-1 that described five colours Soviet Union viroid belongs to viroid.
Experiment of the present invention proves, the examination five colours Soviet Union provided by the invention viroid probe belonged in viroid chip has five colours Soviet Union viroid and belongs to viroid and belong to the highly compatible in level and specificity in belonging to, and 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 to analyze the nucleotide sequence that five colours Soviet Union viroid belongs to viroid, and devise the compatible probe of this genus, standard class viral sample the result proves that probe is respond well.Five colours Soviet Union of the present invention viroid belongs to viroid chip and can be used for five colours Soviet Union viroid and belong to the quarantine identification of viroid.
Accompanying drawing explanation
Fig. 1 is that five colours Soviet Union viroid belongs to viroid examination chip probe dot matrix schematic diagram (10 × 12)
Fig. 2 is the specificity that the viroid 1 sample checking of application five colours Soviet Union belongs to examination chip
Fig. 3 is the sensitivity results that the viroid 1 sample checking of application five colours Soviet Union belongs to examination chip
Fig. 4 is PCR sensitivity technique result
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, examination five colours Soviet Union viroid belong to the preparation of the chip of viroid
1, the design of level highly compatible oligonucleotide probe is belonged to
Download viroid from US National Biotechnology Information center (NCBI) and the international virusology classification council (ICTV) database and belong to full-length genome and nucleotide sequence; Removal more than 90% length and other sequence have the nucleotide sequence of 95% similarity; Using 5 bases as interval, extract the nucleotide sequence of all 40mer continuously; With 40%≤GC content≤60%, single base contents≤50%, repeat base number≤4 continuously, and be that standard is screened the nucleotide sequence extracted without the hairpin structure being greater than 6 bases, and in ncbi database, carry out tetraploid rice to ensure the specificity of institute's acquisition probe.
Devise five colours Soviet Union viroid according to mentioned above principle and belong to 6 probes of viroid, its nucleotide sequence is followed successively by shown in the sequence 1-6 in sequence table respectively.
Can above-mentioned 6 probes of synthetic.
2, the preparation of chip
Spotting buffer (the brilliant core of Boao Biological Co., Ltd used respectively by 6 probes obtained above-mentioned 1
chip sampling liquid, catalog number: 440010) dissolve, concentration is 50 μMs, every bar probe laterally repeats 3 points at aldehyde radical glass chip chip (the brilliant core of Boao Biological Co., Ltd
microassay substrate, catalog number: 420022), often about 0.25nL, spot diameter about 180 μm, dot spacing is 300 μm, and the standard variance of point sample uniformity coefficient is 15%.Chip point is had the one side of probe hydration 10s on 65 DEG C of water-baths, chip is 3cm, in atmosphere natural drying at room temperature apart from water surface distance, is carrying out a hydration.Point there is is the one side of probe upwards, is placed on 250mJ in UV-crosslinked instrument and is cross-linked.Chip is placed on 42 DEG C of preheatings, 0.5%SDS cleans 10min.Chip is transferred in 42 DEG C of pre-hot distilled waters and clean 2min.Chip is placed in 50mL conical centrifuge tube, the centrifugal 1min of 2000rpm, to remove the liquid of chip surface, obtains the chip that examination five colours Soviet Union viroid belongs to viroid.
Examination five colours Soviet Union viroid belongs to the chip of viroid as shown in Figure 1, and in Fig. 1,1-6 is that five colours Soviet Union viroid belongs to the corresponding sequence 1-6 of viroid examination chip probe 1-6(), Hex is that chip fixes positive quality control, and PC is for hybridizing positive quality control, and NC is the negative Quality Control of hybridization.
Embodiment 2, examination five colours Soviet Union viroid belong to the application of the chip of viroid
One, examination chip detection sample
1, the sample Total RNAs extraction for detecting
1) the coleus tissue (five colours Soviet Union viroid 1 latin name Coleus blumeiviroid1(is called for short CbVd-1) infecting five colours Soviet Union viroid 1 is got, be documented in: the detection of Chinese coleus blumei viroid 1 and molecular biological characteristics research .2006 thereof, Plant Pathology 36 (3): 266-231., the public can obtain from China Inst. of Quarantine Inspection Sciences; Coleus is documented in: Beijing, the Efficiency in Buildings in Tianjin Area coleus blumei viroid 1 investigation of epidemic situation and detection, before Soviet Union, the clear Lee's generation of the auspicious Wang Hong of rich Song's method is visited, " plant protection " the 6th phase in 2006, the public can obtain from China Inst. of Quarantine Inspection Sciences) 0.1g sample, powdered by liquid nitrogen grinding, move in the 1.5mL centrifuge tube of sterilizing, then add the Trizol reagent of 1mL, concuss shakes up;
2) 4 DEG C, the centrifugal 10min of 12000rpm, proceeds in a new 1.5mL centrifuge tube by supernatant liquor;
3) add 0.5mL chloroform, thermal agitation 15s, room temperature places 15min; 4 DEG C, the centrifugal 15min of 12000rpm;
4) by upper water phase transition in new 1.5mL centrifuge tube, add equal-volume Virahol, put upside down mixing, room temperature keep 15min;
5) 4 DEG C, the centrifugal 10min of 12000rpm; Outwell supernatant liquor, add the cold washing with alcohol precipitation of 75%, then 4 DEG C, the centrifugal 5min of 12000rpm, abandons ethanol;
7), under precipitating room temperature after abundant drying, be dissolved in 40 μ L distilled waters (DEPC process) ,-20 DEG C save backup, and obtain RNA.
2, sample mark and hybridization
RNA reverse transcription obtained above is obtained cDNA.
Pcr amplification, namely adds cDNA product 2 μ L, upstream primer 0.5 μ L(final concentration is 0.5mmol/L in the reaction tubes of 0.2mL), 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, then increases according to PCR response procedures.
Upstream and downstream primer is made up of the primer for five colours Soviet Union viroid 1 of increasing,
Upstream primer: 5 '-
gGATCC
aGCGCTGCAACGGAATCCA-3 ' (sequence 7);
Downstream primer: 5 '-
gGATCC
gCCAGGGAACCCAGGTAAG-3 ' (sequence 8);
In above-mentioned primer, in square frame, base is protection base, and italicized bases is restriction enzyme site BamH I base, and underscore base is five colours Soviet Union viroid sequence
Its PCR response procedures is: 94 DEG C of 5min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 35 circulations; 72 DEG C of 7min.
Klenow enzyme labelling system is 25 μ L, namely in the PCR reaction tubes of 0.2mL, adds PCR primer 5 μ L, 9N random primer (invitrogen, Cat.No.48190-011) (100 μm of ol/L) 2 μ L and H
2o 12 μ L, 95 DEG C of sex change 3min, ice bath 5min.Then in reaction tubes, add 10 × Klenow enzyme buffer liquid 2.5 μ L, dNTP(2.5mmol/L) 2 μ L, cy3-dCTP 0.5 μ L(Amersham, Cat.No.PA 53021 final concentration is 5nmol/L), klenow enzyme (5U/ μ L) 1 μ L.37 DEG C of reaction 1.5h, 70 DEG C of sex change 5min, ice bath 5min, obtain marking sample.
Hybridization: system is 16 μ L, comprise 2.4 μ L SSC(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 DEG C of sex change 3min, ice bath 5min, brief centrifugation.Be added to by hybridization solution on chip, cover glass is built, 42 DEG C of water-bath hybridized overnight (12h), obtains the chip after hybridizing (five colours Soviet Union viroid 1) respectively.
3, wash, scan
Cleaning system and program as follows:
First washing lotion I, II are placed in microwave oven and are preheating to 42 DEG C, transfer in cleaning box.After hybridization terminates, transferred to by the chip after hybridization and hold in the cleaning box of washing lotion, hybridization surface upwards, is placed on horizontal shaker and slowly cleans.After chip cleaning, be placed in 50mL conical centrifuge tube, the centrifugal 1min of 2000rpm, the liquid of removing chip surface, obtain chip to be detected (five colours Soviet Union viroid 1) respectively.
Above-mentioned chip to be detected (five colours Soviet Union viroid 1) is placed in scanner and carries out scanning analysis; PMT is set to 900, obtains the data such as each point fluorescence intensity and background intensity.
Use Bo Ao biotech firm LuxScan 3.0 chip scanner 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 all signal value medians and background value median in image corresponding points.
If signal value >=600 of probe described at least one and signal to noise ratio >=3, be judged to the positive (infecting for five colours Soviet Union viroid belongs to viroid); The signal value < 600 of probe and signal to noise ratio < 2, be judged to feminine gender (not infecting for five colours Soviet Union viroid belongs to viroid); All the other situations are judged to suspicious, need repeated authentication.
The probe arrangement of Fig. 2 is identical with Fig. 1, and it is 1-6 that five colours Soviet Union viroid belongs to viroid probe.
As shown in Figure 2, the signal value of probe 6 position is 4349 to the result of five colours Soviet Union viroid 1, is greater than 600; Signal to noise ratio is 9, is greater than 3, illustrate the present invention can detect the five colours Soviet Union viroid belong to viroid the five colours Soviet Union viroid 1;
The fixing positive quality control of said chip, hybridize positive quality control and hybridize negative Quality Control performance good, standard model hybridization signal is strong, illustrates that the chip examination program of probe and the foundation designed has good working effect.
Two, examination chip compares with PCR detection sensitivity
Accurately take the coleus tissue that 0.1g infection multicolored Soviet Union viroid is infected, extract total serum IgE, be respectively used to multicolored viroid of reviving and belong to viroid examination chip detection and PCR detection, both RNA used all carry out 10
0, 10
1with 10
2times gradient dilution.
The method of chip detection is with above-mentioned one, and as shown in Figure 3, the probe arrangement of Fig. 3 is identical with Fig. 1, wherein A:10 for result
1dilution; B:10
2dilution.The signal value of A middle probe 6 position is 1471, and signal to noise ratio is 4, can judge that probe 6 is as positive.B no signal.Therefore, five colours Soviet Union viroid belongs to viroid examination chip and object 10 to be checked can be detected
1extension rate.
The primer that PCR detects is
Upstream primer: 5 '-
gGATCC
aGCGCTGCAACGGAATCCA-3 ';
Downstream primer: 5 '-
gGATCC
gCCAGGGAACCCAGGTAAG-3 ',
In above-mentioned primer, in square frame, base is protection base, and italicized bases is restriction enzyme site BamH I base, and underscore base is five colours Soviet Union viroid sequence.
PCR detect result as shown in Figure 4,1:10
0dilution; 2:10
1dilution; 3:10
2dilution; M:MarkerDL2000, can find out, 10
0with 10
1dilution template all obtains the product (the 1-250 position Nucleotide of Genbank DQ178396) of 250bp, can detect 10
1extension rate.
Therefore, five colours Soviet Union viroid to belong to viroid examination chip detection sensitivity suitable with PCR method sensitivity.
Claims (9)
1. a qualification or assistant identification five colours Soviet Union viroid belong to the probe groups of viroid, by probe 1-probe 6 totally 6 probes form, the nucleotide sequence of described probe 1-probe 6 respectively is the sequence 1-6 in sequence table, and it is CbVd-1 that described five colours Soviet Union viroid belongs to viroid.
2. examination five colours Soviet Union viroid belongs to a gene chip for viroid, for probe groups according to claim 1 being fixed on the gene chip that sheet primary surface obtains; It is CbVd-1 that described five colours Soviet Union viroid belongs to viroid.
3. gene chip according to claim 2, is characterized in that: described base is aldehyde radical glass chip.
4. examination five colours Soviet Union viroid belongs to a test kit for viroid, comprises the gene chip described in Claims 2 or 3, and it is CbVd-1 that described five colours Soviet Union viroid belongs to viroid.
5. test kit according to claim 4, it is characterized in that: described test kit also comprises the primer pair belonging to the cDNA of viroid for described five colours Soviet Union viroid of increasing, and described primer pair is specifically made up of the DNA molecular shown in sequence 8 in sequence in sequence table 7 and sequence table.
6. the gene chip described in probe groups, Claims 2 or 3, the test kit described in claim 4 or 5 described in claim 1 are following 1)-4) in application:
1) qualification or assistant identification five colours Soviet Union viroid belong to viroid;
2) characterization or assistant identification five colours Soviet Union viroid belong to viroid product;
3) qualification or the assistant identification plant infection five colours to be measured Soviet Union viroid belong to viroid;
4) characterization or the assistant identification plant infection five colours to be measured Soviet Union viroid belong to viroid product;
It is CbVd-1 that described five colours Soviet Union viroid belongs to viroid.
7. application according to claim 6, is characterized in that: described in treat that measuring plants is coleus.
8. examination or the auxiliary examination plant infection five colours to be measured Soviet Union viroid belong to a method for viroid, comprise the steps:
1) cDNA of the tissue treating measuring plants is marked, obtain marking after product;
2) by step 1) the mark after product that obtains and the gene chip described in Claims 2 or 3 hybridize, obtain hybridizing rear chip;
3) by step 2) chip scanning after the hybridization that obtains,
If the signal absolute value of probe described on described gene chip at least one is not less than 600 and signal to noise ratio is not less than 3.0, then plant infection to be measured or candidate infect the five colours Soviet Union viroid belong to viroid;
It is CbVd-1 that described five colours Soviet Union viroid belongs to viroid.
9. method according to claim 8, is characterized in that:
Step 1) in, described in be labeled as with described cDNA for template, carry out pcr amplification with the described primer pair in test kit according to claim 4, obtain PCR primer, more described PCR primer is carried out Klenow enzyme labelling, obtain mark after product;
Step 2) in, the temperature of described hybridization is 42 DEG C, and the time of described hybridization is 12h;
In described step 2) afterwards with step 3) frontly also comprise the step of being carried out by chip after described hybridization washing;
Probe described in described at least one is any one in described probe groups;
Describedly be organized as blade; Describedly treat that measuring plants is coleus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210193802.XA CN102719563B (en) | 2012-06-12 | 2012-06-12 | Chip for screening coleus scutellarioides viroid and application of chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210193802.XA CN102719563B (en) | 2012-06-12 | 2012-06-12 | Chip for screening coleus scutellarioides viroid and application of chip |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102719563A CN102719563A (en) | 2012-10-10 |
| CN102719563B true CN102719563B (en) | 2015-06-03 |
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| CN201210193802.XA Expired - Fee Related CN102719563B (en) | 2012-06-12 | 2012-06-12 | Chip for screening coleus scutellarioides viroid and application of chip |
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Non-Patent Citations (3)
| Title |
|---|
| 中国锦紫苏类病毒的检测及其分子生物学特征研究;苏前富等;《植物病理学报》;20061231;第36卷(第3期);226-231，图4 * |
| 北京_天津地区锦紫苏类病毒疫情调查与检测;苏前富等;《植物保护》;20061231;第32卷(第6期);116-119 * |
| 生物信息学及其在检验检疫中的应用;张永江;《湖北农业科学》;20070131;第46卷(第1期);152-154 * |
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