CN101857901A - Sepiella maindroni releasing and tagging chip and detection method - Google Patents
Sepiella maindroni releasing and tagging chip and detection method Download PDFInfo
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- CN101857901A CN101857901A CN 201010196300 CN201010196300A CN101857901A CN 101857901 A CN101857901 A CN 101857901A CN 201010196300 CN201010196300 CN 201010196300 CN 201010196300 A CN201010196300 A CN 201010196300A CN 101857901 A CN101857901 A CN 101857901A
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Abstract
The invention discloses a sepiella maindroni releasing and tagging chip and a detection method. The chip comprises a quality-control probe and a detection probe, wherein the quality-control probe comprises an immobilized positive probe, a hybrid positive probe, a positive contrast probe and a negative contrast probe which are artificially synthesized; the detection probe is the nucleotide sequence of two Tai-2009-1 or Zhou-2009-1 releasing groups with high specialty; the releasing and tagging chip can be used for identifying the group taking the two sepias as parents. The releasing and tagging chip has the advantages of bringing convenience to the research of the correlation between the sepiella maindroni releasing groups and descendants and particularly providing a precise data basis for the planning committee for the research of the growth and reproduction of the sepias, along with high tagging specialty and stability, little damage to the sepias and reduced analysis error rate of statistical data of each recaptured group.
Description
Technical field
The present invention relates to the Sepiella maindroni releasing mark, be specifically related to a kind of Sepiella maindroni releasing tagging chip and detection method.
Background technology
Sepiella maindroni (Sepiella maindroni, Rochebrune) be commonly called as cuttlefish, inkfish, cuttle fish, extra large cat etc., be subordinate to Mollusca, Cephalopoda, Decapoda, Sepiidae, because the habit and the anti-dried dew ability of cuttlefish ink-jet, be difficult to for a long time support, rare people sees cuttlefish alive, with live body study the biology of cuttlefish and grow seedlings, cultural technique is global problem.The inventor catches the Sepiella maindroni of 37 work by natural waters, not only supported cuttlefish, and pass through certain measure, make its normal spawning, carry out artificial breeding then and culture research, obtained breakthrough progress, manually bring out surplus the cuttlefish seed 7300, raise 1008 of finished product cuttlefishes, and the ingest habit of live bait of cuttlefish is progressively changed over the dead bait of ingesting, established solid basis for propagating cuttlefish artificially by domestication.
The mark of releasing is one of research living aquatic resources important method, and the mark of releasing mainly contains the method for marking and mark-on method two classes.The method of marking is to do mark at original organ of organism, and as excising fin whole or in part, this method is easy to be fast again, and shortcoming is that the organ of excision can continue regeneration under many circumstances.The mark-on method is that a special mark is attached on the organism, and the mark-on method is divided into two kinds of external tag method and internal tag methods again.The external tag method is the most widely used to be that sign board system is hung on the fish surface, and the internal tag method places biological intravital method with mark (passing as conductor).Aforesaid method is applied to all have bigger difficulty in the marker research of releasing of cuttlefish, at present, the releasing of cuttlefish remains to adopt cuts the simple marking of touching wrist, this method is differentiated the cuttlefish individuality at mark only, can't differentiate with this cuttlefish to be the mark of parent's colony, and touch the stability that wrist regeneration just loses mark, so it is there is bigger error in the data analysis after the recapture, also bigger to the cuttlefish damage.So press for the mark of releasing of a kind of highly sensitive of exploitation and low damage.
Summary of the invention
Technical problem to be solved by this invention provides a kind of Sepiella maindroni releasing tagging chip, this tagging chip of releasing can differentiate with this cuttlefish to be parent's colony, this mark specificity and good stability, few to the cuttlefish damage, the analysis of statistical data specific inaccuracy of each colony after the recapture is reduced.The present invention also provides the detection method of this chip, for the dependency of studying Sepiella maindroni releasing colony and filial generation provides convenience, especially provides than the accurate data foundation for research cuttlefish growth and breeding rule committee.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of Sepiella maindroni releasing tagging chip, comprise solid phase substrate through chemically modified, the surface cure of described solid phase substrate has Quality Control probe point sample and detection probes point sample, described Quality Control probe point sample comprises the positive probe point sample of immobilization, hybridize positive probe point sample, positive control probe point sample, negative control probe point sample and blank sampling liquid, the nucleotide sequence of described detection probes is shown in sequence table SEQ ID NO.1 and SEQ ID NO.2, the nucleotide sequence of the positive probe of described immobilization is shown in sequence table SEQ ID NO.3,5 ' of this nucleotide sequence is modified with by cy3 excited fluorescent element, the nucleotide sequence of the positive probe of described hybridization is shown in sequence table SEQ ID NO.4, the nucleotide sequence of described positive control probe shown in sequence table SEQ ID NO.5 to SEQ IDNO.8, the nucleotide sequence of described negative control probe as the sequence number of Operon company be shown in the H2NC000001 to H2NC000012 (company's site:
Http:// www.operon.com).
The positive probe of immobilization (Hex) is a synthetic, and this sequence compares through we blast and known biotinylated nucleic acid does not have great homology.When the last detection of chip experiment results of hybridization, this sequence should have stronger fluorescent signal under the excitation wavelength of cy3, shown in green on pcolor, shows that the nucleic acid on the chip fixedly is no problem.
Hybridizing positive probe selects for use the artificial design of the animal mitochondria 16SrRNA sequence with higher conservative property synthetic.
The nucleotide sequence that the positive control probe is and laboratory sample is irrelevant is used for the validity of detection chip system, is called outer ginseng; Sequence table SEQ ID NO.5-NO.8 of the present invention gets 4 nucleotide sequences of yeast synthetic as outer the participation in the election, does not have homology through these 4 sequences of comparative analysis and cuttlefish gene order.To join nucleotide sequence outward and be cloned into the PolyA-RNA that joins the nucleotide sequence correspondence in the carrier that has polynucleotide tail (PolyA) outside the method preparation by in-vitro transcription.This PolyA-RNA is incorporated into according to different ratios among the RNA of laboratory sample and goes, together carry out the chip reaction experiment.Hybridization back outer ginseng sequence just has hybridization signal, and the amount of the PolyA-RNA that mixes by adjusting obtains the different hybridization signals of outer ginseng.
The nucleotide sequence of sequence table is arranged------3 ' that be from 5 '.
Every of described solid phase substrate has 4 matrixes, and each matrix is the point sample of 22 row, 22 row.
The positive contrast probe point sample of every row first row, every row last, two rows are made up of the positive probe point sample of negative control probe point sample, immobilization, the positive probe point sample of hybridization and blank sampling liquid, other is the detection probes point sample, and the diameter of point sample is 150 μ m, and the spacing of point sample is 185 μ m.
A kind of detection method of Sepiella maindroni releasing tagging chip comprises the steps:
1) extraction of RNA: with the total RNA in Trizol reagent (Invitrogen company provides) extraction Sepiella maindroni tissue or the cell, concentrate RNA with isopropanol precipitating, spectrophotometer is quantitative, the quality inspection of denaturing formaldehyde gel electrophoresis, and RNA purification column purifying obtains purification of samples;
2) cRNA is synthetic: get above-mentioned purification of samples 1ug, with T7-Oligod (T) (Takara company provides) is primer, with Reverse Transcription System test kit (Promega company provides) synthetic double chain cDNA, synthetic finishing with DNA purification column purifying, use T7 in-vitro transcription enzyme (Takara company provides) that double-stranded cDNA is carried out the synthetic cRNA of in-vitro transcription again, transcribe synthetic back and obtain the cRNA sample with RNA purification column purifying;
3) reverse transcription DNA is synthetic: get above-mentioned cRNA sample 5ug, carry out reverse transcription with ThermoScript II MLV (Takara company provides) and random primer (Takara company provides), with DNA purification column purifying, obtain reverse transcription DNA after the reverse transcription; Random primer is the primer title.
4) fluorescent label DNA is synthetic: with reverse transcription DNA is template, with random primer (Takara company provides) and Klenow fragment (Fermentas company provides) synthetic DNA complementary strand, mix the Cy3-dCTP and the Cy5-dCTP that have fluorophor in the building-up process, the synthetic purifying and quantitative that finishes obtains fluorescent label DNA;
5) test sample preparation: fluorescent label DNA is dissolved in the hybridization solution (CapitalBio company) of 80ul, spend the night in 42 ℃ of hybridization, after hybridization finishes, earlier in the washing lotion I of 42 ℃ of preheatings, wash 4min, in the washing lotion II of 42 ℃ of preheatings, wash 4min again, dry at whizzer then and obtain test sample, described washing lotion I for 2 * salt sour lime solution of containing mass percentage concentration 0.2% sodium lauryl sulphate (SDS) (the SSC original liquid concentration is 20 *, provide by CapitalBio company, 2 * or 0.2 * be the concentration after the dilution), described washing lotion I is 0.2 * salt sour lime solution;
6) retouching instrument detects: test sample is put on the described tagging chip of releasing, the two channels laser scanner that provides with CapitalBio company scans detection, adopt Luxscan 3.0 image analysis software (CapitalBio company provides) that chip image is analyzed, picture signal is converted into numerary signal, then the data on the chip are carried out normalization method with the Lowess method, determine which colony of releasing is test sample belong to.
Compared with prior art, the invention has the advantages that provides a kind of Sepiella maindroni releasing tagging chip and detection method, this chip comprises Quality Control probe and detection probes, the Quality Control probe comprises the positive probe of the immobilization of synthetic, hybridize positive probe, positive control probe and negative control probe, detection probes is the release nucleotide sequence of colony of good Tai-2009-1 of two specificitys or Zhou-2009-1, this tagging chip of releasing can be differentiated with these two colonies that cuttlefish is the parent, this release tagging chip specificity and good stability, few to the cuttlefish damage, the analysis of statistical data specific inaccuracy of each colony is reduced after the recapture, for the dependency of studying Sepiella maindroni releasing colony and filial generation provides convenience, especially provide than the accurate data foundation for research cuttlefish growth and breeding rule committee.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment
1, sets up the colony's high throughput testing system of releasing
Utilization capillary electrophoresis SSR fluorescent mark, set up the release EST-SSR marker detection system of colony of the high-throughout cuttlefish of a cover at aspects such as DNA rapid extraction, pcr amplification and the detections of PCR product, the inventor has set up the cDNA fingerprint base of Sepiella maindroni releasing colony, as two Oligo DNA:SEQ ID NO.1 of the present invention (numbering: Tai-2009-1) and SEQ ID NO.2 (numbering: Zhou-2009-1), the specificity Finger-print labelling method of the cuttlefish colony that obtains for screening.
1.1 extract the DNA of each Sepiella maindroni colony, carry out the PCR reaction, response procedures: 94 ℃ of pre-sex change 5min, 30 circulation (94 ℃ of sex change 30s; Suitable temp annealing 30s; 72 ℃ are extended 45s), 72 ℃ are extended 10min.
1.2 with 30 times of PCR product dilutions, in each hole of 96 orifice plates, add the PCR product behind the mark and 1 μ L dilution in 9.05 μ L deionized formamides, the 0.05 μ LGS3730_500 molecular weight, 95 ℃ of sex change 5min, in 4 ℃ of insulation 10min, the centrifugal 1min of 3000rpm carries out capillary electrophoresis on ABI 3730XLDNA analyser; Prerunning 15KV, 2min; 2KV voltage sample introduction 10s; Electrophoresis 15KV, 20min.Collect raw data with Data Collection software.
1.3 respectively each sample is provided with 3 independently amplification experiments, the result shows that consistence is fine.Data results can carry out cluster by the statistical software analysis.The capillary electrophoresis technique platform has been set up in this experiment, and follow-up work is taken a sample to each colony of releasing, and carries out the SSR capillary electrophoresis behind the sample extraction DNA of acquisition and detects, and the gained data are set up the colony's fingerprint database of releasing.After colony's number reaches certain level, can therefrom screen the specificity fingerprint gene fragment of each colony, be designed to chip probe: two Oligo DNA:Tai-2009-1 of the present invention and Zhou-2009-1, the colony's identification chip exploitation of releasing.
Microsatellite DNA claims that again (simple sequence repeats, SSR), SSR repeats 10～20 times end to end tandem repetitive sequence by 1～6 Nucleotide in the simple sequence repetition.Between the different varieties of same species, there is a large amount of polymorphism marks, all has " fingerprint " of the combination of some DNA fragment specific, have high degree of specificity and stability as this kind.This fingerprint only limits to detect between kind at present, by introducing capillary electrophoresis technique, use high-throughout ssr analysis can obtain more differential gene fragment and genetic information, further set up fingerprint image, can improve the precision and the speed of detection greatly, thereby can identify from colony even individual aspect.
2, chip manufacturing
Design Oligo DNA on each releases colony, every Oligo DNA represents the colony of releasing, with two Oligo DNA SmartArray of the present invention
TM(Capital Bio Corp., Beijing, China) o'clock built in a 75 * 25mm, through on the slide glass of chemically modified, the sample of point on chip also comprises Quality Control probe point sample: the positive probe of immobilization, the positive probe of hybridization, four positive control probes, article 12, negative control probe, and blank sampling liquid, the sampling liquid that sampling liquid is made for gene chip commonly used, commercially available acquisition.
3, chip detecting method
3.1RNA extraction: extract with Trizol reagent (Invitrogen company provides) and to adopt the Sepiella maindroni tissue of catching or the total RNA in the cell, concentrate RNA with isopropanol precipitating, spectrophotometer is quantitative, the quality inspection of denaturing formaldehyde gel electrophoresis, and RNA purification column purifying obtains purification of samples.
3.2cRNA it is synthetic: as to get above-mentioned purification of samples 1ug, with T7-Oligod (T) (Takara company provides) is primer, with Reverse Transcription System test kit (Promega company provides) synthetic double chain cDNA, synthetic finishing with DNA purification column purifying, use T7 in-vitro transcription enzyme (Takara company provides) that double-stranded cDNA is carried out the synthetic cRNA of in-vitro transcription again, transcribe synthetic back and obtain the cRNA sample with RNA purification column purifying.
3.3 reverse transcription DNA is synthetic: get above-mentioned cRNA sample 5ug, carry out reverse transcription, with DNA purification column purifying, obtain reverse transcription DNA after the reverse transcription with ThermoScript II MLV (Takara company provides) and random primer (Takara company provides).
3.4 fluorescent label DNA is synthetic: with reverse transcription DNA is template, with random primer (Takara company provides) and Klenow fragment (Fermentas company provides) synthetic DNA complementary strand, mix the Cy3-dCTP and the Cy5-dCTP that have fluorophor in the building-up process, the synthetic purifying and quantitative that finishes obtains fluorescent label DNA.
3.5 test sample preparation: fluorescent label DNA is dissolved in the hybridization solution of 80ul, spend the night in 42 ℃ of hybridization, after hybridization finishes, earlier in the washing lotion I of 42 ℃ of preheatings, wash 4min, in the washing lotion II of 42 ℃ of preheatings, wash 4min again, dry at whizzer then and obtain test sample, the washing lotion I is the 2 * SSC that contains mass percentage concentration 0.2%SDS, and the washing lotion I is 0.2 * SSC.
3.6 retouching instrument detects: test sample is put on the tagging chip of releasing, the two channels laser scanner that provides with CapitalBio company scans detection, adopt Luxscan 3.0 image analysis software (CapitalBio company provides) that chip image is analyzed, picture signal is converted into numerary signal, then the data on the chip are carried out normalization method with the Lowess method, determine whether test sample belongs to above-mentioned Tai-2009-1 or the Zhou-2009-1 colony of releasing.
Sequence table
＜110〉University Of Ningbo
＜120〉a kind of Sepiella maindroni releasing tagging chip and detection method
<140>
<141>
<160>8
<170>PatentIn?version?3.1
<210>1
<211>60
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＜213〉the cuttlefish Tai-2009-1 of colony that releases
<400>1
TACGCTCCTT?TAATCATCGT?TCCCACCTTA?ATGCAGCTGT?GCGCGCTCTA
AAGGGATTGA?60
<210>2
<211>60
<212>cDNA
＜213〉the cuttlefish Zhou-2009-1 of colony that releases
<400>2
CTTACGTGAC?TGTCCATCGC?TTAACGGCTG?GAGGTCGACA?GATCTGCTAC
TGTCTGAGCT?60
<210>3
<211>50
<212>DNA
＜213〉synthetic
<400>3
GTCACATGCG?ATGGATCGAG?CTCCTTTATC?ATCGTTCCCA?CCTTAATGCA
50
<210>4
<211>50
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＜213〉synthetic
<400>4
CCGGCGAGTT?TATCAAAGGC?GCGAACATCC?GGTCTGAACT?CAGATCACGT
50
<210>5
<211>60
<212>DNA
＜213〉synthetic
<400>5
CCGGCGAAAC?ATCCGGTCTG?AACTCATAAT?GCAGCTGTGC?GGATCACGTC
TGCCTGACAC?60
<210>6
<211>60
<212>DNA
＜213〉synthetic
<400>6
TCTGGGCTGA?GCGATCGCTG?GCTGTGCGAG?CGCGCTACTC?CTACCTCCAA
ACTTCGGCAA?60
<210>7
<211>60
<212>DNA
＜213〉synthetic
<400>7
CCTTACATAA?GGTTGCCAAT?CCTAAGGCTC?ACTCGCTGAC?CACGTGACTC
GCGTTGACTG?60
<210>8
<211>60
<212>DNA
＜213〉synthetic
<400>8
CACTTCTACT?CGCGACGCGC?TCCTGAAGTG?ACGTGAATGT?GACTGCGTCG
TGCACTAGCA?60
Claims (4)
1. Sepiella maindroni releasing tagging chip, comprise solid phase substrate through chemically modified, the surface cure of described solid phase substrate has Quality Control probe point sample and detection probes point sample, described Quality Control probe point sample comprises the positive probe point sample of immobilization, hybridize positive probe point sample, positive control probe point sample, negative control probe point sample and blank sampling liquid, the nucleotide sequence that it is characterized in that described detection probes is shown in sequence table SEQ ID NO.1 and SEQ ID NO.2, the nucleotide sequence of the positive probe of described immobilization is shown in sequence table SEQ ID NO.3,5 ' of this nucleotide sequence is modified with by cy3 excited fluorescent element, the nucleotide sequence of the positive probe of described hybridization is shown in sequence table SEQ ID NO.4, the nucleotide sequence of described positive control probe is shown in sequence table SEQ ID NO.5 to SEQ ID NO.8, and the nucleotide sequence of described negative control probe is shown in the H2NC000001 to H2NC000012 as the sequence number of Operon company.
2. a kind of Sepiella maindroni releasing tagging chip as claimed in claim 1 is characterized in that every of described solid phase substrate has 4 matrixes, and each matrix is the point sample of 22 row, 22 row.
3. a kind of Sepiella maindroni releasing tagging chip as claimed in claim 2, it is characterized in that the positive contrast probe point sample of every row first row, every row last, two rows are made up of the positive probe point sample of negative control probe point sample, immobilization, the positive probe point sample of hybridization and blank sampling liquid, other is the detection probes point sample, the diameter of point sample is 150 μ m, and the spacing of point sample is 185 μ m.
4. the detection method of the described a kind of Sepiella maindroni releasing tagging chip of claim 1 is characterized in that comprising the steps:
1) extraction of RNA: the total RNA with in Trizol reagent extraction Sepiella maindroni tissue or the cell, concentrate RNA with isopropanol precipitating, spectrophotometer is quantitative, the quality inspection of denaturing formaldehyde gel electrophoresis, RNA purification column purifying obtains purification of samples;
2) cRNA is synthetic: get above-mentioned purification of samples 1ug, with T7-Oligod (T) is primer, with Reverse TranscriptionSystem test kit synthetic double chain cDNA, synthetic finishing with DNA purification column purifying, with T7 in-vitro transcription enzyme double-stranded cDNA is carried out the synthetic cRNA of in-vitro transcription again, transcribe synthetic back and obtain the cRNA sample with RNA purification column purifying;
3) reverse transcription DNA is synthetic: get above-mentioned cRNA sample 5ug, carry out reverse transcription with ThermoScript II MLV and random primer, with DNA purification column purifying, obtain reverse transcription DNA after the reverse transcription;
4) fluorescent label DNA is synthetic: with reverse transcription DNA is template, with random primer and Klenow fragment synthetic DNA complementary strand, mixes the Cy3-dCTP and the Cy5-dCTP that have fluorophor in the building-up process, and the synthetic purifying and quantitative that finishes obtains fluorescent label DNA;
5) test sample preparation: fluorescent label DNA is dissolved in the hybridization solution of 80ul, spend the night in 42 ℃ of hybridization, after hybridization finishes, earlier in the washing lotion I of 42 ℃ of preheatings, wash 4min, in the washing lotion II of 42 ℃ of preheatings, wash 4min again, dry at whizzer then and obtain test sample, described washing lotion I is the 2 * salt sour lime solution that contains mass percentage concentration 0.2% sodium lauryl sulphate, and described washing lotion I is 0.2 * salt sour lime solution;
6) retouching instrument detects: test sample is put on the described tagging chip of releasing, scan detection with the two channels laser scanner, adopt Luxscan 3.0 image analysis software that chip image is analyzed, picture signal is converted into numerary signal, then the data on the chip are carried out normalization method with the Lowess method, determine which colony of releasing is test sample belong to.
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| CN2010101963003A CN101857901B (en) | 2010-06-10 | 2010-06-10 | Sepiella maindroni releasing and tagging chip and detection method |
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| CN2010101963003A CN101857901B (en) | 2010-06-10 | 2010-06-10 | Sepiella maindroni releasing and tagging chip and detection method |
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| CN101857901A true CN101857901A (en) | 2010-10-13 |
| CN101857901B CN101857901B (en) | 2012-05-09 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1586188A (en) * | 2004-07-30 | 2005-03-02 | 宁波大学 | Manson's sepilla maindroni fry cultivating method |
| CN101243836A (en) * | 2008-03-07 | 2008-08-20 | 宁波大学 | Method for nursing sepiella maindroni with soft compound feed |
-
2010
- 2010-06-10 CN CN2010101963003A patent/CN101857901B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1586188A (en) * | 2004-07-30 | 2005-03-02 | 宁波大学 | Manson's sepilla maindroni fry cultivating method |
| CN101243836A (en) * | 2008-03-07 | 2008-08-20 | 宁波大学 | Method for nursing sepiella maindroni with soft compound feed |
Non-Patent Citations (5)
| Title |
|---|
| 《中国水产》 20081231 常抗美等 曼氏无针乌贼增养殖开发与利用的研究进展 55-56 1-4 , 第03期 2 * |
| 《水利渔业》 20080731 励迪平等 曼氏无针乌贼遗传多样性的RAPD分析 21-24 1-4 第28卷, 第04期 2 * |
| 《浙江海洋学院学报（自然科学版）》 20091231 王伟定等 东海区适宜增殖放流种类的筛选与应用 379-383 1-4 第28卷, 第4期 2 * |
| 《海洋与湖沼》 20090930 宋微微，王春琳 养殖对曼氏无针乌贼（Sepiella maindroni）群体遗传多样性的影响 590-595 1-4 第40卷, 第5期 2 * |
| 《湛江海洋大学学报》 20050831 尹飞等 曼氏无针乌贼幼体生态因子耐受性的研究 39-43 1-4 第25卷, 第4期 2 * |
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