CN105925711B - A kind of scallop (Chlamys farreri) chip and method detecting polycyclic aromatic hydrocarbon poisonous effect - Google Patents
A kind of scallop (Chlamys farreri) chip and method detecting polycyclic aromatic hydrocarbon poisonous effect Download PDFInfo
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Abstract
A kind of scallop (Chlamys farreri) chip and method detecting polycyclic aromatic hydrocarbon poisonous effect, the chip includes 25 kinds of oligonucleotide probes shown in NO.1~25 SEQ ID that are fixed on solid phase carrier, it is the 25 kinds of metabolic detoxification related genes chosen on to Chlamys farreri metabolic detoxification the related mechanism for many years basis of research, can be used for gene differential expression relevant information of 25 related genes of batch detection Chlamys farreri metabolic detoxification under PAHs stress.Its detection method includes the preparation of Chlamys farreri cDNA sample；The above-mentioned cDNA sample of fluorescent marker；By the cDNA sample and the gene chip hybridization after fluorescent marker；The hybridization signal of Scanning Detction genetic chip obtains result.Using the chip while the high-throughput detection that gene expression information can be achieved, the advantages that also there is easy to operate, testing result high efficient and reliable, a large amount of human and material resources and financial resources can be saved, have broad application prospects.
Description
Technical field
The present invention relates to a kind of detection polycyclic aromatic hydrocarbons to the genetic chip and method of Chlamys farreri poisonous effect, for more
The detection of the variation of related gene expression amount caused by cycloaromatics, belongs to gene chip detecting technique field.
Background technique
Persistence toxic chemical pollutant (Persistent Toxic Substances, PTS) is considered as 21 century shadow
The important environmental problem of human survival and health is rung, wherein polycyclic aromatic hydrocarbon (Polynuclear aromatic
Hydrocarbons, PAHs) 27 kinds of important one of PTS have been classified as by the United Nations.PAHs is mainly derived from the pyrolysis of organic matter
And imperfect combustion has strong stability, high toxicity and the high spy of biological concentration rate as a kind of global environmental contaminants
Point, in more than 500 kinds of carcinogenic substances being currently known, having more than 200 kinds is PAHs and its derivative.With offshore oil and wheel shipping
The development and industry of defeated industry, a large amount of discharges of sanitary sewage, the pollution of PAHs increasingly aggravates in China's marine environment, according to
2011-2013 China marine environmental quality bulletin shows that China's immediate offshore area is different degrees of by PAHs pollution, part
Sea area has been approached medium pollution level.PAHs not only influences the growth and breeding of marine organisms, but also putting step by step by food chain
Seriously threaten the health of the mankind greatly, since pollutant analysis monitoring is relatively difficult in marine environment, biological monitoring in recent years by
To the great attention of ocean toxicologic study person, existing research shows that the variation on organism molecule cellular level can reflect at first
The toxic effect of pollutant provides early warning signal for marine environmental pollution monitoring, these reflection pollutants exist or biology
The index of body response is referred to as biomarker.
The filter-feeding life of bivalve shellfish battalion, metabolic rate are low, have very strong bioconcentration to ocean organic pollutant,
The instruction biology often monitored as marine environmental pollution.Carry out research of the PAHs to bivalve shellfish molecular toxicology mechanism, builds
The PAHs biological monitoring technical system of bivalve shellfish of being based on has become the research topic that current marine pollution monitoring is badly in need of.Originally it grinds
Study carefully and selects China shallow sea distinctive economic shellfish-Chlamys farreri (Chlamys farreri) as research object, it is close in conjunction with China
Extra large PAHs pollution situation studies Chlamys farreri internal related gene expression under PAHs stress using biochip technology
The variation of amount provides efficient, stable monitoring technology to study the polycyclic aromatic hydrocarbons contaminated situation in waters area.
Common detection polycyclic aromatic hydrocarbon mainly has quantitative fluorescent PCR and high-flux sequence to the method for Chlamys farreri poisonous effect
Method.Quantitative fluorescent PCR is that tracking is marked to PCR product by the probe of the specificity of fluorescent dye or fluorescent marker,
Real-time online monitoring reaction process, and corresponding software can be combined to analyze product by experimenter, calculate sample to be tested mould
The initial concentration of plate, but relative to high throughput sequencing technologies and biochip technology, fluorescence quantitative PCR detection scale is smaller, is
Be not suitable for the technology of the detection of large scale experiment.For fluorescent quantitative PCR technique, high throughput sequencing technologies sensitivity compared with
Low and required instrument price is expensive, and testing cost is high, and analysis speed is slow, can not accurately detect polycyclic aromatic hydrocarbon in a short time
To Chlamys farreri poisonous effect.Have not yet to see the scallop (Chlamys farreri) chip for being able to detect polycyclic aromatic hydrocarbon poisonous effect
Report.
Summary of the invention
It is an object of the invention to invent a kind of genetic chip for detecting polycyclic aromatic hydrocarbon to Chlamys farreri poisonous effect, with
It predicts polycyclic aromatic hydrocarbons contaminated situation in environment, is a kind of rapid detection method with high sensitivity and specificity, it is existing to overcome
There is the deficiency of technology.
Technical scheme is as follows:
A kind of detection polycyclic aromatic hydrocarbon is to the genetic chip of Chlamys farreri poisonous effect, it is characterised in that including solid phase carrier and
25 kinds of oligonucleotide probes being fixed on the solid phase carrier, 25 kinds of oligonucleotide probes are detoxified generation to Chlamys farreri
Thank the 25 kinds of metabolic detoxification related genes chosen on the basis of related mechanism years of researches, 25 kinds of oligonucleotide probes
Specific 25 gene orders of Chlamys farreri are derived from, 25 kinds of oligonucleotide probes are shown in NO.1~25 SEQ ID
Nucleotide sequence.
The solid phase carrier is selected from glass slide, silicon wafer, nitrocellulose filter, nylon membrane or polystyrene.
The invention further relates to aforementioned detection detection polycyclic aromatic hydrocarbons to the user of the genetic chip of Chlamys farreri poisonous effect
Method, comprising the following steps:
(a) preparation of Chlamys farreri cDNA sample；
(b) the above-mentioned cDNA sample of fluorescent marker；
Characterized by further comprising following steps:
(c) by the cDNA sample and the gene chip hybridization after fluorescent marker；
(d) hybridization signal of Scanning Detction genetic chip obtains result.If there is gene differential expression, indicate that the comb hole is fanned
Becquerel can then be carried out further quantitative detection by PAHs stress.
The application is after studying many years of the polycyclic aromatic hydrocarbon to the poisonous effect of bivalve shellfish, to grind in original toxicology
On the basis of studying carefully, the novel genetic chip and detection method based on toxicologic study technology developed, this method and fluorescence
For quantitative PCR compared with high-flux sequence method, stability is higher, detection speed faster, detect it is larger, detection sensitivity and
Specificity is higher, and testing cost is lower, has apparent advanced.
The present invention establish for detect Chlamys farreri in the genetic chip of poisonous effect under PAHs stress and
Its method of manufacture and use thereof.Using biological monitoring technology, one kind is provided rapidly and efficiently for monitoring Polycyclic Aromatic Hydrocarbonat Existing in Environment residual
Detection means.
Genetic chip provided in the present invention and detection method through Preliminary Applications in Chlamys farreri, since this method uses
Be polygenes chip technology, have many advantages, such as efficiently, quickly, it is accurate, stablize, can quickly, accurate, specificity detection its
The variation of related gene expression amount under the stress of various concentration polycyclic aromatic hydrocarbon.
Specific embodiment
The present invention is further illustrated by the following examples
One, key instrument:
1. key instrument of table:
Two, main agents:
2. main agents of table:
Three, specific step is as follows for the preparation method of genetic chip of the present invention:
1,25 kinds of specific removing toxic substances the specific probe of design detection: are chosen according to known Chlamys farreri metabolic detoxification mechanism
Metabolism related gene designs specific probe using bioinformatics software Primer Express 2.0；
2, the probe that above-mentioned design is completed is fixed on solid phase carrier nylon membrane by designed array layout after synthesizing
It is upper to constitute genetic chip of the invention.
Four, using said gene chip detection polycyclic aromatic hydrocarbon to the method for Chlamys farreri poisonous effect, comprising the following steps:
(a) preparation of Chlamys farreri cDNA sample, specific as follows:
1, the extraction and quality inspection of sample Total RNA
Experiment BaP (No. CAS: 50-32-8) purity is 97.5%, is purchased from U.S. SIGMA company.BaP mother liquor diformazan
Base sulfoxide (DMSO) is prepared, then is diluted to final 1 μ g/l of experimental concentration with nature seawater, and keep the final concentration of cosolvent DMSO
For 0.001% (v/v).After experiment starts 15 days, 6 scallops are taken respectively, and dissection takes Digestive diverticula to be put into liquid in liquid nitrogen and grinds, claims
Take 80mg in 1.5mL centrifuge tube, sample number into spectrum is C (control group), T (processing group).Using suitable method, such as Trizol
(Invitrogen, Gaithersburg, MD, USA) extracts the total serum IgE in tissue block or cell sample, and further progress is pure
Change and quantitative analysis.
2, Total RNA synthesizes cDNA
1) reverse transcription synthesizes First Strand cDNA
Following reagent is sequentially added in the centrifuge tube of 0.2mL nuclease free:
Take Total RNA 100-500ng, adjustment volume to 4 μ L.
If usingGene expression profile family chip is addedJoin (Cat.No.360030) 1 outside gene expression profile
Otherwise 1 μ L Nuclease-free Water is added in μ L.
It prepares reverse transcription Master Mix (following table 3 show single reaction system dosage), mixes gently, of short duration centrifugation
It is placed on ice bath.5 μ L Master Mix are taken to be transferred in the 0.2mL centrifuge tube containing Total RNA sample respectively.
3. reverse transcription Master Mix of table:
Solution is mixed gently, is reacted 2 hours for 42 DEG C after brief centrifugation.Centrifugation and ice bath after reaction.
2) Second Strand cDNA is synthesized
It prepares Second Strand Master Mix (table 4), mixes gently, ice bath after of short duration centrifugation.In above-mentioned reaction
After each sample cell in 20 μ L Second Strand Master Mix are added.
Table 4.Second Strand Master Mix:
Soft to mix, 16 DEG C are reacted 1 hour, 65 DEG C of 10min.
Reaction tube is placed in after reaction and continues synthetic reaction on ice, or is frozen in -20 DEG C rapidly.
3, synthesis cRNA is transcribed in vitro
CRNA synthesis
Prepare be transcribed in vitro Master Mix (following table 5 show single reaction system dosage), mix gently, it is of short duration from
Solution is collected in tube bottom by the heart, and 30 μ L Master Mix are added into each sample cell that above-mentioned reaction terminates.
Master Mix is transcribed in vitro in table 5.:
It is soft to mix, 40 DEG C reaction 8-14 hours.After the reaction was completed, prepare purifying cRNA.
4, cRNA reverse transcription
1) cRNA reverse transcription generates cDNA
5 μ g of cRNA purified product is taken, adjustment volume to 7.5 μ L is added in 0.2mL nuclease free centrifuge tube, and is added 4
μ L Random Primer, mix, 65 DEG C 5 minutes, ice bath 5 minutes.
It prepares cRNA reverse transcription Master Mix (following table 6 show single reaction system dosage), it is soft to mix, it is of short duration
Solution is collected in tube bottom by centrifugation.8.5 μ L Master Mix are added after to above-mentioned steps in each sample cell.
Table 6.cRNA reverse transcription Master Mix:
It is soft to mix, it is reacted 10 minutes for 25 DEG C after of short duration centrifugation, 37 DEG C are reacted 1.5 hours.
5 μ L of Terminate Solution is added after reaction, mixes.
65 DEG C are reacted 10 minutes, are stored at room temperature 5 minutes.
1 μ L of Neutralize Solution is added, mixes, prepares purifying.
2) purifying, Quantitative Reverse Transcription product
It usesExtract II (MN company, Cat.No.740609.250) kit or other similar
Product is purified.
Using the quantitative cDNA product after purification of ultraviolet specrophotometer, cDNA has characteristic absorption peak at 260nm
(1OD260=40 μ g/ μ L).
(b) the above-mentioned cDNA sample of fluorescent marker, specific as follows:
1, label reaction
The cDNA bulk product purified after reverse transcription is concentrated into 14 μ L, 4 μ L Random Primer are added and mix,
It is denaturalized 3 minutes, ice bath 5 minutes for 95 DEG C after of short duration centrifugation.
Reagent in following table 7 is sequentially added, blows and beats 2-3 mixing using pipettor, 37 DEG C of reactions 1.5 are small after of short duration centrifugation
When, 70 DEG C are reacted 5 minutes.After reaction, prepare purifying.
Table 7. adds reagent:
2, purifying, quantitative mark product
It usesExtract II (MN company, Cat.No.740609.250) kit is pure to marked product
Change.
Product after purification is quantitative using NanoDrop.
Marked product amplifying nucleic acid has characteristic absorption peak (1OD260=50 μ g/ μ L) at 260nm.
(c) specific as follows by the cDNA sample and the gene chip hybridization after fluorescent marker:
1, preparing hybrid system (such as table 8)
It takes the Ep of a 0.5mL nuclease free to manage, sequentially adds following reagent.
8. hybridization solution of table is with tabulation (preparing 100 μ L hybridization solutions):
It is aspirated and is mixed with sample injector, is placed in spare on ice.
2, every chip hybridization system is 81.6 μ L, the hybridization that cDNA and 41.6 μ L including 40 μ L fluorescent markers are newly prepared
Buffer.The DNA sample for being marked with Cy3 and Cy5 fluorescence after purification is separately added into Nuclease-free Water to mend
Sufficient volume (note: if fruit volume alreadys exceed 20 μ L, can be used spin concentration instrument and be concentrated) to 20 μ L.And 41.6 μ L are added
The hybridization solution of mixed preparing, gentle centrifugation are simultaneously mixed with sample injector.
3, sample above is placed in 95 DEG C of incubation 3min, it is spare is then placed into 2min on ice.
4, chip hybridization box is taken, is put into a piece of sizeable filter paper, 100-150 μ L deionized water is added so that keep must
The humidity wanted.
5, the chip that will already be at room temperature is sealed off, and one with bar code is placed in hybridizing box upwardly.On chip
The hybridization solution containing fluorescent marker cDNA of 81.6 μ L is added, then carefully places cover plate (LifterSlipTMCover plate, Themo
Company), it avoids generating bubble.
6, hybridizing box is assembled, is placed on hybridization instrument, general 42 DEG C of hybridized overnights.Rich biochip suggestion difficult to understand is made
With rich production difficult to understand BioMixerTM II chip hybridization instrument (Cat.No.120030), it is uniform to obtain good hybridization
Property；Corresponding hybridization instrument is used to other chip types.
(d) hybridization signal of Scanning Detction genetic chip obtains as a result, specific as follows:
1, after hybridizing, chip is taken out, 0.2%SDS is first contained at 42 DEG C or so, washes 5min in the washing lotion I of 2 × SSC, and
5min is washed in the washing lotion II of 0.2 × SSC of room temperature afterwards, can be used to scan after slide drying.
2, the chip after cleaning is scanned using LuxScan-10KA chip scanner, obtains hybridization picture.
3, data are extracted and are analyzed
Data are extracted to divide chip image using 3.0 image analysis software of LuxScan (CapitalBio company)
Analysis.
As the result is shown inventor choose 25 kinds of metabolic detoxification related genes in, have CYP3A2, CYP2P1, ARSB,
3 seven kinds of genes of GPx1, MT, CLECB, Lysozyme expression quantity in the case where 1 μ g/L BaP coerces 15d has significant up-regulation；And AhR,
Eight kinds of HSP90, GST3, Pgp, SOD, Ferritin 1, COL4, MEGF gene tables have in the 1 μ g/L BaP stress 15d amount of assigning
It is significant to lower, as shown in following table 9.As it can be seen that may determine that experimental group and control group according to the above experimental result there are genes it is poor
Different expression indicates that the Chlamys farreri of experimental group should may then be carried out further quantitative detection by PAHs stress.Cause
This, which, which is applied in actual environment monitoring, can effectively judge in environment with the presence or absence of polycyclic aromatic hydrocarbons contaminated.
The analysis of 9. experimental result data of table:
To sum up, 25 kinds of nucleotide sequences contained by genetic chip of the invention are obtained by strictly screening and comparing
, and its validity is demonstrated in practice, therefore efficiently can quickly be judged in Chlamys farreri body using the biochip technology
The expression quantity situation of change of relevant 25 kinds of metabolic detoxification genes provides one kind for the polycyclic aromatic hydrocarbons contaminated situation in interpretation waters
Efficiently, stable detection technique seems especially great particularly with the monitoring meaning for requiring more areas to detect simultaneously.
Claims (3)
1. a kind of detection polycyclic aromatic hydrocarbon is to the genetic chip of Chlamys farreri poisonous effect, it is characterised in that including solid phase carrier and admittedly
25 kinds of oligonucleotide probes being scheduled on the solid phase carrier, 25 kinds of oligonucleotide probes are derived from Chlamys farreri specific 25
Gene order, 25 kinds of oligonucleotide probes are nucleotide sequences shown in NO.1~25 SEQ ID.
2. a kind of detection polycyclic aromatic hydrocarbon as described in claim 1 exists to the genetic chip of Chlamys farreri poisonous effect, feature
Glass slide, silicon wafer, nitrocellulose filter, nylon membrane or polystyrene are selected from the solid phase carrier.
3. using genechip detection polycyclic aromatic hydrocarbon described in claim 1 to the method for Chlamys farreri poisonous effect, including
(a) preparation of Chlamys farreri cDNA sample；
(b) the above-mentioned cDNA sample of fluorescent marker；
Characterized by further comprising following steps:
(c) by the cDNA sample and the gene chip hybridization after fluorescent marker；
(d) hybridization signal of Scanning Detction genetic chip obtains result.If there is gene differential expression, indicate that the Chlamys farreri can
Further quantitative detection can be then carried out by PAHs stress.
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Non-Patent Citations (5)
| Title |
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| Deep sequencing-based transcriptome profiling analysis of;Yuefeng Cai等;《Gene》;20140904(第551期);261-270 |
| In vitro study of the effect of metabolism enzymes on;Yuefeng Cai等;《Environmental Toxicology and Pharmacology》;20160114(第42期);92-98 |
| Molecular evidence for the existence of an aryl hydrocarbon receptor;Yuefeng Cai等;《Comparative Biochemistry and Physiology, Part B》;20160227;74-84 |
| RNA-seq based on transcriptome reveals differgenetic expressing in Chlamys farreri exposed tocarcinogen PAHs;Qian Jin等;《ScienceDirect》;20141204(第39期);313-320 |
| 多环芳烃(PAHs)在栉孔扇贝体内蓄积特征与毒性效应的研究;修蒙;《中国优秀硕士学位论文全文数据库》;20150215(第2期);B027-198 |
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