CN1244808C - Shellfish sample preparing method suitable for detecting ploidy by flow cytometry - Google Patents
Shellfish sample preparing method suitable for detecting ploidy by flow cytometry Download PDFInfo
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- CN1244808C CN1244808C CN 03133650 CN03133650A CN1244808C CN 1244808 C CN1244808 C CN 1244808C CN 03133650 CN03133650 CN 03133650 CN 03133650 A CN03133650 A CN 03133650A CN 1244808 C CN1244808 C CN 1244808C
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Abstract
The present invention discloses a shellfish sample preparing method suitable for detecting ploidy by flow cytometry. The method acquires biological samples from larvae, juvenile shellfish, young shellfish or grown shellfish; separating liquid of cell nuclei is added into the biological samples, the mixture of the biological samples and the separating liquid is oscillated, ground or cut to prepare homogenate, and the homogenate is precipitated in a still way to obtain supernatant suspension liquid; the supernatant suspension liquid is transferred to a centrifugal tube, the supernatant liquid is removed in a centrifugation way, and dissociative single cell nuclei are collected; cell nucleus fixed liquid I is added into the single cell nuclei, and the cell nuclei are suspended again; then, the supernatant liquid is removed in the centrifugation way, and the dissociative single cell nuclei are collected; cell nucleus fixed liquid II is added into the single cell nuclei, the cell nuclei are suspended, single cell nucleus samples are prepared, and the single cell nucleus samples can be preserved or transported in a long distance under normal temperature and can be used for the detection of the flow cytometry. The present invention is suitable for the detection of the flow cytometer, and has high and stable detection rate.
Description
Technical field
The present invention relates to the ploidy detection technique of shellfish, the preparation method of the shellfish sample that is applicable to the Flow cytometry ploidy is provided especially.
Background technology
Seashells is the leading kind of China's sea-farming, occupies critical role in the mariculture industry of China.The shellfish of China's sea-farming all is a wild type basically at present, is difficult to adapt to the breeding environment that runs down.Therefore, utilizing biotechnology to cultivate good breed variety, is current and even the future development marine shellfish is cultured one of key issue that needs to be resolved hurrily.The shellfish cytogenetics technology of bringing out technology based on polyploid is a field of the most active and tool using value in the present shellfish genetic breeding.The triploid shellfish has many good production traits, is the of great value cultivated shellfish new lines of a class.Usually adopt physico-chemical method artificial induction polyploid at present, a times change rate is difficult to reach 100%, and different with biotic factor because of various environment, and times change rate of polyploid is widely different.Therefore, the detection of polyploid and evaluation are that polyploid is cultivated the requisite crucial ring of research.All need ploidy evaluation fast and accurately in each stage that polyploid shellfish is induced, whether effective to distinguish the artificial induction.Such as, whether biological pollution or other incidents take place in the cultivating process of the larva or the young and cause degradation under the ratio of polyploid; Whether the polyploid adult is at sea in the breeding process, because of causing adhering to of natural seedling that unusual dead or sea area takes place etc. degradation under the polyploid proportion of composing.The detection of ploidy need run through the whole process of polyploid research, and therefore efficient ploidy detection technique has extreme importance.
In colleges and universities or the scientific research institution that China's overwhelming majority is engaged in the marine biotechnology research and development, the ploidy that adopts the chromosome counting method to carry out artificial induction's polyploid is basically identified.But this method specimen preparation process complexity wastes time and energy, and the sample of preparation could be evaluated sample quality through microexamination, often can't obtain enough chromosome division phases because sample quality is not good, causes the ploidy of sample to identify failure.
Flow cytometry is the new technology that individual cells or other biological particulate are carried out fast quantitative analysis and sorting.Its principle is the unicellular measurement zone that passes through one by one that will be suspended in the liquid stream, reaches a series of physical characteristicss and the biochemical characteristic of measuring cell fast, in large quantities with the optical parametric of instrument detecting cell.Since the eighties in 20th century, flow cytometry is extensively utilized in international polyploid shellfish research.The most significant advantage of this technology is to analyze the dna content of a large amount of samples rapidly and accurately, its detection speed is fast, the height of statistics precision is that additive method such as chromosome counting is incomparable, has become one of the most effective research means of polyploid shellfish research.
Influencing one of flow cytometry key of success factor is the specimen preparation quality.The sample that is used for flow cytometry analysis must be a single cell suspension, therefore just needs biomaterial to be detected (larva, the young or adult) is made single cell suspension before detection.Specimen preparation process generation cytoadherence, agglomerate and cell overlap etc. all have a strong impact on the accuracy of recall rate and analysis result.Sample then can cause and analyze failure if produce too much impurity fragment because of degraded takes place in preparation preservation process.Flow cytometer costs an arm and a leg, and at present domestic only R﹠D institution of very few a few family has this equipment, and other unit must be delivered the scientific research institution that has flow cytometer with the sample of preparation and detect.On the other hand, the multiploid induction of seashells is cultivated research, and majority is to carry out in remote experiment field, beach or seed rearing field, sample need be sent back to scientific research institution and detect.In recent years, do not cause the analysis failure to take place repeatedly, fail to give full play to the superiority of flow cytometry, had a strong impact on carrying out of Study on Inducing Polyploid because the specimen preparation quality is good.External polyploid research also faces similar problem.
The preparation of flow cytometry sample at present, require the fixed sample of live body sample or freezing preservation, concerning the larva or the young, keep the harsh traffic condition of its vigor needs, comprise with seawater mediate, oxygenation, low temperature etc., caused significant limitation for transporting sample.The foreign scholar attempts utilizing methyl alcohol/glacial acetic acid immobile liquid or ethanol fixed sample, transportation at normal temperatures, 3～5 days the recall rate of these fixed samples on flow cytometer is very low when discovering traffic expense, be generally 10～30%, to samples such as metacyclic eyespot larvas, can't detect basically.This shows that the biological sample of common fixing means preparation does not far reach the requirement of flow cytometry to test sample.Marine fishery circle all presses for a kind of new flow cytometry sample preparation methods both at home and abroad, is implemented in the high recall rate under the long-time preservation of normal temperature.Up to the present the flow cytometry normal temperature that is applicable to shellfish that yet there are no mature and feasible is both at home and abroad preserved the preparation method's of sample report.
Summary of the invention
In order to solve preparation method's problem of above-mentioned shellfish flow cytometry sample, the purpose of this invention is to provide a kind of sample preparation methods that utilizes flow cytometry to carry out the ploidy detection of shellfish that is applicable to, all kinds of samples of realization shellfish are preserved at normal temperatures and are transported for long-distance, and sample has stable high recall rate on flow cytometer.
To achieve these goals, technical scheme of the present invention is as follows:
Obtain biological sample from shellfish to be detected, the slender karyon suspending liquid of preparation shellfish sample carries out nucleus and fixes; Specifically operation as follows:
1) obtains biological sample from larva, juvenile mollusk, young shellfish or one-tenth shellfish;
2) add every milligram of 1～3mL nucleus parting liquid in the biological sample, vibrate, grind or chop 5～10min, be prepared into the homogenate shape, staticly settle 5～15min, supernatant suspending liquid;
3) described supernatant suspending liquid is transferred to centrifuge tube,, remove supernatant, collect free slender karyon with the centrifugal 1～5min of 1000～3000r/min;
4) add nucleus immobile liquid I in described slender karyon, addition is every milligram of slender karyon 5～15mL, again suspension cell nuclear;
5) with the centrifugal 1～5min of 1000～3000r/min, remove supernatant, collect free slender karyon;
6) add nucleus immobile liquid II in the described slender karyon of step 5), addition is every milligram of slender karyon 5～15mL, suspension cell nuclear, and the slender karyon sample of making can be preserved or transport for long-distance at normal temperatures, to be ready for use on the detection of flow cytometer.
Wherein: the object of detection (biological sample) comprises larva, juvenile mollusk, the young shellfish of shellfish or becomes whole soft bodies or the portion of tissue of shellfish that suitable tissue comprises gill tissue, closed shell flesh and outer embrane; Obtain the larva sample and adopt bolting silk net filtration and centrifugation method, the aperture of used bolting silk net is 20～100 μ m; Obtain young shellfish or become the tissue of shellfish to adopt anatomic method, used dissecting tool can be dissecting scissors or dissecting forceps; When the biological sample that obtains was larva, juvenile mollusk, per 1 milligram of sample added isotonic buffer solution 0.2～1mL in the process of obtaining;
Described nucleus parting liquid, number percent meter by volume, its composition is to contain 1～10 part of Triton X-100 in per 100 parts of isotonic buffer solutions; Described isotonic buffer solution PBS, number percent meter by volume, its composition is by 70～85 parts of Na
2HPO
4With 15～30 parts of NaH
2PO
4Form; Described nucleus immobile liquid I, number percent meter by volume, its composition are that ethanol water and 10～30 parts of isotonic buffer solutions of 70～90 part of 80～95% concentration of volume percent are formed; Potential of hydrogen (pH value) is 7.0～8.0; Described nucleus immobile liquid II, number percent meter by volume, its composition is the ethanol water of 60～80% concentration.
The present invention has following advantage:
1. the inventive method is simple and feasible, and used common reagent, the equipment etc. of being are applicable to that the relatively poor relatively shellfish seedling of experiment condition cultivates.
2. adopt the prepared shellfish sample of the present invention, realized long preservation and transportation at normal temperatures, and the recall rate of sample on flow cytometer high and stable (embodiment can prove that recall rate can reach 100% when preserving 6 months), highly sensitive.The present invention has thoroughly overcome the limitation of the freezing storage and transport of sample needs of commonsense method preparation.
3. applied range.The present invention is applicable to the sample of various shellfishes and each stage of development of shellfish.Can be applicable to comprise kinds such as oyster, scallop, blood clam, clam, quahog, Bao, comprise each budding larva, juvenile mollusk, young shellfish and become shellfish.
4. adopt the biological sample of the present invention's preparation can satisfy the ploidy detection of sample in batches; Can detect becoming shellfish to implement the live body ploidy, the invention provides and carry out the efficient means that the polyploid shellfish ploidy is identified.
Embodiment
Below provide several exemplary embodiments, but the present invention is not confined in following examples.
Embodiment 1
Utilize the inventive method, grow the polyploid larva specimen preparation of oyster, concrete operations are:
1) adopting the aperture is the silk cover filtering of 80 μ m, removes most seawater, obtains each 60000 of the triploid eyespot larvas of oyster dliploid and artificial induction respectively, contains the centrifuge tube in 1.5mL respectively, with the centrifugal 4min of 2000rpm, removes the seawater of supernatant; (its composition is: 70～85 parts of Na to add the isotonic buffer solution PBS 1mL that measures by per 5 milligrams of sample 1mL
2HPO
4With 15～30 parts of NaH
2PO
4, present embodiment adopts Na
2HPO
485 parts, NaH
2PO
415 parts), with the centrifugal 4min of 2000rpm, remove supernatant, collect larva.
2) the nucleus parting liquid 10mL that adds by per 1 milligram of sample 2mL amount (contains 1～10 part of Triton X-100 in per 100 parts of isotonic buffer solutions, present embodiment is the PBS that contains Triton X-100 1%), adopt mill, Potter-Elvehjem Tissue Grinders grinding or vibration or scalpel blade to chop 5～10min, this enforcement adopts mill to grind 5min, organize and cell separation, staticly settle 15min, supernatant suspending liquid;
3) then described supernatant suspending liquid is transferred to the 15mL centrifuge tube,, remove supernatant, obtain free single cell nuclear with the centrifugal 5min of 2000r/min;
4) (composition is: the ethanol water of 70～90 part of 80～95% concentration of volume percent and 10～30 parts of isotonic buffer solution PBS to add nucleus immobile liquid I toward described per 1 milligram of free single cell nuclear of collecting, the pH value is 7.0～8.0, present embodiment adopts 90 parts of the aqueous solution of 80% ethanol, 10 parts of PBS, adjusting the pH value is 7.0,) 5mL, suspension cell nuclear again;
5) with the centrifugal 5min of 2000r/min, remove supernatant, collect slender karyon once more;
6) per 1 milligram of free single cell nuclear of collecting in the described step 5) adds nucleus immobile liquid II, and (composition is: composition is the ethanol water of 60～80% concentration of volume percent, present embodiment adopts 75% ethanol water) 5mL, suspension cell nuclear again.Can obtain to be applicable to the shellfish sample Flow cytometry ploidy, that can preserve at normal temperatures or transport for long-distance.
Described sample retention was utilized machine testing on the flow cytometer after 6 months, and the recall rate of sample is 100%.
Embodiment 2
Utilize the inventive method, carry out the polyploid larva specimen preparation of Chlamys farreri, concrete operations are:
1) adopt 20 μ m silk cover filtering seawater, collect the scallop veliger that concentrates, each 500000 of the triploid larvas of dliploid and artificial induction are contained the centrifuge tube in 2.0mL respectively, with the centrifugal 5min of 3000rpm, remove the seawater of supernatant; (its composition is: Na to add isotonic buffer solution PBS3mL by the amount of per 5 milligrams of sample 1mL
2HPO
480 parts, NaH
2PO
420 parts), with the centrifugal 5min of 3000rpm, remove supernatant, collect larva.
2) larva of past described collection adds nucleus parting liquid 15mL (present embodiment is the PBS that contains Triton X-100 5%) by the amount of per 1 milligram of sample 1mL, and the 6min that vibrates on the vortex oscillator then staticly settles 15min, gets supernatant suspending liquid;
3) then described supernatant suspending liquid is transferred to the 15mL centrifuge tube,, remove supernatant, obtain free single cell nuclear with the centrifugal 5min of 3000r/min;
4) add nucleus immobile liquid I (composition is: 80 parts of the aqueous solution of 90% ethanol, 20 parts of PBS, adjusting pH value is 7.5) 12mL toward described per 1 milligram of free single cell nuclear of collecting, suspension cell is examined again;
5) with the centrifugal 5min of 3000r/min, remove supernatant, collect slender karyon once more;
6) collecting every milligram free single cell nuclear toward described step 5) adds nucleus immobile liquid II (composition is: 12mL 70% ethanol water), suspension cell nuclear again.Can obtain to be applicable to the shellfish sample Flow cytometry ploidy, that can preserve at normal temperatures or transport for long-distance.
Sample retention 6 months.Utilize machine testing on the flow cytometer, the recall rate of sample is 100%.
Embodiment 3
Utilize the inventive method, carry out the specimen preparation of the polyploid children shellfish of haliotis discus hannai Ino, concrete operations are:
1) obtains each 30 sample of the triploid young shellfish of haliotis discus hannai Ino dliploid and artificial induction respectively;
2) in young shellfish, add nucleus parting liquid 20mL (present embodiment is the PBS that contains Triton X-100 2%), adopt scalpel blade to chop young shellfish 10min, organize and cell separation, staticly settle 10min, get supernatant suspending liquid by per 1 milligram of 2mL amount;
3) then described supernatant suspending liquid is transferred to the 15mL centrifuge tube,, remove supernatant, obtain free single cell nuclear with the centrifugal 2min of 1000r/min;
4) add nucleus immobile liquid I (composition is: 90 parts of the aqueous solution of 95% ethanol, 10 parts of PBS, adjusting pH value is 8.0) 10mL toward per 1 milligram of free single cell nuclear of described collection, suspension cell is examined again;
5) with the centrifugal 2min of 1000r/min, remove supernatant, collect slender karyon once more;
6) (composition is the per 1 milligram of free single cell nuclear adding nucleus immobile liquid II that collects toward step 5): 10mL 60% ethanol water), suspension cell nuclear again.Can obtain to be applicable to the shellfish sample Flow cytometry ploidy, that can preserve at normal temperatures or transport for long-distance.
Described sample retention was utilized machine testing on the flow cytometer after 6 months, and the recall rate of sample is 100%.
Embodiment 4
Utilize the inventive method, carry out the polyploid juvenile mollusk specimen preparation of quahog, concrete operations are:
1) adopting the aperture is that the silk cover filtering of 100 μ m is removed most seawater, obtains each 200 of quahog juvenile mollusk dliploid and artificial induction triploids respectively, contains the centrifuge tube in 1.5mL, with the centrifugal 3min of 1500rpm, removes the seawater of supernatant; Amount by per 5 milligrams of 1mL adds isotonic buffer solution PBS 1mL, and (its composition is: 70～85 parts of Na
2HPO
4With 15～30 parts of NaH
2PO
4, present embodiment adopts Na
2HPO
470 parts, NaH
2PO
430 parts), with the centrifugal 3min of 1500rpm, remove supernatant, collect larva.
2) amount by per 1 milligram of 2mL adds nucleus parting liquid 10mL (present embodiment is the PBS that contains TritonX-100 3%), adopts Potter-Elvehjem Tissue Grinders to grind 7min, organizes and cell separation, staticly settles 12min, gets supernatant suspending liquid;
3) then described supernatant suspending liquid is transferred to the 15mL centrifuge tube,, remove supernatant, obtain free single cell nuclear with the centrifugal 3min of 1500r/min;
4) add nucleus immobile liquid I (composition is: 70 parts of the aqueous solution of 85% ethanol, 30 parts of PBS, adjusting pH value is 7.0) 15mL toward per 1 milligram of free single cell nuclear of described collection, suspension cell is examined again;
5) with the centrifugal 3min of 1500r/min, remove supernatant, collect slender karyon once more;
6) add nucleus immobile liquid II (adopting 70% ethanol water) 15mL, suspension cell nuclear again toward the collected per 1 milligram of free single cell nuclear of step 5).Can obtain to be applicable to the shellfish sample Flow cytometry ploidy, that can preserve at normal temperatures or transport for long-distance.
Described sample retention was utilized machine testing on the flow cytometer after 6 months, and the recall rate of sample is 100%.
Embodiment 5
Utilize the inventive method, the polyploid that carries out the mud blood clam becomes the shellfish specimen preparation, and concrete operations are:
1) uses dissecting scissors and dissecting forceps, obtain gill tissue from mud blood clam dliploid and triploid each 20 Cheng Beizhong of artificial induction respectively;
2) toward the amount adding nucleus parting liquid 15mL (present embodiment be the PBS that contain Triton X-100 5%) of the gill tissue that obtains, adopt scalpel blade to chop 10min, organize and cell separation, staticly settle 5min, get supernatant suspending liquid by per 1 milligram of 3mL;
3) then described supernatant suspending liquid is transferred to the 15mL centrifuge tube,, remove supernatant, obtain free single cell nuclear with the centrifugal 3min of 2000r/min;
4) add nucleus immobile liquid I (composition is: 70 parts of the aqueous solution of 90% ethanol, 30 parts of PBS, adjusting pH value is 7.8) 8mL toward per 1 milligram of free single cell nuclear of described collection, suspension cell is examined again;
5) with the centrifugal 3min of 2000r/min, remove supernatant, collect slender karyon once more;
6) add nucleus immobile liquid II (composition is: composition is the ethanol water of 60～80% concentration, and present embodiment adopts 75% ethanol water) 8mL, suspension cell nuclear again toward the collected per 1 milligram of free single cell nuclear of step 5).Can obtain to be applicable to the shellfish sample Flow cytometry ploidy, that can preserve at normal temperatures or transport for long-distance.
Described sample retention was utilized machine testing on the flow cytometer after 6 months, and the recall rate of sample is 100%.
Claims (7)
1. a shellfish sample preparation methods that is applicable to the Flow cytometry ploidy is characterized in that: obtain biological sample from shellfish to be detected, prepare the slender karyon suspending liquid of shellfish sample, carry out nucleus and fix; Specifically operation as follows:
1) obtains biological sample from larva, juvenile mollusk, young shellfish or one-tenth shellfish;
2) add every milligram of 1～3mL nucleus parting liquid in the biological sample, vibrate, grind or chop 5～10min, be prepared into the homogenate shape, staticly settle 5～15min, supernatant suspending liquid;
3) described supernatant suspending liquid is transferred to centrifuge tube,, remove supernatant, collect free slender karyon with the centrifugal 1～5min of 1000～3000r/min;
4) add nucleus immobile liquid I in described slender karyon, addition is every milligram of slender karyon 5～15mL, again suspension cell nuclear;
Wherein: nucleus immobile liquid I is number percent meter by volume, and its composition is that ethanol water and 10～30 parts of isotonic buffer solutions of 70～90 part of 80～95% concentration of volume percent are formed; Potential of hydrogen is 7.0～8.0;
5) with the centrifugal 1～5min of 1000～3000r/min, remove supernatant, collect free slender karyon;
6) add nucleus immobile liquid II in the described slender karyon of step 5), addition is every milligram of slender karyon 5～15mL, and suspension cell nuclear is made and can be preserved at normal temperatures or transport for long-distance, to be ready for use on the slender karyon sample that flow cytometer detects;
Wherein: nucleus immobile liquid II is number percent meter by volume, and its composition is the ethanol water of 60～80% concentration.
2. according to the described shellfish sample preparation methods that is applicable to the Flow cytometry ploidy of claim 1, it is characterized in that: biological sample comprises larva, juvenile mollusk, the young shellfish of shellfish or becomes whole soft bodies or the portion of tissue of shellfish that suitable tissue comprises gill tissue, closed shell flesh and outer embrane.
3. according to the described shellfish sample preparation methods that is applicable to the Flow cytometry ploidy of claim 1, it is characterized in that: obtain larva, juvenile mollusk sample employing bolting silk net filtration and centrifugation method, the aperture of used bolting silk net is 20～100 μ m.
4. according to the described shellfish sample preparation methods that is applicable to the Flow cytometry ploidy of claim 1, it is characterized in that: obtain young shellfish or become the tissue of shellfish to adopt anatomic method, used dissecting tool is for dissecting scissors or dissecting forceps.
5. according to the described shellfish sample preparation methods that is applicable to the Flow cytometry ploidy of claim 1, it is characterized in that: when the biological sample that obtains is larva, juvenile mollusk, add isotonic buffer solution 0.2～1mL by per 1 milligram of sample in the process of obtaining.
6. according to the described shellfish sample preparation methods that is applicable to the Flow cytometry ploidy of claim 1, it is characterized in that: described nucleus parting liquid, number percent meter by volume, its composition is to contain 1～10 part of Triton X-100 in per 100 parts of isotonic buffer solutions.
7. according to claim 5 or the 6 described shellfish sample preparation methods that are applicable to the Flow cytometry ploidy, it is characterized in that: described isotonic buffer solution PBS, number percent meter by volume, its composition is by 70～85 parts of Na
2HPO
4With 15～30 parts of NaH
2PO
4Form.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US11666689B2 (en) | 2016-11-09 | 2023-06-06 | Lilu, Inc. | Automated compression nursing and pumping system |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101858833B (en) * | 2010-05-21 | 2011-11-09 | 王秋艳 | Paralytic shellfish poisoning (PSP) standard sample and preparation method and application thereof |
| CN102706707A (en) * | 2012-05-30 | 2012-10-03 | 徐君怡 | Standard sample of diarrhetic shellfish toxin and preparation method of standard sample |
| CN104568539B (en) * | 2014-12-16 | 2017-05-10 | 何向锋 | Automatic histocyte separator |
| CN106754884B (en) * | 2017-01-10 | 2020-06-09 | 天根生化科技（北京）有限公司 | Kit and application thereof |
| CN106868148B (en) * | 2017-03-08 | 2021-02-26 | 中国科学院苏州生物医学工程技术研究所 | Preparation method of cell nucleus with controllable aggregation state and long-term preservation |
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| US11666689B2 (en) | 2016-11-09 | 2023-06-06 | Lilu, Inc. | Automated compression nursing and pumping system |
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