CN106932330A - A kind of sperm DFI detection methods based on flow cytometry - Google Patents
A kind of sperm DFI detection methods based on flow cytometry Download PDFInfo
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Abstract
The invention discloses a kind of sperm DFI detection methods based on flow cytometry, feature is to comprise the following steps：(1) liquefied seminal fluid is diluted into sperm to 1~2 × 10 with 37 DEG C of buffer solutions6Individual/mL；(2) sperm that 500 μ L diluted is added in flow cytometer loading pipe, adds acidification buffer solution, AO dyeing liquors are added after 30 seconds；(3) calibration of flow cytometer is set, by machine in detection pipe, often twice, often pipe sample is at least recorded 5000 cells and analyzed using DFIView software statistics pipe sample at least METHOD FOR CONTINUOUS DETERMINATION；(4) how result judgement is carried out；(5) after determining, thoroughly removed with bleaching agent, sample feeding pipe cleaning agent and degerming distilled water respectively and residue in cell fragment and fluorescent dye that flow cytometer enters in line-transect, advantage is simple to operate, testing result is accurate, reliable and stable, reproducible and be easy to clinical expansion.
Description
Technical field
The present invention relates to external diagnosis reagent field, more particularly, to a kind of sperm DFI detections based on flow cytometry
Method.
Background technology
, mainly for the DNA fragmentations of sperm, these testing results can not comprehensively, exactly for traditional seminal fluid detection method
Reflection sperm situation.The major function of sperm is the hereditary information for retaining and transmitting former generation, and DNA is present in sperm body, quilt
Nucleoprotamine parcel is in fine and close structure.DNA is a kind of long-chain polymer, and composition unit is four kinds of deoxynucleotides, i.e.,：Gland is fast
Purine deoxynucleotide (dAMP), thymidylic acid (dTMP), deoxycytidylic acid (dCMP), guanine deoxidation
Nucleotides (dGMP).And deoxyribose (pentose) is connected with phosphoric acid molecules by ester bond, its chain backbone long is constituted, arrangement is outside
Side, four kinds of bases are arranged in inner side, it then follows base pair complementarity principle.One of which in each glycan molecule and four kinds of bases
It is connected, the sequence that these bases are arranged along DNA long-chains can constitute genetic code, instruct the synthesis of protein.
DNA stores the hereditary information that organism depends on for existence and multiplies, therefore safeguards the integrality of DNA molecular to cell
It is most important.Factor inside external environment and organism all often causes the damage or change of DNA molecular, and and RNA
And protein can largely synthesize difference in the cell, there was only portion DNA typically in a prokaryotic, in eucaryon dliploid
Also only a pair of identical DNA in cell, if the damage of DNA or the change of hereditary information can not be corrected, can to body cell
Its function or existence can be influenceed, its offspring may then be had influence on to reproduction cell.DNA forms double helix knot in normal state
Structure, therefore, under normal condition, DNA molecular is complete duplex structure, and DNA can cause DNA to damage due to many reasons in vivo
Wound, and procreations of the DNA for damaging to bion produces significant impact；Therefore be to mean a great to sperm DNA integrity detection
's.Traditional sperm DFI detections depend on acridine orange detection method, and by micro- sem observation sperm sample, by recognizing fluorescence
Color and count and calculate DFI values.Our company is processed sperm sample by DFI kits, is dyeed, then by streaming
Cell method carries out automated analysis to sample, and the method testing result excessively relies on manual operation, and testing result subjectivity is strong；Behaviour
Make process cumbersome, detection efficiency is low, poor repeatability, the poor feature of sensitiveness.
The content of the invention
The technical problems to be solved by the invention are to provide that a kind of simple to operate, result is easy to judge, testing result is accurate,
The sperm DFI detection methods based on flow cytometry that are reliable and stable, reproducible and being easy to clinical expansion.
The present invention solve the technical scheme that is used of above-mentioned technical problem for：A kind of sperm DFI based on flow cytometry
Detection method, comprises the following steps：
(1) liquefied seminal fluid is diluted into sperm to 1~2 × 10 with 37 DEG C of buffer solutions6Individual/mL；
(2) sperm that 500 μ L diluted is added in flow cytometer loading pipe, adds acidification buffer solution (main
It is hydrochloric acid to want composition, it is therefore an objective to close DNA is become structure more loose, is carried out in order to follow-up staining procedure), after 30 seconds
Add AO dyeing liquors；
(3) set the calibration of flow cytometer, by machine in detection pipe, often pipe sample at least METHOD FOR CONTINUOUS DETERMINATION twice, every pipe sample
This is at least recorded 5000 cells of sample main group and is analyzed using DFIView software statistics；
(4) dye after spermatoblast suspension by flow cytometer collect sperm red green fluorescence signal (i.e. FITC and
PerCP fluorescence signals), the ratio for collecting sperm sum is accounted for by the red fluorescence sperm being collected into, judge the DNA fragments of sample
Change degree；As a result, in the image collection template set up, the requirement mainly to fluorescence intensity is (according to different instruments for basis for estimation
Collection condition difference sets up corresponding template, specifically includes, and by taking Calibur instruments as an example, FITC fluorescence intensity medians are in
The middle part of capture range is that 400~600, PerCP fluorescence intensity medians are 50~100；
(5) after determining, streaming is residued in the thorough removing of bleaching agent, sample feeding pipe cleaning agent and degerming distilled water respectively thin
Cell fragment and fluorescent dye that born of the same parents' instrument enters in line-transect.
Buffer solution described in step (1) is sodium chloride, potassium dihydrogen phosphate, glucose, potassium chloride, sour magnesium, calcium chloride, N-
The mixture of 2- hydroxyethyl piperazine-N-2- ethyl sulfonic acids disodium salt, sodium acid carbonate, Sodium Pyruvate, bovine serum albumin(BSA) and sodium lactate.
Described acidification buffer solution is 0.08mol/L hydrochloric acid.Acridine orange dye and double-strand are utilized after acidification
DNA combinations green-emitting or yellow fluorescence, the characteristic of hair red fluorescence is combined with single stranded DNA, determines DNA fragment with spermatozoon degree.
Compared with prior art, the advantage of the invention is that：A kind of sperm DFI detections based on flow cytometry of the present invention
Method, is processed sperm sample by DFI kits, is dyeed, including sperm DNA fluorescence dye liquor and dye solution.It is logical
Cross machine testing DNA fragment with spermatozoon rate in fluorescence labeling, streaming, so as to assess the DNA damage degree of sperm, be sterility inspection,
The selection of assisted reproductive therapy scheme provides help.Manual operation is excessively relied on this method avoid traditional technique in measuring result,
Testing result subjectivity is strong；Operating process is cumbersome, and detection efficiency is low, and poor repeatability, the poor feature of sensitiveness realizes sperm
Simple, quick, accurate, the high flux of DFI detections are detected and calculate the purpose of DFI.
Brief description of the drawings
Fig. 1 is spermatoblast group's figure, and spermatoblast main group is with region in door circle in figure；
Fig. 2 is spermatoblast group's luciferase expression figure, and three groups of cells are divided into figure, and the group near FITC (longitudinal axis) is DNA
The good cell mass of integrality, the cell mass of DNA integralities difference is near the cell mass of PerCP (transverse axis), positioned at two groups of centres
Cell mass be the poor cell mass of integrality.
Specific embodiment
The present invention is described in further detail below in conjunction with accompanying drawing embodiment.
Specific embodiment
A kind of sperm DFI detection methods based on flow cytometry, comprise the following steps：
(1) liquefied seminal fluid is diluted into sperm to 1~2 × 10 with 37 DEG C of buffer solutions6Individual/mL；Wherein buffer solution is chlorination
Sodium, potassium dihydrogen phosphate, glucose, potassium chloride, sour magnesium, calcium chloride, N-2- hydroxyethyl piperazine-N-2- ethyl sulfonic acids disodium salt, carbonic acid
The mixture of hydrogen sodium, Sodium Pyruvate, bovine serum albumin(BSA) and sodium lactate；
(2) sperm that 500 μ L diluted is added in flow cytometer loading pipe, addition acidification buffer solution (is
0.08mol/L hydrochloric acid, it is therefore an objective to make close DNA become structure more loose, carried out in order to follow-up staining procedure), 30 seconds
AO dyeing liquors are added afterwards；Acridine orange dye and double-stranded DNA combination green-emitting or yellow fluorescence are utilized after acidification, it is and single-stranded
DNA combines the characteristic of hair red fluorescence, determines DNA fragment with spermatozoon degree；
(3) set the calibration of flow cytometer, by machine in detection pipe, often pipe sample at least METHOD FOR CONTINUOUS DETERMINATION twice, every pipe sample
This is at least recorded 5000 cells of sample main group and is analyzed using DFIView software statistics；
(4) dye after spermatoblast suspension by flow cytometer collect sperm red green fluorescence signal (i.e. FITC and
PerCP fluorescence signals), the ratio for collecting sperm sum is accounted for by the red fluorescence sperm being collected into, judge the DNA fragments of sample
Change degree；As a result, in the image collection template set up, the requirement mainly to fluorescence intensity is (according to different instruments for basis for estimation
Collection condition difference sets up corresponding template, specifically includes, and by taking Calibur instruments as an example, FITC fluorescence intensity medians are in
The middle part of capture range is that 400~600, PerCP fluorescence intensity medians are 50~100；As shown in figure 1, being spermatoblast group
Figure, spermatoblast main group is in figure with region in door circle；Fig. 2 show spermatoblast group's luciferase expression figure, is divided into figure
Three groups of cells, the group near FITC (longitudinal axis) is the good cell mass of DNA integralities, and the cell mass near PerCP (transverse axis) is
It is the cell mass of DNA integralities difference, the cell mass positioned at two groups of centres is the poor cell mass of integrality；
(5) after determining, streaming is residued in the thorough removing of bleaching agent, sample feeding pipe cleaning agent and degerming distilled water respectively thin
Cell fragment and fluorescent dye that born of the same parents' instrument enters in line-transect.
Certainly, described above not limitation of the present invention, the present invention is also not limited to the example above.The art
Change, remodeling, addition or replacement that those of ordinary skill makes in essential scope of the invention, should also belong to protection of the present invention
Scope.
Claims (4)
1. a kind of sperm DFI detection methods based on flow cytometry, it is characterised in that comprise the following steps：
(1) liquefied seminal fluid is diluted into sperm to 1~2 × 10 with 37 DEG C of buffer solutions6Individual/mL；
(2) sperm that 500 μ L diluted is added in flow cytometer loading pipe, acidification buffer solution is added, after 30 seconds
Add AO dyeing liquors；
(3) calibration of flow cytometer is set, by machine in detection pipe, often twice, often pipe sample is extremely for pipe sample at least METHOD FOR CONTINUOUS DETERMINATION
5000 cells of sample main group are recorded less and are analyzed using DFIView software statistics；
(4) the spermatoblast suspension after dyeing collects the red green fluorescence signal of sperm by flow cytometer, by what is be collected into
Red fluorescence sperm accounts for the ratio for collecting sperm sum, judges the DNA degree of fragmentation of sample；
(5) after determining, thoroughly removed with bleaching agent, sample feeding pipe cleaning agent and degerming distilled water residue in flow cytometer respectively
The cell fragment and fluorescent dye entered in line-transect.
2. a kind of sperm DFI detection methods based on flow cytometry according to claim 1, it is characterised in that：Step
(1) buffer solution described in is sodium chloride, potassium dihydrogen phosphate, glucose, potassium chloride, sour magnesium, calcium chloride, N-2- ethoxy piperazines
The mixture of piperazine-N-2- ethyl sulfonic acids disodium salt, sodium acid carbonate, Sodium Pyruvate, bovine serum albumin(BSA) and sodium lactate.
3. a kind of sperm DFI detection methods based on flow cytometry according to claim 1, it is characterised in that：Step
(1) buffer solution described in is sodium chloride, potassium dihydrogen phosphate, glucose, potassium chloride, sour magnesium, calcium chloride, N-2- ethoxy piperazines
The mixture of piperazine-N-2- ethyl sulfonic acids disodium salt, sodium acid carbonate, Sodium Pyruvate, bovine serum albumin(BSA) and sodium lactate.
4. a kind of sperm DFI detection methods based on flow cytometry according to claim 1, it is characterised in that：It is described
Acidification buffer solution be 0.08mol/L hydrochloric acid.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108398407A (en) * | 2018-01-25 | 2018-08-14 | 迈克博（天津）生物科技有限公司 | A kind of human sperm's motility rate and its kit of the two-parameter detection of DNA damage and analysis |
| CN111307696A (en) * | 2020-03-19 | 2020-06-19 | 浙江星博生物科技股份有限公司 | Method and kit for detecting sperm DNA fragmentation rate |
| CN111307694A (en) * | 2020-03-04 | 2020-06-19 | 浙江星博生物科技股份有限公司 | Flow cytometry detection method for DNA fragments of active sperms |
| CN113432959A (en) * | 2021-05-21 | 2021-09-24 | 赛雷纳（中国）医疗科技有限公司 | Preparation method of quality control product for sperm DNA fragmentation detection |
| CN114136866A (en) * | 2021-10-28 | 2022-03-04 | 桂林优利特医疗电子有限公司 | Sperm nucleus DNA integrity detection method and detection kit |
| CN116908078A (en) * | 2023-09-14 | 2023-10-20 | 苏州贝康医疗器械有限公司 | Method, device, storage medium and equipment for detecting sperm DFI |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108398407A (en) * | 2018-01-25 | 2018-08-14 | 迈克博（天津）生物科技有限公司 | A kind of human sperm's motility rate and its kit of the two-parameter detection of DNA damage and analysis |
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| CN111307694A (en) * | 2020-03-04 | 2020-06-19 | 浙江星博生物科技股份有限公司 | Flow cytometry detection method for DNA fragments of active sperms |
| CN111307696A (en) * | 2020-03-19 | 2020-06-19 | 浙江星博生物科技股份有限公司 | Method and kit for detecting sperm DNA fragmentation rate |
| CN113432959A (en) * | 2021-05-21 | 2021-09-24 | 赛雷纳（中国）医疗科技有限公司 | Preparation method of quality control product for sperm DNA fragmentation detection |
| CN114136866A (en) * | 2021-10-28 | 2022-03-04 | 桂林优利特医疗电子有限公司 | Sperm nucleus DNA integrity detection method and detection kit |
| CN116908078A (en) * | 2023-09-14 | 2023-10-20 | 苏州贝康医疗器械有限公司 | Method, device, storage medium and equipment for detecting sperm DFI |
| CN116908078B (en) * | 2023-09-14 | 2023-12-05 | 苏州贝康医疗器械有限公司 | Method, device, storage medium and equipment for detecting sperm DFI |
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