CN107058547A - A kind of detection method of sperm - Google Patents
A kind of detection method of sperm Download PDFInfo
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- CN107058547A CN107058547A CN201710294056.6A CN201710294056A CN107058547A CN 107058547 A CN107058547 A CN 107058547A CN 201710294056 A CN201710294056 A CN 201710294056A CN 107058547 A CN107058547 A CN 107058547A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The present invention provides a kind of detection method of sperm, it is characterised in that comprise the following steps：A. DNA is extracted from seminal fluid using extracts kit;B. FD H capture probes are used to capture the full extron of the DNA, the promoter intron sequences related to genetic disease and build storehouse；C. two generation microarray datasets are used, the DNA is sequenced, the sequence information of the DNA is obtained；D. under sequencing result after machine, the result judgement gene mutation situation according to sequencing；E. compare the information of mutator and database, judge mutator and hereditary disease correlation and risk.This method is using sperm DNA as detection object, for carrying out specifically capture and information analysis with hereditary disease correlated series, incoherent DNA information is have ignored, testing result is accurate, complete detection relative to DNA not only accelerates detection speed, and improves specificity and sensitivity.
Description
Technical field
The present invention relates to technical field of medical equipment, especially a kind of detection method of sperm.
Background technology
Fertility is the sole mode that the mankind multiply and live, passed on from generation to generation.Counted according to the World Health Organization, married couple is about
10-15% suffers from sterility, wherein 1/3 caused by male factor.The infertile number of current China is up to 50,000,000, wherein male
Factor accounting is up to 50%, and this kind of family can be relied on by auxiliary procreation technology after the sperm procreation of anonymous donations in sperm bank
In generation, implement with the landing of " comprehensive two child " policy, the demand of sperm is necessarily ramped.Meanwhile, social civilization constantly enters
At present, people have higher serious hope to the speciality of offspring to step, thus in human sperm bank the quality and quantity of sperm guarantor
Barrier is most important during supplementary reproduction.
The gene of mutation is by changing the quality and quantity of polypeptide chain so that defect occurs for protein, thus triggers disease, is referred to as
Hereditary disease.The generation of disease is only by a pair of alleles control, referred to as single gene inheritance disease.It is transmitted in a simple form, its
Genetic development follows Mendelism, so also known as Mendelian inheritance disease.Dyeing of the single gene inheritance disease according to residing for gene
The difference of body and mode of inheritance can be divided into autosomal dominant（Autosomal Dominant, AD）Hereditary disease, autosome
It is recessive（Autosomal Recessive, AR）Hereditary disease, X are chain dominant（X-linked Dominant）Hereditary disease, X are chain
It is recessive（X-linked Recessive）Hereditary disease and y linkage hereditary disease.The single gene inheritance disease having now been found that is about
Kind more than 3000, the overwhelming majority is rare disease, and is being incremented by every year with the discovery speed of 10-50 kinds, single gene inheritance disease, especially
It is recessive hereditary disease, and the health to modern humans has constituted larger threat.
In addition to clear and definite Mendelian inheritance disease, medical research shows, the formation of a large amount of diseases is nature-nurture factor
Coefficient result.Some common diseases such as heart disease, hypertension, diabetes, rheumatoid arthritis etc., its risk
Genotype with itself is closely related.Meanwhile, some cancers kinds cancer such as breast cancer, colon cancer, prostate cancer, stomach cancer
Generation all have significant familial aggregation and heredity.
Influenceed by heredity and mutation rule, some diseases show as the normal carrier of phenotype, this kind of people carries the base that causes a disease
Cause, itself does not fall ill but Disease-causing gene can be transmitted to offspring.When men and women are Disease-causing gene carrier, filial generation just has hair
The risk of disease.Carrier generally comprises：It is still normal tardy outer that recessive hereditary disease heterozygote, dominant hereditary disease do not show person, phenotype
Aobvious person；Translocation carrier.
Due to subjective or objective factor, contribute in smart volunteer and there may be quite a few hereditary patient or carrier,
Some hereditary diseases such as colour blindness, is just showed at birth, and this kind of disease is relatively easily found by objective judgement method.Some tables
The normal carrier of type by more accurate more sensitive detection means, it is necessary to be judged, with the heredity that south China is more universal
Exemplified by sick thalassemia and G 6 PD deficiency disease, according to data, in α the and β ground of south China each province
Extra large anemia incidence accounts for the 8-12% of crowd.Receive before donations, essence person can be supplied by some by carrying out family history investigation or blood test
Foreclose, but still there are a large amount of " Healthy Peoples " without obvious family history to belong to thalassemia or G 6 PD lacks
Weary disease patient.In addition, some diseases can't just show morbid state but have the property delayed and neurological susceptibility feature at birth, such as
The Hazard ratio ordinary people that the people of carrying BRAC1 gene mutations suffers from breast cancer is higher, by the accumulation of quite a while, waits variation
Be added to certain amount triggering canceration key point can just fall ill, this kind of donor's health by common detection methods generally also
It is difficult to judge.
Therefore, how objective sperm bank is, accurate evaluation sperm donor donations sperm quality, excludes and contributes essence person and suffer from list base
Because of, polygenes and chromosomal disorder or associated morbidity site is carried, hereditary disease risk is suffered to reduction fertilization person's filial generation to closing weight
Will, it is related to the China human mortality prenatal and postnatal care matter of fundamental importance.
At present, sperm bank can carry out following check before sperm Donation is received to contributor：
1) family line investigation：Lineal and collateral line familial inheritance medical history investigation within three generations is carried out, facing for all kinds of hereditary diseases is explained in detail
Bed performance, carries out pedigree analysis on the basis of family line investigation, according to mendel's law to the phenotype of each member and
Genotype is analyzed, and hereditary disease risk is carried to judge to contribute smart person.Hereditary disease has diversity and sporadic, in family line investigation
When, expand the species of investigation disease as far as possible, at utmost to reduce possible hereditary disease risk.
2) auxiliary examination：Essence person is contributed with the presence or absence of colour blindness, anomalous trichromatism or stealthy colour blindness using color vision plate inspection；To build
Overweight people carries out blood pressure and blood glucose measurement, excludes hypertension and hyperglycemic patients；Excluded by physical examination and contribute whether essence person has
The hereditary diseases such as harelip, hypospadia, optic atrophy；Semen efamination, infectious disease inspection.
3) laboratory examination：Using PBLC culture, the aobvious bands of G carry out chromosome karyotype analysis.Chromosome core
Type analysis：Chromosome abnormality is the primary pathogenic event of male sterility, though balance of chromosome dystopy carrier and inversion carrier
Right phenotype is normal, but can cause infertility, miscarriage, stillbirth, neonatal death, congenital abnormality and feeblemindedness etc., therefore if any
This situation, it is necessary to exclude, it is impossible to as tax essence person.Various chromosome morphology Minor variations are also shown in normal population, these are small
Variation to that itself may not cause harm, but the problem of may produce unexpected to offspring, therefore, to any chromosome core
The abnormal tax essence person of type is excluded.
The content of the invention
In order to solve the above-mentioned technical problem, the invention provides a kind of detection method of sperm, it can be to the micro- of sperm
Small variation is judged, and judges whether that offspring can be entailed, and it is more comprehensively more accurate to detect.
A kind of detection method of sperm, comprises the following steps：
A. DNA is extracted from seminal fluid using extracts kit;
B. full extron, promoter and the introne of genetic correlation of the DNA is captured using FD-H capture probes and builds storehouse；
C. two generation microarray datasets are used, the DNA is sequenced, the full length sequence of the DNA is obtained；
D. under sequencing result after machine, the result judgement gene mutation situation according to sequencing；
E. compare the information of mutator and database, judge the influence of mutator.
Further, the two generations microarray dataset is Illumina bis- generations microarray datasets.
Further, the database is HGMD or ClinVar.
Further, introne and startup of the FD-H capture probes comprising full extron full sequence and genetic correlation
Sub- trapping region.
Further, the trapping region quantity that the capture probe is included is N, 350000<N<500000.
Further, step F. is also included after step E according to the influence of mutator, warning prompting message is provided.
Further, the warning prompting message includes the species and risk of hereditary disease, the hereditary disease species bag
Include single gene inheritance disease, disease of multifactorial inheritance and mitochondrial inherited disease；The risk includes person under inspection and offspring suffers from
Sick probability.
Using the above method, the present invention has following technique effect：
1. using full extron, promoter and the introne of genetic correlation in FD-H capture probes capture person under inspection's sperm DNA
Sequence simultaneously builds storehouse, and it is specifically captured for the related site of genetic disease, by analyzing variation information, have ignored
Incoherent DNA information, the complete detection relative to DNA not only accelerates detection speed, and greatly improves specificity
And sensitivity.
2. detection platform is Illumina bis- generations microarray datasets, realize that high flux is quickly sequenced, with reference to FD-H capture probes,
Faster, cost is lower for detection speed.
3. database is HGMD or ClinVar, using internationally recognized authoritative public database, ensureing that information is reliable
Meanwhile, and effectively reduce cost.
4. the trapping region quantity that capture probe is included is N, 350000<N<500000, according to heredity DNA characteristic,
350000<N<500000, faster capture, sequencing speed can be not only ensured, and the comprehensive of hereditary information can be ensured, if
N<350000, then capture region is very few, may detect not comprehensive to the hereditary information of sperm, if N>500000, then may shadow
Ring detection speed, increase cost.
5. when mutator can be hereditary, provide warning prompting message.Remind testing staff's sperm undesirable, from
And prevent from mutator entailing the next generation, effective guarantee contributes sperm quality.
Fig. 1 is the flow chart of embodiment one.
Specific embodiment
The technical scheme of the embodiment of the present invention is explained and illustrated with reference to the accompanying drawing of the embodiment of the present invention, but under
Embodiment only the preferred embodiments of the present invention are stated, and it is not all.Based on the embodiment in embodiment, people in the art
Member obtains other embodiment on the premise of creative work is not made, and belongs to protection scope of the present invention.
Embodiment one：
As shown in figure 1, a kind of detection method of sperm, comprises the following steps：
A. DNA is extracted from seminal fluid using extracts kit；
B. the present embodiment is directed to the characteristic of sperm DNA, devises FD-H capture probes, and the capture probe is whole comprising full extron
The introne and promoter trapping area of sequence and genetic correlation, its total size 70M, comprising 22000 genes of gene, 410000 catch
Area is obtained, full extron, promoter and the introne of genetic correlation of the DNA is captured using the capture probe and storehouse is built；
C. Illumina bis- generations microarray datasets are used, the DNA is sequenced, the full length sequence of the DNA is obtained；
D. under sequencing result after machine, according to the result of sequencing, with reference to bioinformatics, genetic correlation gene mutation situation is analyzed,
Determine whether that gene is undergone mutation, which gene is undergone mutation；
E. compare the information of mutator and HGMD or ClinVar databases, judge whether mutator can be hereditary, if mutation
Gene is in HGMD or ClinVar databases, then the consequence being likely to result according to the mutator, judges shadow of the disease to offspring
The degree of sound.
F. according to influence degree of the disease to offspring, then warning prompting message is provided, the information includes the grade of disease, lost
The species of disease and the gene of variation are passed, hereditary disease species specifically includes single gene inheritance disease, disease of multifactorial inheritance and mitochondria and lost
Pass disease.
Full extron, promoter and the introne of genetic correlation of the DNA is captured using capture probe and storehouse is built, its
Captured for the heredity part of sperm, so that the information to heredity is analyzed, have ignored incoherent DNA information, examine
Survey result accurately and reliably, the complete detection relative to DNA not only accelerates detection speed, and improves specific and sensitive
Degree.When mutator can be hereditary, warning prompting message is provided.Remind testing staff's sperm undesirable, so as to prevent
Mutator is entailed into the next generation.Sequencing analysis are captured by the FD-H target areas of autonomous Design, comprehensively detection detection model
Interior heredity and the disease relevant with inherent cause are enclosed, hereditary variation is transmitted to offspring when reduction for essence by implementing assisted reproduction
Probability, prevent hereditary disease, reach the purpose of prenatal and postnatal care.
The following is the 3 carrier of inheritance disease's embodiments filtered out from 20 male volunteers samples of early stage, using this
The detection product that embodiment is provided is detected and does disease risks assessment, as shown in the table：
| Name | Age | Sex | Disease | Gene | Variation | Disease is parsed |
| YQ | 28 | Man | Galactolipin Mass formed by blood stasis | GALE | p.W336X | Galactosemia is a kind of autosomal recessive inheritance metabolism caused by the functional defect of enzyme in galactose metabolism Property disease.This disease is more to there is symptom in 4~10 days, if cannot correctly rescue in time, will cause serious Nervous system sequelae or death.The mutation Clinvar be not recorded as galactosemia it is doubtful it is pathogenic because Element, thus it is speculated that the mutation may be detrimental mutation.The variation may be hereditary, causes the increase of offspring's risk. |
| TBG | 32 | Man | Heredity Tumour is comprehensive Simulator sickness | BRCA2 | p.Q1037X | The mutation is expressly recited in Clinvar databases is recited as hereditary tumor syndrome pathogenic sites, causes breast The tumours such as gland cancer, oophoroma occur, thus it is speculated that variation can increase risk.The variation may be hereditary, after causing For risk increase. |
| RQ | 20 | Man | Colon and rectum Cancer/stomach cancer | MUTYH | c.934-2A >G | The mutation is recited as the doubtful pathogenic factor of MUTYH correlation polyposises in Clinvar databases, points out meeting Causing the risk of colorectal cancer and stomach cancer increases.The variation may be hereditary, causes offspring's risk to increase Plus. |
It is appreciated that trapping region is not limited to 410000, according to specific detection speed and comprehensive demand, people in the art
Member can suitably be adjusted as needed.Trapping region N can not only ensure faster capture, sequencing speed in 350000-500000
Degree, and comprehensive capture of hereditary information can be ensured, if N<350000, then capture region is very few, may be to the something lost of sperm
Infomation detection is passed not comprehensive, if N>500000, then it may influence detection speed.
It is appreciated that the microarray dataset of the present embodiment is not limited to illumina or 454, Life
The two generations high-flux sequence platform such as Technologies, or from three generations's microarray dataset, such as Pacbio main flows or other
The sequencing data that high-flux sequence platform is produced.In addition it is also possible to be the sequence data that genetic chip sequencing is produced.
As described above, only presently preferred embodiments of the present invention, not for limiting the practical range of the present invention, i.e.,
All equivalent changes and modifications made according to the present invention, are all that scope of the invention as claimed is covered, and no longer one at one stroke here
Example.
Claims (7)
1. a kind of detection method of sperm, it is characterised in that comprise the following steps：、
A. DNA is extracted from seminal fluid using extracts kit;
B. full extron, promoter and the introne of genetic correlation of the DNA is captured using FD-H capture probes and builds storehouse；
C. two generation microarray datasets are used, the DNA is sequenced, the full length sequence of the DNA is obtained；
D. under sequencing result after machine, the result judgement gene mutation situation according to sequencing；
E. compare the information of mutator and database, judge the influence of mutator.
2. the detection method of sperm according to claim 1, it is characterised in that the two generations microarray dataset is Illumina
Two generation microarray datasets.
3. the detection method of sperm according to claim 1, it is characterised in that the database is HGMD or ClinVar.
4. the detection method of sperm according to claim 1, it is characterised in that the FD-H capture probes are comprising complete outer aobvious
The introne and promoter trapping area of sub- full sequence and genetic correlation.
5. the detection method of sperm according to claim 4, it is characterised in that the trapping region number that the capture probe is included
Measure as N, 350000<N<500000.
6. the detection method of sperm according to claim 1, it is characterised in that the detection method is gone back after step E
Including influences of the step F. according to mutator, warning prompting message is provided.
7. the detection method of sperm according to claim 6, it is characterised in that the warning prompting message includes hereditary disease
Species and risk, the hereditary disease species include single gene inheritance disease, disease of multifactorial inheritance and mitochondrial inherited disease；Institute
Stating risk includes person under inspection and offspring's P.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112735518A (en) * | 2020-12-30 | 2021-04-30 | 武汉康圣达医学检验所有限公司 | ROH data analysis system based on chromosome microarray |
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| CN104630362A (en) * | 2015-02-06 | 2015-05-20 | 上海中优医药高科技有限公司 | Gene detection method for transcriptional regulation related gene of spermatoblast |
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| WO2016156353A1 (en) * | 2015-03-30 | 2016-10-06 | General Electric Company | Methods of capturing sperm nucleic acids |
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2017
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642932A (en) * | 2013-12-25 | 2014-03-19 | 上海中优医药高科技有限公司 | Gene detection method of sperm cell apoptosis related gene |
| CN105524981A (en) * | 2014-09-28 | 2016-04-27 | 浙江大学 | Capture kit and method of target gene |
| CN104630362A (en) * | 2015-02-06 | 2015-05-20 | 上海中优医药高科技有限公司 | Gene detection method for transcriptional regulation related gene of spermatoblast |
| WO2016156353A1 (en) * | 2015-03-30 | 2016-10-06 | General Electric Company | Methods of capturing sperm nucleic acids |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112735518A (en) * | 2020-12-30 | 2021-04-30 | 武汉康圣达医学检验所有限公司 | ROH data analysis system based on chromosome microarray |
| CN112735518B (en) * | 2020-12-30 | 2024-04-23 | 武汉康圣达医学检验所有限公司 | ROH data analysis system based on chromosome microarray |
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