CN104846103A - Application of MDPCR (multiple digital PCR (polymerase chain reaction)) technology to chromosome aneuploidy screening - Google Patents
Application of MDPCR (multiple digital PCR (polymerase chain reaction)) technology to chromosome aneuploidy screening Download PDFInfo
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Abstract
The invention provides application of an MDPCR (multiple digital PCR (polymerase chain reaction)) technology to chromosome aneuploidy screening. Concretely, the invention discloses a method for performing NIPS (non-invas ive prenatal screening) including Down's syndrome, Edward's syndrome, Patau syndrome and other chromosome abnormality diseases on pregnant women by the MDPCR technology. The method has the advantages that early, safe, non-invas, accurate and fast effects are achieved, the method is suitable for large-scale detection and clinic application, and the like; intrauterine infection, abortion and death in the pregnancy middle and later periods of the pregnant women can be avoided; the goals of bearing and rearing better children are achieved.
Description
Technical field
The invention belongs to bio-medical technology field, relate to particularly and utilize early stage, safety without wound, accurately, fast, be applicable to extensive detection and multiple digital pcr (the Multiplex digital PCR of clinical application, MDPCR) technology carries out nothing wound Prenatal Screening (Non-invasive prenatalscreening to normal pregnant women, NIPS) mongolism is comprised, Edward's syndrome, the method for handkerchief pottery Cotard and other many times of chromosomal disorders examinations.
Background technology
China is populous nation, and huge population pressure can restrict social development, and can improve the health of the people so carry out prenatal and postnatal care, also can restrict population development, to future, the survival and development of whole nationality play an important role.How unfavorable phenotypic gene frequency in reduction crowd is mainly studied in preventative prenatal and postnatal care, reduces so that eliminate and have the individuality of serious inherited disease and congenital hereditary disease to be born, mainly comprises genetic counseling, Prenatal Screening, utero treatment etc.
Intervene the data of relief fund meeting according to Birth Defects In China, the annual newly-increased inborn defect of China is about 90-120 ten thousand example, accounts for the 4-6% of annual newborn population sum.In existing more than 8,000 ten thousand Disabled personss, about 3,000 ten thousand people are caused by inborn defect.Inborn defect has become children at present and has caused a disease, disables, and even dead major reason, wherein chromosome disease is principal disease.With pregnant early stage health care and Prenatal Screening before pregnant, it is the most important outpost of the tax office of avoiding and reducing newborn infant's defect and carrying out prenatal and postnatal care.In the past, those had the ill high risk Mr. and Mrs of chromosome disease, for breeding healthy offspring, were often in the passive position having no way of selecting.Until 1966, research has found the dependency of pregnant woman's advanced age and Down's syndrome, and after this, Prenatal Screening is developed rapidly.Prenatal Screening refer in utero to the developmental condition of embryo or fetus, whether suffer from disease etc. and carry out checkout and diagnosis.Thus the opportunity of grasp, to can disease therapy, select to carry out utero treatment in time; For untreatable property disease, as chromosome disease etc., informed choice and prevention can be accomplished.
Chromosome disease, refer to chromosomal numerical abnormality and/or Morphology, can betide on every item chromosome, but be mainly in as trisomy 21 (mongolism), 18 3 bodies (Edward's syndrome), 13 3 bodies (handkerchief pottery Cotard) and sex chromosomal abnormality etc., more than 50% is accounted in dying young spontaneous abortion, stillborn foetus, morning, sickness rate about 1% in newborn infant is infertile etc. the major reason of congenital heart disease, mental retardation, sexual abnormality and men and women.Along with chromosome banding technique, round pcr, the fast development of DNA detection technology etc., deepens day by day to the understanding of chromosome aberration and disease relationship, and chromosome disease detects and increasingly increases.The gestation of about 15% is miscarried, and wherein half is caused by chromosome abnormalty, and the embryo being namely about 5%-8% has chromosome abnormalty.But in utero, more than 90% existing spontaneous abortion or stillbirth.Miscarriage more early, has the frequency of chromosome abnormalty higher.Chromosome disease, there is no effective methods for the treatment of so far, the prevention can only being undertaken to a certain degree by means such as Prenatal Screening/diagnosis.But the invasive sampling methods such as antenatal diagnosis many employings amniocentesis traditional at present, the risk of fetal in utero infection or miscarriage etc. increases.
The shortcoming of above-mentioned traditional invasive antenatal diagnosis, causes chromosome disease without the appearance of wound Prenatal Screening/diagnostic method.Method general at present comprises based on cell and method for removing cells, especially in the majority with the latter.The existence of fetus dissociative DNA in pregnant parent population blood, and the updating of DNA detection technology of new generation, make to be more prone to carry out without wound Prenatal Screening/diagnosis.Only need to take pregnant woman's venous blood, utilize DNA detection technology of new generation to carry out detection to the dissociative DNA fragment (comprising fetus dissociative DNA) in maternal peripheral blood plasma and analyze thus detect fetus whether suffer from chromosome disease.But First Trimester, mainly only accounts for the 10-20% of maternal blood DNA from the fetus dissociative DNA of placenta cells.And at present conventional DNA sequencing technology of new generation, detect consuming time longer, with complicated instrument and a large amount of DNA sequence dna of bioinformatics software analysis, detected result need be obtained 1-2 week, and expend much reagent and consumptive material, expensive.Digital pcr (digi tal PCR) is a kind of novel quantitative analytical technology that fast development is in recent years got up, by single sample is divided into more than tens thousand of, even millions of, tens million of part is distributed in different reaction members, each unit comprises the DNA profiling of nothing or one or more copy, respectively pcr amplification is carried out to these DNA profilings, then the fluorescent signal of each reaction member is analyzed.DPCR can not need reference standard sample and curve to realize absolute quantification analysis.Especially it is pointed out that and multiple PCR technique and dPCR technology are organically combined (MDPCR), just significantly can improve dPCR multiplicity and reach more than 20-50.
Therefore this area is in the urgent need to developing a kind of early stage, safety without wound, accurately, fast, be applicable to extensive detection and the novel method of clinical application.
Summary of the invention
The invention provides a kind of method of chromosomal disorders being carried out to early screening.
First aspect present invention, provides a kind of method of detection target chromosome aneuploid (Aneuploidy) of external nondiagnostic, comprises step:
A () provides the pregnant object peripheral blood sample containing fetus dissociative DNA;
B () is left away except cell from described peripheral blood sample one's duty, and from enrichment DNA the described sample removing cell, obtain DNA sample;
C described DNA sample is divided into control group and experimental group by (), wherein,
In experimental group, with described DNA sample for template, with the n1 of amplification target chromosomal gene, multiple digital pcr is carried out to the first primer pair, and record positobe focus number C1 and the ratio of primer pair quantity n1, be designated as a1, wherein a1=C1/n1, and n1 >=2, preferably, n1 >=5;
In control group, with described DNA sample for template, with the n2 of amplification reference chromogene, multiple digital pcr is carried out to the second primer pair, and record positobe focus number C2 and the ratio of primer pair quantity n2, be designated as a2, wherein a2=C2/n2, and n1 >=5, preferably, n1 >=5;
D described a1 value and a2 value compare by (), thus determine the multiple of target chromosome.
In another preference, described is diploid with reference to karyomit(e).
In another preference, described fetus and described pregnant object comprise mouse, rat or people, preferably, are people.
In another preference, in the PCR primer that the first described primer pair and/or the second primer pair increase, there are at least one primer or probe through fluorescent mark.
In another preference, described step (b) also comprises step (b1): described DNA sample is carried out fragmentation process, thus obtains the DNA sample of fragmentation.
In another preference, described fragmentation process comprises physics and biochemical treatment, as fragmentation, supersound process and enzyme are cut.
In another preference, the DNA sample size≤3000bp of described fragmentation.
In another preference, calculate a1/a2 value and be designated as R,
When R is at or about 0.5 (namely close to 0.5), then show that target chromosome described in described sample is monoploid;
When R is at or about 1 (namely close to 1), then show that described in described sample, target chromosome is identical with reference to chromosomal multiple;
When R is at or about 1.025, then show that the target chromosome only having 5% fetus dissociative DNA sample in described sample is triploid, and the target chromosome of all the other 95% pregnant parent population DNA samples is still diploid;
When R is at or about 1.05, then show that the target chromosome only having 10% fetus dissociative DNA sample in described sample is triploid, and the target chromosome of all the other 90% pregnant parent population DNA samples is still diploid;
When R is at or about 1.5 (namely close to 1.5), then show that target chromosome described in described sample is 3 times of bodies;
When R is at or about 2 (namely close to 2), then show that target chromosome described in described sample is 4 times of bodies.
In another preference, when R and 1 discrete time, then show in described sample containing many times of karyomit(e)s.
In another preference, the length of described each amplified production to the first primer is 50-150bp, preferably 60-120bp.
In another preference, described n1 is specific amplification No. 21 chromosomal primers to often pair of primer in the first primer.
In another preference, described target chromosome is the karyomit(e) that Variation in chromosome number disease can occur, and preferably includes No. 21 karyomit(e)s, No. 13 karyomit(e)s, No. 18 karyomit(e)s, sex chromosome.
In another preference, described is normal chromosomal with reference to karyomit(e).
In another preference, described comprises No. 21 karyomit(e)s, No. 13 karyomit(e)s, No. 18 karyomit(e)s with reference to karyomit(e).
In another preference, described target chromosomal gene comprises No. 21 chromosomal Usp25 (NCBI#NM_001283041), C21orf37 (NCBI#LN608865), C21orf58 (NCBI#NM_058180), Setd4 (NCBI#NM_017438) or C21orf62 (NCBI#AF231922) gene; And/or
No. 13 chromosomal Sacs (NCBI#NM_014363), Cenpj (NCBI#NM_018451), Foxo1 (NCBI#NM_002015), Smad9 (NCBI#NM_001127217) or Fgf14 (NCBI#NM_004115); And/or
No. 18 chromosomal Setbp1 (NCBI#NM_015559), Potec (NCBI#NM_001137671), Malt1 (NCBI#AB026118), Dsc3 (NCBI#NM_001941), Cdh19 (NCBI#NM_021153).
In another preference, described target chromosomal gene also comprises Noncoding gene.
In another preference, described is one or more with reference to karyomit(e).
Second aspect present invention, provides a kind of primer set of knowing clearly, and described primer set comprises following primer pair:
In (i) first aspect present invention for the described n1 of the target chromosomal gene that increases to primer pair; With
(ii) in first aspect present invention for the described n2 that increases with reference to chromogene to primer pair;
Wherein, n1, n2 difference >=2.
In another preference, described n1, n2 be at least 3 respectively, 4,5,6,7,8,9,10 right, preferably at least 5 is right.
In another preference, described target chromosome is the karyomit(e) that Variation in chromosome number disease can occur, and preferably includes No. 21 karyomit(e)s, No. 13 karyomit(e)s, No. 18 karyomit(e)s, sex chromosome.
In another preference, described is normal chromosomal with reference to karyomit(e).
In another preference, the template length that in described primer set, the often pair of primer increases is 50-150bp, preferably, is 100-120bp.
In another preference, described target chromosomal gene is selected from and comprises No. 21 chromosomal Usp25 (NCBI#NM_001283041), C21orf37 (NCBI#LN608865), C21orf58 (NCBI#NM_058180), Setd4 (NCBI#NM_017438), C21orf62 (NCBI#AF231922) gene and other gene locuss and non-genomic site; No. 13 chromosomal Sacs (NCBI#NM_014363), Cenpj (NCBI#NM_018451), Foxo1 (NCBI#NM_002015), Smad9 (NCBI#NM_001127217), Fgf14 (NCBI#NM_004115) gene and other gene locuss and non-genomic site; Or No. 18 chromosomal Setbp1 (NCBI#NM_015559), Potec (NCBI#NM_001137671), Malt1 (NCBI#AB026118), Dsc3 (NCBI#NM_001941), Cdh19 (NCBI#NM_021153) gene and other gene locuss and non-genomic site.
Also containing primer fluorescent mark on primer in primer set described in first aspect present invention.
In another preference, described primer fluorescent mark is fluorescence labeling probe.
Described in first aspect present invention for the primer pair sequence of the target chromosomal gene that increases as shown in SEQ ID NO.:1-2; And/or the described primer pair sequence for the reference chromogene that increases is as shown in SEQ ID NO.:4-5; Wherein said target chromosome is No. 21 chromosomal Usp25 genes; Described is No. 18 chromosomal Setbp1 genes with reference to karyomit(e).
In another preference, containing in the primer of fluorescence labeling probe, probe sequence is as shown in SEQ ID NO.:3 or 6.
Third aspect present invention, provides the purposes of primer set described in second aspect present invention, for the preparation of the reagent or the test kit that detect chromosome aneuploid and/or examination karyomit(e) quantity abnormal diseases.
In another preference, described karyomit(e) quantity abnormal diseases comprises monoploid disease or polyploid disease.
In another preference, described monoploid disease comprises 5q-syndrome.
In another preference, described polyploid disease comprises trisomy 21 syndrome, Patau syndrome, Edwards syndrome.
In another preference, described test kit comprises the primer set described in second aspect present invention, and specification sheets.
In another preference, described specification sheets recites following using method, comprises step:
A () provides the pregnant object peripheral blood sample containing fetus dissociative DNA;
B () is left away except cell from described peripheral blood sample one's duty, and from enrichment DNA the described sample removing cell, obtain DNA sample;
C described DNA sample is divided into control group and experimental group by (), wherein,
In experimental group, with described DNA sample for template, with the n1 of amplification target chromosomal gene, multiple digital pcr is carried out to the first primer, and record positobe focus number C1 and the ratio of primer pair quantity n1, be designated as a1, wherein a1=C1/n1, and n1 >=5;
In control group, with described DNA sample for template, with the n2 of amplification reference chromogene, multiple digital pcr is carried out to the second primer, and record positobe focus number C2 and the ratio of primer pair quantity n2, be designated as a2, wherein a2=C2/n2, and n2 >=5;
D described a1 value and a2 value compare by (), thus determine the multiple of target chromosome.
In another preference, described is diploid with reference to karyomit(e).
In another preference, calculate a1/a2 value and be designated as R,
When R is at or about 0.5 (namely close to 0.5), then show that target chromosome described in described sample is monoploid;
When R is at or about 1 (namely close to 1), then show that described in described sample, target chromosome is identical with reference to chromosomal multiple;
When R is at or about 1.025, then show that the target chromosome only having 5% fetus dissociative DNA sample in described sample is triploid, and the target chromosome of all the other 95% pregnant parent population DNA samples is still diploid;
When R is at or about 1.05, then show that the target chromosome only having 10% fetus dissociative DNA sample in described sample is triploid, and the target chromosome of all the other 90% pregnant parent population DNA samples is still diploid;
When R is at or about 1.5 (namely close to 1.5), then show that target chromosome described in described sample is 3 times of bodies;
When R is at or about 2 (namely close to 2), then show that target chromosome described in described sample is 4 times of bodies.
In another preference, the described peripheral blood sample herbal classic fetus dissociative DNA enrichment containing fetus dissociative DNA obtains.
Fourth aspect present invention, provides a kind of test kit detecting target chromosome, containing the primer set described in second aspect present invention in described test kit, and specification sheets.
Fifth aspect present invention, provide a kind of detect target chromosome aneuploid and/or carry out, without the method for wound Prenatal Screening, comprising step to chromosome disease:
A () provides the pregnant object peripheral blood sample containing fetus dissociative DNA;
B () is left away except cell from described peripheral blood sample one's duty, and from enrichment DNA the described sample removing cell, obtain DNA sample;
C described DNA sample is divided into control group and experimental group by (), wherein,
In experimental group, with described DNA sample for template, with the n1 of amplification target chromosomal gene, multiple digital pcr is carried out to the first primer, and record positobe focus number C1 and the ratio of primer pair quantity n1, be designated as a1, wherein a1=C1/n1, and n1 >=5;
In control group, with described DNA sample for template, with the n2 of amplification reference chromogene, multiple digital pcr is carried out to the second primer, and record positobe focus number C2 and the ratio of primer pair quantity n2, be designated as a2, wherein a2=C2/n2, and n2 >=5;
D described a1 value and a2 value compare by (), thus determine the multiple of target chromosome.
In another preference, calculate a1/a2 value and be designated as R,
When R is at or about 0.5 (namely close to 0.5), then show that target chromosome described in described sample is monoploid;
When R is at or about 1 (namely close to 1), then show that described in described sample, target chromosome is identical with reference to chromosomal multiple;
When R is at or about 1.025, then show that the target chromosome only having 5% fetus dissociative DNA sample in described sample is triploid, and the target chromosome of all the other 95% pregnant parent population DNA samples is still diploid;
When R is at or about 1.05, then show that the target chromosome only having 10% fetus dissociative DNA sample in described sample is triploid, and the target chromosome of all the other 90% pregnant parent population DNA samples is still diploid;
When R is at or about 1.5 (namely close to 1.5), then show that target chromosome described in described sample is 3 times of bodies;
When R is at or about 2 (namely close to 2), then show that target chromosome described in described sample is 4 times of bodies.
In another preference, when R and 1 discrete time, then show in described sample containing many times of karyomit(e)s.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1. multiple digital pcr basic procedure.Its flow process comprises sample DNA and is separated and dilution → dPCR sample mix → dPCR sample loading → dPCR amplification → dPCR interpretation of result.
Fig. 2. multiple digital pcr amplification Microarray results sectional drawing.
Fig. 3. high resolving power chromosomal copy number analyzes schematic diagram.Multiple digital pcr amplification chip karyomit(e) absolute quantitation software analysis result is transferred to Excel, shows normal mother body D NA as calculated similar to its theoretical value to different ratios 21-tri-body DNA dyed blended body copy number actual measured value.
Embodiment
The present inventor is through extensive and deep research, propose application MDPCR technology first and in maternal blood liquid, carry out chromosome disease without wound Prenatal Screening, thus develop a kind of early stage, safety without wound, accurately, fast, be applicable to extensive detection and the novel method of clinical application.The method utilizes Multiple techniques enriches fetal dissociative DNA, and utilize different concns fluorescence labeling probe to combine, fluorescent signal can be collected in different zones simultaneously, and different PCR loop number, thus increase MDPCR multiplicity reaches more than 20-50 time.Complete the present invention on this basis.
Definition
" without wound Prenatal Screening " refers to and only needs to take pregnant object peripheral blood, utilize DNA detection technology of new generation to carry out detection to the fetus dissociative DNA fragment in maternal peripheral blood plasma to analyze, thus whether detection fetus suffers from chromosome disease-Down's syndrome (trisomy 21), Edward (18 3 body), handkerchief pottery syndrome (13 3 body) and other chromosome aneuploid abnormal diseases.
" chromosome disease " or " chromosome aneuploid abnormal diseases " refers to chromosomal numerical abnormality and/or Morphology, can betide on every item chromosome.Usually, described karyomit(e) quantity abnormal diseases comprises monoploid disease or polyploid disease.Such as, described monoploid disease comprises 5q-syndrome.Described polyploid disease comprises trisomy 21 (Down's syndrome), 18 3 bodies (Edward), 13 3 bodies (handkerchief pottery syndrome) and sex chromosomal abnormality etc.
Multiple digital pcr
Digital pcr (digital PCR) is a kind of novel quantitative analytical technology that fast development is in recent years got up, by single sample is divided into more than tens thousand of, even millions of, tens million of part is distributed in different reaction members, each unit comprises the DNA profiling of nothing or one or more copy, respectively pcr amplification is carried out to these DNA profilings, then the fluorescent signal of each reaction member is analyzed.DPCR can not need reference standard sample and curve to realize absolute quantification analysis.Digital pcr (digital PCR) is although technological concept just proposes (Sykes PJ et al.Biotechniques 1992 in the nineties, 13:444-449), but be a kind of novel quantitative analytical technology of the rapid rising along with technical development in recent years, by by single Sample Dilution, be divided into more than tens thousand of, even millions of, tens million of part is distributed in different reaction members, each unit comprises the DNA profiling of nothing or a copy, respectively pcr amplification is carried out to these DNA profilings, then the fluorescent signal of each reaction member is analyzed.DPCR can not need reference standard sample and curve to realize absolute quantification analysis.
Multiplex PCR (multiplex PCR) be add in same PCR reaction system two to or more to primer, amplify simultaneously multiple nucleic acid fragment PCR reaction.General PCR only applies pair of primers, produces a nucleic acid fragment by pcr amplification.And multiplex PCR (multiplex PCR) be add in same PCR reaction system two to or more to primer, amplify simultaneously multiple nucleic acid fragment PCR reaction.
The present invention's multiple digital pcr (multiplex digital PCR, MDPCR) used be add in same digital pcr reaction system two to or more to primer, amplify the digital pcr reaction of multiple nucleic acid fragment simultaneously.Especially it is to be noted, by the multiple digital pcr (MDPCR) that multiple PCR technique and dPCR technology organically combine and formed, according to the different fluorescence of DNA probe, the different cycle numbers of DNA cloning and multiple mark fluorescent use simultaneously, just significantly can improve digital pcr multiplicity and reach more than 20-50, in a PCR reaction member, namely carry out 20-50 above digital pcr reaction simultaneously.
The feature of multiple digital pcr used in the present invention has: (1) high efficiency.Detect multiple nucleic acids fragment in same PCR reaction tubes simultaneously, or to there being the goal gene of multiple type to carry out somatotype.(2) systematicness.Multiple digital pcr is suitable for the detection of nucleic acid fragment in groups very much.(3) economical and convenient.Multiple nucleic acids fragment detects in same reaction tubes simultaneously, saves time, reagent consumptive material and funds, provides more Detection Information more accurately for clinical.
Detect the method for chromosome aneuploid
The invention provides a kind of that detect chromosome aneuploid for external nondiagnostic or create Prenatal Screening for carrying out nothing to chromosome disease method.
Method of the present invention comprises step:
A () provides the pregnant object peripheral blood sample containing fetus dissociative DNA;
B () is left away except cell from described peripheral blood sample one's duty, and from enrichment DNA the described sample removing cell, obtain DNA sample;
C described DNA sample is divided into control group and experimental group by (), wherein,
In experimental group, with described DNA sample for template, with the n1 of amplification target chromosomal gene, multiple digital pcr is carried out to the first primer pair, and record positobe focus number C1 and the ratio of primer pair quantity n1, be designated as a1, wherein a1=C1/n1, and n1 >=2, preferably, n1 >=5;
In control group, with described DNA sample for template, with the n2 of amplification reference chromogene, multiple digital pcr is carried out to the second primer pair, and record positobe focus number C2 and the ratio of primer pair quantity n2, be designated as a2, wherein a2=C2/n2, and n1 >=5, preferably, n1 >=5;
D described a1 value and a2 value compare by (), thus determine the multiple of target chromosome.
Wherein, preferably, the cell described in step (b) is the complete or broken hemocyte in blood sample, mainly comprises red corpuscle, white corpuscle, thrombocyte etc., can comprise plasma proteins etc. in addition.Minimizing technology can be undertaken by this area routine techniques.DNA sample through removal cell is the DNA sample of enrichment.
In addition, more preferably, the DNA sample in step (b) is through the sample after fragmentation process.The method that can be used for fragmentation process is not particularly limited, and is method well known to those skilled in the art, and such as physics or biochemical treatment, as fragmentation, supersound process, enzyme are cut etc.The DNA sample of the fragmentation after fragmentation process, its size is no more than 3000bp usually.
In the inventive method, described target chromosome is generally the appearance Variation in chromosome number of existing discovery and causes the karyomit(e) of chromosome disease, preferably includes No. 21 karyomit(e)s, No. 13 karyomit(e)s, No. 18 karyomit(e)s, sex chromosome.Described reference karyomit(e) is then the karyomit(e) of known normal number.Such as, be to adopt other any item chromosome (as No. 13, No. 18) etc. to be reference karyomit(e) when target chromosome is No. 21 karyomit(e)s.In addition, used in the present invention is one or more with reference to karyomit(e).
In the methods of the invention, according to the principle of multiplex PCR and according to the step of the inventive method design, those skilled in the art can obtain the corresponding content of following PCR primer and chromosome G banding result:
Calculate a1/a2 value and be designated as R,
When R is at or about 0.5 (namely close to 0.5), then show that target chromosome described in described sample is monoploid;
When R is at or about 1 (namely close to 1), then show that described in described sample, target chromosome is identical with reference to chromosomal multiple;
When R is at or about 1.5 (namely close to 1.5), then show that target chromosome described in described sample is 3 times of bodies;
When R is at or about 2 (namely close to 2), then show that target chromosome described in described sample is 4 times of bodies.
In another preference, when R and 1 discrete time, then show in described sample containing many times of karyomit(e)s.
When carrying out Prenatal Screening, can also obtain further:
When R is at or about 1.025, then show that the target chromosome only having 5% fetus dissociative DNA sample in described sample is triploid, and the target chromosome of all the other 95% pregnant parent population DNA samples is still diploid.
When R is at or about 1.05, then show that the target chromosome only having 10% fetus dissociative DNA sample in described sample is triploid, and the target chromosome of all the other 90% pregnant parent population DNA samples is still diploid.
Primer set
Present invention also offers a kind of primer set, adopt primer set of the present invention to may be used for that the external nondiagnostic of the present invention detects chromosome aneuploid or create Prenatal Screening for carrying out nothing to chromosome disease method.
In primer set of the present invention, contain the primer pair of amplification target chromosome, and amplification is with reference to chromosomal primer pair, the quantity of the two primer pair be respectively n1 to n2 couple.Wherein, n1 and n2 is respectively the >=positive integer of 2.Preferably, logarithm n1 and n2 that can be used for the primer pair of primer set of the present invention be respectively 3,4,5,6,7,8,9,10 right, preferably at least 5 is right.
Can be used for amplification target chromosome of the present invention and be usually not particularly limited with reference to the primer of chromogene.For the primer pair that can be template according to target chromosomal gene and reference chromogene and design in any prior art.Target chromosome used in the present invention and reference karyomit(e) are not particularly limited, specifically can be as hereinbefore defined.Target chromosomal gene used in the present invention is not particularly limited, and can be arbitrary gene on target chromosome.Those skilled in the art can judge to determine target chromosome and reference karyomit(e) according to method provided by the invention.When determining target and/or with reference to after karyomit(e), corresponding gene can be chosen on described karyomit(e) as template, then by the corresponding primer of conventional design for the described template that increases.
Usually, when target chromosome is No. 21 karyomit(e), available gene comprises Usp25 (NCBI#NM_001283041), C21orf37 (NCBI#LN608865), C21orf58 (NCBI#NM_058180), Setd4 (NCBI#NM_017438), C21orf62 (NCBI#AF231922) etc. and other gene locuss and non-genomic site; When target chromosome is No. 18 karyomit(e), available gene comprises Setbp1 (NCBI#NM_015559), Potec (NCBI#NM_001137671), Malt1 (NCBI#AB026118), Dsc3 (NCBI#NM_001941), Cdh19 (NCBI#NM_021153) etc. and other gene locuss and non-genomic site; When target chromosome is No. 13 karyomit(e), available gene comprises Sacs (NCBI#NM_014363), Cenpj (NCBI#NM_018451), Foxo1 (NCBI#NM_002015), Smad9 (NCBI#NM_001127217), Fgf14 (NCBI#NM_004115) etc. and other gene locuss and non-genomic site.Then only need select the karyomit(e) different from target chromosome with reference to karyomit(e), and select further with reference to corresponding gene on karyomit(e) and other gene locuss and non-genomic site.
Preferably, the gene template length that in primer set, the often pair of primer increases is 50-150bp, preferably, is 100-120bp.Preferably, when target chromosome is No. 21 karyomit(e), for the primer pair sequence of the target chromosomal gene that increases as shown in SEQ ID NO.:1-2, for gene be Usp25; And/or when being No. 18 karyomit(e) with reference to karyomit(e), the described primer pair sequence for increasing with reference to chromogene as shown in SEQ ID NO.:4-5, for gene be Setbp1.
In addition, in the primer pair that multiple digital pcr detects, the absolute value of the form of marker amplified production to measure can usually be adopted and for comparing with contrast.Available marker is not particularly limited, available is preferably, for with reference at least one being connected with fluorescent mark in each primer pair of karyomit(e) and target chromosome, fluorescence labeling probe is such as adopted to carry out the display of primer amplification result, preferably, containing in the primer of fluorescence labeling probe, probe sequence is as shown in SEQ ID NO.:3 (target chromosome) or 6 (with reference to karyomit(e)).
The multiple digital PCR method of the accurate quantification adopting the present invention to propose, at least 5-10 different gene locus to be detected on same karyomit(e) simultaneously, or STR comprises at least 4-6 STR pedestal, and by the DNA molecular of fetus and parent exceed tens thousand of, even millions of, tens million of different PCR unit are analyzed separately, without cross interference, thus in lower concentration fetus dissociative DNA, detect that chromosomal copy number changes, point mutation and single nucleotide polymorphism (SNP).Such as, every 100 microlitres of normal euploid pregnant parent population plasma sample are containing 10 genome equivalent (genome-equivalents, GE) (wherein 1 equivalent comes from fetus dissociative DNA), namely every 100 microlitres are 20 (18 parent copy number+2 fetus copy numbers) containing 21 chromosomal copy number, and trisomy 21 gestation every milliliter containing 21 chromosomal copy number be 21 (18 parent copy number+3 fetus copy numbers), general measure PCR still can not like this explication de texte 20 copy and 21 copy between technicality.But adopt MDPCR method, can Accurate Analysis 20 copy and 21 copy between technicality.
Test kit
Present invention also offers the test kit detecting chromosome aneuploid and/or examination karyomit(e) quantity abnormal diseases.Wherein, containing primer set of the present invention and specification sheets in described test kit.
The specification sheets contained in test kit of the present invention have recorded the present invention and detects the method for target chromosome aneuploid and/or carry out the method without wound Prenatal Screening to chromosome disease.
The present invention has following major advantage:
(1) early detection.In normal pregnancy situation, fetal cell infiltrates into maternal blood through placenta, and cell is destroyed by mother's immune system, and foetal DNA is free in female blood.It is reported, the time that fetus dissociative DNA can measure in female blood is pregnant 5 weeks, and content only has the 5-10% of female blood DNA, increases along with pregnant Zhou Zengjia, can reach 25%, but there is individual difference during second trimester of pregnancy.The dissociative DNA transformation period is short, disappears in point short period of time in puerperium.Add its DNA fragmentation less, average 166bp, conventional PCR method is difficult to detect usually, and multiple digital PCR method is due to tool high sensitivity, can carry out early detection.
(2) safety is without wound.Only need extract 5-10ml maternal peripheral blood to detect, avoid fetal in utero to infect and pregnant woman's miscarriage simultaneously.
(3) accurate.Adopting up-to-date multiple digital round pcr, is the basic reason (DNA change) that chromosomal disorders occur due to what detect, instead of its result (biochemical indicator examination), and recall rate can reach more than 99%, and false positive rate is no more than 1%.According to measuring and calculating, as fetus dissociative DNA accounts for 10% of maternal peripheral blood DNA, only need 10000 PCR element analysises, just can realize recall rate and reach more than 99%, false positive rate is no more than 1% (Evans MI et al.Fetal Diagn Ther 2012,31:244-247).
(4) quick.Detected result obtains in usual 12 hours, and price is 1/2nd of DNA sequencing technology of new generation.
(5) extensive detection and clinical application is applicable to.The multiplicity of multiple digital pcr institute tool, high-throughput and high sensitivity, can detect multiple DNA fragmentation simultaneously, save time, reagent consumptive material and funds in same reaction member.Its indication comprises all gravid woman, especially tube baby's treatment and the pregnant woman repeatedly miscarried, within less than 35 years old, miss the pregnant woman of chromosomal disorders examination chance, within more than 35 years old, exceed the pregnant woman that chromosomal disorders examination adapts to the age, the pregnant woman that refusal amniocentesis or bleeding of the umbilicus puncture and the pregnant woman etc. having puncture contraindication.
Except the above major advantage, the main innovation part of the method is to utilize at least quadruple technology enriches fetal dissociative DNA from maternal peripheral blood DNA, is conducive to early detection fetus dissociative DNA; Different concns fluorescence labeling probe is utilized to combine, optimize loop number that different PCR reacts thus increase MDPCR multiplicity and reach more than 20-50 time, multiple nucleic acids fragment is detected in same reaction tubes simultaneously, save time, reagent consumptive material and funds, provide more Detection Information more accurately for clinical.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number are weight percent and parts by weight.
Experiment material: chemical reagent is purchased from Sigma-Aldrich company; Restriction endonuclease used and other molecular biology reagents are purchased from NEB and Promega company;
ultra 10K device is purchased from Millipour company; Primer is purchased from Invitrogen company, and probe is purchased from Roche company; PCR reagent and consumptive material are purchased from Life Technologies company.
Embodiment 1. is applied MDPCR technology and is carried out trisomy 21 detection
(1) 5ml maternal blood fluid samples is got;
(2) leave away except cell from described peripheral blood sample one's duty, and digest serum or plasma proteins, enrichment DNA from the described sample removing cell, obtain DNA sample;
(3) cut through TaqI enzyme, make DNA segment;
(4) DNA sample that enzyme cuts through is divided into control group and experimental group, applies multiple digital pcr (QuantStudio 3D dPCR) technology and carry out trisomy 21 detection (PCR reaction conditions: 95 DEG C 10 minutes in Pro-Flex PCR system; 60 DEG C 2 minutes, 98 DEG C 30 seconds, repeat 44 times; 60 DEG C 2 minutes).
(5) mixed with trisomy 21 clone genomic dna or 18 heterotrimeric cell system genomic dnas in 10% and 5% ratio respectively by normal gene group DNA, first carry out Taq enzyme and cut, make these genomic DNA pools fragmentations, clip size is about 200bp.To contain 18 3 body DNA biased samples in contrast, carry out multiple digital pcr with these simulation pregnant woman containing trisomy 21 DNA biased sample, parallel sample reaches 8 times.
Wherein, gene selected by 21 karyomit(e)s is Usp25 (NCBI#NM_001283041), C21orf37 (NCBI#LN608865), C21orf58 (NCBI#NM_058180), Setd4 (NCBI#NM_017438), C21orf62 (NCBI#AF231922) etc. and other gene locuss and non-genomic site, Usp25 gene primer is as shown in SEQ ID NO.:1-2, and probe is for shown in SEQ ID NO.:3; Gene selected by 18 karyomit(e)s is Setbp1 (NCBI#NM_015559), Potec (NCBI#NM_001137671), Malt1 (NCBI#AB026118), Dsc3 (NCBI#NM_001941), Cdh19 (NCBI#NM_021153) etc. and other gene locuss and non-genomic site, Setbp1 gene primer is as shown in SEQ ID NO.:4-5, and probe is for shown in SEQID NO.:6.
Result shows: as hybrid dna concentration=5.7ng, Quantstudio 3D digital pcr system can accurately can detect the trisomy 21 foetal DNA (Fig. 2 and Fig. 3) being less than 5% and 10% from pregnant woman blood plasma.These results confirm by 300 routine clinical samples.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a method for the detection target chromosome aneuploid (Aneuploidy) of external nondiagnostic, is characterized in that, comprise step:
A () provides the pregnant object peripheral blood sample containing fetus dissociative DNA;
B () is left away except cell from described peripheral blood sample one's duty, and from enrichment DNA the described sample removing cell, obtain DNA sample;
C described DNA sample is divided into control group and experimental group by (), wherein,
In experimental group, with described DNA sample for template, with the n1 of amplification target chromosomal gene, multiple digital pcr is carried out to the first primer pair, and record positobe focus number C1 and the ratio of primer pair quantity n1, be designated as a1, wherein a1=C1/n1, and n1 >=2, preferably, n1 >=5;
In control group, with described DNA sample for template, with the n2 of amplification reference chromogene, multiple digital pcr is carried out to the second primer pair, and record positobe focus number C2 and the ratio of primer pair quantity n2, be designated as a2, wherein a2=C2/n2, and n1 >=5, preferably, n1 >=5;
D described a1 value and a2 value compare by (), thus determine the multiple of target chromosome.
2. the method for claim 1, is characterized in that, the length of described each amplified production to the first primer is 50-150bp, preferably 60-120bp.
3. the method for claim 1, is characterized in that, described n1 is specific amplification No. 21 chromosomal primers to often pair of primer in the first primer.
4. the method for claim 1, it is characterized in that, described target chromosomal gene comprises No. 21 chromosomal Usp25 (NCBI#NM_001283041), C21orf37 (NCBI#LN608865), C21orf58 (NCBI#NM_058180), Setd4 (NCBI#NM_017438) or C21orf62 (NCBI#AF231922) gene; And/or
No. 13 chromosomal Sacs (NCBI#NM_014363), Cenpj (NCBI#NM_018451), Foxo1 (NCBI#NM_002015), Smad9 (NCBI#NM_001127217) or Fgf14 (NCBI#NM_004115); And/or
No. 18 chromosomal Setbp1 (NCBI#NM_015559), Potec (NCBI#NM_001137671), Malt1 (NCBI#AB026118), Dsc3 (NCBI#NM_001941), Cdh19 (NCBI#NM_021153).
5. the method for claim 1, is characterized in that, described is one or more with reference to karyomit(e).
6. a primer set, is characterized in that, described primer set comprises following primer pair:
In (i) claim 1 for the described n1 of the target chromosomal gene that increases to primer pair; With
(ii) in claim 1 for the described n2 that increases with reference to chromogene to primer pair;
Wherein, n1, n2 difference >=2.
7. primer set as claimed in claim 6, is characterized in that, also containing primer fluorescent mark on the primer in described primer set.
8. primer set as claimed in claim 6, it is characterized in that, the described primer pair sequence for the target chromosomal gene that increases is as shown in SEQ ID NO.:1-2; And/or
The described primer pair sequence for the reference chromogene that increases is as shown in SEQ ID NO.:4-5;
Wherein said target chromosome is No. 21 chromosomal Usp25 genes; Described is No. 18 chromosomal Setbp1 genes with reference to karyomit(e).
9. the purposes of primer set described in claim 6, is characterized in that, for the preparation of the reagent or the test kit that detect chromosome aneuploid and/or examination karyomit(e) quantity abnormal diseases.
10. detect a test kit for target chromosome, it is characterized in that, containing primer set according to claim 6 in described test kit, and specification sheets.
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