CN108504653A - A kind of maternal peripheral blood blood plasma fast separating process and its application - Google Patents
A kind of maternal peripheral blood blood plasma fast separating process and its application Download PDFInfo
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- CN108504653A CN108504653A CN201810326928.7A CN201810326928A CN108504653A CN 108504653 A CN108504653 A CN 108504653A CN 201810326928 A CN201810326928 A CN 201810326928A CN 108504653 A CN108504653 A CN 108504653A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Abstract
The invention discloses a kind of maternal peripheral blood blood plasma fast separating process and its applications.Blood plasma fast separating process includes：The centrifugation of one step is carried out in vitro maternal peripheral blood and obtains blood plasma.Blood plasma after separation is after dissociative DNA extraction, library construction, high-flux sequence, analysis of biological information, compared with the blood plasma of two step centrifugal process, dissociative DNA concentration, the distribution of dissociative DNA segment, foetal DNA concentration, sequencing segment distribution are suitable, it can be applied to dissociative DNA extraction, fetal nucleic acid acquisition of information and noninvasive prenatal gene screening product.The present invention, which breaks traditional blood plasma separation, needs cognition and the way of two step centrifugal process, simplifies the operating process of blood plasma separation and reduces human and material resources input, has good market application value.
Description
Technical field
The present invention relates to free nucleic acids, and field is sequenced, and in particular to a kind of maternal peripheral blood blood plasma fast separating process and its
Using.
Background technology
In the 1940s, extracellular in the presence of being free in French scientist Mandel and Metais discovery peripheral blood
DNA, referred to as peripheral blood dissociative DNA (Circulating DNA in plasma or serum).1997, Lu Yuming etc. existed
Nourish the specific sequence that Y chromosome is detected in the peripheral blood of male tire pregnant woman, it was demonstrated that there is free fetus in maternal blood
Non-invasive prenatal gene screening (NIPT) fetus that is found to be of DNA (cell-free fetal DNA, cffDNA), cffDNA go out
Raw defective disease opens new era.With tradition invasive invasive (chorionic villous sampling, amniocentesis and Cord blood puncture)
Pre-natal diagnosis is compared, and noninvasive prenatal gene screening has noninvasive sampling, advantage that is high-throughput, quickly going out result, presently the most people
Well known application is for assessing common fetal chromosome aneuploid (T21, T18 and T13) abnormal risk.
Noninvasive prenatal gene screening (NIPT) includes the separation of maternal peripheral blood blood plasma, plasma DNA extraction, high pass measurement
Sequence, bioinformatic analysis step.Foetal DNA concentration is to the accuracy of noninvasive antenatal Fetal genome abnormality detection to closing weight
It wants, the crucial Quality Control point of a concentration of noninvasive prenatal gene detection of foetal DNA.Dissociative DNA largely derives from maternal peripheral blood
The Apoptosis of parent itself is only seldom derived partly from fetus, and the cracking of nucleated blood cell influences peripheral blood afterwards in vitro
Total concentration of dissociative DNA and the accuracy of foetal DNA Concentration Testing.Therefore, it is to ensure trip to efficiently separate maternal peripheral blood blood plasma
One of key factor from DNA fetal concentrations.
Maternal peripheral blood blood plasma separation at present is using two classical step centrifugal process：After one step low-speed centrifugal, take blood plasma into
Row second step high speed centrifugation, to ensure to remove parent karyocyte completely.However it is related to a tube behaviour among two step centrifugal process
Make, which increase the probability obscured in blood plasma separation link sample；And centrifugation apparatus need support heparin tube low-speed centrifugal and
Centrifuge tube high speed centrifugation, conditions above is invisible to increase manpower and Material Cost.Therefore, exploitation is a kind of quickly, simplifies, and not shadow
The plasma separation method for ringing fetus dissociative DNA concentration has for obtaining fetal nucleic acid information and carrying out noninvasive prenatal gene detection
Important function.
Invention content
The purpose of the present invention is to provide a kind of maternal peripheral blood blood plasma fast separating process and its applications.
The technical solution used in the present invention is：
A kind of maternal peripheral blood blood plasma fast separating process, the method includes：One is carried out in vitro maternal peripheral blood
Step centrifugation obtains blood plasma.
Preferably, total centrifugal force of 5~10mL maternal peripheral bloods, step centrifugation is 10000~100000 (gmin).
It is further preferred that 5~10mL maternal peripheral bloods, total centrifugal force of step centrifugation is 16000~64000 (g
min)。
It is further preferred that 5~10mL maternal peripheral bloods, the relative centrifugal force of step centrifugation is 1600~3200g, centrifugation
10~20min of Shi Changwei.
Wherein, maternal peripheral blood is selected from the maternal peripheral blood guaranteed the quality；It is molten in normal condition or slightly soluble blood state or moderate
The maternal peripheral blood of blood state.
Above-mentioned maternal peripheral blood blood plasma fast separating process answering in dissociative DNA extraction, fetal nucleic acid acquisition of information
With.
A kind of dissociative DNA extracting method, the method includes：Utilize above-mentioned maternal peripheral blood blood plasma fast separating process
Blood plasma is obtained, dissociative DNA is extracted from blood plasma.
Preferably, dissociative DNA is extracted from blood plasma using ethanol on pellosil post centrifugal process or paramagnetic particle method.
A kind of fetal nucleic acid Information Acquisition System, including：
One step separator of blood plasma：For obtaining blood plasma using above-mentioned maternal peripheral blood blood plasma fast separating process；
Dissociative DNA extraction element：For extracting dissociative DNA from blood plasma；
Library construction device：For carrying out library construction using dissociative DNA；
High-flux sequence device：For carrying out high-flux sequence to library；
Analysis of biological information device：For carrying out analysis of biological information according to high-flux sequence data, fetal nucleic acid is obtained
Information.
Above-mentioned fetal nucleic acid Information Acquisition System is applied to noninvasive foetal chromosome aneuploidy screening system, noninvasive tire
Youngster's microdeletion is micro- to repeat screening system, noninvasive fetus monogenic disease screening system.
The beneficial effects of the invention are as follows：
The present invention maternal peripheral blood blood plasma fast separating process include：One step centrifugation is carried out in vitro maternal peripheral blood
Obtain blood plasma.Blood plasma after separation is after dissociative DNA extraction, library construction, high-flux sequence, analysis of biological information, with two steps
The blood plasma of centrifugal process is compared, and dissociative DNA concentration, the distribution of dissociative DNA segment, foetal DNA concentration, sequencing segment distribution are suitable, can
It is extracted applied to dissociative DNA, fetal nucleic acid acquisition of information and noninvasive prenatal gene screening product.
The maternal peripheral blood blood plasma fast separating process of the present invention, it is in the field of business to generate important benefit：(1) break traditional
Blood plasma detaches the cognition of two step centrifugal process and way；(2) blood plasma separation the experimental implementation time by 30min (to detach 10 Patients with Peripheral
For blood) it reduces to 15min, save the significant effect of operating time；(3) to blood plasma separation device requirement by meet low speed from
The heart and high speed centrifugation condition are kept to meet low-speed centrifugal；(4) in plasma separation, blood plasma separation is grasped by 2 tubes
It is kept to 1 tube operation, secondary mark action in blood plasma separation is reduced, reduces consumptive material demand (pipette tips, the centrifugation of blood plasma separation
Pipe), the probability that sample is artificially obscured in blood plasma separation is reduced, review action when blood plasma separation tube is reduced, also improves blood plasma
The feasibility of lock out operation full-automation detaches full-automatic operating process with blood plasma is simplified；(5) nucleated blood cell is being kept
And while maintaining dissociative DNA integrality, the DNA losses that second step high speed centrifugation is brought are reduced, the rich of fetus dissociative DNA is improved
Degree；(6) the dissociative DNA extraction based on blood plasma quick separating, fetal nucleic acid acquisition of information, noninvasive prenatal gene screening flow all obtain
To simplification, the input of human and material resources is greatly reduced.
Description of the drawings
Fig. 1 is 2100 figure of T001 samples, wherein C1 indicates comparative example 1, and L1~L3 indicates Examples 1 to 3 in order；
Fig. 2 is T001 samples sequencing segment distribution map；
Fig. 3 is 2100 figure of W001 samples, wherein C2 indicates that comparative example 2, L4~L6 indicate embodiment 4~6 in order；
Fig. 4 is W001 samples sequencing segment distribution map；
Fig. 5 is 2100 figure of D001 samples, wherein C1 indicates that comparative example 1, L1 indicate embodiment 1；
Fig. 6 is D001 samples sequencing segment distribution map；
Fig. 7 is 2100 figure of L001 samples, wherein C1 indicates that comparative example 1, L1 indicate embodiment 1；
Fig. 8 is L001 sequencing segment distribution maps.
Specific implementation mode
Inventor is according to containing red blood cell, leucocyte and dissociative DNA in maternal peripheral blood, the cffDNA in maternal peripheral blood
Natural degradation at the DNA fragmentation that main peak is ＜ 313bp, after peripheral blood acquisition cffDNA main sources be cut-off situation with
And centrifugal force (total centrifugal force=relative centrifugal force × centrifugation time), propose a kind of maternal peripheral blood blood plasma fast separating process.
The present invention relates to formula：Total centrifugal force=relative centrifugal force × centrifugation time；
Wherein, relative centrifugal force (RCF, relative centrifugal force), unit is gravity acceleration g；Always
Centrifugal force (TCF, total centrifugal force) unit is gs or gmin；It indicates always to apply in entire centrifugation cycle
The centrifugal force added.
A kind of maternal peripheral blood blood plasma fast separating process, the method includes：One is carried out in vitro maternal peripheral blood
Step centrifugation obtains blood plasma.
Preferably, total centrifugal force of 5~10mL maternal peripheral bloods, step centrifugation is 10000~100000 (gmin).
It is further preferred that 5~10mL maternal peripheral bloods, total centrifugal force of step centrifugation is 16000~64000 (g
min)。
It is further preferred that 5~10mL maternal peripheral bloods, the relative centrifugal force of step centrifugation is 1600~3200g, centrifugation
10~20min of Shi Changwei.
Wherein, maternal peripheral blood is selected from the maternal peripheral blood guaranteed the quality；It is molten in normal condition or slightly soluble blood state or moderate
The maternal peripheral blood of blood state.
Above-mentioned maternal peripheral blood blood plasma fast separating process answering in dissociative DNA extraction, fetal nucleic acid acquisition of information
With.
A kind of dissociative DNA extracting method, the method includes：Utilize above-mentioned maternal peripheral blood blood plasma fast separating process
Blood plasma is obtained, dissociative DNA is extracted from blood plasma.
Preferably, dissociative DNA is extracted from blood plasma using ethanol on pellosil post centrifugal process or paramagnetic particle method.
A kind of fetal nucleic acid Information Acquisition System, including：
One step separator of blood plasma：For obtaining blood plasma using above-mentioned maternal peripheral blood blood plasma fast separating process；
Dissociative DNA extraction element：For extracting dissociative DNA from blood plasma；
Library construction device：For carrying out library construction using dissociative DNA；
High-flux sequence device：For carrying out high-flux sequence to library；
Analysis of biological information device：For carrying out analysis of biological information according to high-flux sequence data, fetal nucleic acid is obtained
Information.
Above-mentioned fetal nucleic acid Information Acquisition System is applied to noninvasive foetal chromosome aneuploidy screening system, noninvasive tire
Youngster's microdeletion is micro- to repeat screening system, noninvasive fetus monogenic disease screening system.
Term " maternal peripheral blood guaranteed the quality " of the present invention refers to that maternal peripheral blood is acquired to heparin tube, as long as meeting phase
It answers and stores and/or transport under the standard of guaranteeing the quality of heparin tube, and be less than the defined quality guarantee period, just belong to the " parent guaranteed the quality
Peripheral blood ", for example, according to this field routine techniques, the maternal peripheral blood acquired using EDTA anticoagulant blood-collecting pipes need to be at 2~8 DEG C
It preserves or transports, and require separated plasma in 8 hours；For another example, stablize the maternal peripheral blood of heparin tube acquisition using free nucleic acid,
It can then expand to room temperature and preserve or transport (4~30 DEG C), it is desirable that separated plasma in 96 hours.In addition, a present invention couple step centrifuges
Temperature be not construed as limiting, the performance of guaranteeing the quality based on heparin tube, using EDTA anticoagulant blood-collecting pipes load peripheral blood need to be at 2~8 DEG C
Lower Cord blood (including centrifugation), and free nucleic acid is stablized heparin tube and can be preserved under normal temperature condition (including centrifugation).
Below in conjunction with specific embodiments, technical scheme of the present invention is clearly and completely described, it is clear that institute
The embodiment of description is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the thinking of the present invention, ability
The every other embodiment that domain those of ordinary skill is obtained without making creative work, belongs to guarantor of the present invention
The range of shield.
Not specified instrument and reagent are commercially available in embodiment, and not specified experimental condition presses this
Field routine operation or manufacturers instruction carry out.
Embodiment 1, a kind of maternal peripheral blood blood plasma fast separating process
Maternal peripheral blood 5mL is acquired, step centrifugation obtains blood plasma, and the concrete operations of the step centrifugation are：It is put into and is equipped with
The heparin tube of maternal peripheral blood is 1600g with relative centrifugal force, is centrifuged 10 minutes, and supernatant is the blood plasma got, in -20 DEG C
It saves backup.
Embodiment 2, a kind of maternal peripheral blood blood plasma fast separating process
Maternal peripheral blood 5mL is acquired, step centrifugation obtains blood plasma, and the concrete operations of the step centrifugation are：It is put into and is equipped with
The heparin tube of maternal peripheral blood is 2500g with relative centrifugal force, is centrifuged 10 minutes, and supernatant is the blood plasma got, in -20 DEG C
It saves backup.
Embodiment 3, a kind of maternal peripheral blood blood plasma fast separating process
Maternal peripheral blood 5mL is acquired, step centrifugation obtains blood plasma, and the concrete operations of the step centrifugation are：It is put into and is equipped with
The heparin tube of maternal peripheral blood is 3200g with relative centrifugal force, is centrifuged 10 minutes, and supernatant is the blood plasma got, in -20 DEG C
It saves backup.
Embodiment 4, a kind of maternal peripheral blood blood plasma fast separating process
Maternal peripheral blood 10mL is acquired, step centrifugation obtains blood plasma, and the concrete operations of the step centrifugation are：It is put into and is equipped with
The heparin tube of maternal peripheral blood is 1600g with relative centrifugal force, is centrifuged 15 minutes, and supernatant is the blood plasma got, in -20 DEG C
It saves backup.
Embodiment 5, a kind of maternal peripheral blood blood plasma fast separating process
Maternal peripheral blood 10mL is acquired, step centrifugation obtains blood plasma, and the concrete operations of the step centrifugation are：It is put into and is equipped with
The heparin tube of maternal peripheral blood is 2500g with relative centrifugal force, is centrifuged 15 minutes, and supernatant is the blood plasma got, in -20 DEG C
It saves backup.
Embodiment 6, a kind of maternal peripheral blood blood plasma fast separating process
Maternal peripheral blood 10mL is acquired, step centrifugation obtains blood plasma, and the concrete operations of the step centrifugation are：It is put into and is equipped with
The heparin tube of maternal peripheral blood is 3200g with relative centrifugal force, is centrifuged 15 minutes, and supernatant is the blood plasma got, in -20 DEG C
It saves backup.
The conventional two step separation methods of comparative example 1, a kind of maternal peripheral blood blood plasma
Maternal peripheral blood 5mL is acquired, the centrifugation of two steps obtains blood plasma, and the concrete operations of the two steps centrifugation are：1) it is put into dress
The heparin tube for having maternal peripheral blood is 1600g with relative centrifugal force, centrifuges 10min, draws supernatant and is transferred to new centrifuge tube
In；2) it is put into the centrifuge tube of supernatant obtained by step, is 16000g with relative centrifugal force, centrifuges 10min, supernatant is the blood got
Slurry, is put in -20 DEG C and preserves.
The conventional two step separation methods of comparative example 2, a kind of maternal peripheral blood blood plasma
Maternal peripheral blood 10mL is acquired, the centrifugation of two steps obtains blood plasma, and the concrete operations of the step centrifugation are：1) it is put into dress
The heparin tube for having maternal peripheral blood is 1600g with relative centrifugal force, centrifuges 15min, draws supernatant and is transferred to new centrifuge tube
In；2) it is put into the centrifuge tube of supernatant obtained by step, is 16000g with relative centrifugal force, centrifuges 10min, supernatant is the blood got
Slurry, is put in -20 DEG C and preserves.
Embodiment 7, dissociative DNA extraction and acquisition fetal nucleic acid information
The plasma separation method that the present embodiment is provided using Examples 1 to 3 and comparative example 1, to coming from Dongguan mother and child care
The in vitro maternal peripheral blood sample of 2, institute (number T001, T002, it was demonstrated that the sample of the male tire in bosom) is detached, and blood plasma is obtained.
The plasma separation method provided using embodiment 4~6 and comparative example 2, to outside 2 in vitro parents of Dongguan healthcare hospital for women ＆ children
All blood samples (number W001, W002, it was demonstrated that the sample of the male tire in bosom) are detached, and blood plasma is obtained.
Plasma DNA extracts
The dissociative DNA in blood plasma, this reality are extracted using modes such as conventional ethanol on pellosil post centrifugal process extraction, paramagnetic particle method extractions
Nucleic acid extraction or purification kit (product article No. S10020) that example selects Dongguan Bo Aomuhua Gene Tech. Company Limited are applied, is carried
Method is taken to be carried out according to product description：First, cracking digestion is carried out to sample with lysate and Proteinase K, dissociative DNA is released
It is put into lysate；Secondly, by free nucleic acid absorption to magnetic bead surfaces；Third washes away unadsorbed albumen using cleaning solution
The impurity such as matter, salinity；4th, the dissociative DNA adsorbed is eluted from magnetic particle, obtains dissociative DNA.
DNA concentration is detected using 3.0 fluorescent quantitation instruments of Qubit, is converted into dissociative DNA total amount, the results are shown in Table 1.
The dissociative DNA total amount (ng) of table 1, each example separated plasma
As it can be seen that the blood plasma fast separating process of Examples 1 to 3 and the conventional two step plasma separation methods of comparative example 1 obtain
Blood plasma in dissociative DNA total amount it is suitable；The conventional two step blood plasma point of the blood plasma fast separating process of embodiment 4~6 and comparative example 2
Dissociative DNA total amount is suitable in the blood plasma obtained from method.
Using 2100 biological analysers of Agilent, select Agilent High Sensitivity DNA Kit to blood plasma
Dissociative DNA carry out segment distribution detection, by taking sample T001 and W001 as an example, as a result as shown in Figure 1, Figure 3 respectively, blood plasma is quick
Separation method is suitable with the dissociative DNA distribution that the blood plasma that two conventional step plasma separation methods obtain extracts.
Library construction
The dissociative DNA of extraction is subjected to library construction using conventional method and is quantified, the present embodiment uses fetal chromosomal
Library agent formulations are built in aneuploid (T21, T18, T13) detection kit (rich biological group difficult to understand)：End reparation buffer solution,
End repair enzyme, DNA connections buffer solution, DNA ligase, PCR amplification reagent, PCR primer, TE solution, P1 connectors, sequence label
X, magnetic bead B1, magnetic bead B2, alternatively, can also use Life Technologies or New England Biolabs,
Enzymatics builds library reagent, sequencing reagent in Proton microarray datasets.
Library constructing method includes：End is repaired, connector connects, PCR amplification, specific as follows：
A. end is repaired
The dissociative DNA of extraction is configured into reaction system according to following table respectively, incubation at room temperature 20min is placed in, is added
100 μ L magnetic beads B1 are combined, and after being clarified on magnetic frame, are shifted and are purified in supernatant to 90 μ L magnetic beads B2,25 μ L TE are molten
Liquid dissolving elution：
B. connector connects
Upper step purified product is configured into reaction system according to following table respectively, incubation at room temperature 30min is placed in, uses 75
μ L magnetic beads B2 is purified, the dissolving elution of 18.2 μ L TE solution：
P1 connectors are the P1-Adapter being made of following two positive and negative sequences：
5'-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3'(SEQ ID NO:1),
3'-T*T*GGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTA-5'(SEQ ID NO:2)。
The Barcode Primer X that sequence label X is made of following positive and negative two sequences are formed：
5'-CCATCTCATCCCT*G*CGTGTCTCCGACTCAGNNNNNNNNNNGAT-3'(SEQ ID NO:3),
3'-CGCACAGAGGCTGAGTCNNNNNNNNNNCTA-5'(SEQ ID NO:4)。
Wherein " N " represents A, T, C, G any one base, and * represents nucleotide and carried out thio-modification.
C.PCR is expanded
Upper step purified product is configured into reaction system according to following table：
PCR primer sequence is：
Primer 1：5’-CCATCTCATCCCTGCGTGTC-3’(SEQ ID NO:5),
Primer 2：5’-CCACTACGCCTCCGCTTTCCTCTCTATG-3’(SEQ ID NO:6).
PCR reaction conditions：72 DEG C, 10min, 95 DEG C, 5min；(95 DEG C, 15s；58 DEG C, 15s；70 DEG C, 1min)
12cycles；70 DEG C, 2min；4 DEG C, holding.
After PCR, purified with 85 μ L magnetic beads B2,50 μ LTE solution dissolving elution, acquisition waits for sequencing library.
High-flux sequence
Emulsion-based PCR reaction, enriching and purifying are carried out to library, primer annealing and sample are loaded into chip, you can are sequenced
Reaction.Ion ProtonTMMicroarray dataset takes single-ended PCR sequencing PCR, and the maximum a length of 300bp of reading is sequenced, obtains sequencing data.Fig. 2 shows
Example property provides each example sequencing fragment length distribution maps of sample T001, and Fig. 4 is exemplary to provide each example sequencing fragment lengths of sample W001
Distribution map, it is seen that dissociative DNA builds library survey in the blood plasma that blood plasma fast separating process is obtained with two conventional step plasma separation methods
The distribution of sequence segment is suitable.
Analysis of biological information
High-flux sequence data are subjected to analysis of biological information, obtain fetal nucleic acid information：The sequence that sequencing is obtained
(reads) it compares and arrives mankind's reference gene group, and remove low-quality comparison result and repetitive sequence, and then calculate 22 pairs of dyes
The sequence number (reads number) and sequence ratio (reads ratio) of colour solid and sex chromosome, according to male tire Y chromosome
With the relationship of foetal DNA concentration, fetus dissociative DNA concentration is obtained, the results are shown in Table 2.
The foetal DNA concentration (%) of table 2, each example separated plasma
As it can be seen that the blood plasma fast separating process of Examples 1 to 3 and the conventional two step plasma separation methods of comparative example 1 obtain
Blood plasma in foetal DNA concentration it is suitable；The conventional two step blood plasma point of the blood plasma fast separating process of embodiment 4~6 and comparative example 2
Foetal DNA concentration is suitable in the blood plasma obtained from method.
Embodiment 8, separated plasma can multigelations
The present embodiment is directed to (to be confirmed to have cherished male tire from 3 in vitro maternal peripheral blood samples of Dongguan healthcare hospital for women ＆ children
Sample, number D001, D002, D003), utilize the plasma separation method that embodiment 1 and comparative example 1 provide to obtain blood plasma,
Multigelation is carried out 3 times to the blood plasma that embodiment 1 detaches：Blood plasma is placed in -20 DEG C of refrigerators after freezing and is taken out,
It after room temperature is thawed completely, then freezes, then thaws completely, be repeated 3 times, be subsequently placed in -20 DEG C of refrigerators and preserve.Testing index when
Between point be arranged in 1st month after multigelation, the 3rd month, with comparative example 1 detach blood plasma be control.
According to the extraction of the plasma DNA of embodiment 7, the method for library construction, high-flux sequence, analysis of biological information,
Obtain the indexs such as corresponding dissociative DNA total amount and fetal nucleic acid concentration.
As a result：
The Circulating DNA fragments distribution map that Agilent 2100 is analyzed is shown by taking sample D001 as an example, as a result such as Fig. 5 institutes
Show, the dissociative DNA distribution and two step blood that the different holding times obtain after the blood plasma multigelation that blood plasma fast separating process obtains
It is suitable to starch the blood plasma that separation method obtains.
Show that segment distribution map is sequenced in dissociative DNA library by taking sample D001 as an example, the results are shown in Figure 6, and blood plasma quickly divides
The dissociative DNA that the different holding times obtain after the blood plasma multigelation obtained from method builds the distribution of library sequencing segment and two step blood plasma
The blood plasma that separation method obtains is suitable.
Table 3 provides the dissociative DNA total amount (ng) and foetal DNA concentration (%) of each example separated plasma.
Testing result after table 3, separated plasma multigelation
As it can be seen that the blood plasma that the blood plasma fast separating process of the present invention obtains after multigelation 3 times, is preserved to 3 months still
It is suitable with free DNA concentration and foetal DNA concentration in the blood plasma that conventional two step separation methods obtain.
Embodiment 9 carries out blood plasma separation to the maternal peripheral blood of slightly soluble blood, Medium hemolysis
The present embodiment is directed to (to be confirmed to have cherished male tire from 3 in vitro maternal peripheral blood samples of Dongguan healthcare hospital for women ＆ children
Sample), sample state is respectively L001 (slightly soluble blood), L002 (slightly soluble blood), L003 (Medium hemolysis), utilizes 1 He of embodiment
The plasma separation method that comparative example 1 provides obtains blood plasma, and according to the extraction of the plasma DNA of embodiment 7, library construction, height
Flux sequencing, analysis of biological information method, obtain the indexs such as corresponding dissociative DNA total amount and fetal nucleic acid concentration.
As a result：
The Circulating DNA fragments distribution map that Agilent 2100 is analyzed is shown by taking sample L001 as an example, as a result such as Fig. 7 institutes
Show, blood plasma fast separating process is suitable with dissociative DNA distribution in the blood plasma that two conventional step plasma separation methods obtain.
Show that segment distribution map is sequenced in dissociative DNA library by taking sample L001 as an example, the results are shown in Figure 8, it is seen that blood plasma is fast
It is suitable that fast separation method with dissociative DNA in the blood plasma that two conventional step plasma separation methods obtain builds library sequencing segment distribution.
Table 4 provides the dissociative DNA total amount (ng) and foetal DNA concentration (%) of each example separated plasma.
Testing result after table 4, slightly soluble blood, Medium hemolysis sample separated plasma
As it can be seen that in the blood plasma that blood plasma fast separating process is obtained with conventional two step plasma separation methods dissociative DNA total amount and
Foetal DNA concentration is suitable.
To sum up, maternal peripheral blood blood plasma fast separating process provided by the invention is to dissociative DNA concentration, dissociative DNA
Segment distribution, foetal DNA concentration, sequencing segment distribution do not make significant difference, and can be applied to dissociative DNA extraction, fetal nucleic acid information
Acquisition and noninvasive prenatal gene screening product, wherein non-invasive screening product includes but not limited to the non-multiple of noninvasive fetal chromosomal
The micro-deleted micro- repetition screening product of body screening product, noninvasive fetal chromosomal, noninvasive fetus monogenic disease screening product.
SEQUENCE LISTING
<110>Dongguan Bo Aomuhua Gene Tech. Company Limited
<120>A kind of maternal peripheral blood blood plasma fast separating process and its application
<130>
<160> 6
<170> PatentIn version 3.5
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<210> 4
<211> 30
<212> DNA
<213>Manual tag
<220>
<221> misc_feature
<222> (4)..(13)
<223> n is a, c, g, or t
<400> 4
atcnnnnnnn nnnctgagtc ggagacacgc 30
<210> 5
<211> 20
<212> DNA
<213>Artificial primer
<400> 5
ccatctcatc cctgcgtgtc 20
<210> 6
<211> 28
<212> DNA
<213>Artificial primer
<400> 6
ccactacgcc tccgctttcc tctctatg 28
Claims (10)
1. a kind of maternal peripheral blood blood plasma fast separating process, the method includes：One step is carried out in vitro maternal peripheral blood
Centrifugation obtains blood plasma.
2. according to the method described in claim 1, it is characterized in that：5~10mL maternal peripheral bloods, total centrifugal force of step centrifugation
For 10000~100000 (gmin).
3. according to the method described in claim 1, it is characterized in that：5~10mL maternal peripheral bloods, total centrifugal force of step centrifugation
For 16000~64000 (gmin).
4. according to the method described in claim 1, it is characterized in that：5~10mL maternal peripheral bloods, the opposite centrifugation of step centrifugation
Power is 1600~3200g, when centrifugation a length of 10~20min.
5. according to the method described in claim 1, it is characterized in that：Maternal peripheral blood is selected from the maternal peripheral blood guaranteed the quality；It is in
The maternal peripheral blood of normal condition or slightly soluble blood state or Medium hemolysis state.
6. Claims 1 to 5 any one of them maternal peripheral blood blood plasma fast separating process is in dissociative DNA extraction, fetal nucleus
Application in sour acquisition of information.
7. a kind of dissociative DNA extracting method, the method includes：Utilize claim 1~6 any one of them maternal peripheral blood
Blood plasma fast separating process obtains blood plasma, and dissociative DNA is extracted from blood plasma.
8. dissociative DNA extracting method according to claim 7, it is characterised in that：Using ethanol on pellosil post centrifugal process or magnetic bead
Method extracts dissociative DNA from blood plasma.
9. a kind of fetal nucleic acid Information Acquisition System, including：
One step separator of blood plasma：For utilizing claim 1~6 any one of them maternal peripheral blood blood plasma quick separating side
Method obtains blood plasma；
Dissociative DNA extraction element：For extracting dissociative DNA from blood plasma；
Library construction device：For carrying out library construction using dissociative DNA；
High-flux sequence device：For carrying out high-flux sequence to library；
Analysis of biological information device：For carrying out analysis of biological information according to high-flux sequence data, fetal nucleic acid information is obtained.
10. the fetal nucleic acid Information Acquisition System described in claim 9 is applied to noninvasive foetal chromosome aneuploidy screening system
The micro-deleted micro- repetition screening system of system, noninvasive fetal chromosomal, noninvasive fetus monogenic disease screening system.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110468180A (en) * | 2019-08-29 | 2019-11-19 | 北京优迅医学检验实验室有限公司 | Plasma dna library and its construction method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016562A (en) * | 2006-02-08 | 2007-08-15 | 陈熹 | Method of concentrating fetus dissociative DNA in pregnant woman blood plasma |
| CN104862301A (en) * | 2014-02-21 | 2015-08-26 | 广州康睿生物医药科技有限公司 | Method for separating and enriching free fetus DNA from maternal blood plasma |
| CN105441426A (en) * | 2015-12-31 | 2016-03-30 | 杭州迪安医学检验中心有限公司 | Method and kit for targeted enrichment of free DNA of peripheral blood |
| CN107354218A (en) * | 2006-05-03 | 2017-11-17 | 香港中文大学 | New fetus mark for pre-natal diagnosis and monitoring |
| CN107699607A (en) * | 2016-08-31 | 2018-02-16 | 刘红彦 | A kind of kit, method and purposes for building fetus dissociative DNA sequencing library |
-
2018
- 2018-04-12 CN CN201810326928.7A patent/CN108504653A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016562A (en) * | 2006-02-08 | 2007-08-15 | 陈熹 | Method of concentrating fetus dissociative DNA in pregnant woman blood plasma |
| CN107354218A (en) * | 2006-05-03 | 2017-11-17 | 香港中文大学 | New fetus mark for pre-natal diagnosis and monitoring |
| CN104862301A (en) * | 2014-02-21 | 2015-08-26 | 广州康睿生物医药科技有限公司 | Method for separating and enriching free fetus DNA from maternal blood plasma |
| CN105441426A (en) * | 2015-12-31 | 2016-03-30 | 杭州迪安医学检验中心有限公司 | Method and kit for targeted enrichment of free DNA of peripheral blood |
| CN107699607A (en) * | 2016-08-31 | 2018-02-16 | 刘红彦 | A kind of kit, method and purposes for building fetus dissociative DNA sequencing library |
Non-Patent Citations (1)
| Title |
|---|
| 琚竹梅: "妊娠早期孕妇外周血中胎儿游离DNA的定量分析", 《中国优秀硕士论文全文数据库(电子期刊)医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110468180A (en) * | 2019-08-29 | 2019-11-19 | 北京优迅医学检验实验室有限公司 | Plasma dna library and its construction method |
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