CN105239164B - Quantitative standard items in a kind of fetus dissociative DNA library and preparation method thereof - Google Patents
Quantitative standard items in a kind of fetus dissociative DNA library and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of based on semiconductor PCR sequencing PCR detection foetal chromosome aneuploidy trisomy 21,18 3 bodies and qPCR plasmid standards for quantitation of library DNA concentration of 13 3 physical examinations and preparation method thereof, belong to the noninvasive antenatal detection category of fetal aneuploidy, quality control is carried out to the detectable concentration for detecting sample.QPCR plasmid standards for quantitation preparation method of the present invention is divided into five steps：(1) joint sequence synthesizes with primer：(2) it is prepared by the library for simulating fetus dissociative DNA；(3) amplified library and purifying；(4) library standard curve is made；(5) pattern detection sheet.The inventive method can provide guarantee with rapid determination of sample library DNA concentration for foetal chromosome aneuploidy trisomy 21, the accuracy of 18 3 bodies and the noninvasive antenatal detection of 13 3 bodies and validity.
Description
Technical field
The invention belongs to biological product technical field, belong to the noninvasive antenatal detection category of foetal chromosome aneuploidy,
It is related to semiconductor PCR sequencing PCR detection fetal chromosomal aneuploid trisomy 21,18 3 bodies, the qPCR of library DNA concentration of 13 3 bodies to determine
Measure standard items and preparation method thereof.
Background technology
Chromosome aneuploid refers to for normal 46 chromosome of people, certain in cell one or several
Chromosome number increases or decreases, morbidity and mortality close relation significant with infantile period.The hair of such disease
Sick rate increases with increasing for pregnant woman age.Be born the neonate about 100,000 of chromosome abnormality every year in China according to the literature
People, chromosome abnormality person accounts for 0.3% in baby living, and wherein trisomy 21,18 3 bodies, Patau syndrome are clinical most common
The chromosome disease most easily occurred.The treatment of trisomy 21,18 3 bodies, Patau syndrome lacks effective ways at present,
Therefore the Prenatal Screening of popularization chromosomal disorders and pre-natal diagnosis, there is very important meaning to reducing inborn defect
Justice, this is advantageous to improve the quality of the population, and realizes prenatal and postnatal care.
The method detected using high throughput sequencing technologies to foetal chromosome aneuploidy is had compared to conventional method
Clear superiority.This method need to only extract maternal peripheral blood and be detected, and can avoid traditional invasive method may be to pregnant woman
The harm come with fetal zone；The DNA sequence dna of mother and fetus is directly detected in addition, compared to detection serum protein markers and is surpassed
Sonic detection, accuracy and sensitivity and reliability all greatly improve.Therefore, can be to pregnant woman blood plasma by deep sequencing
Micro fetus dissociative DNA is accurately detected, and traditional detection method can be overcome to cause to examine because fetus dissociative DNA content is too low
Survey the shortcomings that result false negative is too high.Foetal chromosome aneuploidy trisomy 21,18 3 bodies and 13 3 body detection kits (are partly led
Body PCR sequencing PCR) high-flux sequence principle is based on, these fetus dissociative DNAs are detected, utilize bioinformatic analysis system
Testing result is analyzed, obtains the details of fetal chromosomal number, is realized to trisomy 21 syndrome, 18 3 bodies synthesis
Patau syndrome of seeking peace carries out quickly antenatal auxiliary diagnosis.The DNA data volumes obtained by semiconductor sequencing experiment
(Unique Reads numbers) need to reach 3.5M Reads (original Reads needs 5M or so) could be to foetal chromosome aneuploidy
Symptom is clearly judged.
Sample is detected on semiconductor sequencing system at present, every time experiment can output 80M or so Reads, it is theoretical
Experiment can detect 16 samples every time.But due in sequence testing chip containing 165M micropore, it is necessary to library DNA molecular weight be only
108Individual DNA copy number, i.e., 10-15Mol, it is difficult that equivalent equably mixes that so low concentration, which inherently causes 16 samples, this
The data volume for causing some samples to be sequenced to obtain is not reached into requirement and needs to detect again, this both wastes cost, also delays
Detection time.(semiconductor is sequenced for foetal chromosome aneuploidy trisomy 21,18 3 bodies and 13 3 body detection kits at present
Method) suggest 12 samples of detection every time, therefore on the premise of ensureing that each sample DNA is attained by enough Reads numbers, increase
The sample size of single experiment becomes necessity.
Existing foetal chromosome aneuploidy trisomy 21,18 3 bodies and 13 3 body detection kits (semiconductor PCR sequencing PCR) text
Storehouse concentration Qubit Fluorometric assays, only as DNA and the Molecular of fluorescenceAfter dyestuff combines, ability
Fluorescence signal is sent, therefore this method only detects the concentration of target molecule (rather than pollutant), relative to traditionalSpectrophotometric is more accurate, more accurate.But Qubit detection methods equally have necessarily in high flux gene sequencing process
The defects of, i.e., detection range is 0.1ng/ μ L-10ng/ μ L, for the 10 of addition8Individual DNA molecular is difficult to accurate quantification.Therefore most
The low qPCR technologies that can detect 100 DNA molecular copies are applied undoubtedly to be very important on high-flux sequence.Utilize
DNA standard items establish standard curve, obtain the copy number of library DNA, then carry out upper machine sequencing after mixed in equal amounts.
At present, the qPCR plasmid standards for quantitation for the library DNA concentration that the in the market detection method is related to almost does not have, and this is serious
The Prenatal Screening of chromosomal disorders and the efficiency of diagnosis process are influenceed, adds cost.Detected for increase test kit
Validity in journey, the present invention are used for the chromosome aneuploid of pregnant woman blood plasma free fetal dna with high throughput sequencing technologies
Then human genome DNA is broken into the DNA that length is 160~200bp by (T21, T18 and T13) national standard to trace to the source
Fragment, library preparation is carried out using the kit, it is non-as fetal chromosomal to be diluted to finite concentration after the detections of Qubit 2.0
The qPCR of euploid trisomy 21,18 3 bodies and 13 3 body detection kits (semiconductor PCR sequencing PCR) library DNA Concentration Testing is quantified
Standard items.
The content of the invention
It is an object of the invention to provide a kind of foetal chromosome aneuploidy trisomy 21,18 3 bodies and 13 3 physical examination test agents
QPCR plasmid standards for quantitation of box (semiconductor PCR sequencing PCR) library DNA concentration and preparation method thereof.
In order to realize the object of the invention, a kind of foetal chromosome aneuploidy trisomy 21,18 3 bodies and 13 3 bodies have been invented
The qPCR plasmid standards for quantitation of detection kit (semiconductor PCR sequencing PCR) library DNA concentration.
The qPCR plasmid standards for quantitation of the present invention is to contain the nuclease free solution that a segment length is 260bp or so DNA fragmentations.
The joint sequence of sequencing is contained at the DNA fragmentation both ends of the qPCR plasmid standards for quantitation of the present invention, with library DNA feature phase
Together.
The qPCR plasmid standards for quantitation of the present invention is related to the foundation of library DNA quantitation curves.
QPCR plasmid standards for quantitation preparation method of the present invention：
(1) joint sequence synthesizes with primer：Synthetic linker sequence double-strand Adapter S1 and Adapter S2, and in joint
The primer pair NIPT-Library F and NIPT-Library R of qPCR amplifications are designed in sequence, it is as follows：
Adapter S1：
5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′(+)
3′-C*C*C*GGTAGAGTAGGGACGCACAGAGGCTGAGTC-5′(-)
Adapter S2：
5′-CCA CTA CGC CTC CGC TTT CCT CTC TAT GGG CAG TCG GTG AT-3′(+)
3′-C*C*C*GGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTA-5′(-)
NIPT-Library F：5’-CCATCTCATCCCTGCGTGTC-3’
NIPT-Library R：5’-CCTCTCTATGGGCAGTCGGTGAT-3’
(2) it is prepared by the library for simulating fetus dissociative DNA：Human genome DNA is extracted, by the ultrasonic (condition of its physics：Frequency
The cooling of 0.5min ultrasounds/0.5min stops, 15min/ circulations, 3 circulations) interrupt DNA fragmentation for 160bp~200bp；Utilize
After the DNA fragmentation of Adapter S1 and Adapter S2 sequences and 160bp~200bp is attached by T4DNA ligases, E-
Gel glue reclaim 260bp fragments；
(3) amplified library and purifying：Gathered using NIPT-Library F, NIPT-Library R primers and high-fidelity DNA
The DNA fragmentation of recovery, response procedures are in synthase amplification step (2)：98 DEG C of 2min, 98 DEG C of 15s, 62 DEG C of 15s, 70 DEG C of 1min,
Totally 10 circulations, 70 DEG C of extension 5min；Then the DNA fragmentation that is obtained using magnetic beads for purifying is simultaneously dissolved in nuclease-free water；
(4) library standard curve is made：The library standard items quantitatively built using Qubit 2.0 are examined on sequenator
Survey, obtain Reads numbers more than the dilution for carrying out 10 times of gradients after 80M again, carry out qPCR detections, obtain Ct values and establish standard song
Line；
(5) pattern detection：The library DNA of sample to be checked after the progress concentration determination of library standard curve with sequenator by being examined
Survey, increase the uniformity of data volume between sample.
The qPCR standard items of the present invention are the DNA solution of white clear, have stable, easy to maintain and do not endanger operator and be good for
The advantages of health, at the same qPCR standard items prepare bottle between the poor coefficient of variation it is smaller, this explanation the present invention use, can be used for fetus
The antenatal non-invasive screening of chromosomal disorders and diagnosis.
The present invention can effectively avoid the testing result of same batch library DNA concentration from deviation occur, for increase by 21 3
Homogeneity and detection efficiency between body, 18 3 bodies and the noninvasive antenatal detection sample of 13 3 bodies provide guarantee.
Brief description of the drawings
The Bioanalyzer analysis length distribution maps of Agilent 2100 for the qPCR plasmid standards for quantitation that Fig. 1 displays are established
Spectrum.
Fig. 2 shows that qPCR plasmid standards for quantitation is surveyed using foetal chromosome aneuploidy trisomy 21,18 3 bodies and 13 3 physical examinations
The collection of illustrative plates of machine on kit (semiconductor PCR sequencing PCR).
Fig. 3 shows the amplification curve collection of illustrative plates of qPCR plasmid standards for quantitation.
Fig. 4 shows qPCR quantitation curves.
Fig. 5 shows the quantitative solubility curves of qPCR.
Fig. 6 shows the qPCR quantitative concentrations in same certification storehouse and the comparison of Qubit quantitative concentrations.
Fig. 7 shows that same certification storehouse passes through the quantitative DNA Reads number ratios gone up machine with Qubit after quantitative and be sequenced to obtain of qPCR
Compared with.
Embodiment
Following examples are used to illustrate the present invention, but are not used in the limitation present invention.If technological means used in embodiment
Indicated to be special, be conventional method.If the reagent being related in embodiment and consumptive material without specified otherwise commercially
Obtain.QPCR quantitative experiments in following examples, it is respectively provided with and repeats to test three times, results averaged.
Embodiment 1：The preparation of standard items
First, joint sequence and design of primers synthesis
Design is tried one's best inconsistent sequence with human genome, according to G/C content and design of primers principle, obtains following connect
Header sequence.To prevent joint sequence opposite direction to be connected with purpose fragment, 3 ' ends set cohesive end, to prevent cohesive end from degrading
Prominent C is modified, the minus strand 3 ' that * represents joint holds base C to be modified by thiophosphorylation；Dephosphorization is passed through at another 5 ' both ends
Acidifying modification, to prevent that flat end goes out to occur connection phenomenon to joint sequence in itself, in summary factor synthesizes following joint sequence
Double-strand Adapter S1 and Adapter S2：
Adapter S1：
5′-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-3′
3′-C*C*C*GGT AGA GTA GGG ACG CAC AGA GGC TGA GTC-5′
Adapter S2：
5′-CCA CTA CGC CTC CGC TTT CCT CTC TAT GGG CAG TCG GTG AT-3′
3′-C*C*C*GGT GATGCG GAG GCG AAA GGA GAGATA CCC GTC AGC CAC TA-5′
Primer is designed, the primer sequence of QPCR amplifications is designed on joint sequence, the Tm values of primer are 60 DEG C.
NIPT-Library F：5’-CCA TCT CAT CCC TGC GTG TC-3’
NIPT-Library R：5’-CCT CTC TAT GGG CAG TCG GTG AT-3’
The primer pair being made up of primer NIPT-Library F and primer NIPT-Library R, the target sequence that can be expanded
To connect the DNA library of top connection, for the amplification for plasma DNA library with quantitative, target sequence length is 260bp or so.
2nd, the preparation of standard items
1. extracting human genome DNA, DNA is entered using Bioruptor CD-200 TS Sonication System
3 circulations of row, each 15min physics ultrasounds that circulate interrupt, and interrupt frequency and stop for 0.5min ultrasounds/0.5min coolings, interrupt
Major length for 160bp~200bp simulate fetus dissociative DNA length.
2. Adapter S1 and Adapter S2 sequences and the 180bp fragments after recovery are carried out using T4DNA ligases
Connection, because Adapter S1 and Adapter S2 5 ' ends have carried out dephosphorylation modification, avoid joint itself and connect
Situation about connecing.
3. obtained connection product DNA fragmentation carries out E-Gel glue reclaims, recovery 260bp or so band.
4. utilize NIPT-Library F, NIPT-Library R primers and Phusion DNA Polymerase
DNA fragmentation after (Thermo Scientific) amplification connection.
20 μ L reaction systems：5 × Phusion HF buffer, 4 μ L, 10mM dNTPs 0.4 μ L, Phusion DNA
Polymerase 2U, NIPT-Library F and NIPT-Library R (10 μM) each 1 μ L, DNA profiling 40ng.
Response procedures are：98 DEG C of 2min, 98 DEG C of 15s, 62 DEG C of 15s, 70 DEG C of 1min, totally 10 circulations, 70 DEG C of extension 5min.
5. removing the enzyme added in course of reaction and primer etc. using magnetic beads for purifying method, target DNA is purified.
6. Fig. 1, main peak 260bp are shown in the fragment distribution in the Bioanalyzer of Agilent 2100 analyses library.With
Qubit 2.0 analyzes the concentration of DNA library.
The Qubit concentration of the qPCR plasmid standards for quantitation of table 1.
3rd, the high-flux sequence result of standard items
1. the standard items established using step 2 carry out template preparation, template enrichment is sequenced with upper machine, operate according to 21 3
Body, 18 3 bodies, 13 3 body detection kits (semiconductor PCR sequencing PCR) specification are carried out, and the use of instrument are mainly DA8600 and supporting
Instrument.
2. sequencing result as shown in Fig. 2 loading rates (i.e. the coverage rate of DNA positive magnetic beads in chip micropore) be 87%,
DNA Reads numbers are 87.12M, and it is suitable that total reads numbers, which reach i.e. explainable are sequenced using machine in the concentration of 80M, and
Total reads is higher after standard items sequencing, detects other samples with this standard items, the reads numbers of each sample are also higher, more energy
Meet to require.It can meet that each sample is attained by 3.5M Unique Reads and can provide report using this concentration as standard.
Embodiment 2：The application method of standard items and the foundation of standard curve
First, dilution process
Obtained library is diluted according to following computational methods, is diluted to 100pM first
660 (Da) are the relative molecular mass of mono- base-pair of DNA.
100pM library is diluted 10 times successively, obtains 10pM, 1pM, 0.1pM, 0.01pM and 0.001pM library.
2nd, experimental method
6 library dilutions that step 1 is obtained carry out real-time fluorescence quantitative PCR.Using KAPA SYBR FAST
Universal qPCR kit kits are analyzed.
QPCR reaction systems：Contain 4 μ L, 2 × SYBR mix of template 10 μ L, primer NIPT- in 20 μ L mixed liquors
Library F and each 1 μ L of primer NIPT-Library R (10 μM), the μ L of Nuclease Free Water 4.
QPCR response procedures：95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 30s of 40 circulations, 60 DEG C of annealing extend common 45s.
Solubility curve process：95 DEG C denaturation 1min, 60 DEG C annealing 30s, be increased to 95 DEG C of process medium velocities for 0.5 DEG C/s simultaneously
Continuous acquisition fluorescence.
3rd, interpretation of result
Amplification curve is shown in Fig. 3, and amplification curve is in smooth S type curves.
Standard curve is shown in Fig. 4.Calibration curve equation is y=-3.4361gx+13.93, R2=0.999, y represent critical follow
Number of rings, x representation DNAs library concentration (pM) amount.Lowest detection is limited to 0.001pM, and quantitative section is 0.001pM-100pM, amplification
Efficiency is 98.885%.
Solubility curve is shown in Fig. 5, because sequence is non-single between the positive anti-primer of standard items, therefore does not occur simple spike.
Embodiment 3：The repeatability of qPCR plasmid standards for quantitation detection
By the standard items multigelation 8 times of the 100pM standard items prepared and other 6 dilution factors while carry out qPCR inspections
Survey, obtained period CT values are shown in Table 2, learn the standard items coefficient of variation ＜ 5.00% of 8 each dilution factors of multigelation, relatively
It is smaller, it can be used with the supporting body detection kit of 18 3 body of trisomy 21 13 3 (semiconductor PCR sequencing PCR) (8 RUN).
The result that 2. multigelation of table is 8 times
Embodiment 4：Application of the library plasmid standards for quantitation in fetus dissociative DNA library is quantitative
First, the preparation of library DNA to be measured
16 plasma DNA samples are extracted, surveying (semiconductor PCR sequencing PCR) using the physical examination of 18 3 body of trisomy 21 13 3 is carried out
Library construction, the concentration in library is detected using Qubit 2.0.
2nd, library DNA is diluted
Obtained library DNA is diluted to 10pM (concentration of Qubit methods) according to the formula of embodiment 2 and carries out qPCR inspections
Survey, the concentration of library DNA is drawn by standard curve, sees Fig. 6 compared with the concentration that Qubit 2.0 is directly detected.
3rd, upper machine testing
Obtained library DNA is diluted into 100pM respectively according to qPCR quantitative approach and Qubit quantitative approach to carry out uniformly
Upper machine sequencing, obtained Reads numbers are shown in Fig. 7 after mixing.Some the sample highest reads number generally obtained with it is minimum
Reads numbers differ more than twice and then have sample factor data amount deficiency and need to detect again.Shown according to Fig. 7, qPCR is quantitative
The DNA Reads numbers that machine obtains on library quantitatively obtain evenly than Qubit, and 16 sample standard deviations are between 4M-6M, Unique
Reads is attained by 3.5M, can meet the requirement for providing report；And quantitatively obtain each sample using Qubit methods
Reads highest ＞ 8M reads, minimum only 2M reads, nearly 4 times of difference, sample 11 and sample 16 then need to resurvey.
The plasma DNA that above example 1~4 is related to is extracted according to Jin Maige plasma DNA extracts kits
Carry out.Specially：
1. 600 μ L blood plasma are taken to add 30 μ L Proteinase K, 12 μ L Acryl Carrier, 900 μ L Lysis
Buffer, 60 μ L magnetic beads, overturn mix 10min at room temperature, magnetic bead and nucleic acid is fully combined.Reaction EP pipes are placed in magnetic frame
Upper 4min, after magnetic bead absorption completely, sucking liquid.
2. Wash Buffer I are cleaned：500 μ L Wash Buffer I are added, magnetic bead is resuspended, is placed on magnetic frame
4min, after magnetic bead absorption completely, sucking liquid, remove EP pipes.
3. Wash Buffer II are cleaned：500 μ L Wash Buffer II are added, magnetic bead is resuspended, is placed on magnetic frame
4min, after magnetic bead absorption completely, sucking liquid, EP pipes are still placed on magnetic frame, make magnetic bead continue to adsorb.
4. Wash Buffer III are cleaned：Sucking liquid after 550 μ L Wash Buffer III, 30s is gently added,
Remove EP pipes.
5. dry magnetic bead：EP is managed into of short duration centrifugation.EP pipes are placed on magnetic frame, after magnetic bead absorption completely, suctioned out residual
Solution is stayed, drying 2min makes evaporating totally in pipe.
6. eluted dna：Adding 33 μ L EB buffer, 55 DEG C are incubated 5min in magnetic bead in EP pipes, is resuspended.
7. reclaim DNA sample：EP pipes are placed in 4min on magnetic frame, after magnetic bead absorption completely, supernatant solution suctioned out
In new EP pipes (about 32 μ L).
Although being described in detail with a general description of the specific embodiments to the present invention in above-mentioned,
On the basis of the present invention, it can be modified and improve, this is obvious for art technology.Therefore, not inclined
From the basic modifications or improvements of spirit of the present invention, the scope of protection of present invention is belonged to.
Claims (5)
1. a kind of preparation method of the quantitative standard items in fetus dissociative DNA library, its step are as follows：
(1) joint sequence synthesizes with primer：Synthetic linker sequence double-strand Adapter S1 and Adapter S2, and in joint sequence
The primer pair NIPT-Library F and NIPT-Library R of upper design qPCR amplification, it is as follows：
Adapter S1：
5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′(+)
3′-CCCGGTAGAGTAGGGACGCACAGAGGCTGAGTC-5′(-)
Adapter S2：
5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3′(+)
3′-CCCGGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTA-5′(-)
NIPT-Library F：5’-CCATCTCATCCCTGCGTGTC-3’
NIPT-Library R：5’-CCTCTCTATGGGCAGTCGGTGAT-3’
(2) it is prepared by the library for simulating fetus dissociative DNA：Extract human genome DNA, by its physics ultrasound interrupt for 160bp~
200bp DNA fragmentation；Using T4 DNA ligases by Adapter S1 and Adapter S2 sequences with 160bp~200bp's
After DNA fragmentation is attached, E-Gel glue reclaim 260bp fragments；
(3) amplified library and purifying：Utilize NIPT-Library F, NIPT-Library R primers and high-fidelity DNA polymerase
The DNA fragmentation of recovery in amplification step (2), then using magnetic beads for purifying DNA amplification and it is dissolved in nuclease-free water；
(4) library standard curve is made：The library standard items quantitatively built using Qubit 2.0 are detected on sequenator, are obtained
It is more than the dilution for carrying out 10 times of gradients after 80M again to Reads numbers, Ct values is obtained after qPCR is detected and establish library standard song
Line；
(5) pattern detection：The library DNA of sample to be checked is detected with sequenator after carrying out concentration determination by library standard curve,
The uniformity of data volume between increase sample.
2. preparation method according to claim 1, the Adapter S1 that are further characterized in that in the step (1) and
Adapter S2 sequences, it is the cohesive end modified by thiophosphorylation that its minus strand 3 ', which holds continuous three C bases, and 5 ' ends are passed through
Dephosphorylation is modified.
3. preparation method according to claim 1, the condition for being further characterized in that physics ultrasound in the step (2) are：
Frequency 0.5min ultrasounds/0.5min coolings stop, 15min/ circulations, 3 circulations.
4. preparation method according to claim 1, it is further characterized in that the reaction condition of the step (3) amplified library
For：98 DEG C of 2min, 98 DEG C of 15s, 62 DEG C of 15s, 70 DEG C of 1min, totally 10 circulations, 70 DEG C of extension 5min.
5. preparation method according to claim 1, the standard items of foundation be mainly used in foetal chromosome aneuploidy T21,
T18 and T13 noninvasive Prenatal Screening and diagnosis.
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