CN105132571B - Dissociated the method for mitochondrial DNA content using droplet type digitlization PCR detection peripheral bloods - Google Patents
Dissociated the method for mitochondrial DNA content using droplet type digitlization PCR detection peripheral bloods Download PDFInfo
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Abstract
The present invention establishes a kind of method for the mitochondrial DNA content that dissociates using droplet type digitlization PCR detection peripheral bloods.The method includes the structure of recombinant plasmid, sample preparation, extraction plasma dna and Exosome DNA, blood plasma is pre-processed and using droplet type digitlization PCR analysis recombinant plasmid mtDNA copy numbers.
Description
Technical field
It establishes detection peripheral blood the present invention relates to a kind of using droplet type digitlization round pcr and dissociates mitochondrial DNA content
Method and its kit and the above method or above-mentioned kit in detection peripheral blood dissociates mitochondrial DNA content
Using.
Background technology
In the molecule diagnosis research of human peripheral blood, it is believed that the nucleic acid compositions of Peripheral Circulation can become reflection morbid state
Preferable index.Peripheral Circulation DNA mainly includes core DNA and mitochondrial DNA (mtDNA).It is come from different from core DNA, mtDNA
In cell mitochondrial, for cyclic annular double-strand, the DNA of multicopy, and with proinflammatory effect.Therefore, the detection peripheral blood mtDNA that dissociates contains
Amount potentially contributes to monitoring disease, such as tumour, wound, chronic inflammation.Dissociate mitochondrial DNA currently used for detection peripheral blood
The method of (mitochondrial DNA, mtDNA) content mainly utilizes real-time fluorescence quantitative PCR
Absolute quantitation method (Ajaz S, Czajka A, the Malik A of (quantitativereal-time PCR, qPCR):Accurate
measurement of circulating mitochondrial DNA content from human blood samples
using real-time quantitative PCR.Methods in molecular biology(Clifton,NJ
2015,1264:117-131.).The method expands mitochondrial genomes related gene, such as ND1, COX2 bases by regular-PCR first
Because etc., plasmid vector is cloned into, builds the recombinant plasmid containing chondriogen.It is obtained by concentration mensuration and after calculating
The copy number of plasmid, establishes standard curve, and common a concentration of 10~108copies/μl.In content detection, DNA sample with
Standard plasmid is carried out at the same time qPCR reactions, and sample mtDNA copy numbers are calculated using standard curve.However, this method is in an experiment
It was found that in low copy number, accuracy declines, and it is possible that non-specific amplification, so as to influence the accuracy of experiment.This
Outside, current detection method be both needed to before analyzing sample extraction DNA, however at present commercialization DNA extraction kit
There are the loss situation of extraction process DNA, and yield differs, the difference that result may be caused to judge.Therefore, there is an urgent need to
A kind of higher detection method of accuracy is used for the diagnosis research of peripheral blood DNA molecule.
Droplet type digitlization PCR (droplet digital PCR, ddPCR) is the round pcr of a new generation, different from passing
The qPCR technologies of system, principle are by forming Water-In-Oil droplet, by (nl/ is micro- in PCR reaction distribution to 20000 droplets
Drop), after carrying out PCR reactions, the detection of fluorescence signal is carried out to each droplet one by one, the droplet interpretation containing fluorescence signal is 1,
The droplet interpretation for not having fluorescence signal is 0, and according to Poisson distribution principle and the number and ratio of positive droplet, target point is calculated
The starting copy number of son.Therefore, ddPCR need not establish standard curve, can directly determine to copy the exhausted of target molecule to be checked down to single
To number.
Therefore, ddPCR is in detection, gene copy number variation analysis and gene expression analysis of rare mutation etc.
Application in shown its advantage.It there is no and reported about the research using ddPCR detection mtDNA copy counting methods at present.
Invention content
Dissociated the method for mitochondrial DNA content using droplet type digitlization PCR detection peripheral bloods the present invention relates to a kind of, it is main
Include the following steps：
1) structure of recombinant plasmid：By PCR amplification MT-ND1 genes, MT-ND1PCR products are cloned by T-A
pMDTM18-T carriers, and convert E. coli DH5 α
2) sample preparation：Using the recombinant plasmid of the small extraction reagent kit extraction E.coli DH5 α of plasmid, plasmid copy is adjusted
Number, and it is diluted to 1~106copies/μl。
3) plasma dna and Exosome DNA are extracted：Peripheral blood is acquired to EDTA-Na2Anticoagulant tube, separated plasma.By 500 μ
L blood plasma is divided into two deciles, wherein 250 μ l blood plasma directly extract DNA using paramagnetic particle method, 250 μ l detach exosome first,
1h are incubated to remove the DNA outside exosome in exosome precipitations plus containing resuspension in 1U DNaseI solution and at 37 DEG C.
4) blood plasma is pre-processed：500 μ l blood plasma, 1600g centrifugation 5min are taken, supernatant goes to new 1.5ml EP pipes；
16000g centrifuges 5min, and supernatant is taken to cross 0.22 μm of disposable filter；The blood plasma of pretreatment is taken by 1:1 ratio sequentially adds 1~
104Copies/ μ l MT-ND1-pMD18-T standard plasmids, mixing, mixing sample carry out DD-PCR analyses directly as template.
5) recombinant plasmid mtDNA copy numbers are analyzed using ddPCR, including designing primer and probe, utilizes recombinant plasmid, blood
Starch DNA, blood plasma exosome DNA and through processing or untreated blood plasma as template, amplification system is formed after droplet in PCR
It is expanded on instrument, after amplification, 96 orifice plates for having completed PCR reactions is placed in BIO-RAD DX200Droplet
Reader carries out droplet reading, is analyzed using 1.6 softwares of QuanatSoft.
In one embodiment, the primer sequence of PCR amplification MT-ND1 genes is as follows in the method step 1：Forward direction
Primer is 5'CAGCCGCTATTAAAGGTTCG3', and reverse primer is 5'AGAGTGCGTCATATGTTGTTC 3', amplified fragments
Length 1041bp [Homo sapiens mtDNA (GeneBank No.NC_012920) np3017-4057].
In one embodiment, plasmid copy number is adjusted using the following formula in the method step 2：
MWDNA(daltons)=(daltons/bp)=(2692bp+ of (pMD18-T vector+Insert) × 660
1041bp) × 660 (daltons/bp)=2.46 × 106(daltons)；
CNDNA(copies/ μ l)=CDNA(ng/μl)×6.02×1014/MWDNA=CDNA(ng/μl)×6.02×1014/
2.46×106=CDNA(ng/μl)×2.45×108.
(CNDNA:Plasmid copy number；CDNA:Plasmid DNA concentration；MWDNA:Plasmid molecule amount)
In one embodiment, the primer and probe are as follows in the method step 5：F:
CCCTAAAACCCGCCACATCT；R:GAGCGATGGTGAGAGCTAAGGT；Probe：CCATCACCCTCTACATCACCGCCC,
Using BHQ1 as quenching group.
In one embodiment, blank control and negative control are also included in the amplification system of the method step 5,
Middle blank control is the PCR system without DNA profiling, and negative control is using pMD-18-T plasmids as template.
In one embodiment, the step of the method step 5 generation droplet is as follows：
1) 20 μ l systems are added in the upper Sample holes of droplet generation card, the droplet generation oil of 70 μ l is added in Oil holes, should
Process avoids bubble generation and cross contamination, puts on clean adhesive tape special；
2) droplet generation is placed on drop generators, droplet generating process is carried out, in this process, in Sample holes
System under the action of negative pressure, is mixed to Droplet holes (droplet hole) to form droplet together with droplet generation oil；
3) droplet generation card is taken out, draws in 40 μ l droplets to the clean corresponding hole of 96 orifice plate, covers aluminum foil special paper,
180 DEG C, by after pressure seal mouth, are expanded on regular-PCR instrument.
The present invention utilizes the testing principle of ddPCR technologies, establishes the method for detection mtDNA copy numbers and applied to peripheral blood
The analysis of free mtDNA.The method not only has the characteristics of high sensitivity better than qPCR, specificity and repeatability, and its
QPCR is higher than to chaff interferent (such as anti-coagulants) tolerance.In addition, it is of the invention on the basis of detection method is established, further
Using plasma sample directly as the template of ddPCR, the analysis of blood plasma mtDNA is carried out, is not only eliminated since extraction brings inspection
Uncertainty is surveyed, easily facilitates clinical examination.
Brief description
Fig. 1 shows the qPCR experimental results of recombinant plasmid, and wherein Fig. 1 a are 10~106Copies/ μ l recombinant plasmids
Amplification curve, Fig. 1 b are standard curve.
Fig. 2 shows the ddPCR of recombinant plasmid as a result, wherein Fig. 2 a are 1~104The ddPCR of copies/ μ l recombinant plasmids
One-dimensional droplet figure, Fig. 2 b are plasma dna, recombinant plasmid (102Copies/ μ l), blank control, the ddPCR of negative control it is one-dimensional
Droplet figure；Fig. 2 c are the correlation analysis result of qPCR and ddPCR.Fig. 3 shows ddPCR detection blood plasma mtDNA and exosome
MtDNA's as a result, wherein Fig. 3 a are the one-dimensional droplet figures of ddPCR of blood plasma mtDNA and exosome mtDNA；Fig. 3 b and Fig. 3 c is
18 blood plasma mtDNA and exosome mtDNA copy number results.
Fig. 4 is plasma sample ddPCR testing results, and wherein Fig. 4 a are the one-dimensional droplet figures of ddPCR of plasma sample；Fig. 4 b are
Plasma sample positive droplet number and total droplet number distribution map, P1~P6 are sample number；Fig. 4 c are two kinds of blood plasma pre-treating methods
Results contrast
Fig. 5 is each plasma sample mtDNA copy number analysis results, and grey parts represent the mtDNA copies of former plasma sample
Number, black portions represent the plasmid copy number added in.
Fig. 6 is anti-coagulants EDTA-Na2Influence to ddPCR, wherein Fig. 6 a are that template content is low as a result, Fig. 6 b are mould
The situation of the high droplet saturation of plate content.
Specific embodiment
The structure of 1 recombinant plasmid of embodiment and sample preparation
1st, the structure of recombinant plasmid
First, by PCR amplification MT-ND1 genes, forward direction primer is 5'CAGCCGCTATTAAAGGTTCG3', is reversely drawn
Object is 5'AGAGTGCGTCATATGTTGTTC 3'.Expanding fragment length 1041bp [Homo sapiens mtDNA (GeneBank
No.NC_012920)np3017-4057]。
1) PCR reaction systems are prepared
2) PCR reaction conditions：
3) PCR product electrophoresis：1% Ago-Gel is prepared, the electrophoresis 20min under 120V voltages
MT-ND1PCR products are cloned into pMD by T-ATM18-T carriers, and convert E. coli DH5 α
2. sample preparation
2.1 recombinant plasmid
Using the recombinant plasmid of the small extraction reagent kit extraction E.coli DH5 α of plasmid, plasmid copy is adjusted according to the following formula
Number, and it is diluted to 1~106copies/μl。
MWDNA(daltons)=(daltons/bp)=(2692bp+ of (pMD18-T vector+Insert) × 660
1041bp) × 660 (daltons/bp)=2.46 × 106(daltons)；
CNDNA(copies/ μ l)=CDNA(ng/μl)×6.02×1014/MWDNA=CDNA(ng/μl)×6.02×1014/
2.46×106=CDNA(ng/μl)×2.45×108.
(CNDNA:Plasmid copy number；CDNA:Plasmid DNA concentration；MWDNA:Plasmid molecule amount)
2.2 plasma dnas and Exosome DNA
Peripheral blood is acquired to EDTA-Na2Anticoagulant tube, separated plasma.500 μ l blood plasma are divided into two deciles, wherein 250 μ l blood
Slurry directly extracts DNA using paramagnetic particle method, and 250 μ l detach exosome first, adds in exosome precipitations molten containing 1U DNaseI
It is resuspended in liquid and is incubated 1h at 37 DEG C to remove the DNA outside exosome.
2.3 blood plasma pre-process
1) 500 μ l blood plasma, 1600g centrifugation 5min are taken, supernatant goes to new 1.5ml EP pipes；
2) 16000g centrifuges 5min, and supernatant is taken to cross 0.22 μm of disposable filter；
3) blood plasma of pretreatment is taken by 1:1 ratio sequentially adds 1~104Copies/ μ l MT-ND1-pMD18-T standard matter
Grain, mixing, mixing sample carry out DD-PCR analyses directly as template.
Embodiment 2 is compared using qPCR and ddPCR analysis recombinant plasmid mtDNA copy numbers
3.1 primers and probe
Table 1 is used for the primer and probe of mtDNA copy numbers detection
*：QPCR 3' quenching groups are TAMRA, ddPCR BHQ1
3.2 qPCR
1) PCR reaction systems are prepared
*：DNA profiling is 1~106The recombinant plasmid of copies/ μ l
2) PCR reaction conditions：
3.3 ddPCR
1) PCR system is prepared
*：DNA profiling is respectively：
A) 1~104The recombinant plasmid of copies/ μ l；
B) recombinant plasmid+EDTA-Na2 (5,10,15,20,25,30mM)；
C) plasma dna；
D) blood plasma exosome DNA；
E) pretreated blood plasma and untreated blood plasma.
Blank control：In addition to without DNA profiling, other all sames；
Negative control：PMD-18-T plasmids are as template.
2.3 droplets generate
4) 20 μ l systems are added in the upper Sample holes of droplet generation card, the droplet generation oil of 70 μ l is added in Oil holes, should
Process behaviour avoids bubble generation and cross contamination, puts on clean adhesive tape special；
5) droplet generation is placed on drop generators, droplet generating process is carried out, in this process, in Sample holes
System under the action of negative pressure, is mixed to Droplet holes (droplet hole) to form droplet together with droplet generation oil；
6) droplet generation card is taken out, draws in 40 μ l droplets to the clean corresponding hole of 96 orifice plate, covers aluminum foil special paper,
180 DEG C, by after pressure seal mouth, are expanded on regular-PCR instrument.
2.4 PCR amplification conditions
Often step heating and cooling set 2 DEG C/s above
2.5 read droplet：
96 orifice plates for having completed PCR reactions are placed in BIO-RAD DX200Droplet Reader and carry out droplet reading, profit
It is analyzed with 1.6 softwares of QuanatSoft.
QPCR the and ddPCR results contrasts of 2.6 recombinant plasmids
QPCR analyses 10~106Copies/ μ l recombinant plasmids can reach higher amplification efficiency and present preferable linear
(R2=0.995), but when copy number is less than 10copies/ μ l, Cq values are more than 33 (Fig. 1 a, b), and result precision reduces.But
DdPCR remains to preferably detect mtDNA, and blank control and negative control do not have non-specific amplification in low-copy
(Fig. 2 a, b).
Correlation analysis is carried out to qPCR the and ddPCR results of recombinant plasmid, it is found that the two is linear related, related coefficient
R2=0.996 (Fig. 2 c).
Embodiment 3. detects the copy number of blood plasma mtDNA and blood plasma exosome mtDNA using ddPCR
Exosome is to be present in the outer vesica of various body fluid cells, and blood plasma contains a large amount of exosome.There are many Exosome contains
Protein and nucleic acid, including mtDNA, therefore, exosome mtDNA may be a part of blood plasma mtDNA.
The mtDNA of low-copy in blood plasma and exosome is detected using aforementioned ddPCR technologies, as a result show blood plasma mtDNA with
Exosome mtDNA are respectively 117.61 ± 10.68 (Log10:2.04 ± 0.04) copies/ μ l and 21.06 ± 1.91
(Log10:1.30±0.04)copies/μl.The two ratio (exosome mtDNA/ blood plasma mtDNA) is 0.19 ± 0.018 (figure
3a,b,c)。
Embodiment 4 directly detects the copy number of plasma sample mtDNA using ddPCR
Plasma composition is complicated, includes a large amount of protein and the chaff interferent of other suppression PCRs reaction, and to avoid ddPCR
In the process since the droplet that plasma composition (such as cell fragment, macromolecular immunoglobulin) influences 250 μm of its diameter is formed, because
This directly using blood plasma as template before, the pre-treatment of two methods has been carried out to blood plasma.First, blood plasma 1600g is centrifuged
10min, and then blood plasma is divided into two parts, portion continues 16000g centrifugation 10min, another is through 0.22 μm of disposable filter membrane mistake
Filter, two parts of blood plasma are carried out at the same time ddPCR.
From the one-dimensional figure of droplet as it can be seen that positive droplet and negative droplet can be separated preferably (Fig. 4 a), and total droplet number
Average value is 14,185 (Fig. 4 b), has reached the quantity analyzed as ddPCR.In the two methods handled before relatively blood plasma,
It was found that result and no significant difference, respectively 157.50 ± 24.16 (Log10:2.17 ± 0.06) copies/ μ l and 168.83 ±
31.95(Log10:2.19 ± 0.09) copies/ μ l (Fig. 4 c).
The recovery experiment of 5 recombinant plasmid of embodiment
By recombinant plasmid (325copies/ the μ l, Log of known copy number10:2.498copies/ μ l), add in different blood
Sample is starched, carries out ddPCR analyses, calculates and adds in before and after plasmid, the difference of blood plasma mtDNA copy numbers.As a result, it has been found that calculating is copied
Shellfish number difference is 348.40 ± 34.30 (Log10:2.540 ± 0.041) copies/ μ l connect with the plasmid copy number difference of addition
Closely (Fig. 5), average recovery rate is up to 101.68%.As a result it prompts, carrying out ddPCR analyses as template by the use of blood plasma can reach higher
Accuracy.
6 anti-coagulants EDTA-Na of embodiment2Influence to ddPCR
There are a certain amount of anti-coagulants in blood plasma, EDTA is clinically common anti-coagulants.By various concentration EDTA-Na2
(5,10,15,20,25,30mM) is according to 1:1 adds in recombinant plasmid, carries out ddPCR analyses.As a result, it has been found that when concentration is less than 20mM,
EDTA-Na2It can not inhibit the amplification (Fig. 6 a, b) of ddPCR, and the concentration illustrates considerably beyond normal concentration 5mM
DdPCR is analyzed not by the EDTA-Na of conventional amount used2Interference.
To sum up shown, the new method that this patent is established can be used for low water in the plasma dna of analysis extraction, exosomeDNA
Flat mtDNA contents.Furthermore, it is possible to directly ddPCR analyses are carried out, which not only reduces because of extraction by the use of blood plasma as sample
As a result reliably content loss caused by DNA, and carries out sample analysis convenient for clinical labororatory.
Claims (3)
- A kind of method of mitochondrial DNA content 1. detection peripheral blood nondiagnostic using droplet type digitlization PCR dissociates, including Following steps：1) structure of recombinant plasmid：By PCR amplification MT-ND1 genes, MT-ND1PCR products are cloned by T-A pMDTM18-T carriers obtain MT-ND1-pMD18-T standard plasmids, and convert E. coli DH5 α；2) sample preparation：Using the recombinant plasmid of the small extraction reagent kit extraction E.coli DH5 α of plasmid, plasmid copy number is adjusted, and It is diluted to 1~106copies/μl；3) plasma dna and excretion body DNA are extracted：Peripheral blood is acquired to EDTA-Na2Anticoagulant tube, separated plasma, by 500 μ l blood plasma It is divided into two deciles, wherein 250 μ l blood plasma directly extract DNA using paramagnetic particle method, 250 μ l detach excretion body, sink in excretion body first 1h are incubated to remove the external DNA of excretion in shallow lake plus containing resuspension in 1U DNaseI solution and at 37 DEG C；4) blood plasma is pre-processed：500 μ l blood plasma, 1600g centrifugation 5min are taken, supernatant goes to new 1.5ml EP pipes；16000g 5min is centrifuged, supernatant is taken to cross 0.22 μm of disposable filter；The blood plasma of pretreatment is taken by 1:1 ratio sequentially adds 1~ 104Copies/ μ l MT-ND1-pMD18-T standard plasmids, mixing, mixing sample carry out ddPCR analyses directly as template；5) recombinant plasmid mtDNA copy numbers are analyzed using ddPCR, including designing primer and probe, utilizes recombinant plasmid, blood plasma DNA, blood plasma excretion body DNA and through processing or untreated blood plasma as template, in PCR instrument after amplification system formation droplet On expanded, after amplification, by completed PCR reaction 96 orifice plates be placed in BIO-RAD DX200Droplet Reader Droplet reading is carried out, is analyzed using 1.6 softwares of QuanatSoft,The primer sequence of PCR amplification MT-ND1 genes is as follows in the method step 1)：Forward direction primer is 5' CAGCCGCTATTAAAGGTTCG3', reverse primer 5'AGAGTGCGTCATATGTTGTTC3', expanding fragment length 1041bp is Homo sapiens mtDNA, GeneBank No.NC_012920,3017-4057bp,Plasmid copy number is adjusted using the following formula in the method step 2)：MWDNA=(pMD18-T vector+Insert) × 660=(2692bp+1041bp) × 660=2.46 × 106；CNDNA=CDNA×6.02×1014/MWDNA=CDNA×6.02×1014/2.46×106=CDNA×2.45×108；Wherein, CNDNAIt is plasmid copy number, unit is copies/ μ l；CDNAIt is plasmid DNA concentration, unit is ng/ μ l；MWDNAIt is Plasmid molecule amount, unit daltons；The primer and probe are as follows in the method step 5)：Forward direction primer:CCCTAAAACCCGCCACATCT；Reversely draw Object:GAGCGATGGTGAGAGCTAAGGT；Probe：CCATCACCCTCTACATCACCGCCC, using BHQ1 as quenching group.
- The line grain 2. a kind of detection peripheral blood nondiagnostic using droplet type digitlization PCR according to claim 1 dissociates The method of body DNA content, which is characterized in that the method step 5）Amplification system in also comprising blank control and feminine gender it is right According to wherein blank control is the PCR system without DNA profiling, and negative control is using pMD-18-T plasmids as template.
- The line grain 3. a kind of detection peripheral blood nondiagnostic using droplet type digitlization PCR according to claim 1 dissociates The method of body DNA content, which is characterized in that the method step 5）The step of generating droplet is as follows：20 μ l systems are added in the upper sample well of droplet generation card, the droplet generation oil of 70 μ l is added in oilhole, which avoids Bubble generates and cross contamination, puts on clean adhesive tape special；By droplet generation be placed on drop generators, carry out droplet generating process, in this process, in sample well system with it is micro- Drop generation oil is mixed to droplet hole to form droplet together under the action of negative pressure；Droplet generation card is taken out, draws in 40 μ l droplets to the clean corresponding hole of 96 orifice plate, aluminum foil special paper is covered, at 180 DEG C After pressure seal mouth, expanded on regular-PCR instrument.
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| CN106596487B (en) * | 2016-12-14 | 2019-08-27 | 中国科学院苏州生物医学工程技术研究所 | Intracellular protein detection method based on droplet and namo fluorescence probe |
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