CN109097454A - A kind of detection method of HEK293 gDNA residual quantity - Google Patents
A kind of detection method of HEK293 gDNA residual quantity Download PDFInfo
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Abstract
The present invention provides a kind of detection method of HEK293 gDNA residual quantity, quantitative PCR detection including sample pre-treatments and HEK293 gDNA residual quantity, the sample pre-treatments are the sample-pretreating method based on magnetic bead, digestion process is carried out to sample including cell pyrolysis liquid and Proteinase K, isopropanol is added and magnetic bead is stripped enrichment to nucleic acid, it washes twice to go removing protein and other pollutions, nucleic acid is eluted from magnetic bead；The quantitative PCR detecting method of the HEK293 gDNA residual quantity is using HEK293 gDNA as template, for HEK293 gDNA there is the primer and probe of specificity to carry out quantitative detection using what is designed, the quantitative PCR is the Real-Time Fluorescent Quantitative PCR Technique based on Taqman probe, and the present invention has the advantages that efficient, quick, accurate, high specificity.
Description
Technical field
The present invention provides a kind of detection method of HEK293 gDNA residual quantity, including sample pre-treatments and HEK293 gDNA
The quantitative PCR detection of residual quantity belongs to residual impurity detection field in the control of biological products quality.
Background technique
HEK293 cell, that is, human embryonic kidney cell 293 (ATCC CRL-1573), with transfection efficiency is high, can suspend training
Feeding feature, and HEK293 cell in cultivating system can rapid growth, realize High Density Cultivation on a large scale express, because
This is widely used in production albumen, vaccine, antitumor and anticancer agent and recombined adhenovirus coating etc..Vaccine is carried out with the cell strain of passage
One sixty-four dollar question of production is exactly the remnants of host cell DNA segment.Although being produced by stringent purifying process
It is possible to the DNA fragmentation of residual host cell in product.These residual DNAs may bring infectiousness or oncogenicity risk.Due to
The potential pathogenic risk of residual DNA, many mechanisms have formulated relevant criterion to the residual quantity of DNA in biological products.WHO and Europe
Alliance provides that the residual quantity of DNA in the biological agent product of every person-portion need to be controlled in 10ng or less；U.S. FDA then provides that every person-portion is raw
The residual quantity of DNA is within 100pg in object formulation products.
Residual DNA belongs to trace impurity, and it is a difficult job that quick, efficient, accurate detection is carried out to it.2015
Version " Chinese Pharmacopoeia " third portion detection method remaining for foreign aid's property DNA has only included DNA probe hybrid method and fluorescent staining
Method.DNA probe hybrid method is then to utilize this spy using digoxigenin labeled HEK293 cell DNA to be prepared into DNA hybridization probe
Needle is hybridized to the reference DNA of sample to be tested total DNA and corresponding known concentration, judges sample finally by color signal
The content of residual DNA in product.Although DNA probe hybrid method condition is relatively easy, this method has cumbersome, stable
Property, the disadvantage of sensibility and specificity difference.Fluorescent staining rule is to utilize double-stranded DNA fluorescent dyestuff and double-stranded DNA specificity knot
Conjunction forms compound, to measure the content of residual DNA.When DNA residual quantity is in 1.25-80ng/ml range, fluorescence colour
It is linear preferable.The disadvantages of this method is to interfere vulnerable to ssDNA, dsDNA, while its sensitivity and specificity is difficult to reach detection and want
It asks.
Summary of the invention
The present invention is directed to solve to a certain extent it is above-mentioned in the related technology the technical issues of one of.Therefore, mesh of the invention
Be a kind of detection method of HEK293 gDNA residual quantity is provided, including sample pre-treatments and HEK293 gDNA residual quantity
Quantitative PCR detection has the advantages that efficient, quick, accurate, high specificity.
GDNA (genomic DNA) is carried out to HEK293 cell using QIAGEN DNeasy Blood&Tissue Kit to extract
Purifying, the HEK293gDNA that will have been extracted are dispensed after carrying out integrity detection using agarose gel electrophoresis, deposit in-
80 DEG C of refrigerators are to be used for subsequent experiment.
On the one hand, the present invention provides a kind of method of sample pre-treatments, and sample pre-treatments is before the samples based on magnetic bead
Reason method.
The pre-treatment of the sample, which includes: (1), carries out digestion process, (2) to sample using cell pyrolysis liquid and Proteinase K
Isopropanol and magnetic bead is added, enrichment is stripped to nucleic acid, (3) are washed twice to go removing protein and other pollutions, and (4) are by nucleic acid
It is eluted from magnetic bead to be detected for subsequent quantitation.
Preferably, the constituent of cell pyrolysis liquid are as follows: 6M guanidinium isothiocyanate, 1M DTT, 0.5M sodium acetate, the 2%N- month
Osmanthus acylsarcosine sodium, 50mM EDTA and 0.6M Tris-HCl.
On the other hand, the present invention provides a kind of quantitative PCR detecting method of HEK293 gDNA residual quantity, including is directed to
The design and progress fluorescence quantitative PCR detection of the specific primer and probe of HEK293 gDNA.
The specific primer and probe for HEK293 gDNA of the design are respectively HEK293 qPCR Forward
Primer:5 '-CGWGTANCTRGGAYTAHA-3 ', HEK293qPCR Reverse Primer:5 '-
NGTGARACCCCYYTCTST-3'；HEK293qPCR Probe:5'-FAM-VCCACCSNGCCRGGCTA-MGB-3'.
Preferably, primer concentration is 0.67 μM, and concentration and probe concentration is 0.1 μM.
Preferably, the experiment condition of quantitative fluorescent PCR is 95 DEG C of initial denaturation 5min；Then anneal 95 DEG C of 10s, extends 60
DEG C 1min carries out 40 circulations.
The beneficial effects of the present invention are:
In Sample pretreatment, digestion process is carried out to sample using cell pyrolysis liquid provided by the invention, nucleic acid returns
Yield improves the accuracy of subsequent quantitation analysis 70% or more.
Real-time fluorescence quantitative PCR combines the dual specificity of amplimer and probe, improve detection specificity and
Accuracy.During primer amplification extends, the fluorescent marker of specific bond is hydrolyzed under the hydrolytic enzyme functional catalysis of archaeal dna polymerase
Probe, to discharge fluorescence signal.By fluorescence signal in real time monitoring PCR system, reach to the initial content in sample
Carry out quantitative analysis.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
The integrity detection of Fig. 1 HEK293 gDNA
The remaining whole detection method HEK293 gDNA amplification curve of Fig. 2A HEK293 gDNA
The remaining whole detection method HEK293 gDNA standard curve of Fig. 2 B HEK293 gDNA
The specificity of the remaining whole detection method of Fig. 3 HEK293 gDNA
Specific embodiment:
Main material:
HEK293 cell is purchased from U.S. ATCC (ATCC CRL-1573)；QPCR MIX is purchased from Roche (article No.
2147483647)；Magnetic bead is purchased from Asend (article No. AS316004)；Guanidinium isothiocyanate and N- sodium lauroyl sarcosine are purchased from
Solarbio (article No. G8210 or S8100)；Proteinase K is purchased from Merck (article No. 1245680100).
The extracting and purifying of embodiment 1:HEK293 gDNA
GDNA (genomic DNA) is carried out to HEK293 cell using QIAGEN DNeasy Blood&Tissue Kit to extract
Purifying.Using HEK293 gDNA as positive reference product；CHO gDNA and E.coli gDNA will be extracted pure as negative reference product
HEK293 gDNA after change detects its integrality with agarose gel electrophoresis, sees Fig. 1
Embodiment 2: primer, probe design and optimization
Based on Genebank database and domestic and foreign literature excavation analysis, according to primer, probe design basic principle,
Utilize the multipair primer and probe of Beacon Designer7.9 software design.Using the HEK293 gDNA of extracting as template pair
The multipair primer and probe of design is screened to obtain specificity, sensitivity and reproducible most suitable primer combination of probe,
Sequence composition is as follows:
HEK293 qPCR Forward Primer:5’-CGWGTANCTRGGAYTAHA-3’
HEK293 qPCR Reverse Primer:5’-NGTGARACCCCYYTCTST-3’
HEK293 qPCR Probe:5’-FAM-VCCACCSNGCCRGGCTA-MGB-3’
Embodiment 3: the optimization of quantitative fluorescent PCR reaction system
The 2 most suitable primer combination of probe filtered out in conjunction with the embodiments, other components are not in quantitative fluorescent PCR reaction system
In the case where change, the concentration of primer and probe is respectively set as 0.1 μM, 0.2 μM, 0.4 μM, 0.67 μM and 0.8 μM progress PCR
Reaction.Test result is repeated several times and shows that optimal primer concentration is 0.67 μM, optimal concentration and probe concentration is 0.1 μM.
The 2 most suitable primer combination of probe filtered out in conjunction with the embodiments, in the case that other components are constant in the reaction system,
Annealing elongating temperature is respectively set as 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C of progress PCR reactions.Test result is repeated several times to show
Best annealing elongating temperature is 60 DEG C.The response procedures of experiment are as follows: 95 DEG C of initial denaturation 5min；Then 95 DEG C of 10s, 60 DEG C
1min, 40 circulations.
The drafting of embodiment 4:HEK293 gDNA amplification curve and standard curve
Taking-up is stored in the HEK293 gDNA (30ng/ μ l) for extracting and getting ready in -80 DEG C of refrigerators, carries out 100 times of multiple proportions first
Dilution obtains SD1 (300pg/ μ l), then successively carries out 10 times of multiple proportions gradient dilutions, respectively obtains SD2 (30pg/ μ l), SD3
(3pg/ μ l), SD4 (300fg/ μ l), SD5 (30fg/ μ l).According to the experiment condition after optimization, reaction system is 15 μ l, group
Become: 7.5 μ l, Forward/Reverse primer of qPCR MIX 1/1 μ l, probe 0.15 μ l, DNA 3 μ l, ddH2O
2.35 μ l are then placed in machine operation program, finally obtain the amplification curve of HEK293 gDNA, see Fig. 2A.Use HEK293gDNA
The log value of each dilution concentration maps to Ct (initial cycle number) value of each dilution, and the relationship that the two is linearly successively decreased should
Linearity curve is HEK293 gDNA quantitation curves.The R of curve2Or r value then reflects the amplification efficiency of amplified reaction, R2It answers
Greater than 0.98 or r value is greater than 0.99, i.e. reaction efficiency is shown in Fig. 2 B between 90-105%.
The test of embodiment 5:HEK293 gDNA detection sensitivity
HEK293 gDNA is diluted to 60fg/ μ l, 45fg/ μ l, 30fg/ μ l, 10fg/ μ l respectively, then utilizes embodiment
Reaction system after 3 optimizations is measured to determine that detection sensitivity, each concentration do 6 repetitions.Experimental result is shown: concentration
When down to 10fg/ μ l, still < 1%, therefore, the sensitivity for detecting reaction can be down to 10fg/ μ l for CV value.
Embodiment 6:HEK293 gDNA method for detecting residue accuracy and the experiment of vaccine pattern detection
Different amounts of HEK293 gDNA is added in eluent, basic sample is made, is added in the vaccine sample of different batches
HEK293 gDNA, as a control group without the additionally basic sample of addition HEK293 gDNA and vaccine sample.Take 100 μ l each groups
Sample is separately added into Proteinase K and cell pyrolysis liquid (6M guanidinium isothiocyanate, 1M DTT, 0.5M sodium acetate, 2%N- lauroyl flesh
Propylhomoserin sodium, 50mM EDTA and 0.6M Tris-HCl) in 56 DEG C of processing 30min, be then added isopropanol and magnetic bead to nucleic acid into
Row extracting enrichment, is washed out twice to go removing protein and other pollutions, finally elutes nucleic acid from magnetic bead subsequent to be used for
Quantitative detection simultaneously calculates the rate of recovery.Experimental result is shown: the rate of recovery is 70% or more, and the accuracy of this method is high, experiment parameter
It is shown in Table 1A, table 1B.
The testing result of table 1A HEK293 gDNA method for detecting residue accuracy
The testing result of HEK293 gDNA method for detecting residue accuracy in table 1B vaccine sample
Embodiment 7: Precision Experiment
For repeated experiment, the Sample pretreatment based on magnetic bead is carried out then for the basic sample of two various concentrations
It carries out quantitative detection and calculates its detected level.Every group of sample is independently repeated 8 times, to calculate the CV value of each group sample.Experiment
As the result is shown: the CV value of each group < 15%, shows that experimental repeatability is good, experiment parameter is shown in Table 2A.
The repeatability of the remaining whole detection method of table 2A HEK293 gDNA
Intermediate precision is tested, for the basic sample of three various concentrations, by three different experimenters into
Sample pretreatment and quantitative detection of the row based on magnetic bead.Everyone is independently repeated 3 times every group of sample.Experimental result is shown: each group
CV value<10% and the rate of recovery equal>70%, be shown in Table 2B.
The Intermediate precision of the remaining whole detection method of table 2B HEK293 gDNA
The specificity experiments of the remaining whole detection method of embodiment 8:HEK293 gDNA
CHO gDNA or the E.coli gDNA of equivalent are mixed with HEK293 gDNA respectively and constitute mixing sample, it will be independent
CHO gDNA, E.coli gDNA, HEK293 gDNA and mixing sample carry out respectively gradient doubling dilution then quantified
Detection, and draw curve.Experimental result is shown: for this method just for HEK293 gDNA, specificity is high, sees Fig. 3.
A kind of detection method of HEK293 gDNA residual quantity provided by the present invention is described in detail above.This
Apply that a specific example illustrates the principle and implementation of the invention in text, the explanation of above example is only intended to
It facilitates the understanding of the method and its core concept of the invention.It should be understood that those skilled in the art, not departing from
, can be with several improvements and modifications are made to the present invention under the premise of the principle of the invention, these improvement and modification also fall into this hair
In bright scope of protection of the claims.
Claims (7)
1. a kind of detection method of HEK293 gDNA residual quantity, it is characterised in that: including sample pre-treatments and HEK293 gDNA
The quantitative PCR detection of residual quantity；The sample pre-treatments are a kind of Sample pretreatment method based on magnetic bead, the HEK293
The quantitative PCR detection of gDNA residual quantity includes the design and progress fluorescence for the specific primer and probe of HEK293 gDNA
Quantitative PCR detection.
2. a kind of Sample pretreatment method based on magnetic bead according to claim 1, it is characterised in that: cell pyrolysis liquid
For self-control；
The composition of the further cell pyrolysis liquid are as follows: 6M guanidinium isothiocyanate, 1M DTT, 0.5M sodium acetate, 2% N- laurel
Acylsarcosine sodium, 50mM EDTA and 0.6M Tris-HCl.
3. the design of the specific primer and probe of HEK293 gDNA according to claim 1, it is characterised in that: benefit
HEK293 gDNA is used to screen as multipair primer and probe of the template to design；
The sequence of the further primer and probe combination:
HEK293 qPCR Forward Primer: 5’-CGWGTANCTRGGAYTAHA-3’
HEK293 qPCR Reverse Primer: 5’-NGTGARACCCCYYTCTST-3’
HEK293 qPCR Probe: 5’-FAM-VCCACCSNGCCRGGCTA-MGB-3’。
4. fluorescence quantitative PCR detection according to claim 1, it is characterised in that: other components are not in the reaction system
In the case where change, the concentration of primer and probe is respectively set as 0.1 μM, 0.2 μM, 0.4 μM, 0.67 μM and 0.8 μM progress PCR
Reaction is repeated several times test, determines the reaction density of primer and probe；
Further, the concentration of the primer is 0.67 μM, and the concentration of the probe is 0.1 μM.
5. the fluorescence quantitative PCR detection according to claims 1 or 4, it is characterised in that: reaction system is 15 μ l, group
Become: 7.5 μ l, Forward/Reverse primer of qPCR MIX 1/1 μ l, probe 0.15 μ l, DNA 3 μ l, ddH2O
2.35μl。
6. the fluorescence quantitative PCR detection according to claims 1 or 5, it is characterised in that: the setting of response procedures condition；
Further, procedure condition is set as 95oC initial denaturation 5min；Then 95oC 10s, 60oC 1min, 40 circulations.
7. HEK293 gDNA residual quantity according to claim 1 is the HEK293 gDNA residual quantity in vaccine.
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