CN105861641A - Primer, kit and method for detecting CHO cell DNA residues - Google Patents
Primer, kit and method for detecting CHO cell DNA residues Download PDFInfo
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Abstract
The present invention provides a primer pair for detecting CHO cell DNA residues. The invention uses a nucleotide sequence with GenBank accession number J00052.1 for design, provides a Taqman probe based on the nucleotide sequence, and a corresponding detection method. The method is a real-time PCR method; and based on an obtained standard curve, the DNA amount in CHO cells in a sample to be tested is calculated. The detection method can be used to detect bioproteins using CHO cells as an expression cell line in genetic engineering, such as antibodies, therapeutic proteins and vaccines. The real-time PCR method for detecting DNA of CHO cells and a PCR kit based on the method achieve limit of quantitation as low as 0.1fg / mul for the detection of DNA residues in CHO cells, which shows that the method has good sensitivity and high specificity.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of for detection by quantitative Chinese hamster ovary celI (Chinese
Hamster Ovary, Chinese hamster ovary cell) primer of DNA content, test kit and detection method.
Background technology
In the modern times, large quantities of bioproteins such as monoclonal antibody class medicine, Recombinant protein medicine, vaccine etc. are facing
Be widely applied on Chuan, produce the host cell of these bioproteins be mainly antibacterial, yeast cells,
Mammalian cell.Thus these host cells can be remained in the final protein product obtained through purification
DNA.Although DNA residual quantity is the lowest, but human body can be deposited by the residual DNA in protein product
In potential danger.It is generally acknowledged the DNA may including some the unknown in host cell DNA
Fragment, some may incorporate virus DNA fragmentations human body can be made to be infected, also have some with
The DNA fragmentation of canceration gene may induce generation tumor.Therefore, the DNA in protein product is allowed
Residual quantity is down to the lowest people of being and is known together.
Chinese hamster ovary celI is most widely used eukaryotic expression cell line in bio-pharmaceuticals at present.Exogenous gene
After being transfected into Chinese hamster ovary celI, it is incorporated on chromosomal DNA, can obtain stablizing lasting expression, CHO
Cell is suitable in bioreactor carrying out large-scale culture, high expressed target protein.And complexity can be carried out
Protein translation after fold and modify, the sugar of glycosylation modified and mankind's native protein of expressed albumen
It is high that baseization modifies similarity.Therefore, Chinese hamster ovary celI becomes the reason expressing complex biological high molecular weight protein
The host cell thought, has been widely used for the Expression product of recombinant protein medicine.Produce with Chinese hamster ovary celI
The dosage of various biological protein medicaments, dosage difference can reach tens times to hundreds of times.
For the bioprotein that dosage is little, the requirement of Chinese hamster ovary celI DNA residual is relatively low；For giving
Pharmaceutical quantities is big, and needs the biological protein medicament of frequent drug administration, and Chinese hamster ovary celI DNA residual quantity limits
Strictly, the lowest more good.
In a lot of biological product quality standards, all DNA residual quantity is carried out strict restriction.The U.S.
FDA (Food and Drug Administration) specifies in the guideline of issue in 1997,
In finished product, host cell DNA residual quantity must not be higher than 100pg/dose；Once, non-bowel was given
Host cell DNA residual quantity in the final products of medicine, WHO (World Health Organization)
The relevant criterion announced must not be higher than 100pg/dose, but this standard modification was for not higher than in 1998
10ng/dose；EU (European Union), the relevant criterion that calendar year 2001 announces also is in final products
Host cell DNA residual quantity must not be higher than 10ng/dose；" Chinese Pharmacopoeia " three version in 2010 regulations
Its DNA residual quantity of the biological product of prokaryotic expression is not higher than 10ng/dose, the product of eukaryotic cell expression
DNA residual quantity should be not higher than 100pg/dose.
In order to ensure that the removing of DNA residual reaches standard in each step purifying process, needs sensitivity non-
The highest DNA method for detecting residue.Within current 2010, version " Chinese Pharmacopoeia " three foreign DNAs are residual
DNA hybridization method and fluorescent staining method has been included in allowance detection project.The condition that DNA hybridization method needs
Relatively easy, but the method to there is the time long, complex operation, stability, sensitivity, specificity are poor
Etc. shortcoming.Fluorescent staining method is to utilize this highly sensitive double-stranded DNA fluorescent dyestuff of PicoGreen
DNA content is quantitative determined.The inspection sensitivity of the method is up to 300pg mL-1, DNA contains
Amount is at 1.25～80ng mL-1In the range of linear good (R2≥0.99).The method shortcoming is for being easily subject to
The interference of RNA, ssDNA, dsDNA；For heavy dose of product, such as monoclonal antibody, spirit
Sensitivity is difficult to reach detection demand.
Real-time quantitative PCR is a kind of quick high-throughout detection method, when it is PCR-based amplification,
A specific fluorescent probe is added while adding pair of primers；The increase of product can be by glimmering
Optical signal indicates, by the change of the fluorescence signal in monitoring PCR system in real time, to initial in sample
Template carries out quantitative analysis.The method has the excellent of its uniqueness in terms of specificity, susceptiveness, accuracy
Gesture；In addition residual DNA can not only be carried out quantitatively by quantitative PCR, moreover it is possible to residual DNA is carried out spy
The amplification of the opposite sex, the assessment to residual DNA risk is more objective, and therefore quantitative PCR method is remaining
DNA measures following development trend.
The Chinese patent application of Application No. CN20111006745.4 discloses to be examined with real-time fluorescence PCR
Surveying the test kit of DNA residual in Chinese hamster ovary celI, wherein using EvaGreen is fluorescent dye,
EvaGreen dyestuff can detect strong fluorescence signal after being combined with double-stranded DNA；At double-stranded DNA
Time at most, fluorescence signal is the highest, absworption peak i.e. occurs.EvaGreen dyestuff is for the knot of double-stranded DNA
Merge and there is no specificity, it is impossible to enough identify the DNA fragmentation of separate sources, although in false positive with anti-
Interference aspect is better than original SYBR Green fluorescent dye, but its specificity also cannot be with Taqman
Probe technique compares favourably.
Application No. CN201110099204.1, CN201210475980.1 and CN201210161343.7
Chinese patent application disclose and utilize Taqman probe technique real-time PCR detection Chinese hamster ovary celI
The method of DNA residual, wherein CN201210475980.1 utilizes multiple PCR technique to detect, many
Weight round pcr operating difficulties is loaded down with trivial details, and success rate is low, data processing complex, impacted many factors,
Apply the most extensive.The Chinese hamster ovary celI DNA of CN201110099204.1 and CN201210161343.7 is residual
The quantitative limit of allowance is respectively 3fg/ μ l and 10fg/ μ l, and 2 kinds of methods use different probe sequences respectively,
Then different power of test is drawn.This also the purpose amplified fragments of description selection, primer probe sequence
Design has important impact for PCR power of test.
Summary of the invention
Therefore, for above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of simple, honest and clean
It is thin that the real-time fluorescence PCR technology that valency, accuracy rate are high, quantitative limit is lower carrys out qualitative and quantitative detection CHO
The method of born of the same parents DNA and test kit.Described method can be used for detection using Chinese hamster ovary celI as genetic engineering table
Reach residual DNA in the protein product of cell strain, thus bioprotein is produced and carries out quality-monitoring.
It has been found that the power of test of quantitative PCR method is in addition to closely bound up with fluorescent technique, also
The most relevant to the copy number of the purpose fragment expanded, the application is according to the Chinese hamster ovary celI of high conservative
Alu short tandem repeat fragment designs specific primer probe sequence, and Alu short tandem repeat is at CHO
Cell genomic dna exists in a large number, and copy number is a lot, can significantly improve the sensitivity of detection, it is ensured that
The present processes has a relatively low quantitative limit.
Quantitative limit is the determination limit of a certain analysis method, i.e. under suitable accuracy and precision, energy
Enough quantitative determine the minimum flow of analyte in sample.
The nucleotide sequence that the present invention uses GenBank accession number to be J00052.1, designs special primer
With Taqman probe, carry out quantitative fluorescent PCR with the Chinese hamster ovary celI genomic DNA extracted for template
Method.
The present invention is achieved by the following technical solutions:
On the one hand, the invention provides a kind of primer pair for detecting Chinese hamster ovary celI DNA residual, institute
State primer the specificity of the nucleotide sequence design for using GenBank accession number to be J00052.1 is drawn
Thing；
Described for detect Chinese hamster ovary celI DNA residual nucleotide primer to for:
Forward primer sequence: 5'-CTACCAGAGGTCCTGAGTTCAATT-3'(SEQ ID
NO:1)；
Reverse primer sequences: 5'-GGGCACCAGGTCTCATAACG-3'(SEQ ID NO:2)；
Present invention also offers what the nucleotide sequence using GenBank accession number to be J00052.1 designed
Taqman probe；
Described Taqman probe nucleotide sequence is:
Taqman probe: 5'-CCAGCAACCACATGGTGGCTCAC-3'(SEQ ID NO:3).
Preferably, the 5' end of described Taqman probe is marked with fluorescent reporter group, and 3' end is marked with to be quenched
Go out group；Preferably, described fluorescent reporter group is FAM；Preferably, described quenching group is
TAMRA。
On the other hand, the invention provides a kind of reaction system for detecting Chinese hamster ovary celI DNA residual,
The amplification reaction solution of described reaction system be 2 × Q-PCR react premixed liquid, include Taq enzyme, dNTPs,
Mg2+And PCR buffer, it being usual ingredients, its content is also conventional.Reaction system typically can set
It is 30 μ l.Described DNA profiling can come from sample to be tested, it is also possible to from standard substance or positive internal control
Product.
Another further aspect, the invention provides a kind of method for detecting Chinese hamster ovary celI DNA residual, institute
The method of stating is quantitative real-time PCR, and the response procedures of described real-time fluorescence quantitative PCR is: 95 DEG C
Denaturation 10min；95 DEG C of 15s, 60 DEG C of 1min, 40 circulations；According to the standard curve obtained,
It is calculated the amount of Chinese hamster ovary celI DNA in sample to be checked.
In described real-time fluorescence quantitative PCR, detection is all provided with vertical standard substance, negative quality-control product and sun every time
Property internal control product (Internal Positive Control, IPC).Standard substance are Chinese hamster ovary celI genomic DNA,
Its DNA concentration can be 10-50ng/ μ l, such as 30ng/ μ l.During use, become by DNA diluted
6 Concentraton gradient, respectively 300pg/ μ l, 30pg/ μ l, 3pg/ μ l, 0.3pg/ μ l, 0.03pg/ μ l,
The standard curve gradient concentration of 0.003pg/ μ l.Negative controls is pure water.Wherein IPC template Han IPC,
IPC forward primer, IPC reverse primer, MGB (Minor Groove Binder) probe reagent, its
Middle IPC template is the PGEM-T Easy Vector of Progema.
Described IPC reagent nucleotide primer sequence is:
IPC forward primer sequence: 5'-ACTTGGTCTGACAGTTACCAATG-3'(SEQ ID
NO:4)
IPC reverse primer sequences: 5'-GGAGTCAGGCAACTATGGATG-3'(SEQ ID
NO:5)
The nucleotides sequence of described IPC probe is classified as:
IPC probe: 5'-TAATCAGTGAGGCACCTATCTCAGCGA-3'(SEQ ID
NO:6)
Described IPC probe 5' end be marked with fluorescent reporter group, described fluorescent reporter group be preferably
VIC, it is also possible to be other fluorescent reporter group；3' end is marked with quenching group, described quenching group
It is preferably MGBNFQ, it is also possible to be other fluorescent quenching group.
Described DNA diluent can be conventional.Preferably joined by EDTA, NaOH and Tris-HCl solution
System forms, and wherein Tris-HCl concentration is that the concentration of 5.0-10.0mmol/L, EDTA is
0.5-1.0mmol/L, is 6.0-8.5 with NaOH solution regulation pH.This DNA diluent can be the dilutest
Release the DNA sample of low concentration, and be suitable for PCR amplification.
All measuring samples are both needed to by being dissolved in DNA diluent or pure water after extracting.
Real-time fluorescence quantitative PCR response procedures is preferably: 95 DEG C of denaturations 10min；95 DEG C of 15s,
60 DEG C of 1min, 40 circulations.According to the standard curve obtained, it is calculated CHO in sample to be checked
The amount of cell DNA.
Wherein, the detection sample of described method is using Chinese hamster ovary celI as gene engineering expression cell strain
Bioprotein, it is therefore preferable to antibody, human cytokines or vaccine.
Yet another aspect, the invention provides a kind of test kit for detecting Chinese hamster ovary celI DNA residual,
Described test kit includes that pcr amplification reaction liquid, standard substance, negative quality-control product, IPC and DNA are dilute
Release liquid；
Wherein, described pcr amplification reaction liquid includes Taq enzyme, dNTPs, Mg2+, PCR buffer,
Primer to and Taqman probe；Described primer to for use GenBank accession number be J00052.1's
The specific primer of nucleotide sequence design；Preferably, described primer is to for shown in SEQ ID NO:1
Reverse primer shown in forward primer and SEQ ID NO:2；
Preferably, described Taqman probe be use GenBank accession number be the nucleotide of J00052.1
The Taqman probe of sequential design；Preferably, the sequence SEQ ID NO:3 of described Taqman probe
Shown in；It is further preferred that the 5' end of described Taqman probe is marked with fluorescent reporter group, 3' end
It is marked with quenching group；Preferably, described fluorescent reporter group is FAM；Preferably, described cancellation
Group is TAMRA；
Preferably, described standard substance are Chinese hamster ovary celI genomic DNA；It is highly preferred that described DNA
Concentration is 10-50ng/ μ l, more preferably 30ng/ μ l；It is further preferred that in use, DNA is used
Diluted becomes 6 Concentraton gradient, respectively 300pg/ μ l, 30pg/ μ l, 3pg/ μ l, 0.3pg/ μ l,
0.03pg/ μ l, the standard curve gradient concentration of 0.003pg/ μ l；
Preferably, described negative quality-control product is pure water；
Preferably, described IPC includes IPC template, IPC forward primer, IPC reverse primer, IPC
Probe reagent；Preferably, described IPC template is the PGEM-T Easy Vector of Progema；Preferably
Ground, the sequence of described IPC forward primer as shown in SEQ ID NO:4, the sequence of described IPC reverse primer
Row are as shown in SEQ ID NO:5；Preferably, the sequence such as SEQ ID NO:6 institute of described IPC probe
Show；It is highly preferred that described IPC probe 5' end is marked with fluorescent reporter group, 3' end is marked with cancellation base
Group；Described fluorescent reporter group is preferably VIC；Described quenching group is preferably MGBNFQ；
Preferably, described DNA diluent is formulated by EDTA, NaOH and Tris-HCl solution；
It is highly preferred that described Tris-HCl concentration is 5.0-10.0mmol/L, the concentration of described EDTA is
0.5-1.0mmol/L, the pH NaOH solution of described DNA diluent is adjusted to 6.0-8.5.
The real-time fluorescence PCR technology of the present invention carrys out the method for qualitative and quantitative detection Chinese hamster ovary celI DNA
With the PCR kit prepared according to described method, it is achieved that determining of detection Chinese hamster ovary celI DNA residual
Amount limits as little as 0.5fg/ μ l, shows that this method has extraordinary sensitivity, and has higher specificity.
It addition, IPC reagent used in this method includes the template that IPC expands, primer and VIC-IPC
Probe, the VIC fluorescence signal of generation is distinguished with FAM fluorescence signal, can be to all addition IPC
Whether the Q-PCR reaction tube of reagent plays Quality Control effect, can become with PCR reaction tubes all in evaluation experimental
The generation PCR reaction of merit, it is to avoid false negative phenomenon occurs and can not identification decision.Because each reaction tube
IPC template amount added by is just as, can be by adding up the C of the IPC of each pipeTValue, comes
Amplification efficiency in supervision sample cell and the amplification efficiency in standard curve pipe, IPC in various kinds QC
CTThe C of the IPC of meansigma methods and standard curve pipeTThe ratio of meansigma methods between 90%～110% time, table
Show reliable experiment result；Otherwise, the amplification efficiency in sample cell has with the amplification efficiency in standard curve pipe
Difference, being augmented with of sample cell may be suppressed, and amplification efficiency is inconsistent with standard curve pipe, experiment knot
The most insincere, accuracy cannot ensure.Introduce IPC reagent, can go out during effectively investigating detection
Existing exceptional value, improves the accuracy of detecting system.
The real time fluorescence quantifying PCR method that the present invention provides can detect the pilot process sample of recombiant protein
Chinese hamster ovary celI residual DNA in the samples such as product, semi-finished product, finished product, the most also makees with IPC reagent
Internal reference for PCR reaction.Can be the production reliable quality testing data of offer of recombinant protein medicine,
Research and development and safety in production for recombinant protein medicine provide important support.
Accompanying drawing explanation
Hereinafter, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:
Fig. 1 is the Chinese hamster ovary celI DNA standard substance amplification curve diagram of the present invention, and display amplification curve has bright
Aobvious Exponential growth stage.
Fig. 2 is the Chinese hamster ovary celI DNA standard substance canonical plotting of the present invention, as seen from the figure, described song
The linear equation of line: Y=-3.258lg (X)+29.501, R2=0.999, Chinese hamster ovary celI DNA is described
Standard substance between 1fg to 3000pg in good linear relationship, i.e. CT value and DNA profiling amount
Lg value be good linear relationship, R2All more than 0.99, very low levels CHO can be detected
Cell rests DNA.
Fig. 3 be the present invention Chinese hamster ovary celI specificity test amplification curve diagram, from figure it is known that
Add the lymphoma cell of people, yeast, colibacillary genome and do not add other kinds of genome
The amplification curve height of Chinese hamster ovary celI DNA overlap, the 1st group of amplification curve is for adding and not adding other
The amplification curve diagram (FAM signal) of Chinese hamster ovary celI DNA in the sample of the genome of kind；Second group
Amplification curve is the amplification curve diagram of IPC plasmid in the sample adding and not adding other kinds of genome
(VIC signal).
Fig. 4 is to add the lymphoma cell of people, yeast, colibacillary genome and do not add other kinds
The canonical plotting of the Chinese hamster ovary celI DNA of the genome of class, each standard curve is completely the same, the heaviest
Close.
Detailed description of the invention
Embodiment 1: methodology confirms
1.1 materials and reagent: 2 × Q-PCR premixed liquid is commercially available prod, ViiA 7 quantitative fluorescent PCR
Instrument is Life Technologies product, and DNA diluent is used molecular biosciences level reagent to join by this laboratory
System, genome DNA extracting reagent kit is Life Technologies product；IPC template in IPC reagent
For the PGEM-T Easy Vector series of products of Promage company, the primer related in experiment is with corresponding
Probe is by the synthesis of Life Technologies company.
Primer sequence and probe sequence that methodology confirmation is used are as follows:
Forward primer sequence: 5'-CTACCAGAGGTCCTGAGTTCAATT-3'(SEQ ID
NO:1)
Reverse primer sequences: 5'-GGGCAC CAGGTCTCATAACG-3'(SEQ ID NO:2)
Described Taqman probe nucleotide sequence is:
Taqman probe: 5'-CCAGCAACCA CATGGTGGCT CAC-3'(SEQ ID NO:3)
The fluorescent reporter group of Taqman probe 5' end is FAM；The quenching group of 3' end is TAMRA.
Described IPC reagent nucleotide primer sequence is:
IPC forward primer sequence: 5'-ACTTGGTCTGACAGTTACCAATG-3'(SEQ ID
NO:4)
IPC reverse primer sequences: 5'-GGAGTCAGGCAACTATGGATG-3'(SEQ ID NO:
5)
The nucleotides sequence of described IPC probe is classified as:
IPC probe: 5'-TAATCAGTGAGGCACCTATCTCAGCGA-3'(SEQ ID NO:
6)
The fluorescent reporter group of IPC probe 5' end is VIC；The quenching group of 3' end is MGBNFQ.
The extraction of DNA: extract CHO according to step shown in the description of genome DNA extracting reagent kit
Cell DNA.
The i.e. internal control of IPC reagent compares, containing IPC template, IPC forward primer, IPC reverse primer, IPC
Probe reagent, wherein the template Han IPC is the PGEM-T Easy Vector (Cat#A 1360) of Progema,
Preparation IPC reagent as follows: contain the IPC of the 1fg/ μ l of 1 μ l in the IPC reagent of every 2.5 μ l
Template, the IPC forward primer of 0.5 μ l 10 μMs, the IPC reverse primer of 0.5 μ l 10 μMs, 0.5 μ l5 μM
IPC probe.Above-mentioned each reagent is pre-mixed, when carrying out PCR sample-adding, only need to be at every Guan Zhongjia
Enter 2.5 μ l.
The reagent reacted by PCR takes out: be Q-PCR premixed liquid (2 ×) respectively, forward primer, instead
To primer, Taqman probe, pure water, IPC reagent.Each sample and standard substance do 3 and repeat pipe,
The often reaction system of pipe 30 μ l.Quantity according to sample determines the pipe number of reaction, and according to the form below table 1 prepares
PCR Mix。
Table 1:PCR reaction reagent injection volume table
| Reaction reagent (reaction system 30 μ l) | Injection volume (μ l) |
| Q-PCR premixed liquid (2 ×) | 15 |
| Forward primer | 1 |
| Reverse primer | 1 |
| Taqman | 0.5 |
| IPC reagent | 2.5 |
| DNA profiling | 10 |
| Amount to | 30 |
After PCR reaction system has configured, expand at the ViiA 7 enterprising performing PCR of quantitative real time PCR Instrument.
PCR condition is 95 DEG C of denaturations 10min；95 DEG C of 15s, 60 DEG C of 60s, 40 circulations；Analyze.
1.2 sensitivity experiment
In addition to DNA profiling, the PCR Mix mixed according to 2, table, join in eight unions, often manage
20μl。
Due to the present processes be on the Alu short tandem repeat of Chinese hamster ovary celI genome design draw
Thing, Alu sequence has substantial amounts of repetitive sequence in genome, so the CHO of very low concentrations can be detected
Cell genomic dna.To this end, for the sensitivity verifying this method, Chinese hamster ovary celI DNA is marked
Quasi-product DNA diluent (Tris-HCl 10mmol/L, EDTA 1mmol/L, pH are 8.0) dilutes
Become a series of Concentraton gradient.Take the 1.5ml centrifuge tube without DNA residual, respectively labelling: SD1, SD2,
SD3, SD4, SD5, SD6, SD7 and SD8.Take the Chinese hamster ovary celI DNA standard substance of 30ng/ μ l,
With DNA diluent, ((Tris-HCl 10mmol/L, EDTA1mmol/L, pH are 8) is diluted to 300
pg/μl、30pg/μl、3pg/μl、0.3pg/μl、30fg/μl、3fg/μl、0.5fg/μl、0.1fg/μl。
Amplification curve such as Fig. 1 shows: amplification curve has obvious Exponential growth stage.Standard such as Fig. 2
Curve: linear equation: Y=-3.258lg (X)+29.501, R2=0.999, it is known that, each reaction tube
Middle addition Chinese hamster ovary celI DNA standard substance between 1fg to 3000pg in good linear relationship,
I.e. CT value is good linear relationship with the lg value of DNA profiling amount, R2All more than 0.99, permissible
Detect very low levels Chinese hamster ovary celI residual DNA.
Table 2 is the standard curve of extra three experimental results, and as can be seen from the table, three experiments are at 30fg
To between 3000pg linear very well, R2All can reach more than 0.99, have good linear.
Quantitative limit: from above-mentioned standard curve equation, at DNA profiling amount 0.1fg/ μ l to 300pg/ μ l
Between, have good linear, so this method to quantitative limit LOQ of Chinese hamster ovary celI DNA up to 0.1
fg/μl。
The amplification linear equation of table 2 Chinese hamster ovary celI DNA standard substance
1.3 specificity experiments
Owing to this experiment is the specific primer of design on the Alu repeated sequence of Chinese hamster ovary celI genome,
So primer and probe have the specificity of height, in order to verify the specificity of primer and probe, Wo Men
While adding the template of Chinese hamster ovary celI genome, it is separately added into the lymphoma cell of people, yeast simultaneously
Bacterium, colibacillary genome, carry out pcr amplification reaction.
As it is shown on figure 3, add and the amplification standard curve difference not adding other kinds of genomic DNA
Less, then illustrate that primer and probe will not be non-specific instead in the genomic DNA generation of other species
Should；In each group, IPC template amplification curve difference is less, illustrates that experimental result is reliable.Therefore Fig. 3 explanation
Primer designed by this experiment will not expand other DNA beyond Chinese hamster ovary celI DNA, and this experiment is set
The primer of meter and probe have the specificity of height, and specificity is preferable.
Wherein, in Fig. 3, the A group in first group of amplification curve is the amplification song of 3pg/ μ l CHO DNA
Line, and mix the lymphoma cell of people of 10ng/ μ l, yeast, colibacillary genome respectively
Amplification curve；
In Fig. 3, the B group in first group of amplification curve is the amplification curve of 0.3pg/ μ l CHO DNA, and
Mix the amplification song of the lymphoma cell of people of 10ng/ μ l, yeast, colibacillary genome respectively
Line；
In Fig. 3, the C group in first group of amplification curve is the amplification curve of 30fg/ μ l CHO DNA, and
Mix the amplification song of the lymphoma cell of people of 10ng/ μ l, yeast, colibacillary genome respectively
Line；
In Fig. 3, the D group in first group of amplification curve is the amplification curve of 3fg/ μ l CHO DNA, and point
Can Ru the lymphoma cell of people of 10ng/ μ l, yeast, the amplification curve of colibacillary genome；
In Fig. 3, the E group in first group of amplification curve is the amplification curve of 0.3fg/ μ l CHO DNA, and
Mix the amplification song of the lymphoma cell of people of 10ng/ μ l, yeast, colibacillary genome respectively
Line；
In Fig. 3, second group of amplification curve is IPC matter in the sample adding and not adding other kinds of genome
The amplification curve diagram (VIC signal) of grain.
It is known that add and the amplification standard curve not adding other kinds of genomic DNA from figure
Difference is less, then illustrate that primer and probe will not occur non-specific at the genomic DNA of other species
Property reaction；In each group, IPC template amplification curve difference is less, illustrates that experimental result is reliable.
Fig. 4 is the figure of the amplification Specification Curve of Increasing according to Fig. 3, is the lymphoma being separately added into people in figure
Cell, yeast, colibacillary genome are anti-with the PCR not adding other kinds of genomic DNA
Should, the logarithm value of different Chinese hamster ovary celI amount of DNA and CTThe standard curve drawn.Can from Fig. 4
Arrive, add the lymphoma cell of people, yeast, colibacillary genome and do not add other kinds of base
Because the standard curve height of group DNA overlaps, illustrate that primer and probe will not be at the genes of other species
There is nonspecific reaction in group DNA, illustrates that the primer designed by this experiment and probe have the spy of height
The opposite sex, specificity is preferable.
The conclusion of embodiment 1: (1) this method, with reference to the requirement of FDA and EMA, have developed Gao Ling
The Q-PCR method of sensitivity surveys Chinese hamster ovary celI DNA residual quantity test kit, in each Q-PCR reaction tube
It is provided with internal control reagent, it is ensured that the concordance of the PCR amplification efficiency in all of reaction tube, improves knot
The credibility of fruit.
(2) often in pipe Chinese hamster ovary celI DNA standard substance amount between 1fg to 3000pg (namely
DNA concentration 0.1fg/ μ l to 300pg/ μ l) time, CTValue is good line with the logarithm value of amount of DNA
Sexual relationship, linear equation: Y=-3.258lg (X)+29.501, R2=0.999.The CHO of this method
The quantitative limit of cell DNA can reach 0.1fg/ μ l.Repeat for three times in experiment, CTValue is right with amount of DNA
Numerical value is good linear relationship, linear equation the most relatively, and R2It is above 0.99.
(3) in specificity is tested, human gene group DNA, pastoris genomic dna and large intestine are mixed
Vaccae genomic dna all will not produce non-specific amplification, so, the specificity of this method is fine,
Can specific detection Chinese hamster ovary celI genomic DNA.
The preparation of embodiment 2 test kit
Preparation includes the test kit of consisting of composition:
DNA diluent (5mL/ pipe) 1 pipe, Q-PCR premixed liquid (1.5mL/ pipe) 1 pipe, IPC reagent (250 μ l/
Pipe) 1 pipe, Chinese hamster ovary celI DNA standard substance (40 μ l/ pipe) 1 pipe, forward primer (100 μ l/ pipe) 1 pipe,
Reverse primer (100 μ l/ pipe) 1 pipe, Taqman probe (50 μ l/ pipe) 1 pipe, pure water (1mL/ pipe)
1 pipe.
Chinese hamster ovary celI DNA standard substance, concentration is 3.0 × 107fg/μl。
IPC reagent: containing the 1fg/ μ l IPC template of 1 μ l, 0.5 μ l 10 μMs in the IPC reagent of every 2.5 μ l
IPC-F, the IPC-R of 0.5 μ l 10 μMs, the IPC-Probe of 0.5 μ l5 μM.
Forward primer, reverse primer, Taqman probe are all 10 μMs.
DNA diluent: with deionized water as solvent, Tris-HCl concentration is 10mM/L, EDTA's
Concentration is 0.5mM/L, is 8.0 with NaOH solution regulation pH.
2 × Q-PCR premixed liquid: Q-PCR premixed liquid (2 ×).
Claims (10)
1. one kind for detect Chinese hamster ovary celI DNA residual primer pair, it is characterised in that described in draw
The thing specific primer to the nucleotide sequence design for using GenBank accession number to be J00052.1；
Preferably, described primer is to for the forward primer shown in SEQ ID NO:1 and SEQ ID NO:2
Shown reverse primer.
2. the reaction system being used for detecting Chinese hamster ovary celI DNA residual, it is characterised in that described
Reaction system is real-time fluorescence quantitative PCR reaction system, comprises primer pair as claimed in claim 1.
Reaction system the most according to claim 2, it is characterised in that described system also includes using
GenBank accession number is the Taqman probe of the nucleotide sequence design of J00052.1；
Preferably, shown in the sequence SEQ ID NO:3 of described Taqman probe.
4. reaction system as claimed in claim 2 or claim 3, it is characterised in that described reaction system is also wrapped
Include Taq enzyme, dNTPs, Mg2+, PCR buffer and DNA profiling；
Preferably, described DNA profiling is testing sample, negative quality-control product or IPC.
5. the method being used for detecting Chinese hamster ovary celI DNA residual, it is characterised in that described method
For quantitative real-time PCR, including using primer pair amplifies sequence as claimed in claim 1
Step；
Preferably, described quantitative real-time PCR uses reaction as claimed in claim 2 or claim 3
System is reacted.
Method the most according to claim 5, it is characterised in that described real-time fluorescence quantitative PCR
Response procedures be: 95 DEG C of denaturations 10min；95 DEG C of 15s, 60 DEG C of 1min, 40 circulations；According to
The standard curve obtained, is calculated the amount of Chinese hamster ovary celI DNA in sample to be checked.
7. according to the method described in claim 5 or 6, it is characterised in that the detection sample of described method
For the bioprotein using Chinese hamster ovary celI as gene engineering expression cell strain, it is therefore preferable to antibody, treatment
Property albumen or vaccine.
8. the test kit being used for detecting Chinese hamster ovary celI DNA residual, it is characterised in that described examination
Agent box includes pcr amplification reaction liquid, standard substance, negative quality-control product, IPC and DNA diluent；
Wherein, described pcr amplification reaction liquid includes Taq enzyme, dNTPs, Mg2+, PCR buffer,
Primer to and Taqman probe；Described primer to for use GenBank accession number be J00052.1's
The specific primer of nucleotide sequence design；Described Taqman probe for using GenBank accession number is
The Taqman probe of the nucleotide sequence design of J00052.1.
Test kit the most according to claim 8, it is characterised in that described primer is to for SEQ ID
Forward primer shown in NO:1 and the reverse primer shown in SEQ ID NO:2.
Test kit the most according to claim 8 or claim 9, it is characterised in that described Taqman visits
The sequence of pin is as shown in SEQ ID NO:3.
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| CN109837345A (en) * | 2017-11-24 | 2019-06-04 | 中国食品药品检定研究院 | Detect the primer and method of mouse cell residual DNA |
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| CN112553308A (en) * | 2019-12-13 | 2021-03-26 | 东莞市东阳光生物药研发有限公司 | Method for detecting residual DNA of drug sample |
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| CN109837345A (en) * | 2017-11-24 | 2019-06-04 | 中国食品药品检定研究院 | Detect the primer and method of mouse cell residual DNA |
| CN110305938A (en) * | 2018-03-27 | 2019-10-08 | 天士力生物医药股份有限公司 | A kind of primer pair and detection method for quantitative detection CHO host cell DNA residual quantity |
| CN110305938B (en) * | 2018-03-27 | 2023-02-17 | 天士力生物医药股份有限公司 | Primer pair and detection method for quantitatively detecting DNA residual quantity of CHO host cells |
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| CN111394434B (en) * | 2020-04-17 | 2023-05-23 | 苏州依科赛生物科技股份有限公司 | CHO host cell DNA residue detection kit adopting TaqMan probe method and application thereof |
| CN114075594A (en) * | 2020-08-12 | 2022-02-22 | 四川大学华西医院 | Method for detecting DNA content of Sf9 cell |
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| CN112301028B (en) * | 2020-10-27 | 2021-06-08 | 武汉珈创生物技术股份有限公司 | SCAR marker for identifying CHO cells and construction method and application thereof |
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| CN112592965A (en) * | 2020-12-22 | 2021-04-02 | 依科赛生物科技（太仓）有限公司 | Coli host DNA residue detection kit by TaqMan probe method |
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