CN105648102A - Kit for detecting DNA residues of MDCK cells in vaccines - Google Patents
Kit for detecting DNA residues of MDCK cells in vaccines Download PDFInfo
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- CN105648102A CN105648102A CN201610184173.2A CN201610184173A CN105648102A CN 105648102 A CN105648102 A CN 105648102A CN 201610184173 A CN201610184173 A CN 201610184173A CN 105648102 A CN105648102 A CN 105648102A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention relates to kits, in particular to a kit for detecting the DNA residues of MDCK cells in vaccines. The kit is characterized by comprising a primer group. The primer group comprises an upstream primer as shown in SEQ ID No.1 and a downstream primer as shown in SEQ ID No.2. The circulation amplification of target polynucleotide is achieved through a fluorescent PCR amplification instrument, and accordingly the target polynucleotide can be detected fast and quantitatively.
Description
Technical field
The present invention relates to a kind of minim DNA detection method, be specifically related to a kind of detect the method for mdck cell in vaccine.
Background technology
Mdck cell is the passage cell of a kind of Testis et Pentis Canis (ATCC-CCL34) cell line, have many advantages: viral infection efficiency is high, propagation is fast, and not easily make a variation, mdck cell system (MDCKCellLines) is acknowledged as and is most suitable for one of 3 kinds of cell lines that first, influenza B virus produces. But, the major issue producing vaccine with passage cell strain is its safety, shows as the residual cell DNA having potential oncogenicity. Therefore the DNA of residual in refining vaccine is had strict restriction by WHO, and in mdck cell influenza vaccines as refining in WHO regulation, residual DNA content is less than 10ng/ dosage (being equivalent to 10ng/ml). Remaining mdck cell DNA content is based on one of major quality controlling index of refining vaccine of mdck cell production, it is necessary to carries out strict calibrating purification, makes content not above required standard.
Owing to DNA residual content is non-normally low, it is necessary to adopt more sensitive nucleic acid hybridization technique. And the method being currently used for detection vaccine residual DNA is mainly molecular hybridization, as utilized random priming, with digoxigenin labeled mdck cell DNA, and prepare into DNA probe, on nitrocellulose membrane, carry out dot blot with the reference sample DNA of this probe with testing sample STb gene and concentration known, carry out DNA content in judgement sample finally by color signal intensity.
Compared with molecular hybridization, taqmanPCR technology is applied in detection by quantitative to have the advantage that the analysis by the cycle threshold (ct) to amplification curve and increased logarithmic phase, abandon the molecular hybridization end point analysis method by many factors interference, testing sample can be carried out accurate quantitative analysis, thus effectively monitoring the effect of vaccine purification; DNA cloning and detection process are combined together, it is possible in real time, the overall process of dynamic monitoring DNA cloning, eliminate molecular hybridization last handling process, substantially reduce the interpretation of result time so that the method is more quick, convenient; With the fluorescent probe of template complementary pairing, can effectively combine the dual specificity of PCR and probe hybridization owing to adding one on regular-PCR basis, further increase the specificity of detection target polynucleotide.
Summary of the invention
It is an object of the invention to provide a kind of test kit using real-time fluorescence PCR technology to carry out qualitative and quantitative detection mdck cell DNA, particularly this test kit can be used for detecting mdck cell residual DNA in vaccine sample, thus to vaccine production process carries out quality-monitoring.
The ultimate principle of this test kit is to utilize a pair target polynucleotide specific primer and a target polynucleotide specific probe, containing hot resistant DNA polymerase, dNTPs, containing in magnesium ion buffer, the cyclic amplification of target polynucleotide is realized by fluorescent PCR amplification instrument, thus reaching quickly, the purpose of detection by quantitative target polynucleotide.
One aspect of the present invention provides a kind of test kit, it is characterised in that include primer sets; Described primer sets includes having the forward primer as shown in SEQIDNo.1 and has the downstream primer as shown in SEQIDNo.2.
Further, the probe as shown in SEQIDNo.3 is also included.
Further, primer concentration is 0.2-0.3 ��m of ol/l, and concentration and probe concentration is 0.40-0.60 ��m of ol/l.
Further, a kind of or both mixture above in PCR buffer, deoxynucleotide triphosphates mixture and archaeal dna polymerase are also included.
Another aspect of the present invention provides the application in detection mdck cell DNA residual of the above-mentioned test kit.
Further, described mdck cell DNA residual is the mdck cell DNA residual in vaccine.
Another aspect of the invention non-diagnostic and non-treatment purpose, based on the method for detection mdck cell DNA residual of mentioned reagent box, it is characterised in that take testing sample after pretreatment, extract nucleic acid, amplification, it may be judged whether there is DNA residual.
Further, described sample is vaccine.
Further, the reaction temperature of amplification and time are: 95 DEG C of denaturation 30s; Then 95 DEG C of 5s, 60 DEG C of 34s, 40 circulations.
Accompanying drawing explanation
Fig. 1 is reference material amplification curve, E=99.5%;
Fig. 2 is mdck cell DNA reference material standard curve.
Detailed description of the invention
Main material:
Mdck cell is introduced from ATCC (Unite States Standard DSMZ), MDCKCCL-34, P55, Lot:59681577; QPCRMIX is purchased from takara company of Japan; DNAiso is purchased from this takara company.
The preparation of embodiment 1MDCK cell DNA reference material
Using MDCK genomic DNA as positive reference material; With monkey-kidney cells system Vero, Chinese hamster ovary CHO, influenza virus, DMEM culture medium, PBS, FBS are as negative reference product. Takara extraction agent box is adopted to extract above-mentioned positive reference material stand-by with the DNA of negative reference product.
The screening of embodiment 2 specific primer and probe
By to the Genbank data base whole nucleotide sequence of existing Testis et Pentis Canis and delivered both at home and abroad the nucleic acid sequence alignment analysis of report in document, with the major gene relevant with mdck cell for amplification target site, select the section without secondary structure and high conservative, basic principle according to primed probe design, utilizing software and the multipair primer of engineer and probe, the target-gene sequence highly repeating to guard is core with specific probe.
Detect the genomic DNA of above-mentioned positive reference material and negative reference product with many groups primed probe of above-mentioned design respectively, through repeatedly testing, filter out specificity, sensitivity and the combination of reproducible best primed probe, as follows:
Forward primer is GAGACATAGGCAGAGGGAGAAGSEQIDNO.1;
Downstream primer is GATCCTGGAGACCTGGGATSEQIDNO.2;
Probe is 5FAM-CAGGCTCCATGCACCAGGAGC-3TAMRASEQIDNO.3.
The optimization of embodiment 3 primed probe concentration
In reaction system, other components are constant, the primer from 0.15 ��m of ol/l to 0.5 ��m of ol/l Concentraton gradient and the probe from 0.075 ��m of ol/l to 0.5 ��m of ol/l Concentraton gradient is used to carry out PCR reaction respectively, through repeatedly repeated trials, finally determining that best primer concentration is 0.2-0.3 ��m of ol/l, concentration and probe concentration is 0.40 0.60 ��m of ol/l.
The optimization of embodiment 4 reaction temperature
Activity according to enzyme and the length of target polynucleotide, be mainly optimized the time of annealing temperature and extension, through repeatedly repeated trials, finally determines that the reaction temperature of the best and time is: 95 �� of denaturation 30s;Then 95 �� of 5S, 60 �� of 34s, 40 circulations.
Embodiment 5 sensitivity experiment
Extracting positive reference material and extract the DNA of positive reference material mdck cell, measure OD260 and OD280 numerical value through ultraviolet spectrophotometer, calculating original liquid concentration is 493.6pg/ml, and being diluted to concentration is 10pg/ml, and then 10 times of gradient dilutions become 1.0x100-1.0x10-6, ng/ �� l is as positive sensitivity reference material, and the lower limit according to detection vaccine sample, working concentration is 100-10-4Ng/ �� l, testing result shows, the lower limit sensitivity of this test kit is 1.0x10-6ng/��l��
Embodiment 6 vaccine pattern detection vaccine pattern detection
With vaccine semi-finished product, finished product is as specimen to be checked, after extracting specimen genomic DNA with DNA extraction liquid boiling method respectively, through the nucleic acid amplification system detection that above-mentioned optimization is set up, result shows the mdck cell residual DNA detected in vaccine sample that this test kit can be very sensitive.
The drafting of embodiment 7MDCK cell DNA reference material quantitation curves and real time quantitative PCR method
Foundation
By the log value of the extension rate of MDCKDNA reference material, CT (initial cycle number) value of each dilute sample being mapped, the linear relationship that both taper off, is for mdck cell DNA reference material quantitation curves.
The establishment of real time quantitative PCR method experimental system and optimization. qPCR based on TaqMan probe reacts containing, for example lower composition: PCR reactant liquor, template, primer, probe, PCR reactant liquor containing buffer, archaeal dna polymerase and dNTPs can be bought from multiple producers, primer concentration, concentration and probe concentration is groped according to the amplification efficiency of reaction. temperature conditions is paid special attention in TaqMan experiment, and conventional TaqMan experiment two step method replaces traditional degeneration, annealing, extension three step PCR cycle. two step method: the first step is degeneration, second step is for annealing and extends merging, and temperature is 55-60 ��, and this guarantees that primer probe when extending keeps the combination with target sequence. generally, TaqMan probe is designed at its Tm between 60-70 ��. the TaqMan experiment optimized has high sensitivity and specificity and amplification efficiency good in wide dynamic range. make standard curve with the concentration known template concentrations amplification of gradient dilution, determine amplification efficiency by R2 or the r value of this straight line that successively decreases. reaction efficiency should between 90%--105%, R2 more than 0.980 or r more than 0.99, and repeated sample CT value is close. if experiment can meet above-mentioned requirements, experiment can start at once. if experiment can not meet above-mentioned requirements, it is proposed that experimental implementation person redesigns primer and TaqMan probe. it is to be noted that for making to contain whole sample to be tested template concentrations within the scope of the template concentrations of standard curve, make the amplification of sample to be tested template in the dynamic range of experiment, if sample to be tested reaction result is outside the dynamic range of standard curve, the adjustment of following three steps must be carried out: 1 builds the standard curve covering sample to be tested template concentrations, if analyzing and guaranteeing that the CT value of linear 2 samples to be tested in new dynamic range is lower than the CT value of the standard substance of maximum concentration on standard curve, if reformed after dilution sample to be tested, the CT value of experiment 3 samples to be tested is higher than the CT value of the standard substance of maximum concentration on standard curve, increase experiment of reforming after sample to be tested.
Embodiment 8 specificity verification
Respectively Vero cell DNA, Chinese hamster ovary celI DNA, R-D cell DNA are done and dilute, using the DNA of dilution as sample, detect by the method determined, investigate the cross reaction of institute's construction method and several host cell DNAs.It is shown that with Vero cell DNA, Chinese hamster ovary celI DNA, R-D equal no cross reaction of cell DNA dilute sample. Illustrating that the method is just for mdck cell, specificity is high.
Embodiment 9 accuracy, precision and suitability checking and stability test
Accuracy, according to the method preparing standard working curve prepare high (1ng/ �� l), in (0.1ng/ �� l), low (0.01ng/ �� l) three concentration the dilute sample of cellular matrix influenza vaccines, each concentration prepares 6 parts, every part of sample sets multiple hole, repetition measurement 5 times in not measuring on the same day, calculates the response rate.
Precision, is configured to basic, normal, high by (10 according to the method preparing calibration trace�\6ng����l�\1, 10�\4ng����l�\1, 10�\2ng����l�\1) dilute sample of three concentrations of cells substrate influenza vaccines, and in same eight connecting legs, measure 3 holes by each concentration of the detection method determined, not on the same day different measure in replication 3 times, the concentration gone out by standard working curve regression equation calculation is the detected level of the dilute sample of cellular matrix influenza vaccines, respectively precision in precision and plate between computing board.
The complete genome DNA reference material of preparation preserves at 20 DEG C, the probe of design preserves at 20 DEG C with primer concentration combination MIX, take out weekly a reference material respectively, the sample of 10 times of gradient dilutions is prepared according to the method preparing standard curve, by the method detection determined, investigate 20 DEG C of stability preserving lower reference material. The repeatability of the coefficient of determination of observation caliber curve, amplification efficiency and sample.
Claims (9)
1. a test kit, it is characterised in that include primer sets; Described primer sets includes having the forward primer as shown in SEQIDNo.1 and has the downstream primer as shown in SEQIDNo.2.
2. test kit according to claim 1, it is characterised in that also include the probe as shown in SEQIDNo.3.
3. test kit according to claim 2, it is characterised in that primer concentration is 0.2-0.3 ��m of ol/l, concentration and probe concentration is 0.40-0.60 ��m of ol/l.
4. the test kit according to any one of claims 1 to 3, it is characterised in that also include a kind of or both mixture above in PCR buffer, deoxynucleotide triphosphates mixture and archaeal dna polymerase.
5. the test kit according to any one of Claims 1-4 is in the application of detection mdck cell DNA residual.
6. application according to claim 5, it is characterised in that described mdck cell DNA residual is the mdck cell DNA residual in vaccine.
7. non-diagnostic and non-treatment purpose, based on the method for detection mdck cell DNA residual of the test kit as described in any one of Claims 1-4, it is characterised in that take testing sample after pretreatment, extract nucleic acid, amplification, it may be judged whether there is DNA residual.
8. method according to claim 7, it is characterised in that described sample is vaccine.
9. method according to claim 7, it is characterised in that the reaction temperature of amplification and time are: 95 DEG C of denaturation 30s; Then 95 DEG C of 5s, 60 DEG C of 34s, 40 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201610184173.2A CN105648102A (en) | 2016-03-29 | 2016-03-29 | Kit for detecting DNA residues of MDCK cells in vaccines |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610184173.2A CN105648102A (en) | 2016-03-29 | 2016-03-29 | Kit for detecting DNA residues of MDCK cells in vaccines |
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| CN105648102A true CN105648102A (en) | 2016-06-08 |
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| CN201610184173.2A Pending CN105648102A (en) | 2016-03-29 | 2016-03-29 | Kit for detecting DNA residues of MDCK cells in vaccines |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151412A (en) * | 2021-04-07 | 2021-07-23 | 兰州百源基因技术有限公司 | Specific primer and probe for detecting residual DNA content of MDCK cells and real-time fluorescent quantitative PCR kit |
| CN114634975A (en) * | 2022-05-18 | 2022-06-17 | 北京万泰生物药业股份有限公司 | Method for detecting cell DNA content and kit thereof |
| WO2024011822A1 (en) * | 2022-04-06 | 2024-01-18 | 湖州申科生物技术有限公司 | Primer pair for quantitative detection of dna fragment size distribution in mdck cell, and detection method |
Citations (3)
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| CN101565741A (en) * | 2008-04-25 | 2009-10-28 | 中山大学达安基因股份有限公司 | Kit for detecting DNA remains of Vero cells by real-time fluorescent quantitative polymerase chain reaction (PCR) |
| CN102154503A (en) * | 2011-04-20 | 2011-08-17 | 上海复宏汉霖生物技术有限公司 | Method for detecting DNA (deoxyribonucleic acid) content of CHO (cholesterol) cells by probe |
| CN105018622A (en) * | 2015-08-06 | 2015-11-04 | 云南省动物疫病预防控制中心 | Method for quickly detecting culture medium BHK-21 cell residuals in foot-and-mouth disease vaccine |
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2016
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565741A (en) * | 2008-04-25 | 2009-10-28 | 中山大学达安基因股份有限公司 | Kit for detecting DNA remains of Vero cells by real-time fluorescent quantitative polymerase chain reaction (PCR) |
| CN102154503A (en) * | 2011-04-20 | 2011-08-17 | 上海复宏汉霖生物技术有限公司 | Method for detecting DNA (deoxyribonucleic acid) content of CHO (cholesterol) cells by probe |
| CN105018622A (en) * | 2015-08-06 | 2015-11-04 | 云南省动物疫病预防控制中心 | Method for quickly detecting culture medium BHK-21 cell residuals in foot-and-mouth disease vaccine |
Non-Patent Citations (2)
| Title |
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| APPLIED BIOSYSTEMS: "resDNASEQ® MDCK Residual DNA Quantitation System", 《 HTTPS://WWW.THERMOFISHER.COM/CONTENT/DAM/LIFETECH/GLOBAL/LIFE-SCIENCES/BIOPRODUCTION/PDFS/CO24250-RESDNASEQ-MDCK-DATASHEET.PDF》 * |
| MURIELLE ANDRE等: "Universal real-time PCR assay for quantitation and size evaluation of residual cell DNA in human viral vaccines", 《BIOLOGICALS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151412A (en) * | 2021-04-07 | 2021-07-23 | 兰州百源基因技术有限公司 | Specific primer and probe for detecting residual DNA content of MDCK cells and real-time fluorescent quantitative PCR kit |
| CN113151412B (en) * | 2021-04-07 | 2023-06-16 | 兰州百源基因技术有限公司 | Specific primer and probe for detecting residual DNA content of MDCK cells and real-time fluorescent quantitative PCR kit |
| WO2024011822A1 (en) * | 2022-04-06 | 2024-01-18 | 湖州申科生物技术有限公司 | Primer pair for quantitative detection of dna fragment size distribution in mdck cell, and detection method |
| CN114634975A (en) * | 2022-05-18 | 2022-06-17 | 北京万泰生物药业股份有限公司 | Method for detecting cell DNA content and kit thereof |
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