CN109097454B - Detection method for residual quantity of HEK293gDNA - Google Patents
Detection method for residual quantity of HEK293gDNA Download PDFInfo
- Publication number
- CN109097454B CN109097454B CN201810940792.9A CN201810940792A CN109097454B CN 109097454 B CN109097454 B CN 109097454B CN 201810940792 A CN201810940792 A CN 201810940792A CN 109097454 B CN109097454 B CN 109097454B
- Authority
- CN
- China
- Prior art keywords
- hek293gdna
- residual quantity
- quantitative pcr
- detecting
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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]
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a detection method of residual quantity of HEK293gDNA, which comprises sample pretreatment and quantitative PCR detection of the residual quantity of HEK293gDNA, wherein the sample pretreatment is a sample pretreatment method based on magnetic beads, and comprises the steps of digesting a sample by cell lysate and proteinase K, adding isopropanol and magnetic beads to extract and enrich nucleic acid, washing twice to remove protein and other pollution, and eluting the nucleic acid from the magnetic beads; the quantitative PCR detection method for the residual quantity of the HEK293gDNA takes the HEK293gDNA as a template, and utilizes a designed primer and a probe which have specificity aiming at the HEK293gDNA to carry out quantitative detection, and the quantitative PCR is a real-time fluorescent quantitative PCR technology based on a Taqman probe.
Description
Technical Field
The invention provides a method for detecting the residual quantity of HEK293gDNA, which comprises sample pretreatment and quantitative PCR detection of the residual quantity of HEK293gDNA, and belongs to the field of residual impurity detection in biological product quality control.
Background
The HEK293 cell, namely the human embryonic kidney cell 293(ATCC CRL-1573), has the characteristics of high transfection efficiency and suspension culture, and the HEK293 cell can be rapidly increased in a culture system to realize high-density culture for large-scale expression, so that the HEK293 cell is widely used for producing proteins, vaccines, anti-cancer agents, recombinant adenovirus coating and the like. One of the most important problems in vaccine production using passaged cell lines is the residue of host cell DNA fragments. Although subjected to stringent purification procedures, there is still a possibility that DNA fragments of the host cells may remain in the product. These residual DNA may carry infectious or neoplastic risks. Because of the potential risk of disease caused by residual DNA, many organizations have established relevant standards for the amount of residual DNA in biological products. WHO and European Union stipulate that the residual amount of DNA in each biological preparation product is required to be controlled below 10 ng; the FDA in the united states stipulates that the residual amount of DNA per unit of a biologic product is within 100 pg.
Residual DNA belongs to trace impurities, and the rapid, efficient and accurate detection of the residual DNA is a difficult work. The method for detecting foreign DNA residues in the third section of the "Chinese pharmacopoeia" 2015 edition only includes a DNA probe hybridization method and a fluorescent staining method. The DNA probe hybridization method is characterized in that digoxin is used for marking HEK293 cell DNA to prepare a DNA hybridization probe, then the probe is used for hybridizing with total DNA of a sample to be detected and reference DNA with corresponding known concentration, and finally the content of residual DNA in the sample is judged through a chromogenic signal. Although the DNA probe hybridization method is relatively simple in terms of conditions, it has disadvantages of cumbersome operation, and poor stability, sensitivity and specificity. The fluorescence staining method is to determine the residual DNA content by using the specific binding of a double-stranded DNA fluorescent dye and the double-stranded DNA to form a complex. When the DNA residual quantity is in the range of 1.25-80ng/ml, the fluorescent staining normal is better. The method has the disadvantages of being easily interfered by ssDNA and dsDNA, and simultaneously, the sensitivity and the specificity of the method are difficult to meet the detection requirements.
Disclosure of Invention
The present invention is directed to solving, to some extent, one of the technical problems in the related art described above. Therefore, the invention aims to provide a method for detecting the residual quantity of the HEK293gDNA, which comprises the steps of sample pretreatment and quantitative PCR detection of the residual quantity of the HEK293gDNA and has the advantages of high efficiency, rapidness, accuracy and strong specificity.
Extracting and purifying the gDNA (genome DNA) of the HEK293 cell by using a QIAGEN DNeasy Blood & Tissue Kit, performing integrity detection on the extracted HEK293gDNA by using agarose gel electrophoresis, subpackaging, and storing in a refrigerator at the temperature of-80 ℃ for subsequent experiments.
In one aspect, the invention provides a method for sample pretreatment, which is a magnetic bead-based sample pretreatment method.
The pretreatment of the sample comprises the following steps: (1) digesting a sample by using cell lysate and proteinase K, (2) adding isopropanol and magnetic beads to extract and enrich nucleic acid, (3) washing twice to remove protein and other pollution, and (4) eluting the nucleic acid from the magnetic beads for subsequent quantitative detection.
Preferably, the cell lysate consists of: 6M guanidinium isothiocyanate, 1M DTT, 0.5M sodium acetate, 2% sodium N-lauroylsarcosine, 50mM EDTA and 0.6M Tris-HCl.
On the other hand, the invention provides a quantitative PCR detection method of the residual quantity of the HEK293gDNA, which comprises the steps of designing specific primers and probes for the HEK293gDNA and carrying out fluorescent quantitative PCR detection.
The designed specific primers and probes for HEK293gDNA are respectively HEK293qPCR ForwardPrimer 5 '-CGWGTANCTRGGAYTAHA-3' and HEK293qPCR Reverse Primer 5 '-NGTGARACCCCYYTCTST-3'; HEK293qPCR Probe 5 '-FAM-VCCACCSNGCCRGGCTA-MGB-3'.
Preferably, the primer concentration is 0.67. mu.M and the probe concentration is 0.1. mu.M.
Preferably, the experimental conditions of the fluorescent quantitative PCR are that the denaturation is carried out for 5min at 95 ℃; then annealing at 95 ℃ for 10s, extending at 60 ℃ for 1min, and performing 40 cycles.
The invention has the beneficial effects that:
in the sample pretreatment, the cell lysate provided by the invention is used for digesting the sample, the recovery rate of nucleic acid is above 70%, and the accuracy of the subsequent quantitative analysis is improved.
The real-time fluorescent quantitative PCR combines the dual specificity of the amplification primer and the probe, and improves the specificity and the accuracy of detection. During the primer amplification and extension process, the specifically bound fluorescent labeled probe is hydrolyzed under the catalysis of the hydrolase function of the DNA polymerase, so that a fluorescent signal is released. The initial content in the sample is quantitatively analyzed by monitoring the fluorescent signal in the PCR system in real time.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 integrity test of HEK293gDNA
FIG. 2A HEK293gDNA residue integral detection method HEK293gDNA amplification curve
FIG. 2B HEK293gDNA residue integral detection method HEK293gDNA standard curve
FIG. 3 specificity of the Overall detection method for HEK293gDNA residues
The specific implementation mode is as follows:
the main materials are as follows:
HEK293 cells were purchased from American ATCC (ATCC CRL-1573); qPCR MIX was purchased from Roche (cat No. 2147483647); magnetic beads were purchased from Asend (cat. No. AS 316004); guanidinium isothiocyanate and sodium N-lauroyl sarcosinate were purchased from Solarbio (cat # G8210 or S8100); proteinase K was purchased from Merck (cat # 1245680100).
Example 1: extraction and purification of HEK293gDNA
HEK293 cells were purified by gDNA (genomic DNA) extraction using QIAGEN DNeasy Blood & Tissue Kit. HEK293gDNA is used as a positive reference substance; coli gDNA as negative reference, and the integrity of the extracted and purified HEK293gDNA was checked by agarose gel electrophoresis, see FIG. 1
Example 2: primer and probe design and optimization
Based on the Genebank database and the mining analysis of domestic and foreign documents, multiple pairs of primers and probes are designed by utilizing Beacon design 7.9 software according to the basic principle of primer and probe design. The extracted HEK293gDNA is used as a template to screen a plurality of pairs of designed primers and probes to obtain an optimal primer probe combination with good specificity, sensitivity and repeatability, and the 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’
example 3: optimization of fluorescent quantitative PCR reaction system
With reference to the optimum primer probe combination selected in example 2, PCR reactions were carried out while the concentrations of the primers and probes were set to 0.1. mu.M, 0.2. mu.M, 0.4. mu.M, 0.67. mu.M and 0.8. mu.M, respectively, in the fluorescent quantitative PCR reaction system without changing the other components. The results of multiple repeated experiments show that the optimal primer concentration is 0.67 mu M, and the optimal probe concentration is 0.1 mu M.
With the combination of the optimal primer probe combination selected in example 2, the annealing extension temperature was set to 50 ℃, 55 ℃, 60 ℃ and 65 ℃ respectively for PCR reaction without changing other components in the reaction system. The repeated test results show that the optimal annealing extension temperature is 60 ℃. The reaction procedure for the experiment was: pre-denaturation at 95 ℃ for 5 min; then 95 ℃ for 10s, 60 ℃ for 1min, 40 cycles.
Example 4: drawing of HEK293gDNA amplification curve and standard curve
The HEK293gDNA (30 ng/. mu.l) extracted and prepared in a refrigerator stored at-80 ℃ was taken out, and 100-fold dilution was performed to obtain SD1(300 pg/. mu.l), and then 10-fold gradient dilution was performed to obtain SD2(30 pg/. mu.l), SD3(3 pg/. mu.l), SD4(300 fg/. mu.l), and SD5(30 fg/. mu.l), respectively. According to the optimized experimental conditions, the reaction system is 15 mul, and the reaction system comprises the following components: qPCR MIX 7.5. mu.l, Forward/Reverse primer 1/1. mu.l, probe 0.15. mu.l, DNA 3. mu.l, ddH2O2.35. mu.l, and then put into a machine running program to finally obtain an amplification curve of HEK293gDNA, as shown in FIG. 2A. The log values of the respective dilutions of HEK293gDNA were plotted against the Ct (initial cycle) value for each dilution in a linearly decreasing relationship, i.e., a HEK293gDNA quantitation standard curve. R of the curve2Or R is a value reflecting the amplification efficiency of the amplification reaction, R2It should be greater than 0.98 or r-value greater than 0.99, i.e., reaction efficiency between 90-105%, see FIG. 2B.
Example 5: HEK293gDNA detection sensitivity test
HEK293gDNA was diluted to 60 fg/. mu.l, 45 fg/. mu.l, 30 fg/. mu.l, 10 fg/. mu.l, respectively, and then assayed using the reaction system optimized in example 3 to determine the detection sensitivity, 6 replicates for each concentration. The experimental results show that: the CV value is still < 1% at concentrations as low as 10 fg/. mu.l, and thus the sensitivity of the detection reaction can be as low as 10 fg/. mu.l.
Example 6: HEK293gDNA residue detection method accuracy and vaccine sample detection experiment
Different amounts of HEK293gDNA were added to the eluate to prepare a base sample, HEK293gDNA was added to the vaccine samples of different batches, and the base sample and the vaccine sample without additional HEK293gDNA were used as controls. 100 mu l of each group of samples are respectively added with proteinase K and cell lysis solution (6M guanidine isothiocyanate, 1M DTT, 0.5M sodium acetate, 2% N-lauroyl sarcosine sodium, 50mM EDTA and 0.6M Tris-HCl) to be processed for 30min at 56 ℃, then isopropanol and magnetic beads are added to extract and enrich nucleic acid, then the nucleic acid is washed twice to remove protein and other pollution, and finally the nucleic acid is eluted from the magnetic beads to be used for subsequent quantitative detection and the recovery rate is calculated. The experimental results show that: the recovery rate is above 70%, the method has high accuracy, and the experimental parameters are shown in tables 1A and 1B.
TABLE 1 detection results of HEK293gDNA residue detection method accuracy
Detection result of HEK293gDNA residue detection method accuracy in vaccine sample shown in Table 1B
Example 7: precision experiment
For the repeatability experiment, the sample pretreatment based on the magnetic beads is carried out on two basic samples with different concentrations, then the quantitative detection is carried out, and the detection amount is calculated. Each set of samples was independently repeated 8 times to calculate the CV value for each set of samples. The experimental results show that: the CV values of each group are less than 15%, which shows good experimental repeatability, and the experimental parameters are shown in Table 2A.
TABLE 2 repeatability of the Overall detection method for HEK293gDNA residues
For the intermediate precision experiment, for three basic samples with different concentrations, three different experimenters carry out sample pretreatment and quantitative detection based on magnetic beads. Each individual group of samples was independently repeated 3 times. The experimental results show that: CV values for each group were < 10% and recoveries were > 70%, see table 2B.
TABLE 2 intermediate precision of the Overall detection method for HEK293gDNA residues
Example 8: specificity experiment of HEK293gDNA residue integral detection method
Equal amounts of CHO gDNA or E.coli gDNA were mixed with HEK293gDNA to form a mixed sample, and individual CHO gDNA, E.coli gDNA, HEK293gDNA and the mixed sample were diluted by gradient fold ratio, respectively, and then subjected to quantitative detection, and a curve was drawn. The experimental results show that: the method only aims at the HEK293gDNA, and has high specificity, as shown in figure 3.
The method for detecting the residual quantity of the HEK293gDNA provided by the invention is described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the principle of the present invention, and these changes and modifications also fall into the protection scope of the appended claims.
Claims (6)
1. A detection method of the residual quantity of HEK293gDNA is characterized in that: comprises the steps of sample pretreatment and quantitative PCR detection of the residual quantity of HEK293 gDNA; the sample pretreatment is a sample pretreatment method based on magnetic beads, and the quantitative PCR detection of the HEK293gDNA residual quantity comprises the design of specific primers and probes for the HEK293gDNA and the fluorescent quantitative PCR detection; screening multiple pairs of designed primers and probes by using HEK293gDNA as a template;
the sequences of the primer and probe combinations obtained after screening were:
HEK293 qPCR Forward Primer: 5’-CGWGTANCTRGGAYTAHA-3’
HEK293 qPCR Reverse Primer: 5’-NGTGARACCCCYYTCTST-3’
HEK293 qPCR Probe: 5’-FAM-VCCACCSNGCCRGGCTA-MGB-3’。
2. the method for detecting the residual quantity of HEK293gDNA according to claim 1, which is characterized in that: the cell lysis solution in the sample pretreatment method based on the magnetic beads is self-made;
the composition of the cell lysate is: 6M guanidinium isothiocyanate, 1M DTT, 0.5M sodium acetate, 2% sodium N-lauroylsarcosine, 50mM EDTA and 0.6M Tris-HCl.
3. The method for detecting the residual quantity of HEK293gDNA according to claim 1, which is characterized in that: the concentration of the primer and the reaction concentration of the probe in the reaction system for the fluorescent quantitative PCR detection are respectively 0.67 mu M and 0.1 mu M.
4. The method for detecting the residual quantity of HEK293gDNA according to claim 1 or 3, which is characterized in that: the reaction system for the fluorescent quantitative PCR detection is 15 mul, and the reaction system comprises the following components: qPCR MIX 7.5. mu.l, Forward/Reverse primer 1/1. mu.l, probe 0.15. mu.l, DNA 3. mu.l, ddH2O 2.35μl。
5. The method for detecting the residual quantity of HEK293gDNA according to claim 4, which is characterized in that: the reaction program conditions of the fluorescent quantitative PCR detection are as follows: 95oC, pre-denaturation for 5 min; then the95oC 10s， 60oC1 min, 40 cycles.
6. The method for detecting the residual amount of the HEK293gDNA according to claim 1, wherein the method for detecting the residual amount of the HEK293gDNA is used for detecting the residual amount of the HEK293gDNA in a vaccine sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810940792.9A CN109097454B (en) | 2018-08-17 | 2018-08-17 | Detection method for residual quantity of HEK293gDNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810940792.9A CN109097454B (en) | 2018-08-17 | 2018-08-17 | Detection method for residual quantity of HEK293gDNA |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109097454A CN109097454A (en) | 2018-12-28 |
| CN109097454B true CN109097454B (en) | 2020-11-06 |
Family
ID=64850185
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810940792.9A Active CN109097454B (en) | 2018-08-17 | 2018-08-17 | Detection method for residual quantity of HEK293gDNA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109097454B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021115399A1 (en) * | 2019-12-13 | 2021-06-17 | 东莞市东阳光生物药研发有限公司 | Method for detecting residual dna in drug sample |
| CN115786326B (en) * | 2022-11-04 | 2023-10-03 | 重庆医科大学 | Virus nucleic acid extraction kit and method for extracting complete virus particle nucleic acid by using same |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857902A (en) * | 2010-06-21 | 2010-10-13 | 西北农林科技大学 | Method for screening effective shRNA of lipoprotein lipase gene |
| CN102367476A (en) * | 2011-10-14 | 2012-03-07 | 太湖瑞晶生物科技有限公司 | Detection method for slow virus infected cell |
| WO2012028740A1 (en) * | 2010-09-03 | 2012-03-08 | Confarma France | Quantification of residual host cell dna by real-time quantitative pcr |
| CN103421885A (en) * | 2012-05-23 | 2013-12-04 | 中国食品药品检定研究院 | Establishment of method for detection of DNA residual quantity of CHO cells through quantification PCR method and standard product |
| CN104694640A (en) * | 2015-02-15 | 2015-06-10 | 内蒙古必威安泰生物科技有限公司 | Quantitative detection method for residual quantity of DNA in host cell of foot and mouth disease vaccine |
| CN108103245A (en) * | 2018-01-15 | 2018-06-01 | 南京驯鹿医疗技术有限公司 | Detect the method and its application of slow virus quality index combination |
-
2018
- 2018-08-17 CN CN201810940792.9A patent/CN109097454B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857902A (en) * | 2010-06-21 | 2010-10-13 | 西北农林科技大学 | Method for screening effective shRNA of lipoprotein lipase gene |
| WO2012028740A1 (en) * | 2010-09-03 | 2012-03-08 | Confarma France | Quantification of residual host cell dna by real-time quantitative pcr |
| CN102367476A (en) * | 2011-10-14 | 2012-03-07 | 太湖瑞晶生物科技有限公司 | Detection method for slow virus infected cell |
| CN103421885A (en) * | 2012-05-23 | 2013-12-04 | 中国食品药品检定研究院 | Establishment of method for detection of DNA residual quantity of CHO cells through quantification PCR method and standard product |
| CN104694640A (en) * | 2015-02-15 | 2015-06-10 | 内蒙古必威安泰生物科技有限公司 | Quantitative detection method for residual quantity of DNA in host cell of foot and mouth disease vaccine |
| CN108103245A (en) * | 2018-01-15 | 2018-06-01 | 南京驯鹿医疗技术有限公司 | Detect the method and its application of slow virus quality index combination |
Non-Patent Citations (2)
| Title |
|---|
| 康柏西普制品中CHO 细胞DNA 残留量的检测;牛冬云等;《中国生物制品学杂志》;20130730;第26卷(第7期);摘要、第1024页1.6 Q-PCR方法的建立及标准曲线的绘制 * |
| 重组腺相关病毒载体中残余HEK293 细胞蛋白含量检测方法的建立;曲伟红等;《中国药学杂志》;20160130;第51卷(第1期);摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109097454A (en) | 2018-12-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106947838B (en) | African swine fever virus non-structural gene real-time fluorescence LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method | |
| EP3636769B1 (en) | Sample nucleic acid measurement test kit, reagent, and application thereof | |
| EP3792364A1 (en) | Method for detecting nucleic acid based on prokaryotic argonaute protein and application thereof | |
| US20230340624A1 (en) | Detection reagents for severe acute respiratory syndrome coronavirus 2 and detection methods | |
| CN111471804B (en) | Kit for detecting novel coronavirus with high sensitivity and high throughput and application thereof | |
| CN107475252B (en) | Nucleic acid releasing agent, method for rapidly releasing nucleic acid and application thereof | |
| CN103074334B (en) | Method for detecting CHO cell DNA | |
| CN105861641A (en) | Primer, kit and method for detecting CHO cell DNA residues | |
| CN111020031A (en) | Method for detecting tumor gene mutation by combining sequence specific blocker with specific PCR (polymerase chain reaction) program | |
| CN109097454B (en) | Detection method for residual quantity of HEK293gDNA | |
| CN113462820A (en) | Multiplex RT-PCR primer probe set for real-time fluorescent quantitative detection of four porcine diarrhea viruses, kit and detection method thereof | |
| CN112111609A (en) | Universal nucleic acid detection kit for enteroviruses | |
| CN112813151A (en) | Whole blood direct fluorescence PCR detection method and application of whole blood sample treatment fluid in preparation of gene detection kit | |
| CN105586438B (en) | GeXP multiple rapid detection primers and detection method for detecting akabane virus, foot-and-mouth disease virus and bluetongue virus | |
| CN105803050B (en) | Primer and method for detecting CHO cell DNA | |
| CN108300804B (en) | Material and method for HBV miR-3 fluorescent quantitative PCR detection | |
| US20220372464A1 (en) | System and method for automatic nucleic acid extraction and quialitative analysis | |
| CN114591945A (en) | DNA virus nucleic acid extraction detection reagent, kit, method and application thereof | |
| CN113930418A (en) | Nucleic acid releasing agent and method for releasing nucleic acid | |
| CN113388700A (en) | Kit for detecting FCV (FCV), FPV (FPV) and FHV-1 viruses by using nucleic acid hand-free triple fluorescence RT-LAMP (reverse transcription loop-mediated isothermal amplification) | |
| CN110951924A (en) | One-step multiple RT-PCR detection method for simultaneously detecting 3 pathogens of sugarcane mosaic disease | |
| CN111088396A (en) | Triple real-time fluorescence PCR method for simultaneously detecting haemophilus parasuis, porcine parvovirus and porcine circovirus type 2 | |
| CN105586437B (en) | Establishment of detection method for GeXP virus carried by four entry cows | |
| CN106676163B (en) | Primer and method for detecting pichia pastoris cell DNA | |
| CN117660701B (en) | LAMP primer group, kit and method for detecting Liquorice pangolin virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |