WO2023029278A2 - 一种快速检测病毒基因组大小的方法及试剂盒 - Google Patents
一种快速检测病毒基因组大小的方法及试剂盒 Download PDFInfo
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- 238000000034 method Methods 0.000 title claims description 30
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 44
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 24
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- 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
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- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
Definitions
- the invention belongs to the field of molecular biology, and in particular relates to a kit for rapidly detecting virus genome and a detection method thereof.
- rAAV Recombination adeno-associated virus
- the current production methods of recombinant adeno-associated virus gene drugs have greatly improved compared with 20 years ago, but the improvement of their quality control methods is very limited, which may be related to the slow commercialization of gene therapy in the past.
- AAV consists of a protein capsid and single-stranded genomic DNA.
- genomic DNA of adeno-associated virus is extracted by conventional methods, the obtained DNA is positive single-stranded DNA and negative single-stranded DNA.
- the traditional detection of the genome size of adeno-associated virus is mainly realized by Southern blot. This method has complicated steps, high cost, and must use radioactive isotopes, which is not suitable as a quality control method for gene medicines.
- the present invention aims to overcome the deficiencies of the prior art and provide a fast, efficient, easy, under conventional experimental conditions, can detect the rapid detection kit and detection kit of the genome size of Parvoviridae family (Parvoviridae family) virus method.
- the present invention provides the following technical solutions:
- a method for rapidly detecting the size of the viral genome which involves changing the genomic single-stranded DNA of the virus into a more stable double-stranded DNA and then performing agarose gel electrophoresis detection; the virus is selected from the Parvoviridae family (Parvoviridae family) viruses.
- the method includes adding a lysate or its dilution to the virus sample, cracking the virus capsid in the sample under appropriate conditions, and then using agarose gel for electrophoresis analysis;
- the lysate uses water as a solvent, selected from any of the following:
- the final concentration of the detergent in the system is 0.01-0.1%
- the final concentration of NaCl in the system is 0-0.2M
- the final concentration of the chelating agent in the system is 0-6mM.
- described detergent is selected from mild detergent, preferably sodium dodecyl sulfate (SDS), polysorbate 20 (Tween-20), TritonX 100, Proteinase K or its Mixture; more preferably SDS.
- mild detergent preferably sodium dodecyl sulfate (SDS), polysorbate 20 (Tween-20), TritonX 100, Proteinase K or its Mixture; more preferably SDS.
- the chelating agent is EDTA.
- the final concentration of SDS in the system is 0.03-0.1%, most preferably 0.05%.
- the final concentration of NaCl in the system is 0.02-0.08M, preferably 0.05M.
- the final concentration of EDTA in the system is 3-6 mM, preferably 5 mM.
- the lysate consists of 0.5% SDS, 0.5M NaCl and 50mM EDTA.
- the suitable condition is cooling on ice or at room temperature after heat treatment at 70-100°C.
- the heating temperature in the present invention is an appropriate temperature for cleaving the protein.
- the suitable conditions are heating at 90-100°C for 10-15 minutes and cooling at room temperature for 10-15 minutes; preferably heating at 95°C for 10 minutes and cooling at room temperature for 10 minutes.
- the virus described in the present invention is preferably an adeno-associated virus.
- a virus lysate selected from any of the following:
- described detergent is selected from mild detergent, preferably sodium dodecyl sulfate (SDS), polysorbate 20 (Tween-20), TritonX 100, Proteinase K or Mixtures thereof; still more preferably SDS.
- mild detergent preferably sodium dodecyl sulfate (SDS), polysorbate 20 (Tween-20), TritonX 100, Proteinase K or Mixtures thereof; still more preferably SDS.
- the chelating agent is EDTA.
- the SDS concentration is 0.3% to 1%, most preferably 0.5%.
- the NaCl concentration is 0.2-0.8M, more preferably 0.5M.
- the concentration of EDTA is 30-60 mM, more preferably 50 mM.
- described lysate is made up of 0.5% SDS, 0.5M NaCl and 50mM EDTA.
- a kit for detecting the size of a virus genome is characterized by comprising the virus lysate of the present invention.
- the lysate is composed of 0.5% SDS, 0.5M NaCl and 50mM EDTA.
- the kit also includes 6X DNA gel loading buffer dye.
- the kit of the present invention may additionally contain tubes for mixing components, microplates, test strips, instruction materials describing usage methods, and the like as needed.
- the detection system can be an instrument with functions such as fluorescence readout, chemiluminescence readout, and radioactive readout.
- the detection system also includes a computer and detection and analysis software.
- kits of the present invention are mainly: fast, efficient, convenient, and capable of maintaining the stability of single-stranded DNA; the present invention uses common experimental reagents, materials and methods, and can detect the presence of adeno-associated virus under conventional experimental conditions. Genome size, this method is currently the only effective means to detect the genome size of the adeno-associated virus gene drug.
- the adeno-associated virus genome detection method and kit provided by the present invention can provide clearer electrophoretic imaging results, and have the advantages of brightening bands, reducing background values, and reducing sample residual pores.
- the materials used in the adeno-associated virus genome detection method and kit provided by the present invention are simple and easy to obtain, and at the same time have low requirements on the experimental environment, and have the advantages of rapidity, simplicity, effectiveness, and practicability.
- Figure 1 includes Figure 1A and Figure 1B, showing the results of agarose gel electrophoresis after AAV2-GFP samples were treated with lysates 1, 11, 12, and 13, and water (H 2 O) was used as a negative control, AAV2 capsid served as a negative control.
- Figure 1A is the result of electrophoresis
- Figure 1B is the band quantification result of the experimental group (AAV2-GFP) in Figure 1A.
- Figure 2 Comprising Figure 2A and Figure 2B, showing the results of agarose gel electrophoresis after AAV2-GFP samples were treated with lysates 1, 2, and 14, and AAV2 capsid was used as a negative control.
- Figure 2A is the result of electrophoresis;
- Figure 2B is the band quantification result of the experimental group (AAV2-GFP) in Figure 2A.
- Figure 3 Comprising Figure 3A and Figure 3B, showing the results of agarose gel electrophoresis after AAV2-GFP samples were treated with lysates 2, 3, 4, and 5, and AAV2 capsid was used as a negative control.
- Figure 3A is the result of electrophoresis;
- Figure 3B is the band quantification result of the experimental group (AAV2-GFP) in Figure 3A.
- Figure 4 Comprising Figure 4A and Figure 4B, showing the results of agarose gel electrophoresis after AAV2-GFP samples were treated with lysates 2, 6, and 7, and AAV2 capsid was used as a negative control.
- Fig. 4A is the result of electrophoresis;
- Fig. 4B is the band quantification result of the experimental group (AAV2-GFP) in Fig. 4A.
- Figure 5 includes Figure 5A and Figure 5B, showing the results of agarose gel electrophoresis for samples that were warmed at 4°C (on ice), 25°C (room temperature) or 37°C after being treated with lysate 5, and AAV2 capsid was used as a negative control.
- Fig. 5A is the result of electrophoresis;
- Fig. 5B is the band quantification result of the experimental group (AAV2-GFP) in Fig. 5A.
- Figure 6 Comprising Figure 6A and Figure 6B, showing the results of agarose gel electrophoresis for samples heated at 95°C or not, and AAV2 capsid was used as a negative control.
- Figure 6A is the result of electrophoresis, in which the sample conditions from left to right are: (1) H 2 O treatment, no heating, (2) H 2 O treatment, heating at 95°C for 10 minutes, no cooling, (3) H 2 O treatment, heating at 95°C for 10 minutes, cooling at room temperature for 10 minutes, (4) treatment with lysate 2, heating at 95°C for 10 minutes, cooling at room temperature for 10 minutes;
- Figure 6B is the band of the experimental group (AAV2-GFP) in Figure 6A Quantify results.
- Figure 7 Comprising Figure 7A and Figure 7B, showing the results of agarose gel electrophoresis after AAV2-GFP samples were treated with lysates 4, 8, 9, and 10, and AAV2 capsid was used as a negative control.
- Fig. 7A is the result of electrophoresis;
- Fig. 7B is the band quantification result of the experimental group (AAV2-GFP) in Fig. 7A.
- Figure 8 The results of agarose gel electrophoresis after AAV2-GFP samples were treated with lysates 15, 16, and 17, and the AAV2 capsid was used as a negative control electrophoresis results.
- Figure 9 is the results of agarose gel electrophoresis after the AAV2-GFP samples were treated with 2X lysate 10 (lane 1) and undiluted lysate 10 (lane 2), and the AAV2 capsid was used as the negative control electrophoresis results picture.
- the kit for rapid detection of adeno-associated virus genome of the present invention comprises:
- Lysis buffer (lysis buffer), the composition is shown in the table below.
- Step 1 Prepare Lysis Solution 1, 11, 12, 13.
- Step 2 Return the sample to room temperature and turn on the metal bath.
- Step 3 Prepare 1% Agarose Gel
- Step 4 Processing the adeno-associated virus to be detected
- Step 5 Perform Agarose Gel Electrophoresis
- the lysates 1, 2, and 14 were prepared, and the experimental conditions and methods were referred to in Example 1.
- FIG. 2A The results obtained by agarose gel electrophoresis of samples treated with lysates 1, 2, and 14 respectively are shown in Figure 2A.
- lysates 2 and 14 had more obvious strips than lysate 1.
- the background value of the band in lysate 14 is relatively high, showing the situation of band dragging;
- Figure 2B is the result of quantifying the bands of the electrophoresis results of the AAV2-GFP experimental group in Figure 2A, calculated based on lysate 1 Ratio, it can be found that lysate 2 and 14 are significantly different from lysate 1, and the effect of lysate 14 is slightly better than that of lysate 2, but considering the imaging results of electrophoresis, 0.05% SDS (lysate 2) is more appropriate concentration.
- the lysate 2-7 was prepared, and the experimental conditions and methods were referred to Example 1.
- Embodiment 4 sample cooling condition test
- Step 1 Prepare lysate 4.
- Step 2 Return the sample to room temperature and turn on the metal bath.
- Step 3 prepare 1% agarose gel, the method is the same as that in Example 1.
- Step 4 Process the sample to be tested
- step (4) The sample in step (4) was cooled on ice for 10 minutes, at room temperature (25° C.) for 10 minutes, or at 37° C. for 10 minutes.
- Step 5 Perform Agarose Gel Electrophoresis
- Figure 5A The results obtained by agarose gel electrophoresis of samples treated with lysate 4 are shown in Figure 5A. Clear bands can be obtained;
- Figure 5B is the result of quantification of the electrophoresis bands of the AAV2-GFP experimental group in Figure 5A, and the ratio is calculated based on the condition of 4°C. It can be found that the three cooling conditions have no significant difference. difference, thus it can be proved that the effect of the lysate of the present invention is not affected by the temperature of the cooling sample environment.
- Step 1 Prepare lysate 2.
- Step 2 Return the sample to room temperature and turn on the metal bath.
- Step 3 prepare 1% agarose gel, the method is the same as that in Example 1.
- Step 4 Process the sample to be tested
- Step 5 Perform Agarose Gel Electrophoresis
- the results obtained by agarose gel electrophoresis are shown in Figure 6A.
- the sample conditions from left to right are: (1) H 2 O treatment, no heating; (2) H 2 O treatment, heating at 95°C for 10 minutes, Uncooled; (3) H 2 O treatment, heating at 95°C for 10 minutes, cooling at room temperature for 10 minutes; (4) treatment with lysate 2, heating at 95°C for 10 minutes, cooling at room temperature for 10 minutes.
- the clearest bands can be obtained by using lysate 2 and heating at 95°C for 10 minutes, and there is no large amount of sample remaining in the channel;
- Figure 6B shows the AAV2-GFP experimental group in Figure 6A
- the ratio was calculated based on the H 2 O + unheated condition. It can be found that heating the sample can cause a significant difference, and the treatment with lysate 2 + heating at 95°C has the best effect, thus It can be proved that the high temperature of 95°C can destroy the viral protein capsid and release the single-stranded genomic DNA.
- Embodiment 6 EDTA optimal concentration test
- Example 1 The lysates 4, 8, 9, and 10 were prepared, and the experimental conditions and methods were referred to in Example 1.
- lysate 4 The effect of 10 is relatively good, which proves that the addition of EDTA promotes the formation of double-stranded DNA, making the bands clearer and brighter, and adding 5mM EDTA to 0.05% SDS and 0.05M NaCl (lysate 10) is the best solution .
- Step 1 Prepare lysate 10, 15, 16, 17.
- Step 2 Return the sample to room temperature and turn on the metal bath.
- Step 3 Prepare 1% Agarose Gel
- Step 4 Processing the adeno-associated virus to be detected
- Step 5 Perform Agarose Gel Electrophoresis
- Step 1 Prepare lysate 10.
- Step 2 Return the sample to room temperature and turn on the metal bath.
- Step 4 Processing the adeno-associated virus to be detected
- sample in channel 1 take 5 ⁇ l of AAV2-GFP sample (1 ⁇ 10 11 ) warmed at room temperature, and mix with 5 ⁇ l of 2X lysate 10; sample in channel 2: take 9 ⁇ l of AAV2-GFP sample warmed at room temperature GFP sample (1 ⁇ 10 11 ), mixed with 1 ⁇ l of undiluted lysate 10;
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Description
Claims (18)
- 一种快速检测病毒基因组大小的方法,其特征在于,将病毒的基因组由单链DNA变为更加稳定的双链DNA后进行琼脂糖凝胶电泳检测;所述的病毒选自细小病毒科(Parvoviridae family)病毒。
- 根据权利要求1所述的方法,其特征在于,所述方法包括向病毒样品中加入裂解液或其稀释液,在合适的条件下使样品中的病毒衣壳裂解,再使用琼脂糖凝胶进行电泳分析;所述的裂解液以水为溶剂,选自以下任意一种:(1)去污剂;(2)去污剂和NaCl组成;(3)由去污剂、NaCl以及螯合剂组成;其中,加入裂解液后,使去污剂在系统中的终浓度为0-0.1%,NaCl在系统中的终浓度为0-0.2M,螯合剂在系统中的终浓度为0-6mM。
- 根据权利要求2所述的方法,其特征在于,所述的去污剂选自温和去污剂,优选十二烷基硫酸鈉(SDS)、吐温-20、TritonX 100、Proteinase K或其混合物;更进一步优选SDS。
- 根据权利要求2所述的方法,其特征在于,所述的螯合剂为EDTA。
- 根据权利要求3所述的方法,其特征在于,SDS在系统中的终浓度为0.03%-0.1%,优选0.05%。
- 根据权利要求2所述的方法,其特征在于,NaCl在系统中的终浓度为0.02-0.08M,优选0.05M。
- 根据权利要求2所述的方法,其特征在于,EDTA在系统中的终浓度为3-6mM,优选5mM。
- 根据权利要求1~7中任一项所述的方法,其特征在于,所述裂解液由0.5%SDS、0.5M NaCl和50mM EDTA组成。
- 根据权利要求1~7中任一项所述的方法,其特征在于,所述合适的条件为70~100℃加热处理之后在冰上或室温冷却。
- 根据权利要求9所述的方法,其特征在于,所述合适的条件为90~100℃加热10~15min之后在室温冷却10~15min;优选95℃加热10min之后在室温冷却10min。
- 一种病毒裂解液,其特征在于,以水为溶剂,选自以下任意一种:(1)0.01%~1%去污剂;(2)由0.01%~1%去污剂和0.01M~1M NaCl组成;(3)由0.01%~1%去污剂、0.01M~1M NaCl以及1.5mM~100mM螯合剂组成。
- 根据权利要求11所述的病毒裂解液,其特征在于,所述的去污剂选自温和去污剂,优选十二烷基硫酸鈉、聚山梨醇酯20(吐温-20)、TritonX 100、Proteinase K或其混合物;更进一步优选SDS。
- 根据权利要求1所述的病毒裂解液,其特征在于,所述的螯合剂为EDTA。
- 根据权利要求12所述的病毒裂解液,其特征在于,SDS浓度为0.3%~1%,优选0.3%~0.6%,进一步优选0.5%。
- 根据权利要求11所述的病毒裂解液,其特征在于,NaCl浓度为0.2~0.8M,优选0.5M。
- 根据权利要求11所述的病毒裂解液,其特征在于,EDTA浓度优选30~60mM,优选50mM。
- 根据权利要求11~16中任一项所述的病毒裂解液,其特征在于,所述裂解液由0.5%SDS、0.5M NaCl和50mM EDTA组成。
- 一种检测病毒基因组大小的试剂盒,其特征在于包含权利要求11~17中任一项所述的病毒裂解液;所述的病毒选自细小病毒科(Parvoviridae family)病毒。
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