CN115216561A - VSVG gene copy number detection kit, and use method and application thereof - Google Patents
VSVG gene copy number detection kit, and use method and application thereof Download PDFInfo
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- CN115216561A CN115216561A CN202210558433.3A CN202210558433A CN115216561A CN 115216561 A CN115216561 A CN 115216561A CN 202210558433 A CN202210558433 A CN 202210558433A CN 115216561 A CN115216561 A CN 115216561A
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- 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
- C12Q1/706—Specific hybridization probes for hepatitis
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- 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
- C12Q1/6851—Quantitative amplification
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention relates to C12N15, in particular to a VSVG gene copy number detection kit, and a using method and application thereof. The VSVG gene copy number detection kit comprises a VSVG quantitative reference substance, a VSVG Primer \10939x (Primer probe mixed liquid), a Q-PCR Reaction Buffer and a DNA diluent. The kit simplifies the operation steps of the qPCR reaction operation during reaction liquid preparation, the prepared reagent is contained in the kit and is directly used for experiments, the workload of operators is reduced, the whole operation time is saved, and if the kit is used for continuous detection, the detection result is more comparative. The kit provided by the invention has the advantages of rapid detection, strong specificity and reliable performance, the lowest detection limit can reach the fg level (Feike level), the kit accords with the CDE (national drug administration drug evaluation center) guiding principle, and the kit can accurately detect the VSVG gene copy number of the DNA fragment with the size of 200bp.
Description
Technical Field
The invention relates to C12N15, in particular to a VSVG gene copy number detection kit, and a using method and application thereof.
Background
Lentiviral (LVs) vectors, one of the most commonly used transfer vectors for the transmission of genetic information, are also the first choice vectors for cell engineering. When gene therapy is carried out, the lentiviral vectors used are all non-replicating, however, replication Competent Lentiviruses (RCL) may occur during this process due to recombination mechanisms or other reasons. If the RCL is not detected, a large number of uncontrolled copies of the RCL can cause harm to the human body. The replication-competent lentivirus vector is a vector developed by using the coat protein of VSVG, a herpesvirus, based on HIV-1 (human immunodeficiency virus I), and thus detection of the VSVG gene is considered as a standard for the detection of RCL.
Patent No. CN113789345A provides a kit for detecting replication-competent lentivirus (RCL) and its application, using NL4 strain as positive control, improving the sensitivity of detection, increasing the amount of addition experiment to reduce false positive of the detection result. The patent No. CN110117675B provides a reagent and a method for real-time fluorescent quantitative PCR detection of RCL, and the amplification efficiency and specificity of RCL primers and detection probes are improved.
However, currently, there is no kit capable of detecting RCL by real-time fluorescence quantitative PCR method in the market, and in practical operation, an operator is required to perform configuration of PCR reaction solution before on-line detection, which not only causes problems in consistency of different batches of reaction solution configuration, but also complicates the configuration process, and therefore, reliability of data for detecting VSVG gene copy number is questioned.
Disclosure of Invention
In order to solve the above problems, a first aspect of the present invention provides a VSVG gene copy number detection kit including a VSVG quantitative reference, a VSVG Primer & Probe MIX (Primer-Probe MIX), a Q-PCR Reaction Buffer (Reaction Buffer), and a DNA diluent.
As a preferable technical scheme of the invention, the concentration of the VSVG quantitative reference substance is 27-34ng/mL.
Preferably, the VSVG quantitative reference is e.
As a preferred technical scheme of the invention, the Q-PCR Reaction Buffer comprises a Q-PCR Reaction premixed solution, a VSVG mutant, hALB (human serum albumin) and DNase/RNase-Free deionized water.
As a preferred technical scheme of the invention, in Q-PCR Reaction Buffer, the volume ratio of Q-PCR Reaction premix, VSVG mutant, hALB (human serum albumin) and DNase/RNase-Free deionized water is (9-14): (1.4-2): (0.7-1.3): (1.5-2).
Preferably, in the Q-PCR Reaction Buffer, the volume ratio of the Q-PCR Reaction premix, the VSVG mutant, hALB (human serum albumin) and DNase/RNase-Free deionized water is 12:1.6:1.1:1.7.
VSVG refers to a regulatory element after transcription of the hepatitis B virus of woodchuck, is a DNA sequence with a length of about 600bp, is widely used in viral vectors and can enhance the expression of transgenes.
Preferably, the VSVG mutant is selected from one or more of VSVG-F7, VSVG-R7, VSVG-P7 and VSVG-03.
Further preferably, the VSVG mutants are VSVG-F7, VSVG-R7 and VSVG-P7.
Still further preferably, in the VSVG mutant, the volume ratio of VSVG-F7, VSVG-R7 and VSVG-P7 is (0.4 to 0.8): (0.4-0.8): (0.1-0.6).
Still more preferably, in the VSVG mutant, the volume ratio of VSVG-F7, VSVG-R7 and VSVG-P7 is 0.6:0.5:0.5.
the applicant finds out through a large number of experiments that a specific regulating element is added into the system, and the proportion of the regulating element is further controlled, so that the kit is favorable for having excellent accuracy after being placed for a long time, and the stability of the kit is improved. The applicants speculate that this may be due to the fact that different types of regulatory elements have specific base sequences, and when acting with albumin and Escherichia coli, the pairing efficiency and the pairing stability are improved.
Preferably, the hALB is selected from one or more of hALB-F5, hALB-R5 and hALB-probe.
Further preferably, hALB is hALB-F5, hALB-R5 and hALB-probe, and the volume ratio of hALB-F5, hALB-R5 and hALB-probe in hALB is (0.4-1.2): (0.1-0.5): (0.1-0.5).
More preferably, the volume ratio of hALB-F5, hALB-R5 and hALB-probe in hALB is 0.5:0.3:0.2.
DNase/RNase-Free deionized water is water which is treated and has no nuclease or protease activity, and can be used in nuclease-sensitive molecular experiments such as cDNA synthesis and RNA extraction.
The second aspect of the invention provides a method for using a VSVG gene copy number detection kit, which comprises the following steps: (1) diluting; (2) adding a sample; and (3) setting a program.
In a preferred embodiment of the present invention, the step (1) is performed by diluting the VSVG quantitative reference substance with a DNA diluent.
Preferably, the step (1) is specifically: diluting the E.coli DNA quantitative reference substance by 0.1 time by using a DNA diluent to obtain ST0; then, the DNA dilutions were gradually diluted by 5 gradients 0.1-fold to obtain STD1, STD2, STD3, STD4 and STD5.
As a preferred technical scheme of the invention, the DNA diluent is pretreated before the step (1).
Preferably, the pretreatment specifically comprises: melting the DNA diluent at 1-5 ℃, shaking after 8h, and centrifuging at 3000-7000r/min for 10-50s.
As a preferred technical scheme of the invention, the sample adding is to add 9-14 mu L of qPCR MIX into reaction wells 1-7 of a pore plate respectively.
As a preferred technical scheme of the invention, the qPCR MIX is obtained by mixing VSVG Primer & Probe MIX (Primer Probe mixed liquid) and Q-PCR Reaction Buffer.
Preferably, in qPCR MIX, the volume ratio of VSVG Primer & Probe MIX and Q-PCR Reaction Buffer is (11.8-13.2): (1.7-2.5).
Preferably, reaction wells 1-5 are used for standard curve determination. 3-8. Mu.L of STD1, STD2, STD3, STD4 and STD5 were added to the reaction wells 1-5, respectively.
Preferably, the reaction well 6 is used for performing a No Template Control (NTC) experiment, and 3-8. Mu.L of a DNA dilution is added to the reaction well 6.
Preferably, the reaction well 7 is used for performing an experiment of a sample to be tested, and 3 to 8 μ L of the sample to be tested is added to the reaction well 7.
Further preferably, the sample to be tested contains VSV-G gene, and the size of the DNA fragment of the VSV-G gene is 200bp.
Further preferably, after the above steps are completed, the well plate is shaken for 2-5s and then placed into a qPCR instrument.
The qPCR instrument utilizes the principle of a PCR fluorescent probe method, and PCR reaction products are accumulated continuously along with the progress of PCR reaction, so that the intensity of a fluorescent signal is increased in equal proportion. After each cycle, a fluorescence intensity signal is collected and the change in product amount is monitored by the change in fluorescence intensity.
As a preferred embodiment of the present invention, the program setting includes setting of a creation program and setting of a reaction program.
Preferably, the setting of the creation program is specifically to set the reaction volume to be 20 μ L, and the pre-denaturation is carried out for 10min at 95 ℃; reaction at 95 ℃ for 15s, reaction at 60 ℃ for 1min, cycling the process from pre-denaturation at 95 ℃ to reaction at 60 ℃ for 1min, and cycling for forty times.
Preferably, the reaction program is set by selecting the fluorescence FAM mode of Select fluorescence, and naming the output file with fg.
The third aspect of the invention provides an application of a VSVG gene copy number detection kit, and the VSVG gene copy number detection kit is applied to the cell field and the gene field.
Compared with the prior art, the invention has the following beneficial effects:
in the Q-PCR Reaction Buffer, the volume ratio of the Q-PCR Reaction premix, VSVG mutant, hALB (human serum albumin) and DNase/RNase-Free deionized water is controlled to be (9-14): (1.4-2): (0.7-1.3): (1.5-2), mixing with VSVG Primer & Probe MIX, which is beneficial to improving the stability of the kit and the precision of the precision measurement level; the volume ratio of VSVG-F7, VSVG-R7 and VSVG-P7 is (0.4-0.8): (0.4-0.8): (0.1-0.6) not only is the kit favorable for having excellent accuracy after being placed for a long time, but also the stability of the kit is improved; in qPCR MIX, VSVG Primer & Probe MIX and Q-PCR Reaction Buffer were set in a volume ratio of (11.8-13.2): (1.7-2.5), so that the reagent kit is not easy to generate the problem of reagent kit deterioration such as color change and the like after the reagent kit is placed for a long time and is vibrated during sample adding. The kit simplifies the operation steps of the qPCR reaction operation during reaction liquid preparation, the prepared reagent is contained in the kit and is directly used for experiments, the workload of operators is reduced, the whole operation time is saved, and if the kit is used for continuous detection, the detection result is more comparative. The kit provided by the invention has the advantages of rapid detection, strong specificity and reliable performance, the lowest detection limit can reach the fg level (Feike level), the kit conforms to the CDE (national drug administration drug evaluation center) guiding principle, and the kit can accurately detect the VSVG gene copy number of the DNA fragment with the size of 200bp.
Detailed Description
Examples
Coli DNA was purchased from Guangxi Biotechnology Inc., having a product number of A004449-0010 and a concentration of 29ng/ml, Q-PCR reaction premix was purchased from Guilin Youri Biotechnology Inc., having a product number of YST0038, DNase/RNase-Free deionized water was purchased from Beijing Yaada Biotechnology Inc., having a product number of RT121, DNA universal diluent was purchased from Roche, VSVG-F7, VSVG-R7, VSVG-P7, hALB-F5, hALB-R5, hALB-Probe, VSVG Primer & Probe MIX was purchased from Jiangsu Spectrum New Bio-medicine Inc., and the sample to be tested was a CAR-T/TCR-T cell genome prepared by HIV-1 lentivirus vector technology, containing VSV-G gene, which was purchased from Jiangsu Spectrum New Bio-medicine Inc., PCR instrument was purchased from BIO-qqqx, CFX model 96.
Example 1
The present example provides a VSVG gene copy number detection kit comprising a VSVG quantitative reference, a VSVG Primer & Probe MIX, a Q-PCR Reaction Buffer, and a DNA diluent.
Coli (e.coli) DNA.
The Q-PCR Reaction Buffer comprises Q-PCR Reaction premixed liquid, VSVG mutant, hALB (human serum albumin) and DNase/RNase-Free deionized water.
In Q-PCR Reaction Buffer, the volume ratio of the Q-PCR Reaction premix, VSVG mutant, hALB (human serum albumin) and DNase/RNase-Free deionized water was 12:1.6:1.1:1.7.
the VSVG mutants are VSVG-F7, VSVG-R7 and VSVG-P7. In the VSVG mutant, the volume ratio of VSVG-F7, VSVG-R7 and VSVG-P7 was 0.6:0.5:0.5.
hALB is hALB-F5, hALB-R5 and hALB-probe, and in the hALB, the volume ratio of hALB-F5 to hALB-R5 to hALB-probe is 0.5:0.3:0.2.
the embodiment also provides a using method of the VSVG gene copy number detection kit, which comprises the following steps: (1) diluting; (2) sample adding; and (3) setting a program.
The step (1) is specifically to dilute the VSVG quantitative reference substance by using a DNA diluent.
The step (1) is specifically as follows: diluting an E.coli DNA quantitative reference substance to a concentration of 0.1 time by using a DNA diluent to obtain ST0; then, the DNA diluent was used to dilute the DNA in 5 steps 0.1 times to obtain STD1, STD2, STD3, STD4 and STD5.
The DNA diluent is pretreated before step (1). The pretreatment specifically comprises the following steps: and melting the DNA diluent at 5 ℃, oscillating after 8 hours, and centrifuging for 20s at 5000 r/min.
The sample adding is specifically to add 14.7. Mu.L of qPCR MIX into the reaction wells 1-7 of the well plate respectively.
The qPCR MIX was obtained by mixing VSVG Primer & Probe MIX (Primer Probe mixture) and Q-PCR Reaction Buffer.
In qPCR MIX, the volume ratio of VSVG Primer & Probe MIX and Q-PCR Reaction Buffer was 12.7:2.
reaction wells 1-5 were used for standard curve determination. mu.L of STD1, STD2, STD3, STD4 and STD5 were added to each of the reaction wells 1 to 5.
Reaction well 6 was used for a No Template Control (NTC) experiment, and 5.3. Mu.L of a DNA dilution was added to reaction well 6.
The reaction well 7 is used for carrying out an experiment of a sample to be tested, and 5.3. Mu.L of the sample to be tested is added into the reaction well 7.
After the above steps were completed, the well plate was shaken for 3s and placed in a qPCR instrument.
The program setting includes setting of a creation program and setting of a reaction program.
Setting a creating program to set the reaction volume to be 20 mu L and carrying out pre-denaturation at 95 ℃ for 10min; reaction at 95 ℃ for 15s, reaction at 60 ℃ for 1min, cycling the process from pre-denaturation at 95 ℃ to reaction at 60 ℃ for 1min, and cycling for forty times.
Specifically, the reaction program is set by selecting the fluorescence FAM mode of Select fluorescence, and naming the output file with the unit of fg.
Example 2
This example provides a VSVG gene copy number detection kit, which is different from example 1 in that the VSVG mutant has a VSVG-F7, VSVG-R7 and VSVG-P7 volume ratio of 0.4:0.4:0.2.hALB is hALB-F5, hALB-R5 and hALB-probe, and the volume ratio of hALB-F5, hALB-R5 and hALB-probe in hALB is 0.5:0.2:0.3.
this example also provides a method of using a VSVG gene copy number detection kit, similar to example 1.
Example 3
This example provides a VSVG gene copy number detection kit, which is different from example 1 in that the volume ratio of the Q-PCR Reaction premix, the VSVG mutant, hALB (human serum albumin) and DNase/RNase-Free deionized water in the Q-PCR Reaction Buffer is 11:1.5:0.9:1.6.
this example also provides a method of using a VSVG gene copy number detection kit, similar to example 1.
Example 4
This example provides a VSVG gene copy number detection kit, which differs from example 1 in that the volume ratio of VSVG-F7, VSVG-R7 and VSVG-P7 is 0.1:0.1:0.5.hALB is hALB-F5, hALB-R5 and hALB-probe, and the volume ratio of hALB-F5, hALB-R5 and hALB-probe in hALB is 0.4:0.2:0.2.
this example also provides a method of using a VSVG gene copy number detection kit, similar to example 1.
Example 5
This example provides a VSVG gene copy number detection kit, which differs from example 1 in that in qPCR MIX, the volume ratio of VSVG Primer & Probe MIX and Q-PCR Reaction Buffer is 9:2.
this example also provides a method of using a VSVG gene copy number detection kit, similar to example 1.
And (4) performance testing:
1. and (3) testing accuracy: VSVG gene copy number test was performed using the kit obtained in examples 1 to 5, and correlation coefficient R was obtained by qPCR 2 The results are shown in Table 1:
TABLE 1
| Examples | R 2 |
| 1 | 0.999 |
| 2 | 0.999 |
| 3 | 0.999 |
| 4 | 0.99 |
| 5 | 0.999 |
2. Testing the detection limit: obtaining the kit according to the embodiment 1, testing the kit by a qPCR instrument, replacing the sample to be tested with deionized water, analyzing the deionized water by the qPCR instrument to obtain the relative standard deviation RSD, calculating the limit of detection LOD, wherein LOD =3RSD, and obtaining the LOD value of 1.00 multiplied by 10 according to the result 1 fg/μl。
3. And (3) testing the recovery rate: the VSVG gene copy number test was performed using the kit obtained in examples 1 to 5, and the test sample was STD1, to obtain the recovery rates of the test samples of examples 1 to 5, as shown in Table 2:
TABLE 2
4. And (3) stability testing: after the kit obtained in example 1 was placed in a 37 ℃ incubator for 7 days to perform an accelerated stability destruction test, the VSVG gene copy number test was performed again, and the sample to be tested was STD1, the recovery rate was obtained, and the results are shown in table 3:
TABLE 3
| Examples | Recovery rate |
| 1 | 100.72％ |
| 2 | 101.50％ |
| 3 | 97.01％ |
| 4 | 61.11％ |
| 5 | 115.03％ |
Claims (10)
1. A VSVG gene copy number detection kit is characterized by comprising a VSVG quantitative reference substance, a VSVG Primer & Probe MIX, a Q-PCR Reaction Buffer and a DNA diluent.
2. The VSVG gene copy number detection kit of claim 1, wherein the concentration of the VSVG quantitation reference is 27-34ng/mL.
3. The VSVG gene copy number detection kit of claim 2, wherein the Q-PCR Reaction Buffer comprises a Q-PCR Reaction premix, a VSVG mutant, hALB, and DNase/RNase-Free deionized water.
4. The VSVG gene copy number detection kit of claim 3, wherein in the Q-PCR Reaction Buffer, the volume ratio of the Q-PCR Reaction premix, the VSVG mutant, the hALB and the DNase/RNase-Free deionized water is (9-14): (1.4-2): (0.7-1.3): (1.5-2).
5. A method of using the VSVG gene copy number detection kit according to any one of claims 1 to 4, characterized in that the method of use is as follows: (1) diluting; (2) sample adding; and (3) setting a program.
6. The method for using a VSVG gene copy number detection kit according to claim 5, wherein the step (1) is a step of diluting the VSVG quantitation reference substance with a DNA diluent.
7. The VSVG gene copy number detection kit of claim 5, wherein the DNA diluent is pre-treated prior to step (1).
8. The VSVG gene copy number detection kit of claim 7, wherein the sample application is performed by applying 9 to 14 μ L of qPCR MIX to each of reaction wells 1 to 7 of a well plate.
9. The VSVG gene copy number detection kit of claim 8, wherein the qPCR MIX is obtained by mixing a VSVG Primer & Probe MIX and a Q-PCR Reaction Buffer.
10. Use of the VSVG gene copy number detection kit according to any one of claims 1 to 4, wherein the VSVG gene copy number detection kit is applied to the cellular field and the genetic field.
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