CN112176100A - General method for detecting copy number of target gene and virus titer and application - Google Patents
General method for detecting copy number of target gene and virus titer and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a universal method for detecting copy number of a target gene, a universal method for detecting titer of lentivirus, application of the universal method, a PCBP2 gene detection kit and a universal kit for detecting copy number of the target gene and titer of the virus. The calibration factor adopts PCBP2 gene, and the nucleotide sequence of the PCBP2 gene template is shown as SEQ ID NO1 or SEQ ID NO 2. The PCBP2 gene solves the problem that the traditional method can only detect human samples and has universality; and the traditional method for detecting the virus titer is poor in coincidence with the method for detecting the virus titer by the flow cytometry, and the universality of the virus titer detection in practical clinical application is not high, while the PCBP2 gene is adopted as a calibration gene, so that the result stability is good, the slow virus titer detection is more accurate, the detection result is more coincident with the detection result of the flow cytometry, and the clinical application is facilitated.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a universal method for detecting copy number of a target gene, a universal method for detecting titer of lentivirus, application of the universal method, a PCBP2 gene detection kit and a universal kit for detecting copy number of the target gene and titer of the virus.
Background
Lentivirus vectors have been widely used in the research of animal model gene therapy and the preparation of transgenic animals, and the accurate determination of the titer and infection efficiency of recombinant lentiviruses is a key step, which makes the detection of virus titer especially important because the transduction efficiency of the viral vectors mainly depends on the virus titer.
The common methods for detecting the virus titer or copy number are an ELISA method, a Q-PCR (Q-polymerase chain reaction), a flow cytometry method, an RNA-Dot-blot, a TCID50 assay method and a Real-time PCR method (DNA quantitative detection method), wherein the ELISA method, the Q-PCR and the RNA-Dot-blot belong to methods for detecting the virus physical titer, and only the quantity of virus particles or nucleic acid can be reflected and the infection capacity of infected cells can not be reflected; the flow cytometry method and the Real-time PCR method can detect the infection titer of the active virus, and are methods close to the Real infection titer. However, the flow detection workload is large, and the virus titer is influenced by the antibody affinity, the batch and the like, so that the repeatability and the error value of the detected virus titer are large, and meanwhile, for the condition that the virus titer is underestimated in a high-titer virus, multiple times of condition exploration is needed, and the method is only suitable for the condition that the expression gene can be detected by a flow method; the titer detection is carried out by a DNA quantitative method based on RT-PCR to detect the copy number of the vector DNA after the infected cells, the result is more stable and is closest to the real infection titer, the prior RT-PCR method based on the detection of the virus titer or the copy number of gene therapy has more detection researches on human cells or human samples, and has less researches on non-human animal samples such as mouse samples. In preclinical screening of gene therapy or modified cell therapy, animal experiments are needed to evaluate the in vivo maintenance and residual conditions of virus vectors such as gene vectors, CAR expression vectors or TCR-T expression vectors, and currently found internal references for detecting virus copy number do not exist in non-human genomes such as mice, so that the detection can be carried out only by using a method without the internal references, but the method without the internal references is poor in stability and accuracy.
Therefore, there is an urgent need for a method for using a universal gene highly conserved among various mammals such as human, mouse, hamster, etc. as a calibrator gene and detecting the titer of a target gene.
Disclosure of Invention
In view of this, the inventors found in previous studies that the gene PCBP2 (poly-cytosine binding protein 2), which encodes a protein that appears to be multifunctional, is one of the major cellular poly (rC) binding proteins; the protein coded by the gene and PCBP-1 are used as a translation coactivator of poliovirus RNA to promote the replication of the poliovirus RNA. It is also involved in the translational control of 15-lipoxygenase mRNA (human papillary cells), and current research is less focused, mainly on early diagnosis of neurological tumors and some solid tumors such as colorectal cancer, breast cancer. In the previous research of the inventor, PCBP2 is expressed in various mammals such as human, mouse, hamster and the like, is highly conserved, and can be used in a method for detecting virus titer and copy number by RT-PCR.
Therefore, an object of the present invention is to provide a universal method for detecting copy number of a target gene, which combines with RT-PCR detection technology to rapidly and universally detect copy number of the target gene in vivo and in vitro after gene modification, ensure that the sample loading amount of a sample to be detected during detection is corrected during detection, improve the detection versatility of the sample to be detected and the accuracy of the result, and can improve the versatility of DNA quantitative method for detecting copy number, and can be applied to animal experiments (mice, monkeys, etc.) to accurately detect copy number to provide stable data support for the development of clinical experiments.
In order to achieve the purpose, the invention adopts the following scheme:
the primer group and the probe are designed aiming at the PCBP2 Gene, and the 144 nucleotides of the PCBP2 Gene (Gene ID:5094) template are shown as SEQ ID: NO1 at the 12623-nd 12766 position or 104 nucleotides of the 22951-nd 23054 position are shown as SEQ ID: NO 2. The nucleotide sequence of the upstream primer of the primer group is designed to be shown as SEQ ID NO3 or SEQ ID NO6, the nucleotide sequence of the corresponding downstream primer is designed to be shown as SEQ ID NO4 or SEQ ID NO7, and the nucleotide sequence of the probe is designed to be shown as SEQ ID NO5 or SEQ ID NO 8.
In the prior art, no internal reference is provided, VCN detection can be performed only in the 293T, Hela cell line even after the CDKN1A internal reference is provided, detection limitations exist, and accurate experimental data cannot be obtained in animal experiments. The PCBP2 gene solves the problem that the traditional method can only detect human samples and has universality; and the method for detecting the virus titer by the traditional method is poor in coincidence with the method for detecting the virus titer by the flow cytometry, the universality of the virus titer detection in practical clinical application is not high, the PCBP2 gene is adopted as a calibration gene, the result stability is good, the slow virus titer detection is more accurate, the method is more coincident with the detection result of the flow cytometry, and the clinical application is facilitated.
Further, the method comprises the steps of:
1) designing upstream and downstream primers according to the PCBP2 gene; the nucleotide sequence of the PCBP2 gene template is shown as SEQ ID NO1 or SEQ ID NO 2;
2) extracting a genome template;
3) and detecting and calculating the copy number and amplification efficiency of the target gene by adopting any one of a PCR SYBR dye method and a PCR TaqMan probe method.
Further, the data were analyzed by analysis software to determine the copy number of the target gene, and the copy number was calculated from the standard curve, i.e., (copy number of the target gene/copy number of the calibration gene PCBP 2) × 2.
Furthermore, the nucleotide sequence of the upstream primer is shown as SEQ ID NO3 or SEQ ID NO6, and the nucleotide sequence corresponding to the downstream primer is shown as SEQ ID NO4 or SEQ ID NO 7.
Furthermore, the nucleotide sequence of the probe is shown as SEQ ID NO5 or SEQ ID NO 8.
Specifically, the nucleotide sequence of the PCBP2 Gene template is 144 nucleotides at positions 12623-12766 and 104 nucleotides at positions 22951-23054 of the PCBP2 Gene (Gene ID: 5094).
Specifically, the method for extracting the target genome template comprises the following steps: adding a proper amount of tissue lysate and proteinase K into the cell sample, and incubating the cell sample at 55 ℃ overnight for digestion and lysis; then, removing protein by using RNase and 5M NaCl solution, and transferring the supernatant into a new 2ml centrifuge tube; adding precooled absolute ethyl alcohol to precipitate DNA; after DNA precipitation, the supernatant was discarded, 70% ethanol was added to remove salt ions, and then TE buffer was used to resuspend and dissolve DNA, and the DNA was stored at 4 ℃ for a short period or at-20 ℃ for a long period.
The invention also aims to provide a universal method for detecting the titer of the lentivirus, which is real and accurate in detecting the titer of the non-human virus and is superior to a flow detection method.
In order to achieve the purpose, the invention adopts the following scheme:
the method comprises the following steps:
1) designing upstream and downstream primers according to the PCBP2 gene: the nucleotide sequence of the PCBP2 gene template is shown as SEQ ID NO1 or SEQ ID NO 2;
2) extracting a genome template of an infected virus cell;
3) determination of viral titer the data were analyzed with analytical software and the viral titer, expressed as TU/mL, was calculated from the standard curve (number of plated cells x copy number/cell)/amount of virus added.
Furthermore, the nucleotide sequence of the upstream primer is shown as SEQ ID NO6, and the nucleotide sequence corresponding to the downstream primer is shown as SEQ ID NO 7.
Furthermore, a probe can be designed according to the PCBP2 gene, and the nucleotide sequence of the probe is shown as SEQ ID NO 8.
The fourth purpose of the invention is to provide the universal method for detecting the copy number of the target gene, the universal method for detecting the titer of the lentivirus and the application of a primer group designed aiming at the PCBP2 gene, in particular to the application of improving the universality of detecting the copy number by a DNA quantitative method and detecting the titer of the virus.
In order to achieve the purpose, the invention adopts the following scheme:
the nucleotide sequence of the upstream primer of the primer group is shown as SEQ ID NO3 or SEQ ID NO6, and the nucleotide sequence of the corresponding downstream primer is shown as SEQ ID NO4 or SEQ ID NO 7.
Further, the test object includes a human and an animal.
Further, the detection of copy number includes in vivo copying and in vitro copying.
The fifth purpose of the invention is to provide three kits, and the kit can be used for conveniently and accurately detecting the gene copy number and the virus titer of the PCBP2 gene and/or the target gene in vitro and in vivo.
In order to achieve the purpose, the invention adopts the following scheme:
the PCBP2 gene detection kit comprises a primer group designed aiming at the PCBP2 gene, wherein the nucleotide sequence of an upstream primer of the primer group is shown as SEQ ID NO3 or SEQ ID NO6, and the nucleotide sequence of a corresponding downstream primer is shown as SEQ ID NO4 or SEQ ID NO 7.
A universal kit for detecting copy number of a target gene and virus titer comprises a primer group and a dye, wherein the primer group is designed aiming at PCBP2 gene, the nucleotide sequence of an upstream primer of the primer group is shown as SEQ ID NO3 or SEQ ID NO6, and the nucleotide sequence of a corresponding downstream primer is shown as SEQ ID NO4 or SEQ ID NO 7.
A universal kit for detecting copy number of a target gene and virus titer comprises a primer group and a probe, wherein the primer group and the probe are designed aiming at PCBP2 gene, the nucleotide sequence of an upstream primer of the primer group is shown as SEQ ID NO3 or SEQ ID NO6, the nucleotide sequence of a corresponding downstream primer is shown as SEQ ID NO4 or SEQ ID NO7, and the nucleotide sequence of the probe is shown as SEQ ID NO5 or SEQ ID NO 8.
The invention has the beneficial effects that:
1) the primer group and the probe for detecting the target gene can improve the universality of detecting copy number by a DNA quantitative method; the detection method can be applied to animal experiments (such as mice and monkeys) to accurately detect the copy number so as to provide stable data support for the development of clinical tests; the detection method has the advantages that the titer and the copy number obtained by the detection method are relatively close to the real infection efficiency of the virus, and the detection result is very stable, so that the titer and the copy number of the virus can be detected in a flux and universal manner;
2) the detection method can not only carry out Vector Copy Number (VCN) detection in a commonly used 293T, Hela cell line, but also carry out VCN detection in a CHO tool cell line;
3) the primer group and the probe ensure that the primer group and the probe can be applied to RT-PCR detection by a dye method or a probe method, ensure accurate amplification among different templates and ensure the accuracy of a detection result;
4) the detection method and the kit have the advantages of accurate detection and good stability and repeatability.
Drawings
FIG. 1 shows a standard curve for detection of PCBP2-75 primers in RT-PCR using dye method using plasmid, human genome and mouse genome as templates.
FIG. 2 shows a standard curve for detection of PCBP2-144 primers in RT-PCR using dye method using plasmid, human genome and mouse genome as templates.
FIG. 3 shows a standard curve for the detection of PCBP2-75 primers in RT-PCR by a probe method using plasmids, human genomes and mouse genomes as templates.
FIG. 4 shows a standard curve for the detection of PCBP2-144 primers in RT-PCR by a probe method using plasmids, human genomes and mouse genomes as templates.
FIG. 5 shows the detection of lentivirus titer of a sample derived from mouse cells using PCBP2 as an internal reference.
FIG. 6 shows the detection of lentivirus titers in human cell-derived samples using PCBP2 as the internal reference.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1 versatility of PCBP2 in copy number detection by PCR SYBR stain method
Two pairs of primers were designed for 144 nucleotides at positions 12623 and 12766 of the PCBP2 Gene (Gene ID:5094) as shown in SEQ ID: NO1 and 104 nucleotides at positions 22951 and 23054 as shown in SEQ ID: NO2, respectively, as shown in the following table.
Plasmid extraction
Adding a proper amount of tissue lysate and proteinase K into the cell sample, and incubating the cell sample at 55 ℃ overnight for digestion and lysis; then, removing protein by using RNase and 5M NaCl solution, and transferring the supernatant into a new 2ml centrifuge tube; adding precooled absolute ethyl alcohol to precipitate DNA; after DNA precipitation, the supernatant was discarded, 70% ethanol was added to remove salt ions, and then TE buffer was used to resuspend and dissolve DNA, and the DNA was stored at 4 ℃ for a short period or at-20 ℃ for a long period.
Genome extraction
Adding a proper amount of tissue lysate and proteinase K into a human or mouse cell sample, and incubating the cell sample at 55 ℃ overnight for digestion and lysis; then, removing protein by using RNase and 5M NaCl solution, and transferring the supernatant into a new 2ml centrifuge tube; adding precooled absolute ethyl alcohol to precipitate DNA; after DNA precipitation, the supernatant was discarded, 70% ethanol was added to remove salt ions, and then TE buffer was used to resuspend and dissolve DNA, and the DNA was stored at 4 ℃ for a short period or at-20 ℃ for a long period.
1) Fluorescent quantitative PCR amplification by PCR SYBR dye method
Diluting the primers to 100 x with sterilized purified water, and diluting the upstream and downstream primers to prepare 10 x premixed solution for use; secondly, the plasmid is diluted by 10 times or 5 times of gradient with sterilized pure water, and recombinant plasmids with different concentration titers are used as positive standard templates of the fluorescence quantitative PCR reaction. The PCR system was (10 ul): template 4.5ul, upstream and downstream primer mix (10X) 0.4ul, CXR 0.1ul,
qPCR Master Mix 5 ul. The reaction conditions are that pre-denaturation is carried out for 5min at 95 ℃, then annealing and extension are carried out for 1min at 95 ℃ for 15s and 60 ℃ for 40 cycles in total, and a cycle is carried out in a dissolution curve program at 95 ℃ for 15s, 60 ℃ for 15s and 95 ℃ for 15s, and a fluorescence quantitative PCR amplification instrument is used for carrying out experiments.
The experimental results are shown in fig. 1-2 and the following table, fig. 1 shows the fluorescence quantitative primer amplification efficiency of PCBP2-75 by SYBR dye method using plasmid (a), human genome (B) and mouse genome (C) as templates, respectively; FIG. 2 shows the efficiency of primer amplification by the SYBR dye method using PCBs 2-144 as templates for fluorescence quantification using plasmids (A), human genomes (B), and mouse genomes (C), respectively.
The result shows that when two groups of designed primers are adopted and plasmid, human genome and mouse genome are used as templates for amplification, the amplification efficiency of the primers reaches over 88 percent and even 99.594 percent, which indicates that the amplification efficiency of the primers is good, and the primers can be used for fluorescence quantitative PCR of samples from the plasmid, the human genome and the mouse genome by using a dye method and have universality.
Example 2 versatility of PCBP2 in copy number detection by the singleplex PCR TaqMan probe method
1) A primer and a probe were designed for 144 nucleotides at positions 12623 and 12766 of the PCBP2 Gene (Gene ID:5094) as shown in SEQ ID: NO1 and 104 nucleotides at positions 22951 and 23054 as shown in SEQ ID: NO2, the primer used was the primer of example 2, and the nucleotide sequence of the probe was shown in SEQ ID: NO 7.
2) Plasmid extraction
Adding a proper amount of tissue lysate and proteinase K into the cell sample, and incubating the cell sample at 55 ℃ overnight for digestion and lysis; then, removing protein by using RNase and 5M NaCl solution, and transferring the supernatant into a new 2ml centrifuge tube; adding precooled absolute ethyl alcohol to precipitate DNA; after DNA precipitation, the supernatant was discarded, 70% ethanol was added to remove salt ions, and then TE buffer was used to resuspend and dissolve DNA, and the DNA was stored at 4 ℃ for a short period or at-20 ℃ for a long period.
3) Genome extraction
Adding a proper amount of tissue lysate and proteinase K into a human or mouse cell sample, and incubating the cell sample at 55 ℃ overnight for digestion and lysis; then, removing protein by using RNase and 5M NaCl solution, and transferring the supernatant into a new 2ml centrifuge tube; adding precooled absolute ethyl alcohol to precipitate DNA; after DNA precipitation, the supernatant was discarded, 70% ethanol was added to remove salt ions, and then TE buffer was used to resuspend and dissolve DNA, and the DNA was stored at 4 ℃ for a short period or at-20 ℃ for a long period.
4) PCR TaqMan probe method for fluorescent quantitative PCR amplification
The primer dry powder is diluted to 100 x by sterilization and purification, when in use, the upstream primer and the downstream primer are diluted to 10 x premixed solution for use, then the probe is diluted to 10 x working solution for use by sterilization and purification, the plasmid is diluted by 10 times or 5 times of gradient by sterilization and pure water, and the recombinant plasmid with different concentration titers is used as a positive standard template of the fluorescence quantitative PCR reaction. The PCR system was (20 ul): template 4.5ul, upstream and downstream primer Mix (10X) 0.8ul, probe (10X) 0.5ul, TaqMan Fast Advanced Master Mix 10ul, Nuclear-free water 2.9 ul. The reaction conditions are that the denaturation is carried out for 10min at 50 ℃ and pre-denaturation is carried out for 10min at 95 ℃, then the denaturation is carried out for 15s at 95 ℃, the annealing and the extension are carried out for 1min at 60 ℃, and the total number is 40. The reaction was performed on a fluorescent quantitative PCR amplification instrument.
The experimental results are shown in fig. 3, fig. 4 and the following table, fig. 3 shows the amplification efficiency of PCBP2-75 primer and probe set when the primer and probe set are amplified by using a plasmid as a template; FIG. 4 shows the amplification efficiency of the PCBP2-144 primer and probe set when the plasmid (A), the human genome (B) and the mouse genome (C) are used as templates for amplification respectively; the result shows that when the primer and the probe are adopted and plasmid, human genome and mouse genome are taken as templates for amplification, the amplification efficiency of the primer reaches 96.234-106.082%, the amplification of the templates from different sources is stable, and the primer and the probe can be applied to copy number detection.
Example 3 Lentiviral titer assay
1) The prepared virus was infected into CHO cells (mouse ovarian cell line) and genomic extraction was carried out 72h after infection to obtain 10 different samples of transduced virus, numbered: 174. 205, 206, 207, 209, 215, 216, 80, 87 and 89, using the multiplex TaqMan probe method, the virus titer was determined by analyzing the data with analytical software, and the virus titer was calculated according to the standard curve (number of cells plated x number of copies/cell)/amount of virus added, and the result was expressed in TU/mL.
2) The traditional internal reference-free method, the CDKN1A and the detection method using the PCBP2 as the internal reference are respectively adopted to compare the differences of different methods of 17 virus samples transduced into a CHO cell line, and finally, the PCBP2 is used as the internal reference to detect the virus titer of the mouse-derived sample to be superior to the traditional internal reference-free method and the latest method using the CDKN1A internal reference.
The experimental results (table below) show that the method for detecting the virus titer by using the PCBP2 as the internal reference, which is developed by the invention, can detect the titer in a sample derived from a mouse cell line, the titer detected by the titer detection method is consistent with the method for detecting the real infection efficiency of the virus by using a flow cytometer, and the accuracy of the titer detection by using the method for using the PCBP2 as the internal reference is significantly higher than that of the conventional method for detecting the titer and that of the method for using the CDKN1A as the internal reference.
Example 4 copy number detection, accuracy, stability verification
1) The prepared viruses infected PBMC cells (human peripheral blood-derived monocytes, 6 samples of different batches in total), were subjected to genome extraction, numbered: 20180701-21, 20180701-25, 20180701-19, 20180702-21, 20180702-25 and 20180702-28, the virus copy number was measured by the TaqMan probe method, and the data was analyzed by an analysis software, and the virus copy number was calculated from a standard curve, wherein the copy number per cell is (target gene copy number/calibration gene PCBP2 copy number) × 2.
2) The TaqMan probe method is used for detection, and primers are designed as shown in the following table, wherein the primers and the probes of the PCBP2 adopt the primers and the probes of the example 2:
as shown in FIGS. 5-6 and the following table, the designed primer sequences have good amplification efficiency, the detected copy number is related to the positive expression rate of the target gene carried by the lentiviral vector on the surface of the transduced cell, and the detection result is very stable. The copy number of the target gene detected by the method is between 1.4 and 2.0 when the target gene is expressed in 50 to 60 percent, the detected copy number is relatively stable, the numerical value of the copy number is only related to the positive rate of gene expression, and the large deviation of the copy number caused by batch and detection time can be avoided.
| Plasmid numbering | Copy number/cell | Positive rate (%) |
| 20180701-21 | 1.6 | 62.2 |
| 20180701-25 | 1.4 | 50 |
| 20180701-19 | 1.6 | 65.7 |
| 20180702-21 | 1.9 | 56.5 |
| 20180702-25 | 1.4 | 67.3 |
| 20180702-28 | 1.8 | 60.5 |
Therefore, PCBP2 is selected as a correction gene, the gene PCBP2 is highly conserved in sequences of the part among more than 20 species such as human, mouse, hamster and the like, a primer and a probe are designed in a preserved area, and the primer and the probe are verified on the basis of TaqMan qPCR (quantitative polymerase chain reaction), so that the result shows that PCBP2 can show good amplification efficiency in different templates such as human, mouse, hamster and plasmid, and further the correction of the detection sample loading quantity of different species by using one universal correction gene is realized, and the titer detection work on different tool cells 293T or CHO is satisfied; and good amplification efficiency is still kept for Luc and PCBP2 in a multiple PCR system, titer and copy number obtained by using PCBP2 as an internal reference are relatively close to the real infection efficiency of the virus, and a detection result is very stable, so that flux and universal virus titer detection is realized.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (13)
1. A universal method for detecting the copy number of a target gene is characterized in that a PCBP2 gene is adopted as a calibration factor, and the nucleotide sequence of a PCBP2 gene template is shown in SEQ ID NO1 or SEQ ID NO 2.
2. The method as claimed in claim 1, wherein the primer set used is designed for PCBP2 gene, the nucleotide sequence of the upstream primer of the primer set is shown as SEQ ID NO3 or SEQ ID NO6, and the nucleotide sequence of the corresponding downstream primer is shown as SEQ ID NO4 or SEQ ID NO 7.
3. The method of claim 2, wherein a probe with a nucleotide sequence shown in SEQ ID NO5 or SEQ ID NO8 is designed against the PCBP2 gene.
4. Method according to claim 1, characterized in that it comprises the following steps:
1) designing an upstream primer and a downstream primer according to a calibration factor PCBP2 gene; the nucleotide sequence of the PCBP2 gene template is shown in SEQ ID NO1 or SEQ ID NO 2;
2) extracting a genome template;
3) and detecting and calculating the copy number and amplification efficiency of the target gene by adopting any one of a PCR SYBR dye method and a PCR TaqMan probe method.
5. A universal method for detecting lentivirus titer is characterized in that the used calibration factor adopts PCBP2 gene, and the nucleotide sequence of the PCBP2 gene template is shown as SEQ ID NO1 or SEQ ID NO 2.
6. The method of claim 5, characterized in that it comprises the steps of:
1) designing upstream and downstream primers according to the PCBP2 gene: the nucleotide sequence of the upstream primer is shown as SEQ ID NO6, and the nucleotide sequence corresponding to the downstream primer is shown as SEQ ID NO 7;
2) extracting a genome template;
3) determination of viral titer the data were analyzed with analytical software and the viral titer, expressed as TU/mL, was calculated from the standard curve (number of plated cells x copy number/cell)/amount of virus added.
7. The method of claim 6, wherein the probe is further designed based on the PCBP2 gene, and the nucleotide sequence of the probe is shown in SEQ ID NO 8.
8. The method of claim 1 and claim 5, and the use of the primer set designed for PCBP2 gene for improving the versatility of DNA quantitation method for detecting copy number and detecting virus titer, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO3 or SEQ ID NO6, and the nucleotide sequence of the corresponding downstream primer is shown in SEQ ID NO4 or SEQ ID NO 7.
9. The use of claim 8, wherein the test objects include humans and animals.
10. The use of claim 8, wherein said detection of copy number comprises in vivo copying and in vitro copying.
The PCBP2 gene detection kit is characterized by comprising a primer group designed aiming at the PCBP2 gene, wherein the nucleotide sequence of an upstream primer of the primer group is shown as SEQ ID NO3 or SEQ ID NO6, and the nucleotide sequence of a corresponding downstream primer is shown as SEQ ID NO4 or SEQ ID NO 7.
12. A universal kit for detecting copy number of a target gene and virus titer is characterized by comprising a primer group and a dye, wherein the primer group is designed for PCBP2 gene, the nucleotide sequence of an upstream primer of the primer group is shown as SEQ ID NO3 or SEQ ID NO6, and the nucleotide sequence of a corresponding downstream primer is shown as SEQ ID NO4 or SEQ ID NO 7.
13. A universal kit for detecting copy number of a target gene and virus titer is characterized by comprising a primer group and a probe, wherein the primer group and the probe are designed aiming at PCBP2 gene, the nucleotide sequence of an upstream primer of the primer group is shown as SEQ ID NO3 or SEQ ID NO6, the nucleotide sequence of a corresponding downstream primer is shown as SEQ ID NO4 or SEQ ID NO7, and the nucleotide sequence of the probe is shown as SEQ ID NO5 or SEQ ID NO 8.
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