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CN-122012808-A - Primer and probe for detecting BaEV genes in cells, and detection method and application thereof

CN122012808ACN 122012808 ACN122012808 ACN 122012808ACN-122012808-A

Abstract

The invention belongs to the technical field of molecular biology, and mainly provides a primer and a probe for detecting BaEV genes in cells, and a detection method and application thereof. Specifically disclosed is a primer composition for amplifying Beav genes, which comprises a Beav-F2 primer with a nucleotide sequence of SEQ ID NO. 1 and a Beav-R2 primer with a nucleotide sequence of SEQ ID NO. 2. The kit can sensitively and accurately detect the gene residues of BaEV in HSC, NK, CAR-NK and resting T cells, well evaluate the potential risk of RCL at the cell level of BaEV-LVs, and can be used for industrial production.

Inventors

  • ZHANG YUHAN
  • LI HONGJIAN
  • ZHANG DAN
  • ZHAO YUAN

Assignees

  • 江苏谱新生物医药有限公司

Dates

Publication Date
20260512
Application Date
20251223

Claims (10)

  1. 1. A primer composition for amplifying Beav genes, wherein the composition comprises a Beav-F2 primer and a Beav-R2 primer, respectively; the nucleotide sequence of the Beav-F2 primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the Beav-R2 primer is shown as SEQ ID NO. 2.
  2. 2. A composition for detecting Beav genes in a test sample, which comprises the primer composition as claimed in claim 1 and a probe BaEV-P2, wherein the nucleotide sequence of the probe BaEV-P2 is shown as SEQ ID NO. 3.
  3. 3. The composition of claim 2, wherein the 5 'end of the probe BaEV-P2 is connected with a fluorescent group or a quenching group, the 3' end of the probe BaEV-P2 is connected with a fluorescent group or a quenching group, and preferably the 5 'end of the probe BaEV-P2 is connected with a fluorescent group, and the 3' end of the probe BaEV-P2 is connected with a quenching group.
  4. 4. The composition of claim 3, wherein the fluorophore is selected from at least one of FAM, VIC, HEX, TRT, CY, CY5, ROX, JOE, FITC, TET, NED, alexa Fluor series, BODIPY series, LCRED640, LCRED705, quasar705, or TexasRed, preferably the fluorophore is selected from FAM; and/or the quenching group is selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB and Dabcy1, preferably, the quenching group is selected from TAMRA; And/or, the 1 st nucleotide A of the SEQ ID NO. 3 is connected with a fluorescent group, and the 22 nd nucleotide A of the SEQ ID NO. 3 is connected with a quenching group; And/or the sample to be tested is selected from a nucleic acid extract or a cell culture substance, preferably the nucleic acid extract is prepared from an animal cell, more preferably the animal cell comprises a somatic cell, a stem cell and/or an immune cell, even more preferably the somatic cell comprises a fibroblast, a keratinocyte, a liver cell, an islet beta cell, a chondrocyte, a cardiomyocyte, a retinal pigment epithelial cell, a dopaminergic neuron, even more preferably the immune cell comprises an alpha beta T cell, a gamma delta T cell, a regulatory T cell, a tumor-infiltrating lymphocyte, a double negative T cell, a virus-specific T cell, a dendritic cell, a macrophage, a cytokine-induced killer cell, a lymphokine-activated killer cell, a B lymphocyte, an NK cell, a constant NKT cell, a CAR-NK cell, a CAR-T cell, a TCR-T cell, a CAR-Macrophage, CAR-Treg cell, an engineered DC cell and/or a resting T cell, even more preferably the stem cell comprises an embryonic stem cell, an induced pluripotent stem cell, a mesenchymal stem cell, a dental mesenchymal stem cell, an amniotic membrane, an endothelial stem cell, a mesenchymal stem cell, an endothelial cell, a mesenchymal stem cell, a mesenchymal cell, a stem cell or a mesenchymal cell, the metabolites include primary metabolites and/or animal cell secondary metabolites; And/or the mass ratio of Beav-F2 primer to Beav-R2 primer is 1:0.2-5, preferably, the mass ratio is 1:1; And/or the mass ratio of the Beav-F2 primer to the Beav-R2 primer to the probe BaEV-P2 is 1:0.2-5:0.1-2.5, preferably the mass ratio is 2:2:1.
  5. 5. A kit for detecting the copy number of Beav genes in a test sample, said kit comprising a composition according to any one of claims 1-4.
  6. 6. The kit of claim 5, wherein the kit further comprises a DNA polymerase, preferably the DNA polymerase comprises any one or more of Taq, bst, vent, phi, pfu, tru, tth, tl1, tac, tne, tma, tih, tf1, pwo, kod, sac, sso, poc, pab, mth, pho, ES4, klenow; And/or the kit further comprises a PCR reaction liquid, wherein the PCR reaction liquid preferably comprises dNTPs, mg2+ solution and/or nuclease-free water; And/or the kit further comprises Beav gene standard and/or reference gene, preferably, the concentration of Beav gene in Beav gene standard is 2E+06 to 20 copies/. Mu.L, preferably, the concentration is 2E+06 copies/. Mu.L, 2E+05 copies/. Mu.L, 2E+04 copies/. Mu.L, 2E+03 copies/. Mu.L, 2E+02 copies/. Mu.L and 20 copies/. Mu.L; And/or the kit further comprises a DNA extracting solution, preferably, the DNA extracting solution comprises SDS (sodium dodecyl sulfate), naCl (sodium chloride), tris-HCl, EDTA ethylenediamine tetraacetic acid) and/or DTT (dithiothreitol).
  7. 7. A method for detecting BaEV gene residues in a test sample, comprising the step of amplifying the test sample using the composition of any one of claims 1-4 or the kit of claim 5 or 6.
  8. 8. The method of claim 7, wherein the method comprises the steps of: (1) Obtaining a sample to be tested; (2) Preparing a reaction system and performing qPCR; (3) A step of evaluating the residual condition of the BaEV gene in the test sample based on the fluorescence signal of the test sample to be evaluated: a. If two or more thirds of the tested samples are not amplified signals or the average value of the copy number of the BaEV genes in the tested samples is less than 10 copies/reaction, judging that the detection result of the BaEV genes is negative; b. if the copy number of the BaEV genes in two thirds of the tested samples to be evaluated in the tested samples is not less than 10 copies/reaction, judging that the BaEV gene detection result is positive; Preferably, the number of samples to be evaluated is 3n, n being 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, the number of samples to be evaluated is the same as the number of samples to be evaluated.
  9. 9. The method according to claim 7 or 8, further comprising the step c. If the BaEV gene test result is positive, performing step a and/or b evaluation on parallel samples of the test sample, taking the evaluation result as the final result; And/or, the amplification comprises PCR, preferably, the PCR comprises conventional PCR, fluorescent quantitative PCR, reverse transcription PCR, nested PCR, multiplex PCR, digital PCR, more preferably, the PCR comprises fluorescent quantitative PCR, and the PCR amplification procedure comprises the steps of pre-denaturation at 95-98 ℃ for 35min, denaturation at 95-98 ℃ for 15-30 s, circulation for 40-50 times, annealing and extension at 60-65 ℃ for 60-65 s, circulation for 40-50 times; And/or the test sample is selected from a nucleic acid extract or a cell culture material; preferably, the nucleic acid extract is prepared from animal cells; more preferably, the animal cells include somatic cells, stem cells, and/or immune cells; even more preferably, the somatic cells include fibroblasts, keratinocytes, hepatocytes, islet beta cells, chondrocytes, cardiomyocytes, retinal pigment epithelial cells, dopaminergic neurons, even more preferably, the immune cells include alpha beta T cells, gamma delta T cells, regulatory T cells, tumor-infiltrating lymphocytes, double negative T cells, virus-specific T cells, dendritic cells, macrophages, cytokine-induced killer cells, lymphokine-activated killer cells, B lymphocytes, NK cells, constant NKT cells, CAR-NK cells, CAR-T cells, TCR-T cells, CAR-Macrophage, CAR-Treg cells, engineered DC cells, and/or resting T cells, even more preferably, the stem cells include embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, dental pulp stem cells, placental mesenchymal stem cells, amniotic membrane/membrane chorionic mesenchymal stem cells, menses-derived mesenchymal stem cells, hematopoietic stem cells, endothelial stem cells, endothelial cells, and/or tissue-derived cells, and more preferably, stem cells including extracts of the human tissue, and/or cell precursors of the animal cells, more preferably, the tissue cells, and/or more preferably, the tissue cell or the tissue cell culture of the tissue of the human tumor cells, the cell culture material also comprises animal cell culture medium, metabolites of animal cells, and more preferably, the metabolites comprise primary metabolites and/or secondary metabolites of animal cells.
  10. 10. Use of the composition of claims 1-4, the kit of claim 5 or 6 for the preparation of a product for detecting Beav genes, quality control or cell therapy in a test sample.

Description

Primer and probe for detecting BaEV genes in cells, and detection method and application thereof Technical Field The invention belongs to the technical field of molecular biology, and particularly relates to a primer and a probe for detecting BaEV genes in cells, and a detection method and application thereof. Background In recent years, cell therapy technology has developed faster because of its safety and wide applicability, and in practical application, there is still a certain potential safety hazard in cell therapy products using lentiviruses as vectors, and the main reason is that it is still possible to generate viruses with specific replication ability as lentiviruses, i.e. to generate replication-competent lentiviruses (replication competent lentivirus, RCL). In view of the potential risk of RCL, CDE, FDA, european union and NMPA have issued related documents, requiring control of RCL at different stages of production and application, including cell banks, terminal cells, viral vectors and transduced cells for the production of viruses. There are two common choices on the envelope protein of the lentiviral vector, the first envelope protein is vesicular stomatitis virus envelope protein pseudotyped lentivirus (VSV-G-LVs) which is mainly used for T cell transformation, and the second envelope protein is baboon endogenous retrovirus (BaEV-LVs) which is mainly used for genetic modification of difficult-to-transduce cells such as Hematopoietic Stem Cells (HSC), natural killer cells (NK), resting T cells and the like. There are many methods for reference to the potential hazard research of RCL of VSV-G-LVs at the cell level. Patent number CN118621064a provides a method for detecting RCL in a sample by using a conventional indicator cell culture method in combination with an RTQPCR method. However, there is little research on methods for detecting RCL at the level of BaEV-LVs cells. Therefore, how to provide a detection method for RCL on the BaEV-LVs cell level is a technical problem facing the person skilled in the art. Disclosure of Invention In view of the above-described drawbacks of the prior art, an object of the present invention is to provide a specific BaEV gene target, in particular, a primer and a probe for the BaEV gene target, which can sensitively and accurately detect the gene residues of BaEV in HSC, NK, CAR-NK resting T cells, and well evaluate the potential risk of RCL at the BaEV-LVs cell level. In a first aspect the present application provides a primer composition for amplifying Beav genes, said composition comprising a Beav-F2 primer and a Beav-R2 primer, respectively; the nucleotide sequence of the Beav-F2 primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the Beav-R2 primer is shown as SEQ ID NO. 2. In another aspect, the application provides a composition for detecting Beav genes in a test sample, which comprises the primer composition according to claim 1 and a probe BaEV-P2, wherein the nucleotide sequence of the probe BaEV-P2 is shown as SEQ ID NO. 3. In another aspect the application provides a method of detecting BaEV gene residue in a test sample, the method comprising amplifying the test sample using a composition as described in any one of the above or a kit as described in any one of the above. In another aspect, the application provides the use of the composition, the kit, or the kit for detecting Beav genes, quality control, or cell therapy products in a test sample. The application has the beneficial effects that: according to the application, through a specific BaEV gene target, the BaEV gene residue in HSC, NK, CAR-NK and resting T cells can be sensitively and accurately detected, and the potential risk of RCL at the BaEV-LVs cell level can be well estimated. The amplification efficiency of BaEV gene residue detection is 85% -110%, R2 is more than or equal to 0.990, and the detection result is stable and reliable. The method has the advantages of complete methodological verification, strong specificity, high accuracy and good repeatability, can be used for quality research, release detection, stability research, comparability research and the like of cell therapy products, and has wide market application prospect. Drawings FIG. 1 shows an amplification curve of Beav gene primer probes. FIG. 2 shows a standard curve for Beav gene assays. Detailed Description Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure of the present invention, which is to be read in light of the specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. Before further describing embodiments of the present invention, it is to be understood that the scope of the invention is not limited to the specific embodiments desc