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CN-121992090-A - Detection method for detecting Sf9 host DNA residues by using fluorescent probe and application

CN121992090ACN 121992090 ACN121992090 ACN 121992090ACN-121992090-A

Abstract

The invention provides a detection method for detecting Sf9 host DNA residues by using a fluorescent probe and application thereof, belonging to the technical field of biological detection, wherein the method designs a specific primer pair (such as SEQ ID NO: 1/2) and a fluorescent probe (such as SEQ ID NO: 11) aiming at a high-copy conserved sequence of Sf9 host DNA, and is matched with a kit comprising a gene amplification solution, a standard substance and a quality control substance, extracts DNA by a liquid nitrogen grinding-column extraction method, and detects the DNA after real-time fluorescent quantitative PCR amplification. The method has the detection sensitivity of 0.003 pg/mu L and the linear range of 300 pg/mu L-0.003 pg/mu L, has no cross reaction on irrelevant DNA, has the repeatability CV percent of less than or equal to 10 percent, and the reagent is stable after freezing and thawing for 10 times, and is completed in 1.5 hours in the whole process, thereby solving the problems of the existing method such as specificity, insufficient sensitivity, complex operation and the like, and being applicable to the quality control of biological products such as gene therapy drugs, cell vaccines and the like.

Inventors

  • HUANG YI
  • XIE LIQI
  • YAO JIE
  • YAN MENGDI

Assignees

  • 上海探实生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260226

Claims (10)

  1. 1. The specific primer pair for detecting the Sf9 host DNA residues is characterized by comprising an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2; The upstream primer and the downstream primer are designed aiming at a high copy conserved sequence of Sf9 host DNA, and the absolute value of the difference of the Tm temperatures of the upstream primer and the downstream primer is less than or equal to 1 ℃.
  2. 2. A specific primer pair for detecting Sf9 host DNA residues, characterized in that the primer pair is any combination of: the upstream primer sequence of the combination 1 is shown as SEQ ID NO. 3, and the downstream primer sequence is shown as SEQ ID NO. 4; the upstream primer sequence of the combination 2 is shown as SEQ ID NO. 5, and the downstream primer sequence is shown as SEQ ID NO. 6; The upstream primer sequence of the combination 3 is shown as SEQ ID NO. 7, and the downstream primer sequence is shown as SEQ ID NO. 8; the upstream primer sequence is shown as SEQ ID NO. 9, and the downstream primer sequence is shown as SEQ ID NO. 10.
  3. 3. A fluorescent probe for detecting Sf9 host DNA residues is characterized in that the fluorescent probe is matched with the specific primer pair according to claim 1 or 2, the nucleotide sequence of the fluorescent probe is any one of SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15, a fluorescent reporter group is marked at the 5 'end of the probe, and a fluorescent quenching group is marked at the 3' end of the probe.
  4. 4. The fluorescent probe of claim 3, wherein the fluorescent reporter group is selected from FAM, NEX, ROX, TET, JOE, VIC, CY, CY5, and Texas Red, the fluorescent quenching group is selected from BHQ, TAMRA, eclipse, dabcyl, lowa BlackTM RQ, and Lowa BlackTM FQ, preferably the fluorescent reporter group is FAM, and the fluorescent quenching group is TAMRA.
  5. 5. A kit for detecting Sf9 host DNA residues, comprising the specific primer pair of claim 1 or 2, the fluorescent probe of claim 3 or 4, and further comprising a gene amplification solution, enzyme-free water (DEPC water), sf9 DNA standard, positive quality control, and negative quality control; the gene amplification solution is 2X TAQMAN QPCR Mix and contains Taq DNA polymerase, dNTPs and magnesium ions; The Sf9 DNA standard substance has the concentration of 30 ng/mu L, and the purity of the single non-impurity band is more than or equal to 95 percent through agarose gel electrophoresis verification, and the A260/A280 ratio is 1.823-1.826; the positive quality control product is a DNA solution containing a Sf9 host DNA specific conserved region sequence, and the conserved region sequence is shown as SEQ ID NO. 16; the negative quality control product is enzyme-free water (DEPC water).
  6. 6. The kit according to claim 5, wherein the components of the kit are added to a 30. Mu.L reaction system in an amount of 2X TAQMAN QPCR Mix 15.0. Mu.L, upstream primer 0.6. Mu.L, downstream primer 0.6. Mu.L, fluorescent probe 0.3. Mu. L, DEPC water 3.5. Mu.L, and template 10.0. Mu.L; the final concentration of the upstream primer is 0.2 mu M, the final concentration of the downstream primer is 0.2 mu M, and the final concentration of the fluorescent probe is 0.1 mu M; when the primer set of claim 1 is used in combination with the probe set forth in SEQ ID NO. 11, it is an optimal detection combination.
  7. 7. A method for detecting Sf9 host DNA residues, the method comprising the steps of: S1, sample pretreatment, namely extracting DNA (deoxyribonucleic acid) of a biological product sample to be detected by adopting a liquid nitrogen grinding and column extraction method, wherein the specific steps are 4000g centrifugation for 5min, collecting thalli, grinding liquid nitrogen into powder, adding Buffer GSL and RNase A, cracking in a 70 ℃ water bath for 10min, adding Buffer PSS for purification, isopropanol precipitation, buffer GW1/GW2 washing and Buffer TB elution to obtain a DNA solution to be detected; The Sf9 DNA standard substance is diluted into 300 pg/mu L, 30 pg/mu L, 3 pg/mu L, 0.3 pg/mu L, 0.03 pg/mu L and 0.003 pg/mu L standard line solution by DEPC water gradient, and negative quality control substances are arranged at the same time; S2, preparing a reaction system, namely preparing 30 mu L of the reaction system according to the proportion of claim 6 by adopting the kit of claim 5 or 6; S3, PCR amplification, namely placing the reaction system in a real-time fluorescence quantitative PCR instrument, and executing the following procedures of pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15S, annealing at 60 ℃ for 1min (reading fluorescent signals), wherein the total time is 40 cycles; S4, result analysis, namely drawing a standard curve according to the amplification result of the standard substance series solution, carrying out linear regression analysis by adopting a least square method by taking the logarithm of the standard substance concentration as the abscissa and the corresponding Ct value as the ordinate, wherein the correlation coefficient R 2 of the standard curve is more than or equal to 0.999, the amplification efficiency is 90% -110%, and calculating the residual concentration of Sf9 host DNA in the sample to be detected according to the Ct value of the sample to be detected and combining the standard curve.
  8. 8. The method for detecting Sf9 host DNA residues according to claim 7, wherein the detection sensitivity of the method is 0.003 pg/μl (fg level), the linear range is 0.003 pg/μl-300 pg/μl; The Ct value of detection on the independent DNA of CHO cells, HEK293 cells, E.coli, pichia pastoris and the like is more than 35 or is not detected, and no cross reaction exists; CV% of three parallel detection of the same sample is less than or equal to 10%, CV% of detection results after 10 times of repeated freezing and thawing of the nucleic acid amplification solution is less than or equal to 13.9%, and the method has good repeatability and durability.
  9. 9. The method for detecting Sf9 host DNA residues according to claim 7, comprising the step of quality control: S11, the negative quality control product needs to meet the requirements that the Ct value of the FAM channel is 0 or is not detected, and no obvious S-shaped amplification curve exists; S12, positive quality control products are required to meet the requirements that the FAM channel has a Ct value which is less than or equal to 40 and has an obvious S-shaped amplification curve, the amplification efficiency is 90-110%, and the correlation coefficient R 2 is more than or equal to 0.999; S13, if the negative quality control product and the positive quality control product do not meet the requirements, or the standard curve amplification efficiency exceeds the range of 90% -110%, and R 2 is less than 0.999, judging that the experiment is invalid and detecting again; S14, judging the result of the sample to be detected, wherein the Ct value is less than or equal to 40, and the sample has an obvious S-shaped amplification curve, and judging that the Sf9 host DNA residue is positive; ct value >40 or undetected, and is judged as negative, ct value 40< 44 or less, repeated detection is recommended, and the result after repeated is still 40< Ct value 44 or less is judged as positive, and the result after no amplification is judged as negative.
  10. 10. Use of a specific primer pair according to claim 1 or 2, a fluorescent probe according to claim 3 or 4, a kit according to any one of claims 5-6 for quality control of a biotherapeutic agent comprising a gene therapeutic agent, a cellular vaccine, a recombinant protein agent, said agent comprising Sf9 cells as expression system or production material.

Description

Detection method for detecting Sf9 host DNA residues by using fluorescent probe and application Technical Field The invention relates to the technical field of biological detection, in particular to a detection method for detecting Sf9 host DNA residues by using a fluorescent probe and application thereof. Background In the development and production of biotherapeutic drugs such as gene therapy and cell vaccines, sf9 cell lines are widely used due to the high-efficiency recombinant protein expression capability and the advantage of viral vector preparation. However, residual Sf9 host cell DNA in biologicals may carry oncogenes or elicit immune responses, presenting a potential biosafety risk, and thus strict detection of its residual amount is a central element in biologicals quality control. At present, the detection method of host cell DNA residues mainly comprises a molecular hybridization method, a common PCR method and a real-time fluorescent quantitative PCR method. The conventional real-time fluorescent quantitative PCR method has the problems of weak specificity of a primer probe, insufficient detection sensitivity, poor durability and the like when detecting Sf9 host DNA, cannot effectively distinguish the interference of irrelevant DNA, and cannot easily meet the strict requirement of biological therapeutic drugs on the detection method; Therefore, a detection method for detecting Sf9 host DNA residues by using a fluorescent probe and application thereof are provided. Disclosure of Invention In view of the above, the present invention provides a detection method and application for detecting Sf9 host DNA residues using fluorescent probes, so as to solve or alleviate the technical problems existing in the prior art, and at least provide a beneficial choice. The technical scheme is that the specific primer pair for detecting the Sf9 host DNA residues comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, the upstream primer and the downstream primer are designed aiming at a high-copy conserved sequence of the Sf9 host DNA, and the absolute value of a Tm temperature difference value of the upstream primer and the downstream primer is less than or equal to 1 ℃. Further, any combination of the following: The upstream primer sequence of the combination 1 is shown as SEQ ID NO. 3, and the downstream primer sequence is shown as SEQ ID NO. 4; the upstream primer sequence of the combination 2 is shown as SEQ ID NO. 5, and the downstream primer sequence is shown as SEQ ID NO. 6; the upstream primer sequence of the combination 3 is shown as SEQ ID NO. 7, and the downstream primer sequence is shown as SEQ ID NO. 8; The upstream primer sequence is shown as SEQ ID NO. 9, and the downstream primer sequence is shown as SEQ ID NO. 10. The fluorescent probe for detecting the Sf9 host DNA residues is matched with the specific primer pair, the nucleotide sequence of the fluorescent probe is any one of SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15, a fluorescent report group is marked at the 5 'end of the probe, and a fluorescent quenching group is marked at the 3' end of the probe. Further, the fluorescence reporting group is selected from FAM, NEX, ROX, TET, JOE, VIC, CY, CY5 or Texas Red, the fluorescence quenching group is selected from BHQ, TAMRA, eclipse, dabcyl, lowa BlackTM RQ or Lowa BlackTM FQ, preferably, the fluorescence reporting group is FAM, and the fluorescence quenching group is TAMRA. A kit for detecting Sf9 host DNA residues, which comprises any one of the specific primer pairs and any one of the fluorescent probes, and further comprises a gene amplification solution, enzyme-free water (DEPC water), an Sf9 DNA standard substance, a positive quality control substance and a negative quality control substance; the gene amplification solution is 2X TAQMAN QPCR Mix and contains Taq DNA polymerase, dNTPs and magnesium ions; The Sf9DNA standard substance has the concentration of 30 ng/mu L, and the purity of the single non-impurity band is more than or equal to 95 percent through agarose gel electrophoresis verification, and the A260/A280 ratio is 1.823-1.826; The positive quality control product is a DNA solution containing a Sf9 host DNA specific conserved region sequence, and the conserved region sequence is shown as SEQ ID NO. 16; the negative quality control product is enzyme-free water (DEPC water). Further, the addition amount of each component in the kit in a 30 mu L reaction system is 2X TAQMAN QPCR Mix 15.0 mu L, 0.6 mu L of an upstream primer, 0.6 mu L of a downstream primer, 3.5 mu L of fluorescent probe 0.3 mu L, DEPC water and 10.0 mu L of a template, the final concentration of the upstream primer is 0.2 mu M, the final concentration of the downstream primer is 0.2 mu M, the final concentration of the fluoresce