CN106119411A - A kind of detection method of CAR T cell virus efficiency of infection - Google Patents

A kind of detection method of CAR T cell virus efficiency of infection Download PDF

Info

Publication number
CN106119411A
CN106119411A CN201610482950.1A CN201610482950A CN106119411A CN 106119411 A CN106119411 A CN 106119411A CN 201610482950 A CN201610482950 A CN 201610482950A CN 106119411 A CN106119411 A CN 106119411A
Authority
CN
China
Prior art keywords
cell
infection
virus
car
efficiency
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610482950.1A
Other languages
Chinese (zh)
Inventor
凌发忠
徐华栋
许国贞
刘振云
熊伍平
王维
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sinobioway Cell Therapy Co Ltd
Original Assignee
Sinobioway Cell Therapy Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sinobioway Cell Therapy Co Ltd filed Critical Sinobioway Cell Therapy Co Ltd
Priority to CN201610482950.1A priority Critical patent/CN106119411A/en
Publication of CN106119411A publication Critical patent/CN106119411A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses the detection method of a kind of CAR T cell virus efficiency of infection, comprise the steps: virus infection CAR T cell；Treat that cytotostatic is expressed, the complete genome DNA of extracting CAR T cell；Fluorescent quantitative PCR technique is used to detect the viral copy number being incorporated in complete genome DNA；Data process: by the Poisson regularity of distribution, calculate the efficiency of infection of this virus；Wherein efficiency of infection formula: P (K)=1 P (0)；Wherein, P (0)=e‑m；E is natural constant；M is MOI value, i.e. infection multiplicity.The present invention can calculate the efficiency of infection of virus, and computational methods are easy, and degree of accuracy is high, it is simple to practical operation.

Description

A kind of detection method of CAR-T cell virus efficiency of infection
Technical field
The present invention relates to CAR-T cell virus efficiency of infection technical field, particularly relate to a kind of CAR-T cell virus and infect The detection method of efficiency.
Background technology
The cell therapy of tumor starts from the eighties in last century.It is nonspecific carefully that first generation cell therapy technology belongs to employing Born of the same parents treat, and main method includes LAK, NK, CIK etc..Second filial generation cell therapy technology is risen in the nineties in last century, main representative For DC-CIK, this treatment technology belongs to inducing tumor-specific cell therapy.The first generation and second filial generation cell therapy technology by In reasons such as offer limited effectiveness, the most almost no longer carry out in major countries such as USA and Europes.Third generation cell therapy technology starts from this century Just, belong to gene-recombinated cell treatment, mainly have CAR-T, CAR-NK etc..Wherein, up-to-date CAR-T technology not only solves T The targeting of cell, anti-personnel problem, also solve a difficult problem for its internal Effective multiplication.
In the cultivation flow process of CAR-T cell, the therapeutic effect of cell is had very important by the infection ability of virus Effect, the most effectively detects CAR-T cell virus efficiency of infection, becomes technical problem the most urgently to be resolved hurrily.
Summary of the invention
The present invention proposes the detection method of a kind of CAR-T cell virus efficiency of infection, can calculate the efficiency of infection of virus, Computational methods are easy, and degree of accuracy is high, it is simple to practical operation.
The detection method of a kind of CAR-T cell virus efficiency of infection that the present invention proposes, comprises the steps:
S1, by virus infection CAR-T cell；
S2, treat that cytotostatic is expressed, the complete genome DNA of extracting CAR-T cell；
S3, utilization fluorescent quantitative PCR technique detect the viral copy number being incorporated in complete genome DNA；
S4, data process:
By the Poisson regularity of distribution, calculate the efficiency of infection of this virus；
Wherein efficiency of infection formula: P (K)=1-P (0)；
Wherein, P (0)=e-m；
P (0): the percentage rate of the most infected cell；
P (K): the percentage rate of infected cell；
E: natural constant；
M is MOI value, i.e. infection multiplicity.
Preferably, the computing formula of m is m=viral copy number: cell number；
Wherein viral copy number unit is copies/ μ g, and cell number unit is individual/μ g, 2.5～3.5 × 105Individual cell contains Having 1ugDNA, in 1 μ g DNA, the copy number of virus is known by fluorescent quantitative PCR technique detection.
Preferably, 3 × 105Individual cell contains 1ugDNA.
In S2, the most stably expressed by regular-PCR identification of cell.
In the present invention, detected the viral copy number being incorporated in cell genomic dna, root by fluorescent quantitative PCR technique The infection multiplicity of this virus is calculated, owing to virus infected cell is a kind of Random event according to the ratio meter of viral copy number Yu cell number Part, it then follows the Poisson regularity of distribution, thus can calculate the efficiency of infection of virus.Computational methods are easy, and degree of accuracy is high, it is simple to Practical operation.
Detailed description of the invention
Below, by specific embodiment, technical scheme is described in detail.
Embodiment 1
The detection method of a kind of CAR-T cell virus efficiency of infection, comprises the steps:
S1, by virus infection CAR-T cell；
S2, treat that cytotostatic is expressed, the complete genome DNA of extracting CAR-T cell；
S3, utilization fluorescent quantitative PCR technique detect the viral copy number being incorporated in complete genome DNA；At S4, data Reason:
By the Poisson regularity of distribution, calculate the efficiency of infection of this virus；
Wherein efficiency of infection formula: P (K)=1-P (0)；
Wherein, P (0)=e-m；
P (0): the percentage rate of the most infected cell；
P (K): the percentage rate of infected cell；
E is natural constant；
M is MOI value, i.e. infection multiplicity.
Embodiment 2
The detection method of a kind of CAR-T cell virus efficiency of infection, comprises the steps:
S1, by virus infection CAR-T cell；
S2, treat that cytotostatic is expressed, the complete genome DNA of extracting CAR-T cell；
S3, utilization fluorescent quantitative PCR technique detect the viral copy number being incorporated in complete genome DNA；At S4, data Reason:
By the Poisson regularity of distribution, calculate the efficiency of infection of this virus；
Wherein efficiency of infection formula: P (K)=1-P (0)；
Wherein, P (0)=e-m；
P (0): the percentage rate of the most infected cell；
P (K): the percentage rate of infected cell；
E is natural constant；
M is MOI value, i.e. infection multiplicity.
The computing formula of m is m=viral copy number: cell number；
Wherein viral copy number unit is copies/ μ g, and cell number unit is individual/μ g, 3.5 × 105Individual cell contains 1ugDNA, in 1 μ g DNA, the copy number of virus is known by fluorescent quantitative PCR technique detection.
Embodiment 3
The detection method of a kind of CAR-T cell virus efficiency of infection, comprises the steps:
S1, by virus infection CAR-T cell；
S2, treat that cytotostatic is expressed, the complete genome DNA of extracting CAR-T cell；
S3, utilization fluorescent quantitative PCR technique detect the viral copy number being incorporated in complete genome DNA；
S4, data process:
By the Poisson regularity of distribution, calculate the efficiency of infection of this virus；
Wherein efficiency of infection formula: P (K)=1-P (0)；
Wherein, P (0)=e-m；
P (0): the percentage rate of the most infected cell；
P (K): the percentage rate of infected cell；
E is natural constant；
M is MOI value, i.e. infection multiplicity.
The computing formula of m is m=viral copy number: cell number；
Wherein viral copy number unit is copies/ μ g, and cell number unit is individual/μ g, 2.5 × 105Individual cell contains 1ugDNA, in 1 μ g DNA, the copy number of virus is known by fluorescent quantitative PCR technique detection.
Embodiment 4
The detection method of a kind of CAR-T cell virus efficiency of infection, comprises the steps:
S1, by virus infection CAR-T cell；
S2, treat that cytotostatic is expressed, the complete genome DNA of extracting CAR-T cell；
S3, utilization fluorescent quantitative PCR technique detect the viral copy number being incorporated in complete genome DNA；
S4, data process:
By the Poisson regularity of distribution, calculate the efficiency of infection of this virus；
Wherein efficiency of infection formula: P (K)=1-P (0)；
Wherein, P (0)=e-m；
P (0): the percentage rate of the most infected cell；
P (K): the percentage rate of infected cell；
E is natural constant；
M is MOI value, i.e. infection multiplicity.
The computing formula of m is m=viral copy number: cell number；
Wherein viral copy number unit is copies/ μ g, and cell number unit is individual/μ g, 3 × 105Individual cell contains 1ugDNA, in 1 μ g DNA, the copy number of virus is known by fluorescent quantitative PCR technique detection.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto, Any those familiar with the art in the technical scope that the invention discloses, according to technical scheme and Inventive concept equivalent or change in addition, all should contain within protection scope of the present invention.

Claims (3)

1. the detection method of a CAR-T cell virus efficiency of infection, it is characterised in that comprise the steps:
S1, by virus infection CAR-T cell；
S2, treat that cytotostatic is expressed, the complete genome DNA of extracting CAR-T cell；
S3, utilization fluorescent quantitative PCR technique detect the viral copy number being incorporated in complete genome DNA；
S4, data process:
By the Poisson regularity of distribution, calculate the efficiency of infection of this virus；
Wherein efficiency of infection formula: P (K)=1-P (0)；
Wherein, P (0)=e-m；
P (0): the percentage rate of the most infected cell；
P (K): the percentage rate of infected cell；
E is natural constant；
M is MOI value, i.e. infection multiplicity.
The detection method of CAR-T cell virus efficiency of infection the most according to claim 1, it is characterised in that the calculating of m is public Formula is m=viral copy number: cell number；
Wherein viral copy number unit is copies/ μ g, and the unit of cell number is individual/μ g, 2.5～3.5 × 105Individual cell contains 1ugDNA, in 1 μ g DNA, the copy number of virus is known by fluorescent quantitative PCR technique detection.
The detection method of CAR-T cell virus efficiency of infection the most according to claim 2, it is characterised in that 3 × 105Individual carefully Born of the same parents contain 1ugDNA.
CN201610482950.1A 2016-06-24 2016-06-24 A kind of detection method of CAR T cell virus efficiency of infection Pending CN106119411A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610482950.1A CN106119411A (en) 2016-06-24 2016-06-24 A kind of detection method of CAR T cell virus efficiency of infection

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610482950.1A CN106119411A (en) 2016-06-24 2016-06-24 A kind of detection method of CAR T cell virus efficiency of infection

Publications (1)

Publication Number Publication Date
CN106119411A true CN106119411A (en) 2016-11-16

Family

ID=57265876

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610482950.1A Pending CN106119411A (en) 2016-06-24 2016-06-24 A kind of detection method of CAR T cell virus efficiency of infection

Country Status (1)

Country Link
CN (1) CN106119411A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019129048A1 (en) * 2017-12-28 2019-07-04 上海细胞治疗研究院 Method and kit for determining car copy number by using dual fluorescence quantitative pcr
CN111394316A (en) * 2020-04-09 2020-07-10 济南宜明医疗科技有限公司 Preparation of standard substance for detecting CAR-T gene copy number
CN113151593A (en) * 2020-12-31 2021-07-23 肇庆大华农生物药品有限公司 Method for determining content of virus difficult to observe whether tissue cells are infected or not

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104404002A (en) * 2014-12-12 2015-03-11 江苏省农业科学院 Composition for improving infection efficiency of porcine circovirus type 2, and application of composition

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104404002A (en) * 2014-12-12 2015-03-11 江苏省农业科学院 Composition for improving infection efficiency of porcine circovirus type 2, and application of composition

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
李影等: "用荧光定量PCR方法检测转染细胞中外源基因的拷贝数", 《中山医科大学学报》 *
肖薇等: "荧光定量PCR法用于转染CHO细胞中人凝血因子Ⅶ基因拷贝数检测的研究", 《中国输血杂志》 *
马海燕等: "应用荧光实时定量PCR方法检测重组慢病毒滴度及其感染效率", 《生命科学研究》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019129048A1 (en) * 2017-12-28 2019-07-04 上海细胞治疗研究院 Method and kit for determining car copy number by using dual fluorescence quantitative pcr
CN111394316A (en) * 2020-04-09 2020-07-10 济南宜明医疗科技有限公司 Preparation of standard substance for detecting CAR-T gene copy number
CN113151593A (en) * 2020-12-31 2021-07-23 肇庆大华农生物药品有限公司 Method for determining content of virus difficult to observe whether tissue cells are infected or not

Similar Documents

Publication Publication Date Title
Jafarinejad-Farsangi et al. High affinity of host human microRNAs to SARS-CoV-2 genome: An in silico analysis
Yang et al. Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice
Ni et al. Epidemiological and etiological characteristics of hand, foot, and mouth disease in Ningbo, China, 2008–2011
Huang et al. Establishment of an H6N2 influenza virus lineage in domestic ducks in southern China
Grey et al. Identification and characterization of human cytomegalovirus-encoded microRNAs
Song et al. Cellular microRNAs inhibit replication of the H1N1 influenza A virus in infected cells
Dimitrov et al. Newcastle disease viruses causing recent outbreaks worldwide show unexpectedly high genetic similarity to historical virulent isolates from the 1940s
Sakamoto et al. Next‐generation sequencing of miRNAs in clinical samples of Epstein–Barr virus‐associated B‐cell lymphomas
Terrier et al. Host microRNA molecular signatures associated with human H1N1 and H3N2 influenza A viruses reveal an unanticipated antiviral activity for miR-146a
Du et al. Patterns of accumulation of miRNAs encoded by herpes simplex virus during productive infection, latency, and on reactivation
Li et al. Postexposure treatment with the live-attenuated rabies virus (RV) vaccine TriGAS triggers the clearance of wild-type RV from the Central Nervous System (CNS) through the rapid induction of genes relevant to adaptive immunity in CNS tissues
Lu et al. Naturally occurring reassortant infectious bursal disease virus in northern China
Kang et al. Phylogenetic relationships and pathogenicity variation of two Newcastle disease viruses isolated from domestic ducks in Southern China
Kolb et al. Genomic, phylogenetic, and recombinational characterization of herpes simplex virus 2 strains
CN106119411A (en) A kind of detection method of CAR T cell virus efficiency of infection
Monne et al. Reassortant avian influenza virus (H5N1) in poultry, Nigeria, 2007
Tombácz et al. Strain Kaplan of pseudorabies virus genome sequenced by PacBio single-molecule real-time sequencing technology
Li et al. Role of MicroRNAs in host defense against infectious bursal disease virus (IBDV) infection: a hidden front line
Chang et al. Enterovirus 71 antagonizes the antiviral activity of host STAT3 and IL-6R with partial dependence on virus-induced miR-124
Mukherjee et al. Infection with influenza A viruses causes changes in promoter DNA methylation of inflammatory genes
Hemida et al. Exploiting the therapeutic potential of microRNAs in viral diseases: expectations and limitations
Valli et al. Evolutionary pattern of pandemic influenza (H1N1) 2009 virus in the late phases of the 2009 pandemic.
Wu et al. MicroRNA profile analysis of Epithelioma papulosum cyprini cell line before and after SVCV infection
Bian et al. Prediction of signaling pathways involved in enterovirus 71 infection by algorithm analysis based on miRNA profiles and their target genes
Chen et al. Phylodynamic analyses of class I Newcastle disease virus isolated in China

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20161116