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Method for quantitatively detecting recombinant lentivirus titers

A recombinant lentivirus, quantitative detection technology, applied in microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc., can solve problems such as inconvenience and cumbersome operation

Active Publication Date: 2017-11-21
SHENZHEN GENTARGET BIOTHERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although this method is feasible in the titer determination of recombinant lentiviruses without marker genes, after obtaining the copy number of the lentiviral genome, it still needs to rely on the recombinant lentiviruses with fluorescent markers as a control to accurately calculate the recombinant lentiviruses in the samples to be tested. The titer of the virus, the operation is relatively cumbersome and inconvenient
[0007] In summary, traditional detection methods cannot accurately measure the titer of recombinant lentiviruses without the help of fluorescent markers

Method used

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  • Method for quantitatively detecting recombinant lentivirus titers
  • Method for quantitatively detecting recombinant lentivirus titers
  • Method for quantitatively detecting recombinant lentivirus titers

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Experimental program
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preparation example Construction

[0075] The preparation method of the WPRE component standard product is simple, easy and economical, so that a large number of WPRE component standard products can be obtained.

[0076] Of course, in other embodiments, WPRE element standard products can also be obtained by direct base synthesis.

[0077] S124. Put the Ct value of the amplified hTERT gene obtained in S122 into the standard curve established by the hTERT gene standard to obtain the content of the hTERT gene in the genomic DNA.

[0078] The standard curve established by the hTERT gene standard product has a corresponding relationship between the Ct value and the hTERT gene content. According to the Ct value of the amplified hTERT gene, the content of the hTERT gene in the genomic DNA can be quantitatively calculated, that is, the content of the hTERT gene.

[0079] In one embodiment, the standard curve established by the hTERT gene standard product is obtained by the following operations: the hTERT gene standard ...

Embodiment 1

[0105] Preparation of WPRE element standard and hTERT gene standard

[0106] Referring to the sequence of the WPRE element template synthesized with the accession number NC_004107.1 in the GenBank database, the upstream primer of the WPRE element template shown in SEQ ID No.5 and the upstream primer of the WPRE element template shown in SEQ ID No.6 were added according to the system recommended by the PrimeSTAR HS (Premix) kit. Show the downstream primers of the WPRE element template, and carry out PCR amplification reaction. The reaction conditions are 98°C for 10s, 1 cycle; 98°C for 10s, 55°C for 10s, 72°C for 60s, 30 cycles; 72°C for 5min, 1 cycle.

[0107] Referring to the sequence of the hTERT gene template synthesized with the accession number NG_009265.1 in the GenBank database, the upstream primer of the WPRE element template shown in SEQ ID No.7 and the upstream primer of the WPRE element template shown in SEQ ID No.8 were added according to the system recommended by t...

Embodiment 2

[0111] Establish a standard curve for the WPRE element standard and a standard curve for the hTERT gene standard

[0112] Take 2 μL of the WPRE component standard with a concentration of 0.5ng / μL (the quality is 1ng), and perform a gradient dilution with a factor of 8, dilute 7 gradients (the minimum content is diluted 2097152 times), and obtain a total of 8 WPRE component standards . Using these 8-content WPRE element standard products as templates, respectively add the WPRE element upstream primer shown in SEQ ID NO: 1 and the WPRE element downstream primer shown in SEQ ID NO: 2 to perform real-time fluorescent quantitative PCR amplification reaction. Reaction conditions It is: 95°C for 30 seconds, enter the cycle; 95°C for 5 seconds, 60°C for 30 seconds, end-point plate reading, a total of 40 cycles. get as Figure 4 The amplification curve shown and as Figure 5 Melting curve shown. according to Figure 4 Amplification curves are shown to obtain Ct values ​​for each l...

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Abstract

The invention relates to a method for quantitatively detecting recombinant lentivirus titers. The method includes: infecting target cells with samples to be detected; extracting a genome DNA (deoxyribonucleic acid) in the infected target cells; then detecting the copy number of WPRE elements in the genome DNA and the copy number of hTERT genes; calculating the number of particles in recombinant lentivirus in the average target cells according to the copy number of the WPRE elements and the copy number of the tTERT genes; calculating the number of the particles of the recombinant lentivirus contained in the samples to be detected in unit volume according to the sum of the target cells and the number of the particles of the recombinant lentivirus in the average target cells to obtain the titers of the recombinant lentivirus in the samples to be detected. The method is simple in operation, and the recombinant lentivirus titers can be detected accurately under the condition of not using fluorescent markers.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for quantitatively detecting the titer of recombinant lentivirus. Background technique [0002] Recombinant lentivirus (Lentivirus) vector is a gene therapy vector developed on the basis of HIV-1 (Human Immunodeficiency Virus Type I), which has the ability to infect both dividing cells and non-dividing cells, and is currently the most widely used gene delivery tool One of them is widely used in the preparation of transgenic animals and gene therapy research. [0003] In the process of recombinant lentivirus preparation, virus titer is usually used to measure the success of recombinant lentivirus preparation and the quality of recombinant lentivirus, so titer determination is an important step. In the prior art, the main methods for determining the titer of recombinant lentiviruses include flow cytometry (FACS), enzyme-linked immunoassay (ELISA) method, gold standard ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q2561/113C12Q2531/113C12Q2563/107
Inventor 易吉辉熊霞辉刘恒李扬兮许春莲毛侃琅
Owner SHENZHEN GENTARGET BIOTHERAPEUTICS CO LTD
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