Quantitative determination method of vaccinia virus lister strain based on ddPCR

A technology for quantitative determination of vaccinia virus, applied in the field of PCR detection, can solve problems such as the inability to know the proportion of vaccinia virus lister strains, insufficient sensitivity of ordinary PCR detection, and influence on experimental results, etc., to achieve the benefits of process development, low detection limit, The effect of high sensitivity

Pending Publication Date: 2021-12-31
TOT BIOPHARM CO LTD
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Problems solved by technology

Among them, ordinary PCR detection is not sensitive enough to be quantitative, and can only qualitatively detect the content of vaccinia virus lister strains in the modified vaccinia virus, and cannot know the proportion of vaccinia virus lister strains in the modified vaccinia virus; although qPCR can quantitatively detect, but the detection The lower limit is high, and it is not enough for accurate quantification when the content of vaccinia virus lister strain is very small, which affects the experimental results. At the same time, the accuracy of qPCR detection results depends on the quantification of standards, the accuracy of standard concentrations, and the operation of diluted standards. The correctness of all affects the accuracy of qPCR results

Method used

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  • Quantitative determination method of vaccinia virus lister strain based on ddPCR
  • Quantitative determination method of vaccinia virus lister strain based on ddPCR
  • Quantitative determination method of vaccinia virus lister strain based on ddPCR

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: A kind of method (ddPCR) of vaccinia virus lister strain in quantitative determination sample

[0031] This embodiment provides a method for quantitatively determining the vaccinia virus lister strain in a sample, the sample is a modified vaccinia virus, and the modified vaccinia virus is obtained through the vaccinia virus lister strain (Genbank number of the complete genome sequence is: KX061501.1 ) in the original TK fragment (the nucleotide sequence is SEQ ID NO: 4), which is obtained by deleting the specific fragment (the nucleotide sequence is SEQ ID NO: 1) and inserting the target fragment. The specific steps are as follows:

[0032] Extraction steps: use the viral DNA extraction kit to extract the viral DNA in the modified vaccinia virus, use 100 μL of virus for extraction each time, and elution with 50 μL of elution buffer;

[0033] Detection steps: use the specific fragment as a probe, use the extracted viral DNA as a template, use ddPCR to quant...

experiment example 1

[0071] Experimental Example 1: Quantitative determination of vaccinia virus lister strain in modified vaccinia virus

[0072] This experimental example provides the quantitative determination experiment of the vaccinia virus lister strain in the modified vaccinia virus, and the experimental process is as follows:

[0073] The methods of Example 1 and Comparative Examples 1-5 were respectively used to transform the modified vaccinia virus (the modified vaccinia virus was synthesized by Dongyao Pharmaceutical Co., Ltd., the Genbank number of the complete genome sequence of the vaccinia virus lister strain is: KX061501.1, and the target fragments are in order The GM-CSF sequence and the HSP-70 sequence in series, wherein, the Genebank number of the GM-CSF sequence is: MA883124.1, and the Genebank number of the HSP-70 sequence is: AX811495.1) were determined for the vaccinia virus lister strain.

[0074] The experimental result of comparative example 1 (ordinary PCR) sees figure ...

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Abstract

The invention relates to a quantitative determination method of a vaccinia virus lister strain based on ddPCR, and belongs to the technical field of PCR detection. The invention provides a method for quantitatively determining the vaccinia virus lister strain in a sample. The method comprises the following steps of: firstly extracting virus DNA in the sample, then taking a specific fragment as a probe, taking the extracted virus DNA as a template, and performing quantitative analysis on the vaccinia virus lister strain in the sample by using the ddPCR. The method is based on the ddPCR, the ddPCR can perform absolute quantification on nucleic acid molecules and is high in sensitivity, and meanwhile, the ddPCR does not need a standard substance, is less in variables, higher in accuracy and lower in detection lower limit, and is more suitable for quantitative determination of the vaccinia virus lister strain with a low copy number, so that the method for quantitatively determining the vaccinia virus lister strain in the sample has the advantages of high sensitivity, high accuracy and low detection lower limit.

Description

technical field [0001] The invention relates to a method for quantitative determination of vaccinia virus lister strain based on ddPCR, belonging to the technical field of PCR detection. Background technique [0002] Vaccinia virus (vaccinia virus) belongs to poxvirus (Poxvirus), which is closely related to smallpox virus and vaccinia virus in serology and immunology. In the past, vaccinia virus has been used as a vaccine against smallpox. With the development of molecular biology, people have discovered its new value—a favorable carrier of genetic engineering. [0003] The transformation of vaccinia virus is to delete the specific fragment and insert the exogenous target fragment in the original TK fragment of the vaccinia virus lister strain (wild type). The TK gene of vaccinia virus encodes thymidine kinase, and the virulence of the modified vaccinia virus that deletes a specific segment in the TK gene and inserts an exogenous target segment is significantly lower than ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/159C12Q2545/114Y02A50/30
Inventor 程欣张妍孙菁韩佳迪单艺玮林凡佳
Owner TOT BIOPHARM CO LTD
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