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Kit for detecting human diploid cell dna and its application

A technology of human diploid cells and kits, applied in the field of PCR detection, can solve the problems of no kits for rapid detection of residual human DNA content, no detection methods, etc., and achieve good sensitivity, high sensitivity, and good specificity Effect

Active Publication Date: 2019-05-21
SINOVAC RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The U.S. FDA has approved some PCR diagnostic kits for the quantitative detection of pathogens, and my country has also approved hepatitis B, hepatitis C, enterovirus, HIV and SARS, etc. There is no corresponding detection method, and there is no kit for rapid detection of residual human DNA content by TaqManPCR technology, which can meet the needs of vaccine production and quality control, so as to reduce the production cost of biological products enterprises and improve the economic benefits of enterprises

Method used

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  • Kit for detecting human diploid cell dna and its application
  • Kit for detecting human diploid cell dna and its application
  • Kit for detecting human diploid cell dna and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The selection of embodiment 1 human diploid cell conserved sequence and the design of specific primers and probes

[0038] By comparing the human diploid cell gene sequences reported in the NCBI database, the present invention finds the Alu sequence by comparing the human diploid cell gene sequences reported in the NCBI database. Using the conserved domains analysis function in NCBI online BLAST, the genomic Alu sequence is a member of the SINE family in mammalian genomes, with about 500,000 copies. There is an Alu sequence in an average of 4-6kb. The human Alu sequence is a highly conserved sequence, the conserved target sequence of human cells was found, and the sequence was used as a genetic marker for detecting human diploid cell DNA. The genetic marker of the conserved region of the genome is highly homologous in various human embryonic lung diploid cells, and has the sequence shown in SEQ ID NO.4 or a specific fragment thereof.

[0039] The sequence of the speci...

Embodiment 2

[0043] Embodiment 2 detects the establishment of human diploid cell DNA reaction system

[0044] 1. Sample selection: According to the needs of each link of vaccine production, select samples that require quality control of human diploid cell DNA, such as vaccine stock solution, semi-finished products and finished products.

[0045] 2. Establishment and optimization of the reaction system:

[0046] Sample preparation: Human diploid cell SV-1 was used as positive reference; Chinese hamster ovary cells CHO, HEK293, VERO, rabies virus, enterovirus 71, Mycoplasma pneumoniae, and Staphylococcus aureus were used as negative reference. Extract the DNA of the above-mentioned positive reference product and negative reference product separately for use.

[0047] The optimization of the concentration of primer and probe: under the condition that other components in the reaction system remain unchanged, use concentration gradient from 0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μ...

Embodiment 3

[0061] Example 3 Detection of Human Diploid Cell DNA in Vaccine Finished Samples by Taqman Fluorescent Quantitative PCR

[0062] The vaccine samples come from vaccines based on human diploid cells, which are inactivated polio vaccine (vero), rabies vaccine (vero), rabies vaccine (human diploid cells), polio vaccine (human diploid cells), enterotype 71 virus (diploid cells), hepatitis A inactivated vaccine (diploid cells), sample processing and DNA extraction, PCR reaction and result analysis refer to the method in Example 2.

[0063] After the reaction, save the detection data file. Adjust the analysis parameters according to the curve graph obtained from the PCR amplification results, so that the standard curve under the standard curve (Std curve) window reaches the best, that is, the absolute value of the correlation value is >0.97, and then analyze the samples. The test results of 7 samples are shown in Table 1: 2 samples have no Ct value, which can be clearly negative; th...

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Abstract

The invention provides a kit for detecting human diploid cell DNA (deoxyribonucleic acid) and an application of the kit. According to human cell genomic sequence comparison, a highly conserved Alu sequence is determined and is highly homologous in human embryo lung diploid cells with different sources, a primer pair and a probe for detecting human diploid cells are designed by adopting a conserved region as a target sequence, and nucleotide sequences of the primer pair and the probe are represented as SEQ ID NO.1-3 respectively. The invention further provides a method for performing real-time fluorescent quantitative PCR (polymerase chain reaction) detection on the human diploid cell DNA and a detection kit. The detection kit has the advantages of accurate detection, high sensitivity, high specificity, simplicity, convenience and rapidness, is suitable for DNA detection of all currently known human embryo lung diploid cells, has low cost, can be used for clinical detection as well as quality control of a biological product production process and has good economic value and application prospect.

Description

technical field [0001] The invention relates to the technical field of PCR detection, in particular to a target sequence for detecting human diploid cell DNA in biological products, fluorescent quantitative PCR primers and TaqMan probes, and the invention also relates to using the target sequence to detect human diploid cell DNA Detection methods and kits. Background technique [0002] Human diploid cells are the safest cell matrix and have many advantages: Compared with primary cells, they do not contain any foreign contamination factors, can establish cell banks, and can be expanded and maintained from a cell seed culture frozen in an ampoule Good uniformity. The risk of tumorigenicity and carcinogenicity is very small compared to immortalized cells such as vero cells. In my country and most developed countries, human diploid cells are usually used as the most commonly used cell matrix for vaccine production. However, with the improvement of science and technology and t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6876C12Q1/686C12Q1/6851C12Q1/04
CPCC12Q1/6851C12Q1/6888C12Q2561/113C12Q2561/101C12Q2545/113C12Q2531/113
Inventor 金亚明吕哲王奚瑶王琳张星星高强尹卫东
Owner SINOVAC RES & DEV