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Primer for specifically detecting human genome DNA and application thereof

A genome and human-derived technology, applied in the direction of recombinant DNA technology, DNA/RNA fragments, microbial measurement/testing, etc., can solve the problems of insufficient sequence sensitivity, inconsistent results, inaccurate results, etc.

Pending Publication Date: 2019-09-10
SHANGHAI AISAER BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But sequences designed from these genes yielded inaccurate results due to insufficient sensitivity
In order to increase the sensitivity of detection, primers targeting highly repetitive sequences in the human genome (such as α-satellite, Alu) were used in qPCR experiments (10), but due to the highly active mobility of these sequences in the human genome, the same amount of Template DNA may contain highly variable amounts of target sequence, thus leading to inconsistent results

Method used

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  • Primer for specifically detecting human genome DNA and application thereof
  • Primer for specifically detecting human genome DNA and application thereof
  • Primer for specifically detecting human genome DNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0079] The sensitivity of embodiment 2SRGAP2 qPCR detection method

[0080] The primers and probes for the specific fragments of human SRGAP2 and FOX2A were designed and synthesized, and the primers and probes for SRGAP2 and FOX2A were as described in Example 1.

[0081] Genomic DNA extracted from human mesenchymal stem cells (MSC) was used as a standard, starting with 100ng of human mesenchymal stem cell genomic DNA, using New Zealand rabbit liver tissue genomic DNA as a diluent, and 5-fold gradient dilution to prepare 7 concentration samples (STD -1 to STD-7, wherein the content of human-derived genomic DNA is 100000pg, 20000pg, 4000pg, 800pg, 160pg, 32pg, 6.4pg per 5μl sample), and the primers and probes of SRGAP2 and FOX2A were used to pair these 7 Concentration samples were subjected to qPCR experiments to compare the specificity and sensitivity of the two primers. The result is as figure 2 Shown, where *p<0.05; **p<0.01. The results showed that the specificity of t...

Embodiment 3

[0082] Example 3 Formulate the standard curve of SRGAP2 qPCR detection method

[0083] Using New Zealand rabbit liver genomic DNA as the diluent, the concentration of human mesenchymal stem cell genomic DNA was serially diluted, and qPCR detection was performed with the SRGAP2 primer probe as described in Example 1 until the lowest limit of stable detection was 32.00pg / 5μl is set as the lower limit of detection (or lower limit of quantification); the highest limit that can be stably detected is 90000.00pg / 5μl, which is set as the upper limit of detection (or upper limit of quantitation); concentrations higher than the upper limit of detection can also be detected , but false positive results may occur.

[0084] With the upper detection limit and lower detection limit as the limit, human mesenchymal stem cell genomic DNA and New Zealand rabbit liver genomic DNA were mixed to prepare samples of standard concentration, and qPCR detection was performed using the SRGAP2 primer p...

Embodiment 4

[0094] Example 4 SRGAP2 qPCR method precision, accuracy detection

[0095] The lower limit of quantification (LLOQ, Lower limit of quantification), low concentration quality control (LQC, Low Quality control), high concentration quality control (HQC, High Quality control), Samples with 5 concentrations including middle concentration quality control (MQC, Middle Quality control) and upper limit of quantification (ULOQ, Upper limit of quantification) were subjected to qPCR with the SRGAP2 primer probe as described in Example 1. The concentration of each sample was as described for the corresponding sample in Example 3. Amplification curve such as Figure 4 shown.

[0096] The intra-assay and inter-assay precision and accuracy of samples with different concentrations were investigated. Accept the Guidelines for the Validation of Quantitative Analytical Methods for Biological Samples in the Standard Reference Pharmacopoeia (14-16), as follows:

[0097]

[0098] Remark:

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Abstract

The invention discloses a method for detecting human DNA by specifically detecting the human SRGAP2 gene, a primer and probe for specifically detecting the human SRGAP2 gene, and a TaqMan qPCR methodcapable of being used for detecting the human DNA in animal organs, tissue, cells, body fluid and blood. The method is suitable for detecting the human DNA in any animal sample and includes but not limited to biochores, metabolism and residues of cell and gene treatment products in the body of an animal receiving treatment.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and more specifically relates to the detection of human DNA. Background technique [0002] Cell therapy is the transplantation of healthy stem cells into patients or themselves to achieve the purpose of repairing lesions or rebuilding cells and tissues with normal functions. Studies have found that transplantation of human-derived cells into immunodeficient mice has been widely used to study the function of stem cells in tissue repair and regeneration or cancer cell metastasis (1), while transplantation of established cell therapy products (human-derived cells) After entering the host body, its biodistribution, migration or residue over time, therapeutic effect and biosafety in the host are important steps in pre-clinical testing (2). [0003] For cell therapy products, a series of experiments (such as quantitative experiments) are required to evaluate the effect of cell transplantatio...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2563/107C12Q2561/101C12Q2545/114
Inventor 高歌周安宇
Owner SHANGHAI AISAER BIOTECH CO LTD
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