Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as standard substance and detection kit for real-time fluorescent quantitative PCR detection

A technology of real-time fluorescence quantification and detection kit, which is applied in the field of biomedicine, can solve the problems of difficult control of the concentration ratio between the target gene and the reference gene, the large error range of the standard concentration, and the high difficulty of absolute quantification of the standard, so as to reduce the cost of detection , easy operation and high repeatability

Inactive Publication Date: 2017-02-15
上海康派尼恩医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The inventors of the present invention aimed at the relative quantitative detection method of the gene copy number in the existing method, because the absolute quantification of the standard itself is difficult and the concentration error range of the standard is relatively large, which makes it difficult to control the concentration ratio relationship between the target gene and the reference gene, resulting in Errors in experimental results, complex detection methods, and high time-consuming and costly problems provide a simple and efficient real-time fluorescent quantitative PCR detection method and its standard products and detection kits

Method used

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  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as standard substance and detection kit for real-time fluorescent quantitative PCR detection
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as standard substance and detection kit for real-time fluorescent quantitative PCR detection
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as standard substance and detection kit for real-time fluorescent quantitative PCR detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Human Cell Line Genomic DNA Extraction

[0036] The genomic DNA used for the construction of the plasmid standard product of the present invention comes from the K562 cell line cultured in vitro (purchased from ATCC).

[0037] Taking Qiagen's Allprep DNAmini KIT as an example to introduce the DNA extraction steps of cell lines:

[0038] (1) Count the number of cells, if the number of cells is 6 , add 350μL Buffer RLT Plus; if the number of cells is ≤1×10 7 , add 600μL Buffer RLT Plus (if the volume is too large, extract in several times).

[0039] (2) After mixing, transfer to the Allprep DNA adsorption column (placed in a 2mL collection tube), and centrifuge at the highest speed for 30 seconds.

[0040] (3) Replace with a new 2mL collection tube, add 500μL Buffer AW1, centrifuge at the highest speed for 15 seconds, and discard the liquid in the collection tube.

[0041] (4) Add 500 μL Buffer AW2 and centrifuge at the highest speed for 2 minutes.

[0042] ...

Embodiment 2

[0043] Example 2 Human Paraffin Embedded Tissue Genomic DNA Extraction

[0044] The samples tested in the present invention are paraffin-embedded human breast cancer tissues (from Changhai Hospital Affiliated to Second Military Medical University).

[0045] Taking Qiagen's GeneRead DNA FFPE KIT as an example to introduce the sample DNA extraction steps:

[0046] (1) Scrape the tissue on the paraffin section into a 1.5mL centrifuge tube.

[0047] (2) Add 160 μL of deparaffinization solution, vortex for 10 seconds, centrifuge briefly, and incubate at 56°C for 3 minutes.

[0048] (3) Add freshly prepared 100 μL incubation buffer / proteinase K solution (prepared from 55 μL RNase-free water, 25 μL FTB buffer, and 20 μL proteinase K) after restoring equilibrium at room temperature. After vortex centrifugation, incubate at 56°C for 1 hour, and then directly transfer to 90°C for further incubation for 1 hour.

[0049] (4) After simple centrifugation, take the clear bottom liquid, pu...

Embodiment 3

[0058] Embodiment 3 prepares special standard

[0059] 1. Preparation of the carrier

[0060] The TA cloning vector pMD18-T was purchased from Promega Company.

[0061] 2. Preparation for inserting fragments

[0062] 2.1. Preparation of target gene and reference gene amplification region

[0063] The PCR method was used to prepare gene fragments of the target gene and the reference gene. The PCR template was the cellular genomic DNA extracted in Example 1, and the reaction system was shown in Table 1 (PCR reaction system 1).

[0064] Table 1. Reaction system for preparing target gene fragments by PCR amplification (50 μL / tube, PCR reaction system 1)

[0065]

[0066] The HER2 gene fragment can be prepared by adding the outer forward and reverse primer sequences of HER2 to the PCR amplification system; the RPPH1 gene fragment can be prepared by adding the outer forward and reverse primer sequences of RPPH1 to the amplification system, and the sequence is shown in Table 2....

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Abstract

The invention discloses a real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as a standard substance and a detection kit for real-time fluorescent quantitative PCR detection. The special standard substance is used for drawing standard curves, real-time fluorescent quantitative PCR detection is performed on an HER2 gene of a to-be-detected sample and a reference gene, and the detection method is used for clinical targeted therapy of breast cancer and stomach cancer. The invention further provides a plasmid vector containing a reference gene segment and a to-be-detected target gene segment in an equal proportion and taken as the detection standard substance. The invention also provides the real-time fluorescent quantitative PCR kit for detecting the amplification state of the HER2 gene. The kit comprises a PCR reaction system for detecting the amplification state of the HER2 gene, the standard substance and negative and positive controls. The detection method and the detection kit have the advantages of being convenient to use, high in detection efficiency, cost-saving and the like. Great significance in diagnosis of HER2 positive breast cancer as well as research and development, quality control and clinical application of breast cancer resistant drugs is realized.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a real-time fluorescence quantitative PCR detection method, a standard product and a detection kit. Background technique [0002] Copy number variation (Copy Number Variation, CNV) is an important part of gene structure variation, caused by genome rearrangement, generally refers to the increase or decrease of the copy number of large genome fragments with a length of more than 1kb, mainly manifested as submicroscopic Horizontal deletions and duplications. CNV is closely related to tumorigenesis. Disklin et al. found that CNV at 1q21.1 site is closely related to neuroblastoma; and CNV of tumor-related genes can have a great impact on tumorigenesis and metastasis, such as CNV on GSTM1 gene It will lead to an increased risk of bladder cancer; CNV on the tumor suppressor gene WWOX will lead to a decrease in the activity of the tumor suppressor gene, making lung cance...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114C12Q2531/113C12Q2545/101C12Q2545/113
Inventor 唐向荣陈建鹤吴思敏唐志君姜为为杨水兵赵氏璧娄竞
Owner 上海康派尼恩医疗科技有限公司
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