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Real-time fluorescence quantitative PCR detection method based on quantum dot fluorescence quenching

A real-time fluorescence quantification and fluorescence quenching technology, applied in the field of bioengineering, can solve the problems of high detection background, poor photostability, wide emission spectrum, etc., and achieve good sensitivity and specificity

Inactive Publication Date: 2009-06-03
上海赛安生物医药科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the shortcomings of SYBRGreen organic fluorescent dyes such as instability, high temperature degradation, easy decomposition when exposed to light, wide spectrum of emitted light, poor photostability, and high detection background, quantum dots just make up for their shortcomings and have significant advantages.

Method used

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  • Real-time fluorescence quantitative PCR detection method based on quantum dot fluorescence quenching
  • Real-time fluorescence quantitative PCR detection method based on quantum dot fluorescence quenching
  • Real-time fluorescence quantitative PCR detection method based on quantum dot fluorescence quenching

Examples

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Effect test

Embodiment 1

[0018] Dissolve cadmium chloride in an alkaline (pH10) aqueous solution containing thioglycolic acid, add ionic tellurium source NaHTe (prepared from tellurium powder and sodium borohydride), and react at 100°C for 30 to 240 minutes. The reaction time is different to obtain quantum dots. With different particle sizes, the color of the solution changes from green to orange and finally red. CdTe quantum dots with carboxyl groups on the surface are synthesized. CdTe quantum dots with different particle sizes in the range of 1.2nm to 2.8nm can be obtained by this method. Here, quantum dots with a particle size of about 2.8 nanometers are selected, and the concentration of the obtained quantum dot solution is 3.5 mg / mL. When excited at any wavelength less than 500 nanometers, fluorescence with an emission wavelength of 554 nm can be obtained.

[0019] Take breast cancer-associated antigen gene (brcaal gene) as template DNA 3 μL, dNTP 3 μL, buffer 5 μL, primer A 1 μL, primer B 1 μL, ...

Embodiment 2

[0021]Dissolve cadmium chloride in an alkaline (pH10) aqueous solution containing thioglycolic acid, add ionic tellurium source NaHTe (prepared from tellurium powder and sodium borohydride), and react at 100°C for 30 to 240 minutes. The reaction time is different to obtain quantum dots. The particle size is different, the color of the solution is from green to orange and finally red, and CdTe quantum dots with carboxyl groups on the surface are synthesized. CdTe quantum dots with different particle sizes in the range of 1.6nm to 3nm can be obtained by this method. Here, quantum dots with a particle size of 3 nanometers are selected, excited at any wavelength less than 500 nanometers, and fluorescence with an emission wavelength of 560 nm can be obtained.

[0022] Take breast cancer-associated antigen gene (brcaa1 gene) as template DNA 3 μL, dNTP 3 μL, buffer 5 μL, primer A 1 μL, primer B 1 μL, magnesium ion 2 μL, Taq DNA polymerase 1 μL, quantum dots synthesized above 1 μL, and...

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Abstract

This invention relates to a real-time fluorescence quantitative PCR detection method based on quantum dot fluorescence quenching. As the super fluorescent character of quantum dot and the PCR dual-chain product can combine with quantum dot and quench the fluorescent signal of combined quantum dot by quenching level positive correlated with the product quantity, adding quantum dot with negative charge on surface into normal PCR amplification system with final quantum dot concentration within 0.007mg / ml~1.33mg / ml; putting the PCR reaction tube into PCR tester, detecting the PCR product at different time. This invention is fast, sensitive and well specificity.

Description

technical field [0001] The invention relates to a method in the technical field of bioengineering, in particular to a real-time fluorescence quantitative PCR detection method based on quantum dot fluorescence quenching. Background technique [0002] PCR is polymerase chain reaction technology. Fluorescent quantitative PCR technology uses fluorescent dyes or fluorescently labeled specific probes to mark and track PCR products, monitor the reaction process online in real time, and analyze the results with corresponding software. The initial template amount of the sample to be tested. Compared with common PCR, fluorescent quantitative real-time PCR has many advantages, so it has been widely used. So far, there are two main types of fluorescent quantitative PCR labeling methods: internal dyes such as SYBR Green I and SYBR Gold, sequence-specific probes such as Taqman probes, molecular beacon probes (Molecular Beacons) and fluorescence resonance energy transfer Probes [Dual Pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76G01N21/64C12Q1/68
Inventor 崔大祥高峰贺蓉潘碧峰
Owner 上海赛安生物医药科技股份有限公司