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Method for detecting HBV DNA and single base mutation based on quantum dot resonance energy transfer

A technology of resonance energy transfer and single-base mutation, which is applied in microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms. Not high problems, to achieve the effect of high resolution, simple operation and strong signal

Active Publication Date: 2012-04-25
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods generally have some defects, such as complex operation process, low sensitivity, or lack of multi-target high-throughput detection capability, which is time-consuming

Method used

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  • Method for detecting HBV DNA and single base mutation based on quantum dot resonance energy transfer
  • Method for detecting HBV DNA and single base mutation based on quantum dot resonance energy transfer
  • Method for detecting HBV DNA and single base mutation based on quantum dot resonance energy transfer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Preparation of CdSe / ZnS core / shell quantum dots coated with mercaptopropionic acid

[0037] First, trioctyl was synthesized according to the method of Yang et al. Phosphorous oxide (TOPO) wrapped CdSe / ZnS core / shell quantum dots with a fluorescence emission peak of 605 nm, then take 0.5 mL of the toluene solution of TOPO-QDs synthesized above, add chloroform to it, and then add mercaptopropionic acid ( MPA) solution (dissolved in methanol, adjusted to pH ~ 10 with tetramethylammonium hydroxide in advance); the mixture was stirred at room temperature for 30 minutes, then 5 mL of water was added thereto, and after repeated shaking, the aqueous layer was removed from the organic After the layers were separated, acetone was added to precipitate the QDs in the aqueous layer. After centrifuging the mixed solution, the precipitated QDs were dissolved in Milli-Q ultrapure water to prepare water-soluble CdSe / ZnS coated with MPA. QDs: Ultrasonic the obtained QDs aqueous solu...

Embodiment 2

[0047] (1) Preparation of CdSe / ZnS core / shell quantum dots coated with mercaptopropionic acid

[0048] The method is as described in Example 1.

[0049] (2) Preparation of QDs-DNA probe crosslinks

[0050] The method is as described in Example 1.

[0051] (3) Design and synthesis of HBV DNA detection probes

[0052] The method is as described in Example 1.

[0053] (4) Detection of HBV DNA single base mutation based on quantum dot resonance energy transfer

[0054] Mix 70 μL of QDs-DNA probe, 10 μL of signal DNA (10 μM), 10 μL of rtM204I / V (YMDD) or single base mutated rtV204 (YVDD) target DNA (10 μM), 5 units of Taq DNA ligase and Taq 210 μL mixture of coupling buffer (115 μL, 20 mM) was reacted at 42° C. for 15-30 minutes. After the ligation reaction was completed, the reaction mixture was heated and denatured at 85° C. for 5 min on an Eppendorf PCR instrument to dissociate the double strands into single strands. Immediately after denaturation, the reaction mixture was...

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Abstract

The invention relates to a method for detecting HBV DNA and single base mutation based on quantum dot resonance energy transfer, which comprises the following steps: preparing an MPA-coated CdSe / ZnS nucleus / shell structural quantum dot; designing and synthesizing a HBV DNA detection probe; preparing a QDs-DNA probe cross-linking agent; and detecting high flux of complementary target HBV DNA and single base mutation. Since Cy5 can not directly generate fluorescence signals under 485nm lighter excitation, the method has simple operation, can be compatible with various instruments without separating unlinked DNA in a hybridization system, has little background interference and strong signal; and the invention, also has the characteristics of specificity, speediness, high resolution, high sensitivity and high flux, and can be applied to detection of the target HBV DNA and single base mutation in clinical medicine.

Description

technical field [0001] The invention belongs to the detection field of target DNA or single base mutation, in particular to a method for detecting HBV DNA and single base mutation based on quantum dot resonance energy transfer. Background technique [0002] Hepatitis B is a worldwide disease caused by hepatitis B virus (HBV). There are nearly 350 million HBV chronic carriers in the world, and 120 million HBV carriers in my country, and it is on the rise. HBV infection can cause a variety of diseases, such as: chronic hepatitis, cirrhosis and primary liver cancer. Therefore, HBV infection is a global health problem. In terms of the treatment of HBV, the most important and effective way to treat chronic hepatitis B (CHB) is to inhibit virus replication by anti-HBV drugs. Drugs such as lamivudine and adefovir have been widely used in the treatment of anti-HBV. However, after long-term use of drugs, the virus produces genetic mutations, which in turn cause mutations and drug ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/93C12Q1/70G01N21/64
Inventor 毛红菊王祥钟新华赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI