Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Series G-quadruplex-heme DNA enzyme label-free signal amplification method based on rolling-circle amplification

A technology of rolling circle amplification and signal amplification, applied in the field of molecular biology, which can solve the problems of requiring special markers, relying on large instruments, and high cost

Active Publication Date: 2017-02-01
HUNAN INSTITUTE OF ENGINEERING
View PDF3 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with traditional detection methods, the currently used rapid detection technology for Salmonella shows many advantages, but at the same time, it also has certain defects, such as complicated operation, high cost, dependence on large-scale instruments, and the need for special labels, etc.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Series G-quadruplex-heme DNA enzyme label-free signal amplification method based on rolling-circle amplification
  • Series G-quadruplex-heme DNA enzyme label-free signal amplification method based on rolling-circle amplification
  • Series G-quadruplex-heme DNA enzyme label-free signal amplification method based on rolling-circle amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Feasibility Analysis:

[0088] Preparation of pretreatment microspheres: take 100 μL streptavidin functionalized microspheres, wash twice with 100 μL affinity eluent, centrifuge at 3500 rpm for 5 min, remove the supernatant, add 49.4 μL affinity eluent and 0.6 μL 100 μmol / L of biotinylated capture probe was shaken at 37°C for 1 hour, centrifuged and washed twice with affinity eluent to remove unbound probes, and finally 100 μL of affinity eluent was added and incubated at 4°C Save the pretreated beads.

[0089] Preparation of circularized DNA: Take a sterilized dry centrifuge tube, measure 74.5 μL of deionized water with a pipette gun, add 10 μL of 10× ligase reaction buffer solution, 5 μL of 5’ phosphorylated 1 μmol / L of circular DNA, 10 μL of synthetic Salmonella DNA sequence (10 pmol / L), 0.5 μL of high temperature ligase in a total volume of 100 μL. After the solution was mixed, the ligation cycle amplification technique was adopted, and the steps were as follows:...

Embodiment 2

[0093] sensitivity analysis:

[0094] Preparation of pretreated microspheres: take 100 μL of streptavidin functionalized microspheres and wash twice with 100 μL of affinity eluent, centrifuge at 3500 rpm for 5 min, remove the supernatant, add 49.4 μL of affinity eluent and 0.6 μL of 100 μmol / L biotinylated capture probe was shaken at 37°C for 1 hour, centrifuged and washed twice with affinity eluent to remove unbound probes, and finally 100 μL of affinity eluent was added and incubated at 4°C Save the pretreated beads.

[0095]Preparation of circularized DNA: Take a sterilized dry centrifuge tube, measure 74.5 μL of deionized water with a pipette gun, add 10 μL of 10× ligase reaction buffer solution, 5 μL of 5’ phosphorylated 1 μmol / L of circular DNA, 10 µL of synthetic Salmonella DNA-seq, 0.5 µL of high-temperature ligase in a total volume of 100 µL. After the solution was mixed, the ligation cycle amplification technique was adopted, and the steps were as follows: place t...

Embodiment 3

[0098] Performance analysis for the detection of Salmonella in real samples:

[0099] Preparation of pretreated microspheres: 100 μL of streptavidin functionalized microspheres were washed twice with 100 μL of affinity eluent, and centrifuged at 3500 rpm for 5 min. Remove the supernatant, add 49.4 μL of affinity eluent and 0.6 μL of 100 μmol / L biotinylated capture probe, shake at 37°C for 1 hour, centrifuge and wash twice with affinity eluent to remove unbound Finally, add 100 μL of affinity eluent and store the pretreated microspheres at 4°C.

[0100] Cultivation of Salmonella and digestion of genomic DNA: Salmonella was cultured in LB medium, and the genomic DNA of Salmonella was extracted using a commercial general bacterial genomic DNA extraction kit. Treat the extracted Salmonella genomic DNA with RsaI restriction endonuclease, the conditions are: add 10U of RsaI and its reaction buffer solution to 50ng of Salmonella genomic DNA, with a total volume of 50μL, react at 37°...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The application provides a series G-quadruplex-heme DNA enzyme label-free signal amplification method based on rolling-circle amplification. The series G-quadruplex-heme DNA enzyme label-free signal amplification method comprises the steps of: 1) using affinity eluent and a biotinylated capture probe to treat functional microballs of streptavidin to obtain pretreated microballs; 2) cyclically amplifying and purifying the sample genomic DNA to obtain cyclic DNA; 3) mixing the pretreated microballs obtained in step 1) and the prepared cyclic DNA obtained in step 2) and then amplifying them in the rolling-circle to obtain rolling-circle amplification products; 4) mixing the rolling-circle amplification products with the heme to obtain the rolling-circle amplification products with signals amplified, and forming an amplified detection signal by the rolling-circle amplification products with signals amplified. In the step 1) and step 2) there is no time sequence. The detection limit of salmonella is 0.03 pmol / L, the detection cost is low, the operation is simple, the sensitivity is extremely high, the detection is rapid, and whether the salmonella is contained in the sample can be judged by the naked eye.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a rolling circle amplification tandem G-quadruplex-heme DNA enzyme-free labeling signal amplification method. Background technique [0002] Salmonella is a Gram-negative enteric bacterium in the Enterobacteriaceae family that can cause disease in humans or animals. According to relevant statistical reports, among all kinds of bacterial food poisoning in various countries in the world, food poisoning caused by Salmonella often ranks first. The largest salmonella food poisoning in the world was Salmonella typhimurium poisoning caused by pork in Sweden in 1953. 7,717 people were poisoned and 90 died. Therefore, the detection of Salmonella has always been the core issue of Salmonella research. [0003] The traditional method currently used to detect Salmonella is the bacterial culture method. This culture method can be divided into three different stages in general. The w...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12Q1/10C12R1/42
CPCC12Q1/682C12Q1/689C12Q2531/125C12Q2563/155C12Q2563/125
Inventor 张何傅昕陈人可
Owner HUNAN INSTITUTE OF ENGINEERING
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com