Method and kit for detecting ribonuclease

A ribonuclease and ribonucleotide technology, which is applied in the field of a method for detecting ribonuclease and a kit thereof, can solve problems such as the inability to meet the needs of routine quality control in laboratories, and achieves low cost, high sensitivity, The effect of small reaction volume

Active Publication Date: 2016-05-11
GUANGZHOU RIBOBIO
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of current commercial detection methods is only in the RNaseA range of 10-100pg / ml, which cannot meet the needs of routine quality control in laboratories

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
  • Method and kit for detecting ribonuclease
  • Method and kit for detecting ribonuclease
  • Method and kit for detecting ribonuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, be used to detect the RNA probe synthesis of ribonuclease

[0069] 1. Design principles of RNA probes

[0070] 1) The RNA probe consists of a single-stranded RNA, an energy donor group and an energy acceptor group;

[0071] Single-stranded RNA consists of 2-30 ribonucleotides, preferably 4-10 ribonucleotides; single-stranded RNA specifically consists of 4, 5, 6, 9 or 10 nucleotides.

[0072] The single-stranded RNA comprises at least one ribonuclease cleavage site, at least 1 G and at least 1 C, preferably the single-stranded RNA comprises at least one ribonuclease cleavage site and at least 1 GC sequence. Preferably, the nucleotide next to the G of the GC sequence or the CG sequence is A or U;

[0073] The energy donor group and the energy acceptor group are connected to the nucleotides at the two ends of the single-stranded RNA, the energy donor group is a fluorescent emitting group, and the energy acceptor group is a fluorescent quencher corresponding...

Embodiment 2

[0082] Embodiment 2, detect RNaseA

[0083] 1. Reagents for detection

[0084] 1. Preparation of RNA probes and reagents

[0085] The RNA probes shown in Table 1 were synthesized.

[0086] 2. RNase probe preparation

[0087] RNase probe stock solution preparation (100pmol / ul): RNA probe dry powder added RNasefreeddH 2 O, make the concentration of the RNA probe 100 pmol / ul, obtain the stock solution, and store it in the dark at -20°C.

[0088] RNase probe working solution preparation (10pmol / ul): take RNase-free 1.5ml tube, add 90ulddH 2 O and 10ul RNase probe stock solution, mix well, and store at -20°C in the dark.

[0089] 3. Preparation of 10× reaction buffer

[0090] Table 2 for 10x reaction buffer

[0091] components

scope

specific value

NaCl

0-5M, and not 0

50mM

KCl

0-5M, and not 0

20mM

Tris

20mM-1M

20mM

MgCl 2

10-100mM

10mM

pH

6.5-9.0

8.0

[0092]Prepare a 10× r...

Embodiment 3

[0107] Embodiment 3, the sensitivity research of RNA probe detection RNaseA

[0108] 1. Preparation of reaction system

[0109] Configure according to Table 2 and Table 3 of Example 2, wherein the RNA probe is the SEQ9 probe to obtain the reaction system.

[0110] 2. Detection

[0111] The samples to be tested are RNaseA diluted with water according to different dilution factors.

[0112] Add the above-mentioned RNaseA diluted with water at different dilution times to the reaction system of different tubes, so that the concentration of RNaseA in the reaction system is 10 6 pg / ml, 10 5 pg / ml, 10 4 pg / ml, 10 3 pg / ml, 10 2 pg / ml, 10pg / ml, 1pg / ml, 0.1pg / ml, 0.01pg / ml, 0.001pg / ml.

[0113] Detection method is the same as that of Example 2.

[0114] The result is as image 3 A, shows that adopting the detection method of the present invention, by using SEQ9 to detect RNaseA of different concentrations, the lower limit of detection of RNaseA is 0.1 pg / ml.

[0115] Will im...

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 invention discloses a method and kit for detecting ribonuclease. The method includes the following steps of firstly, designing and synthesizing an RNA probe; secondly, evenly mixing a to-be-detected sample and a reaction system with the RNA probe to obtain reaction liquid; thirdly, detecting the fluorescence intensity of the reaction liquid, and determining whether ribonuclease is contained in the to-be-detected sample according to the fluorescence intensity. The method has the advantages that flexibility is high (0.1 pg / ml RNase A, 100 times higher than Ambion); real-time observation can be achieved; thirdly, high-throughput detection can be conducted, wherein a large number of samples can be detected at the same time; reaction is small in volume, low in cost, easy and rapid (about 1 hour), and the method is suitable for conventional quality control of laboratories; a board spectrum is achieved, wherein content and relative content of different types of ribonuclease in different samples can be detected; safety is achieved, wherein no harmful compounds are used.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting ribonuclease and a kit thereof. Background technique [0002] Ribonuclease (RNase) can hydrolyze RNA substrates into small molecular nucleic acids. Most ribonuclease activities require divalent cations (such as Ca 2+ , Mg 2+ Wait). Among them, RNaseA is a widely used endonuclease. RNaseA hydrolyzes RNA but not DNA. [0003] RNaseA efficiently and specifically catalyzes the cleavage of phosphodiester bonds on the backbone of the RNA chain at the 3′ end of pyrimidine nucleotide residues C and U, forming oligomers with 2′,3′-cyclic phosphate derivatives Nucleotides. [0004] RNase is a ubiquitous environmental pollutant in the laboratory, and the monitoring of RNase activity is a routine quality control (QC) step. On the other hand, the method for detecting RNase can also be applied to disease diagnosis, such as cancer detection. In 1970, Reddi confirmed f...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/44
CPCC12Q1/44C12Q1/6818C12Q2525/131C12Q2525/117C12Q2563/107
Inventor 张必良刘霭珊曹亮克雷格·梅洛
Owner GUANGZHOU RIBOBIO
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap