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

Method for preparing RNase-resistant dengue virus nucleic acid detection quality control product

A technology for dengue virus and quality control material, applied in the field of infectious disease detection, can solve the problems of cumbersome purification method and general purification effect.

Inactive Publication Date: 2015-06-17
张瑾
View PDF2 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, armored RNA quality control products are mostly purified by polyethylene glycol precipitation or cesium chloride ultracentrifugation. The purification method is cumbersome and the purification effect is average. Therefore, the establishment of an easy and efficient purification method is also a key factor for the preparation and promotion of armored RNA quality control products.

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 for preparing RNase-resistant dengue virus nucleic acid detection quality control product
  • Method for preparing RNase-resistant dengue virus nucleic acid detection quality control product
  • Method for preparing RNase-resistant dengue virus nucleic acid detection quality control product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Obtaining and identification of armored RNA containing histidine tag and dengue virus 3' URT gene

[0023] 1. Materials

[0024] Type 1 dengue virus RNA, D-pET32a plasmid with protein tag removed, and MS2 phage RNA are all stored in our laboratory.

[0025] 2. Method

[0026] 1) Primer design

[0027] According to the comparative analysis of the whole genome sequence of dengue virus types I-IV published by GenBank, the sequence of the highly conserved 3' non-coding region of the virus was selected as the target fragment for amplification. Primer 5.0 software designed primers DFVF / DFVR with a fragment size of 320 bp, and HindIII / NotI restriction sites were introduced into the amplification primers. In order to amplify the coat protein gene and mature enzyme protein gene of MS2 phage, the primer pair MSF and MSR were designed, and BamHI and Hind III restriction sites were added at the 5' end, respectively. The primer pair MS-HISF / MS-HISR was used to insert ...

Embodiment 2

[0050] Embodiment 2: Armored RNA quality control product stability verification

[0051] 1. Stability test under different storage conditions

[0052] The virus-like particle solution purified in Example 1 was diluted to 105 copies / mL with a buffer containing 15% glycerol, and 50uL per tube was placed in room temperature, 4°C, and -20°C for 5d, 30d, 60d, and 90d. , 120d, 150d, 180d, sampling and testing to observe its stability. Another tube of 600uL virus particles was placed at -70°C and 37°C for 10 times of repeated freezing and thawing to test its stability. The t-test analysis of the test group and the control group (-70°C) data shows that there is no statistically significant difference between the test group and the control group (P>0.5), and the standard substance can be stored at -20°C and 4°C for at least 6 months Above, the storage period at room temperature is at least 14 days, and repeated freezing and thawing has little effect on the stability of the pseudoviru...

Embodiment 3

[0055] Embodiment 3: the practical application of quality control product in dengue fever nucleic acid detection

[0056] 1. Reagents

[0057] Nucleic acid extraction reagents: Viral RNA Mini RNA Extraction Kit

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 for preparing a RNase-resistant dengue virus nucleic acid detection quality control product. An armored RNA quality control product carrying a histidine tag and a dengue virus 3'URT gene is prepared by utilizing an MS2 phage armor technology and a site-directed mutagenesis technology; a dengue virus armored RNA quality control product is obtained through nickel column affinity chromatography purification, the quality control product is easy to purify, stable, high in concentration and free of biological infectivity, resists RNase, and can be used for carrying out quality control on the whole processes of virus detection, reverse transcription, PCR and the like in dengue virus nucleic acid detection.

Description

technical field [0001] The invention relates to the technical field of infectious disease detection, and relates to a preparation method of an RNase-resistant dengue virus nucleic acid detection quality control product. Background technique [0002] Dengue virus belongs to the Flaviviridae family (Flaviviridae). It is a single-stranded positive-sense RNA virus and is the most important arbovirus for humans. It can cause dengue fever and dengue hemorrhagic fever, and is mainly transmitted by Aedes aegypti and Aedes albopictus. The disease is widely prevalent in tropical and subtropical regions, and is a human infectious disease with wide distribution, frequent incidence and great harm. In recent years, global warming, population flow and other factors have led to a significant increase in the incidence of dengue virus infection in tropical and subtropical regions of Asia, Africa and South America, and there are often small-scale outbreaks in southern my country . Therefore, ...

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): C12Q1/70C12Q1/68C12N15/70C12N15/40C12N7/04C12R1/93
CPCC12Q1/701C12Q2600/166Y02A50/30
Inventor 张瑾薛晓宁徐翮飞陈晓光张娟张齐林元朱可
Owner 张瑾
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