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

Novel coronavirus SARS-CoV-2 ORF1ab gene and N gene isothermal amplification primer group as well as kit

A sars-cov-2orf1ab, coronavirus technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of no real-time fluorescence constant temperature, and achieve the effect of low test cost

Pending Publication Date: 2021-02-26
DALIAN NATIONALITIES UNIVERSITY +1
View PDF10 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solve the problem that for the nucleic acid detection of the new coronavirus SARS-CoV-2, only real-time fluorescent quantitative RT-PCR kits are used to detect ORF1ab and N genes, and constant temperature amplification chip kits are used to detect S genes and N genes, but there is no real-time fluorescent constant temperature RT-PCR Problems with kit detection of ORF1ab and N gene

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
  • Novel coronavirus SARS-CoV-2 ORF1ab gene and N gene isothermal amplification primer group as well as kit
  • Novel coronavirus SARS-CoV-2 ORF1ab gene and N gene isothermal amplification primer group as well as kit
  • Novel coronavirus SARS-CoV-2 ORF1ab gene and N gene isothermal amplification primer group as well as kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 ORF1ab and N gene real-time fluorescent constant temperature RT-PCR detection primer set specificity verification

[0060] 1.1 Specificity test of constant temperature amplification primer set

[0061] 1.1.1 Preparation of positive control substance

[0062] According to the SARS-CoV-2 ORF1ab and N gene nucleic acid sequences published by the Novel Coronavirus National Science and Technology Resource Service System (http: / / nmdc.cn / # / nCoV), the ORF1ab gene and N gene plasmids were artificially synthesized, and the in vitro transcription RNA was positive Control samples were aliquoted and stored at -20°C for later use.

[0063] 1.1.2 Specific amplification test

[0064] Use other pathogens with the same infection site or similar infection symptoms as the new coronavirus pneumonia to verify specific cross-reactions. Since these pathogen samples cannot be obtained during the outbreak, they are artificially synthesized gene fragments. They are: coronavirus 229E, ...

Embodiment 2

[0091] Example 2 Optimization of ORF1ab and N gene real-time fluorescent constant temperature RT-PCR detection system

[0092] 2.1 Establishment of real-time fluorescence constant temperature RT-PCR reaction system

[0093] The 25 μL reaction system includes 2.0 μL of RNA template, 12.5 μL of reaction solution RM-SCI (Guangzhou Double Helix Gene Technology Co., Ltd., China, containing 2.8mM dNTPs, 40mM Tris-HCl, 20mM KCl, 16mM MgSO 4 , 20mM (NH 4 ) 2 SO 4 , 1.6M betaine), 0.5 μL of 1000×SYBR Green I (Thermo Scientific), 0.8 μL of 8U / μL Bst polymerase (NEB), 0.2 μL of 5U / μL AMV enzyme (TAKARA, China, Dalian), 8 μL of Ultrapure water, 1.0 μL of inner primer, outer primer and loop primer. Finally, add 1 drop (about 20 μL) of paraffin to seal the tube.

[0094] Using self-designed and confirmed the most specific primer sets (inner primers, outer primers and loop primers) for the detection of novel coronavirus SARS-CoV-2 ORF1ab and N gene, respectively prepared real-time assay...

Embodiment 3

[0103] Example 3 Sensitivity verification of ORF1ab and N gene real-time fluorescent constant temperature RT-PCR detection

[0104] 3.1 Sensitivity test method

[0105] The ORF1ab and N gene in vitro transcription RNA plasmid positive control samples were diluted with a 10-fold difference, and the gradient concentrations were 1×10^6 copies / μL, 1×10^5 copies / μL, 1×10^4 copies / μL, 1× 10^3copies / μL, 1×10^2copies / μL, 10copies / μL, tested by the optimized fluorescent constant temperature RT-PCR reaction system to verify the constant temperature of the new coronavirus SARS-CoV-2 ORF1ab and N gene designed in this case Sensitivity of the real-time fluorescence constant temperature RT-PCR detection method established by amplifying the inner and outer loop primer sets detected by RT-PCR.

[0106] 3.2 Sensitivity test results

[0107]The sensitivity test was carried out according to the method in 3.1, and the results showed that ORF1ab and N genes were all detected in three parallel sa...

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

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Login to View More

Abstract

The invention relates to a novel coronavirus SARS-CoV-2 ORF1ab gene and N gene isothermal amplification primer group as well as a kit, and belongs to the technical field of biology. By closely analyzing novel coronavirus SARSCoV2 whole genome sequences published internationally, performing intraspecific conservative comparison, extraspecific homology comparison and difference site analysis on thenovel coronavirus SARSCoV2 ORF1ab gene and N gene, as well as designing, screening and determining 2 inner primers, 2 outer primers and 2 loop primers for each gene as a core technology, the specificity and sensitivity of a real-time fluorescence constant-temperature RTPCR detection kit are enhanced. The RTPCR kit has performance indexes at the same levels as those of a fluorescent quantitative RTPCR kit, and enables detection to be completed within 30-45 minutes; and moreover, the kit is simple, convenient and rapid to operate.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel coronavirus SARS-CoV-2 ORF1ab and N gene constant temperature amplification primer set and kit. Background technique [0002] To provide immediate public health support, China has released 43 sets of SARS-CoV-2 genome sequences in the Novel Coronavirus National Science and Technology Resource Service System (http: / / nmdc.cn / # / nCoV), and the Global Shared Avian Influenza Database Initiative ( The genome sequence of the virus was published in the GISAID database. At the time of writing, the extent of human-to-human transmission of SARS-CoV-2 is unknown, but there is evidence of some human-to-human transmission, as well as evidence of gastrointestinal transmission of SARS-CoV-2 (separation in feces, gastrointestinal broken mucosa, bleeding Out of the virus), remind all walks of life to pay attention to prevent the spread of the new coronavirus caused by sewage pollution. [000...

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): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2561/113C12Q2563/107C12Q2521/107C12Q2545/114Y02A50/30
Inventor 曹际娟郑文杰郑秋月季超
Owner DALIAN NATIONALITIES UNIVERSITY
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