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

Establishment and application of novel E-LAMP

A loop-mediated isothermal, RT-LAMP technology, applied in the field of temperature amplification technology, can solve the problems of high price, high equipment requirements, RNase and nucleic acid amplification pollution, etc., to reduce pollution opportunities, facilitate quality control work, Detects the effect of improved specificity

Inactive Publication Date: 2008-05-14
北京金迪克生物技术有限责任公司
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0021] The present invention is a novel enzyme-linked-loop-mediated isothermal gene designed to overcome the disadvantages of complex operation, serious contamination by RNase and nucleic acid amplification, high requirements for experimental hardware equipment, and high price in the current virus gene detection process. Amplification technology (E-LAMP)

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
  • Establishment and application of novel E-LAMP
  • Establishment and application of novel E-LAMP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0048] Embodiment 1: E-LAMP detection of hepatitis C virus gene (HCV)

[0049] (1) Coat streptavidin at a coating concentration of 5 μg / ml on a microplate or a microwell reaction plate, add 100 μl to each well, overnight at 4°C, pat dry the microplate, and set aside.

[0050] (2) Wash the plate with PBS, then block with 1% BSA at 37°C for 1 hour, wash the plate, and store at -20°C for later use.

[0051] (3) Add the E-LAMP reaction system with labeled primers, and amplify at 63° C. for 1.5 h.

[0052] The reaction system of E-LAMP is as follows: F3 and B3, 5pmol; BIP and FIP, 40pmol; Loop-1 and Loop-2, 20pmol; the final concentration of other reaction components is 1.0mM dNTP, 1mM betaine, 6mMMgSO4, 2.5μl 10× Bst-DNA Polymerase Buffer, 8U Bst-DNA polymerase, 1U AMV reverse transcriptase, 5 μl HCV RNA sample, replenish water to 25 μl, mix well, 63°C, water bath for 1.5h.

[0053] (4) Remove the reaction solution in the well and wash the plate.

[0054] (5) Add HRP-labeled an...

Embodiment approach 2

[0058] Implementation Option 2: Sensitivity Test for E-LAMP Solution

[0059] In order to verify the semi-quantitative effect of E-LAMP and the feasibility of routine gene detection, a 10-fold serial dilution was performed on a quantified HCV RNA sample, and the sensitivity of the E-LAMP solution was tested using the diluted sample. The specific operating procedures were as follows: The E-LAMP detection step is performed. The test results show that the sensitivity of this method can theoretically detect 10 copies of HCV cDNA molecules, which is the same as that of ordinary RT-LAMP, which proves that the sensitivity of this technology has not been affected after the modification.

Embodiment approach 3

[0060] Embodiment 3: E-LAMP detects the specificity of HCV virus

[0061] The method is the same as the E-LAMP detection of hepatitis C virus gene (HCV), and the RNA samples used are changed to HIV and HBV. The experimental results show that the OD values ​​of HIV and HBV tend to be negative, which proves that the specificity of this detection method is high.

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 belongs to the application field of biotechnology, relates to human medicine and veterinary medicine, and includes the detection of RNA viruses in various biological products, blood donors, patient blood samples and the like. Specifically, the enzyme-linked-loop-mediated isothermal amplification technique (E-LAMP) was used to analyze the conserved and virus-specific gene sequences of the HCV RNA virus through computer software, and six primers were designed to completely match the eight binding regions in the identified target sequence. , just one step to complete the cyclic displacement amplification reaction in an isothermal environment. At the same time, the common enzyme-linked immunosorbent technique is organically combined with RT-LAMP, and the intermediate product of RT-LAMP is solid-phased by biotin-labeling different primers. On a microwell reaction plate coated with streptavidin, the HRP enzyme-labeled anti-Digoxin antibody specifically binds to Digoxin labeled on another primer to finally present a color reaction. The entire detection process from amplification to result presentation is realized in a single tube. Compared with the traditional RT-PCR method, it has the advantages of safety, specificity, sensitivity and convenience.

Description

technical field [0001] The invention is an enzyme-linked-loop-mediated isothermal amplification technique (E-LAMP) used for HCV RNA virus detection. It is used for the monitoring of RNA viruses in various biological products, blood donors, patient blood samples, etc. Applications include human, veterinary and other biological defense. Background technique [0002] 1. Current status of virus gene detection technology [0003] Since the in vitro amplification of nucleic acid has become a reality, various nucleic acid amplification enzymes and nucleic acid amplification methods have emerged in an endless stream, and genetic diagnostic technology has been more and more widely used in disease monitoring, gene mutation, and gene feature analysis. So far, the established target gene amplification methods mainly include PCR, bDNA, SDA, 3SR, NASBA, etc., which are most widely used. Although these methods have strong nucleic acid amplification capabilities, there are many disadvant...

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): G01N21/78C12Q1/70C12Q1/68
Inventor 李启明马学军高寒春孙梅生侯云德
Owner 北京金迪克生物技术有限责任公司
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