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

A novel high-throughput method for quantification of HBV cccDNA from cell lysate by real-time PCR

A primer pair, reverse primer technology, applied in the high-throughput field

Inactive Publication Date: 2017-08-29
F HOFFMANN LA ROCHE & CO AG
View PDF8 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because of this, those qPCR-based methods all amplify the DNA signal from the chromosomal insert of the 1.1× HBV genome (Guo et al., JVirol. 2007 Nov, 81(22):12472-84), and thus failed to be used in HepDES19 Selective amplification of cccDNA in cells

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
  • A novel high-throughput method for quantification of HBV cccDNA from cell lysate by real-time PCR
  • A novel high-throughput method for quantification of HBV cccDNA from cell lysate by real-time PCR
  • A novel high-throughput method for quantification of HBV cccDNA from cell lysate by real-time PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1: Comparison of a qPCR assay using a conventional cccDNA primer pair against extracted viral genomic DNA and a one-step qPCR assay

[0136] The source of cell material was HepDES19 with no induction of cccDNA (background, due to the presence of 1 μg / mL tetracycline) or induction (cccDNA expression, due to the absence of tetracycline) after six days of culture. Test the regular group. DNA extraction methods and one-step qPCR assays were performed following the procedures described above.

[0137] figure 2 The results shown in describe the range between DNA from Tet-on (background) conditions and DNA from Tet-off (cccDNA expression) conditions. For the extracted DNA method, the conventional group showed an 11.3-fold difference in the qPCR assay. For the one-step qPCR assay, there was only a 1.3-fold difference. By comparing the results of the extracted DNA method and the one-step qPCR assay, the conventional panel could not be used to establish a one-step cc...

Embodiment 2

[0138] Example 2: Test of new cccDNA primers for detection of extracted DNA from HepDES19 or direct lysate without extraction (one-step qPCR assay)

[0139]The source of cell material was HepDES19 with no induction of cccDNA (background, due to the presence of 1 μg / mL tetracycline) or induction (cccDNA expression, due to the absence of tetracycline) after six days of culture. The following materials were used in this example: conventional set, PAIR0-P0, PAIR1-P0, PAIR2-P0, PAIR3-P0 and PAIR4-P0. The dye used in P0 was TAMRA and the quencher used in P0 was BHQ2. A one-step qPCR assay was performed following the procedure described above.

[0140] image 3 The results shown in a show that the range between extracted DNA from background and extracted DNA from cccDNA expression in HepDES19 cells was improved from 11.3-fold in conventional group to PAIR0-P0, PAIR1-P0, PAIR2-P0, PAIR3- 50-80 times in P0 and PAIR4-P0. This indicates that the new primer pairs and probes, namely PA...

Embodiment 3

[0141] Example 3: Experimentation of new cccDNA probes for detection of extracted DNA from HepDES19 or direct lysates of a one-step qPCR assay without extraction (one-step qPCR assay).

[0142] Cell material was derived from HepDES19 with no cccDNA induction (background, due to the presence of 1 μg / mL tetracycline) or induction (cccDNA expression, due to the absence of tetracycline) after six days of culture.

[0143] For DNA extraction studies, the probes used were P0, P1, P2 and P3, where in each probe the dye used was TAMRA and the quencher BHQ2 was used. The primer used for all reactions was PAIR4.

[0144] For the one-step qPCR assay, the probe used was P2, and the dye used in P2 was TAMRA and the quencher used in P2 was BHQ2. The primers used were PAIR1 and PAIR4, respectively. A conventional group was also used for control.

[0145] Figure 4 The results shown in a describe the design of new probes and a comparison of their ability to detect HBVcccDNA in extracted v...

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 present invention provides (1) a set of novel DNA primers that can be used for high efficient real-time qPCR of Hepatitis B virus (HBV) circular DNA (cccDNA) and (2) a novel high throughput method for quantification of HBV cccDNA from cells using this set of novel DNA Primers by real-time qPCR. It also provides a use of this method in monitoring cccDNA level in experimentally infected liver cells and evaluation of therapeutic effect on HBV.

Description

field of invention [0001] The present invention relates to (1) a novel DNA primer set that can be used for highly efficient real-time qPCR of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and (2) using this new DNA primer set to obtain A new high-throughput method for the quantification of HBV cccDNA in cells. It also relates to the use of this method to monitor cccDNA levels in experimentally infected hepatocytes and to evaluate the effect of treatment against HBV. Background of the invention [0002] Since cccDNA plays an important role in HBV biology, many experimental systems have been established to study potential drug candidates targeting cccDNA. To provide a controllable cell model of cccDNA, Ladner et al. (Ladner, S.K., Otto, M.J., Barker, C.S., Zaifert, K., Wang, G.H., Guo, J.T., Seeger, C., King, R.W., Antimicrob Agents Chemother . 1997 Aug;41(8):1715-20) has inserted into the HBV viral genome under the transcriptional control of the tet promot...

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): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/706C12Q1/6851C12Q1/686C12Q2600/16
Inventor W·恩格林赵虎N·秦
Owner F HOFFMANN LA ROCHE & CO AG
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