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

Primer probes and identification method for enterovirus typing identification

An enterovirus and probe technology, applied in the field of primers, probes and identification for enterovirus typing and identification, can solve the difficulties of primer and probe design, the increase of reaction system configuration and identification result reporting time, the amount of multiple samples, etc. It can reduce the configuration of the reaction system, facilitate the application, and simplify the detection steps.

Active Publication Date: 2018-08-21
SHANGHAI BIOGERM MEDICAL TECH CO LTD
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When using the rRT-PCR method to detect viral nucleic acids, one of the difficulties is that the mutation of the virus brings difficulties to the design of primers and probes. Sequence comparison and analysis, if the primers and probes for typing and identification are no longer suitable for popular strains, the primers and probe sequences need to be updated
The second is to use the same fluorescently-labeled serotype identification probe. In the same detection reaction system, only one serotype of enterovirus can be typed and identified. If multiple serotypes need to be typed and identified, more Larger sample volume, the corresponding increase in the configuration of the reaction system and the reporting time of identification results

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
  • Primer probes and identification method for enterovirus typing identification
  • Primer probes and identification method for enterovirus typing identification
  • Primer probes and identification method for enterovirus typing identification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, enterovirus and the design and synthesis of enterovirus VP1 gene primers and probes of CA16 and EV71 serotypes become

[0048] Download the VP1 genes of all serotypes of enteroviruses in China from 2015 to 2017 in the GenBank database, and the VP1 genes of CA16 and EV71 serotypes. The software compares and analyzes the consistency of the gene sequences of different serotypes, and selects relatively conserved regions to design a complete Genome-specific amplification primer sequences. In primer design, 4 or less degenerate bases are allowed at the same variable site. The extracted candidate primers were screened to meet the following requirements: ①The position of the probe is close to that of the same-strand primer, and the size of the PCR product is between 100bp and 150bp. ②The GC% of the primer and probe is between 25% and 75%; ③The primer length is about 20bp, the Tm value is between 58°C and 60°C, the probe length is 20bp to 30bp, and the Tm valu...

Embodiment 2

[0053] Embodiment 2, detect unknown virus

[0054] 1. Extraction of viral RNA:

[0055] Take 140 μL sample (throat swab, anal swab or processed stool specimen), add it to 560 μL AVL lysate containing 5.6 μg CarrierRNA, and extract according to the instructions of QIAamp Viral RNA Mini Handbook (Qiagen, catalog #52904 / 52906) Viral RNA, elution volume 50 μL.

[0056] 2. rRT-PCR reaction

[0057] 1) System configuration: using Bioline's SensiFAST TM Probe No-ROX 0ne-Step Kit (BIO-76001) reaction solution, the configuration components are as follows:

[0058] Table 2

[0059]

[0060] 2) rRT-PCR: put the reaction tube with the above reaction system in the PCR machine for rRT-PCR, the reaction procedure is as follows:

[0061] table 3

[0062]

[0063] 4. Judgment of results: judge the results when the results of the negative and positive control are established, that is, the negative reference has no Ct value, while the positive reference has a Ct value. Under norm...

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 primer probes and an identification method for enterovirus typing identification. The method designs three groups of primer probes shown as SEQ ID No.1-SEQ ID No.9 directed atall serotypes of VP1 genes in group A, group B and group C of enteroviruses, and the VP1 gene conserved regions of coxsackie virus group A type 16 and enterovirus type 71, selects different fluorescent markers for the three probes, achieves identification of enteroviruses and two serotype enteroviruses in one tube reaction system simultaneously, and simplifies the enterovirus prevalent serotype laboratory detection procedure. At the same time, the invention discloses treatment of to-be-detected samples, a fluorescence RT-PCR reaction system and reaction conditions. The primer probes and the identification method provided by the invention can realize detection of pandemic enteroviruses in the Chinese mainland and two common serotypes, are easy to operate and convenient to apply, thus providing a feasible technical method for study of enterovirus infection etiology and clinical epidemiology.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer probe and an identification method for typing and identifying enteroviruses. Background technique [0002] Typically, enteroviruses cause mild symptoms, but serious conditions such as encephalitis, myocarditis, polio, acute heart failure and sepsis have also been reported. The disease activity showed a seasonal epidemic trend, with peaks in summer and early autumn in temperate regions. [0003] Enteroviruses belong to the small RNA virus family, which can be divided into four serotype groups according to the different tissue culture and disease forms of infected experimental animals, including poliovirus, Coxsackie virus group A, and Coxsackie virus group B , Echo virus. Enteroviruses are further divided into multiple serotypes based on genetic and phenotypic similarities. Up to now, there are more than 110 serotypes. Enteroviruses are error-prone during genom...

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): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2521/107
Inventor 朱兆奎李晓丹赵百慧
Owner SHANGHAI BIOGERM MEDICAL TECH CO LTD
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