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

EV71 single-stranded DNA aptamer and chemiluminescent detection kit for detecting enterovirus 71 using double aptamers

A chemiluminescence reagent, EV71 technology, applied in the field of EV71 virus qualitative detection, achieves high specificity, simple and fast detection operation

Active Publication Date: 2022-04-26
ZHENJIANG NO 1 PEOPLES HOSPITAL
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant report on the detection of EV71 based on aptamer technology.

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
  • EV71 single-stranded DNA aptamer and chemiluminescent detection kit for detecting enterovirus 71 using double aptamers
  • EV71 single-stranded DNA aptamer and chemiluminescent detection kit for detecting enterovirus 71 using double aptamers
  • EV71 single-stranded DNA aptamer and chemiluminescent detection kit for detecting enterovirus 71 using double aptamers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Screening of EV71 aptamers:

[0036] 1) Main reagents:

[0037] Binding Buffer: 1*PBS, 5mM MgCl 2 , 1% BSA, 1μg / ml tRNA, 0.02% tween-20, 5mM imidazole Washing Buffer: 1*PBS, 5mM MgCl 2 , 0.02% tween-20

[0038] Table 1 Screening library and primer sequences

[0039]

[0040] 2) The first round of SELEX

[0041] a.library was dissolved in water to a final concentration of 20 μM.

[0042] b. Configuration extension system

[0043]

[0044] The extension program is as follows: 95°C for 60s, 58°C for 60s, 72°C for 60min

[0045] c. Add the product to SA agarose bead and incubate at room temperature for 10 minutes to wash away unbound sequences.

[0046] d. Add 150mM NaOH and incubate at room temperature for 5min, aspirate the supernatant, and add 300mM HCl to neutralize.

[0047] e. Using water as a control, Nanodrop measures the concentration of modified single-stranded DNA.

[0048] f. Denaturation at 95°C for 5 minutes, add Binding Buffer to 1ml after ice ...

Embodiment 2

[0082] Aptamer affinity assay (ELISA):

[0083] a. Dilute the protein with PBS to a final concentration of 200ng / ml, set 2 replicate wells for each experimental group, add 50μl to each well. After covering the plate with sealing film, keep overnight at 4°C.

[0084] b. Washing: fill each well with Washing Buffer, let it stand for 5 seconds, pour off the washing solution, and repeat 3 times. Pat dry one last time.

[0085] c. Add 150 μl of PBS containing 1% BSA to each well to block, and incubate at room temperature for 1 hour.

[0086] d. Plate washing: the operation is the same as step b.

[0087] e. Dilute aptamer to the target concentration with Binding Buffer.

[0088] f. Take the coated strip and add 50 μl to each well. Incubate at room temperature for 1h.

[0089] g. Plate washing: the operation is the same as step b.

[0090] h. Dilute Streptavidin-HRP with Aassay Diluent 1:200. Add 50 μl to each well. Incubate at room temperature for 0.5h.

[0091] i. Washing...

Embodiment 3

[0096] Aptamer Specificity Assay (ELISA):

[0097] Referring to the experimental method in Example 2, four coating concentrations of 100ng / ml, 200ng / ml, 500ng / ml, and 1000ng / ml were set up, and the amount of aptamer fixed in each well was 200nM. The result is as Figure 5 As shown, the BSA protein at each coating concentration did not bind to the aptamer. Under the condition of higher coating concentration, the aptamer can also bind to Coxsackie virus A16 (cox) to a certain extent, but the binding ability is significantly lower than that of enterovirus 71 (EV).

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

This patent relates to an EV71 single-stranded DNA aptamer and a chemiluminescent detection kit for detecting enterovirus 71 using a double aptamer. The aptamers are aptamer V11 having a sequence such as SEQ ID NO.1, aptamer V21 having a sequence such as SEQ ID NO.2, and aptamer V7 having a sequence such as SEQ ID NO.3. The kit of the present invention uses aptamer V21 as a capture probe and aptamer V11 as a signal probe. Specifically include: capture probe-coupled immunomagnetic beads, analysis buffer, signal probe-coupled enzyme conjugate, SA-HRP solution, and luminescent substrate solution. The kit has high specificity for the detection of EV71; it can qualitatively detect EV71 from samples such as cerebrospinal fluid, herpes fluid, throat swabs and anal swabs of suspected hand, foot and mouth disease patients; the detection operation of the kit of the present invention is simple and fast, The detection process only takes about 45 minutes.

Description

technical field [0001] The invention relates to a single-stranded DNA aptamer for EV71, and a chemiluminescent kit for detecting enterovirus 71 (EV71) using a double aptamer and an application thereof, which is suitable for qualitative detection of EV71 virus. Background technique [0002] Hand, foot and mouth disease (HFMD) is an infectious disease caused by enterovirus, which has occurred sporadically in various places in recent years. The disease is common in preschool children, especially infants, and can cause fever, rashes, and ulcers on the hands, feet, and mouth. Generally, the prognosis is good. Individual patients can cause complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis. disease. The source of infection of hand, foot and mouth disease is patients and latent infections, mainly through close contact with the virus in the feces, oral secretions, and skin herpes fluid of infected persons, through the fecal-oral route or the respira...

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 Patents(China)
IPC IPC(8): C12N15/115G01N33/58G01N33/543
Inventor 茅凌翔邹欣然吴静顾佳奇贡佳怡沈俐
Owner ZHENJIANG NO 1 PEOPLES HOSPITAL
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