Primer and kit for detecting coxsackievirus A6 type RT-LAMP (Reverse Transcription Loop-mediated Isothermal Amplification) nucleic acid

A technology of RT-LAMP and Coxsackie virus, which is applied in the application field of biological detection technology, can solve the problems of simple, fast and accurate diagnosis, unfavorable on-site sample detection, unfavorable grass-roots promotion, etc., to meet the requirements of rapid on-site detection and low cost Low, fast detection effect

Inactive Publication Date: 2012-12-12
何雅青 +2
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the classic methods for detecting enteroviruses mostly use cell separation and culture and specific antibody detection. This method has cumbersome steps and a long detection cycle, and some enteroviruses are difficult to proliferate in cells, and are prone to missed detection; based on CA6 Nucleic acid amplification molecular biology diagnostic techniques such as PCR, RT-PCR, and real-time fluorescent quantitative RT-PCR (real-time fluorescent RT-PCR) play an important role in the detection of pathogenic microorganisms and disease diagnosis, but these me

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 and kit for detecting coxsackievirus A6 type RT-LAMP (Reverse Transcription Loop-mediated Isothermal Amplification) nucleic acid
  • Primer and kit for detecting coxsackievirus A6 type RT-LAMP (Reverse Transcription Loop-mediated Isothermal Amplification) nucleic acid
  • Primer and kit for detecting coxsackievirus A6 type RT-LAMP (Reverse Transcription Loop-mediated Isothermal Amplification) nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Design and synthesis of LAMP primers

[0023] According to the CA6VP1 gene sequence published by GenBank, its relatively conserved region was analyzed using the biological software CLAST, and the LAMP online design software PrimerExplorer V4 ( http: / / primerexplorer.jp / e / ) designed six primers for relatively conserved sequence regions, including two inner primers FIP (F2+F1C) and BIP (B2+B1C), two outer primers F3 and B3, and two loop primers LF and LB, respectively Eight binding regions in the target sequence were matched, and the primers were evaluated using Oligo 6 software and BLAST.

[0024] The primer sequences are:

[0025] F3 (Forward Outward Citation): 5'-ACTCGCTGTGTGATGAATCG-3'; that is, SEQ ID NO:1

[0026] B3 (reverse external reference): 5'-GCGTTGTGCTATCATTGAGG-3'; ie SEQ ID NO:2

[0027] FIP(F1C+F2, forward introductory):

[0028] 5'-CCTTCACCTCCACAACTCCTACTGAGGCGAGTGTGGAACA-3'; namely SEQ ID NO:3

[0029] BIP (B1C+B2, reverse internal citati...

Embodiment 2

[0037] Embodiment 2: the extraction of RNA

[0038] The RNA extraction of virus samples uses Roche High Pure Viral RNA Kit, and the steps are carried out according to its operating instructions. Specific steps: 1. Add 400 μl Binding Buffer to 200 μl sample treatment solution, mix well, transfer to a filter column, and centrifuge at 8000 g / min 15s; 2. Discard the filtrate, replace the collection tube, add 500μl Inhibitor Removal Buffer, centrifuge at 8000g / min for 1min; 3. Discard the filtrate, replace the collection tube, add 450μl Wash Buffer, centrifuge at 8000g / min for 1min; 4. Repeat step 3 once; 5. Discard the filtrate, replace the collection tube, and centrifuge at the maximum speed of the centrifuge for 10s; 6. Transfer the filter column to a 1.5ml EP tube, add 50μl Elution Buffer, and centrifuge at 8000g / min for 1min. The eluate is the purified nucleic acid. Virus cell culture and herpes fluid are directly extracted according to the above method; fecal samples, anal sw...

Embodiment 3

[0039] Example 3: Establishment of CA6RT-LAMP amplification method

[0040] 1. CA6RT-LAMP reaction system

[0041] Use the sample RNA to be tested as a template for RT-LAMP reaction.

[0042] Table 1 CA6RT-LAMP reaction system

[0043] components

volume

RT-LAMP reaction solution

23μl

RNA

2μl

total capacity

25μl

[0044] Wherein, the preparation of RT-LAMP reaction solution is shown in Table 2.

[0045] Formulation of table 2CA6RT-LAMP reaction solution

[0046] components

Final concentration

working concentration

volume

10×buffr

1×

2.5μl

MgCl 2

5mM

25mM each

5μl

dNTPs

1.4mM each

10mM each

3.5μl

FIP

1.6μM

40μM

1μl

BIP

1.6μM

40μM

1μl

F3

0.2μM

10μM

0.5μl

B3

0.2μM

10μM

0.5μl

LF

0.8μM

40μM

0.5μl

LB

0.8μM

40μM

0.5μl

AMV

...

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 relates to the application field of biological detection technologies, and in particular relates to a primer pair and a kit for coxsackievirus A6 type RT-LAMP (Reverse Transcription Loop-mediated Isothermal Amplification) nucleic acid detection. The primer pair comprises two outer primers F3 and B3, two inner primers FIP and BIP and two loop primers LF and LB; and the kit comprises the primer pair. The CA6RT-LAMP has the characteristics of strong specificity, high sensitivity, simplicity in operation methods, quick detection, easiness in result judgment, low cost and the like, can better satisfy the requirement of on-site quick detection, is easy to popularize and apply in grassroots units, can be used for early rapid diagnosis of epidemic outbreaks, such as hand-foot-and-mouth diseases and the like and clinical cases in disease prevention and control organizations, hospitals, kindergarten units, and has broad market prospect and enormous economic and social benefits.

Description

technical field [0001] The invention relates to the application field of biological detection technology, in particular to primers and kits for nucleic acid detection of Coxsackievirus A6 (Coxsackievirus A6, CA6 for short) various specimens (feces, anal swab, throat swab, herpes fluid, cerebrospinal fluid). Background technique [0002] Coxsackievirus A6 is a single-stranded positive-sense RNA virus belonging to the Picoranviridae family. CA6 can cause diseases such as hand, foot and mouth disease and herpetic angina. Although CA6 is generally not considered to be the main pathogen of HFMD, there have been reports of outbreaks of HFMD caused by CA6 in countries and regions such as Singapore, Finland, Taiwan, and Japan in recent years, and HFMD caused by CA6 is related to other types of intestinal Enteroviruses caused by enteroviruses are more likely to cause symptoms such as Beau's lines and nail shedding. The mechanism is unclear, but it may indicate that CA6 has a wider s...

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): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 何雅青陈惠玲舒柏华
Owner 何雅青
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap