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Kit and oligonucleotide sequences for detecting astrovirus

A nucleotide sequence and astrovirus technology, applied in the biological field, can solve problems such as the detection of unseen astroviruses, achieve high specificity, improve detection sensitivity, and simple detection

Inactive Publication Date: 2010-03-17
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant reports on the detection of astroviruses using RT-LAMP technology at home and abroad

Method used

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  • Kit and oligonucleotide sequences for detecting astrovirus
  • Kit and oligonucleotide sequences for detecting astrovirus
  • Kit and oligonucleotide sequences for detecting astrovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Design of RT-LAMP primers

[0044] Referring to the highly conserved nucleic acid sequence (GenBank accession No.L23513) at the 5' end of the capsid protein coding gene (ORF2) of the type 1 astrovirus Oxford strain, use the Primer Explorer V4.0 online software (http: / / primerexplorer.jp / elamp4.0.0 / index.html) to design astrovirus-specific RT-LAMP primers, the primers are shown in Table 1. The Tm value of the primers is between 59°C and 65°C; the terminal free energy (G) is less than -4cal / mol, which can ensure the stability of the primers; the GC content is about 50%; there is no complementarity at the 3' end of the primers sequence, which can effectively prevent the formation of secondary structures inside the primer. The astrovirus-specific circular upstream primer (LF) and circular downstream primer (LB) designed in the present invention help to improve detection sensitivity and shorten reaction time. 6 primers recognize 8 different sites of the target g...

Embodiment 2

[0045] Example 2: Establishment and Optimization of Astrovirus RT-LAMP Detection System

[0046] 1. Method:

[0047] 1) Mg 2+ Concentration: Prepare the reaction mixture according to the reaction system in Table 2, and adjust the Mg 2+ Concentration to 2mM, 4mM, 6mM, 8mM, 10mM, 12mM, after incubation at 65°C for 90min, react at 85°C for 2min to terminate the reaction. Comparing different Mg 2+ Effect of concentration on amplification efficiency.

[0048] 2) Concentration of dNTPs: prepare the reaction mixture according to the reaction system in Table 2, adjust the final concentration of dNTPs to 0mM, 0.2mM, 0.6mM, 1.0mM, 1.4mM, 1.8mM respectively, and keep it at 65°C for 90min, then react at 85°C for 2min Stop the reaction. The effects of different concentrations of dNTPs on the amplification efficiency were compared.

[0049] 3) Betaine concentration: prepare the reaction mixture according to the reaction system in Table 2, and adjust the concentration of betaine to 0M,...

Embodiment 3

[0055] Embodiment 3: the composition of the test kit that detects Astrovirus

[0056] 1. The composition of the kit (stored at -20°C)

[0057] (1) TRIZOL lysate: purchased from Invitrogen, product number: 15596-026;

[0058] (2) DEPC water: purchased from Shanghai Sangong, item number: D1005;

[0059] (3) 2×RT-LAMP reaction solution: its components are: 2×ThermoPol buffer, 1.6M betaine, 3.6mM dNTPs, 8mM MgSO 4 , 0.4 μM Outer Upstream Primer (F3), 0.4 μM Outer Downstream Primer (B3), 3.2 μM Inner Upstream Primer (FIP), 3.2 μM Inner Downstream Primer (BIP), 1.6 μM Loop Upstream Primer (LF) and 1.6 μM Loop Like downstream primer (LB); Wherein the outer upstream primer (F3) is the nucleotide sequence shown in the sequence table SEQ ID No.1, and the outer downstream primer (B3) is the nucleotide sequence shown in the sequence table SEQ ID No.2 Sequence, the inside upstream primer (FIP) is the nucleotide sequence shown in the sequence table SEQ ID No.3, the inside downstream prim...

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Abstract

The invention discloses a kit and oligonucleotide sequences for detecting astrovirus, in particular to a group of oligonucleotides which are used for detecting astrovirus and have the oligonucleotidesequences shown from the sequence table SEQ ID No.1 to the sequence table SEQ ID No.6, the kit containing the oligonucleotides and a detection method thereof. The kit of the invention has high sensitivity, strong specificity, low cost and simple and convenient operation. During detection, when lots of nucleic acid is synthesized, a by-product magnesium pyrophosphate precipitate is generated, whichhas high specificity. Whether the products are amplified or not can be judged only by observing the turbidity of the products with naked eyes.

Description

technical field [0001] The invention relates to a test kit and oligonucleotide for detecting astrovirus, more specifically, a rapid detection kit for reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for detecting astrovirus , specifically refers to an oligonucleotide sequence, a kit and a method for rapidly detecting astroviruses by utilizing the RT-LAMP principle, and belongs to the field of biotechnology. Background technique [0002] Astrovirus is one of the important pathogens causing acute viral enteritis in infants, the elderly and immunocompromised persons, and can be sporadic or outbreaks. Virus particles are spherical, without envelope, with a diameter of 28nm to 30nm, with 5 to 6 small angles, making the whole virus particle a star-shaped structure; it is a single-stranded positive-sense RNA virus without a capsid, and the viral gene is 6.8kb long, including A 5' non-coding region (5' non-coding region, NCR), three open reading frames (openre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 周琦曾静魏海燕王金花张西萌
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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