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Kit and oligonucleotide sequences for detecting rotavirus A

A nucleotide sequence, rotavirus technology, applied in the biological field, can solve problems such as unseen rotavirus detection, and achieve the effects of improving detection sensitivity, low cost, and shortening reaction time

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 rotavirus using RT-LAMP technology at home and abroad

Method used

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  • Kit and oligonucleotide sequences for detecting rotavirus A
  • Kit and oligonucleotide sequences for detecting rotavirus A
  • Kit and oligonucleotide sequences for detecting rotavirus A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Design of RT-LAMP primers

[0043] Select the highly conserved sequence of the NSP3 gene (GenBank accession No.X81436) of group A rotaviruses, and use Primer Explorer V4.0 online software (http: / / primerexplorer.jp / elamp4.0.0 / index.html) to design group A rotavirus The specific RT-LAMP primers of virus, the primers are shown in Table 1. The Tm value of the primers is between 59°C and 62°C; the terminal free energy (G) is less than -4cal / mol, which can ensure sufficient stability of the primers; the GC content is about 40%; 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 rotavirus-specific loop primer (Loop B) designed in the present invention helps to improve the detection sensitivity and shorten the reaction time. 5 primers recognize 7 different sites of the rotavirus NSP3 gene, which fully guarantees the specificity of the amplification reaction. ...

Embodiment 2

[0043] Select the highly conserved sequence of the NSP3 gene (GenBank accession No.X81436) of group A rotaviruses, and use Primer Explorer V4.0 online software (http: / / primerexplorer.jp / elamp4.0.0 / index.html) to design group A rotavirus The specific RT-LAMP primers of virus, the primers are shown in Table 1. The Tm value of the primers is between 59°C and 62°C; the terminal free energy (G) is less than -4cal / mol, which can ensure sufficient stability of the primers; the GC content is about 40%; 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 rotavirus-specific loop primer (Loop B) designed in the present invention helps to improve the detection sensitivity and shorten the reaction time. 5 primers recognize 7 different sites of the rotavirus NSP3 gene, which fully guarantees the specificity of the amplification reaction. Primers were entrusted to Dalian Bao Biological...

Embodiment 3

[0052] Embodiment 3: detect the composition of the kit of A group rotavirus

[0053] 1. The composition of the kit (stored at -20°C)

[0054] (1) TRIZOL lysate: purchased from Invitrogen, product number: 15596-026;

[0055] (2) DEPC water: purchased from Shanghai Sangong, item number: D1005;

[0056] (3) 2×RT-LAMP reaction solution: its components are: 2×ThermoPol buffer, 1.6M betaine, 3.6mMdNTPs, 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 primer (LB); wherein the outer upstream primer ( F3) is the nucleotide sequence shown in the sequence listing SEQ ID No.1, the outer downstream primer (B3) is the nucleotide sequence shown in the sequence listing SEQ ID No.2, and the inner upstream primer (FIP) is the sequence listing SEQ The nucleotide sequence shown in ID No.3, the inner downstream primer (BIP) is the nucleotide sequence shown in the sequence t...

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Abstract

The invention discloses a kit and oligonucleotide sequences for detecting rotavirus A, in particular to a group of oligonucleotides which are used for detecting rotavirus A and have the oligonucleotide sequences shown from the sequence table SEQ ID No.1 to the sequence table SEQ ID No.5, 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, which has 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 present invention relates to a kind of test kit and oligonucleotide for detecting group A rotavirus, more specifically, a kind of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for detecting group A rotavirus The rapid detection kit specifically refers to an oligonucleotide sequence, a kit and a method for rapidly detecting group A rotavirus by utilizing the RT-LAMP principle, and belongs to the field of biotechnology. Background technique [0002] Rotavirus (Rotavirus) belongs to the family Reoviridae and the genus Rotavirus. Spherical, 60-80nm in diameter, double-layered capsid, no envelope, observed under the electron microscope after negative staining, the shape of the virus is wheel-shaped, hence the name rotavirus. It is a double-stranded RNA virus, about 18550bp, composed of 11 gene segments, each segment contains an open reading frame, encoding 6 structural proteins (VP1, VP2, VP3, VP4, VP6, VP7) and 5 non-structura...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 魏海燕曾静王金花周琦张西萌
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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