A set of isothermal amplification primers for rapid detection of bovine and sheep Akabane disease virus and its application

A technology for isothermal amplification of Akabane disease virus, applied in the field of warm amplification primers, can solve the problems of insufficient sensitivity and high cost, and achieve the effects of good sensitivity, high specificity, and high amplification efficiency

Inactive Publication Date: 2016-01-13
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the technical deficiencies such as insufficient sensitivity and high cost of the existing methods for detecting Akabane disease virus in cattle and sheep, and to provide a safe, simple, fast and more sensitive ring-mediated isothermal amplification method for detecting cattle, sheep, and sheep. Method for sheep Akabane disease virus

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  • A set of isothermal amplification primers for rapid detection of bovine and sheep Akabane disease virus and its application
  • A set of isothermal amplification primers for rapid detection of bovine and sheep Akabane disease virus and its application
  • A set of isothermal amplification primers for rapid detection of bovine and sheep Akabane disease virus and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The design and synthesis of embodiment 1 primer

[0045] According to the conserved fragment sequence of Akabane disease virus S gene, two pairs of specific outer primers and inner primers were designed using PrimerExplorerV4 (https: / / primerexplorer.jp / lamp4.0.0 / index.html) gene analysis software, without the need to design loop primers. The outer primers are F3 and B3, and the nucleotide sequences are shown in SEQ ID NO.1-2; the inner primers are FIP and BIP, and the nucleotide sequences are shown in SEQ ID NO.3-4.

[0046] SEQ ID NO.1 (F3):

[0047] 5'–TGCAAATCCAGTGTCAGACA-3';

[0048] SEQ ID NO.2 (B3):

[0049] 5'–GGGGCAAATCCCAGGTAC-3';

[0050] SEQ ID NO.3 (FIP):

[0051] 5'-CTTGCACTGCTCAGCAACCCATGCCTTTACGCTTCACCG-3';

[0052] SEQ ID NO.4 (BIP):

[0053] 5'-GCAGAGGCAGCTGCCACAATTGCATACCCCGTCACTCCAG-3'.

[0054] All primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

Embodiment 2

[0055] The preparation of embodiment 2 template

[0056] 1. Viral RNA extraction

[0057] After the bovine and sheep Akabane disease virus (the virus was donated by the MacArthur Institute of Agriculture, Australia) was propagated by cell culture, 200 μL of the culture solution was added to a clean 1.5mL centrifuge tube, and the virus was extracted according to the instructions of the TIANGEN nucleic acid extraction kit. RNA.

[0058] Viral RNA was directly reverse-transcribed or frozen at -20°C for later use.

[0059] 2. Reverse transcription

[0060] According to the instruction manual of ReverseTranscriptaseXL (AMV) reverse transcriptase from Treasure Bio (Dalian) Co., Ltd., the first-strand cDNA synthesis (RT) of the target gene was carried out. Add the following components to the 20 μL reverse transcription system:

[0061] 5×AMVBuffer 4μL;

[0062] dNTPs (10 pmol / μL) 2 μL;

[0063] Reverse transcription primer (30pmol / μL) 1μL;

[0064] Rnasin (20U) 1 μL;

[0065]...

Embodiment 3

[0068] Embodiment 3 The establishment of ring-mediated isothermal amplification reaction system

[0069] 1. Through a large number of experimental explorations, an optimal loop-mediated isothermal amplification reaction system and reaction conditions were established.

[0070] The specific components and content of the optimal reaction system are as follows (25 μL):

[0071] BstDNA polymerase (8U / μL) 1 μL;

[0072] BstDNA polymerase buffer (10×) 2.5 μL;

[0073] Template 2 μL;

[0074] MgSO4 (25mmol / μL) 2.5μL;

[0075] Betaine (0.4mol / μL) 2.5μL;

[0076] dNTP (25mmol / μL) 2.5μL;

[0077] BIP / FIP (20pmol / μL) 2μL;

[0078] F3 / B3 (10 pmol / μL) 0.5 μL;

[0079] h 2 O9.5 μL.

[0080] The reaction conditions are as follows: 55 min at 63°C, 1 min at 98°C.

[0081] After the reaction, electrophoresis detection was carried out, and the electrophoresis was carried out at a constant voltage of 120V for 20-30 minutes, and the results were observed.

[0082] 2. Amplification resul...

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Abstract

The invention discloses a group of isothermal amplification primers for rapid detection of cattle and sheep Akabane disease virus and application thereof. The nucleotide sequences of the primers are respectively as SEQ? ID? Shown in NO.1~4. In the present invention, the cDNA obtained by sample RNA reverse transcription is used as a template, and the above-mentioned primers are used for loop-mediated isothermal amplification detection. After the reaction, the result can be judged in two ways: one is that the amplified product is subjected to electrophoresis, and a specific ladder-like appearance occurs. The sample of the strip is judged as positive; the second is by adding calcein to the reaction system, and judged by naked eyes, the sample with color change is judged as positive for bovine and sheep Akabane disease virus. The isothermal amplification method for detecting Akabane disease virus in cattle and sheep by using the primers in the present invention has low requirements on instruments and equipment, is fast, safe, has good specificity, and has strong sensitivity. The quarantine unit has provided technical support for the rapid detection of cattle and sheep, which has very good promotion and application value.

Description

technical field [0001] The invention belongs to the technical field of virus molecular biology detection. More specifically, it relates to a group of isothermal amplification primers for rapid detection of cattle and sheep Akabane disease virus and its application. Background technique [0002] Akabane disease (Akabane virus, AKAD), also known as Akabane disease, is a polymorphic viral infectious disease of cattle, sheep and goats caused by Akabane virus (AKAV). Symptoms of the disease mainly include miscarriage, premature birth, stillbirth, fetal malformation, mummification, etc., which can cause congenital arthrogryposis and hydrocephalus syndrome. The disease is an insect-borne infectious disease, and the vectors are blood-sucking mosquitoes and Culicoides. Akabane disease is a legally notifiable disease of the World Organization for Animal Health (OIE), and it is also one of the seven diseases that must be tested for cattle and sheep imported from abroad in my country....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/70C12Q2531/119C12Q2563/173
Inventor 鱼海琼贾坤罗长保赵吟田纯见吴晓薇陈芳罗琼刘志玲张敏
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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