Isothermal amplification primer group capable of rapidly detecting cattle and sheep akabane disease viruses and application thereof

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

Inactive Publication Date: 2014-06-04
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

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  • Isothermal amplification primer group capable of rapidly detecting cattle and sheep akabane disease viruses and application thereof
  • Isothermal amplification primer group capable of rapidly detecting cattle and sheep akabane disease viruses and application thereof
  • Isothermal amplification primer group capable of rapidly detecting cattle and sheep akabane disease viruses and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Design and synthesis of primers

[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 PrimerExplorer V4 (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] Example 2 Preparation of 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 Reverse Transcriptase XL (AMV) reverse transcriptase from Bao Biology (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×AMV Buffer 4μL;

[0062] dNTPs (10pmol / μL) 2μL;

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

[0064] Rnasin (20U) 1μL;

[0065] V...

Embodiment 3

[0068] Example 3 Establishment of a loop-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 contents of the optimal reaction system are as follows (25 μL):

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

[0072] Bst DNA 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 (10pmol / μL) 0.5μL;

[0079] h 2 O 9.5 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 result...

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Abstract

The invention discloses an isothermal amplification primer group capable of rapidly detecting cattle and sheep akabane disease viruses and application thereof. Nucleotide sequences of the isothermal amplification primer group are respectively shown as SEQIDNo.1-4. According to the invention, cDNA obtained through reverse transcription on RNA (ribonucleic acid) of a sample is taken as a template, the isothermal amplification primer is utilized for carrying out loop-mediated isothermal amplification detection, results can be judged in the following two ways after reaction is finished: electrophoresis is carried out on amplification products, and the sample with a specific stair-step strip is judged to be positive; calcein is added into a reaction system, judging is carried out by virtue of naked eyes, and the sample with change in colour is judged to carry positive cattle and sheep akabane disease viruses. The isothermal amplification method for detecting cattle and sheep akabane disease viruses by utilizing the isothermal amplification primer is low in requirement on instrument and equipment, rapid, safe, good in specificity and strong in sensitivity, provides a technical support for cattle and sheep akabane disease detection in the animal husbandry, especially rapid detection on cattle and sheep in a basic quarantine department, and has good popularization 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 (AKAD), also known as Akabane spot 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. Studies ...

Claims

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Application Information

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Patent Type & Authority Applications(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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