Swine acute diarrhea syndrome coronavirus primer combination and kit and method thereof

A primer composition, a technology for acute diarrhea, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve problems such as only 86% consistency, and achieve low cost of the instrument, improved sensitivity, and extended expansion. high efficiency

Inactive Publication Date: 2018-08-17
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The genome sequence analysis of SADS coronavirus provides clues for finding the source of the virus: SADS virus is highly similar to the bat coronavirus HKU2 genome sequence first discovered by the University of Hong Kong in 2007, with a full-length sequence identity of 95%, but the envelope protein ( S protein) amino acid sequence identity is only 86%

Method used

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  • Swine acute diarrhea syndrome coronavirus primer combination and kit and method thereof
  • Swine acute diarrhea syndrome coronavirus primer combination and kit and method thereof
  • Swine acute diarrhea syndrome coronavirus primer combination and kit and method thereof

Examples

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Effect test

Embodiment 1

[0036] A kind of porcine acute diarrhea syndrome coronavirus primer composition provided by the present invention comprises forward inner primer SADS-FIP, reverse inner primer SADS-BIP, forward outer primer SADS-F3, reverse outer primer SADS-B3, Composition of forward loop primer SADS-LF and reverse loop primer SADS-LB;

[0037] The nucleotide sequence of the primer SADS-F3 is shown in SEQ ID NO: 1;

[0038] The nucleotide sequence of the primer SADS-B3 is shown in SEQ ID NO: 2;

[0039] The nucleotide sequence of the primer SADS-FIP is shown in SEQ ID NO: 3;

[0040] The nucleotide sequence of the primer SADS-BIP is shown in SEQ ID NO: 4;

[0041] The nucleotide sequence of the primer SADS-LB is shown in SEQ ID NO:5.

[0042] The nucleotide sequence of the primer SADS-LF is shown in SEQ ID NO: 6;

Embodiment 2

[0044] An RT-LAMP detection kit for SADS of the present invention, the kit contains the RT-LAMP primer composition of Example 1.

Embodiment 3

[0046] The method for detecting porcine acute diarrhea syndrome coronavirus provided by the invention, used material is as follows:

[0047] DNA / RNA Nucleic Acid Extraction Kit TGUide Virus DNA / RNA Kit Kit (Catalog #Q5724 Tiangen Biochemical Technology Co., Ltd.)

[0048] Nucleic acid automatic extractor T-Guide OSE-M48 (Tiangen Biochemical Technology Co., Ltd.)

[0049] Constant temperature fluorescence detector DEAOU-308C (Guangzhou Diao Biotechnology Co., Ltd.)

[0050] Bst2.0 Polymerase Bst 2.0 (WarmStart DNA Polymerase Cat. No. #M5038L NEW ENGLANGBioLaBs)

[0051] 100mM magnesium sulfate solution 100mM (MgSO 4 solution Cat. No. #B1003S NEW ENGLANG BioLaBs)

[0052] 10× reaction buffer Isothermal Amplification Buffer (product number #B0537S NEW ENGLANGBioLaBs company), components: 20mM Tris-HCl, 10mM (NH4)2SO4, 2mM MgSO4, 50mM KCl, 0.1% Tween20, pH 8.8@25°C.

[0053] dNTP Mix (Product No. D7373 Biyuntian Biotechnology Co., Ltd.)

[0054] Betaine (Product No. BCBS087V SI...

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PUM

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Abstract

The invention discloses a swine acute diarrhea syndrome coronavirus primer combination and a kit and method thereof. The swine acute diarrhea syndrome coronavirus primer combination and the kit and method thereof aim at providing an SADS (swine acute diarrhea syndrome) detecting primer and method high in sensitivity and specificity. The swine acute diarrhea syndrome coronavirus primer combinationis composed of a forward inner primer of SADS-FIP, a backward inner primer of SADS-BIP, a forward outer primer of SADS-F3, a backward outer primer of SADS-B3, a forward loop primer of SADS-LF and a backward loop primer of SADS-LB. The detecting method of the swine acute diarrhea syndrome coronavirus primer combination comprises 1) extracting RNA (ribonucleic acid) of virus from a sample; 2) takingthe extracted RNA as a template, and establishing an RT-LAMP(reverse transcription-loop-mediated isothermal amplification) system through the RT-LAMP primer combination of SADS to perform RT-LAMP ata constant temperature of 60-65 DEG C; 3) detecting amplification products through a DEAOU-308C thermostatic fluorescence detector. The swine acute diarrhea syndrome coronavirus primer combination andthe kit and method thereof belong to the field of biological detecting technology.

Description

technical field [0001] The invention discloses a primer composition, specifically, a RT-LAMP primer composition for detecting porcine acute diarrhea syndrome coronavirus (SADS), and the invention also discloses a method for detecting porcine acute diarrhea syndrome coronavirus Kit and method; belonging to the technical field of biological detection. Background technique [0002] At the end of October 2016, a lethal piglet disease broke out in a pig farm in Qingyuan, Guangdong Province. The piglets showed severe acute diarrhea, vomiting, and rapid weight loss. The mortality rate of piglets under 5 days old was as high as 90%. Three other farms subsequently had outbreaks. As of May 2017, a total of 24,693 piglets had died. According to the clinical symptoms, the researchers tested the samples of sick pigs for porcine epidemic diarrhea virus, transmissible gastroenteritis virus and other known porcine diarrhea-related viruses. However, at the height of the outbreak, all test...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107Y02A50/30
Inventor 王华南黄韧丛锋曾繁文郭鹏举王乐烽
Owner ZHEJIANG UNIV
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