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RT-HDA (Reverse Transcription-Helicase-Dependent Isothermal Amplification) kit and primer for detecting foot-and-mouth disease virus

A foot-and-mouth disease virus and kit technology are applied in the field of inspection and quarantine to achieve the effects of simple equipment requirements, easy popularization and application, easy mastery and operation

Active Publication Date: 2014-05-28
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

[0007] At present, international research on HDA technology is still in its infancy, and there is no research on HDA detection of animal viruses at home and abroad.

Method used

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  • RT-HDA (Reverse Transcription-Helicase-Dependent Isothermal Amplification) kit and primer for detecting foot-and-mouth disease virus
  • RT-HDA (Reverse Transcription-Helicase-Dependent Isothermal Amplification) kit and primer for detecting foot-and-mouth disease virus
  • RT-HDA (Reverse Transcription-Helicase-Dependent Isothermal Amplification) kit and primer for detecting foot-and-mouth disease virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Design and synthesis of HDA primers

[0052] According to the foot-and-mouth disease virus gene sequence registered in Genbank, DNAMAN software was used for comparison and analysis, and the highly conserved region of FMDV-5'UTR gene was selected, and the online software Primer3 (http: / / frodo.wi.mit.edu / ) was used for primers Design, analyze and evaluate primers with Oligo6.0 software, and design general primers for foot-and-mouth disease virus. Primer sequences are the same as Table 1. Primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

Embodiment 2

[0053] Embodiment 2: the extraction of foot-and-mouth disease virus RNA

[0054] FMDV RNA was extracted using RNA extraction reagents.

[0055] The specific operation is as follows:

[0056] ①Take n 1.5mL sterilized Eppendorf tubes, where n is the sum of the number of samples to be tested, one tube of positive control and one tube of negative control, and mark each tube with a number.

[0057] ② Add 600 μL TRIZOL lysate to each tube, then add 200 μL each of the sample to be tested, negative control and positive control, and use a tip for each sample; then add 200 μL of chloroform, shake and mix for 5 seconds on a mixer. Centrifuge at 12000r / min for 15min at 4°C.

[0058] ③ Take the same number of 1.5mL sterilized Eppendorf tubes as in ①, add 500 μL of isopropanol (pre-cooled at -20°C), and number each tube. 5.3.2 After centrifugation, transfer the supernatant in each tube to the corresponding tube, absorb at least 500 μL of the supernatant, be careful not to suck out the mi...

Embodiment 3

[0063] Embodiment 3: the establishment of RT-HDA

[0064] 1. Concentration of primers, MgSO 4 The concentration, NaCl and reverse transcriptase concentration were optimized respectively, and the reaction system of RT-HDA was established.

[0065] Obtain foot-and-mouth disease virus RNA according to embodiment 2 method, template concentration is about 10 4 copy / microliter, perform RT-HDA, and optimize the reaction system. The optimized range of each component is shown in Table 2, the reaction system is shown in Table 3, and the primer sequences used are shown in Table 1.

[0066] Table 2 The optimal range of each component in the FMDV RT-HDA reaction system

[0067] ingredient name

concentration range

increment interval

Primer I and Primer II (both 5 μM)

50~200nM

25nM

MgSO 4 (100mM)

3.0~4.5mM

0.5mM

NaCl (500mM)

20~50mM

5mM

[0068] Table 3 Optimization of FMDV RT-HDA reaction system

[0069]

[0070]...

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Abstract

The invention discloses an RT-HDA (Reverse Transcription-Helicase-Dependent Isothermal Amplification) kit and primers for detecting a foot-and-mouth disease virus. The s for detecting a foot-and-mouth disease virus are oligonucleotide sequences shown in the sequence list SEQ ID No:1 and the sequence table SEQ ID No:2. In the invention, the novel isothermal amplification technology HDA is applied to the detection of the foot-and-mouth disease virus for the first time. Compared with the traditional FDMV detection method, the HDA detection method is quicker, more convenient, safer and more reliable. The traditional virus isolation and serological method has long time consumption and biosafety risk. The HDA method imitates the natural replication mechanism in the body and has high safety and quick response, and only 2 h are needed at most to complete detection. Compared with PCR (Polymerase Chain Reaction), the HDA method does not need any PCR instrument, the equipment requirement is simple, only one water bath kettle is needed, and the HDA method is easier to master and operate speaking from the technology level, is easier to be popularized and applied to regions with insufficient conditions and is more conducive to the diagnosis and monitoring of the foot-and-mouth disease virus.

Description

technical field [0001] The invention relates to an RT-HDA kit and primers for detecting foot-and-mouth disease virus, belonging to the field of inspection and quarantine. Background technique [0002] Foot-and-mouth disease (FMD) is an acute febrile highly contagious infectious disease caused by Foot-and-mouth disease virus (FMDV). It mainly infects artiodactyls. The mouth, tongue, and Blisters occur on lips, hooves, breasts and other parts, and ulceration forms rotten spots. Humans and non-cloven-hoofed animals can also be infected with the disease, but the symptoms are milder. Because pigs, cattle, sheep and other major domestic animals can be infected with the disease, and can form a large-scale global epidemic, the World Organization for Animal Health (OIE) and the Food and Agriculture Organization of the United Nations (FAO) have listed the disease as a category A zoonotic disease. In my country, foot-and-mouth disease is listed as a class of animal infectious disease...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 蒲静乔彩霞张伟高志强刘环汪琳谷强张鹤晓
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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