Real-time fluorescence RT-HDA (Reverse Transcriptase-Helicase-Dependent Isothermal Amplification) kit and primer for detecting foot-and-mouth disease virus

A foot-and-mouth disease virus and real-time fluorescence technology, applied in the field of inspection and quarantine, to achieve the effect of fast response, easy to grasp and operate, and simple program setting

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

At present, international research on real-time fluorescent HDA technology is still in its infancy, and there is no research on real-time fluorescent HDA detection of animal viruses at home and abroad.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Design and synthesis of HDA primers

[0054] 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

[0055] Embodiment 2: the extraction of foot-and-mouth disease virus RNA

[0056] FMDV RNA was extracted using RNA extraction reagents.

[0057] The specific operation is as follows:

[0058] 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.

[0059] 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.

[0060] 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

[0065] Example 3: Establishment of real-time fluorescent RT-HDA

[0066] 1. Concentration of primers, MgSO 4 The concentration and NaCl concentration were optimized respectively, and the reaction system of real-time fluorescent RT-HDA was established.

[0067] Obtain foot-and-mouth disease virus RNA according to embodiment 2 method, template concentration is about 10 4 copy / microliter, perform real-time fluorescent 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.

[0068] Table 2 The optimal range of each component in the FMDV RT-HDA reaction system

[0069] ingredient name

concentration range

increment interval

[0070] 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

[0071] Table 3 Optimizati...

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Abstract

The invention discloses a real-time fluorescence RT-HDA (Reverse Transcriptase-Helicase-Dependent Isothermal Amplification) kit and primer for detecting foot-and-mouth disease virus. A group of primers for detecting the foot-and-mouth disease virus have an oligonucleotides sequence shown by a sequence table SEQ ID No:1 and a sequence table SEQ ID No:2. The kit and the primer have the following advantages that the novel HAD technology is applied to the detection of the foot-and-mouth disease virus, and compared with the existing FMDV (Foot-And-Mouth Disease Virus) detecting method, the HDA detecting method has the advantages of fastness, more convenience, safety and reliability; for the conventional virus separation and serology method, long time is consumed, the bio-safety risk exists; for the HDA method, a natural reproduction mechanism in a body is simulated, the safety is high, the reaction speed is quick, and the detection can be completed by only 2 hours at most; and compared with fluorescence PCR (Polymerase Chain Reaction), the HDA detecting method has the advantages that the program setting is easier, the reaction conditions such as annealing temperature and the like do not need to be explored, and the HDA detecting method is easier to master and operate in the aspect of technical level. Due to the optimization of reaction conditions, according to the real-time fluorescence RT-HDA method, at least 10copies / reaction of viral nucleic acid can be detected. The technology has a great application prospect in the monitoring and diagnosis of the foot-and-mouth disease virus, and the kit and the primer are diagnosis tools with good promotion and application values.

Description

technical field [0001] The invention relates to a real-time fluorescent 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 anim...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 蒲静高志强张鹤晓谷强乔彩霞刘环张伟汪琳
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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