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RT-HDA kit and primer for detecting avian influenza virus

An avian influenza virus and kit technology, applied in the field of inspection and quarantine, can solve the problems of being easily affected by various factors and the high cost of fluorescent PCR instruments, and achieve the effects of being beneficial to diagnosis and monitoring, easy to popularize and apply, and quick to respond.

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

However, there are still some shortcomings in the traditional PCR technology: expensive equipment is needed, especially the fluorescent PCR instrument is expensive; it is easily affected by many factors; the amplification time often takes several hours
[0008] 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 kit and primer for detecting avian influenza virus
  • RT-HDA kit and primer for detecting avian influenza virus
  • RT-HDA kit and primer for detecting avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Design and synthesis of HDA primers

[0053] According to the gene sequence of avian influenza virus registered in Genbank, DNAMAN software was used for comparative analysis, and the highly conserved region of AIV-M gene was selected, and the online software Primer3 (http: / / frodo.wi.mit.edu / ) was used for primer design , and combined with Oligo6.0 software to analyze and evaluate the primers, and design general primers for avian influenza virus. Primer sequences are the same as Table 1. Primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

Embodiment 2

[0054] Embodiment 2: the extraction of avian influenza virus RNA

[0055] Avian influenza virus RNA was extracted using RNA extraction reagents.

[0056] The specific operation is as follows:

[0057] 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.

[0058] 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.

[0059] 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 ...

Embodiment 3

[0064] Embodiment 3: the establishment of RT-HDA

[0065] 1. Concentration of primers, MgSO 4 The concentration and NaCl concentration were optimized respectively, and the reaction system of RT-HDA was established.

[0066] Obtain bird flu 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 sequences of the primers and probes used are shown in Table 1.

[0067] The optimized range of each component in the reaction system of table 2AIV RT-HDA

[0068]

[0069] The optimization of table 3AIV RT-HDA reaction system

[0070]

[0071] 2. Results:

[0072] After many experiments, the final primer concentration was determined to be 100nM; MgSO 4 The concentration was determined to be 4.0 mM; the NaCl concentration was 40 mM.

[0073] figure 1 After optimizing the RT-HDA...

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Abstract

The invention discloses an RT-HDA kit for detecting avian influenza virus and a primer. The primer for detecting the avian influenza virus is oligonucleotides sequences shown in a sequence table SEQ ID (Sequence ID) NO: 1 and a sequence table SEQ ID NO:2. According to the RT-HDA kit, a novel constant-temperature amplifying technology HDA is used for detecting the avian influenza virus for the first time; compared with a conventional detection method of AIV (Avian Influenza Virus), the RT-HAD kit has the advantages that: (1) an HDA detection method is faster, more convenient, safer and more reliable. A conventional method of separating virus and a conventional serology method need more time, and bring a risk to safety of an organism; the HDA method simulates a natural replication mechanism in vivo, so that the safety is high, a reaction is fast to carry out, and detection can be accomplished by only two hours; and (2) compared with a PCR (Polymerase Chain Reaction), the HDA detection method has the advantages that PCR instruments are saved, a requirement on equipment is simple, only one water bath kettle is needed, so that the technology is convenient to master and operate, the detection method can be convenient to popularize and use in regions with insufficient conditions, and benefits are provided for diagnosing and monitoring the avian influenza virus.

Description

technical field [0001] The invention relates to an RT-HDA kit and primers for detecting avian influenza virus, belonging to the field of inspection and quarantine. Background technique [0002] Avian Influenza (AI) is an infection and / or disease syndrome in poultry caused by Orthomyxoviridae, Influenzavirus, and Influenza A viruses. Avian influenza has a long history. It was first discovered in 1878. Perroncito first reported the occurrence of avian influenza in Italy. It has been listed as a severe infectious disease by the International Veterinary Office (OIE). As one of the most important diseases with potential threats, my country has also listed highly pathogenic avian influenza (HPAI) as a first-class animal infectious disease for prevention and control. In recent years, there have been avian influenza epidemics in many areas of my country, which have brought different degrees of blows to the poultry industry. So far, there have been many fatal incidents of highly path...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 蒲静张伟刘环乔彩霞高志强汪琳谷强
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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