Nucleic-acid sequence-based amplification (NASBA) method for detecting swine influenza virus (SIV)

A swine flu virus and a pair of technology, applied in the field of animal health inspection, can solve the problems of exacerbation of disease and increase of fatality rate

Inactive Publication Date: 2012-07-04
天津市宁河原种猪场有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The case fatality rate is low when it is infected alone, but the disease is often mixed with other infectious disea...

Method used

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  • Nucleic-acid sequence-based amplification (NASBA) method for detecting swine influenza virus (SIV)
  • Nucleic-acid sequence-based amplification (NASBA) method for detecting swine influenza virus (SIV)
  • Nucleic-acid sequence-based amplification (NASBA) method for detecting swine influenza virus (SIV)

Examples

Experimental program
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Effect test

Embodiment 1

[0102] (1) Extraction of viral RNA: Take 200 mL of chicken embryo allantoic fluid of swine influenza virus and add it to a 1.5 mL centrifuge tube, add 600 μl Trizol solution, shake and mix, let stand at room temperature for 5 minutes, then add 200 μl chloroform, shake and mix, and let stand at room temperature 5min, centrifuge at 12000rpm at 4℃ for 10min; take the supernatant into a 1.5mL centrifuge tube, add 800μl of isopropanol containing 1‰DEPC, mix by inverting, centrifuge at 12000rpm for 10min, discard the supernatant, add 500μl of isopropanol containing 1‰DEPC 70% ethanol, centrifuge at 12000rpm at 4°C for 10min, discard the ethanol, dry at room temperature, add 20 μl DEPC water to dissolve, and store at -80°C for later use.

[0103] (2) NASBA amplification system:

[0104] 3 μl RNA of the sample to be tested

[0105] Buffer 4μl (containing 40mM / L PH8.5 Tris-HCL, 70mM / L KCl, 12mM / L MgCl 2 , 5mM / L DTT buffer)

[0106] 10mM / L dNTP 2μl

[0107] 20mM / L NTP 2μl

[0108] ...

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Abstract

The invention discloses a nucleic-acid sequence-based amplification (NASBA) method for detecting a swine influenza virus (SIV). The NASBA method mainly comprises the following steps of: extracting the ribonucleic acid (RNA) from the SIV, preparing an NASBA system, preparing an NASBA program, carrying out identification on an NASBA product and carrying out PCR (Polymerase Chain Reaction) amplification. According to the NASBA method, the NASBA rapid detection method is established by taking the NP gene of the SIV as a target gene and is used for the diagnosis of the SIV. The method has higher specificity and sensitivity so as to detect a virus solution with the dilution factor of 10<-5>, so that the method can be used for the rapid detection of clinically-suspected SIV samples, meanwhile, the technical level of China in the diagnosis, epidemic surveillance, inspection and quarantine of swine influenza can be improved so as to ensure the healthful and rapid development of swine industry.

Description

Technical field [0001] The invention is the field of animal health inspection technology, involving a NASBA method for detecting pig influenza viruses. Background technique [0002] Swine Influenza (SI) is caused by the influenza virus (SIV) in the influenza virus (SIV). It is one of the pork tract diseases that are commonly existed and difficult to eradicate.The typical clinical symptoms of the disease are fever, anorexia, weight loss, mental slump, runny nose, cough, and difficulty breathing.The mortality rate is low during infection, but the disease is often mixed with other infectious diseases, such as infectious pleural pneumonia, which increases the disease and greatly improves the mortality of the disease. [0003] There are two types of common influenza viruses: one is salivary acid α-2,3 lactose (SAα-2,3-Gal), and the other is salivary acid α-2,6 galactose (SAα-2, 6-Gal).HA receptor specificity of different influenza viruses is different. Most bird flu viruses are prefer...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 鄢明华张莉张荣升崔尚金李秀丽吴莹高荣玲张国伟任卫科王志军
Owner 天津市宁河原种猪场有限责任公司
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