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Nucleic acid sequence-based amplification-enzyme-linked immunosorbent assay (NASBA-ELISA) kit for type A swine influenza viruses

A technology of swine influenza virus and kit, applied in the field of animal health inspection, can solve problems such as reproductive barriers of sows, slowing down of fattening pigs' weight gain, and economic losses caused by pig farming

Active Publication Date: 2013-06-19
天津市农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, swine flu can also cause reproductive disorders in sows, slow down in fattening pigs, etc., causing huge harm and economic losses to the pig industry.

Method used

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  • Nucleic acid sequence-based amplification-enzyme-linked immunosorbent assay (NASBA-ELISA) kit for type A swine influenza viruses
  • Nucleic acid sequence-based amplification-enzyme-linked immunosorbent assay (NASBA-ELISA) kit for type A swine influenza viruses
  • Nucleic acid sequence-based amplification-enzyme-linked immunosorbent assay (NASBA-ELISA) kit for type A swine influenza viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Reagent composition

[0087] (1) Primers and probes

[0088] NP-DET: 5'-GATGCTAGGTCGCATATGAGTC-3' 2μl

[0089] NP-T7: 5'-AATTCTAATACGACTCACTATAGGGCTGCGGATGCCTTCTGTT-3' 2μl

[0090] Capture probe NP-CP:BIO-TAA GGA CCA GAA GG G 1μl

[0091] Detection probe NP-DP:DIG-GATGCTAGGTCGCATATGAG 1μl

[0092] (2) NASBA Amplification Reagent

[0093] BA (buffer): 40mM / L PH8.5 Tris-HCL, 70mM / L KCl, 12mM / L MgCl 2 , 5mM / L DTT 4μl

[0094] DA: 10mM / L dNTP 2μl

[0095] NA: 20mM / L NTP 2μl

[0096] MA (enzyme mixture): 1~2UAMV, 0.2~0.5URNaseH, 30~50U T7 RNA polymerase, 2~6μl 1mg / mlBSA 4μl

[0097] SA: DMSO 1 μl

[0098] (3) NASBA product detection reagent

[0099] 96-well ELISA plate: streptavidin-coated ELISA plate

[0100] Hybridization buffer: 20×SSPE (pH7.4), 50mM / L Tris-HCl (pH8.8), 1% BSA 43μl

[0101] Enzyme-labeled antibody: alkaline phosphatase anti-digoxigenin antibody 100μl

[0102] Chromogenic solution: 3mg pNPP dissolved in 1mLddH 2 Store 100μl in O, protected fr...

Embodiment 2

[0106] NASBA testing process

[0107] (1) Amplification system: Add 3 μl template RNA, 4 μl BA, 2 μl DA, 2 μl NA, 1 μl SA, 2 μl primer NP-T7, and 2 μl primer NP-DET to the amplification tube.

[0108] (2) NASBA amplification program: heat on a DNA amplification instrument at 65°C for 5 minutes, cool at 41°C for 5 minutes, quickly add 4 μl MA, react at 41°C for 90 minutes; stop the reaction at 4°C.

[0109] (3) Detection of NASBA amplification products: Add 43 μl of hybridization buffer, 1 μl of capture probe, 1 μl of detection probe and 5 μl of NASBA product into the enzyme-labeled well, shake and mix well, and put it in a 41°C incubator for 1~2h reaction . Add 150 μl washing solution to each well, place at room temperature for 3~5min, discard the solution, and wash the plate 3 times repeatedly. Combined with enzyme-labeled antibody Add 100 μl of enzyme-labeled antibody to each well, incubate at room temperature for 30 minutes, and wash the plate 3 times. Color development ...

Embodiment 3

[0110] Embodiment 3: comparative experiment:

[0111] Adopt the contrast of the test kit of measuring swine influenza virus NASBA-ELISA of the present invention and RT-PCR assay method:

[0112] The swine influenza virus A / Swine / Tianjin / TJ1 / 2004 (H1N1) (can be replaced by the swine influenza virus standard strain A / Swine / Coloraelo / 1 / 77 (H1N1) of the Key Laboratory of Animal Influenza, Ministry of Agriculture, Harbin Veterinary Research Institute) 10 -1 -10 -6 RNA was extracted for each dilution, and the kit and RT-PCR were used for detection at the same time.

[0113] 1. Extraction of viral RNA

[0114] (1) Take 200 μl of swine flu venom at each dilution, add 600 μl Trizol Reagent, vortex for 30 seconds, and stand at room temperature for 5 minutes.

[0115] (2) Add 0.2mL of chloroform, vortex for 30s, and stand at room temperature for 5min.

[0116] (3) Centrifuge at 12,000 rpm at 4°C for 10 min, take the upper layer, put it in a new centrifuge tube, add 800 μL of 100% ...

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Abstract

The invention relates to a kit for a nucleocapsid protein (NP) gene of type A swine influenza viruses. The kit consists of a pair of amplification primers, a capture probe, a detection probe, a nucleic acid sequence-based amplification (NASBA) amplification reagent, and an NASBA product detection reagent. An NASBA rapid detection method is established by taking the NP gene of the type A swine influenza viruses as a target gene, and is used for diagnosing the type A swine influenza viruses, so that the technical levels of the diagnosis, epidemic monitoring, and inspection and quarantine of swine influenza in China are improved, and the healthy and rapid development of pig husbandry is guaranteed.

Description

technical field [0001] The invention belongs to the technical field of animal sanitation inspection, relates to a nucleotide and its application, in particular to a type A swine influenza virus NASBA-ELISA detection kit. Background technique [0002] Swine Influenza (SI) is an acute, highly contagious mass respiratory disease caused by Swine Influenza Virus (SIV), clinically characterized by sudden onset, high fever, cough, and dyspnea . The disease spreads fast, and in pigs of all ages and strains, the incidence rate is as high as 100%, and the lethality rate is higher for piglets with imperfect immune systems. In addition, swine flu can also cause reproductive disorders in sows, slow down in fattening pigs, etc., causing huge harm and economic losses to the pig industry. Since swine flu was first reported in the United States in 1918, it has caused multiple pandemics worldwide, and has so far spread to Europe, the United States, Asia, Africa, Australia and other parts of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 鄢明华李秀丽张莉王荣升王东孙英峰韩伟张国伟路超张健池晶晶
Owner 天津市农业科学院
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