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Classical swine fever virus detecting method and kit thereof by nucleic acid isothermal amplication

The technology of a detection kit and detection method, which is applied in the field of nucleic acid amplification detection of swine fever virus, achieves the effects of improved detection sensitivity and specificity, simple and intuitive result observation, and reasonable technical scheme design

Inactive Publication Date: 2010-06-30
SHANGHAI FOSUN PHARMA (GROUP) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no public report on the application of NITAG technology to detect CSFV RNA. Through detection, it can quickly and effectively diagnose swine fever virus, which is suitable for use in the field of inspection and quarantine

Method used

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  • Classical swine fever virus detecting method and kit thereof by nucleic acid isothermal amplication
  • Classical swine fever virus detecting method and kit thereof by nucleic acid isothermal amplication

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation and composition of embodiment 1 swine fever virus NITAG detection kit

[0045] Kit composition

[0046] (1) 2xNITAG reaction buffer

[0047] The concentration of each component in every 10μl 2×NITAG reaction buffer is: 80mM Tris-HCl (pH8.3), 150mM KCl, 24mM MgCl 2 , 2mM dNTPs, 4mM NTPs, 10mM DTT, 30% (v / v) dimethyl sulfoxide (DMSO), two primers (SEQ ID No: 1 and SEQ ID No: 2) each 0.4 μmol / L.

[0048] (2) Mixed enzyme

[0049] Each 5 μl mixed enzyme contains 6.5U reverse transcriptase, 32U T7 RNA polymerase, 0.1U RNaseH enzyme, 25U RNasin Inhibitor and 2.1 μg bovine serum albumin (BSA).

[0050] (3) Probe

[0051] It includes 10 μl of 4 μmol / L biotin-labeled capture probe (SEQ ID No: 5), 20 μl of gold nanoparticle-labeled detection probe (10 μl each of SEQ ID No: 3 and SEQ ID No: 4).

[0052] (4) Chromatographic membrane immobilized with avidin labeling

[0053] Add 2 mg / ml avidin to the fiber membrane or nylon membrane in an amount of 2 μl / cm, and d...

Embodiment 2

[0057] The CSFV RNA NITAG detection of embodiment 2 classical swine fever virus culture

[0058] The implementation of the present invention is exemplified below by introducing the RNA NITAG detection of the classical swine fever virus culture extraction.

[0059] RNA extraction : adopt classical swine fever virus CSFV39 wild strain, through inoculating and cultivating in porcine kidney cell PK-15, the virus liquid measurement titer of harvest (about 10 6 ~10 7 TCID50 / mL, used after half tissue culture infection dose rapid assay method). A certain volume of virus liquid was diluted 10 times with normal saline, and 250 μl was added to 500 μl Trizol extraction reagent, mixed repeatedly with a pipette, left at room temperature for 5 minutes, added 200 μl chloroform, and oscillated back and forth by hand for 15 seconds. Leave at room temperature for 5 minutes, centrifuge at 12,000 rpm at 4°C for 15 minutes, transfer the supernatant to a new centrifuge tube, add 200 μl isopropa...

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Abstract

The present invention relates to a classical swine fever virus (CSFV) detecting method and kit thereof by nucleic acid isothermal amplication. The detection includes the steps in sequence of unwinding the RNA template of CSFV under 60 to 70 DEG C and then cooling, adding with reverse transcriptase, ribonuclease H, RNA polymerase, performing the isothermal amplication under 37 to 42 DEG C conducted by primer to obtain RNA amplicon, then detecting the RNA amplicon by gold nanometer probe and capture probe, and obtaining the detecting result through chromatography cross-chromogenic reaction. The RNA is positive if chromogenic stripe appears on the cross film and the RNA is negative or else. The kit of the method can detect the RNA of CSFV quickly and accurately.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection technique for nucleic acid amplification of swine fever virus. Background technique [0002] Classical Swine Fever (CSF), also known as Hog Cholera (HC), is an acute, febrile, highly contagious infectious disease caused by Classical Swine Fever Virus (CSFV). Extensive hemorrhage, infarction, and necrosis are caused by acute course, high fever retention, high mortality, and degeneration of small vessel walls. Since swine fever was first discovered in Ohio, USA in 1833, reports of swine fever have gradually increased around the world. In recent years, although various measures have been taken to control swine fever to a certain extent, new features have appeared in its prevalence and incidence, and the harm to the pig industry is still very serious. At present, the early specific diagnosis of swine fever disease, the killing of infected animals and vaccination...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/76
Inventor 吴大治夏懿
Owner SHANGHAI FOSUN PHARMA (GROUP) CO LTD
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