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Kit for detecting wild strain of classical swine fever virus

A technology of swine fever virus and wild strains, applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of specificity, poor sensitivity, low sensitivity, time-consuming and labor-intensive, etc., to achieve highly specific effect

Active Publication Date: 2019-12-03
BEIJING UNIV OF AGRI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the isolation and identification of viruses is cumbersome, time-consuming and labor-intensive, and there are cross-reactions in serum experiments such as ELISA, and the sensitivity is low.
Although colloidal gold test paper is convenient, its specificity and sensitivity are poor, and it is prone to false positives
[0005] Conventional PCR technology has the following disadvantages: 1) The instrument is expensive; 2) The operation is cumbersome; 3) It takes a long time, thus limiting its promotion and use at the grassroots level
Hybridization chip method, high cost, complicated operation, relying on more expensive instruments

Method used

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  • Kit for detecting wild strain of classical swine fever virus
  • Kit for detecting wild strain of classical swine fever virus
  • Kit for detecting wild strain of classical swine fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, screening and preparation of primer combinations

[0078] 1. Design multiple sets of primers and probe combinations for the envelope glycoprotein E2 gene of the wild strain of classical swine fever virus. Through experiments, the combination of primers and probes for CSFV-5 was selected as the group with the best detection effect. Synthesized by Industrial Bioengineering (Shanghai) Co., Ltd.

[0079] Table 2 is used to detect 5 sets of primers and probe set sequences of CSFV wild strain

[0080]

[0081]

[0082] In the combination of primers and probes mentioned above, each single-stranded DNA is packaged independently.

[0083] In the above combination of primers and probes, the final concentrations of primers, probes, and agarose are 0.2 μM, 50 nM, and 0.1% (mass percentage).

[0084] 2. Using the RNA of the wild strain of CSFV as a template, use the primers and probe sets prepared in step 1 to carry out isothermal amplification detection on the...

Embodiment 2

[0089] Embodiment 2, the kit specificity analysis that is used to detect swine fever virus wild strain

[0090] Using CSFV field strain (CSFV), CSFV vaccine strain (CSFV-C), highly pathogenic porcine blue ear virus (HP-PRRSV) and porcine blue ear virus classic strain (PRRSV) as templates, the The CSFV-3 primer probe set was used for detection.

[0091] Mix 7 μL constant temperature amplification buffer, 1 μL constant temperature amplification enzyme solution, and 2 μL sample RNA solution to be tested to form 10 μL reaction solution, vortex and oscillate to mix, then place in a real-time fluorescent PCR instrument, set at 41°C, and perform constant temperature amplification reaction for 1 hour. At the same time, real-time fluorescence scanning is completed.

[0092] If a positive amplification curve occurs within 45 minutes (that is, the amplification curve is a typical inverted "S-type" amplification curve), it indicates that there is CSFV in the reaction system.

[0093] If...

Embodiment 3

[0095] Embodiment 3, the kit sensitivity analysis that is used to detect classical swine fever virus wild strain

[0096] 1. Preparation of reference substance templates with different concentrations

[0097] The above-mentioned purified classical swine fever virus wild virus RNA template was quantified and serially diluted to obtain 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 copies / μL template diluent.

[0098] The copy number calculation formula is as follows:

[0099] (6.02×10 23 copies / mole)×(x ng / μl×10 -9 ) / (RNA base number×340)=copies / μl.

[0100] Wherein, x ng / μl=(OD260)×(dilution factor)×(40).

[0101] 2. Reaction system preparation

[0102] Take 7 μL of constant temperature amplification buffer, 1 μL of constant temperature amplification enzyme solution, and 2 μL of sample RNA solution to be tested (diluted template) and mix them into 10 μL of reaction solution, and vortex to mix.

[0103] 3. Isothermal amplification reaction and dete...

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Abstract

The invention relates to a method and kit for detecting a wild strain of a classical fever virus. The method for detecting the wild strain of the classical swine fever virus includes the following steps that (1), a detection sample is obtained, specifically, wild strain RNA of the classical swine fever virus is extracted, and the RNA concentration is diluted to 10 copies / [mu]L after extraction; (2), a reaction system is prepared, specifically, 7[mu]L of a constant temperature amplification buffer solution, 1[mu]L of a constant temperature amplification enzyme solution and 2[mu]L of a RNA solution diluted in the step (1) are taken and mixed into 10 [mu]L of a reaction solution, and the reaction solution is evenly mixed through vortex vibration; (3), nucleic acid sequence based amplification(NASBA) and detection are carried out, specifically, the reaction solution is put into a real-time fluorescent PCR instrument, an NASBA primer group and a probe group are used, the temperature is setto be 41 DEG C, constant temperature amplification reaction is carried out for 1h, and meanwhile, real-time fluorescent scanning is completed; and (4) a result is judged, specifically, when a typicallying-S type amplification curve is detected, the detecting result is positive.

Description

Technical field: [0001] The invention relates to a virus detection method, in particular to a detection method of a wild strain of swine fever virus and a kit thereof. Background technique: [0002] Classical swine fever (CSF) is an acute, febrile, highly contagious infectious disease caused by classical swine fever virus (CSFV), commonly known as "hog cholera". Swine fever is distributed worldwide and has a high degree of harm, which has brought serious economic losses to the pig industry. The World Organization for Animal Health (OIE) listed it as a class A infectious disease and stipulated it as an international key quarantine object. Listed as a first-class infectious disease, it is one of the four major diseases that are subject to compulsory immunization in my country. [0003] Classical swine fever virus is a member of the genus Pestivirus of the family Flaviviridae, and its genome is a single-stranded positive-strand RNA with a length of about 12.3 kb. The two ends ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2561/113C12Q2563/107Y02A50/30
Inventor 张岩李建东马银平牟海青
Owner BEIJING UNIV OF AGRI
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