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Kit for rapidly detecting Seneca virus and detection method of kit

A kit and virus technology, which is applied in the field of rapid detection of Seneca virus kit and its detection, can solve the problems of difficult differential diagnosis, disease diagnosis, treatment and prevention and control, and achieve accurate diagnosis, easy operation and sensitive sex high effect

Active Publication Date: 2018-11-13
CHINA AGRI VET BIO SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The object of the present invention is to provide a kind of test kit and detection method thereof for rapid detection of Seneca virus, aiming to solve the above-mentioned background technology after pigs are infected with A-type Seneca virus, the clinical symptoms caused by it are related to foot-and-mouth disease and pig blisters. The clinical symptoms caused by porcine vesicular disease, swine herpes and vesicular stomatitis infection are similar, which makes it difficult to differentiate clinically and brings great difficulties to the diagnosis, treatment, prevention and control of diseases.

Method used

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  • Kit for rapidly detecting Seneca virus and detection method of kit
  • Kit for rapidly detecting Seneca virus and detection method of kit
  • Kit for rapidly detecting Seneca virus and detection method of kit

Examples

Experimental program
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Effect test

Embodiment 1

[0022] A kit for rapid detection of Seneca virus, comprising:

[0023] RPA primer probe mixture, PBST solution, flow chromatography test strips (MileniaHybridtech1strips, Milenia Biotec), RPA lyophilized enzyme and RPA reaction premix, RPA reaction premix is ​​29.5μL RehydrationBuffer and 2.5μL 280mM Magnesium Acetate mixture .

[0024] The concrete preparation method of above-mentioned kit is:

[0025] 1. The RNA of SVA is extracted, and the extraction method is carried out according to the existing technology.

[0026] 2. Synthesize cDNA by reverse transcription, and the reverse transcription method is carried out according to the existing technology.

[0027] 3. Design and synthesis of RPA primers and probes

[0028] Referring to the Seneca virus VP1 gene sequence in GenBank, specific conserved regions were selected, and RPA primers and probes were designed. All primers and probes were synthesized by Shanghai Sangong, and the gene sequences of the primers and probes are...

Embodiment 2

[0032] The method for rapidly detecting Seneca virus using the above kit specifically comprises the following steps:

[0033] (1) Recombinase polymerase amplification (RPA) reaction:

[0034] Using TwistAmp TM Prepare 50 μL real-time RPA reaction system with nfo kit, add 2.1 μL of upstream primers with a concentration of 10 μM, 2.1 μL of downstream primers with a concentration of 10 μM, 0.6 μL of probes with a concentration of 10 μM, 29.5 μL of buffer, and 9.2 μL of distilled water. , recombinant enzyme and single-chain binding protein lyophilized preparation, 4 μL of cDNA template and 2.5 μL of MgAc with a concentration of 280 mM were added to the reaction tube to obtain the amplification product.

[0035] (2) Detection of amplification products:

[0036] Prepare PBST solution: dissolve 1mL Tween-20 in 1000mL PBS solution and mix well. After the amplification reaction, draw 2 μL of RPA amplification product and mix with 198 μL PBST solution to form a mixed solution, then ...

Embodiment 3

[0038] Sensitivity test of the kit of the present invention:

[0039] The experimental process is as follows: Seneca virus cDNA is subjected to RPA amplification reaction, and the amount of Seneca virus cDNA added in each amplification reaction is 400pg, 40pg, 4pg, 400fg, 40fg, 4fg, 0.4fg, and the reaction ends Carry out flow chromatography test strip detection according to the Seneca virus quick detection method of the present invention afterward, detection result is as follows: figure 1 As shown, it can be seen that the method can detect 40 fg of Seneca virus cDNA added to the reaction system, and the detection sensitivity is high.

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Abstract

The invention discloses a kit for rapidly detecting Seneca viruses and a detection method of the kit. The kit comprises an RPA (Recombinase Polymerase Amplificatio) primer probe mixed liquid, a PBST (Phosphate Buffered Solution), a flow measurement chromatography test paper strip, RPA lyophozyme and an RPA reaction premixed liquid, wherein the primer in the RPA primer probe mixed liquid has gene sequences as follows: an upstream primer: 5'-AATTCCTGTTTGACCGATCTCGGCTACTGA-3'; a downstream primer: 5'-biotin-CCAGTAAAGTGGTAGGGTACGTGCAGTTGGTC3'; the RPA primer probe has a gene sequence as follows: 5'-FAM-GGCCTGTACGTCAACCCGTCTGACAGTGGCG-THF-TCTCGCTAACACTTC-Spacer C3-3'. The kit for rapidly detecting Seneca viruses, which is disclosed by the invention, is good in specificity and accurate in diagnosis, has sensitivity better than that of a conventional PCR (Polymerase Chain Reaction), the Seneca virus detection method disclosed by the invention is very simple, convenient and rapid to operate, and is not only applicable to clinical diagnosis, but also applicable to multiple fields such as food security detection, on-site detection in breeding fields and environment assessment.

Description

technical field [0001] The invention belongs to the technical field of gene detection, in particular to a kit for rapidly detecting Seneca virus and a detection method thereof. Background technique [0002] Senecavirus A (Senecavirus A, SVA) is the only member of the Picornaviridae Seneca genus, which can cause severe blistering disease in sows and death of newborn piglets, causing huge losses to the breeding industry. Seneca virus disease is an animal infectious disease caused by SVA. It mainly infects pigs, and pigs of different ages are susceptible to infection. Vesicular disease caused by SVA infection and foot-and-mouth disease (foot-and-mouth disease, FMD), swine vesicular disease (swine vesicular disease, SVD), swine vesicular exanthema of swine (VES) and vesicular stomatitis (vesicular stomatitis, The lesions caused by VS) are very similar, and it is difficult to make a clinical differential diagnosis, which brings great difficulties to the diagnosis, treatment, pre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101C12Q2565/625C12Q2565/137
Inventor 王会宝董金杰吕宏亮王凡张涛杨锐刘萍邓瑞雪陈苗苗王超英张云德
Owner CHINA AGRI VET BIO SCI & TECH
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