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Primer, probe and kit for detecting bovine viral diarrhea virus

A technology for bovine viral diarrhea and detection reagents, applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve high sensitivity, strong specificity, and high sensitivity

Inactive Publication Date: 2015-09-09
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the research on RPA technology is still in its infancy, and there is no report on the application of lateral flow chromatography RPA technology in BVDV detection at home and abroad.

Method used

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  • Primer, probe and kit for detecting bovine viral diarrhea virus
  • Primer, probe and kit for detecting bovine viral diarrhea virus
  • Primer, probe and kit for detecting bovine viral diarrhea virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Design and screening of primers and probes

[0043] Effective design of primers and probes is the most critical link in determining the success of experiments. However, RPA technology is in its infancy, and there is no dedicated primer and probe design software, nor is there a large amount of data to provide a basis for its primer design principles. At present, in the experiment, it is necessary to design multiple pairs of primers from both ends of the target sequence for optimization and screening. The primer design requirements of this technology are extremely strict, and the substitution or increase or decrease of individual bases will have an important impact on the experimental results. The following factors usually need to be considered in the design: (1) The length of the primer is required to be 30-35bp, the length of the probe is required to be 46-52bp, and the GC content is 40%-60%. Repeat sequence. (2) Detection of amplified fragments less tha...

Embodiment 2

[0049] Example 2: Establishment and investigation of laboratory BVDV RPA amplification detection method

[0050] 1. Establishment of laboratory BVDV RPA amplification detection method

[0051] 1.1 Synthesis of BVDV genome cDNA

[0052] Take 200 μL of cell culture virus supernatant, refer to the instructions of the viral RNA extraction kit, extract virus RNA, and dissolve it in 50 μL of RNAase-free water. According to Fermentas RevertAid TM First Strand cDNA Synthesis Kit instructions for RT-PCR reaction to obtain BVDV cDNA.

[0053] 1.2 RPA amplification

[0054] In the present embodiment, the PRA method is used to amplify the specific sequence of BVDV, and the specific steps are:

[0055] (1) Add 2.1 μL (10 μmol / L) of the upstream and downstream primers designed in Example 1, 0.6 μL (10 μmol / L) of the LF probe, 29.5 μL of Rehydration buffer, and 2 μL of template DNA into the centrifuge tube. 2 O was made up to a volume of 47.5 μL, vortexed and centrifuged briefly; see Ta...

Embodiment 3

[0072] Embodiment 3: Clinical sample BVDV detection

[0073] 1. Synthesis of BVDV genome cDNA

[0074] Take 16 samples of diarrhea feces samples and 3 parts of negative samples identified as BVDV-positive cattle by BVDV fluorescent quantitative RT-PCR established in this laboratory, and use the method of "1.1 Synthesis of BVDV genome cDNA" in Example 2 to synthesize BVDV genome cDNA , for RPA template amplification.

[0075] 2. Lateral flow chromatography RPA detection of clinical samples

[0076] The genomic cDNAs of the prepared clinical stool samples were respectively used as templates, and amplified according to the RPA detection method described in step 1 in Example 2. After RPA amplification, the hybridization product was displayed on the lateral flow chromatography test strip, and the control band and the detection band were displayed in the reaction area of ​​the test strip of 16 samples, indicating that 16 clinical samples contained BVDV, and the results were consis...

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Abstract

The invention discloses a primer and a probe combination for detecting bovine viral diarrhea virus by a recombinase polymerase application (RPA) technology. The forward primer sequence is as shown in SEQ ID No.1; the reverse primer sequence is as shown in SEQ ID No.2; and the probe sequence is as shown in SEQ ID No.3. The invention also discloses a kit for bovine viral diarrhea virus detection. When the primer and the probe are used for detecting, a clinical sample only needs the treatment steps of virus RNA extraction, reverse transcription by a one-step method to form cDNA, and isothermal amplification, thermal cycle reaction is not needed, amplification does not need to be performed in a polymerase chain reaction (PCR) instrument, and the result can be displayed clearly on a lateral flow assay test strip, so that the primer and the probe have the advantages of high sensitivity, high specificity, simple reaction procedure, short detection time and the like, and are applicable to quick, accurate, simple and convenient diagnosis and treatment on the diseases in cattle farms.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a primer and a probe for detecting bovine viral diarrhea virus by applying a recombinase polymerase amplification technique (Recombinase Polymerase Amplification, RPA) combined with a lateral flow chromatography technique (Lateral Flow Assay) Combinations and their kits. Background technique [0002] Bovine viral diarrhea virus (Bovine Viral Diarrhea virus, BVDV) caused bovine viral diarrhea mucosal disease, is an acute, febrile, contact infectious disease of cattle, clinically characterized by fever, diarrhea, mucosal erosion, ulcers, white blood cells Reduction, persistent infection and immune tolerance, immunosuppression, abortion of pregnant cows, stillbirths and deformities, and fatal mucosal disease are the main features. The disease is distributed worldwide, the infection situation is complex, the individual symptoms of persistent infection are hidden, and the animal infectio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70
Inventor 何洪彬侯佩莉王洪梅
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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