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Primer, probe and kit for detecting infectious bovine rhinotracheitis viruses

A technique for rhinotracheitis virus and detection reagent, which is applied in the biological field and achieves the effects of high sensitivity, simple detection and high sensitivity

Active Publication Date: 2015-08-26
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

[0004] At present, the research on RPA technology is still in its infancy, and there are no reports on the application of RPA technology to IBRV detection at home and abroad.

Method used

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  • Primer, probe and kit for detecting infectious bovine rhinotracheitis viruses
  • Primer, probe and kit for detecting infectious bovine rhinotracheitis viruses
  • Primer, probe and kit for detecting infectious bovine rhinotracheitis viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Design and screening of primers and probes

[0042] 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

[0048] Example 2: Establishment and investigation of laboratory IBRV RPA amplification detection method

[0049] 1. Establishment of laboratory IBRV RPA amplification detection method

[0050] 1.1 Extraction of IBRV genomic DNA

[0051] Take 200 μL of the cell culture virus supernatant, apply the Rapid Extraction of Viral Genomic DNA Kit to extract IBRV DNA, and finally dissolve it in 50 μL of deionized water.

[0052] 1.2 RPA amplification

[0053] In this embodiment, the PRA method is used to amplify the IBRV specific sequence, and the specific steps are:

[0054] (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 Make up O to a volume of 47.5 μL (Table 2), vortex and briefly centrifuge;

[0055] Table 2 RPA amplification system

[0056]

[0057] (2) Transfer 47.5 μL of the above mixture to a 0.2 mL TwistAmp nfo...

Embodiment 3

[0071] Embodiment 3: clinical sample IBRV detection

[0072] 1. Crude extraction of IBRV genomic DNA

[0073] Take a total of 20 nasal swabs from bovines identified as IBRV positive and negative by BVDV fluorescent quantitative RT-PCR established in our laboratory, and add 50 μLTES (10 mmol Tris-HCl, pH8.0, 5 mmol / LEDTA, 0.5% SDS) and 150 μg / mL proteinase K, 65°C, act for 15min, take the supernatant for RPA template amplification.

[0074] 2. Lateral flow chromatography RPA detection of clinical samples

[0075] The genomic DNA of the prepared clinical samples was 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 14 samples, indicating that 14 clinical samples contained IBRV, and the results were...

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Abstract

The invention relates to a primer, a probe and a kit for detecting infectious bovine rhinotracheitis viruses, and discloses a primer and probe combination used for detecting the infectious bovine rhinotracheitis viruses according to an RPA technology, the forward primer sequence of the primer and probe combination is shown as SEQ ID No.1; the reverse primer sequence is shown as SEQ ID No.2; the probe sequence is shown as SEQ ID No.3. The invention further discloses the kit for detecting the infectious bovine rhinotracheitis viruses. The primer and the probe are adopted for detection, a clinical sample is only subjected to virus DNA crude extraction and RPA isothermal amplification, the result can be displayed on a lateral flow chromatography test strip, a heat circular reaction is not needed, and amplification needs not to be performed in a PCR instrument; the primer, the probe and the kit have the advantages of being high in sensitivity, strong in specificity, simple in reaction procedure, short in detection time, and suitable for clinical field detection in a non-lab environment, and have a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer and a primer for detecting bovine infectious rhinotracheitis virus by applying a recombinase polymerase amplification technique (Recombinase Polymerase Amplification, RPA) combined with a lateral flow chromatography technique (Lateral Flow Assay). Probe combinations and their kits. Background technique [0002] Infectious bovine rhinotracheitis virus (Infectious bovine rhinotracheitis virus, IBRV) is an acute, febrile, contagious infectious disease of cattle. The disease can cause immunosuppression, and secondary bacterial infection can lead to more severe respiratory disease. At present, the disease is widely prevalent in the world, and the disease seriously affects the international cattle production and trade, and has been listed as a B-type communicable disease by the World Organization for Animal Health (OIE). my country detected IBRV in dairy cows importe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/70C12Q2521/507C12Q2522/101C12Q2531/119C12Q2565/625
Inventor 何洪彬侯佩莉王洪梅
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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