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Detection method for infectious bovine rihinotracheitis virus in aerosol

A rhinotracheitis virus and infectious technology, applied in the field of detection of bovine infectious rhinotracheitis virus in aerosol, can solve the problems of difficult collection of virus aerosol, limited research, low sensitivity, etc., and achieve reliable results and high sensitivity , the effect of easy operation

Active Publication Date: 2014-08-13
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

[0006] At present, there are many research reports on the collection and diagnosis methods of air microbial aerosols such as bacteria. Due to the difficulty of collecting virus aerosols, low concentration, and the low sensitivity of traditional detection methods, the research is limited.
So far, there is no research report on the application of SYBR GreenⅠreal-time fluorescent quantitative PCR technology to detect aerosol-borne IBRV

Method used

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  • Detection method for infectious bovine rihinotracheitis virus in aerosol
  • Detection method for infectious bovine rihinotracheitis virus in aerosol
  • Detection method for infectious bovine rihinotracheitis virus in aerosol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The design and synthesis of embodiment 1 primer

[0036] According to the conserved sequence of bovine infectious rhinotracheitis virus gD gene (GenBank: NC001847) in GeneBank, use PrimerPremier5.0 to design full-length amplification primers gD-F, gD-R (Table 1, sequence 1 and sequence 2, such as SEQIDNO. 1 and 2), used for the construction of positive standard plasmids; using PrimerExpress3.0 software, design a pair of specific primers IBRV-F, IBRV-R (table 1, sequence 3 and sequence 4, SEQIDNO.3, 4 institute shown), for the detection of bovine infectious rhinotracheitis virus.

[0037] Table 1 Primer oligonucleotide sequence

[0038]

Embodiment 2

[0039] Example 2 Establishment of SYBRGreenⅠreal-time fluorescent quantitative PCR detection method

[0040] (1) Establishment of SYBRGreenⅠreal-time fluorescent quantitative PCR detection method

[0041] 1. Preparation of samples to be tested

[0042] Take 200 μL of the cell culture virus of bovine infectious rhinotracheitis virus, bovine viral diarrhea virus, bovine parainfluenza type 3 virus, bovine coronavirus, and bovine rotavirus, and extract viral DNA / RNA according to the instructions of the viral DNA / RNA extraction kit. RNA, viral RNA obtained according to Fermentas RevertAid TM First Strand cDNA SynthesisKit instructions for reverse transcription.

[0043] 2. Preparation of standard plasmid positive template

[0044] The DNA of bovine infectious rhinotracheitis virus obtained in step 1 is used as a template to carry out PCR amplification, and the reaction system is 50 μ L: 2 × GCBufferI (Mg 2+ Plus) 25 μL, 2.5mMdNTPMixture 8 μL, LATaq 0.5 μL (5 U / μL), template 2 μ...

Embodiment 3

[0069] Embodiment 3, the detection of aerosol sample

[0070] (1) Aerosol sample collection

[0071]Select different areas of the dairy farm (dairy barn, sports field, milking parlor, etc.), apply the MS-I air microbial sampling box, and pass the international standard all-glass-impinger (AGI for short), A Porton sampler collects air samples. First install the tripod, place the sampler at a height of 1.5m from the ground, then inject 10mL of sterile phosphate buffer saline (PBS) into the Proton sampler, add a drop of olive oil at the same time, and put it into the tripod fixing bracket to fix it. Connect one end of the Porton impact regulator to the air inlet of the main unit, and connect the other end to the air outlet of the Proton sampler with a rubber tube. The sampling flow rate is 12.5L / min, and the sampling time is 20min. After sampling, the collected liquid in the sampler was collected into a 15mL centrifuge tube and stored for detection.

[0072] (2) Template DNA ...

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Abstract

The invention discloses a detection method for the infectious bovine rihinotracheitis virus in an aerosol. The method comprises the following steps: (1) acquisition of an aerosol sample; (2) extraction of genome total DNA of the aerosol sample; (3) detection: a step of carrying out PCR amplification; (4) establishment of a standard curve and a melting curve: a step of establishing the standard curve of positive standard plasmid and the melting curve of an amplification system; and (5) judgment: a step of judging whether the aerosol sample contains the infectious bovine rihinotracheitis virus. The invention further discloses specific primers (as shown in SEQ ID No. 3 and 4) and a kit (composed of the specific primers, the infectious bovine rihinotracheitis virus positive standard plasmid pEASY-T3-D, a SYBRGreenI real-time fluorescent quantitative PCR reagent and ddH2O). The method provided by the invention is applicable to detection of an infectious bovine rihinotracheitis virus aerosol sample and to detection of samples like clinical blood, milk and tissue and has wide application prospects.

Description

technical field [0001] The invention relates to a method for detecting bovine infectious rhinotracheitis virus in aerosol, belonging to the field of biotechnology. Background technique [0002] Infectious Bovine Rhinotracheitis Virus (IBRV) is a pantropic virus, which can infect and lie latent in multiple parts after invading cattle, and can cause multi-system infection in cattle, making sick cattle infected for a long time and can be Detoxification within a certain period of time, resulting in a large-scale infection of the herd. The main source of infection of the disease is sick cattle and asymptomatic infected cattle. Its transmission route is the respiratory tract and reproductive tract, and the virus is excreted through the nose, eyes, vaginal secretions, and semen. The disease can infect cattle through polluted air droplets, and it is easier to spread rapidly in overcrowded and closed environments, which has a great impact on the fattening rate, milk production and r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 何洪彬宋玲玲
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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