Detection method for infectious bovine viral diarrhea virus in aerosol

A bovine viral diarrhea and aerosol technology, which is applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve the problem of low virus aerosol concentration, insufficient research on virus aerosol, and the impact of There are many factors, etc., to achieve the effect of a wide range of application prospects

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

Air bioaerosol research mainly focuses on the detection of airborne bacteria, fungi and their toxins, endotoxins, etc. in the animal house environment, while the virus aerosol concentration is low, difficult to collect, sample processing and virus identification, etc. Compared with other microbial research methods

Method used

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  • Detection method for infectious bovine viral diarrhea virus in aerosol
  • Detection method for infectious bovine viral diarrhea virus in aerosol
  • Detection method for infectious bovine viral diarrhea virus in aerosol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Design and Synthesis of Primer of Example 1

[0047] According to the conserved sequence of bovine viral diarrhea virus 5'-UTR gene (GenBank: AF091605.1,) in GeneBank, use PrimerPremier5.0 to design full-length amplification primers B-F, B-R (Table 1, sequence 1 and sequence 2, such as SEQ ID NO. 1, 2), used for the construction of positive standard plasmids; using PrimerExpress3.0 software, design a pair of specific primers RT-F, RT-R (Table 1, sequence 3 and sequence 4, such as SEQ ID NO. 3, 4 shown) for universal bovine viral diarrhea virus detection. Based on the sequences of different types of bovine viral diarrhea virus in GeneBank, PrimerExpress 3.0 software was used to design specific identification and detection primers for type I bovine viral diarrhea virus and type II bovine viral diarrhea virus (Table 1, sequences 5-8, As shown in SEQ ID NO. 5, 6, 7, 8).

[0048] Table 1 Primer oligonucleotide sequences

[0049]

Embodiment 2

[0050] Example 2 Establishment of the universal SYBRGreen I real-time fluorescence quantitative RT-PCR detection method for bovine viral diarrhea virus

[0051] (1) Determination of SYBRGreen Ⅰ real-time quantitative RT-PCR detection method

[0052] 1. Preparation of samples to be tested

[0053] Take 200 μL of cell culture virus of bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine enterovirus, bovine coronavirus, and bovine rotavirus, and extract RNA according to the instructions of the viral RNA extraction kit. TM FirstStrandcDNASynthesisKit instructions for RT-PCR reaction.

[0054] 2. Preparation of standard products

[0055] PCR amplification was carried out using the cDNA of bovine viral diarrhea virus obtained in step 1 as the template. The reaction system was 50 μL: 10×LAPCRBufferII (Mg2+Plus) 5 μL, 2.5mM dNTP Mixture 8 μL, LATaq 0.5 μL (5U / μL), template 2 μL, 10 μmol / L of B-F and B-R gene full-length upstream and downstream primers, 2 μL each...

Embodiment 3

[0078] Example 3 Establishment of a real-time fluorescent quantitative RT-PCR typing detection method for bovine viral diarrhea virus SYBRGreen I

[0079] 1. Establishment of amplification conditions for genotyping detection of bovine viral diarrhea virus by SYBRGreen I real-time fluorescence quantitative RT-PCR

[0080] Optimize the optimal primer concentration, annealing temperature and cycle conditions for different genotypes of bovine viral diarrhea virus SYBRGreenⅠ real-time quantitative RT-PCR reaction according to the steps of SYBRGreenⅠ real-time quantitative RT-PCR for universal detection of bovine viral diarrhea virus Wait. The optimal final concentration of upstream primer and downstream primer in the reaction system was determined to be 0.25 μmol / L, and the optimal annealing temperature was 65 °C.

[0081] 2. Determination of standard curve and analysis of melting curve

[0082] According to the analysis method of step 4 fluorescent quantitative RT-PCR standard c...

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Abstract

The invention discloses a detection method for the bovine viral diarrhea virus in an aerosol. The method comprises the following steps: (1) acquisition of an aerosol sample; (2) extraction of RNA and cDNA of the virus in 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 bovine viral diarrhea virus. The invention further discloses specific primers (as shown in SEQ ID No. 3 to 8) and a kit (composed of the specific primers, the bovine viral diarrhea virus positive standard plasmid pEASY-T3-B1, a SYBRGreenI real-time fluorescent quantitative PCR reagent and ddH2O). The method provided by the invention is applicable to typing detection of a bovine viral diarrhea 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 viral diarrhea virus in aerosol, and belongs to the field of biotechnology. Background technique [0002] Bovine viral diarrhea (BVD) is an endemic infectious disease caused by bovine viral diarrhea virus (BVDV). The disease can be transmitted by contact and aerosol. The respiratory tract, eye secretions, milk and feces of sick and latently infected cows contain the virus, which can be outbreaks in closed herds. Industrial production and related business areas have caused huge economic losses. [0003] BVDV is a single-stranded positive-stranded RNA virus with an envelope. The genome consists of a 5' untranslated region (5'NTR), a long open reading frame, which encodes viral structural proteins, including viral capsid proteins and envelope proteins. (Erns, E1 and E2) and non-structural proteins, and 3' untranslated region (3'NTR). BVDV is divided into bovine viral diarrhea virus type I (BVDV-1) ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 何洪彬侯佩莉
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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