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BVDV (bovine viral diarrhea virus) internal control typing fluorescent PCR (polymerase chain reaction) detection kit and preparation thereof

A technology of bovine viral diarrhea and detection kit, which is applied in the directions of fluorescence/phosphorescence, material excitation analysis, microbe determination/inspection, etc., to achieve the effect of high detection sensitivity, good sensitivity and short detection time

Inactive Publication Date: 2014-04-16
XINJIANG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] After retrieval, there is no relevant detection with high sensitivity, good specificity, and convenient operation at home and abroad, and through the addition of internal standards, indicating and calibrating false negative results, and there is no detection that can simultaneously detect bovine viral diarrhea virus genotype I (BVDV I ) and Bovine Viral Diarrhea Virus Genotype II (BVDV II) Internal Standard Typing Fluorescent PCR Method Report

Method used

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  • BVDV (bovine viral diarrhea virus) internal control typing fluorescent PCR (polymerase chain reaction) detection kit and preparation thereof
  • BVDV (bovine viral diarrhea virus) internal control typing fluorescent PCR (polymerase chain reaction) detection kit and preparation thereof
  • BVDV (bovine viral diarrhea virus) internal control typing fluorescent PCR (polymerase chain reaction) detection kit and preparation thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1: Preparation of Template and Construction of Positive Recombinant Plasmid

[0046] 1. Design and synthesis of primers and probes

[0047] The primer information of the present invention is shown in Table 1. Among them, referring to the 5′-UTR conserved region of BVDV I with the accession number M31182.1 and the BVDV II with the accession number AY149216.1 in GenBank, and the mRNA conserved region of bovine GAPDH with the accession number NM001034034.2, a pair of specificity was designed for each Primers, the lengths of the amplified target fragments are 127bp, 169bp, and 152bp respectively. For details, see the attached gene sequence list.

[0048] Table 1 Design results of fluorescent PCR primers for internal standard typing

[0049]

[0050] 2. Preparation of template RNA and construction of positive recombinant plasmid

[0051] Whole blood, serum, tissue, and anal swab samples were extracted with Trizol conventional reagents; milk was centrifuged at ...

Embodiment 2

[0060] Example 2: Establishment of an internal standard typing fluorescent PCR amplification system

[0061] Single-plex conditions were optimized in a two-step method. In the first step, the concentrations of primers BR1, BR2, and GR were 0.6 μM, 0.75 μM, and 0.4 μM, respectively, and the RNA template was 10 μL. The above reactions were performed in a water bath at 70°C for 10 minutes, and placed on ice for 2 minutes. Solution 6μL, add 50uM-MLV and 10u RNase inhibitor, add dNTPs to a final concentration of 0.5mM, make up to 10μL with deionized water, incubate at 42°C for 1h, and complete the cDNA preparation; the second step, 20μL system, take the cDNA2 obtained in the first step -5 μL, the final concentrations of the upstream and downstream primers of BVDV I, BVDV II and GAPDH are 0.05 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM and 0.4 μM for optimization, 2×SYBR Premix Dimer Eraser 10 μL, the fluorescent quantitative PCR reaction program was: pre-denaturation at ...

Embodiment 3

[0065] Embodiment three: Specific detection

[0066] The kit constructed in Example 1 respectively amplified positive nucleic acid samples such as BVDV I, BVDV II, FMDV, BTV, BDV, CSFV, etc. Except for the amplification of BVDV I, BVDV II and GAPDH, the rest of the tested samples were all negative , indicating that the method has good specificity, see attached figure 2 .

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Abstract

The invention discloses a BVDV (bovine viral diarrhea virus) internal control typing fluorescent PCR (polymerase chain reaction) detection kit and a preparation thereof. By means of the primer design, a BVDV I, a BVDV II and a PCR template for monitoring internal control are obtained through single PCR amplification, a BVDV internal control typing fluorescent PCR detection system is established through the primer design, PCR amplification and optimization of reaction conditions, the BVDV I and the BVDV II can be independently detected or synchronously detected, and quality monitoring can be performed on the detection result. The prepared kit comprises two parts of a cDNA (complementary deoxyribonucleic acid) synthesis system and an SYBR fluorescent PCR amplification system. The minimum detection limit of the kit to three types of genes is 102 copy, the kit has better specificity and repeatability, the detection quality is ensured, and the kit has a better application value.

Description

field of invention [0001] The invention relates to the field of molecular biology. Specifically, the invention relates to a bovine viral diarrhea virus (BVDV) internal standard typing fluorescent PCR detection kit and the technical field of preparation thereof. Background technique [0002] BVDV is a member of the Pestivirus genus in the family Flaviviridae, which is more harmful to the cattle industry. Among the existing detection standards in my country, the detection methods for BVDV include fluorescent antibody method based on cell culture technology, immunoperoxidase monolayer detection method, trace serum neutralization test, antigen capture ELISA and PCR, the first three detection methods The operation is complicated, and the requirements for personnel and the environment are high, which is not convenient for the application of large-scale import and export inspection and quarantine. The sensitivity of antigen capture ELISA detection method is low. With the rapid dev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
CPCC12Q1/686C12Q1/70C12Q2545/101C12Q2531/113
Inventor 冉多良季新成王克栋史茜刘建华王科珂
Owner XINJIANG AGRI UNIV
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