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RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification primer for quickly detecting bovine parainfluenza 3 virus, and application thereof

A technology of RT-PCR and bovine parainfluenza virus, applied in the direction of microorganisms, methods based on microorganisms, biochemical equipment and methods, etc., can solve the problems of affecting the detection effect, long reaction time, and many materials, and achieve less time-consuming, The effect of low cost and high sensitivity

Inactive Publication Date: 2018-05-04
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

International methods for diagnosing the virus include cell culture, tissue immunofluorescence detection, neutralization test, ELISA, agglutination test, etc. These tests have disadvantages, such as long reaction time, many materials, and obvious lag in detection time
Moreover, there are cross-reactions and non-specific reactions between species in serological diagnostic methods, which greatly hinder the application of serological methods and affect the detection effect.

Method used

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  • RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification primer for quickly detecting bovine parainfluenza 3 virus, and application thereof
  • RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification primer for quickly detecting bovine parainfluenza 3 virus, and application thereof
  • RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification primer for quickly detecting bovine parainfluenza 3 virus, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Establishment of the RT-PCR method for rapid detection of bovine parainfluenza virus type 3

[0040] 1. Preparation of materials

[0041] Bovine parainfluenza virus type 3, Cryptobacterium crypticus, Klebsiella pneumoniae, Mycoplasma bovis, bovine infectious rhinotracheitis virus, and Mannella hemolyticus were isolated, identified and preserved by Guangxi Veterinary Research Institute, and tissue samples were obtained from veterinary clinics. 10×RT-PCRBuffer, dNTPs, ES-Taq DNA polymerase, viral genomic DNA / RNA extraction kit, bacterial genomic DNA extraction kit, reverse transcription kit HiFiScript cDNA Synthesis Kit were purchased from Kangwei Century Biotechnology Co., Ltd.

[0042] 2. Design and synthesis of RT-PCR primers

[0043] According to the homology comparative analysis of the bovine parainfluenza virus type 3 HN gene sequence in GenBank, the conserved sequence region was selected as the amplification region, and the specific amplification primers w...

Embodiment 2

[0074] Example 2 Rapidly detects the annealing temperature test of bovine parainfluenza virus type 3 RT-PCR method

[0075] Perform RT-PCR amplification at annealing temperatures of 50 °C, 52 °C, 54 °C, 56 °C, 58 °C, 60 °C, and 62 °C to determine the optimal annealing temperature. The results showed that the designed primers had a large tolerance to the annealing temperature, and could amplify well under the reaction programs with annealing temperatures of 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃ and 62 ℃. Add a single destination strip ( figure 1 ). For this purpose, the RT reaction program used was 45 °C for 30 min, 85 °C for 5 min; the PCR reaction program was 95 °C for 5 min; followed by 30 cycles of 95 °C for 35 s, 58 °C for 40 s, and 72 °C for 50 s; finally Extend at 72°C for 10 min.

Embodiment 3

[0076] Embodiment 3 detects the specific detection result of bovine parainfluenza virus type 3 RT-PCR method

[0077] Extraction of genomic DNA / RNA from bovine parainfluenza virus type 3, Cryptobacterium crypticus, Klebsiella pneumoniae, Mycoplasma bovis, bovine infectious rhinotracheitis virus, and Mannella hemolyticus (RNA viruses are first reverse-transcribed into cDNA) , using the optimized reaction system and reaction program to carry out RT-PCR amplification to detect the specificity of the detection method of the present invention, the results show that only the bovine parainfluenza virus type 3 sample amplifies the target fragment band, which is a positive result, There was no amplification in the 5 control strain reaction tubes and the water control reaction tube, which was a negative result ( figure 2 ), indicating that the method has good specificity.

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Abstract

The invention discloses a RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification primer for quickly detecting a bovine parainfluenza 3 virus, and application thereof, which belong to the field of animal bacteriology and molecular biology. The RT-PCR amplification primer is formed by an upstream primer with a nucleotide sequence as shown in an SEQ ID NO:1 and a downstream primer with a nucleotide sequence as shown in an SEQ ID NO:2; the primer is used for preparing a RT-PCR amplification kit for detecting the bovine parainfluenza 3 virus. An RT-PCR detection method provided by the kit has high specificity and sensibility, good repeatability, and high reliability, can specifically detect the bovine parainfluenza 3 virus and quickly and accurately obtain a detection result, islow in price, simple and convenient to operate, and suitable for a basic level at the same time, and can be used as a quick, accurate, simple and convenient detection tool for quick identification ina bovine parainfluenza 3 virus laboratory and large-scale epidemiological investigation.

Description

technical field [0001] The invention relates to the technical fields of animal virology and molecular biology, in particular to a rapid, simple and low-cost RT-PCR amplification primer for detecting bovine parainfluenza virus type 3 and its application. Background technique [0002] Bovine parainfluenza virus 3 (BPIV-3) is one of the main pathogens causing acute respiratory diseases in cattle and other ruminants. The clinical manifestations of sick animals are loss of appetite, depression, fever, dyspnea, runny nose, cough, etc. Along with the exacerbation of the disease, the secondary hemolytic Mannella, Pasteurella multocida, Cryptobacterium cryptica, Mycoplasma bovis, bovine infectious rhinotracheitis infection, thus causing severe pneumonia, leading to respiratory disease syndrome, greatly increased the number of animals Mortality rate brings huge economic loss to the breeding industry. The virus is an RNA virus with an envelope and is a member of the Paramyxovirus gen...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2565/125
Inventor 彭昊李军吴翠兰冯世文潘艳陶立马春霞谢永平钟舒红胡帅杨威陈泽祥贺会利李常挺
Owner GUANGXI VETERINARY RES INST
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