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Multiplex-PCR (polymerase chain reaction) detection kit for bovine respiratory disease complex and preparation method thereof

A technology for detection kits and respiratory tracts, applied in biochemical equipment and methods, microbe determination/inspection, etc., can solve the problem that the four pathogens of bovine respiratory disease syndrome cannot be effectively detected at the same time

Inactive Publication Date: 2014-01-08
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem that the existing multiplex PCR method for detecting bovine respiratory disease syndrome cannot effectively detect four pathogens of bovine respiratory disease syndrome at the same time, the invention provides a bovine respiratory disease syndrome multiplex PCR detection kit and its Preparation

Method used

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  • Multiplex-PCR (polymerase chain reaction) detection kit for bovine respiratory disease complex and preparation method thereof
  • Multiplex-PCR (polymerase chain reaction) detection kit for bovine respiratory disease complex and preparation method thereof
  • Multiplex-PCR (polymerase chain reaction) detection kit for bovine respiratory disease complex and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0112] Embodiment 1 The assembly of bovine respiratory disease syndrome multiplex PCR detection kit of the present invention

[0113] (1) Prepare PCR reaction solution:

[0114] ①Design four pairs of specific primers according to the conserved gene sequence of the virus genome, which are primers P for identifying bovine infectious rhinotracheitis virus (IBRV) 1 and P 2 , a primer P for the identification of bovine respiratory syncytial virus (BRSV) 3 and P 4 , a primer P for the identification of bovine viral diarrhea virus (BVDV) 5 and P 6 And the primer P for identifying bovine parainfluenza virus type 3 (BPI3) 7 and P 8 ; The sequences of these eight primers are as follows:

[0115] P 1 : 5'-GCTCGCCAACTTCTTTCAGGG-3',

[0116] P 2 : 5'-GCGTCAAACTCCTCCCTCTTCCTC-3',

[0117] P 3 : 5'-TATGCTATGTCCCGATTGG-3',

[0118] P 4 : 5'-ACTGATTTGGCTAGTACACCC-3',

[0119] P 5 : 5'-GGTAGCAACAGTGGTGAGTTC-3',

[0120] P 6 : 5'-CTCAGGTTAAGATGTGCTGTG-3',

[0121] P 7 : 5'-GC...

Embodiment 2

[0131] Embodiment 2 The use method of bovine respiratory disease syndrome multiplex PCR detection kit of the present invention

[0132] (1) Extract sample DNA from the samples to be tested, including cell cultures, secretions, tissue cultures, nasal swabs, and fecal swabs;

[0133] (2) Determination of multiplex PCR reaction conditions

[0134] After optimizing the conditions of reaction concentration and annealing temperature, it was determined that in a 25μL reaction system, MightyAmp DNA polymerase 0.625U, 2xBuffer Mix buffer 12.5μL, primer P 1 ,P 2 ,P 3 ,P 4 ,P 5 ,P 6 ,P 7 ,P 8 0.5 μL (10 pmol) each, 2.0 μL of the sample to be tested, and make up the rest to 25 μL with sterile double distilled water. The PCR reaction conditions are: 98°C for 2min; 98°C for 10s, 57°C for 15s, 68°C for 1min, 35 cycles ; Total extension at 68°C for 10 minutes;

[0135] (3) Using the extracted sample DNA as a template, add the sample DNA, positive control plasmid, and negative control...

Embodiment 3

[0138] Embodiment 3 The specificity experiment of the multiplex PCR detection kit of the present invention

[0139] ①Using IBRV, BRSV, BVDV and BPI3 positive control recombinant plasmids and negative control (distilled water) as templates respectively, the specificity experiment of the multiplex PCR detection kit was carried out;

[0140] ②Primer P for identification of bovine parainfluenza virus type 3 (BPI3) was added to the mixture of IBRV, BRSV and BVDV positive control recombinant plasmids 7 and P 8 , carry out the specificity experiment of the multiplex PCR detection kit on its mixed template;

[0141] ③Primer P for identifying bovine viral diarrhea virus (BVDV) was added to the mixture of IBRV, BRSV and BPI3 positive control recombinant plasmids 5 and P 6 , carry out the specificity experiment of the multiplex PCR detection kit on its mixed template;

[0142] ④ Add the primer P for identifying bovine respiratory syncytial virus (BRSV) to the mixture of IBRV, BVDV an...

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Abstract

The invention discloses a multiplex-PCR (polymerase chain reaction) detection kit for bovine respiratory disease complex and a preparation method of the multiplex-PCR detection kit and relates to the field of detection of main viruses of bovine respiratory diseases. The problem that four pathogens of the bovine respiratory disease complex cannot be simultaneously and effectively detected in a multiplex-PCR method for detecting the bovine respiratory disease complex is solved. The kit comprises MightyAmp DNA polymerase, a 2xBuffe Mix buffer solution, sterile double distilled water, and four pairs of specific primers for identifying infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus and bovine parainfluenza 3 virus respectively, and also comprises positive control plasmids of the four viruses. The kit can simultaneously detect nucleic acids containing the four viruses in the same reaction system, is high in specificity and sensitivity and can accurately detect hosts and pathogenetic animals which suffer from invisible infection or continuously take viruses in the group of cattle, infectivity is avoided, the safety is high, the result can be detected in a short time, and time and labor are saved.

Description

technical field [0001] The invention relates to the technical field of detection of main viruses of bovine respiratory disease, in particular to a bovine respiratory disease syndrome multiplex PCR detection kit and a preparation method thereof. Background technique [0002] With the rapid development of my country's import and export trade and cattle industry, bovine systemic diseases occur frequently and cause serious harm. Studies have shown that Bovine respiratory disease compLex (BRDC) is caused by cattle production and transportation and early weaning. One of the main systemic diseases in the world, it is caused by the joint action of multiple pathogenic factors or pathogens including stress, viruses and bacteria, so the potential infection of viruses is an important cause of the disease, and it is also the initiator of respiratory diseases in cattle . Viruses isolated from bovine respiratory diseases mainly include bovine infectious rhinotracheitis virus (IBRV), bovine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2531/113
Inventor 郭利王炜杨艳玲武华张淑琴李光玉陈立志程世鹏杨福合李春义温永俊王凤雪孙娜王建科易立
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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