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Primer combination for identifying ChPV (Chicken Parvovirus) and ARV (Avian Reoviruses) and application thereof

A technology for avian reovirus and chicken parvovirus, which is applied to microorganisms, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of lack of rapid differential diagnosis, low sensitivity, and long time consumption.

Active Publication Date: 2016-05-11
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the methods for laboratory diagnosis and detection of the above two pathogens are mainly traditional virus isolation, agar diffusion test, indirect fluorescence test and enzyme-linked immunosorbent assay (ELISA), etc., but these detection methods are time-consuming, low sensitivity, Standardization is difficult and other shortcomings have certain limitations in practical application, so more and more scholars are committed to PCR detection methods
Compared with conventional PCR, multiplex PCR has the characteristics of simultaneous detection and identification of multiple pathogens, and has unique advantages and high clinical practical value in the differential diagnosis of mixed infection of multiple pathogens. However, there is no rapid differential diagnosis at present. Multiplex PCR detection method for ChPV and ARV

Method used

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  • Primer combination for identifying ChPV (Chicken Parvovirus) and ARV (Avian Reoviruses) and application thereof
  • Primer combination for identifying ChPV (Chicken Parvovirus) and ARV (Avian Reoviruses) and application thereof
  • Primer combination for identifying ChPV (Chicken Parvovirus) and ARV (Avian Reoviruses) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Embodiment 1, primer design

[0096] A large number of sequence analyzes and comparisons were carried out to obtain several primers for identifying chicken parvovirus and several primers for identifying avian reovirus. Preliminary experiments were carried out on each primer to compare performances such as sensitivity and specificity, and finally a pair of primers for identifying chicken parvovirus and a pair of primers for identifying avian reovirus were obtained.

[0097] The specific primer pair used to identify chicken parvovirus consists of the following two primers (5'→3'):

[0098] ChPV-F (Sequence 1 of the Sequence Listing): CGAAGAAGAGGAACCCACC;

[0099] ChPV-R (SEQ ID NO: 2 of the Sequence Listing): TCTGGCTCGTCTGGTAATC;

[0100] The specific primer pair for identifying avian reovirus consists of the following two primers (5'→3'):

[0101] ARV-F (Sequence 3 of the Sequence Listing): TCTATGAACGGCTGACCAA;

[0102] ARV-R (SEQ ID NO: 4 of the Sequence Listing): C...

Embodiment 2

[0106] Embodiment 2, double PCR reaction condition optimization

[0107] 1. Template preparation

[0108] 1. Extract the genomic DNA of chicken parvovirus to obtain sample A.

[0109] 2. Extract the total RNA of avian reovirus and reverse transcribe it into cDNA to obtain sample B.

[0110] 3. Mix sample A and sample B to obtain a mixed sample.

[0111] 2. Optimization of primer concentration

[0112] Take the mixed sample obtained in step 1 as a template, and use the primer combination prepared in Example 1 to perform double PCR.

[0113] Reaction system for double PCR (25.0 μL): Contains 2×PCRMix12.5 μL, 2.0 μL of the mixed sample obtained in step 1 (in the 2.0 μL mixed sample, the genomic DNA content of chicken parvovirus is 0.58 ng, avian reovirus The content of cDNA is 0.53ng), primer pair I and primer pair II, and finally use ddH 2 O to make up to 25.0 μL.

[0114] According to the concentration of primer pair I and primer pair II in the reaction system, set up 9 dif...

Embodiment 3

[0145] Embodiment 3, specificity

[0146] 1. Extract the genomic DNA of the sample to be tested. The samples to be tested are: chicken parvovirus (ChPV), Marek virus (MDV), and infectious laryngotracheitis virus (ILTV).

[0147] 2. Extract the total RNA of the sample to be tested and reverse transcribe it into cDNA. The samples to be tested are: avian reovirus (ARV), Newcastle disease virus (NDV), H9 subtype avian influenza virus (AIVH9), and infectious bronchitis virus (IBV).

[0148] 3. Use each genomic DNA sample obtained in step 1, each cDNA sample obtained in step 2, and the mixed sample obtained in step 1 of embodiment 2 as templates, and perform double PCR using the primer combination prepared in embodiment 1.

[0149] Reaction system for double PCR (25.0 μL): 2×PCRMix 12.5 μL, template 2.0 μL, primer pair I and primer pair II, and ddH at the end 2 O to make up to 25.0 μL. In the reaction system of double PCR, the concentrations of ChPV-F and ChPV-R were both 0.4pmo...

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Abstract

The invention discloses a primer combination for identifying ChPV (Chicken Parvovirus) and ARV (Avian Reoviruses) and application of the primer combination. The primer combination consists of a primer pair I and a primer pair II, wherein the primer pair I consists of ChPV-F and ChPV-R which are respectively shown in sequence tables 1 and 2; the primer pair II consists of ARV-F and ARV-R which are respectively shown in sequence tables 3 and 4. The invention protects the application of the primer combination in identifying the ChPV and the ARV, the application of the primer combination in identifying whether a to-be-detected virus is ChPV or ARV or not and the application of the primer combination in identifying whether a to-be-detected sample is infected with the ChPV and / or the ARV or not. According to the primer combination for identifying the ChPV and the ARV and the application of the primer combination, disclosed by the invention, a duplex PCR (Polymerase Chain Reaction) established by the invention can be used for rapidly detecting mixed infection of the ChPV and the ARV and is suitable for large-batch detection, the cost and the time are saved, pollution is also reduced, and very high practical value is obtained.

Description

technical field [0001] The invention relates to a combination of primers for identifying chicken parvovirus and avian reovirus and its application Background technique [0002] Chicken parvovirus (Chinckenparvovirus, ChPV) is a single-stranded DNA virus, which is one of the main pathogens causing intestinal diseases in chickens, which can cause diarrhea, mental depression, thermoregulation disorders, growth retardation, increased feed consumption, etc. Acute or chronic intestinal diseases, short stature syndrome, malnutrition syndrome. ChPV is ubiquitous in chicken flocks and mainly affects chicks, but the infection rate of commercial broilers is higher, followed by laying hens or breeders, causing huge economic losses to the chicken industry. Since 2010, the disease has broken out in North America, Poland, Hungary, Croatia, South Korea and other countries, causing great economic losses to the chicken industry. Avian reoviruses (ARV) are double-stranded RNA viruses that ca...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 谢芝勋奉彬邓显文张艳芳黄娇玲王盛范晴谢志勤黄莉谢丽基曾婷婷罗思思刘加波
Owner GUANGXI VETERINARY RES INST
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