Primer group for identifying avian adenovirus type 4 and chicken infectious anemia virus and application of primer group

A technology for chicken infectious anemia and poultry adenovirus, applied in the direction of microorganisms, recombinant DNA technology, and methods based on microorganisms

Inactive Publication Date: 2019-12-13
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Compared with conventional PCR, multiplex PCR has the characteristics of simultaneous detection and identification of multiple pathogens, and has unique advantages and high clinical practi

Method used

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  • Primer group for identifying avian adenovirus type 4 and chicken infectious anemia virus and application of primer group
  • Primer group for identifying avian adenovirus type 4 and chicken infectious anemia virus and application of primer group
  • Primer group for identifying avian adenovirus type 4 and chicken infectious anemia virus and application of primer group

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0028] Example 1. Primer design

[0029] A large number of sequence analyses and comparisons have obtained several primers for identifying avian adenovirus type 4 and several primers for identifying chicken infectious anemia virus. Preliminary experiments were performed on each primer, and the sensitivity and specificity were compared, and finally primer pair I and primer pair II for identifying avian adenovirus type 4 and chicken infectious anemia virus were obtained.

[0030] The specific primer pair used to identify avian adenovirus type 4 (primer pair I for short) consists of the following two primers (5'→3'):

[0031] FadV-4F (SEQ.ID.NO.1): TTCGCCAAGTCTCAGTACAAT;

[0032] FadV-4R (SEQ.ID.NO.2): GGAGTCGTGATACAGCAGGTT;

[0033] The specific primer pair used to identify chicken infectious anemia virus (primer pair II for short) consists of the following two primers (5’→3’):

[0034] CIAV-F (SEQ.ID.NO.3): CGACATCGGAGGAGACAGA;

[0035] CIAV-R (SEQ.ID.NO.4): AGGGTCATTTGCTTAGGGT.

[0036] T...

Example Embodiment

[0037] Example 2. Optimization of double PCR reaction conditions

[0038] 1. Preparation of template

[0039] 1. Extract the genomic DNA of avian adenovirus type 4 to obtain sample A.

[0040] 2. Extract the DNA of chicken infectious anemia virus to obtain sample B.

[0041] 3. Mix sample A and sample B to obtain a mixed sample.

[0042] 2. Optimization of primer concentration

[0043] Take the mixed sample obtained in step 1 as a template, and use the primer combination prepared in Example 1 to perform double PCR.

[0044] Double PCR reaction system (25.0μL): contains 2×PCR Mix 12.5μL, the mixed sample obtained in step 1 is 2.0μL (in the 2.0μL mixed sample, the content of genomic DNA of avian adenovirus type 4 is 1.0ng, which is infected by chicken The DNA content of anemia virus is 1.0ng), primer pair I and primer pair II, and finally ddH 2 Make up O to 25.0 μL.

[0045] According to the concentration of primer pair I and primer pair II in the reaction system, 8 different reaction syste...

Example Embodiment

[0074] Example 3. Specificity

[0075] 1. Extract the genomic DNA of the sample to be tested. The samples to be tested are: avian adenovirus type 1-12 (FadV1-12), chicken infectious anemia virus (CIAV), chicken parvovirus (ChPV), Marek virus (MDV), infectious laryngotracheitis virus (ILTV) .

[0076] 2. Extract the total RNA of the sample to be tested and reverse transcribed into cDNA. The samples to be tested are: avian nephritis virus (ANV), chicken Newcastle disease virus (NDV), H9 subtype avian influenza virus (AIV-H9), and infectious bronchitis virus (IBV).

[0077] 3. Using each genomic DNA sample obtained in step 1, each cDNA sample obtained in step 2, and the mixed sample obtained in step 1 of Example 2 as templates, the primer combination prepared in Example 1 is used to perform double PCR.

[0078] Double PCR reaction system (25.0μL): contains 2×PCR Mix 12.5μL, template 2.0μL, primer pair I and primer pair II, and finally ddH 2 Make up O to 25.0 μL. In the double PCR reac...

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Abstract

The invention discloses a primer group for identifying avian adenovirus type 4 and a chicken infectious anemia virus. The primer group comprises a primer pair I and a primer pair II; the primer pair Iconsists of a primer FadV-4 F and a primer FadV-4R; the primer pair II consists of a primer CIAV-F and a primer CIAV-R; the primers FadV-4F, FadV-4R, CIAV-F and CIAV-R have base sequences of SEQ. ID. NO. 1 to SEQ. ID. NO. 4 respectively. Accordingly, an inventor further establishes a corresponding detection and identification method and develops a corresponding kit. Established double PCR can beused for rapidly detecting mixed infection of the avian adenovirus type 4 and the chicken infectious anemia virus, is suitable for mass detection, can save the cost and the time, can further reduce pollution and has very high practical value.

Description

technical field [0001] The invention belongs to the technical field of detecting poultry adenovirus type 4 and chicken infectious anemia virus, and in particular relates to a primer set for identifying poultry adenovirus type 4 and chicken infectious anemia virus and its application. Background technique [0002] Adenovirus type 4 (FadV-4) is a non-enveloped double-stranded DNA virus. Since the second half of 2015, Shandong, Jiangsu, Henan, Hebei, Liaoning, Jilin, Anhui, Zhejiang, Hubei and Shanxi An acute infectious disease characterized by pericardial effusion and hepatosplenomegaly has occurred in chicken flocks in other places. The disease can cause high infectivity and high mortality. Infecting 3-5 week-old chickens can lead to sudden death, death rate as high as 80%. The disease has no obvious clinical symptoms, and individual chickens have clinical manifestations such as depression and curly feathers before death. The lesions of the autopsy of the dead chickens are ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 谢芝勋张艳芳邓显文刘加波谢志勤张民秀谢丽基罗思思曾婷婷
Owner GUANGXI VETERINARY RES INST
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