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Primer group, kit and method for simultaneously detecting pigeon circovirus, pigeon adenovirus and pigeon herpes virus type 1

A pigeon circovirus and pigeon herpes virus technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of low isolation rate, easy to cause missed diagnosis, long cycle, etc., to achieve High sensitivity, reduced labor costs, high specificity

Active Publication Date: 2020-05-01
JINLING INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005]At present, there are not many methods for diagnosing the above-mentioned infections, especially for mixed infections, simple virus isolation and identification is not only costly, long-term, but also difficult, and the isolation rate is not high. easy to cause misdiagnosis

Method used

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  • Primer group, kit and method for simultaneously detecting pigeon circovirus, pigeon adenovirus and pigeon herpes virus type 1
  • Primer group, kit and method for simultaneously detecting pigeon circovirus, pigeon adenovirus and pigeon herpes virus type 1
  • Primer group, kit and method for simultaneously detecting pigeon circovirus, pigeon adenovirus and pigeon herpes virus type 1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Extraction of viral DNA

[0042] Scrape the mucous membrane of the oral cavity, sinuses, and esophagus to be tested, and put 10-20mg of the sample into a 1.5mL centrifuge tube. Centrifuge at 12,000×g for 3 minutes, and transfer the supernatant to a new 1.5mL centrifuge tube. Add 230 μL BufferGA and 20 μL PK working solution, and vortex to mix. Water bath at 55°C until the tissue is completely enzymatically hydrolyzed. Add 250μl BufferGB to the digestion solution, vortex mix at the highest speed for 20sec, and bathe in 70℃ water for 10min. Then column purification: add 250 μL absolute ethanol to the digestion solution, vortex and mix for 15-20sec. Put the gDNA Columns adsorption column into the Collection Tubes 2mL collection tube. Transfer the mixed solution (including the precipitate) obtained in the previous step to the adsorption column. Centrifuge at 12,000×g for 1 min. Discard the filtrate and place the adsorption column in a collection tube. Add ...

Embodiment 2

[0044] Example 2: Design and synthesis of primers and effectiveness testing

[0045] 1. Design and synthesis of primers: based on Hexon gene of PiAdV (GenBank: MF576429.1), Rep and capsid gene of PiHV-1 (GenBank: JX901125.1) and DNA polymerase of PiCV (GenBank: KJ995972.1) in Genbank Gene nucleic acid sequences were compared with DNA-Star software, and conserved regions were screened out. Specific primers were designed with Primer5.0 software, and then verified with NCBI BLAST. The following three pairs of primers were screened out, as shown in Table 1.

[0046] Table 1 is used to detect the primer design sequence of three kinds of pathogens

[0047]

[0048] Among them, the predicted amplified fragment sizes are: PiAdV product is 616bp, PiHV-1 product is 239bp and PiCV product is 418bp. Primers were synthesized by Nanjing Sipujin Biotechnology Co., Ltd.

[0049] 2. Effectiveness detection: single PCR detection and triple PCR detection of clinical samples

[0050] (1) A ...

Embodiment 3

[0066] Embodiment 3: kit and detection method

[0067] 1. The composition of this kit: primers for amplifying PiAdV, PiHV-1 and PiCV; 2×Es Taq MasterMix; double distilled water; positive DNA templates for PiAdV, PiHV-1 and PiCV; DNA genome extraction kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., model DC102); PCR reaction reagents and electrophoresis detection reagents.

[0068] 2. The content of each component:

[0069] (1) Primers: Primers were synthesized by Nanjing Sipujin Biotechnology Co., Ltd. The total amount of each primer for amplifying PiAdV, PiHV-1, and PiCV is 2OD when synthesized. Divide 1OD into 1 tube, then add double-distilled water to dissolve it to 10mM, and then pack into 100μL tube, freeze at -20℃, Grab and go.

[0070] (2) DNA genome extraction kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., model (DC102-01),

[0071] ① Product use: This product is suitable for animal tissues from 1-20mg and less than 5×10 6 Extract genomi...

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Abstract

The invention discloses a primer group, a kit and a method for simultaneously detecting a pigeon circovirus, a pigeon adenovirus and a pigeon herpes virus type 1. Compared with the prior art, the invention has the following advantages: (1) the kit can be used for simultaneously detecting the pigeon circovirus, the pigeon adenovirus and the pigeon herpes virus type 1, i.e., detection results of three pathogenic bacteria can be obtained by an experiment, so compared with traditional detection methods, the method saves cost and time, and also reduces labor expenditure; (2) the primer group is high in sensitivity, and the lower detection limits of the primer group on the DNAs of three viruses, namely PiAdV, PiHV-1 and PiCV, are 32 pg / [mu]L, 46 pg / [mu]L and 55 pg / [mu]L respectively; and (3) products obtained after amplification by primers are 239 bp, 418 bp and 616 bp respectively, and can be remarkably separated through electrophoresis, so observation is facilitated.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a primer set, a kit and a method for simultaneously detecting pigeon circovirus, pigeon adenovirus and pigeon herpes virus type 1. Background technique [0002] Pigeon herpesvirus type Ⅰ (PHV-1) belongs to the family Herpesviridae. Pigeons are the natural host of PHV-1 and are in a state of latent infection. After the pigeons are infected with PHV-1, the adult pigeons become asymptomatic carriers and will shed the virus from time to time; the virus cannot be transmitted through eggs, but the young pigeons will be infected by eating latently infected parent pigeon milk shortly after hatching . Young pigeons are protected against severe disease or death by acquiring maternal immunity through the yolk; although they survive, they can become asymptomatic carriers of the virus. So far, there is no commercially available vaccine to prevent PHV-1 infection. Vaccin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12Q1/6806C12N15/11
CPCC12Q1/705C12Q1/701C12Q1/686C12Q1/6806C12Q2600/16C12Q2600/166C12Q2537/143C12Q2565/125C12Q2545/113C12Q2527/125Y02A50/30
Inventor 陈俊红戴鼎震蒋加进方光远陈涛陆雨楠黄翔宇
Owner JINLING INST OF TECH
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