Bird polyoma virus PCR (polymerase chain reaction) diagnostic kit and detection method thereof

A diagnostic kit and polyoma virus technology are applied in the field of molecular biology diagnosis of animal diseases in veterinary medicine, which can solve problems such as the inability to quickly control the epidemic situation, and achieve the effects of good practicability and applicability, easy operation and high stability

Inactive Publication Date: 2018-07-24
XIANYANG VOCATIONAL TECHN COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is a lack of diagnostic technology for rapid diagnosis of polyomavirus infection in birds, and it is impossible to quickly control the epidemic

Method used

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  • Bird polyoma virus PCR (polymerase chain reaction) diagnostic kit and detection method thereof
  • Bird polyoma virus PCR (polymerase chain reaction) diagnostic kit and detection method thereof
  • Bird polyoma virus PCR (polymerase chain reaction) diagnostic kit and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0035] A PCR diagnostic kit for avian polyomavirus, comprising: lysate, amplification reaction mixture, negative control and positive control, specifically as follows:

[0036] 1) Lysis solution: the composition is DNAiso Reagent, 20 reactions total 20mL, packed into 1 bottle.

[0037] 2) Amplification reaction mixture: sterilized triple distilled water, 10×PCR Buffer, 2.5mmol L -1 dNTP, 25μmol·L -1 APV-F upstream primer, 25 μmol L -1 APV-R downstream primer and 5U / μL rTaq DNA polymerase, the volume ratio of each reaction is 17.0︰2.5︰2︰0.5︰0.5︰0.5, a total of 23μL, 20 reactions totaling 460μL, packed into 1 tube; the design of the primers and prepared as follows:

[0038] Referring to the avian polyomavirus genome sequence published on GenBank, after comprehensive analysis and comparison, 2 primers were designed for the avian polyomavirus gene. The sequences of the primers are as follows:

[0039]The sequence of the upstream primer of APV-F is: 5'-GTATGTATGACCCATAGAA-3'; t...

Embodiment 2

[0068] 1. DNA extraction

[0069] 1.1 Processing and DNA extraction of feather pulp samples

[0070] Pull out 3-5 wing feathers from suspected cases, cut the feather pulp, add 800 μL of lysate, mix by inversion, let stand for lysis for 10 minutes, centrifuge at 4°C and 12000 r / min for 10 minutes, draw 600 μL of supernatant and put it into another centrifuge tube, Then add 600 μL of ice-cold absolute ethanol, mix it upside down, let it settle for 10 minutes, centrifuge at 12,000 r / min at 4°C for 10 minutes, discard the supernatant, wash once with 75% ethanol, discard the ethanol, and invert the EP tube in the air Dry naturally, use 40μL 8mmol·L -1 NaOH was dissolved to obtain the feather pulp sample DNA extract.

[0071] 1.2 Negative control sample extraction: draw 100 μL sterilized three-distilled water into a sterile centrifuge tube, add 800 μL lysate, mix evenly by inversion, let stand for lysis for 10 minutes, add 600 μL ice-cold absolute ethanol, mix upside down, and let...

Embodiment 3

[0084] 1. Processing of serum samples

[0085] The wing vein blood was collected from the parrots of suspected cases, and it was naturally coagulated. After the serum was separated, the serum was separated for use.

[0086] 2. DNA extraction

[0087] 2.1 DNA extraction from serum samples: Pipette 100 μL of the obtained supernatant into a 1.5 mL sterile EP tube, add 800 μL of lysate, mix evenly by inversion, let stand for lysis for 10 minutes, add 600 μL of ice-cold absolute ethanol, mix by inversion, and let stand for precipitation 10min, centrifuge at 12000r / min for 10min, discard the supernatant, wash once with 75% ethanol, discard the ethanol, invert the EP tube, dry naturally in the air, and use 40μL 8mmol·L -1 NaOH was dissolved to obtain the DNA extract of the serum sample.

[0088] 2.2 Negative control sample extraction: draw 100 μL sterilized three-distilled water into a sterile centrifuge tube, add 800 μL lysate, mix evenly by inversion, let stand for lysis for 10 m...

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Abstract

The invention provides a bird polyoma virus PCR (polymerase chain reaction) diagnostic kit and a detection method thereof. The bird polyoma virus PCR diagnostic kit comprises a lysate, an amplified reaction mixture, a negative control and a positive control, wherein the lysate is prepared from a DNAiso Reagent; the amplified reaction mixture is prepared from sterilized tri-distilled water, PCR buffer, dNTP, an APV-F upstream primer, an APV-R downstream primer and rTaq DNA polymerase; the negative control is the sterilized tri-distilled water; the positive control is bird polyoma virus positiveplasmids. The kit is high in sensitivity, good in specificity, high in stability, and intuitive in result; the detection method is easy to operate, convenient, fast, and capable of greatly reducing the detection time, provides a forceful technological means for clinically controlling bird polyoma virus infection, and particularly has a significance on rapid diagnosis of parrot bird polyoma virusinfection.

Description

technical field [0001] The invention relates to the technical field of molecular biology diagnosis of animal diseases in veterinary medicine, in particular to an avian polyomavirus PCR diagnostic kit and a detection method thereof. Background technique [0002] Avian polyoma virus (Avian Polyoma Virus, APV) was first discovered in budgie infection cases, also known as Francis feather drop disease. The main symptom of infected budgerigars is the absence of down feathers on the abdomen and back. Later, it was discovered that parrots such as macaws, conures, moon rings, kekes, lovebirds, lorikeets, Amazon parrots, gray parrots, eagle heads, and Badan can also be infected, becoming an important pathogen of parrots. [0003] APV is Polyomavirus (Polyomavirus), a member of Polyomaviridae (Polyomaviridae), with 20-sided symmetry and circular double-stranded DNA. The virus can be found in the blood, feathers, feces, and crop secretions of sick parrots, among which feathers are the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2565/125
Inventor 朱小甫吴旭锦
Owner XIANYANG VOCATIONAL TECHN COLLEGE
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