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Identification primer, identification method and kit of parrot Borna virus 4

An identification method and kit technology, applied in the field of biological detection and disease diagnosis, can solve the problems of few research reports, infection of parrots, etc.

Pending Publication Date: 2020-10-02
广州动物园
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, parrots have been infected with PaBV-4 and died from time to time in my country, but there are few relevant research reports in China

Method used

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  • Identification primer, identification method and kit of parrot Borna virus 4
  • Identification primer, identification method and kit of parrot Borna virus 4
  • Identification primer, identification method and kit of parrot Borna virus 4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] SYBR Green Ⅰ real-time fluorescence quantitative PCR sensitive test.

[0052] 1. Gradient dilution of known positive samples: perform 10-fold serial dilution of known positive samples to prepare 6.7×10 6 ~6.7×10 1 6 serial dilutions of positive samples in copies / μL.

[0053] 2. SYBR Green Ⅰ real-time fluorescent quantitative PCR detection:

[0054] The real-time fluorescence quantitative PCR detection used the cDNA of the 6 samples in 1 as the template, PaBV4M-F2 and PaBV4M-R2 as primers, using TB Green Premix Ex Taq II (product of TaKaRa Company); DreamTaq Green PCR Master Mix (2×) (Thermo Scientific company product) to carry out PCR reaction, amplification is carried out in CFX96 real-time fluorescent quantitative PCR instrument (Bio-Rad company).

[0055] The amplification system is: the total volume is 25 μL, as follows: 12.5 μL of TB Green Premix Ex TaqⅡ, 1 μL of PaBV4M-F, 1 μL of PaBV4M-R2, 9.5 μL of ddH2O, 1 μL of cDNA template, ddH for negative control cDNA ...

Embodiment 2

[0062] SYBR Green Ⅰ real-time fluorescent quantitative PCR specificity test.

[0063] 1. Sample preparation: avian influenza virus cDNA (including H5N6, H7N9 and H9N2 subtypes), Newcastle disease virus cDNA (NDV), chicken infectious bronchitis virus cDNA (IBDV) are provided by this laboratory; 6.7 x 10 4 Copy / μL samples were used as detection samples for sensitivity experiments.

[0064] 2. Real-time fluorescent quantitative PCR detection of cDNA of H5N6, H7N9, H9N2, NDV, IBDV in 1 and 6.7×10 4 Copy / μL samples were used as templates, PaBV4M-F2 and PaBV4M-R2 were used as primers, and TB Green Premix Ex TaqII (product of TaKaRa Company); DreamTaq Green PCR Master Mix (2×) (product of Thermo Scientific Company) was used for PCR reaction. The amplification was performed on a CFX96 real-time fluorescent quantitative PCR instrument (Bio-Rad). The amplification system is: the total volume is 25 μL, as follows: TB Green Premix Ex TaqⅡ 12.5 μL, PaBV4M-F2 1 μL, PaBV4M-R 21 μL, ddH 2...

Embodiment 3

[0068] Sample testing and validation experiments.

[0069] 1. Disease tissue collection: Aseptically collected 8 tissues such as glandular stomach and brain from parrots with suspected clinical symptoms and dead parrots raised in different zoos.

[0070] 2. Treatment of diseased tissue

[0071] Add 5ml of PBS solution, use a hand-held high-speed homogenizer (Jingxin Technology), grind the diseased tissue, and take 1.5ml of the supernatant for later use.

[0072] 3. Viral RNA extraction and reverse transcription

[0073] AxyPrep Body Fluid Viral DNA / RNA Miniprep Kit 250-prep RNA extraction kit was used to complete the extraction of viral RNA according to the extraction method and steps of the kit, and PeimeScript reverse transcription kit was used for viral reverse transcription to prepare viral cDNA for later use.

[0074] 4. Primer design and synthesis

[0075] Taking the M gene fragment 428bp (SEQ ID No.5) as the reference sequence, the fluorescent quantitative PCR primer...

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Abstract

The invention provides an identification primer, an identification method and a kit of parrot Borna virus 4, and relates to the field of biological detection and disease diagnosis. The sequences of the fluorescent quantitative PCR identification primer are as shown in SEQ ID No.1 and SEQ ID No.2. The identification method comprises the following steps of S1, extracting virus RNA of a parrot disease material tissue, and reversely transcribing the virus RNA into cDNA; S2, taking the cDNA obtained in the step S1 as a template, and carrying out SYBR Green I fluorescent quantitative PCR amplification by using the identification primer; and S3, analyzing an amplification curve, determining the PaBV-4 to be positive when the ct value is smaller than or equal to 30 and the amplification curve is S-shaped, and determining the PaBV-4 to be negative when the ct value is larger than 30. The primer and the identification method disclosed by the invention have the characteristics of high sensitivity, strong specificity and good repeatability, can be used for detecting the PaBV-4, and are particularly suitable for detecting and identifying early period and low inapparent virus infection content of the PaBV-4.

Description

technical field [0001] The invention relates to the field of biological detection and disease diagnosis, in particular to an identification primer, identification method and kit for Borna virus type 4 in psittaciform. Background technique [0002] Parrot bornavirus (PaBV) is a non-segmented single-stranded negative-sense RNA virus with a genome size of about 8900 bp, which replicates and proliferates in the nucleus and has an envelope. In the 1970s, a wasting disease called Proventricular dilatation disease (PDD) was reported in macaws. The clinical manifestations of parrots with PDD are mainly severe vomiting, loss of appetite, depression, loose feathers, and neurological symptoms such as ataxia, blindness or tremors in severe cases. Virus infection invades the nervous system of the digestive tract, causing gastrointestinal dysfunction, diarrhea symptoms and inability to digest food normally, undigested feed in the stool, and sometimes even blood in the stool. Sick parrot...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107
Inventor 单芬黎金荣焦培荣李婉萍翟俊琼代军威陈武
Owner 广州动物园
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