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Real-time fluorescent PCR detection method of duck pox virus and primers and probes for detection

A real-time fluorescence, duck pox virus technology, applied in the detection of duck pox virus primers and probes, duck pox virus real-time fluorescent PCR detection, duck pox virus detection field, can solve the lack of targeted detection technology and method, can not Carry out etiology and epidemiology, lose the opportunity to prevent and treat diseases, etc., to achieve the effect of high degree of automation, high accuracy and high sensitivity

Active Publication Date: 2017-01-04
广西悦牧生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many professional duck farmers have never encountered duck pox virus, so even if they see the symptoms, they cannot make a judgment and lose the opportunity to prevent and cure the disease
[0004] Since duckpox is a newly discovered disease in my country, there is still a lack of targeted detection techniques and methods, and relevant etiological and epidemiological studies cannot be carried out to scientifically evaluate the impact of the disease on domestic shelduck ducks, other breeds of poultry and wild poultry. Harm and its degree, and formulate targeted prevention and control measures

Method used

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  • Real-time fluorescent PCR detection method of duck pox virus and primers and probes for detection
  • Real-time fluorescent PCR detection method of duck pox virus and primers and probes for detection
  • Real-time fluorescent PCR detection method of duck pox virus and primers and probes for detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Effectiveness test of real-time fluorescent PCR detection method for duck pox virus

[0028] 1. Design primers and probes

[0029] Specific primers and probe sequences capable of detecting duckpox virus were designed for duckpox virus P4b gene:

[0030] Upstream primer: 5'-TGCTAAACAACAAAGGCCTATGC-3',

[0031] Downstream primer: 5'-TCCAGAATCTGGGACACTTTTACA-3'.

[0032] Fluorescent probe: 5'-CTTCGCAGTAGCAAACAAAAGCCCGAT-3'

[0033] Both upstream and downstream primers and fluorescent probes were synthesized by Life Company, in which the 5' end of the fluorescent probe was labeled with the fluorescent group FAM, and the 3' end was coupled with the quencher group BHQ1.

[0034] 2. Extract duckpox virus DNA

[0035] TakaRa MiniBEST ViralDNA / RNA Extraction Kit (Dalian Bao Biological Co., Ltd.) was used to extract the sample DNA.

[0036]

[0037] 3. Real-time fluorescent PCR detection

[0038] The reaction system was 20 µL, including 10 µL of TaqMan® Universal Master ...

Embodiment 2

[0042] Specificity Test of Real-time Fluorescent PCR Detection Method for Duck Pox Virus

[0043] 1. Viruses and samples used in the experiment

[0044] Duckpox tissue samples (TD1) were collected from the infected shelducks in Tiandong County, Guangxi, and provided by the Veterinary Laboratory of the Guangxi Animal Disease Prevention and Control Center.

[0045] Other waterfowl-derived viruses or poxviruses: H9N2 subtype avian influenza virus (AIV), H5N2 subtype avian influenza virus (AIV), duck flavivirus (Duck Flavivirus, DFV) and duck hepatitis virus (DuckHepatitis Virus, DHV) ) was provided by the Veterinary Laboratory of Guangxi Animal Disease Prevention and Control Center; Newcastle Disease Virus (Newcastle Disease Virus, NDV) live vaccine (Lasota strain), goat pox virus (Goatpox Virus, GTPV) live vaccine (AV41 strain), chickenpox virus ( Fowlpox Virus, FPV) live vaccine (282E4 strain), duck plague virus (DuckEntertitis Virus, DEV) live vaccine (C-KCE strain) and goose...

Embodiment 3

[0053] Sensitivity test of real-time fluorescent PCR detection method for duck pox virus

[0054] 1. Samples required for the experiment

[0055] After the duckpox virus p4b gene clone was sequenced correctly, the plasmid was extracted, and the concentration of the plasmid was determined and diluted to 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 copies / µL.

[0056] 2. Sensitivity test of real-time fluorescent PCR detection method

[0057] The diluted gradient plasmid was used as a sample, and the fluorescent PCR detection method provided in Example 1 was used for detection to determine the sensitivity of the method.

[0058] 3. Test results

[0059] The fluorescent PCR instrument collects the fluorescent signal and automatically generates the sample amplification curve. like image 3 As shown, the amplification curve C of duckpox P4b gene plasmid copy number is 1 and 10 t Greater than 32, the copy number is 10 2 、10 3 、10 4 、10 5 、10 6 The plasmid amplific...

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Abstract

The invention discloses a real-time fluorescence PCR detection method for duck pox virus as well as a primer and a probe for detection. A specific primer pair and the probe are designed according to a P4b gene sequence in a genome DNA of poultry pox virus, and the real-time fluorescence PCR detection method for the duck pox virus is established. The real-time fluorescence PCR detection method has a target sequence which is double controlled by the primer and the fluorescence probe, is good in the specificity, low in false positive, high in sensitivity, simple and convenient to operate and high in automation degree, is capable of quantifying product and rapidly and accurately detecting and identifying the duck pox virus by one step of amplification and detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting duck pox virus, in particular to a real-time fluorescent PCR detection method for duck pox virus; in addition, the invention also relates to primers and probes for detecting duck pox virus. Background technique [0002] Duck poxvirus is a member of the family Poxviridae, the subfamily Chordopoxvirinae, and the genus Avipoxvirus. Duckpox is a viral infectious disease of domestic ducks or wild waterfowl caused by duckpox virus. Fibrinous necrosis and hyperplastic lesions (diphtheria type) appear in oral and esophageal mucosa, and systemic infection may also occur concurrently. When mild cutaneous duckpox occurs in poultry flocks, the mortality rate is low, but it affects animal feeding and growth; while systemic infection, especially diphtheria duckpox, or concurrent infection or poor feeding management, the mortality rate is relatively low. High, can cause ser...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/70C12Q2561/113C12Q2561/101
Inventor 郑敏曹慧慧孙文超韦显凯蒙振亩郑列丰苏娇秀梁晟李军
Owner 广西悦牧生物科技有限公司