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Triple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for H9 subtype avian influenza virus, duck Tembusu virus and duck circovirus

A duck Tembusu virus and RT-PCR technology, applied in the field of RT-PCR detection, can solve problems such as early diagnosis and rapid control of unfavorable animal diseases, unfavorable prevention and control of duck disease, time-consuming and other problems

Inactive Publication Date: 2018-08-10
江西省农业科学院畜牧兽医研究所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Methods such as virus isolation, agar diffusion test and enzyme-linked immunosorbent assay are time-consuming, which is not conducive to the prevention and treatment of duck diseases
[0006] The traditional methods for the diagnosis of animal infectious diseases are mainly through the isolation of pathogens, electron microscope observation, serological identification and other methods, but this method is not conducive to the early diagnosis and rapid control of animal diseases

Method used

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  • Triple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for H9 subtype avian influenza virus, duck Tembusu virus and duck circovirus
  • Triple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for H9 subtype avian influenza virus, duck Tembusu virus and duck circovirus
  • Triple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for H9 subtype avian influenza virus, duck Tembusu virus and duck circovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, design and synthesis of primers

[0041] Three pairs of specific primers ( Table 1).

[0042] Table 1 identifies the primer sequences of H9AIV, DTMUV and DuCV

[0043]

Embodiment 2

[0044] Example 2. Triple RT-PCR identification of H9 subtype avian influenza virus (H9AIV), duck tambushu virus (DTMUV) and duck circovirus (DuCV)

[0045] Step 1. Establishment of triple RT-PCR system

[0046] (1) Preparation of samples to be tested

[0047] According to the instructions of the viral RNA / DNA rapid purification kit, the H9 subtype avian influenza virus RNA, the duck Tembusu virus RNA and the duck circovirus DNA were extracted. The concentration and purity of the nucleic acid were determined according to the Sambrook method, and stored at -70°C for later use.

[0048] (2) Optimization of triplex RT-PCR reaction conditions

[0049] RT-PCR was performed in one step using the One Step RT-PCR kit. Optimize each cycle parameter and each primer concentration of RT-PCR to determine the best RT-PCR mode.

[0050] By optimizing the concentration of RT-PCR primers, each reaction temperature, time and number of cycles, etc., it was finally determined that the best wor...

Embodiment 3

[0064] Embodiment 3, the assembly of detection kit

[0065] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.

[0066] H9 Subtype Avian Influenza Virus, Duck Tembusu Virus and Duck Circovirus Triple RT-PCR Detection Kit, including PCR reagents for detecting H9 Subtype Avian Influenza Virus, Duck Tembusu Virus and Duck Circovirus, One- StepRT-PCR kit, positive control solution and negative control solution.

[0067] The PCR reagent includes a PCR primer set consisting of H9AIV upstream and downstream primers, DTMUV upstream and downstream primers and DuCV upstream and downstream primers;

[0068] The H9AIV upstream and downstream primers are respectively the primer 1 and the primer 2;

[0069] The DTMUV upstream and downstream primers are respectively the primer 3 and the primer 4;

[0070] The DuCV upstream and downstream primers are respectively the primer 5 and the primer 6;

[0071] The One-Step RT-PCR kit includes 2×one-...

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Abstract

The invention discloses a triple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for an H9 subtype avian influenza virus, a duck Tembusu virus and a duck circovirus. The kit comprises three pairs of specific primers. Tests show that by adopting the kit, three pathogens of the H9 subtype avian influenza virus, the duck Tembusu virus and the duck circovirus can be simultaneously detected through only one time of RT-PCR reactions, and the kit has the advantages of good specificity, high sensitivity, low cost, high efficiency and the like; in addition, in primer designing, amplification results can be directly judged according to different lengths of amplification segments, and the kit is relatively simple, convenient, visible and practical. By adopting the kit, 1pg of H9 subtype avian influenza virus RNA (Ribonucleic Acid), 50pg of duck Tembusu virus RNA and 10ng of duck circovirus DNA can be simultaneously detected, an accurate detection method is provided for early morbidity stages of the three viruses, and the kit has a great significance on cutting propagation of viruses, has wide application prospects and has great practical significances for sustainable development of the duck breeding industry.

Description

technical field [0001] The invention relates to the technical field of RT-PCR detection, in particular to a triple RT-PCR detection kit for H9 subtype avian influenza virus, duck Tembusu virus and duck circovirus. Background technique [0002] Avian influenza is a general term for poultry infections and diseases caused by different subtypes of influenza A viruses in the Orthomyxoviridae family. H9 subtype avian influenza virus (H9AIV) generally has low to moderate virulence, but it is widely distributed and can cause immunosuppression of ducks and lead to mixed infection with other duck diseases, and infection of laying ducks can also cause egg production rate drop The obvious decline has greatly affected the economic benefits of laying ducks, so its harm to the duck industry cannot be ignored. [0003] Duck Tambusu virus (DTMUV) was first discovered in 2010 in Fujian, Zhejiang, Jiangsu and other eastern coastal areas of China, and gradually spread to most parts of the coun...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 李海琴傅光华韦启鹏黄瑜季华员谭美芳康昭风唐维国曾艳兵黄江南傅秋玲
Owner 江西省农业科学院畜牧兽医研究所
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