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Primer probe combination and kit for simultaneously detecting duck circovirus, duck plague virus and duck adenovirus 3

A technology for duck circovirus and duck plague virus is applied in the field of livestock and poultry disease diagnosis to achieve the effect of rapid detection of pathogens, saving time and reducing workload

Active Publication Date: 2020-12-11
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is a single PCR method for detecting duck circovirus, duck plague virus and duck type 3 adenovirus, and a triple PCR detection method for duck circovirus, duck plague virus and another virus, but it has not been seen that only one pair of primers is used. A report on simultaneous detection and differentiation of duck circovirus, duck plague virus and duck adenovirus type 3

Method used

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  • Primer probe combination and kit for simultaneously detecting duck circovirus, duck plague virus and duck adenovirus 3
  • Primer probe combination and kit for simultaneously detecting duck circovirus, duck plague virus and duck adenovirus 3
  • Primer probe combination and kit for simultaneously detecting duck circovirus, duck plague virus and duck adenovirus 3

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Embodiment 1

[0048] (1) Design and preparation of hybridization probe amplification primers

[0049] Referring to duck circovirus Rep protein gene, duck plague virus gH protein gene, duck type 3 adenovirus DNA polymerase protein gene conserved sequence and green fluorescent protein gene sequence, three pairs were designed for the synthesis of duck circovirus, duck Three specific hybridization probes and amplification primers for pestivirus and duck type 3 adenovirus nucleic acids. The primer information is shown in Table 1. The sequence in bold is the sequence encoding green fluorescent protein. The above primers were sent to Fuzhou Boshang Biotechnology Co., Ltd. for synthesis, diluted with sterile ultrapure water to a concentration of 10 μM, and stored at -20°C for later use.

[0050] Table 1 Primer Information

[0051]

[0052]

[0053] (2) Hybridization probe preparation and purification

[0054] The green fluorescent protein particle (pIRES2-EGFP, Takara Biotechnology Co., Ltd...

Embodiment 2

[0056] Establishment of a PCR detection method for multiple viruses

[0057] 1. Design and preparation of hybridization probe detection primers With reference to the green fluorescent protein gene sequence in the hybridization probe, design 1 pair of reverse amplification primers for detecting the amplification of the hybridization probe, and the upstream primer is SEQ ID No.1 ( JC-GFPF): 5'-GCAGAA CAC CCC CAT CGG CGA-3', the downstream primer is SEQ ID No.2 (JC-GFPR): GTT CTT CTG CTT GTCGGC CAT G-3'. When using this pair of primers for detection, a specific fragment of 466 bp should be amplified for duck circovirus, a specific fragment of 316 bp for duck plague virus and a specific fragment of 202 bp for duck type 3 adenovirus.

[0058] 2. PCR detection of duck circovirus Take preserved duck circovirus, centrifuge briefly, take 200 μL supernatant, and extract sample DNA according to the operation manual of QIAGEN virus DNA extraction kit. The nucleic acid concentration of th...

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Abstract

The invention provides a primer probe combination for simultaneously detecting duck circovirus, duck plague virus and duck adenovirus 3, and belongs to the technical field of livestock and poultry disease diagnosis. The primer probe combination comprises a primer pair and a probe, a nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, and a nucleotide sequence of adownstream primer of the primer pair is shown as SEQ ID No.2; the probe comprises a duck circovirus probe, a duck plague virus probe and a duck adenovirus 3 probe, a nucleotide sequence of the duck circovirus probe is shown as SEQ ID No.3, a nucleotide sequence of the duck plague virus probe is shown as SEQ ID No.4, and a nucleotide sequence of the duck adenovirus 3 probe is shown as SEQ ID No.5.By adopting the primer probe combination provided by the invention, the duck circovirus, the duck plague virus and the duck adenovirus 3 can be simultaneously detected.

Description

technical field [0001] The invention belongs to the technical field of livestock and poultry disease diagnosis, and in particular relates to a combination of primers and probes and a kit for simultaneously detecting duck circovirus, duck plague virus and duck type 3 adenovirus. Background technique [0002] Duck circovirus (DuCV) is an important pathogen discovered in recent years that seriously affects the growth performance and immune response of ducks. It is known that the virus exists widely in ducks, and ducks of different breeds and growth stages can be infected. Meat ducks infected with circovirus often show growth retardation and messy coat. The weight of infected ducks is more than 35% lower than that of uninfected ducks of the same age. Clinically, it is also found that cases of duck circovirus are positive, and there are many other pathogens mixed. The phenomenon of infection. [0003] Duck plague virus (DPV) is an acute, septic, and highly fatal infectious dise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16
Inventor 傅光华黄瑜程龙飞傅秋玲刘荣昌万春和陈红梅施少华林建生
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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