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Primer pair for detecting pigeon torque teno viruses and application of primer pair

A lenovirus and primer pair technology, applied in the field of primer pairs for detection of pigeon lenovirus, achieves high specificity and stability, rapid and accurate detection

Active Publication Date: 2013-07-17
靖江市华信科技创业园有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no domestic report on pigeon fine ring virus. Under the background of the obviously high infection rate of pigeon fine ring virus in our country, the development of PCR detection method and kit for pigeon fine ring virus in my country should be established as soon as possible in order to investigate and prevent and control the disease It is a matter of urgency, and it has very important practical significance and market value for the stable development of my country's pigeon market

Method used

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  • Primer pair for detecting pigeon torque teno viruses and application of primer pair
  • Primer pair for detecting pigeon torque teno viruses and application of primer pair
  • Primer pair for detecting pigeon torque teno viruses and application of primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Preparation of the kit.

[0033] 1. Source and treatment of viruses and disease materials

[0034] Pigeon fine ring clinical disease materials were collected in October 2009 from internal organs of diseased pigeons in Jiangsu Danyang, Anhui and other places. Take 0.5-2.0 g of visceral tissue in a sterile mortar, add physiological saline to grind, freeze and thaw repeatedly 2-3 times, centrifuge at 5000 rpm for 5 min, and take the supernatant and store it at -20°C. Pigeon ring-positive disease materials, chicken anemia-positive disease materials, and pig ring-positive disease materials are kept by the laboratory.

[0035] 2. Reagents

[0036] PCR pre-mix and proteinase K were purchased from Dalian Takara Company; DNA Marker and GoldView were purchased from Nanjing Shanggao Biological Company; primers were synthesized by Shanghai Yingjun. Other reagents are imported or domestic analytical reagents.

[0037] 1. Design and synthesis of primers

[0038] Accor...

Embodiment 2

[0043] Embodiment 2: detection method

[0044] Follow the steps below to check:

[0045] (1) Sample processing: The clinical disease materials of pigeon fine ring were collected in October 2009 from internal organs of diseased pigeons in Jiangsu Danyang, Anhui and other places. Take 0.5-2.0g of internal organs in a sterile mortar, add normal saline to grind, repeat After freezing and thawing for 2-3 times, centrifuge at 5000rmp for 5min, and store the supernatant at -20°C. Pigeon ring-positive disease materials, chicken anemia-positive disease materials, and pig ring-positive disease materials are kept by the laboratory.

[0046](2) Extraction of viral DNA: Freeze and thaw the tissue suspension of suspected disease material 3 times, centrifuge to obtain 450 μL of supernatant, add proteinase K to a final concentration of 500 μg / mL, add SDS to a final concentration of 10 g / L, and bathe in water at 55°C for 30 minutes. Extract once each with phenol, phenol-chloroform (volume ra...

Embodiment 3

[0050] Embodiment 3: specificity experiment

[0051] Using the same method as in Example 2 to extract the DNA of the pigeon fine ring positive disease material, the pigeon ring positive disease material, the chicken anemia positive disease material, and the pig circular ring positive disease material, and use this as a template to carry out PCR with the designed primers Amplify. Using 1% agarose for gel electrophoresis, only a fragment of about 500bp was amplified by the pigeon leiocircle virus (see figure 1 ), the gene sequencing of the PCR product of the pigeon leptochram-positive disease material was carried out, and the sequence was compared with the published sequence in GenBank. The result was 55-75% homologous to the published human and porcine leptochram gene sequences, Considering the high degree of heterogeneity among different species of pigeon leovirus, it can be shown that the amplified fragment is pigeon leovirus, while pigeon circovirus disease material, chicke...

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Abstract

The invention discloses a primer pair for detecting pigeon torque teno viruses. The nucleotide sequence of the primer pair is represented by SEQID NO:2 and SEQ ID NO:3. The invention also discloses a nucleotide sequence of the pigeon torque teno viruses and a preparation method for the nucleotide sequence. The nucleotide sequence of the virus is obtained by performing polymerase chain reaction (PCR) on the primer pair and is represented by SEQ ID NO:1. The invention also discloses a detection kit which comprises the primer pair and is used for detecting the pigeon torque teno viruses and a detection method. According to the primer pair for detecting the pigeon torque teno viruses, on the basis of a specific primer pair, a PCR condition is optimized; the nucleotide sequence of the pigeon torque teno viruses is amplified; and a detection condition and a detection system are optimized, so that the kit for detecting the virus is prepared. The kit is relatively high in specificity and stability; the sensitivity of the kit is up to 5 pg; and the kit can quickly and accurately detect the pigeon torque teno viruses. The kit can be used as an effective tool for quickly detecting the pigeon torque teno viruses.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to a pair of primers for detecting pigeon serovirus and an application thereof. Background technique [0002] Torque teno virus (TTV) is a new type of zoonotic DNA virus discovered in recent years. It was first discovered in human hepatitis in 1997. It is widely present in humans, Domestic animals, wild animals and some companion animals. Leptoviruses belong to the genus Leptoviruses of the family Leptoviridae, which are spherical, non-enveloped, and circular single-stranded negative-sense DNA viruses. TTV genomic DNA is highly poor in quality, and because TTV is a single-stranded DNA virus (letrocyclovirus), the titer in the serum of infected animals is quite different, which may also be due to the current TTV complete gene sequence on GenBank. number of reasons. The position of the open reading frame (OFR) of TTV and the function of each part of the coding gene are not well under...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 张志成庄林林蒋加进戴鼎震
Owner 靖江市华信科技创业园有限公司
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