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Primer group and kit for detecting avian Borna virus, and usage method thereof

A virus detection and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc., can solve problems such as death, animal septicemia, and inability to move normally.

Inactive Publication Date: 2013-05-01
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the expansion of the glandular stomach is affected, and the normal peristalsis cannot be performed, and the microorganisms multiply in it, resulting in sepsis and death of the animal

Method used

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  • Primer group and kit for detecting avian Borna virus, and usage method thereof
  • Primer group and kit for detecting avian Borna virus, and usage method thereof
  • Primer group and kit for detecting avian Borna virus, and usage method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] The preparation of embodiment 1 sample and the selection of primer

[0110] 1. Sample preparation

[0111] 1.1. Sample preparation Cell culture

[0112] Porcine Borna virus-positive samples were inoculated into well-grown monolayer porcine testis (ST) cells, cultured and passaged at 37°C, and the cytopathic changes were observed.

[0113] Cell culture passage and virus detection

[0114] Use parrot "Liuli 363" and "Hui Hue" glandular stomach disease materials (among them, parrot "Liu Li 363" and "Hui Hue" are the names of the sampling birds) after aseptic treatment, inoculate ST cell monolayer, and culture at 37°C for 5-7 days. After 5 consecutive passages, the cells appeared to shrink and fall off ( figure 1 ), which were positive by RT-PCR ( Figure 3B ).

[0115] 1.2. Total RNA extraction:

[0116] Various ABV detection samples were carried out according to the instructions of the total RNA extraction kit RNAiso Plus / MiniBEST (Takara) and the viral total nuclei...

Embodiment 2

[0167] Example 2 sample detection test

[0168] 1. RT-LAMP (reverse transcription-loop mediated constant temperature amplification) experiment

[0169] Prepare RT-LAMP reaction system, including 12.5 μl of reaction buffer (2×), 1 μl of inner primer (FIP / BIP) (final concentration 0.4-0.8 μM), outer primer (F3 / B3) (final concentration 0.1-0.2 μM) 1 μl, Bst polymerase 0.8 μl, reverse transcriptase 0.2 μl, RNA template (1-100ng) 2.0 μl, DEPC water 7.5 μl, total volume 25 μl. Mix the above reagents evenly, add 20 μl of sealing solution, put 1 μl of chromogenic solution in the middle of the cap of the reaction tube, and cap tightly, and pay attention that the chromogenic solution cannot fall into the reaction solution. Then, put the reaction tube into the Loopamp turbidimeter LA-100, take out the reaction tube, pop the dye into the reaction solution, and observe the color change after mixing. Green indicates nucleic acid amplification (positive +), orange indicates no nucleic acid...

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Abstract

The invention provides a primer group for detecting avian Borna virus. The primer group comprises two pairs of primers, inner primers FIP / BIP and outer primers F3 / B3, designed by using an avian Borna virus matrix protein gene as a target gene, and based on a loop-mediated isothermal amplification technology. The invention also discloses a kit for detecting avian Borna virus and a usage method thereof. The technical scheme provided by the invention can efficiently, accurately detect avian Borna virus, can be widely used in the inspection and quarantine of source of such disease, and lays foundation for study on an animal model of pathogenic mechanism of the RNA virus in a host.

Description

technical field [0001] The invention relates to a biological detection reagent, in particular to a primer, a kit and a use method for detection of avian Borna virus. Background technique [0002] Avian Bornavirus (ABV) is a new Bornavirus (BDV) isolated from parrots with Proventricular Dilatation Disease (PDD). BDV can persist in the host's central nervous system, and its entire life cycle is completed in the nucleus of neurons. After virus infection, the gene fuses with the host cell chromosome, and the viral DNA sequence can be detected in the host genome. Poultry is an important natural foci of BDV, and virus strains have been detected in fecal samples collected in bird activity ponds. PDD was first reported in the 1970s, initially primarily in captive parrots in North America. Due to extensive commercial activities, PDD continues to spread among pet birds. It has been reported all over the world that more than 50 species of parrots have been infected. In addition, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 林志雄田纯见王宏罗琼赵吟罗长保鱼海琼刘志玲陈茹吴晓薇朱道中
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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