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Method for cloning complete sequence of muscovy duck origin goose parvovirus genome encoding area

A technology for gosling plague virus and virus genome, which is applied in the field of cloning the entire coding region of the gosling plague virus genome in Muscovy ducks, can solve the problems of misreading of sequence bases, high cost, heavy workload, etc., and achieves high accuracy, Small workload and high efficiency

Inactive Publication Date: 2014-12-24
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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Problems solved by technology

[0004] At present, there is little research in this field on how to effectively obtain the full sequence of the MDGPV genome coding region. As a traditional method, the segmented PCR cloning method is used to amplify the MDGPV genome coding region There are many defects in this method. The target gene must be divided into several segments to be cloned separately. When divided into several segments, several subclonings are required, and then subcloning sequencing and splicing are performed, resulting in high cost, heavy workload, and the possibility of sequence base misreading.

Method used

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  • Method for cloning complete sequence of muscovy duck origin goose parvovirus genome encoding area

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Embodiment 1

[0030] Step 1, virus isolates:

[0031] The new genotype GPV Fujian representative strain MDGPV-PT was isolated, identified and preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0032] Step 2, virus DNA extraction:

[0033] Genomic DNA of MDGPV-PT strain is extracted by AxyPrep Body Fluid Virus DNA / RNA Mini Kit. The specific operation steps are as follows:

[0034] a. Collect 200 μL of allantoic fluid of muscovy duck embryos with MDGPV virus, and transfer to a 1.5 mL centrifuge tube.

[0035] b. Add 200 μL Buffer V-L, vortex to mix evenly, and let stand for 5 min.

[0036] c. Add 75 μL Buffer V-N, vortex to mix well, and centrifuge at 12,000×g for 5 min.

[0037] d. Transfer the supernatant to a new 2 mL centrifuge tube (provided in the kit), add 300 μL isopropanol (1% glacial acetic acid), invert up and down 6-8 times, and mix well.

[0038] e. Put the preparation tube in a 2 mL centrifuge tube (provided in ...

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Abstract

The invention discloses a method for cloning a complete sequence of a muscovy duck origin goose parvovirus genome encoding area. A viral genome DNA is extracted from muscovy duck embryo allantoic fluid containing a muscovy duck origin goose parvovirus; with the viral genome DNA as a template, a gene segment containing the complete sequence of the encoding area is amplified by virtue of designed specific primers; a target gene segment is connected with a carrier; and positive colonies are selected to be subjected to sequencing, so as to directly obtain the complete sequence of the muscovy duck origin goose parvovirus genome encoding area. Compared with a traditional method, the method disclosed by the invention has the characteristics of long amplified target segment, small experimental workload, low cost and high efficiency.

Description

technical field [0001] The invention relates to a method for cloning the complete sequence of the genome coding region of Muscovy duck origin goose parvovirus (MDGPV). Background technique [0002] Gosling plague (Gosling Plague, GP), also known as Goose Parvovirus (GPV) infection or Derzsy's disease, is a highly contagious disease caused by GPV that mainly affects goslings and young muscovy ducks. Exudative enteritis, small intestinal mucosal surface necrosis, and intestinal embolism are characteristic lesions. Clinically, Muscovy duck parvovirus (MDPV) and Muscovy duck parvovirus (MDPV) disease (commonly known as "three-week disease") are often prevalent in the same duck flock in the same area, and young Muscovy ducks are susceptible to both GPV and MDPV. Goslings are only susceptible to GPV. Muscovy duck and gosling plague is an epidemic that has caused diarrhea of ​​varying degrees in young muscovy ducks in Putian and other places in Fujian since 1997, and some diseas...

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Application Information

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IPC IPC(8): C12N15/70C12N15/10C12R1/93
Inventor 王劭陈少莺程晓霞林锋强陈仕龙王锦祥
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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