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Goose astrovirus, goose parvovirus and goose calico virus multiple nano PCR detection primer pair, kit and application method

The technology of goose parvovirus and astrovirus is applied in the field of molecular biology diagnosis of animal diseases, and achieves the effects of easy operation, rapid detection and good amplification effect.

Active Publication Date: 2021-12-24
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE JIANGXI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since 2018, researchers have successively detected a new GoCV strain from geese suffering from gout. This virus strain has a high homology with goose enteritis type GoCV H146 strain. Research on channels and fashion trends is still in its infancy
At present, there is no technical application of a multiplex nano-PCR method with good sensitivity, specificity and stability that can simultaneously detect goose astrovirus, goose parvovirus and goose calicivirus

Method used

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  • Goose astrovirus, goose parvovirus and goose calico virus multiple nano PCR detection primer pair, kit and application method
  • Goose astrovirus, goose parvovirus and goose calico virus multiple nano PCR detection primer pair, kit and application method
  • Goose astrovirus, goose parvovirus and goose calico virus multiple nano PCR detection primer pair, kit and application method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1 Materials and methods

[0047] 1.1 Virus strains Goose Astrovirus (GoAstV), Goose Parvovirus (GPV), Goose Calicivirus (GoCV), Tambusu Virus (TMUV), Duck Plague Virus (DVE), Goose Paramyxovirus (GPMV), Goose viral hepatitis virus (DVH), reovirus (DRV), inactivated H9 subtype avian influenza inactivated virus (H9 AIV), inactivated H7 subtype avian influenza virus (H7 AIV), inactivated H5 subtype avian influenza virus (H7 AIV);

[0048] 1.2 Main Reagents Spin Column Type Viral RNA / DNA Extraction Kit PrimeScript IV RTase, 5×PrimeScript IV Buffer, RNase Inhibitor, dNTP Mixture, rTaq DNA Polymerase were purchased from Bio-Technology (Beijing) Co., Ltd.; DL2000 DNA Marker purchased from Beijing Qingke Xinye Biotechnology Co., Ltd.

[0049] 1.3 Experimental method

[0050]1.3.1 Design and synthesis of primers This implementation refers to the complete genome sequences of GoAstV, GPV, and GoCV viruses published on GenBank, and is comprehensively analyzed and compared by MEG...

Embodiment 2

[0067] A kind of goose astrovirus, goose parvovirus and goose calicivirus multiplex nano-PCR detection primer pair, kit and method application thereof obtained after the optimization of embodiment 1 were tested for specificity, stability and sensitivity, and passed the field test The practicability and applicability of the multiple nano-PCR diagnostic kits for goose astrovirus, goose parvovirus and goose calicivirus obtained in the present invention are verified as follows:

[0068] (1) Specificity test

[0069] GoAstV, GPV, GoCV, Tambusu virus (TMUV), duck plague virus were extracted by the detection method of a kind of goose astrovirus, goose parvovirus and goose calicivirus multiplex nano-PCR diagnostic kit obtained after the optimization of Example 1 (DVE), goose paramyxovirus (GPMV), goose viral hepatitis virus (DVH), reovirus (DRV), inactivated H9 subtype avian influenza inactivated virus (H9 AIV), inactivated H7 subtype Type avian influenza virus (H7 AIV), inactivated ...

Embodiment 3

[0081] 1 Processing of visceral tissue samples

[0082] After the visceral tissue was quick-frozen in liquid nitrogen, it was quickly transferred to a mortar pre-cooled in liquid nitrogen until it was ground into a powder. Grind and centrifuge at a mass ratio of visceral tissue to normal saline of 1:3, and use a spin-column viral DNA / RNA extraction kit to extract viral DNA and RNA in the tissue to obtain the RNA / DNA extract of the sample to be tested;

[0083] 2 reverse transcription reaction

[0084] 2.1 Reverse transcription reaction of visceral tissue samples: take 18 μL of reverse transcription reaction mixture, add 2 μL of RNA extraction solution of visceral tissue samples, mix well, and perform reverse transcription reaction. React for 15 minutes at ℃ and 5 minutes at 95℃ to inactivate the enzyme, then cool on ice to obtain the cDNA product of the visceral tissue sample:

[0085] 3 PCR amplification reaction

[0086] 3.1 PCR amplification reaction of visceral tissue s...

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Abstract

The invention relates to a goose astrovirus (GoAstV), goose parvovirus (GPV) and goose calico virus (GoCV) multiple nano PCR detection primer pair, a kit and an application method thereof. The kit comprises a reverse transcription mixed solution, an amplification reaction mixed solution, a negative control and a positive control. According to the method, a nano PCR detection application method for the goose astrovirus, the goose parvovirus and the goose calico virus is established, a multiple nano PCR reaction system is optimized, and the primer concentration, the annealing temperature, the rTaq DNA polymerase concentration, the nano particle diameter and the nano particle concentration are determined, so that the amplification effects of the three viruses of GoAstV, GPV and GoCV are better; and the method is high in sensitivity, good in specificity and high in stability, the detection method is easy to operate, convenient and rapid, the detection time can be greatly shortened, the diagnosis problem of GoAstV, GPV and GoCV mixed infection can be thoroughly solved, and the method is suitable for rapid detection in clinical laboratories.

Description

technical field [0001] The invention relates to the field of molecular biology diagnosis of animal epidemic diseases, in particular to a pair of multiple nano-PCR detection primers for goose astrovirus, goose parvovirus and goose calicivirus, a kit and an application method thereof. Background technique [0002] In recent years, new waterfowl diseases continue to appear around the world, which is an important reason for the heavy losses of the waterfowl industry. Therefore, it is of great significance to establish a rapid detection method for the prevention and control of emerging and reappearing mutant virus diseases in waterfowl. Since 2017, an increasing number of cases of astrovirus-associated goose gout disease have been detected in many provinces of China, such as Anhui, Shandong, Jiangsu, Guangdong, Liaoning, Heilongjiang, Jiangxi, and Henan, with a mortality rate of up to 50%, and the new goose astroviruses (GoAstV) can be transmitted vertically, which brings greate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2527/143C12Q2527/149C12Q2537/143C12Q2563/155C12Q2565/125Y02A50/30
Inventor 张帆帆李海琴韦启鹏杨群康昭风谭美芳曾艳兵谭佳
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE JIANGXI ACAD OF AGRI SCI
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