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Method for identifying new goose parovovirus

A goose parvovirus, a new type of technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of economic loss, difficult differential diagnosis, and shortening of the upper beak in the duck industry. Achieve rapid analysis, high sensitivity and good repeatability

Inactive Publication Date: 2019-07-12
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in recent years, a new type of parvovirus disease has appeared in young Muscovy ducks and Cherry Valley ducks in Fujian, Zhejiang, Anhui and Jiangsu provinces. The incidence rate of the disease is 10-30%, and the fatality rate is less than 3%. The symptoms are mainly broken feet and shortened upper beak, which has caused serious economic losses to the duck industry in my country
Through whole-genome sequencing and phylogenetic tree analysis of the pathogen of the disease, it was found that the pathogen is closely related to goose parvovirus. ) The similarity of the whole gene sequence to GPV is 89.7%-96.7%, and the similarity to MDPV is 79.6%-83.9%, so it is difficult to differential diagnosis by ordinary PCR diagnostic methods

Method used

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  • Method for identifying new goose parovovirus
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  • Method for identifying new goose parovovirus

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Experimental program
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Effect test

Embodiment 1

[0049] According to the nucleic acid sequences of NGPV, MDPV and GPV genomes, the present invention designs a pair of primers for fluorescent quantitative PCR; after a large number of screenings, the present invention screens out a group of primers with high sensitivity and strong specificity, the sequence of which is as follows:

[0050] NGPV-F: 5'-CACTTCCTGGCGCGCAAAATATTT-3' (SEQ ID NO: 1);

[0051] NGPV-R: 5'-TGCGTCACCGGAAGCAC-3' (SEQ ID NO: 2).

Embodiment 2

[0052] Embodiment 2 Positive standard substance preparation, fluorescent quantitative PCR detection method

[0053] 1) Preparation of standard samples

[0054] In order to draw the standard curve of fluorescent quantitative PCR, we extracted the DNA of the new goose parvovirus as a PCR template, used primers to amplify the target sequence, and connected the amplified product into the vector to obtain the vector of the target sequence of the new goose parvovirus , the positive standard of the new goose parvovirus.

[0055] 2) Fluorescent quantitative PCR detection of positive standard samples

[0056] The new goose parvovirus positive standard in the above steps was diluted for detection.

[0057]The diluted plasmid standard was amplified under optimal amplification conditions, and the fluorescence quantitative PCR amplification reaction system was:

[0058]

[0059] The PCR amplification reaction program was: pre-denaturation at 95°C for 3min; denaturation at 95°C for 15...

Embodiment 3

[0062] Embodiment 3 A kind of fluorescent quantitative PCR detection method

[0063] 1) Nucleic acid extraction from samples: the samples to be extracted contained 2 livers positive for novel goose parvovirus, 3 livers positive for classical goose parvovirus and 2 livers positive for muscovy duck parvovirus.

[0064] 2) Using the extracted nucleic acid as a template to carry out fluorescent quantitative PCR, the reaction system is:

[0065]

[0066] The PCR amplification reaction program was: pre-denaturation at 95°C for 3min; denaturation at 95°C for 15s; annealing at 60°C for 60s; cycle 39 times.

[0067] At the same time, a negative control group and a positive control group were set up, wherein the negative control was physiological saline. The positive control group uses the solution containing the positive standard product of the new goose parvovirus gene as the nucleic acid template.

[0068] 3) Result analysis: The PCR product is tagged and tracked with the fluore...

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Abstract

The invention discloses a method for identifying new goose parovovirus. The fluorescent quantitation PCR detection method is high in flexibility and capable of detecting 10 copy number of gene of thenew goose parovovirus while an ordinary PCR only can detect 104 copy number, and the flexibility is higher 1000 times; the specificity is high, according to the difference region between classic gooseparovovirus and the new goose parovovirus and the difference region between muscovy duck parvovirus and the new goose parovovirus, a pair of specificity primers are designed and synthesized, and thegene fragments of the new goose parovovirus can be specifically amplified; the repeatability is high, analysis can be accurately and quickly conducted, and application and popularization in clinical practices are facilitated.

Description

technical field [0001] The invention belongs to the field of molecular detection of animal pathogens, and relates to a method for identifying novel goose parvovirus. Background technique [0002] Duck parvovirus is an infectious disease caused by goose parvovirus (GPV) or muscovy duck parvovirus (Duckparvovirus, MDPV). The main symptoms of sick ducks caused by classic MDPV and GPV strains are diarrhea, soft feet, and exudative enteritis. The morbidity and mortality of infected ducklings within three weeks of age are very high. However, in recent years, a new type of parvovirus disease has appeared in young Muscovy ducks and Cherry Valley ducks in Fujian, Zhejiang, Anhui and Jiangsu provinces. The incidence rate of the disease is 10-30%, and the fatality rate is less than 3%. The main symptoms are easily broken feet and shortened upper beak, which have caused serious economic losses to the duck industry in my country. Through whole-genome sequencing and phylogenetic tree ana...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701
Inventor 卞国志马海彬袁建丰龚凤平罗梦萍
Owner SOUTH CHINA AGRI UNIV
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