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Dual-PCR (Polymerase Chain Reaction) assay kit for duck circovirus and duck hepatitis virus

A duck circovirus and duck hepatitis virus technology, applied in the biological field, can solve the problems of immunosuppression, damage to the immune system of sick ducks, economic losses in the duck farming industry, etc., and achieve good specificity, important practical significance, and important practical value. Effect

Active Publication Date: 2012-07-25
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two diseases are widely prevalent, spread rapidly, have high morbidity and mortality, and are easy to be mixed with infection. At present, they are more harmful to waterfowl worldwide and bring huge economic losses to the duck industry.
Duck circovirus destroys the immune system of sick ducks, causing immunosuppression. Duck hepatitis virus is widespread and highly lethal. Sick ducks are easily infected with these two viruses at the same time. Once infected at the same time, it will cause great losses.
At present, the differential diagnosis of DuCV and DHV mainly relies on traditional pathogen isolation and serological tests, but these methods have the disadvantages of long diagnostic time, poor specificity, low sensitivity, and cumbersome operations, which are not conducive to rapid diagnosis of these two diseases

Method used

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  • Dual-PCR (Polymerase Chain Reaction) assay kit for duck circovirus and duck hepatitis virus
  • Dual-PCR (Polymerase Chain Reaction) assay kit for duck circovirus and duck hepatitis virus
  • Dual-PCR (Polymerase Chain Reaction) assay kit for duck circovirus and duck hepatitis virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, design and synthesis of primers

[0047] According to the published gene libraries of duck circovirus DuCV and duck hepatitis virus DHV, DNA Star software was used to design 2 pairs of specific primers for DuCV and DHV gene sequences. The nucleic acid sequences of the primers are shown in Table 1. Primers were synthesized by Shanghai Invitrogen Company.

[0048] Table 1 is the nucleotide sequence of DuCV and DHV primers

[0049]

Embodiment 2

[0050] Embodiment 2, double PCR detection

[0051] 1. Sample preparation

[0052] Propagation and collection of the virus: The seed virus was properly diluted and inoculated into 10-day-old SPF chicken embryos and specific pathogen-free healthy duck embryos, and the allantoic fluid of dead embryo bodies was collected after 120 hours of propagation.

[0053] The above-mentioned seed virus is duck type I hepatitis virus (DHV I) AV2111 strain (hereinafter referred to as DHV I), muscovy duck parvovirus, Newcastle disease virus, goose plague virus and H9 subtype avian influenza virus.

[0054]Extract the RNA from the above-mentioned proliferated DHV I, Newcastle disease virus and H9 subtype avian influenza virus according to the instruction manual of TRIzol LS Reagent, and then transcribe them respectively. Using 10 μL system, add 2 μL 5×RT Buffer, 10 mmol / L dNTP 1μL, 5U / μL AMV 0.5μL, 20U / μL RNase inhibitor 0.5μL, free primer 0.5μL, total RNA template to be tested 2μL, make up to...

Embodiment 3

[0071] Embodiment 3, double PCR detection sample to be tested

[0072] The collected 12 copies (numbered 1-12) of duck disease materials (collected liver) were ground into a suspension, and then the virus was isolated and identified, and the DNA and RNA of the duck disease materials were extracted respectively, and the RNA was reverse-transcribed to obtain cDNA. The DNA and cDNA of each sample were mixed (volume ratio 1:1) to obtain mixed samples numbered 1-12.

[0073] The above-mentioned mixed samples numbered 1-12 were respectively used as templates, and double PCR detection was performed according to the PCR reaction system and reaction mode of Example 2-2.

[0074] If a 245bp fragment is obtained, the sample contains DuCV, otherwise it does not;

[0075] If a 569bp fragment is obtained, the sample contains DHV I, otherwise it does not;

[0076] If the fragments of 245bp and 569bp are obtained, the sample contains DuCV and DHV I, otherwise there is no.

[0077] The resu...

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Abstract

The invention discloses a dual-PCR assay kit for duck circovirus and duck hepatitis virus. The dual-PCR assay kit provides a primer group for assaying duck circovirus and duck hepatitis virus, which consists of a primer 1, a primer 2, a primer 3 and a primer 4; and the nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are sequence 1, sequence 2, sequence 3 and sequence 4 sequentially in a sequence table. The dual-PCR technique which can simultaneously assay and identify DuCV (duck circovirus) and DHV (duck hepatitis virus) as two pathogens by establishing PCR reaction just once has good specificity, moreover, the experiment utilizes the difference of amplified fragment lengths to directly determine an amplification result in the primer design, and thereby the method is simpler, more visual and more practical during result determination.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double PCR detection kit for duck circovirus and duck hepatitis virus. Background technique [0002] Duck circovirus (Duck Circovirus, DuCV) and duck hepatitis virus (DHV) are both common in ducks. Duck circovirus (DuCV) can cause symptoms such as growth retardation, messy feathers, and weight loss in ducks, and can infect the immune system of poultry, causing Immunosuppressive. Duck viral hepatitis caused by duck hepatitis virus (DHV) is a rapidly spreading viral disease that can make sick ducks highly lethal. The disease course is short and death is fast, and the mortality rate can be as high as 100%. These two diseases are widely prevalent, spread rapidly, have high morbidity and mortality, and are easy to be mixed with infection. At present, they are more harmful to waterfowl worldwide and bring huge economic losses to the duck industry. Duck circovirus destroys the immune sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 谢芝勋赵光远谢丽基谢志勤刘加波庞耀珊邓显文范晴
Owner GUANGXI VETERINARY RES INST
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