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Triple polymerase chain reaction (PCR) kit for duck hepatitis virus type I, duck circoviruses and Muscovy duckling parvovirosis and application of triple PCR kit

A technology for Muscovy duck parvovirus and duck circovirus, applied in the biological field, can solve the problems of long time, low sensitivity, and difficulty in standardization, and achieve the effects of low cost, high sensitivity and strong specificity

Active Publication Date: 2012-10-10
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the traditional detection methods for these three viruses include serum neutralization test, agar diffusion test and ELISA, etc., but these tests have the disadvantages of time-consuming, low sensitivity, and difficult standardization, which have certain limitations in practical application

Method used

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  • Triple polymerase chain reaction (PCR) kit for duck hepatitis virus type I, duck circoviruses and Muscovy duckling parvovirosis and application of triple PCR kit
  • Triple polymerase chain reaction (PCR) kit for duck hepatitis virus type I, duck circoviruses and Muscovy duckling parvovirosis and application of triple PCR kit
  • Triple polymerase chain reaction (PCR) kit for duck hepatitis virus type I, duck circoviruses and Muscovy duckling parvovirosis and application of triple PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, design and synthesis of primers

[0039] Three pairs of specific primers (Table 1).

[0040] Table 1 identifies the primer sequences of MDPV, DuCV and DHV I

[0041]

Embodiment 2

[0042] Example 2, Triple RT-PCR Identification of Duck Type I Hepatitis Virus, Duck Circovirus and Muscovy Duck Parvovirus

[0043] 1. Establishment of triple RT-PCR system

[0044] 1. Preparation of samples to be tested

[0045] According to the instructions of the virus RNA / DNA rapid purification kit, the RNA of the duck type I hepatitis virus AV2111 strain, the DNA of the Muscovy duck parvovirus and the DNA of the duck circovirus were extracted. The concentration and purity of nucleic acid were determined according to the Sambrook method, and stored at -20°C for later use.

[0046] 2. Optimization of triple RT-PCR reaction conditions

[0047] RT-PCR was performed in one step using the One Step RT-PCR Kit. Optimize each cycle parameter and each primer concentration of RT-PCR to determine the best RT-PCR mode.

[0048] By optimizing the concentration of RT-PCR primers, each reaction temperature, time and number of cycles, etc., it was finally determined that the optimal w...

Embodiment 3

[0064] Embodiment 3, triple RT-PCR detection clinical disease material

[0065] Using the triple RT-PCR rapid detection method for duck type I hepatitis virus, duck circovirus and Muscovy duck parvovirus established in Example 2, 180 disease materials collected in duck groups in Guangxi in 2011 were detected, and the PCR products Clonal sequencing analysis was performed to evaluate its clinical utility.

[0066] 1. Preparation of samples to be tested

[0067] The collected 180 duck disease materials (liver harvesting) were ground into a suspension, and the supernatant was collected by centrifugation after repeated freezing and thawing for 3 times. The DNA and RNA of the duck disease materials were extracted, and 180 samples to be tested were obtained.

[0068] 2. Triple RT-PCR

[0069] The 180 samples to be tested prepared in step 1 were respectively used as templates, and amplified according to the optimized PCR reaction conditions obtained in step 1-2 of embodiment 2.

[...

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Abstract

The invention discloses a primer pair group and a kit for performing identification or auxiliary identification on duck-related viruses and application of the primer pair group and the kit. The primer pair group consists of three primer pairs, and the three primer pairs consist of two single-stranded deoxyribonucleic acids (DNA) which are shown as a sequence 1 and a sequence 2, a sequence 3 and asequence 4, and a sequence 5 and a sequence 6 respectively; and the duck-related viruses are at least one of Muscovy duckling parvovirosis, duck circoviruses and duck hepatitis virus type I. A triplepolymerase chain reaction (PCR) technology that three kinds of pathogens, namely the Muscovy duckling parvovirosis, the duck circoviruses and the duck hepatitis virus type I can be simultaneously detected and identified through one-time PCR is established, and has the advantages of high specificity and sensitivity (the lowest detection line is 1pg), low cost, high efficiency and the like; and moreover, an amplification result is directly determined by utilizing the difference of the length of amplified fragments in the aspect of primer design, so that the method is relatively simple, convenient, intuitive and practical during result determination.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a primer pair set, kit and application thereof for identifying or assisting the identification of duck-associated viruses, in particular to a method for identifying or assisting the identification of Muscovy duck parvovirus, duck circovirus and duck type I hepatitis Primer pair sets, kits and applications of viruses. Background technique [0002] Duck Hepatitis Virus type I (DHV I) is the pathogen of a highly lethal viral infectious disease in ducklings. The disease mainly affects ducklings within 4 weeks of age, and the mortality rate is as high as 90%. It is one of the most serious infectious diseases that endanger the duck industry. Duck circovirus (Duck Circovirus, DuCV) is one of the newly discovered viruses in recent years. It was first discovered and sequenced by German scholar Hattermann et al. in 2003, and then discovered and reported in various parts of the world. Soike et ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 谢芝勋谢丽基邓显文谢志勤庞耀珊范晴刘加波
Owner GUANGXI VETERINARY RES INST
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