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Duplex PCR (polymerase chain reaction) detection kit for duck Newcastle disease virus and duck circovirus

A technology for Newcastle disease virus and duck circovirus, applied in the biological field, can solve the problems that there are no reports on the detection and diagnosis of Newcastle disease virus and duck circovirus

Active Publication Date: 2012-08-01
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there have been no reports on the detection and diagnosis of Newcastle disease virus and duck circovirus by duplex PCR

Method used

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  • Duplex PCR (polymerase chain reaction) detection kit for duck Newcastle disease virus and duck circovirus
  • Duplex PCR (polymerase chain reaction) detection kit for duck Newcastle disease virus and duck circovirus
  • Duplex PCR (polymerase chain reaction) detection kit for duck Newcastle disease virus and duck circovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, design and synthesis of primers

[0052] The F gene of Newcastle disease virus and the V1 / rep gene sequence of duck circovirus in GenBank were compared using Lasergene software, and primers were designed in the conserved regions using the online software Primer Premier 5.0. Primers were synthesized by Shanghai Invitrogen Company. The primer sequences are listed in Table 1.

[0053] Table 1 is the double PCR primer sequence

[0054]

Embodiment 2

[0055] Embodiment 2, double PCR detection

[0056] 1. Extraction of nucleic acid

[0057] Refer to the TIANamp blood / cell / tissue gene DNA extraction kit to extract duck circovirus, Muscovy duck parvovirus, duck plague virus AV1221, gosling plague virus, Riemerella anatipestifer, Escherichia coli, avian multocida Bacillus DNA;

[0058] Refer to the instruction manual of TRIzol LS Reagent to extract the RNA of duck Newcastle disease virus, Newcastle disease virus NDV-F48E9, Newcastle disease virus NDV-Lasota, DHV I, H9 subtype avian influenza virus respectively, reverse transcribe into cDNA respectively, and store at -70℃ Save for later.

[0059] 2. Establishment of double PCR amplification system

[0060] 1. Reaction system optimization

[0061] The amount of duck Newcastle disease virus primers, duck circovirus primers, templates, etc. was optimized, and the optimal reaction amount was determined after repeated experiments. The final double PCR reaction system was determin...

Embodiment 3

[0078] Embodiment 3, double PCR detection sample to be tested

[0079] 38 copies (numbered 1-38) of duck disease materials (liver collection) submitted for inspection in Yulin, Nanning, and Haikou were extracted from duck disease materials DNA and RNA respectively, and the RNA was reverse transcribed to obtain cDNA. The DNA and cDNA of each sample were Mix (volume ratio 1:1) to obtain mixed samples numbered 1-38.

[0080] The above-mentioned mixed samples numbered 1-38 were respectively used as templates, and double PCR amplification was performed according to the optimized PCR reaction system and optimized reaction conditions in Example 2.

[0081] If a 493bp fragment is obtained, the sample contains Newcastle disease virus, otherwise it does not;

[0082] If a 218bp fragment is obtained, the sample contains duck circovirus, otherwise it does not;

[0083] If the fragments of 493bp and 218bp are obtained, the sample contains Newcastle disease virus and duck circovirus, othe...

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PUM

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Abstract

The invention discloses a duplex PCR (polymerase chain reaction) detection kit for duck Newcastle disease virus and duck circovirus, and particularly relates to a primer group for detecting Newcastle disease virus and duck circovirus, which comprises a first primer, a second primer, a third primer and a fourth primer. Nucleotide sequences of the first primer, the second primer, the third primer and the fourth primer are respectively a first sequence, a second sequence, a third sequence and a fourth sequence in a sequence list. Testing shows that the two pairs of primers are designed to establish a duplex PCR detection method for Newcastle disease virus and duck circovirus, and the duplex PCR technique capable of detecting two pathogens, namely the Newcastle disease virus and the duck circovirus has the advantages of simplicity in operation, high sensitivity, high specificity, high repeatability and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double PCR detection kit for duck Newcastle disease virus and duck circovirus. Background technique [0002] Duck Newcastle disease is an acute, highly contagious and fatal infectious disease caused by Duck Newcastle Diseases duck NDV. Early studies have found that ducks have strong resistance to pathogenic APMV-1, and only behave as healthy carriers, even if they are infected with strong viruses, they will not cause disease. However, in recent years, it has been found that the natural prevalence of duck-derived Newcastle disease virus, which causes high pathogenicity and mortality, is seriously harmful to the duck industry. Hebei, Fuzhou, Zhejiang, Shanxi, Shandong, Henan and other places have successively reported the occurrence and prevalence of the disease. The above studies show that the infection of duck-derived Newcastle disease virus is on the rise, which will cause great ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 谢芝勋许宗丽谢丽基庞耀珊谢志勤刘加波邓显文范晴
Owner GUANGXI VETERINARY RES INST
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