Double-PCR (Polymerase Chain Reaction) detection kit for newcastle disease virus and duck plague virus

A technology of Newcastle disease virus and duck plague virus, applied in the biological field, can solve the problems of cumbersome operation, low sensitivity, and long diagnosis time, and achieve high application value, good specificity, and high sensitivity

Active Publication Date: 2013-04-10
GUANGXI VETERINARY RES INST
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  • Abstract
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  • Application Information

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Problems solved by technology

At present, the differential diagnosis of NDV and DPV mainly relies on traditional pathogen isolation and serological tests, but these methods have the disadv

Method used

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  • Double-PCR (Polymerase Chain Reaction) detection kit for newcastle disease virus and duck plague virus
  • Double-PCR (Polymerase Chain Reaction) detection kit for newcastle disease virus and duck plague virus
  • Double-PCR (Polymerase Chain Reaction) detection kit for newcastle disease virus and duck plague virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the design of primer

[0042] For the conserved genes of Newcastle Diseases (ND for short) virus and Duck Plague (DP for short) virus (ND conserved gene is the 1676-2094th nucleotide of F gene genbank number JN872152.1, DP conserved gene It is the 55471-56072 nucleotide of EU08288.2), combined with the comprehensive analysis results of DNAstar, Primer5 and NCBI-blast biological software, designed two pairs of specific primers, the primers were synthesized by Shanghai Invitrogen Company, and the primer sequence parameters are shown in the table 1:

[0043] Table 1 is the nucleotide sequence of NDV, DPV primer

[0044]

Embodiment 2

[0045] Embodiment 2, the specificity and sensitivity test of primer in double PCR detection

[0046] 1. Double PCR specificity test

[0047] Newcastle disease virus NDV-F48E9 was purchased from China Veterinary Drug Administration;

[0048] Newcastle disease virus NDV-Lasota was purchased from China Veterinary Drug Administration;

[0049] Newcastle disease virus C 30 The strain was purchased from China Veterinary Drug Administration;

[0050] Duck plague local isolate 1 (being duck plague virus, hereinafter referred to as DPV) is recorded in "Research on the detection of duck plague virus by polymerase chain reaction", Chinese Journal of Veterinary Medicine 2000, 34 (4): 10-12, the public can download from Guangxi Obtained by Zhuang Autonomous Region Veterinary Research Institute;

[0051] Duckling hepatitis virus is recorded in "Research on Detection of Duck Plague Virus by Polymerase Chain Reaction", Chinese Journal of Veterinary Medicine, 2000, 34(4): 10-12, and the pu...

Embodiment 3

[0083] Embodiment 3, double PCR detection of clinical sample

[0084] For the collected 7 copies (numbered 1-7) of duck disease materials, 1-3 livers, 4-5 spleens, 6-7 lungs were collected, and they were ground into suspensions for virus isolation and identification, and the duck disease samples were extracted respectively. The samples were DNA and RNA, 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 1-7.

[0085] The above-mentioned mixed samples numbered 1-7 were respectively used as templates, and the double PCR detection was carried out according to the method of 3) of 1 of Example 2.

[0086] If a 419bp fragment is obtained, the sample contains NDV, otherwise it does not;

[0087] If a 602bp fragment is obtained, the sample contains DPV, otherwise it does not;

[0088] If fragments of 419bp and 602bp are obtained, the sample contains NDV and DPV, and vice versa.

[0089] ...

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Abstract

The invention discloses a double-PCR (Polymerase Chain Reaction) detection kit for newcastle disease virus and duck plague virus. A primer group provided by the invention is composed of a primer 1, a primer 2, a primer 3 and a primer 4. Nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are respectively a sequence 1, a sequence 2, a sequence 3 and a sequence 4 in a sequence table sequentially. Experiments show that newcastle disease and duck plague can be detected and identified simultaneously by one-time PCR reaction. Compared with the conventional pathogen separation identification and serological experiments, double PCRs have the characteristics of good specificity, high sensibility, fastness, simplicity, convenience, and the like, and has a high clinical application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double PCR detection kit for Newcastle disease virus and duck plague virus. Background technique [0002] Newcastle Diseases (ND for short) and Duck Plague (DP for short) are common acute, septic and highly contagious infectious diseases of ducks, geese and other birds of the order Anseridae. These two diseases are widespread and spread rapidly , with high morbidity and mortality, is currently one of the most serious diseases for waterfowl in the world. Bring huge economic losses to the waterfowl industry. These two diseases both affect the nervous system in the course of acute sepsis, with respiratory symptoms and green feces at the same time. Their clinical manifestations, pathological changes, and epidemiology are very similar. When these two diseases occur, it is easy to confuse them. At present, the differential diagnosis of NDV and DPV mainly relies on traditional pathogen ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 谢芝勋陈安莉谢丽基刘加波谢志勤庞耀珊邓显文范晴
Owner GUANGXI VETERINARY RES INST
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