Primer and reagent kit of differentiating virulent and attenuated Newcastle disease viruses and differential detection method

A technology for identification and detection of Newcastle disease virus, which is applied in the field of identification and detection, primers and kits for distinguishing the strength of Newcastle disease virus, and can solve the problems that cannot meet the needs of actual production, so as to facilitate rapid differentiation and diagnosis and save time. , the effect of high sensitivity

Inactive Publication Date: 2017-07-21
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] With the widespread application of attenuated Newcastle disease vaccine in clinical practice, the detection of NDV alone cannot meet the needs of actual production. It is necessary to establish a detection technology to quickly distinguish between strong and attenuated vaccine strains in order to take appropriate clinical measures. preventive measures

Method used

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  • Primer and reagent kit of differentiating virulent and attenuated Newcastle disease viruses and differential detection method
  • Primer and reagent kit of differentiating virulent and attenuated Newcastle disease viruses and differential detection method

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Embodiment 1

[0034] 1. Primer design

[0035] Four primers (QF1, QF2, QF3, QF4) were designed according to the sequence difference of the fusion gene cleavage site of strong and weak strains of NDV. The primers were synthesized by Huada Gene Company and purified at PAGE plus level; among them, QF1 and QF2 were used for attenuated strains The specific fragment of QF2 and QF4 is used for the specific fragment of virulent strains, the length is 281bp; the specific fragment of QF2 and QF4 is 478bp, see Table 1 for details.

[0036] Table 1

[0037]

[0038] 2. RT-PCR

[0039] 2.1 RNA extraction

[0040] Three representative strains were selected: attenuated strain LaSota, strong virus strain F48E9, genotype 200 μL of allantoic fluid (containing the virus) was used to extract RNA. The extraction of viral RNA was carried out according to Bioer’s Simple RNA kit, and finally 30 μL of DEPC water was used to dissolve the RNA;

[0041] 2.2 RT-PCR

[0042] RT: RT reaction system (20μL): 10mM ...

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Abstract

The invention belongs to the technical field of animal pathogeny detection and particularly relates to primer and a reagent kit of differentiating virulent and attenuated Newcastle disease viruses, and a differential detection method. The differential detection method particularly comprises the steps of extracting RNA (ribonucleic acid) of strains to be detected, dissolving RNA with 30 microliter DECP (diethyl chlorophosphite) water, performing RT-PCR (reverse transcription-polymerase chain reaction), and conducting agarose gel electrophoresis test on a DNA (deoxyribonucleic acid) amplification result. The primer designed according to different nucleotide sequences of F gene cracking sites of the virulent and attenuated NDV (Newcastle disease virus) strains has good specificity and can accurately differentiate the virulent and attenuated Newcastle disease virus strains; the reagent kit is high in sensitivity, short in consumed time, and accurate and effective in detection, can detect to-be-detected samples, with nucleic acid not less than 100 ng/microliter, of the Newcastle disease viruses and facilitates quick clinical differential diagnosis.

Description

technical field [0001] The invention belongs to the technical field of animal pathogen detection, and in particular relates to a primer for distinguishing the intensity of Newcastle disease virus, a kit thereof, and a method for identification and detection. Background technique [0002] Newcastle disease (ND), commonly known as chicken plague, is a highly contagious acute infectious disease caused by Newcastle disease virus (NDV). It is characterized by enteritis, severe diarrhea and neurological symptoms. It is one of the most important diseases, causing great economic losses. At present, this disease is prevalent in many countries and regions in the world. The World Organization for Animal Health (OIE) lists it as a statutory reportable disease (listed as a class A disease before 2005), and my country lists it as a first-class animal disease. It is a priority disease species determined in the "National Medium and Long-term Animal Disease Prevention and Control Plan (2012-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 王友令袁小远孟凯张玉霞艾武亓丽红
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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