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Double RT-PCR kit for newcastle disease virus and avian pneumovirus, and application thereof

A chicken Newcastle disease virus, RT-PCR technology, applied in the field of double RT-PCR kits, can solve problems such as difficult to distinguish and easy to confuse

Active Publication Date: 2014-05-28
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The respiratory symptoms caused by the two viruses are sometimes difficult to distinguish clinically and are easily confused

Method used

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  • Double RT-PCR kit for newcastle disease virus and avian pneumovirus, and application thereof
  • Double RT-PCR kit for newcastle disease virus and avian pneumovirus, and application thereof
  • Double RT-PCR kit for newcastle disease virus and avian pneumovirus, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, design and synthesis of primers

[0056] According to the conserved sequences of chicken Newcastle disease virus F gene (GenBank: JX840454.1, sequence 5 in the sequence listing) and avian pneumonia virus F gene (GenBank: AF187154, sequence 6 in the sequence listing) in GenBank, primers were designed using DNAStar primer design software, The designed primers were verified by Blast comparison in GenBank, and finally 2 pairs of specific primers for the amplification of AIV H9 and APV were finally determined and synthesized (Table 1).

[0057] Table 1 identifies the primer oligonucleotide sequences of NDV and APV

[0058]

Embodiment 2

[0059] Embodiment 2, double RT-PCR identification chicken Newcastle disease virus and poultry pneumonia virus

[0060] 1. Establishment of double RT-PCR system

[0061] 1. Preparation of samples to be tested

[0062] Refer to the instructions of the TIANamp virus genome DNA / RNA extraction kit to extract APV / MN strain of avian pneumonia virus, chicken Newcastle disease virus (Lasota), chicken Newcastle disease virus (F48E9), chicken Newcastle disease virus (GX7 / 02), chicken Newcastle disease virus (GX9 / 03), chicken Newcastle disease Virus (GX10 / 03), H9N2 subtype avian influenza virus (A / Chicken / Guangxi / 067C4 / 2010), chicken infectious bronchitis virus (Mass41), avian encephalomyelitis virus (AE Van), avian reovirus (Reo S1133) RNA; Extraction of chicken infectious laryngotracheitis virus (Beijing strain), chicken Marek's disease (MDV) vaccine virus, mycoplasma gallisepticum (MG) and avian Escherichia coli (E.coli O 2 ) DNA.

[0063] The concentration and purity of the nucleic...

Embodiment 3

[0086] Example 3, double RT-PCR identification kit for chicken Newcastle disease virus and avian pneumonia virus detection of clinical sample accuracy

[0087] 1. Extraction of RNA from clinical tissue samples

[0088] Take 5g of lung and trachea tissues from clinically diseased chickens (25-60 day-old Sanhuang chickens with head subcutaneous edema, sneezing, conjunctival flushing, exhalation symptoms, and neurological symptoms) stored at -70°C, respectively. For C20120402, C20120427, C20120820, C20130504, C20130516, C20120314, C20130609, C20130715, C20130811, C20131016, put it into a grinding bowl for grinding, add 10 mL of sterilized PBS solution for 30 times to mix, freeze and thaw repeatedly After 5 minutes, collect the supernatant, store at -70°C for later use or extract sample RNA according to the method of Example 2 step-1, and simultaneously extract the RNA of chicken Newcastle disease virus (Lasota) and avian pneumonia virus APV / MN according to the method of Example 2...

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Abstract

The invention discloses a double RT-PCR kit for newcastle disease virus (NDV) and avian pneumovirus (APV), and an application thereof. The kit contains a set of primer pairs for determining or assistantly determining NDV and APV: a primer pair 1 (sequence 1 and sequence 2) and a primer pair (sequence 3 and sequence 4). Experiments show that two pathogens of NDV and APV can be detected and determined at the same time by only one-time PCR. The kit has the advantages of high specificity, high sensitivity, low cost and high efficiency. The kit can directly determine amplification results by using different amplification fragment lengths in primer design; and the kit is simple and convenient, intuitive and practical. The kit can simultaneously detect 10 pg of NDV RNA and 10 pg of APV RNA at least. Sensitivity of the kit provides rapid and accurate diagnosis results for early onset caused by the two viruses. The kit has great significance for preventing and controlling the two diseases, and has significant realistic significance for sustainable and healthy development of poultry industry.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a double RT-PCR kit for chicken Newcastle disease virus and avian pneumonia virus and its application. Background technique [0002] Newcastle disease virus (NDV) and avian pneumonia virus (APV) are two major viruses that harm the poultry industry. Avian pneumonia virus mainly causes upper respiratory tract symptoms, such as sneezing, conjunctival flushing, lacrimal gland swelling, and subcutaneous edema on the head, commonly known as swollen head; infection of laying hens mainly causes egg production to drop by 2%-40%, and hatching rate decreases; As the disease progressed, neurological symptoms also appeared. Chickens infected with Newcastle disease virus can also cause upper respiratory tract symptoms, mainly coughing and respiratory rales, which can cause a large number of deaths in severe cases. The respiratory symptoms caused by the two viruses are sometimes difficult to distin...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2537/143
Inventor 谢芝勋谢志勤刘加波庞耀珊邓显文谢丽基范晴罗思思
Owner GUANGXI VETERINARY RES INST
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