Avian pneumovirus Taqman probe fluorescent quantitative RT-PCR detection kit

An avian pneumonia virus, fluorescence quantitative technology, applied in the direction of fluorescence/phosphorescence, microbial determination/inspection, DNA/RNA fragment, etc.

Active Publication Date: 2013-09-25
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the application of Taqman fluorescent quantitative RT-PCR technology to the detection and diagnosis of poultry pneumonia virus

Method used

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  • Avian pneumovirus Taqman probe fluorescent quantitative RT-PCR detection kit
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  • Avian pneumovirus Taqman probe fluorescent quantitative RT-PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, design and synthesis of primers

[0057]According to the conserved sequence of the F gene of avian pneumonia virus in GenBank (GenBank: AF187154, sequence 4 in the sequence listing), a pair of specific primers and a Taqmam probe were designed using Primer Express3.0 software (Table 1).

[0058] Table 1 Primers and oligonucleotide sequences

[0059]

Embodiment 2

[0060] Embodiment 2, Taqman probe fluorescent quantitative RT-PCR detection

[0061] 1. Determination of Taqman probe fluorescent quantitative RT-PCR detection method

[0062] 1. Preparation of samples to be tested

[0063] According to the instructions of the TIANamp virus genome DNA / RNA extraction kit, extract avian pneumonia virus APV / MN, chicken Newcastle disease virus (Lasota), H9 subtype avian influenza virus, chicken infectious bronchitis virus (Mass41), avian encephalomyelitis virus (AE Van), avian reovirus (Reo S1133) RNA; Extract chicken infectious laryngotracheitis virus (Beijing strain), chicken Marek's disease (MDV) vaccine virus, Mycoplasma gallisepticum (MG), Pasteurella avian (C48-1) and avian Escherichia coli (E.coli O 2 ) DNA.

[0064] 2. Preparation of standard products

[0065] Use the cDNA of avian pneumonia virus APV / MN obtained in step 1 as a template for PCR amplification, and the reaction system is 50 μL: 2×Premix Taq TM mix 25 μL (product of Tak...

Embodiment 3

[0099] Embodiment 3, the detection of clinical disease material

[0100] The samples to be tested were 54 chicken lungs and trachea with swollen heads and respiratory symptoms collected from chicken farms in different regions of Guangxi (numbered C1-C54), and the total RNA of these diseased materials were extracted respectively.

[0101] The total RNA extracted from 54 disease samples was used as a template, and the Taqman probe fluorescence quantitative RT-PCR method established according to the two-step reaction method in Step 1 4 of Example 2 was used for detection.

[0102] If the reaction result under 530nm excitation light (FAM) is an S-shaped curve, the sample to be tested contains avian pneumonia virus; if the reaction result is a straight line, the sample to be tested does not contain avian pneumonia virus.

[0103] The test results are as follows:

[0104] It has been determined that under 530nm excitation light, there are 2 samples with S-shaped curves (numbered C1...

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Abstract

The invention discloses an avian pneumovirus Taqman probe fluorescent quantitative RT-PCR detection kit and an application thereof. The kit contains a composition for detecting the avian pneumovirus, and the composition is composed of a primer pair and a probe; the primer pair is composed of a primer 1 and a primer 2, the primer is single-stranded DNA represented by sequence 1 in a sequence table, and the primer 2 is single-stranded DNA represented by sequence 2 in the sequence table; and the sequence of the probe is sequence 3 in the sequence table. Experiments prove that the kit has the advantages of short detection time, strong specificity, high sensitivity, simple operation and low price when the kit is used for the avian pneumovirus Taqman probe fluorescent quantitative RT-PCR detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an avian pneumonia virus Taqman probe fluorescent quantitative RT-PCR detection kit and application thereof. Background technique [0002] Avian pneumonia virus (avian pneumovirus, APV) is a member of the avian metapneumovirus genus and belongs to the family Avian Paramyxoviridae. , commercial broilers and eggs, causing these chickens to become ill. The age at which the virus causes chickens to become ill is generally at the age of 4-7 weeks, and it is most likely to occur at the age of 5-6 weeks. Mainly cause symptoms of upper respiratory tract, such as sneezing, conjunctival flushing, lacrimal gland swelling, the obvious symptoms are subcutaneous edema of the head, and head swelling; infection of laying hens with the virus mainly causes a drop in egg production, with a drop rate of 2%. Between -40%, the hatching rate of breeding eggs is reduced; with the development of the disease,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
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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