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Duplex fluorescence quantitative rt-PCR detection kit for chicken mycoplasma synovium and avian reovirus and its primer set

A technology for mycoplasma synovialis and avian reovirus is applied in the field of double fluorescence quantitative RT-PCR detection kits and primer sets, which can solve the problems of complexity, high requirements for reagents and primers, and achieve high detection sensitivity. Effect

Active Publication Date: 2016-08-24
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is much more complicated to establish a multiplex fluorescent PCR method than a single-plex one, and it has higher requirements on reagents and primers. At the same time, it is necessary to ensure that there is no mutual interference between the fluorophores labeled by different probes. The fluorescent quantitative PCR instrument used with corresponding multiple detection channels
At present, there are no reports on the detection and diagnosis of mycoplasma synovialis and avian reovirus using multiplex fluorescent quantitative PCR technology

Method used

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  • Duplex fluorescence quantitative rt-PCR detection kit for chicken mycoplasma synovium and avian reovirus and its primer set
  • Duplex fluorescence quantitative rt-PCR detection kit for chicken mycoplasma synovium and avian reovirus and its primer set
  • Duplex fluorescence quantitative rt-PCR detection kit for chicken mycoplasma synovium and avian reovirus and its primer set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, design and synthesis of primers and Taqman probes

[0041] According to the conserved sequences of Mycoplasma gallisovus and Avian Reovirus in GenBank, multiple sets of probes and primers were designed using PrimerExpress 3.0 software, and two pairs of specific primers were selected by analyzing the dimer between the primers and two Taqmam probes (Table 1).

[0042] Table 1 Primers and TaqMan probe sequences (5'-3')

[0043]

Embodiment 2

[0044] Embodiment 2, establishment of fluorescence quantitative RT-PCR detection

[0045] 1. Determination of fluorescent quantitative RT-PCR detection method

[0046] 1. Sample preparation

[0047] 1), nucleic acid extraction

[0048] Referring to the TRizol RNA extraction kit instructions, extract the RNA of avian reovirus, Newcastle disease virus, infectious bronchitis virus, chicken infectious laryngobronchitis virus, H9 subtype avian influenza, and refer to the method of Xie Zhixun (Detection of Avian Adenovirus by Polymerase Chain Reaction [J]. Avian Deseases, 1999,43:98O1051) extracted the DNA of Mycoplasma gallisepticum, Mycoplasma gallisepticum S6 strain, Mycoplasma gallisepticum K-2221 strain, Mycoplasma gallisepticum and Mycoplasma turkey.

[0049] 2), reverse transcription of RNA

[0050] Avian reovirus S1133 RNA was reverse transcribed to synthesize cDNA, as follows: Establish the following reverse transcription system, the total reaction volume is 20 μL, the a...

Embodiment 3

[0082] Embodiment 3, the assembly of detection kit

[0083] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.

[0084] Solution A: PCR amplification buffer, primer 1, primer 2, probe A, primer 3, primer 4, probe B; among them, the PCR amplification buffer includes 2×Premix Ex Taq (Probe qPCR) (Dalian Bao Biological Engineering Co., Ltd., catalog number: RR390A); the final concentrations of primer 1, primer 2, and probe A are all 0.3 μM, and the final concentrations of primer 3, primer 4, and probe B are all 0.3 μM;

[0085] Liquid B: MS+ARV template, as a positive control;

[0086] Solution C: sterilized distilled water, as a negative control.

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Abstract

The invention discloses a bi-fluorescence quantitative RT-PCR detection kit for mycoplasma synoviae and avian reovirus. The kit comprises a primer group and a probe group, wherein the primer group comprises a primer 1, a primer 2, a primer 3 and a primer 4, which respectively have base sequences of sequence tables SEQ.ID.No.1 and 2, and SEQ.ID.No.4 and 5; and the probe group comprises a probe A and a probe B which respectively have the base sequences of sequence tables SEQ.ID.No.3 and 6. Experiments prove that the kit disclosed by the invention has the advantages of short detection time, high detection sensibility and strong specificity, two pathogens of the mycoplasma synoviae and the avian reovirus can be detected and discriminated at the same time, and the corresponding pathogeny content can be quantified, so that the kit can be used for assessing the curative effects of vaccines and medicines on the mycoplasma synoviae and the avian reovirus and researching pathogenic mechanisms and other respects; therefore, the kit disclosed by the invention has an important significance on prevention and control of the mycoplasma synoviae and the avian reovirus.

Description

technical field [0001] The invention belongs to the technical field of RT-PCR detection, and in particular relates to a dual fluorescent quantitative RT-PCR detection kit and a primer set for mycoplasma synovial sac and avian reovirus. Background technique [0002] Mycoplasma synoviae (MS) and avian reovirus (ARV) are one of the important pathogens of poultry, which can cause a variety of syndromes and cause huge economic losses to the poultry industry. Mycoplasma gallinarum mainly causes chickens, ducks, geese, quail, pigeons, etc., an acute or chronic infectious disease characterized by joint exudative synovial meningitis and tenosynovitis. It is ubiquitous and mainly affects commercial broiler chickens, laying hens or breeder chickens. Avian reovirus is an RNA virus of the Reoviridae family, which mainly harms chickens and turkeys, and can cause intestinal diseases, chronic respiratory diseases, myocarditis, arthritis / dwarf syndrome, malnutrition syndrome, etc. Co-infec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/06C12R1/93C12R1/35
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2537/143C12Q2561/101
Inventor 谢芝勋黄莉谢丽基邓显文罗思思谢志勤庞耀珊刘加波
Owner GUANGXI VETERINARY RES INST
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