Bi-fluorescence quantitative RT-PCR detection kit for mycoplasma synoviae and avian reovirus, and primer group thereof
A technology for mycoplasma synovialum and avian reovirus, which is applied in the field of double fluorescent quantitative RT-PCR detection kits and primer sets, can solve the problems of complexity, high requirements for reagents and primers, and achieve high detection sensitivity Effect
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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 Primer Express 3.0 software, and two pairs of specificity were selected by analyzing the dimer between the primers. 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 fluorescent 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, Mycoplasma gallisepticum K-2221, 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 above 1) avian reov...
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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