Composition for detecting mycoplasma ovipneumoniae by taking transketolase gene as target kit and method thereof
A technology of mycoplasma pneumoniae and transketolase, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as high technical requirements, complicated operation, and difficulty in development, and achieve excellent technical effects and operation Simplicity, repeatability, and improved stability
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Embodiment 1
[0050] This embodiment is a detection system for the first amplification reaction of a specific transketolase gene, that is, a process of detecting a specific gene by polymerase chain reaction. Specifically, it includes the following:
[0051] (1) The detection system of the first amplification reaction of the present invention:
[0052]
[0053] (2) Reaction conditions:
[0054]
[0055] (3) Test result: After the reaction, take 5 μL of the reaction product and test it by agarose gel (1.5%) electrophoresis. The test result is shown in figure 1 shown.
Embodiment 2
[0057] This embodiment is a detection system for the second amplification reaction of specific gene transketolase, that is, a basic RPA detection system. Specifically, it includes the following:
[0058] (1) The detection system of the second amplification reaction of the present invention:
[0059]
[0060] After the above reaction solution was mixed, 1 μL of the template to be tested and 1.25 μL of MgoAC were added, vortexed and briefly centrifuged, and the reaction solution was placed in a 39°C water bath for 20 minutes. Result detection: after the reaction, 5 μL of the reaction product was taken and detected by agarose gel (1.5%) electrophoresis.
[0061] (2) Exploration of the best response time
[0062] After establishing the reaction system, set the time gradient from 0 min to 45 min to explore the optimal reaction time. To determine the optimal reaction time, use the positive standard plasmid 1.9×10 8 Copies / μL is used as the template for the amplification reacti...
Embodiment 3
[0073] This embodiment is a system based on the third amplification reaction of specific gene transketolase, namely nfo RPA.
[0074] details as follows:
[0075] (1) detection system of the present invention comprises:
[0076] The first detection system:
[0077]
[0078] Among them, Primer free Rehydration buffer is a known commercially available product.
[0079] The second detection system:
[0080] The first oligonucleotide (10mmoL / L) 1~2μL
[0081] Second oligonucleotide (10mmoL / L) 1~2μL
[0082] The third oligonucleotide (10mmoL / L) 0.5~1μL
[0084] Basic mixture 14~20μL
[0085] wxya 2 O. margin.
[0086] This base mix contains all the enzymes and reagents needed for the reaction, and only the template and corresponding oligonucleotides need to be added to it. Before the reaction, for example, 1.25 μl of magnesium acetate (MgOAc) should be added to the mixture system to start the reaction. After the reaction, an amplification...
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