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

Active Publication Date: 2019-12-13
NINGXIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the polymerase chain reaction method is fast and can replace the culture method in a certain sense, its high sensitivity also makes the polymerase chain reaction method relatively demanding on the experimental environment and instruments, and there are high technical requirements, complicated operation, and It is not easy to popularize, and it is difficult for general grassroots laboratories to carry out, and the price is relatively high

Method used

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  • Composition for detecting mycoplasma ovipneumoniae by taking transketolase gene as target kit and method thereof
  • Composition for detecting mycoplasma ovipneumoniae by taking transketolase gene as target kit and method thereof
  • Composition for detecting mycoplasma ovipneumoniae by taking transketolase gene as target kit and method thereof

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Experimental program
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Effect test

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

[0083] DNA or RNA template

[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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Abstract

The invention discloses a composition for detecting mycoplasma ovipneumoniae by taking a transketolase gene as a target, a kit and a method thereof. A detection target of the composition is the transketolase gene of mycoplasma ovipneumoniae; various different fragments in the gene can be targeted, the experiments prove that the composition disclosed by the invention can be used for designing various specific combination situations so as to be applied to various amplification methods such as traditional PCR, basic RPA, nfo RPA and exo RPA; in addition, the experimental results can be observed by means of agarose gel electrophoresis, a lateral flow chromatography test strip and a constant-temperature fluorescence amplification instrument. The optimal reaction time and the optimal reaction temperature in the detection process and the specificity, the sensitivity, the repeatability and the stability of detection are explored; the optimal reaction condition is found, the mycoplasma ovipneumoniae rapid detection method which is good in specificity, high in sensitivity and capable of being stably repeated is established, and the composition has the advantages of being easy to operate, convenient to implement, and capable of saving time and free of large experimental instruments and equipment.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a composition, a kit and a method for rapid detection of ovine mycoplasma pneumoniae. Background technique [0002] Mycoplasma pneumonia of sheep and goats (MPSG) is a highly contagious infectious disease transmitted through air, droplets, drinking water, etc. The main clinical symptoms are panting, coughing, high fever, progressive weight loss, and chronic hyperplastic stroma pneumonia etc. The pathogenic bacteria that have been found to cause mycoplasma pneumonia include Mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae, MO), Mycoplasma capricolum subsp.capricolum (Mcc), Mycoplasma mycoides subsp.capri (Mmc) Among them, MO is one of the common pathogenic bacteria. The disease is widely distributed and popular in my country, and the infection rate is high, which seriously affects the sheep breeding industry. Therefore, it is very important to establish an accurate det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/35
CPCC12Q1/689
Inventor 李敏郝秀静金华马春骥王玉炯
Owner NINGXIA UNIVERSITY
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