Kit for loop-mediated isothermal amplification detection of Mycoplasma ovipneumoniae and preparation and usage methods thereof

A Mycoplasma pneumoniae, ring-mediated isothermal technology, applied in biochemical equipment and methods, microbial-based methods, microbial measurement/inspection, etc., can solve problems such as difficulty, high diagnostic cost, and time-consuming, and reduce detection. cost, rapid pathogen detection, and the effect of reducing high costs

Active Publication Date: 2012-08-15
GUIZHOU UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a ring-mediated isothermal amplification detection kit for Mycoplasma ovis pneumoniae and its preparation and use methods, which can avoid the blindness and cumbersomeness of clinical pathogen detection for suspected cases of Mycoplasma ovis pneumoniae infection in goats, A

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  • Kit for loop-mediated isothermal amplification detection of Mycoplasma ovipneumoniae and preparation and usage methods thereof
  • Kit for loop-mediated isothermal amplification detection of Mycoplasma ovipneumoniae and preparation and usage methods thereof

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Embodiment Construction

[0026] Embodiment of the present invention: the raw materials and formula of the ring-mediated isothermal amplification detection kit for Mycoplasma ovis pneumoniae are: 1000-2000 μL reaction solution, 100-200 μL Bst DNA polymerase, 50~100μL positive control, 50~100μL negative control, 1mL-2 mL liquid paraffin, 1mL-2 mL ultrapure water.

[0027] The reaction solution includes an inner primer mixture, an outer primer mixture, a LAMP reaction buffer, Mg 2+ And dNTP, the volume ratio of the five liquids in the reaction solution is 2:2:2.5:2:1.

[0028] The inner primers and outer primers are specific inner primers FIP and BIP, outer primers F3 and B3 for the ring-mediated isothermal amplification test of the Mycoplasma ovipneumoniae 16S rRNA gene, and the concentration of the inner primers is 20 μmol / L, and the concentration of the outer primers is 5 μmol / L. L, the primer sequence is shown in Table 1:

[0029] Table 1 Internal and external primer sequences

[0030] ...

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Abstract

The invention discloses a raw material composition of a kit for loop-mediated isothermal amplification (LAMP) detection of Mycoplasma ovipneumoniae and a preparation method and a usage method of the kit. The raw material composition comprises 1000-2000muL of reaction solution, 100-200muL of Bst (Bacillus stearothermophilus) DNA polymerase, 50-100muL of positive control, 50-100muL of negative control, 1-2mL of liquid paraffin and 1-2mL of ultrapure water. The reaction solution comprises an inner primer mixture solution, an outer primer mixture solution, an LAMP reaction buffer, Mg<2+> and dNTPs (deoxyribonucleotide triphosphates), wherein the volume ratio of the five liquids in the reaction solution is 2:2:2.5:2:1. The preparation method comprises the following steps: 1) determination of an optimum reaction temperature and an optimum reaction time; 2) specific detection, sensibility test and clinical application detection and 3) kit packaging. The kit disclosed by the invention has rapid, simple and accurate characteristics for pathogen detection of goat suspected cases of mycoplasma ovipneumoniae infection in goat farms.

Description

technical field [0001] The invention relates to a Mycoplasma pneumoniae ring-mediated isothermal amplification detection kit based on Mycoplasma ovipneumoniae 16S rRNA gene and a preparation and use method. Background technique [0002] Mycoplasma ovis pneumoniae ( Mycoplasma ovipneumoniae , Mo) for goat infectious pleuropneumonia ( Contagious Caprine Plneuropneumonia, CCPP) is one of the main pathogens, which can infect sheep and goats. After the infection of goats, the clinical manifestations are respiratory disorder, emaciation, high fever, lying down and other symptoms, and the mortality rate is high, causing serious economic losses to the goat breeding industry. [0003] At present, the detection of Mo at home and abroad mainly relies on the isolation and identification of pathogens, serological tests and polymerase chain reaction techniques. Compared with the three diagnostic methods, the traditional separation and identification of mycoplasma has high requirements ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/35
Inventor 程振涛张双翔周碧君文明王开功
Owner GUIZHOU UNIV
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