Visual LAMP detection kit for streptococcic mastitis pathogenic bacteria

A detection kit and streptococcal detection technology, applied in the field of visual LAMP detection kits for streptococcal mastitis pathogens, can solve the problem of establishing streptococcal mastitis pathogens, aerosol pollution laboratories, It is difficult to eliminate interference factors and other problems, and achieve the effect of low cost, high sensitivity and simple operation method

Inactive Publication Date: 2014-05-14
GUANGXI UNIV
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Problems solved by technology

Most of the currently established LAMP reaction methods use agarose gel electrophoresis, ultraviolet analysis and imaging, or add fluorescent dyes after the reaction is completed to determine the results. They can only analyze the final results of the LAMP reaction, and there is a risk of aerosol contamination of the laboratory. Due to the lac

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  • Visual LAMP detection kit for streptococcic mastitis pathogenic bacteria
  • Visual LAMP detection kit for streptococcic mastitis pathogenic bacteria
  • Visual LAMP detection kit for streptococcic mastitis pathogenic bacteria

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Embodiment Construction

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the materials and reagents used can be obtained from commercial sources.

[0025] Bst DNA polymerase (large fragment) was purchased from New England Biolabs; PCR reagents were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0026] 1. Preparation of visual LAMP detection kit for streptococcal mastitis pathogens

[0027] Reaction solution A is 23 μl per tube, and its composition is: 2.5 μl 10×Bst buffer, 1.0 μl Bst DNA polymerase (8U / μl), 3.0 μl 10mM dNTPs, 2 μl 100mM MgSO 4 , 0.75 μl 40 μM inner primer 1, 0.75 μl 40 μM inner primer 2, 1 μl 5 μM outer primer 1, 1 μl 5 μM outer primer 2, 4 μl 5M Betaine, 1 μl 625 μM calcein, 1 μl 12.5 mM MnCl2 and 5 μl ultrapure water.

[0028] Among them, 10×Bst buffer contains 200mM Tris-HCl (pH8.8), 100mM KCl, 100mM (NH 4 ) 2 SO 4 , 20mM MgSO 4 , 1%Triton X-100;

[0029] The sequence...

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Abstract

The invention discloses a visual LAMP (Loop-Mediated Isothermal Amplification) detection kit for streptococcic mastitis pathogenic bacteria, wherein the inventor designs two specific inner primers and two specific outer primers according to the common gene conservative area of the streptococcic mastitis pathogenic bacteria by virtue of adopting the loop-mediated isothermal amplification technique, so that the visual LAMP detection kit is obtained. A method for detecting the streptococcic mastitis pathogenic bacteria by using the LAMP detection kit only needs a common water bath kettle in stead of expensive PCR (Polymerase Chain Reaction) instrument, and the method is simple to operate and low in cost, but higher in sensitivity than PCR detection; the detection results are visual and can be obtained through judgment by naked eyes, and the results do not need to be observed through gel electrophoresis ultraviolet analysis and development. The visual LAMP detection kit can be used for overcoming the defects of long time taken, high workload, easy cross contamination, complex operation and the like of the existing detection technology, and is more specific and sensitive than the conventional detection method, and thus applicable to quick detection in basic-level veterinary stations and dairy farms; besides, the visual LAMP detection kit has excellent application prospect.

Description

technical field [0001] The invention belongs to the field of detection of streptococcal mastitis pathogenic bacteria, in particular to a visual LAMP detection kit for streptococcal mastitis pathogenic bacteria. Background technique [0002] Mastitis is one of the most common diseases in dairy farms and causes the most serious economic losses. It restricts the healthy development of dairy farming and dairy processing industries, and endangers public health and human health. How to effectively prevent and control this disease has become an important issue at home and abroad. focus of attention. Staphylococcus aureus, Streptococcus, and Escherichia coli, the incidence of these three pathogenic bacteria accounted for more than 90% of the total incidence of mastitis in dairy cows. Common Streptococcus pathogenic bacteria (Streptococcus) of mastitis in dairy cows mainly include Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis, among which Streptococcu...

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/465
Inventor 杜玉兰贺婷何宝祥
Owner GUANGXI UNIV
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