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Loop-mediated isothermal amplification fast detecting reagent kit and method for O157:H7 coliform

A ring-mediated isotherm, detection kit technology, applied in the direction of microbial-based methods, microbial determination/testing, biochemical equipment and methods, etc.

Inactive Publication Date: 2009-07-22
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Notomi et al. developed a new DNA loop-mediated isothermal amplification method (loop-mediated isothermal amplification, LAMP) in 2000. Because of its simple operation and easy interpretation of results, it can be promoted in grassroots laboratories. This method has been widely used in some countries and regions, but domestic research on this technology has just started, and there is no literature report on the detection of O157:H7 coliform bacteria using LAMP technology

Method used

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  • Loop-mediated isothermal amplification fast detecting reagent kit and method for O157:H7 coliform
  • Loop-mediated isothermal amplification fast detecting reagent kit and method for O157:H7 coliform
  • Loop-mediated isothermal amplification fast detecting reagent kit and method for O157:H7 coliform

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Embodiment 1

[0102] Utilize the LAMP amplification detection method described in the present invention to detect 56 strains O157 bacterial strains (comprising standard strain O157:H7, local O157 isolates and other intestinal experimental strains) detect, carry out this result with ordinary PCR, quantitative PCR method result comparison to verify the specificity of this method. In the experiment, the fliC gene LAMP results of E. coli O28ac84-1373, LT-85-44 and 84-135 were positive, and the results of common PCR and quantitative PCR were also positive. O157: Related to the fliC gene of H7 flagellar antigen H antigen.

[0103] The reaction system is as follows:

[0104] 10× buffer 2.5μL

[0105] Primers rfbE FIP, stx2 FIP, flic FIP each 1.6μM

[0106] Primers rfbE BIP, stx2 BIP, flic BIP each 1.6μM

[0107] Primers rfbE F3, stx2 F3, flic F3 each 0.2μM

[0108] Primers rfbE B3, stx2 B3, flic B3 0.2 μM each

[0109] Primer rfbE LB 0.4 μM

[0110] dNTP mix (1:1:1:1) total 0.6mM

[0111] Be...

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Abstract

The invention provides a quick detecting kit that is used for loop-mediated isothermal augmentation of O157:H7 coliform, designs LAMP primers respectively for O157:H7 O-antigen encoding rfbE, Shiga-like toxin stx2 and H flagellar antigen encoding flic gene, provides a quick detecting kit and a detecting method with strong specificity and high sensitivity that are used for loop-mediated isothermal augmentation of O157:H7 coliform, and has following essential advantages: (1) expensive and precise instruments and equipments are unnecessary, and only a metal pyrolyzer / temperature-constant water bathing device is required; (2) the detecting kit and the detecting method consume less time, and can finish reaction in about 1 hour at constant temperature; (3) a result can be determined by observing color changes of a reaction system with naked eyes under white light, thus having simple and convenient operation; and (4) the method is simple, easy, quick, specific and sensitive, and has wider application prospect in quick detection aspects.

Description

(1) Technical field [0001] The invention relates to a rapid detection kit and detection method for loop-mediated isothermal amplification of O157:H7 coliform bacteria. (2) Background technology [0002] The newly discovered and re-emerged pathogenic microorganisms are new issues facing the world's medicine, and enterohaemorrhagic coli O157:H7 is one of them. O157:H7 is the most important serotype of Enterohemorrhagic Escherichia coli (EHEC), which was first discovered in the United States in 1982 and is a new pathogen. Its pathogenicity is strong, and the infectious dose is extremely low. Ingestion of less than 10 bacteria can cause disease. The bacterium mainly causes hemorrhagic enteritis (HC), hemolytic uremic syndrome (HCS) and thrombotic thrombocytopenic purpura (TTP), and the fatality rate is as high as 30%. It is a worldwide public health problem of global concern. Since the first strain of EHEC O157:H7 was detected in Xuzhou, Jiangsu Province in 1988 in our country...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/19
Inventor 朱水荣
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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