Escherichia coli detection method based on time-resolved fluorescence method and DNA hybridization

Abstract

The invention discloses an escherichia coli detection method based on a time-resolved fluorescence method and DNA hybridization. A capture probe DNA1 and an identifying probe DNA 2 are designed according to a DNA target sequence of escherichia coli; the capture probe DNA1 is fixedly marked on a glass sheet; and the identifying probe DNA 2 is jointed with long-life luminescent rare-earth europium complex. When the capture probe DNA1 and the identifying probe DNA 2 are connected in series and can generate base-pair complementary hybridization reaction with the target DNA (DNA of biology to be detected); and a DNA detection model based on two serial-connected probes on the surface of the glass sheet is established. The long-life luminescent rare-earth europium complex is used as a marker distinguishing the probe DNA 2; and the time-resolved fluorescence method can effectively eliminate the disturbance of fluorescent sources in a biological system and background light of a solid matrix. The detection method takes little time, is visual, flexible, accurate in reaction results and has very obvious advantages.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and relates to a detection method of Escherichia coli based on time-resolved fluorescence method and DNA hybridization. Background technique [0002] Escherichia coli (E.coli), commonly known as Escherichia coli, was discovered by Escherich in 1885 and has been considered as a common prokaryotic microorganism for quite a long time. The normal flora in the gut are non-pathogenic bacteria. It was not until the middle of the 20th century that it was recognized that some special serotypes of E. coli were pathogenic to humans and animals, often causing severe diarrhea and sepsis. Since it was first discovered in the United States in 1982, it has been reported in many countries, including my country, and it is increasing day by day. Japan was particularly notable in 1996 for several major outbreaks caused by food contamination with the bacteria. It has been ranked among the top three intestina...

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Problems solved by technology

[0003] Existing Escherichia coli detection methods include traditional bacterial culture methods, biochemical identification, and immunological methods. These methods have the disadvantages of cumbersome operation, time-consuming and laborious, and low detection efficiency; fluorescence in situ hybridization (FISH, fluorescencein site hybridization) ) is one of the most widely used and most effective technologies for the detection of common pathogenic microorganisms in the environment, but the fluorescent probes in the FISH method are easily interfered by the background of the endogenous fluorescence of the biological system and the background of the fixed microbial matrix, and the sensitivity is limited. Restrictions; Gene chip technology is a new technology developed rapidly in recent years. It has the advantages of fast, accurate and efficient detection of bacteria, but its high equipment costs and operating costs are difficult for most testing institutions to bear, and its versatility is extremely limited. big constraints

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