Time-resolved fluoroimmunoassay (TRFIA) of Klebsiella oxytoca

Abstract

The invention discloses a TRFIA of Klebsiella oxytoca, relating to detection of pathogenicbacteria. The invention aims at providing a simple, convenient and rapid TRFIA of Klebsiella oxytoca with high sensitivity and strong specificity. The method comprises the steps of: microbially verifying the Klebsiella oxytoca strains; preparing a Klebsiella oxytoca antibody; performing Eu3+ marking and purifying of IgG; and creating a Klebsiella oxytoca TRFIA detection method. The sensitivity is 1.0x10<5> cfu / pore, which is higher than that of a fluorescence anti-body method. If the fluorescence anti-body method is used for performing entire pathogenicbacteria thallus detection, the fluorescence strength is necessary for visual observation by a microscope, so the subjectivity is large; besides, the detection sensitivity is low resulting from the interference of background fluorescence, and a specific sensitivity value cannot be figured out. As shown in the detection of CPS values of Klebsiella oxytoca with different concentrations by the TRFIA method, the method has the advantages of wide range and capability of quantitative detection.

Description

technical field [0001] The invention relates to a detection of pathogenic bacteria, in particular to a time-resolved fluorescent immunodetection method for Klebsiella oxytoca. Background technique [0002] Klebsiella oxytoca is an important opportunistic pathogen that can cause human and animal diseases, food poisoning and other diseases (Dong Jie, et al. Clinical analysis of food poisoning caused by Klebsiella oxytoca. Zhonghua Journal of Infectious Diseases. 2002, 20: 315-316). The establishment of sensitive, specific and rapid detection technology to monitor Klebsiella oxytoca can play a positive guiding role in the early prevention of diseases. [0003] Due to the different serotypes of Klebsiella oxytoca, the traditional detection methods of bacterial morphology and physiological and biochemical characteristics cannot meet the needs of disease prevention and control. Molecular biology methods such as quantitative PCR detection have begun to be applied in the detection...

AI Technical Summary

Problems solved by technology

ELISA uses enzymes as markers, but enzymes are biological macromolecules, which are easy to inactivate and difficult to store. Macromolecular enzymes tend to cause steric hindrance to immune reactions. At the same time, this method must use color development for signal detection

The fluorescent antibody method uses fluorescent substances as markers. This marker has a small molecular weight and is stable. After being labeled with an antigen or antibody, it has little effect on the immune response. It can directly measure the fluorescent signal without color development, which makes up for the shortcomings of the ELISA method. However, because the wavelength of the background fluorescence is similar to that of the marker during measurement, it interferes with the determination of the sample, resulting in low determination sensitivity

Time-resolved fluorescence immunoassay (TRFIA) uses rare earth ion chelate as a marker, which is a long-lived fluorescent substance, which can be separated from short-lived background fluorescence by a time-resolved fluorometer, effectively Eliminate the interference of background fluorescence, this method has been used in the fields of chemistry, medicine, food and environment (Wu F B, et al.Synthesis of a highly fluorescent beta-diketone-europium chelate and its utility in time-resolved fluoroimmunoassay of serum total thyroxine. Anal Chem, 2002, 74:5882-5889) has received widespread attention, but the detection of Klebsiella oxytocia has never been reported

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