Method for detecting Salmonella on basis of technique for amplifying nanogold-labeled and silver-enhanced signals

Abstract

The invention relates to a method for detecting Salmonella on the basis of the technique for amplifying nanogold-labeled and silver-enhanced signals. The method comprises the following steps: conducting the DNA (deoxyribonucleic acid) hybridization among a Salmonella specific oligonucleotide capture probe, a nanogold-labeled specific oligonucleotide display probe and the target nucleotide sequences of pathogenic bacteria, or alternatively, conducting the direct immunoaffinity among Salmonella polyclonal antibodies, nanogold-labeled polyclonal antibodies and Salmonella, so as to form sandwiched complexes; and adding silver-enhancement solution to the sandwiched complexes to accelerate the deposition of silver particles by the nanogold particles anchored onto the enzyme-labeled pores through the hybridization or immunoaffinity, so that the sensitive detection of Salmonella can be achieved by the absorbance detection. The detection limit of the method reaches 33fmol / L (DNA hybridization analysis) or 50cfu / mL (immunoaffinity analysis); and moreover, the detection limit thereof reaches 0.3fmol / L (DNA hybridization analysis) or 5cfu / mL (immunoaffinity analysis) according to the luminal-based chemiluminescence detection by further chemically dissolving out the deposited silver. The food-borne pathogenic bacteria detection technology established by the invention is accurate, sensitive and rapid, thereby attaching great significance to food safety.

Description

technical field [0001] The present invention relates to the DNA hybridization between the Salmonella sequence-specific oligonucleotide capture probe, the gold-labeled Salmonella oligonucleotide display probe and the Salmonella target nucleic acid sequence to form a sandwich complex; or the mouse anti-Salmonella polyclonal antibody directly Capture the target Salmonella, and then combine with nano-gold-labeled mouse anti-Salmonella polyclonal antibody to form a sandwich complex; then add silver enhancement solution, and hybridize or immunoaffinity-anchor the nano-gold particles on the enzyme-labeled well to control the quantitative deposition of silver particles , combined with optical density detection technology or dissolution chemiluminescence detection technology, a new ultra-sensitive detection system for Salmonella was established. The invention belongs to the technical field of biological cytology and microbial fungus detection methods. Background technique [0002] S...

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Problems solved by technology

[0003] At present, the detection method of Salmonella mainly relies on the traditional bacterial isolation and identification method, which generally takes 4-5 days, which is time-consuming and laborious. However, immunoenzyme test, immunodiffusion method, latex agglutination test, immunofluorescence method and enzyme-linked immunosorbent assay ( ELISA) and other methods are not ideal due to operating procedures, detection time, specificity detection, etc.

Although the polymerase chain reaction (PCR) technology has the advantages of simplicity, rapidity, high sensitivity and strong specificity, it has been widely used in many fields such as microbial detection and genetic disease diagnosis, but it causes pollution in the laboratory environment and the use of Stains have carcinogenicity and other shortcomings, which are attracting more and more attention from scholars

Although these methods can shorten the detection time, they require special equipment or certain experimental conditions and experimental skills, and are difficult to promote at the grassroots level

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