Method for detecting Salmonella on basis of technique for amplifying nanogold-labeled and silver-enhanced signals
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A Salmonella, signal-enhancing technology, applied in the field of sandwich complexes, can solve the problems of operating procedures, unsatisfactory specific detection of detection time, time-consuming and laborious, difficult to promote, etc.
Inactive Publication Date: 2010-07-14
JIANGNAN UNIV
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[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
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example 1
[0055] 1 material
[0056] 1.1 Instrument
[0057] Multiskan MK3 Microplate Reader (Thermo Company, USA), DF-101S Collective Magnetic Heating Stirrer (Yuhua Instrument Factory, Gongyi City, Henan Province), TU-1900 UV-Vis Spectrophotometer (Beijing Puxi General Instrument Co., Ltd.) , TecnaiG220 transmission electron microscope (TEM) (US FEI company), LH586-2 constant temperature water tank (Shanghai Jingke Industrial Co., Ltd.), 5804R high-speed refrigerated centrifuge (Shanghai Anting Scientific Instrument Factory), 96 microplate (Canada BBI company) ).
[0058] 1.2 Reagents and samples
[0059] Bovine serum albumin (BSA) (Beijing Dingguo Bioengineering Co., Ltd.), chloroauric acid (HAuCl 4 ) (Shanghai Jiuyue Chemical Co., Ltd.), hydroquinone, silver nitrate, phosphoric acid (China Pharmaceutical Shanghai Chemical Reagent Company), avidin (product of Amresco, USA), and ultrapure water were prepared by Milli-Q system.
[0060] Oligonucleotide sequences were purchased from...
example 2
[0085] 1 Reagents and materials
[0086] Mouse anti-Salmonella polyclonal antibody was purchased from Shanghai Sangong. Nanogold and mouse anti-Salmonella polyclonal antibody-nanogold complex were prepared and synthesized in our laboratory. The Salmonella strains used and other related strains are preserved in this laboratory. The tested food samples were purchased from 6 different local farmers' markets. Other reagent materials used, apparatus and equipment are identical with example 1.
[0087] 2 methods
[0088] 2.1 Operation steps
[0089] Add 100 μL of target bacteria to the microplate wells coated with mouse anti-Salmonella polyclonal antibody, incubate at 37°C for 20 min, and then wash with PBST vigorously for 3 times to remove unbound bacteria. Then add 100 μL mouse anti-Salmonella polyclonal antibody-nanogold complex to each well, incubate at room temperature for 20 min, and then wash with PBST vigorously for 3 times to remove unbound antibody-nanogold complex. ...
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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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