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Method for detecting Salmonella typhimurium

A technology for Salmonella and Typhimurium, which is applied in the field of analytical chemistry and can solve the problems of sensitivity limitation and high background signal

Active Publication Date: 2017-09-05
QINGDAO UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional card-issuing structure will open the card-issuing structure under normal circumstances. Even if there is no trigger chain, it will also form the card-issuing self-assembly, that is, non-specific self-assembly, which will cause the background signal to be too high, so the improvement of sensitivity is limited. limits

Method used

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  • Method for detecting Salmonella typhimurium
  • Method for detecting Salmonella typhimurium
  • Method for detecting Salmonella typhimurium

Examples

Experimental program
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Effect test

example 1

[0041] Example 1: A method for detecting Salmonella typhimurium

[0042] 1. Experimental part

[0043] 1.1 Instruments and reagents

[0044] 1.1.1 Instruments and equipment

[0045] DHG blast drying oven (Shanzhi Instrument Equipment Co., Ltd., Shanghai); AR224CN Ohaus analytical balance (Qingdao Zhonghe Hengxin Electronics Co., Ltd., Qingdao); THZ constant temperature oscillation box (Jiayuan Xingye Technology Co., Ltd., Beijing) ; RFL-1 ultra-weak chemiluminescence detector (Ruimai Analytical Instrument Co., Ltd., Xi'an); Anke-TGL-16C Feiweng brand high-speed centrifuge (Anting Scientific Instrument Factory, Shanghai).

[0046] 1.1.2 Reagents

[0047] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl 4 ) was purchased from Sigma; the carboxyl magnetic beads with a particle size of 0.5 μm and a concentration of 10 mg / mL were purchased from Tianjin Beisi Le Chromatography Technology Development Center; Lu...

example 2

[0071] Example 2: Sensitivity comparison of methods

[0072] Using non-mismatched probe DNA:

[0073] 5’-AGA TGA GTG GAT TAA ACC CGG TAG ACT CAT CTT TGA ATA ACT ACC GGGTTT AAT CCC ACG AGA TAC TGT TCC-SH-3’;

[0074] One mismatch base probe DNA:

[0075]5’-AGA TGA GTG GAT TAA ACC CGG TAG ACT CAT CTT TGA ATA AGT ACC GGGTTT AAT CCC ACG AGA TAC TGT TCC-SH-3’;

[0076] Two mismatch base probe DNA:

[0077] 5'-AGA TGA GTG GAT TAA ACC CGG TAG ACT CAT CTT TGA ATA TGT ACC GGGTTT AAT CCC ACG AGA TAC TGT TCC-SH-3' instead of probe DNA, other steps are the same, according to the method of the present invention for Salmonella typhimurium Detection, the detection limit of determination is 10000cfu / mL, 4500cfu / mL and 1980cfu / mL respectively, all of which are higher than the detection limit of three mismatched base probe DNA. It shows that a method for detecting Salmonella typhimurium proposed by the present invention has high sensitivity.

example 3

[0078] Example 3: Impact of mismatches on the system

[0079] In order to weaken the background signal and improve the signal-to-noise ratio, the influence of the catalytic hairpin self-assembly technology and the mismatch catalytic hairpin self-assembly technology of this application on the experiment was explored. In the catalytic hairpin self-assembly technology, the capture DNA adopts the above-mentioned sequence, and the probe DNA is a non-mismatched probe DNA; in this application, the DNA sequence in the 3' end 2 region of the probe DNA is mismatched by three bases, that is, the detection Needle DNA. Such as Figure 4 As shown, under the same conditions, when there is no Salmonella typhimurium, the chemiluminescence signal is 373 when the non-mismatched probe DNA is used, while the signal is only 147 when the probe DNA is used. Although the signal of the hairpin self-assembly technique was higher than that of the mismatch-catalyzed hairpin self-assembly technique under...

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Abstract

The invention specifically relates to a method for detecting Salmonella typhimurium, belonging to the field of analytical chemistry. The method comprises the following steps: modifying LumAuNPs with probe DNA so as to obtain a chemiluminescence probe; modifying a magnetic bead by using captured DNA with a hairpin structure so as to obtain a captured DNA-modified magnetic bead; modifying another magnetic bead by using aptamer DNA and aptamer complementary sequence DNA so as to obtain a DNA-modified magnetic beads; adding a Salmonella typhimurium solution containing a target into a solution of the DNA-modified magnetic bead, carrying out magnetic separation, sucking up obtained clear liquid, adding the clear liquid into a solution of the captured DNA-modified magnetic bead, and connecting the chemiluminescence probe with the surface of the magnetic bead via catalytic hairpin self-assembly technology under the action of hybridization of the probe DNA on the chemiluminescence probe; and after magnetic separation and adding hydroxylamine-O-sulfonic acid for generation of chemiluminiscence so as to realize determination of Salmonella typhimurium. According to the invention, LumAuNPs-hydroxylamine-O-sulfonic acid is used as a chemiluminescence system, so the intensity of a detection signal is improved; and mismatched bases are introduced into the probe DNA, so background signals are reduced, and determination sensitivity is improved. The method provided by the invention has the advantages of easiness and high sensitivity.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and in particular relates to a method for detecting Salmonella typhimurium. Background technique [0002] Salmonella typhimurium (Salmonella typhimurium) is a Gram-negative bacterium and one of the main pathogens causing acute gastroenteritis (Cossu A, Levin R E. Rapid conventional PCR and real-time-qPCR detection of low numbers of from ground beef without enrichment[J].FoodBiotechnology,2014,28(2):96-105;Torlak E,Akan I M,Inal M.Evaluation of Rapidchek select for the screening of Salmonella in meat and meat products[J]. Journal of Microbiological Methods, 2012, 90(3):217-219). Salmonella typhimurium has a wide range of hosts, and can survive on almost all living tissues. Many poultry, livestock, mice, birds and cold-blooded animals are their natural hosts. Flies and fleas can carry the bacteria, and they are often transmitted by contaminated water, milk and Food travels between people and a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N21/76
CPCG01N21/76G01N33/56916G01N2333/255
Inventor 混旭钟华孟妍王周平
Owner QINGDAO UNIV OF SCI & TECH
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