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A method for detecting Salmonella

A technology for Salmonella and target bacteria, applied in the field of microbial detection, can solve the problems of high cost, low specificity and sensitivity, long detection period, etc., and achieve the effects of low detection limit, improved specific identification, and fast detection speed.

Active Publication Date: 2019-06-11
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of relatively low specificity and sensitivity, high cost and long detection period of the method for detecting Salmonella in the above prior art, the present invention provides a heme-based method with high specificity and sensitivity, fast detection speed and low cost. / G-quadruplex-induced photoelectron transfer of DNA silver nanoclusters for the detection of Salmonella

Method used

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  • A method for detecting Salmonella
  • A method for detecting Salmonella
  • A method for detecting Salmonella

Examples

Experimental program
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Effect test

preparation example Construction

[0042] Described preparation method, the synthetic operation step of preferred DNA silver nanocluster is as follows:

[0043] ① Add 15 μL of 100 μM H3 and 73 μL of 20 mM PB buffer (pH 7.0) into the EP tube wrapped in tinfoil, and then add 6 μL of 1.5 mM AgNO 3 solution (make sure the Ag + The ratio of H3 to H3 is 6:1), shake for 1 min, and place at 4°C for 30 min.

[0044] ② After 30 min, continue to add 6 μL of 1.5 mM NaBH to the EP tube 4 , shaken for 1 min, and placed in the dark at 4 °C for more than 6 h for later use.

[0045] Described preparation method, preferred homogeneous reaction operating steps are as follows:

[0046] H1 (3 μL, 20 μM), Primer (3 μL, 5 μM), Phi 29 polymerase (0.5 U), dNTPs (5 μL), Nt.AlwI endonuclease (0.5 U), H2 (9 μL, 50 μM), heme (5 μL) and H3 (9 μL, 50 μM), 10× buffer solution (buffer10 μL) and 5 μL sterilized water were added to the EP tube wrapped in foil paper, and then 5 μL target After mixing evenly, they were placed in a water bath ...

Embodiment 1

[0050] The preparation method of described fluorescent biosensor comprises the following steps:

[0051] The synthetic operation steps of DNA silver nanoclusters are as follows:

[0052] ① Add 15 μL of 100 μM H3 and 73 μL of 20 mM PB buffer (pH 7.0) into the EP tube wrapped in tinfoil, and then add 6 μL of 1.5 mM AgNO 3 solution (make sure the Ag + The ratio of H3 to H3 is 6:1), shake for 1 min, and place at 4°C for 30 min.

[0053] ② After 30 min, continue to add 6 μL of 1.5 mM NaBH to the EP tube 4 , shaken for 1 min, and placed in the dark at 4 °C for more than 6 h for later use.

[0054] The main steps of the reaction process in homogeneous solution are as follows:

[0055] a. Mix H1 (final concentrations are 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.2 μM, 1.4 μM), Primer (3 μL, 5 μM), Phi 29 polymerase (0.5 U), dNTPs (5 μL ), Nt.AlwI endonuclease (0.5 U), H2 (9 μL, 50 μM), heme (5 μL) and H3 (9 μL, 50 μM), 10× buffer (buffer10 μL) and 5 μL Add sterilized water to the EP tub...

Embodiment 2

[0063] The preparation method of described fluorescent biosensor comprises the following steps:

[0064] The synthetic operation steps of DNA silver nanoclusters are as follows:

[0065] ① Add 15 μL of 100 μM H3 and 73 μL of 20 mM PB buffer (pH 7.0) into the EP tube wrapped in tinfoil, and then add 6 μL of 1.5 mM AgNO 3 solution (make sure the Ag + The ratio of H3 to H3 is 6:1), shake for 1 min, and place at 4°C for 30 min.

[0066] ② After 30 min, continue to add 6 μL of 1.5 mM NaBH to the EP tube 4 , shaken for 1 min, and placed in the dark at 4 °C for more than 6 h for later use.

[0067] The main steps of the reaction process in homogeneous solution are as follows:

[0068] a. Mix H1 (3 μL, 20 μM), Primer (3 μL, 5 μM), Phi 29 polymerase (0.1U, 0.2 U, 0.3U, 0.4 U, 0.5 U, 0.6 U, 0.7 U), dNTPs ( 5 μL), Nt.AlwI endonuclease (0.5 U), H2 (9 μL, 50 μM), heme (5 μL) and H3 (9 μL, 50 μM), 10× buffer (buffer10 μL) and Add 5 μL of sterilized water to the EP tube wrapped in tin ...

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Abstract

The invention provides a method for detecting salmonellae. The method comprises the following steps: synthesizing a DNA silver nano cluster; and carrying out index enlargement under the combined action of Phi 29 polymerase and Nt.AlwI endonuclease and homogeneous reaction of strand displacement cycle amplification, and then carrying out fluorescence detection. The method provided by the invention has the advantages that specific recognition of an aptamer is utilized, and the aptamer is designed into a hairpin structure, so that high specificity detection on a target object salmonellae is realized; and cyclic utilization of Trigger is realized by utilizing the combined action of the Phi 29 polymerase and Nt.AlwI endonuclease as well as strand displacement reaction, signal simplifying effect is achieved, and detection sensitivity is effectively improved.

Description

technical field [0001] The invention relates to a method for detecting salmonella, belonging to the technical field of microorganism detection. Background technique [0002] Salmonella is a common foodborne pathogen, a Gram-negative, intracellular parasitic enteric bacterium. The bacteria widely exist in nature, not only can cause acute, chronic or latent infection of livestock, poultry and other animals, but also cause food poisoning by contaminating food, posing a great threat to human beings. According to statistics, among the types of bacterial food poisoning in various countries in the world, food poisoning caused by Salmonella often ranks first. Salmonella sickens about 40,000 Americans and kills about 600 each year. Salmonella is the most common pathogenic bacteria in food poisoning in my country, accounting for the first place in food poisoning. The clinical symptoms of salmonellosis mainly include headache, abdominal pain, fever, etc., and the mortality rate is 1...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 刘素冷雪琪王玉裴倩倩崔雪君韩聪徐成功刘学娇秦艺菲
Owner UNIV OF JINAN
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