Salmonella typhimurium detecting method based on RCA (rolling circle amplification) method

A rolling circle replication, Salmonella technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. Convenient, Sensitive Detection of Effects

Inactive Publication Date: 2016-03-02
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the major disadvantage of these methods...

Method used

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  • Salmonella typhimurium detecting method based on RCA (rolling circle amplification) method
  • Salmonella typhimurium detecting method based on RCA (rolling circle amplification) method
  • Salmonella typhimurium detecting method based on RCA (rolling circle amplification) method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] (1) Put 2 μL phi29 buffer solution in a centrifuge tube and add 15 μL sterilized water;

[0041] (2) Put 1 μL, 10 μM HP in a centrifuge tube;

[0042] (3) Add 1 μL of the target substance to the centrifuge tube, and react fully at 37°C for 2 hours, so that the hairpin can recognize and capture the target substance;

[0043] (4) Add 1 μL of padlock probe to the centrifuge tube and react at 37°C for 1 hour;

[0044] (5) Add 1 μL T4 DNA and react at 16°C for 3 hours, in order to link the padlock probes into a circle;

[0045] (6) Denature and inactivate T4 DNA ligase at 65°C to prevent the ligase from affecting subsequent reactions;

[0046] (7) Add 0.5 μL ExoI and 0.5 μL ExoIII to the system for 30 minutes to hydrolyze other DNA except circular DNA;

[0047] (8) Heat the system to 80°C to inactivate the two exonucleases and prevent them from being affected in the subsequent process;

[0048] (9) Add 1 μL, 10 μM primer, then add phi29, dNTP and BSA and react at 37°C fo...

Embodiment 2

[0054] (1) Incubate completely in Salmonella typhimurium solution at 37°C.

[0055] (2) After putting 2 μL of phi29 buffer solution into 5 centrifuge tubes, add 15 μL of sterilized water respectively.

[0056] (3) Put 1μL, 1μM, 2μM, 5μM, 10μM, 20μM HP in a centrifuge tube

[0057] (4) Add 1 μL of the target substance to the centrifuge tube, and react fully at 37°C for 2 hours, so that the hairpin can recognize and capture the target substance.

[0058] (5) Add 1 μL of padlock probe to the centrifuge tube and react at 37°C for 1 hour to make the padlock probe and the hairpin structure base-pair complementary to form a DNA double strand with a cut. .

[0059] (6) Add 1 μL T4 DNA ligase, react at 16°C for 3 hours, under the action of T4 DNA ligase, the nick can be repaired, and the padlock probe is connected into a circle.

[0060] (7) Denature and inactivate T4 DNA ligase at 65°C to prevent it from affecting subsequent reactions. .

[0061] (8) Add 0.5 μL ExoI and 0.5 μL Ex...

Embodiment 3

[0069] (1) Incubate completely in Salmonella typhimurium solution at 37°C.

[0070] (2) After putting 2 μL of phi29 buffer solution into 5 centrifuge tubes, add 15 μL of sterilized water respectively.

[0071] (3) Put 1μL and 10μM HP in a centrifuge tube respectively.

[0072] (5) Add 1 μL of the target substance to the centrifuge tube, and react fully at 37°C for 2 hours, so that the hairpin can recognize and capture the target substance.

[0073] (5) Add 1 μL of 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM padlock probes to the centrifuge tube, and react at 37°C for 1 hour to make the padlock probes and the hairpin structure base-pair complementary to form a DNA double strand with a cut . .

[0074] (6) Add 1 μL T4 DNA ligase, react at 16°C for 3 hours, under the action of T4 DNA ligase, the nick can be repaired, and the padlock probe is connected into a circle.

[0075] (7) Denature and inactivate T4 DNA ligase at 65°C to prevent it from affecting subsequent reactions. .

[007...

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PUM

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Abstract

The invention provides a Salmonella typhimurium detecting method based on an RCA (rolling circle amplification) method. The RCA technology is adopted, a large quantity of sequences capable of being bound with Ag clusters are formed in a short time, the Ag clusters are wrapped in a DNA (deoxyribonucleic acid) strand more firmly under the action of a G-rich auxiliary strand, and fluorescence signals are generated. According to the method, the RCA and the Ag clusters are combined for application, so that signal detection is more sensitive and fast, and a prepared biosensor has the characteristics of simplicity in operation, fastness, high sensitivity and the like.

Description

technical field [0001] The invention relates to a detection method for Salmonella typhimurium based on nucleic acid aptamer and RCA technology. Background technique [0002] Salmonella typhimurium is one of the main pathogens causing acute gastroenteritis. is a very important bacterium for the development of microbial genetics. Salmonella typhimurium is a group of non-adaptive or pantropic Salmonella with a wide range of hosts, and it is one of the strains with the highest isolation rate in the world. Salmonella typhimurium can invade the human body through many ways, but mainly through contamination of food and water, oral transmission, direct contact or indirect transmission. For example, nosocomial infection is mainly transmitted indirectly through the hands of medical staff, medical equipment, diapers and pads. Pathogen-contaminated air can also spread through the respiratory tract. Generally susceptible, infants and young children are mostly dominant infections, and...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10
CPCC12Q1/6844C12Q2521/501C12Q2525/205C12Q2531/125C12Q2563/137Y02A50/30
Inventor 黄加栋王玉崔洁刘素王虹智郭玉娜邱婷婷许颖崔雪君裴倩倩冷雪琪韩聪
Owner UNIV OF JINAN
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