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Kit for detecting shiga-toxigenic escherichia coli in food

A technology for producing Shiga toxin and Escherichia coli, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of long detection period, many false positives, low sensitivity, etc., and achieve short detection time, short research and development period, and simple operation. Effect

Inactive Publication Date: 2016-10-12
杨国林
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In order to solve the problems of long detection period, low sensitivity, and many false positives in the existing detection of Escherichia coli, the present invention provides a kit for specifically identifying Shiga toxin-producing Escherichia coli STEC and its application

Method used

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  • Kit for detecting shiga-toxigenic escherichia coli in food
  • Kit for detecting shiga-toxigenic escherichia coli in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The preparation of embodiment 1 Shiga toxin-producing escherichia coli STEC bacterium liquid

[0061] Take the clinically isolated Shiga toxin-producing Escherichia coli STEC O157:H7 strain, put it in an LB liquid medium test tube, shake it overnight at 37 degrees Celsius and 180 r / min, and set aside.

Embodiment 2

[0062] The acquisition of embodiment 2 aptamers

[0063] 1. The random single-stranded DNA library and primers were synthesized by Shanghai Bioengineering Co., Ltd.

[0064] Random single-stranded DNA library: 5'-TTGGACAGTGGACGTGAAGC(N36)GACCAAGTGACAGTGACGAG-3' (Note: n36 represents 36 sets of any one of 36 A, T, C, and G bases).

[0065] Primer Ⅰ: 5’–TTGGACAGTGGACGTGAAGC-3’

[0066] Primer Ⅱ: 5'-CTCGTCACTGTCACTTGGTC-3'

[0067] Primer III: 5’-Digoxigenin–TTGGACAGTGGACGTGAAGC-3’

[0068] Primer IV: 5'-biotin-CTCGTCACTGTCACTTGGTC-3'.

[0069] 2. SELEX screening to obtain Shiga toxin-producing Escherichia coli STEC-specific oligonucleotide aptamers

[0070] 1) SELEX screening process:

[0071] a. For the first round of screening, take 10 μg of synthetic random single-stranded DNA and add it to 400ul 1×binding buffer, denature at 95°C for 5 minutes, and then quickly place it on ice for 10 minutes;

[0072] b. Add 1 mL of Shiga toxin-producing Escherichia coli STEC suspensio...

Embodiment 3

[0086] Embodiment 3 Combination characteristic verification

[0087] Take 1.5 μg of aptamers, digest them with calf intestinal alkaline phosphatase (CIP) at 37°C for 1 h, purify and recover the dephosphorylated DNA; label [γ-32P]ATP on the dephosphorylated DNA by T4 polynucleotide kinase The end of a DNA molecule. 10nmol of radioactively labeled DNA aptamers were incubated with bacteria of different concentrations at 37°C for 30min, the reaction solution of each group was filtered through a nitrocellulose membrane, the filter membrane was washed, dried, and the residual amount on the filter membrane was measured by a liquid scintillation counter. For radiation dose, the same sample was measured twice in parallel. Calculate the dissociation constant of each aptamer and bacteria. The result is as follows:

[0088]

[0089]

[0090] It can be seen from the above results that the 21 aptamers of the present invention have very strong binding properties, and there are no ap...

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Abstract

The invention relates to a kit for detecting shiga-toxigenic escherichia coli in food. The shiga-toxigenic escherichia coli is a novel high-pathogenicity foodborne pathogen carrying one or two kinds of prophage-encoded shiga toxin genes. In a detection method in the prior art, time and labor are consumed, and detection is inconvenient; the kit for specifically detecting the shiga-toxigenic escherichia coli contains an aptamer specifically combined with the shiga-toxigenic escherichia coli, wherein the aptamer is single-chain DNA. The kit has the advantages of being short in detection time and development cycle, stable in quality, easy to operate and the like and is widely applied to food and sanitary safety detection.

Description

field of invention [0001] The invention belongs to the technical field of food detection, and in particular relates to a kit for detecting Shiga toxin-producing Escherichia coli in food. Background technique [0002] Shiga toxin-producing Escherichia coli (STEC for short) is a new class of highly pathogenic foodborne pathogens carrying prophage encoding one or two Shiga toxin genes, including Escherichia coli O26, and more than 150 other serotypes of Escherichia coli such as O157, O45, O103, O104, O111, O121, and O145. The bacterium is a Gram-negative bacillus with no spores and flagella. It can grow at 10-65°C, and the optimum growth temperature is 33-42°C. It has strong acid resistance (pH2.5-3.0) and can resist gastric acid. of digestion. [0003] According to incomplete statistics, 70% of non-O157 STEC infections in the United States from 1983 to 2002 were caused by O26, O45, O103, O121, O111, and O145 serotypes; in September 2011, the United States Department of Agric...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56916
Inventor 杨国林
Owner 杨国林
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