Kit for detecting enteroaggregative escherichia coli in food
A technology of Escherichia coli and kits, which is applied in the field of kits for the detection of enteroaggregative Escherichia coli in food, can solve the problems of long detection cycle, low sensitivity, and many false positives, and achieve short detection time, short research and development cycle, and high quality. stable effect
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Embodiment 1
[0052] The preparation of embodiment 1 enteroaggregative Escherichia coli bacterial liquid
[0053] Take the enteroaggregative Escherichia coli isolated clinically, place them in LB liquid medium test tubes, shake overnight at 37 degrees Celsius and 180 r / min, and set aside.
Embodiment 2
[0054] Example 2 Obtaining of aptamers
[0055] 1. The random single-stranded DNA library and primers were synthesized by Shanghai Bioengineering Co., Ltd.
[0056] Random single-stranded DNA library: 5'-TTGGACAGTGGACGTGAAGC(N35)GACCAAGTGACAGTGACGAG-3' (Note: n35 represents any 35 sets of 35 A, T, C, G bases).
[0057] Primer Ⅰ: 5’–TTGGACAGTGGACGTGAAGC-3’
[0058] Primer Ⅱ: 5'-CTCGTCACTGTCACTTGGTC-3'
[0059] Primer III: 5’-Digoxigenin–TTGGACAGTGGACGTGAAGC-3’
[0060] Primer IV: 5'-biotin-CTCGTCACTGTCACTTGGTC-3'.
[0061] 2. SELEX screening to obtain enteroaggregative Escherichia coli-specific oligonucleotide aptamers
[0062] 1) SELEX screening process:
[0063] 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;
[0064] b. Add 1mL enteroaggregative Escherichia coli suspension 1.5×10 8 cells / mL, combine in a shaker ...
Embodiment 3
[0078] Example 3 Verification of Combination Characteristics
[0079] Take 1.5 μg of the 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:
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[0081]
[0082] It can be seen from the above results that the 18 aptamers of the present invention have very strong binding properties, and there are ...
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