Detection method for sensitively and rapidly detecting staphylococcus aureus enterotoxin B

A technology for the detection of staphylococcus enterotoxin B, which is applied in the field of sensitive and rapid detection of Staphylococcus aureus enterotoxin B, and can solve the problems of limiting the application of molecular beacons

Inactive Publication Date: 2018-12-11
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The premise that the above method can be realized is that the aptamer has a unique secondary structure, but most of the nucleic acid aptamers are current...

Method used

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  • Detection method for sensitively and rapidly detecting staphylococcus aureus enterotoxin B
  • Detection method for sensitively and rapidly detecting staphylococcus aureus enterotoxin B
  • Detection method for sensitively and rapidly detecting staphylococcus aureus enterotoxin B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Design of hairpin probe and determination of denatured conditions

[0029] In order to ensure the rationality of the sequence design, the hairpin probe was designed using NUPACK software. In the Analysis work interface, the type of nucleic acid was adjusted to DNA, and the working temperature was set to 37°C. At the same time, in the Advanced options option box, set Na + 0.4M, Mg 2+is 5mM. First, use NUPACK software to verify whether the designed H1 and H2 sequences can normally form the hairpin structure. Secondly, set Number of strand species to 2, Maxium complex size to 4, perform hybridization operation on H1 and H2 at equal concentrations, and verify whether H1 and H2 hybridize without priming strand H0. Finally, set the Number ofstrand species to 3, the Maxium complex size to 5, and other parameters unchanged, import the sequences of H0 or SEBaptamer into the software respectively, and introduce the sequences of H1 and H2 as shown in Table 1 and Table...

Embodiment 2

[0030] Example 2: Gel electrophoresis characterization confirms the feasibility of the SEB system detected by competitive fluorescent hybridization chain reaction

[0031] (1) Pretreatment of hairpin DNA H1 and H2 and SEB nucleic acid aptamers: Before the reaction, H1 and H2 with a concentration of 5 μM were heated and denatured at 95°C for 5 minutes, and then slowly cooled and kept at 4°C for 1 hour, 1 μM The SEB nucleic acid aptamer was heated and denatured at 95°C for 5 minutes, and then rapidly cooled in an ice-water bath.

[0032] (2) Gel electrophoresis characterization verification HCR amplification: in Tris-HCl buffer (20mM, 400mM NaCl, 5mMMgCl 2 , pH 7.2) were added 10 μL of H1 (5 μM), 10 μL of H2 (5 μM), 10 μL of priming chain (0, 0.63, 1.25, 2.5, 5, 10 μM), mixed the reaction solution and reacted in a metal bath at 37 ° C for 60 min .

[0033] (3) Gel electrophoresis characterization verification Competitive fluorescence hybridization chain reaction detection SEB:...

Embodiment 3

[0042] Embodiment 3: Measuring HCR dynamics process by fluorescence method

[0043] (1) Before the reaction, H1 and H2 at a concentration of 5 μM were heated and denatured at 95° C. for 5 minutes, and then cooled slowly and kept at 4° C. for 1 hour. See Table 1 for the sequences used.

[0044] (2) To prepare a reaction system, take 1136 μL hybridization buffer, add 80 μL H1 (5 μM) and 80 μL H2 (5 μM) and mix well to prepare a mixed reaction system.

[0045] (3) Take 20 μL of priming strand H0 with a concentration of 10 μM, and dilute to 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.3125 μM respectively, each concentration being at least 10 μL.

[0046] (4) Put the reaction system in a constant temperature water bath at 37° C., and react in the dark for 2 hours.

[0047] Since H1 is designed in the form of a molecular beacon, when the hairpin structure is opened, the fluorescence recovers. At this time, in order to study whether there is a linear or nonlinear dynamic law of the corre...

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Abstract

The invention relates to a detection method for sensitively and rapidly detecting staphylococcus aureus enterotoxin B, comprising the following steps: heating H1 and H2 with concentration of 5Mum at 95 DEG C for denaturation for 5min, heating an SEB aptamer at 85-95 DEG C for denaturation for 5-10min, then taking 6.7-33.3muL of SEB aptamer with concentration of 1muM, adding 100muL of SEB, mixing and then adding 80-100muL of hybrid buffer solution, and reacting for 30min at 37 DEG C; adding 6.7-33.3muL of 1muM H0, and reacting for 30min at 37 DEG C; adding 10-30muL of mixed reaction system of 100muL of H1 with concentration of 5muM and 100muL of H2 with concentration of 5muM; reacting for 45min at 37 DEG C constant temperature, and measuring the fluorescence value; compared with a conventional detection technology based on nucleic acid aptamer, the detection sensitivity is reduced by 2 orders of magnitudes.

Description

technical field [0001] The invention relates to the technical field of food safety detection and analysis, in particular to a detection method for sensitive and rapid detection of Staphylococcus aureus enterotoxin B. Background technique [0002] Staphylococcus aureus is an important food-borne pathogen that causes bacterial food poisoning. It exists widely on the body surface of animals, human nasal cavity and spoiled food. It is not pathogenic in itself, but it secretes enterotoxin, etc. Extracellular toxins can cause toxic reactions. Among them, Staphylococcal enterotoxin B (Staphylococcal enterotoxin B, SEB) is an example. It has heat resistance, low pH resistance, and resistance to trypsin decomposition. Ingestion or inhalation can cause poisoning such as nausea, vomiting, and abdominal cramps. Phenomenon. Due to its low production cost, large polluted area, and various modes of transmission, SEB is listed as a Class B potential biological warfare agent by the US CDC,...

Claims

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Application Information

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IPC IPC(8): C12Q1/6818
CPCC12Q1/6818C12Q2525/205C12Q2525/301C12Q2563/107
Inventor 徐艳阳霍冰洋张铁华高志贤李薇茹王紫微王雪松
Owner JILIN UNIV
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