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A dual-aptamer functional nucleic acid thermostatic microfluidic chip sensor for microbial detection

A microfluidic chip and functional nucleic acid technology, which is applied in the fields of biological material analysis, laboratory containers and instruments, etc., can solve the problems of time-consuming, inability to detect on-site, cumbersome operation, etc., to improve the capture ability and efficient on-site detection. Effect

Active Publication Date: 2022-06-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Provide a research basis for the detection of Bacillus cereus based on nucleic acid aptamers, and solve the problems of high dependence on antibodies and genomes, time-consuming, cumbersome operations, low specificity, low sensitivity, and inability to detect on-site

Method used

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  • A dual-aptamer functional nucleic acid thermostatic microfluidic chip sensor for microbial detection
  • A dual-aptamer functional nucleic acid thermostatic microfluidic chip sensor for microbial detection
  • A dual-aptamer functional nucleic acid thermostatic microfluidic chip sensor for microbial detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 Microfluidic chip biosensor composition, installation instructions and use of each part

[0090] The microfluidic chip sensor is composed of figure 1 As shown, it includes an observation dark box 1 for observing fluorescence, a microfluidic chip 2 for detecting Bacillus cereus, and a capillary microvalve control device 3 for controlling the flow of liquid in the microfluidic chip. Insert the microfluidic chip capillary valve control device 3 with the microfluidic chip 2 installed into the fluorescence collection dark box 1 .

[0091] Observation obscura 1 for observing fluorescence such as figure 2 As shown, it is composed of a box body 101 , a dark box top cover 102 , a lamp bead partition 103 and a lamp bead 104 . The lamp beads 104 are installed in the 9×9 array holes 105 reserved on the lamp bead separator 103 , and welded to form a lamp bead array 104 that generates sufficient fluorescent intensity. Install the lamp bead spacer 103 on which the lamp b...

Embodiment 2

[0095] Example 2 Screening of recognition elements in microfluidic chip biosensors

[0096] The nucleic acid aptamer of Bacillus cereus is obtained by whole-cell screening technology, thereby realizing the specific recognition of Bacillus cereus in the present application. The nucleic acid sequences used in the experiment are shown in Table 1. The specific whole-cell screening process is as follows: (a) Take 1 nmol of the synthesized random library, denature it at 95°C for 5 minutes, immediately place it on ice, and place it for 5 minutes. Add 600 μL of bacterial suspension and excess tRNA and BSA to the treated library. Incubate at 35°C with shaking at 120rpm for 90min. (b) After incubation, centrifuge at 4°C for 5 min at 8000 rpm. The ssDNA bound to the target cell is settled down with the cell, while the ssDNA that is not bound to the target cell is in the supernatant. Wash the pellet twice with 1×BB to remove ssDNA with weak binding ability. (c) Add 100 μL of 1×TE buffe...

Embodiment 3

[0102] Example 3 Cutting optimization of identification elements in microfluidic chip biosensors

[0103] On the basis of the results of Example 2, cutting optimization was carried out, using IDT to predict the secondary structure of the preferred aptamer, selecting the predicted result with the lowest minimum free energy for subsequent cutting operations, and reserving 2-3 nt on both sides of the hairpin structure. At the end, the trimmed aptamer is called a trimmed aptamer. Then, the cutting aptamer is cut again. The purpose of the second cutting is to disassemble and subdivide the complete stem-loop structure in the cutting aptamer, so as to accurately locate the core sequence of the aptamer participating in the interaction, and A secondary cropped aptamer is called a secondary cropped aptamer. The IDT website was used to predict the secondary structure of the tailored aptamer and the secondary tailored aptamer, and the sequence with suitable cutting length and lower minim...

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Abstract

The invention discloses a nucleic acid thermostatic microfluidic chip sensor with double aptamer function for microorganism detection. The sample solution, magnetic capture complex-dumbbell-shaped probe, RCA-luminescent system, and buffer solution are added dropwise to the microfluidic chip, and the separation, enrichment and Detection; the aptamer of Bacillus cereus is obtained through whole-cell screening and tailoring, and has high affinity and specificity. At the same time, the sensor has the advantages of complete functions, simple and portable, can complete the detection within 1 hour, and does not need to be expanded and cultivated.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a dual-aptamer functional nucleic acid thermostatic microfluidic chip sensor for microbial detection. Background technique [0002] Bacillus cereus is a Gram-positive bacterium that produces mesophytic spores. It is often detected in protein-rich foods such as vegetables, dairy products, and meat products, and can produce toxins such as enterotoxins and vomitoxins, which can lead to food poisoning events. The endospores of Bacillus cereus will germinate after proper heat treatment, and the heat treatment can destroy other microorganisms. When the spores germinate without competition, Bacillus cereus can reproduce better. The spores of Bacillus cereus are hydrophobic and can adhere to epithelial cells, which can easily cause intestinal infections in humans, as well as local tissue and systemic infections. Therefore, rapid detection methods for Bacillus cereus are in u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N21/64B01L3/00
CPCG01N33/56911G01N21/6486B01L3/5027B01L3/502761G01N2333/32
Inventor 许文涛周子琦朱龙佼张文强杜再慧
Owner CHINA AGRI UNIV
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