Dual-aptamer functional nucleic acid constant-temperature micro-fluidic chip sensor for microbiological detection

A technology of microfluidic chips and biosensors, which is applied in the fields of biomaterial analysis, laboratory containers, instruments, etc., can solve the problems of inability to detect on-site, take a long time, and have high dependence on antibody genomes, and achieve efficient on-site detection and improve The effect of capture ability

Active Publication Date: 2022-05-13
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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  • Dual-aptamer functional nucleic acid constant-temperature micro-fluidic chip sensor for microbiological detection
  • Dual-aptamer functional nucleic acid constant-temperature micro-fluidic chip sensor for microbiological detection
  • Dual-aptamer functional nucleic acid constant-temperature micro-fluidic chip sensor for microbiological detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 Composition, Installation Instructions and Uses of Each Part of Microfluidic Chip Biosensor

[0090] Microfluidic chip sensors consist 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 liquid flow in the microfluidic chip. Insert the microfluidic chip capillary microvalve control device 3 with the microfluidic chip 2 installed into the fluorescence collection dark box 1 .

[0091] Obscura 1 for observing fluorescence 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 partition 103 , and welded to form the lamp bead array 104 that produces sufficient fluorescence intensity. Install the lamp bead partition 103 with the lamp bead array 104 installed int...

Embodiment 2

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

[0096] The nucleic acid aptamer of Bacillus cereus was obtained by whole-cell screening technology, and then the specific recognition of Bacillus cereus in this application was realized. The nucleic acid sequences used in the experiment are shown in Table 1. The specific whole-cell screening process is as follows: (a) Take 1nmol of the synthetic random library, denature at 95°C for 5 minutes, place it on ice immediately, and let it stand for 5 minutes. Add 600 μL of bacterial suspension, and excess tRNA and BSA to the processed library. Incubate with shaking at 35°C and 120rpm for 90min. (b) After incubation at 8000rpm, centrifuge at 4°C for 5 minutes, the ssDNA combined with the target bacteria will be sedimented along with the bacteria, while the ssDNA not combined with the target bacteria will be in the supernatant, discard the supernatant as much as possible, and use Wash the pellet twic...

Embodiment 3

[0102] Example 3 Tailoring optimization of recognition elements in microfluidic chip biosensors

[0103] On the basis of the results in Example 2, carry out tailoring optimization, use IDT to predict the secondary structure of the preferred aptamer, select the predicted result with the lowest minimum free energy for subsequent cutting operations, and reserve 2-3 nt on both sides of the hairpin structure At the end, the trimmed aptamers are called trimmed aptamers. Then perform secondary trimming on the tailoring aptamer. The purpose of the secondary trimming is to disassemble and subdivide the complete stem-loop structure in the trimming aptamer, so as to accurately locate the core sequence of the aptamer involved in the interaction, and Aptamers that are trimmed twice are called secondary trimmed aptamers. Use the IDT website to predict the secondary structure of the tailoring aptamer and the secondary tailoring aptamer, and select a sequence with a suitable cutting length a...

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Abstract

The invention discloses a dual-aptamer functional nucleic acid constant-temperature micro-fluidic chip sensor for microbiological detection. The method comprises the following steps: dropwise adding a sample solution, a magnetic capture complex-dumbbell-shaped probe, an RCA-luminescent system and a buffer solution onto a micro-fluidic chip, and realizing separation, enrichment and detection of bacillus cereus through control of a valve and an external magnetic field; wherein the aptamer of the bacillus cereus is obtained through whole-cell screening and cutting, and has high affinity and specificity. Meanwhile, the sensor has the advantages of complete functions, simplicity, portability, capability of completing detection within one hour, no need of enlarged culture and the like.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a nucleic acid constant temperature microfluidic chip sensor with double aptamer function for microbial detection. Background technique [0002] Bacillus cereus is a Gram-positive bacterium that produces mesozoic spores. It is often detected in protein-rich foods such as vegetables, dairy products, and meat products, and can produce toxins such as enterotoxin and vomitoxin, which can cause food poisoning incidents. The endophytic spores of Bacillus cereus will germinate after proper heat treatment, and at the same time, heat treatment can eliminate other microorganisms. In the absence of competition, the spores germinate and Bacillus cereus can reproduce better. The spores of Bacillus cereus are hydrophobic and can adhere to epithelial cells, which can easily cause human intestinal infection, local tissue and systemic infection. Therefore, rapid detection methods for...

Claims

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Application Information

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Patent Type & Authority Applications(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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