Self-suction type multichannel pathogenic bacterium detection micro-fluidic chip based on LAMP (loop-mediated isothermal amplification)

A pathogenic bacteria detection and microfluidic chip technology, applied in the field of microbial detection, can solve problems that have not yet been combined with multi-channel pathogenic bacteria detection microfluidic chips

Active Publication Date: 2018-05-04
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this method has not been combined with LAMP-

Method used

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  • Self-suction type multichannel pathogenic bacterium detection micro-fluidic chip based on LAMP (loop-mediated isothermal amplification)
  • Self-suction type multichannel pathogenic bacterium detection micro-fluidic chip based on LAMP (loop-mediated isothermal amplification)
  • Self-suction type multichannel pathogenic bacterium detection micro-fluidic chip based on LAMP (loop-mediated isothermal amplification)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Selection of SA and VP characteristic nucleic acid sequences and design and screening of corresponding LAMP primers

[0053] According to species specificity, SA's nuc 300bp conserved nucleic acid fragment and VP in the gene tlh The 400bp conserved nucleic acid fragment in the gene was selected as the respective characteristic sequence. 6 LAMP primers corresponding to the SA signature sequence (F3- nuc 、B3- nuc , LF- nuc , LB- nuc , FIP- nuc , BIP- nuc ) self-designed by using computer software; 6 primers corresponding to the VP characteristic sequence (F3- tlh 、B3- tlh , LF- tlh , LB- tlh , FIP- tlh , BIP- tlh ) was obtained based on literature review (Yamazaki, W., Ishibashi, M., Kawahara, R., Inoue, K., 2008. BMC Microbiol. 8, 163.). All primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The characteristic sequences and primer sequences are shown in Table 1.

[0054] Table 1. List of characteristic sequences and pri...

Embodiment 2

[0056] Example 2 Preparation of LAMP-based self-priming multi-channel pathogen detection microfluidic chip

[0057] Three-dimensional schematic diagram of self-priming multi-channel pathogen detection microfluidic chip based on LAMP figure 1 As shown, the actual picture is as figure 2 As shown, the exploded diagram is as image 3 As shown, the chip includes a sampling layer 1, a reaction layer 2 and a support layer 3 from top to bottom; the sampling layer 1 includes a sampling port 4, a sampling channel 5 and a nanoscale anti-evaporation film 8; The reaction layer 2 includes a reaction chamber 6 and a circular filter paper sheet 7;

[0058] In the sampling layer 1, the sampling channel 5 is located at the bottom of the layer, one end converges at the sampling port 4, and the other end communicates with the reaction chamber 6; the sampling channel 5 has three channel walls in the sampling layer 1, and the bottom Hollow out; when the sampling layer 1 and the reaction layer ...

Embodiment 3

[0067] Example 3 SA plasmid template detection of self-priming multi-channel pathogen detection microfluidic chip based on LAMP

[0068] 1. Configuration of LAMP reaction solution

[0069] every 8 mu L LAMP reaction solution contains the following components: 0.8M betaine, 1.4 mM dNTP solution, 0.8 mu L 10 × Isothermal Amplification Buffer, 6 mM Magnesium Sulfate Solution, 0.16 mu L 10 × SYBR Green I fluorescent dye, 300 mu M Hydroxynaphthol Blue Solution, 2.56 U Bst 2.0 Hot Start DNA Polymerase, 430 copies / mu L Plasmid template containing SA signature sequence; if there are N reaction chambers on the chip, configure 8×N mu L reaction solution.

[0070] 2. Chip degassing

[0071] Use scotch tape to seal the injection port of the prepared chip; place the chip in a vacuum desiccator, and degas it under 10kPa pressure for 1 hour.

[0072] 3. Self-priming sample injection

[0073] Use a pipette to draw the LAMP reaction solution, and insert the tip with the react...

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Abstract

The invention discloses a self-suction type multichannel pathogenic bacterium detection micro-fluidic chip based on LAMP (loop-mediated isothermal amplification). The self-suction type multichannel pathogenic bacterium detection micro-fluidic chip comprises a sample injection layer, a reaction layer and a support layer, wherein the sample injection layer comprises a first sample injection layer, an anti-evaporation membrane and a second sample injection layer; a sample injection opening is formed in the center of the sample injection layer; the second sample injection layer is provided with aplurality of sample injection channels; a plurality of reaction chambers are arranged in the reaction layer; round filter paper sheets are arranged in the reaction chambers; the first sample injectionlayer, the second sample injection layer and the reaction layer are made of PDMS materials. The invention also discloses application of the micro-fluidic chip in the aspect of pathogenic bacterium detection. The self-suction type multichannel pathogenic bacterium detection micro-fluidic chip has the advantages that the micro-fluidic chip technology and the LAMP technology are combined; the defectthat it is difficult for the traditional LAMP reaction to perform simultaneous detection of multiple target objects is overcome; the characteristic of high gas solubility of the PDMS materials is utilized, so that the automatic sample injection without the help of any instrument is realized, the operation errors caused by manual sample injection is avoided and the operation steps of the chip aresimplified; the simultaneous detection of multiple pathogenic bacteria is realized.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and in particular relates to a LAMP-based self-priming multi-channel pathogen detection microfluidic chip and a detection method. Background technique [0002] Pathogenic bacterial diseases are always threatening human health. Realizing efficient and accurate detection of pathogenic bacteria is the key to controlling such diseases. [0003] At present, the most commonly used detection methods in China and even in the world are plate culture method or polymerase chain reaction (Polymerase Chain Reaction, PCR). Point of care testing). Loop-mediated isothermal amplification (LAMP) is a nucleic acid isothermal amplification technology that has emerged in recent years. LAMP enables rapid exponential amplification of target sequences without the thermal cycling of traditional PCR methods. Compared with conventional PCR, thermal denaturation of the template, temperature cycling, electrophoresi...

Claims

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Application Information

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IPC IPC(8): C12M1/00C12Q1/6844C12Q1/689C12Q1/14C12Q1/04C12R1/445C12R1/63
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2565/629C12Q2563/107
Inventor 李娟庞博赵超牟颖徐坤宋秀玲王娟
Owner JILIN UNIV
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