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Self-driven microfluidic detection chip and preparation method and application thereof

A detection chip and microfluidic technology, applied in the field of microbial detection, can solve the problems of puncture, small amount of sample detection at one time, high detection cost, etc., and achieve the effect of preventing nucleic acid cross-contamination, aerosol pollution, and external pollution

Active Publication Date: 2019-05-10
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practical application, there are certain deficiencies: (1) problems such as aerosol pollution, detection specificity and sensitivity are prominent; (2) based on complex primer design, it is difficult to detect multiple target genes; (3) one sample The amount of testing is small, and the testing cost is too high
However, its microfluidic chip must maintain a negative pressure environment in the fluid channel, and the sealing membrane is pierced during use, which increases the difficulty of chip preparation, storage and use.

Method used

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  • Self-driven microfluidic detection chip and preparation method and application thereof
  • Self-driven microfluidic detection chip and preparation method and application thereof
  • Self-driven microfluidic detection chip and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Such as figure 1 , 2 As shown, the self-driven microfluidic detection chip of this embodiment includes several detection units, and each detection unit includes an upper chip layer and a lower chip layer arranged in sequence:

[0060] The upper layer of the chip is provided with a sampling pool 1 and an air vent 2, and the sampling pool 1 adopts an inverted tapered design, and the sampling pool 1 and the air vent 2 all run through the upper layer of the chip; the lower layer of the chip is provided with a fluid channel 4, The detection pool 5 and the waste liquid pool 3; the fluid channel 4 is respectively connected to the waste liquid pool 3 and the detection pool 5, the upper layer of the combined chip and the lower layer of the chip, and the sampling pool 1 is connected to the detection pool 5 and the waste liquid pool through the fluid channel 4 3 connected, the waste liquid pool 3 communicates with the exhaust hole 2, and the surface of the fluid channel 4 is prov...

Embodiment 2

[0078] Step 1. Specificity analysis of target gene detection

[0079] The prawn tissues infected with WSSV, IHHNV, SHIV, AHPND, EHP, IMNV, CMNV, TSV, YHV-1 and YHV-8 preserved in the laboratory were selected as the samples to be tested, and the nucleic acid targeted release agent (volume ratio of isothiocyanate Guanidinium acid salt: polyacrylamide: BSA: gelatin = 6:2:1:1) quickly extract sample nucleic acid as a detection template, and operate according to the instruction manual.

[0080] Take the self-driven microfluidic chip prepared in Example 1, add 6 groups of sample detection units in sequence: DNA containing equal proportions of infected WSSV, IHHNV, SHIV, AHPND, EHP shrimp tissues (final concentration is 10ng / μL ); containing equal proportions of RNA infected with IMNV, CMNV, TSV, YHV-1, and YHV-8 shrimp tissues (final concentration: 10 ng / μL); all repeated 3 times. Take SPF Litopenaeus vannamei tissue nucleic acid (final concentration 10ng / μL) and add it to the nega...

Embodiment 3

[0087] Step 1. Extraction of sample nucleic acid:

[0088] 1) The Marine Aquaculture Biological Disease Control and Molecular Pathology Laboratory of the Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences collected 6 samples of diseased prawns from different regions across the country, and the prawn tissues were cut into 0.5×0.5cm 2 Tissue pieces of different sizes were put into correspondingly numbered 1.5mL EP tubes and kept on ice. Add 150 μL of water to the sample tube, immerse the tissue sample, homogenize for 1-3 minutes, until no tissue remains, and let stand at room temperature for 5 minutes.

[0089] 2) Prepare nucleic acid targeted release agent, the volume ratio is guanidinium isothiocyanate:polyacrylamide:BSA:gelatin=6:2:1:1.

[0090] Add 5 μL nucleic acid targeted release agent to a 0.2mL PCR tube, add 20 μL of the above-mentioned tissue grinding supernatant and mix well; place in a metal bath, keep warm at 99°C for 10 minutes; tak...

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Abstract

The invention belongs to the field of microorganism detection and particularly relates to a self-driven microfluidic detection chip and a preparation method and application thereof. The self-driven microfluidic detection chip comprises an upper chip layer and a lower chip layer which are sequentially arranged; the upper chip layer is provided with a sample introduction pool and an exhaust hole, the upper chip layer is provided with a fluid channel which is communicated with a detection pool and a waste liquid pool, and the surface of the fluid channel is modified by a hydrophilic material to achieve liquid flow self-driving. The invention also discloses the application of the self-driven microfluidic detection chip in shrimp pathogen detection. The chip is high in automation degree, operators can rapidly, sensitively and microscopically detect multi-purpose nucleic acids only by single-time sample addition, the consumption of detection reagents is greatly reduced, and the production cost is saved.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and in particular relates to a self-driven microfluidic detection chip and a preparation method and application thereof. Background technique [0002] In recent years, the development of shrimp farming is mainly manifested in the substantial expansion of the scale, the continuous innovation of the model and the rapid growth of the speed, all of which have brought great economic benefits to the whole society, but at the same time, due to the lack of healthy and sustainable farming The concept of biosecurity, the emergence of new pathogens and mutant strains of epidemics frequently, the abuse of drugs lead to the increase of microbial resistance, the destruction of ecological diversity, and the food safety problems are becoming more and more serious. Therefore, it is an inevitable trend to adjust the healthy development of my country's shrimp farming industry under the guidance of biosecurity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/34C12M1/00
Inventor 曹志黄倢董宣万晓媛李晨
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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