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Ring mediated isothermal amplification microfluidic chip for detectinganimal and plant DNA virus and application of ring mediated isothermal amplification microfluidic chip

A microfluidic chip, ring-mediated isothermal technology, applied in the direction of microorganism-based methods, specific-purpose bioreactor/fermenter, bioreactor/fermenter combination, etc., can solve the problem of cumbersome detection steps and multiple toxic reagents to avoid workload and sample contamination, improve sensitivity, and improve detection efficiency

Pending Publication Date: 2019-11-12
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention aims to solve the current technical problems of cumbersome detection steps and many toxic reagents for animal and plant DNA viruses, and provides a ring-mediated isothermal amplification microfluidic chip, which can be used to detect DNA viruses, which can save conventional DNA virus detection Many steps (including DNA extraction, polymerase chain reaction, product electrophoresis, result display, etc.), so that sample addition, amplification, and result characterization are all placed on a microfluidic chip, which has the advantages of miniaturization, integration, detection Advantages of speed

Method used

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  • Ring mediated isothermal amplification microfluidic chip for detectinganimal and plant DNA virus and application of ring mediated isothermal amplification microfluidic chip
  • Ring mediated isothermal amplification microfluidic chip for detectinganimal and plant DNA virus and application of ring mediated isothermal amplification microfluidic chip

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Detection of African Swine Fever Virus

[0032] 1) Adding samples: Take clinical samples from sows with reproductive disorders or piglets with symptoms of respiratory disorders and aborted fetuses, separate serum, take 50 microliters of sample serum, put it in a 1.5ml centrifuge tube, add 200 microliters of sterilized of ultrapure water, smashed with a glass rod and mixed evenly. Draw 50 microliters of the sample mixture with a pipette gun, pierce the tip of the pipette into the sealing tape of the sample hole, and inject the mixture into the reaction channel as the reaction solution.

[0033] 2) Amplification: After the reaction solution flows into the reaction chamber and is evenly mixed with the powder of the LAMP kit, the semiconductor heating component is used to heat at a constant temperature of 65°C under the reaction chamber, and the reaction product is amplified for 20 minutes. The reaction system refers to the method of using the general-purpose LAMP kit. The...

Embodiment 2

[0041] Detection of Sugarcane Baculovirus

[0042] 1) Adding samples: Select suspected susceptible sugarcane plants, take the diseased leaves (about 1 mm × 1 mm), put them in a 1.5 ml centrifuge tube, add 200 microliters of sterilized ultrapure water, mash and mix with a glass rod uniform. Draw 50 microliters of the sample mixture with a pipette gun, pierce the tip of the pipette into the sealing tape of the sample hole, and inject the mixture into the reaction channel as the reaction solution.

[0043] 2) Amplification: After the reaction solution flows into the reaction chamber and is evenly mixed with the powder of the LAMP kit, the semiconductor heating component is used to heat at a constant temperature of 65°C under the reaction chamber, and the reaction product is amplified for 20 minutes. The reaction system refers to the method of using the general-purpose LAMP kit. The LAMP kit contains 25 microliters of 2×LAMP Mix, FIP primer (final concentration of 2 micromole per...

Embodiment 3

[0051] Detection of Staphylococcus aureus

[0052] 1) Adding samples: select foie gras and intestines, grind them thoroughly after mixing, add 10 times of PBS diluent, mix thoroughly, use a pipette gun to absorb 50 microliters of the mixed solution, and pierce the tip of the pipette into the sealing tape of the sample hole, The mixed liquid is injected into the reaction channel as the reaction liquid. All reagents and instruments are sterilized before use to prevent bacterial contamination.

[0053] 2) Amplification: After the reaction liquid flows into the reaction chamber and mixes evenly with the powder of the LAMP kit, use a semiconductor heating component under the reaction chamber to heat at 62°C for 45 minutes at a constant temperature to amplify the reactant. The LAMP kit contains 5 microliters of 10×Bst buffer, MgSO 4 (25 mmol per liter) 8 microliters, dNTP (10 mmol per liter) 8 microliters, betaine (5 moles per liter) 8 microliters, inner primer (20 micromol per li...

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Abstract

The invention discloses a ring mediated isothermal amplification microfluidic chip for detectinganimal and plant DNA virus. The microfluidic chip comprises a double-layer polydimethylsiloxane (PDMS) plate with upper and lower layers of PDMS plates, the upper PDMS plate is provided with three penetrating holes which are a sample adding hole, a blocking hole and a venthole sequentially; and the lower layer PDMS plate engraved with a channel for the flow of reaction liquid, a reaction cavity for isothermal amplification and a channel for the display of results which are corresponding to the threepenetrating holes of the upper layer PDMS plate. The microfluidic chip is used for detecting the animal and plant DNA virus, DNA does not need to be extracted, other instruments are not relied on, the detection speed is high, specificity is high, operation is simple and convenient, and the detection results are directly observed with naked eyes.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a loop-mediated isothermal amplification microfluidic chip for detecting animal and plant DNA viruses and its application. Background technique [0002] Animal and plant viruses are a class of intracellular parasitic pathogens with infective activity. It brings tens of billions of dollars of losses to the global agricultural and animal husbandry economy every year, and seriously hinders the healthy, rapid and sustainable development of agriculture and animal husbandry. In the current production, the rapid and accurate detection and diagnosis of viral diseases caused by animal and plant-related materials is of great significance. However, the commonly used methods for animal and plant virus detection mainly include biological methods, immunological methods, electron microscopy methods and molecular biological methods. Among them, the biological method is more...

Claims

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Application Information

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IPC IPC(8): C12M1/34C12M1/00C12Q1/70C12Q1/689C12Q1/6844C12Q1/14C12R1/94C12R1/93C12R1/445
CPCC12Q1/701C12Q1/689C12Q1/6844C12Q2565/629C12Q2531/119
Inventor 谢雨彤李涛常荣山刘玲俐赵亚楠
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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