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Method for carrying out nucleic acid isothermal amplification by using paper-based microfluid

A paper-based microfluidics and isothermal amplification technology, applied in the field of microfluidics, can solve the problems of complex process, cumbersome process, and the cost cannot be disposable, and achieve the effect of simplifying the preparation process, fast analysis, and light weight.

Active Publication Date: 2015-01-21
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, microfluidic devices are generally realized by semiconductor or integrated circuit processing mode, the process is cumbersome and complicated, and the cost is still far from reaching the goal of disposable

Method used

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  • Method for carrying out nucleic acid isothermal amplification by using paper-based microfluid
  • Method for carrying out nucleic acid isothermal amplification by using paper-based microfluid
  • Method for carrying out nucleic acid isothermal amplification by using paper-based microfluid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Loop-mediated isothermal amplification detection of seven bacteria simultaneously using paper microfluidics

[0027] Use paper microfluidics to simultaneously detect whether the sample contains the following seven bacteria: Pseudomonas aeruginosa, Vibrio parahaemolyticus, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae , Acinetobacter baumannii, using loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP), according to the following steps:

[0028] 1. The DNA solution of the extracted sample is ready for use, and the nucleic acid is quickly extracted by boiling.

[0029] 2. Precipitate the LAMP primers of seven kinds of bacteria on the seven reaction zones 22 of paper microfluidics respectively. The genes detected by different bacteria and the primers used are shown in Table 1: through biotinylated primers and avidinized microcoagulation The gel was coupled, dried at room temperature and ...

Embodiment 2

[0040] Example 2 Loop-mediated isothermal amplification detection of seven bacteria simultaneously using paper microfluidics heated by resistance wire

[0041] Others are the same as in Example 1, except that a heating layer 3 is pasted under the reaction layer 2 through a double-sided adhesive layer 4 without any patterns and holes. The heating layer 3 is made of filter paper, and its lower surface is provided with a resistance wire 31, preferably the resistance wire 31 is formed by spraying and depositing metal powder, and the resistance wire 31 and the variable resistor 5 are connected in series with a power supply and a switch through a wire. The magnitude of the current is changed through the adjustment of the variable resistor 5 , so as to regulate the temperature of the heating layer 3 . The metal indicator used is not HNB but orange-yellow Calcein.

[0042] When in use, drop the sample to be tested mixed with the metal indicator Calcein into the sample injection hole ...

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Abstract

The invention discloses a method for carrying out nucleic acid isothermal amplification by using paper-based microfluid. The method comprises the following steps: (1) preparing a sample DNA solution; (2) coating a primer on a reaction zone of paper-based microfluid, wherein the microfluid comprises a reaction layer made of filter paper and at least one sampling layer; sampling holes are formed at the centers of the sampling layers; a plurality of reaction holes are evenly distributed around the sampling holes; each orifice is coated by a hydrophobic material; the sampling layers are stacked above the reaction layer; the reaction layer is provided with a sample zone, a plurality of reaction zones and reaction channels; the sample zone is drawn by the hydrophobic material and corresponds to the sampling hole; the plurality of reaction zones and reaction channels correspond to the reaction holes; the reaction zones are the same as the reaction holes in diameter; the diameter of the sample zone is smaller than those of the sampling holes; and the reaction zones are communicated with the sample zones through the reaction channels of which the widths are gradually reduced from the reaction zones to the sample zones; (3) dropwise adding the reaction liquid into the sampling holes, heating the microfluid to reach amplification temperature; and (4) observing the color change of the reaction zones, so as to obtain the detection result. The method is low in cost and accurate in result.

Description

technical field [0001] The invention relates to the field of microfluid technology, in particular to a method for nucleic acid isothermal amplification using paper microfluid and a device for implementing the method. Background technique [0002] Nucleic acid amplification technology represented by PCR has been widely used in scientific research and clinical work. Based on the rapid development of molecular biology, many new nucleic acid isothermal amplification methods have been developed, such as: nucleic acid sequence-dependent amplification method (NASBA), self-sequence replication (3SR), loop-mediated isothermal amplification (LAMP) , Strand Displacement Amplification (SDA), etc. However, traditional PCR amplification requires expensive specialized amplification equipment, and operators also need professional training, which results in high testing costs. Due to the large size and heavy weight of the amplification equipment, the detection site cannot be moved flexibly....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2563/131C12Q2565/513
Inventor 王欢府伟灵黄庆王云霞张立群黄君富
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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