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Continuously mobile nest type PCR (polymerase chain reaction) microfluidic method

A technology of microfluidic control and constant temperature zone, which is applied in the field of microfluidic PCR analysis and detection, can solve the problems of limited timeliness, time-consuming increase, etc., and achieve the effect of high-sensitivity detection

Inactive Publication Date: 2012-07-25
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, because nested PCR includes two rounds of PCR amplification, it takes twice as much time compared to conventional PCR, especially nested PCR usually takes 2.5-4 hours to complete on a traditional thermal cycler, resulting in time-sensitive detection. Sex is extremely restricted

Method used

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  • Continuously mobile nest type PCR (polymerase chain reaction) microfluidic method

Examples

Experimental program
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Effect test

Embodiment 1

[0061] The continuous flow nested PCR microfluidic method of the present invention, the specific steps are as follows:

[0062] (1) Extract the genomic DNA of the sample to be tested.

[0063] (2) Primer design: Design nested primers consisting of two pairs of primers for the specific gene DNA sequence of the target detection substance.

[0064] (3) Preparation of PCR mixture: Prepare 25 μL premixed system containing 1× SybrGreen I fluorescent dye outer primer and inner primer PCR respectively, and the concentration of the outer primer is 50% to 75% of the primer concentration of the general PCR system. The concentration of internal primers in the PCR premix system containing internal primers is 75% to 100% of that of the general PCR system.

[0065] (4) Add the DNA sample of the analyte to the PCR premix system containing the outer primers, and introduce the PCR reaction solution from the inlet end of the capillary 3 into the capillary microchannel on the continuous flow nes...

Embodiment 2

[0084] Continuous Flow Nested PCR Microfluidic Method Amplification Cycle Number Test

[0085] 1. PCR reaction system: Taking the detection of Listeria monocytogenes as an example, the concentration of the DNA sample to be tested is 5×10 6 copies / μL, the PCR reaction primers and reaction system were the same as in Example 1, and the negative control used sterilized water instead of the template.

[0086] 2. PCR amplification reaction: set the number of cycles of the first round of PCR amplification reaction to 20, 25, 30, and 35 respectively. Other reaction conditions and reaction steps are the same as in Example 1, and different cycle numbers are tested. For the impact on the PCR reaction results, see the experimental results Figure 5 .

[0087] Depend on Figure 5 It can be seen that within a certain range, increasing the cycle number of PCR reaction increases the fluorescence intensity of PCR products. Therefore, in order to obtain the desired amount of PCR amplificat...

Embodiment 3

[0089] Continuous Flow Nest PC R Amplification time test of microfluidic method

[0090] 1. PCR reaction system: Taking the detection of Listeria monocytogenes as an example, the concentration of the DNA sample to be tested is 5×10 6 copies / μL, the PCR reaction primers and reaction system were the same as in Example 1, and the negative control used sterilized water instead of the template.

[0091] 2. PCR amplification reaction: Set the linear flow rate of the first round of PCR amplification reaction to:

[0092] 2 mm / s (the circulation time in the three constant temperature zones of melting zone, annealing zone and elongation zone is about 10 s, 10 s and 22 s respectively);

[0093] 3.5 mm / s (the circulation time in the three constant temperature zones is about 6 s, 6 s and 14 s respectively);

[0094] 5 mm / s (the circulation time in the three constant temperature zones is about 4 s, 4 s and 10 s respectively);

[0095] 7.5 mm / s (the circulation time in the three cons...

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Abstract

The invention discloses a continuously mobile nest type PCR (polymerase chain reaction) microfluidic method. A nest type primer consisting of two pairs of primers is designed according to a DNA (deoxyribonucleic acid) sequence of a target detected object, after DNA of a to-be-detected sample is added into a PCR reaction system containing an outer primer the DNA of the to-be-detected sample and the PCR reaction system are mixed uniformly, the mixture is injected into a continuously mobile nest type PCR microfluidic device, PCR reaction liquid completes 20-35 PCR expansion circulations along capillary tubes spirally wound on three independent temperature-control constant-temperature copper cylinders, and fluorescence detection and analysis systems at the tail ends of the capillary tubes are used for acquiring and analyzing fluorescent images of expanded products. The expanded products are added into a PCR reaction system containing an inner primer, so that nest type PCR second-step expansion and product detection is completed as the above. By means of analyzing the intensity of the fluorescent images of the expanded products in two turns, whether the to-be-detected sample has the target detected object or not and the relative quantity of the target detected object can be determined. The continuously mobile nest type PCR microfluidic method can realize low-cost, high-sensitivity and fast detection for low-abundance DNA samples, detection limit can achieve 1 copy number / total volume, and consumed time is only 35-45min.

Description

technical field [0001] The invention belongs to the technical field of microfluidic PCR analysis and detection, in particular to a continuous flow nested PCR microfluidic method. Background technique [0002] Since Manz and Widmer first proposed the concept of miniature total analysis system in the early 1990s, microfluidic technology, as its core technology, has developed into one of the most cutting-edge scientific and technological fields in the world. At present, the hotspots and key application areas of micro-analysis systems are mainly concentrated in the field of life sciences. The advantages of microfluidic technology in promoting the miniaturization, integration, automation and portability of analytical instruments make it an important tool in biomedicine and high-throughput drug synthesis. Screening, environmental monitoring and protection, health quarantine, health care diagnosis, forensic identification, detection of biological and chemical warfare agents and man...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 章春笋邢达舒博文
Owner SOUTH CHINA NORMAL UNIVERSITY
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