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Rapid PCR amplification and CRISPR visual detection device and method

A detection device and fast technology, applied in the field of nucleic acid amplification and visual detection, can solve the problems of inactivation, complicated and cumbersome operation, large amplicon yield, etc., and achieve the effects of shortened detection time, accurate test results, and convenient detection

Pending Publication Date: 2021-01-01
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the detection sensitivity of the CRISPR method alone is not high, it is usually combined with nucleic acid amplification
However, because the CRISPR system is not compatible with the nucleic acid amplification system or the reaction temperature is different from the nucleic acid amplification system, the cleavage efficiency of the CRISPR / Cas enzyme is greatly reduced or inactivated. Therefore, researchers usually divide nucleic acid amplification and CRISPR detection into two steps. After the nucleic acid amplification is completed, the amplification product is transferred to the CRISPR system for detection
The operation is complicated and cumbersome, and due to the large output of amplicons, opening the cover after the amplification is completed can easily cause the problem of amplicon aerosol pollution, resulting in false positives in subsequent detection results, making the detection results unreliable

Method used

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  • Rapid PCR amplification and CRISPR visual detection device and method
  • Rapid PCR amplification and CRISPR visual detection device and method
  • Rapid PCR amplification and CRISPR visual detection device and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] refer to figure 1 , figure 2 , image 3 , Figure 4 , Figure 5 , the illustration is only a schematic representation and does not represent the actual size.

[0104]The PCR amplification and CRISPR visual detection device includes: an airtight module 1 and a heating module 2. The airtight module 1 includes: a lower carrier 11, an upper cover 12, a plurality of PCR amplification chambers 13, a plurality of CRISPR reaction chambers 14 corresponding to the number of a plurality of PCR amplification chambers 13, and a plurality of PCR amplification chambers The connecting channel 15 that communicates between the chamber 13 and each CRISPR reaction chamber 14. That is, one PCR amplification chamber 13 corresponds to one CRISPR reaction chamber 14, and the two chambers are communicated through a connecting channel 15. The function of the connecting channel is to block the PCR amplification chamber 13 and the CRISPR reaction without external force. The substances in th...

Embodiment 2

[0116] The schematic diagram of airtight module 1 of embodiment 2 still refers to figure 1 , figure 2 As shown, the difference from Example 1 is that the depth of the PCR amplification chamber 13 is 2 mm, and the cross-sectional diameter is 10 mm; the depth of the CRISPR reaction chamber 14 is 3 mm, and the cross-sectional diameter is 10 mm. The connecting channel is a cuboid with a width of 0.6 mm. In this embodiment, the upper cover 12 adopts a transparent hard high-temperature resistant cover, and an external thread is provided at the connection between the cover and the heating module.

[0117] refer to Image 6 As shown, the heating module 2 includes a CRISPR heating module 21 and two PCR heating modules, which are respectively a denaturing PCR heating module 22 and an annealing and extending PCR heating module 25, wherein the CRISPR heating module 21 is circular, and the denaturing PCR heating module The module 22 and the annealing and extension PCR heating module 25...

Embodiment 3

[0127] The difference between embodiment 3 and embodiment 1 is that, as Figure 7 , Figure 8 As shown, each of the PCR amplification chambers 13 is arranged near the center of the disc-shaped carrier and evenly arranged on the same circumference, the depth of the PCR amplification chamber 13 is 5mm, and the cross-sectional diameter is 10mm; The CRISPR reaction chamber 14 is arranged close to the edge of the disc-shaped carrier and evenly arranged on the same circumference. The depth of the CRISPR reaction chamber 14 is 5 mm, and the cross-sectional diameter is 10 mm. In practical application, after adding samples to the PCR amplification chamber 13 and the CRISPR reaction chamber 14, the upper cover 12 is used to seal the two chambers and the connecting channel 15. The connecting channel is a cuboid with a width of 1 mm. In this embodiment, the upper cover body 12 adopts a polystyrene sealing film.

[0128] Such as Figure 9 , Figure 10 , Figure 11 As shown, the heatin...

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Abstract

The invention relates to a rapid PCR amplification and CRISPR visual detection device and method. The visual detection device comprises a closed module and a heating module, wherein the closed modulecomprises a lower carrier, an upper cover body, a plurality of PCR amplification chambers arranged in the lower carrier and a plurality of CRISPR reaction chambers of the corresponding number; the amplification chambers are communicated with the corresponding CRISPR reaction chambers through connecting channels; the upper cover body is connected with the lower carrier in a sealed manner and all the chambers and the connecting channels are enabled to be sealed; and the heating module is used for heating the PCR amplification chambers and / or the CRISPR reaction chambers. Compared with the priorart, the device has the advantages that the specific surface area is large, and the heat transfer efficiency is improved, so that the PCR amplification time is greatly shortened, and the total detection time is further shortened to be within a few minutes. According to the invention, amplification of a plurality of PCR systems and CRISPR visual detection can be realized, and the temperature-controllable heating module is integrated in the detection device, so that the detection is more convenient, and the detection time is shortened.

Description

technical field [0001] The invention belongs to the field of nucleic acid amplification and visual detection, and in particular relates to a rapid PCR amplification and CRISPR visual detection device and detection method. Background technique [0002] Since the birth of polymerase chain reaction (PCR) technology in 1985, nucleic acid amplification detection technology has been rapidly applied in various fields such as medical treatment, agriculture, and environmental sanitation due to its high detection sensitivity. For the detection of nucleic acid amplification products, the visual detection method has great advantages in the field and rapid detection of nucleic acids because it does not require equipment and can interpret the results with naked eyes. Existing visual detection methods usually use high-concentration fluorescent intercalating dyes (SYTO 9, SYBR GREEN I, ethidium bromide, propidium iodide, etc.) Fluorescence to achieve visual detection; or use calcein, methy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/38C12M1/34C12M1/02C12M1/00C12Q1/686
CPCB01L7/525C12Q1/686C12Q2525/161C12Q2521/327C12Q2563/107
Inventor 李富友王瑞徐明
Owner FUDAN UNIV
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