CN-120349875-B - Nucleic acid detection method based on gel self-interference and microfluidic detection platform

CN120349875BCN 120349875 BCN120349875 BCN 120349875BCN-120349875-B

Abstract

The application provides a nucleic acid detection method based on gel self-interference and a microfluidic detection platform. The method comprises the steps of mixing a sample to be detected with a lysis solution, magnetic beads, a magnetic bead cleaning solution and a nucleic acid eluent in sequence through a digital microfluidic chip after the sample to be detected is added, extracting sample nucleic acid droplets, mixing the sample nucleic acid droplets with a first round of RPA reagent and a second round of RPA reagent through the digital microfluidic chip to carry out two rounds of RPA amplification to obtain sample product droplets, mixing the sample product droplets with a CRISPR reagent through the digital microfluidic chip, moving the sample product droplets to a solid phase gel position to carry out sequence identification and cutting reaction to obtain solid phase gel after the cutting reaction, carrying out self-interference signal detection on the solid phase gel in the solution after the cutting reaction through an optical detection system, and outputting a nucleic acid detection result whether the sample to be detected contains a nucleic acid sequence to be detected. The method realizes high-specificity and full-automatic non-labeled PRA detection.

Inventors

FU RONGXIN
ZHAO QINGCHEN
AI XIN
ZHANG HANZHI
ZHOU TIANQI
ZHANG SHUAILONG
LI HANG
HU HANQI
LIU HAOBING
ZHAO TAO

Assignees

北京理工大学

Dates

Publication Date: 20260508
Application Date: 20250428

Claims (7)

1. The method is characterized in that the method is applied to a microfluidic detection platform, the microfluidic detection platform comprises a digital microfluidic chip and an optical detection system, the digital microfluidic chip comprises an upper chip and a lower chip, solid-phase gel is embedded in the upper chip, the lower chip comprises a sample adding electrode, a waste liquid storage electrode, an electrode array and a plurality of liquid storage electrodes, the liquid storage electrodes are respectively used for storing lysate, magnetic beads, magnetic bead cleaning liquid, nucleic acid eluent, a first round of RPA reagent, a second round of RPA reagent and CRISPR reagent, the sample adding electrode is used for storing a sample to be detected, the thickness of a dielectric layer of the digital microfluidic chip is 5 mu m, the electrode array is designed to be 4 multiplied by 16, and the digital microfluidic chip is placed on a stage of the optical detection system, and the method comprises the following steps: After a sample to be detected is added into the lower chip, the sample to be detected is sequentially mixed with the lysis solution, the magnetic beads, the magnetic bead cleaning solution and the nucleic acid eluent through the digital microfluidic chip so as to extract sample nucleic acid droplets from the sample to be detected; Mixing the sample nucleic acid droplets with the first round of RPA reagent and the second round of RPA reagent through the digital microfluidic chip to perform two rounds of RPA amplification to obtain sample product droplets containing two rounds of nucleic acid amplification products; The method for performing two rounds of RPA amplification by mixing the sample nucleic acid droplets with the first round of RPA reagent and the second round of RPA reagent through the digital microfluidic chip to obtain sample product droplets containing two rounds of nucleic acid amplification products comprises the following steps: Mixing the first round of RPA reagents with the sample nucleic acid droplets through the electrode array to obtain a first round of amplification products; Mixing the second round of RPA reagents with the first round of amplification products via the electrode array to obtain the sample product droplets comprising two rounds of nucleic acid amplification products; Mixing the sample product liquid drops with the CRISPR reagent through the digital microfluidic chip, and moving to the solid phase gel to perform sequence recognition and cutting reaction to obtain solid phase gel after the cutting reaction; Detecting a self-interference signal of solid-phase gel in the solution after the cleavage reaction by the optical detection system, and determining and outputting a nucleic acid detection result of whether the sample to be detected contains a nucleic acid sequence to be detected; the optical detection system is used for detecting self-interference signals of solid phase gel in the solution after the cleavage reaction, determining and outputting a nucleic acid detection result of whether the sample to be detected contains a nucleic acid sequence to be detected, and the method comprises the following steps: The optical detection system is used for detecting self-interference signals of solid-phase gel in the solution after the cutting reaction to obtain target interference signals; if the phase of the target interference signal changes, determining that the solid phase gel changes in thickness through the optical detection system; And if the thickness of the solid phase gel changes, outputting the nucleic acid detection result containing the nucleic acid sequence to be detected in the sample to be detected through the optical detection system.
2. The method according to claim 1, wherein the detecting the self-interference signal of the solid phase gel in the solution after the cleavage reaction by the optical detection system to obtain the target interference signal comprises: The optical detection system is used for detecting self-interference signals of solid-phase gel in the solution after the cutting reaction so as to obtain synthetic reflection signals; and performing fast Fourier transform on the synthesized reflected signals through the optical detection system, and separating the target interference signals from the synthesized reflected signals in the microfluidic chip.
3. The method according to claim 1, wherein the method further comprises: If the sample to be detected contains the nucleic acid sequence to be detected, analyzing a phase change value of the target interference signal through the optical detection system; And determining and outputting the nucleic acid concentration of the nucleic acid sequence to be detected based on the phase change value through the optical detection system.
4. The method of claim 1, wherein the step of determining the position of the substrate comprises, The step of sequentially mixing the sample to be detected with the lysis solution, the magnetic beads, the magnetic bead cleaning solution and the nucleic acid eluent through the digital microfluidic chip to extract sample nucleic acid droplets from the sample to be detected comprises the following steps: Mixing the sample to be tested and the lysate with magnetic beads through the electrode array to release sample nucleic acid in the sample to be tested, wherein the sample nucleic acid is enriched on the magnetic beads; Mixing the magnetic beads with the magnetic bead cleaning liquid through the electrode array to clean the magnetic beads; mixing the cleaned magnetic beads with the nucleic acid eluent through the electrode array to obtain sample nucleic acid droplets;
5. The microfluidic detection platform is characterized by comprising a digital microfluidic chip and an optical detection system, wherein the digital microfluidic chip comprises an upper chip and a lower chip, solid-phase gel is embedded in the upper chip, the lower chip comprises a sample adding electrode, a waste liquid storage electrode, an electrode array and a plurality of liquid storage electrodes, the plurality of liquid storage electrodes are respectively used for storing lysate, magnetic beads, magnetic bead cleaning liquid, nucleic acid eluent, a first round of RPA reagent, a second round of RPA reagent and CRISPR reagent, the sample adding electrode is used for storing a sample to be detected, the thickness of a dielectric layer of the digital microfluidic chip is 5 mu m, the electrode array is designed to be 4 multiplied by 16, and the digital microfluidic chip is placed on a stage of the optical detection system; The digital microfluidic chip is used for sequentially mixing the sample to be detected with the lysate, the magnetic beads, the magnetic bead cleaning solution and the nucleic acid eluent after the sample to be detected is added into the lower chip so as to extract sample nucleic acid droplets from the sample to be detected; the digital microfluidic chip is also used for mixing the sample nucleic acid droplets with the first round of RPA reagent and the second round of RPA reagent for two rounds of RPA amplification to obtain sample product droplets containing two rounds of nucleic acid amplification products; The digital microfluidic chip is further used for mixing the sample product liquid drops with the CRISPR reagent, moving to the solid phase gel to perform sequence recognition and cutting reaction, and obtaining solid phase gel after the cutting reaction; The digital microfluidic chip is specifically used for mixing the first round of RPA reagent with the sample nucleic acid liquid drops through the electrode array to obtain a first round of amplification product; Mixing the second round of RPA reagents with the first round of amplification products via the electrode array to obtain the sample product droplets comprising two rounds of nucleic acid amplification products; the optical detection system is used for detecting the self-interference signals of the solid phase gel after the cutting reaction, and determining and outputting a nucleic acid detection result of whether the sample to be detected contains a nucleic acid sequence to be detected; The optical detection system is specifically used for detecting a self-interference signal of solid-phase gel in the solution after the cutting reaction to obtain a target interference signal, determining that the solid-phase gel has thickness change if the phase of the target interference signal changes, and outputting the nucleic acid detection result containing the nucleic acid sequence to be detected in the sample to be detected if the solid-phase gel has thickness change.
6. A nucleic acid detecting apparatus comprising: processor, and A memory in which a program is stored, Wherein the program comprises instructions which, when executed by the processor, cause the processor to perform the gel self-interference based nucleic acid detection method according to any one of claims 1-4.
7. A non-transitory computer-readable storage medium storing computer instructions for causing the computer to perform the gel self-interference-based nucleic acid detection method according to any one of claims 1-4.

Description

Nucleic acid detection method based on gel self-interference and microfluidic detection platform Technical Field The application relates to the technical field of nucleic acid detection, in particular to a gel self-interference-based nucleic acid detection method and a microfluidic detection platform. Background Nucleic acid detection is a method by which a biological sample (e.g., a blood sample) is analyzed for the presence of a specific nucleic acid sequence with a detection object under laboratory conditions. The current mainstream RPA nucleic acid detection technology mainly adopts fluorescent probes or lateral flow test paper to characterize amplicons, for example, exo fluorescent probes are adopted to detect the amplicons, the lowest detectable concentration of 7.74 copies of RNA molecules in 1 microliter of template solution, or RPA amplified products with FAM and biotin labels are combined on FAM-resistant gold particles, and biotin ligands on the lateral flow test paper and the FAM-resistant probes are utilized to detect the amplified products, wherein the detection limit is 1.0 copies/microliter. However, in the existing RPA nucleic acid detection technology, fluorescent or other markers are mostly used for reading out amplicons, so that the detection cost and the complexity of a detection flow are increased, and the detection of non-labeled RPA amplicons with high integration level is to be developed. Disclosure of Invention The application provides a nucleic acid detection method based on gel self-interference and a microfluidic detection platform, which realize a high-specificity and full-automatic universal method for non-labeled PRA detection. The technical scheme is as follows: According to one aspect of the present application, there is provided a gel self-interference-based nucleic acid detection method applied to a microfluidic detection platform including a digital microfluidic chip and an optical detection system, the digital microfluidic chip including an upper chip and a lower chip, a solid phase gel being embedded in the upper chip, a lysate and magnetic beads, a magnetic bead cleaning solution, a nucleic acid eluent, a first round of RPA reagent, a second round of RPA reagent and a CRISPR reagent being stored in the lower chip, the digital microfluidic chip being placed on a stage of the optical detection system, the method comprising: After a sample to be detected is added into the lower chip, the sample to be detected is sequentially mixed with the lysis solution, the magnetic beads, the magnetic bead cleaning solution and the nucleic acid eluent through the digital microfluidic chip so as to extract sample nucleic acid droplets from the sample to be detected; mixing the sample nucleic acid droplets with the first round of RPA reagent and the second round of RPA reagent through the digital microfluidic chip for two rounds of RPA amplification to obtain sample product droplets containing two rounds of nucleic acid amplification products; Mixing the sample product liquid drops with the CRISPR reagent through the digital microfluidic chip, and moving to the solid phase gel to perform sequence recognition and cutting reaction to obtain solid phase gel after the cutting reaction; And detecting the self-interference signal of the solid gel in the solution after the cleavage reaction by the optical detection system, and determining and outputting a nucleic acid detection result of whether the sample to be detected contains a nucleic acid sequence to be detected. According to another aspect of the present application, there is provided a microfluidic detection platform, the microfluidic detection platform including a digital microfluidic chip and an optical detection system, the digital microfluidic chip including an upper chip and a lower chip, the upper chip having a solid phase gel embedded therein, the lower chip having a lysis solution and magnetic beads, a magnetic bead cleaning solution, a nucleic acid eluent, a first wheel of RPA reagent, a second wheel of RPA reagent, and a CRISPR reagent stored therein, the digital microfluidic chip being placed on a stage of the optical detection system; The digital microfluidic chip is used for sequentially mixing the sample to be detected with the lysate, the magnetic beads, the magnetic bead cleaning solution and the nucleic acid eluent after the sample to be detected is added into the lower chip so as to extract sample nucleic acid droplets from the sample to be detected; the digital microfluidic chip is further used for mixing the sample nucleic acid droplets with the first round of RPA reagent and the second round of RPA reagent for two rounds of RPA amplification to obtain sample product droplets containing two rounds of nucleic acid amplification products; The digital microfluidic chip is further used for mixing the sample product liquid drops with the CRISPR reagent, moving to the solid phase gel to perform sequence recognition and cu