CN-121978323-A - High-robustness high-signal-strength digital immunodetection kit and method

CN121978323ACN 121978323 ACN121978323 ACN 121978323ACN-121978323-A

Abstract

The invention relates to the field of quarantine detection, and discloses a high-robustness high-signal-strength digital immunodetection kit and a method, wherein the kit comprises an inert nucleic acid blocking agent and a nucleic acid intercalating dye; the inert nucleic acid blocking agent is coated on the surface of the solid phase carrier or is contained in the rolling circle amplification reaction liquid, the nucleic acid intercalating dye is contained in the rolling circle amplification reaction liquid, and the emission spectrum of the nucleic acid intercalating dye is compatible with the emission spectrum of the sequence specific probe with fluorescent label. The strong and uniform signal of the invention needs to rely on accurate single magnetic bead counting to be converted into accurate quantitative results. Meanwhile, clear signal separation reduces the difficulty of flow design, so that single magnetic bead analysis in the presence of a sealing agent is simpler and more reliable. The two function together, so that the kit is particularly suitable for the steady and accurate quantification of the extremely low abundance protein markers in complex clinical samples.

Inventors

LIN MENGJIE
ZHANG WENJING
DU WEI
TIAN JINGQING
LI DONG

Assignees

成都玛雅光年科技有限公司
无锡博奥玛雅医学科技有限公司

Dates

Publication Date: 20260505
Application Date: 20260407

Claims (10)

1. A high robustness high signal strength digital immunoassay kit, comprising: a solid phase carrier coated with capture molecules; A detection molecule bearing a first affinity tag; A circular DNA template with a second affinity tag, the first affinity tag being capable of specifically binding to the second affinity tag; rolling circle amplification enzyme; A rolling circle amplification reaction solution comprising deoxynucleoside triphosphates and a sequence-specific probe with a fluorescent label; the kit further comprises an inert nucleic acid blocking agent and a nucleic acid intercalating dye; The inert nucleic acid blocking agent is coated on the surface of the solid phase carrier or contained in the rolling circle amplification reaction liquid; The nucleic acid intercalating dye is contained in the rolling circle amplification reaction solution, and the emission spectrum of the nucleic acid intercalating dye is compatible with the emission spectrum of the sequence-specific probe with fluorescent label.
2. The digital immunodetection kit according to claim 1, wherein the inert nucleic acid blocking agent is selected from at least one of a naturally derived nucleic acid comprising at least one of salmon sperm DNA and calf thymus DNA and an artificially synthesized nucleic acid comprising at least one of Poly adenine nucleotide Poly (a), random sequence single stranded DNA.
3. The digital immunodetection kit of claim 1, wherein the nucleic acid intercalating dye is a fluorescent dye capable of binding to double-stranded DNA and single-stranded DNA, and the nucleic acid intercalating dye is selected from at least one of SYBR Green I, SYBR Green II, SYBR Gold, and EvaGreen.
4. The digital immunodetection kit according to claim 1, wherein the solid phase carrier is selected from the group consisting of a magnetic bead, a microsphere and a chip, the capture molecule is a capture antibody, the detection molecule is a detection antibody, and the kit is used for detecting a protein, a nucleic acid or an exosome target.
5. The digitized immunoassay kit according to claim 1, wherein the first affinity tag is biotin, the second affinity tag is streptavidin, the fluorescent substance labeled with the sequence-specific probe with fluorescent label is fluorescein isothiocyanate FITC, and the rolling circle amplification enzyme is phi29 enzyme.
6. The digital immunodetection method with high robustness and high signal intensity is characterized by comprising the following steps of: step S1, mixing and incubating a sample to be detected with a solid-phase carrier coated with capture molecules; Step S2, adding a detection molecule with a first affinity tag into a reaction system for incubation and washing, and then adding a circular DNA template with a second affinity tag for incubation and washing, wherein the first affinity tag can be specifically combined with the second affinity tag; s3, adding rolling circle amplification enzyme and rolling circle amplification reaction liquid into a reaction system to perform rolling circle amplification reaction and washing, wherein the rolling circle amplification reaction liquid contains deoxynucleoside triphosphate and a sequence specific probe with fluorescent marks; Step S4, re-suspending the solid-phase carrier after the reaction in the step S3, detecting by an input flow cytometer, collecting forward scattering light signals, side scattering light signals and fluorescent channel signals, calculating to obtain signal values AMP, and substituting the signal values AMP into a calibration curve to calculate the concentration of the target in the sample to be detected; The surface of the solid phase carrier is pre-coated with an inert nucleic acid blocking agent, or the inert nucleic acid blocking agent is added into the rolling circle amplification reaction liquid in the step S3; A nucleic acid intercalating dye is also added into the rolling circle amplification reaction liquid in the step S3, and the emission spectrum of the nucleic acid intercalating dye is compatible with the emission spectrum of the sequence specific probe with the fluorescent mark; In the step S3, the sequence-specific probe with fluorescent label is hybridized with the single-stranded DNA product generated by rolling circle amplification, and the nucleic acid intercalating dye is intercalated into the double-stranded structure region and the single-stranded structure region of the single-stranded DNA product to jointly perform double fluorescent labeling.
7. The method according to claim 6, wherein the inert nucleic acid blocking agent is at least one selected from the group consisting of a naturally derived nucleic acid comprising at least one of salmon sperm DNA and calf thymus DNA, and an artificially synthesized nucleic acid comprising at least one of Poly adenine nucleotide Poly (A) and random sequence single-stranded DNA, wherein the nucleic acid intercalating dye is a fluorescent dye capable of binding to double-stranded DNA and single-stranded DNA, and wherein the nucleic acid intercalating dye is at least one selected from the group consisting of SYBR Green I, SYBR Green II, SYBR Gold, evaGreen.
8. The digital immunodetection method of claim 6, wherein said capture molecule is a capture antibody, said detection molecule is a detection antibody, said first affinity tag is biotin, said second affinity tag is streptavidin, and said rolling circle amplification enzyme is phi29 enzyme; The specific operation condition of the step S2 is that the detection molecules with the first affinity tag are added and then are incubated for 1 hour at 37 ℃ under the vibration, and the circular DNA template with the second affinity tag is added and then are incubated for 15 minutes at 37 ℃ under the vibration; The specific operation condition of the step S3 is that the rolling circle amplification enzyme and the rolling circle amplification reaction liquid are added and then incubated for 1 hour at 37 ℃ under shaking.
9. The digitized immunoassay method of claim 6, wherein in step S4, the step of substituting the signal value AMP into a calibration curve to calculate the concentration of the target in the sample to be measured specifically comprises: obtaining signal values AMP calculated after the calibration products with different known concentrations are detected by the flow cytometer; Generating the calibration curve by four-parameter or five-parameter fitting by taking the known concentration of the calibrator as an X axis and a corresponding signal value AMP as a Y axis; Substituting the signal value AMP obtained by detection and calculation of the sample to be detected into the calibration curve, and calculating the concentration of the target in the sample to be detected.
10. The digitized immunoassay method of claim 9, further comprising the step of calculating a minimum detection limit as follows: repeatedly measuring the zero-concentration calibrator serving as a sample to obtain a signal value AMP corresponding to a measurement result; calculating an average value M and a standard deviation SD of a signal value AMP corresponding to the measurement result to obtain a target signal value corresponding to a value M+2.5SD; Performing two-point regression fitting according to the concentration between the zero concentration calibrator and the adjacent concentration calibrator and the signal value AMP result to obtain a primary equation; substituting the target signal value into the primary equation, and calculating a corresponding concentration value as the lowest detection limit.

Description

High-robustness high-signal-strength digital immunodetection kit and method Technical Field The invention relates to the field of quarantine detection, in particular to a high-robustness high-signal-strength digital immunodetection kit and a method. Background In the prior art, the ultra-high sensitivity digital immunodetection method of Rolling Circle Amplification (RCA) and flow cytometry comprises capturing single target protein molecules on antibody coated magnetic beads, amplifying in-situ signals of each binding event by RCA to generate a long single-stranded DNA concatemer, hybridizing by a sequence specific fluorescent probe, and finally performing digital counting by using flow cytometry. However, when this technology is converted for clinical practical application, two key problems are found that affect its detection performance and reliability: 1. the magnetic bead aggregation problem is that RCA amplification produces single-stranded DNA tandem repeats of thousands of bases in length, and the long-chain nucleic acid is easy to induce nonspecific aggregation of immune magnetic beads through base complementary pairing, electrostatic action and hydrophobic action. The aggregation of magnetic beads can cause abnormal forward/side scattering signals during flow detection, the single particle gating efficiency is reduced, the detection variation Coefficient (CV) is obviously increased, the quantitative result is biased, and even the flow cytometer pipeline is blocked, so that the stability, the repeatability and the clinical applicability of the detection method are seriously affected. Signal intensity and homogeneity problems hybridization labelling is performed only on sequence specific probes, the signal intensity of which may be affected by probe hybridization efficiency, local folding or steric hindrance of the RCA product. Clausson et al (2015) indicated that RCA products (RCPs) may exhibit irregular signal foci due to spontaneous folding of single stranded DNA, even scattered into multiple signal spots, which can affect the uniformity and intensity of single bead fluorescent signals in flow assays, and thus the signal-to-noise ratio and interpretation accuracy of low concentration sample detection. Disclosure of Invention The invention aims to solve the technical problems of (1) the problem of magnetic bead aggregation caused by RCA long-chain products and (2) the problem of insufficient or nonuniform signal intensity of the RCA products on a single magnetic bead in the application of the IVD kit by the existing digital immunodetection method based on RCA amplification, thereby comprehensively improving the robustness, accuracy and sensitivity of the kit detection. In order to solve the technical problems, the invention provides a digital immunodetection kit with high robustness and high signal intensity, which comprises: a solid phase carrier coated with capture molecules; A detection molecule bearing a first affinity tag; A circular DNA template with a second affinity tag, the first affinity tag being capable of specifically binding to the second affinity tag; rolling circle amplification enzyme; A rolling circle amplification reaction solution comprising deoxynucleoside triphosphates and a sequence-specific probe with a fluorescent label; the kit further comprises an inert nucleic acid blocking agent and a nucleic acid intercalating dye; The inert nucleic acid blocking agent is coated on the surface of the solid phase carrier or contained in the rolling circle amplification reaction liquid; The nucleic acid intercalating dye is contained in the rolling circle amplification reaction solution, and the emission spectrum of the nucleic acid intercalating dye is compatible with the emission spectrum of the sequence-specific probe with fluorescent label. The invention also provides a high-robustness high-signal-strength digital immunodetection method, which comprises the following steps: step S1, mixing and incubating a sample to be detected with a solid-phase carrier coated with capture molecules; Step S2, adding a detection molecule with a first affinity tag into a reaction system for incubation and washing, and then adding a circular DNA template with a second affinity tag for incubation and washing, wherein the first affinity tag can be specifically combined with the second affinity tag; s3, adding rolling circle amplification enzyme and rolling circle amplification reaction liquid into a reaction system to perform rolling circle amplification reaction and washing, wherein the rolling circle amplification reaction liquid contains deoxynucleoside triphosphate and a sequence specific probe with fluorescent marks; Step S4, re-suspending the solid-phase carrier after the reaction in the step S3, detecting by an input flow cytometer, collecting forward scattering light signals, side scattering light signals and fluorescent channel signals, calculating to obtain signal values AMP, and substituting t