Porous flat-bottom container and sample imaging detection method

Abstract

The invention discloses a porous flat-bottom container which comprises an open container body and a surrounding assembly, the open container body comprises a bottom plate and an edge outer wall, the edge outer wall is arranged around the edge of the bottom plate, and a plurality of flat-bottom sample pools which are sequentially arranged are arranged in the open container body; each of the flat-bottom sample pools is formed by surrounding a bottom plate, edge reinforcing ribs and inner hole reinforcing ribs, or is formed by surrounding the bottom plate and the inner hole reinforcing ribs, andthe edge reinforcing ribs and the inner hole reinforcing ribs are located on the bottom plate; and the surrounding assembly is arranged along the top of the edge outer wall. The porous flat-bottom container provided by the invention has excellent bottom flatness, mechanical strength, heat conduction uniformity, sealing and heat insulation properties and anti-pollution performance, can meet the requirements of micro-droplet type digital nucleic acid amplification accurate temperature control reaction and detection, and realizes the rapid and accurate program temperature control of micro-droplets in the flat-bottom sample pools and large-area distortionless upright imaging detection.

Description

technical field [0001] The application relates to the field of molecular biology, in particular to a porous flat-bottom container and a sample temperature-controlled reaction and imaging detection method. Background technique [0002] Droplet digital polymerase chain reaction (ddPCR) technology is a method for absolute quantification of nucleic acid molecules. Compared with quantitative PCR (quantitative PCR, qPCR), it has significant advantages in precision, accuracy and sensitivity; at the same time, ddPCR eliminates the dependence on the quantitative standard curve and improves the tolerance to amplification inhibitors ; Therefore, ddPCR is called the third generation PCR technology. Based on the similar principle of ddPCR, a series of digital isothermal nucleic acid amplification (dNAA) technologies have been developed, including digital RPA (Recombinase Polymerase Amplification, RPA), digital LAMP (digital loop-mediated amplification, dLAMP), etc. The above technologi...

AI Technical Summary

Problems solved by technology

[0006] (1) When adding droplets to a multi-well plate, it is difficult to precisely control the number of droplets added to the wells, making it difficult to form a monolayer of tiled droplet arrays at the bottom of wells with a fixed area

Too many droplets will overlap, resulting in unsatisfactory imaging analysis results; too few droplets will cause holes in the single-layer droplet array, which will affect the precision of digital PCR detection

[0007] (2) When performing digital PCR temperature control, the plastic materials used in most commonly used orifice plates often have poor thermal conductivity, resulting in slow temperature rise and fall speeds when performing program temperature control operations on the orifice plate; Metal materials are often not transparent, and metal materials will reflect the light emitted by the bright field light source and the fluorescence excitation light source, which will affect the upright imaging analysis

[0008] (3) When performing digital PCR temperature control, the porous plate made of metal material with good thermal conductivity will quickly conduct heat downward when it contacts the high-temperature heat cover, resulting in uneven temperature between the holes of the porous plate; in addition, the metal material with good thermal conductivity If the structural design of the porous plate made of materials is unreasonable, stress release and bending often occur during the molding preparation process, and the bottom surface is difficult to maintain a high level, so there is a gap between it and the temperature-controlled heating module, resulting in uneven temperature between the holes of the porous plate

[0009] (4) When performing digital PCR temperature control, it is usually necessary to apply a sealing film or a cover for sealing and pollution control, but high temperature heating causes a large number of air bubbles in the oil phase and water phase in the orifice plate to stay on the surface of the sealing film or cover , which seriously affects the quality of subsequent upright imaging; in addition, after the temperature control process is over, due to the shrinkage of air cooling, the sealing film or cover of the orifice plate collapses or the flatness decreases, which affects the direct upright imaging through the sealing film or cover the quality of

[0010] (5) When performing upright imaging analysis on the micro-droplet array in the flat-bottomed well, the reflective smooth hole wall of the porous plate material will reflect the droplet sample in the sample cell, seriously affecting the imaging quality and statistical analysis of data

[0011] (6) When performing upright imaging analysis on the micro-droplet array in the hole, because the oily liquid in the orifice plate tends to infiltrate the hole wall, the contact position between the oily liquid and the hole wall will produce a curved liquid surface, resulting in blurred edge of the hole wall during imaging

At the same time, the volume of the oily liquid in different holes is difficult to keep consistent, resulting in inconsistent focal lengths for imaging analysis of different holes, and it is difficult to guarantee the imaging clarity of different holes without refocusing

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