阅读说明：本技术 一种用于微滴式数字pcr仪的计数校准装置、制备方法及使用方法 (Counting calibration device for droplet type digital PCR instrument, preparation method and use method ) 是由 林挺 于祥春 冯晓燕 黄慧 龚建 于 2020-05-09 设计创作，主要内容包括：本发明提出的一种用于微滴式数字PCR仪的计数校准装置,采用封闭式的单层腔体,即腔体1、腔体2、腔体3、腔体4内分别放入效仿数字PCR中微滴结构的、具有固定数量的FITC标记的基底微球、红色荧光标记的微球、绿色荧光标记的微球、蓝色荧光标记的微球。通过单层腔体内具有固定数量并标记有不同颜色荧光的微球对数字PCR的计数系统先进行计数校准,形成校准文件,从而进一步提高对样本进行检测时的准确度及精密度。(The invention provides a counting and calibrating device for a micro-drop digital PCR instrument, which adopts a closed single-layer cavity, namely a cavity 1, a cavity 2, a cavity 3 and a cavity 4 are respectively filled with a fixed number of FITC marked substrate microspheres, red fluorescence marked microspheres, green fluorescence marked microspheres and blue fluorescence marked microspheres which imitate the micro-drop structure in digital PCR. The counting system of the digital PCR is firstly counted and calibrated through the microspheres which are fixed in number and marked with different colors of fluorescence in the single-layer cavity to form a calibration file, so that the accuracy and precision of the sample in detection are further improved.)

1. A count calibrating device for droplet formula digital PCR appearance which characterized in that: comprises a cavity 1, a cavity 2, a cavity 3 and a cavity 4; polymethyl methacrylate sheets are placed in the cavities 1 to 4, and a fixed number of microspheres with different colors and fluorescent marks are sealed in the polymethyl methacrylate sheets; wherein the content of the first and second substances,

the cavities 1 to 4 are single-layer closed cavities, and the heights of the cavities are 90-130 micrometers; 30000 FITC-labeled substrate microspheres are sealed in a sheet which is placed in the cavity 1 and takes polymethyl methacrylate as a carrier; 27000 substrate microspheres marked by FITC, 1000 microspheres marked by blue fluorescence, 1000 microspheres marked by green fluorescence and 1000 microspheres marked by red fluorescence are sealed in a sheet which is placed in the cavity 2 and takes polymethyl methacrylate as a carrier; 24000 FITC-labeled substrate microspheres, 2000 blue fluorescence-labeled microspheres, 2000 green fluorescence-labeled microspheres and 2000 red fluorescence-labeled microspheres are sealed in a sheet which is placed in the cavity 3 and takes polymethyl methacrylate as a carrier; 18000 pieces of FITC-labeled substrate microspheres, 4000 pieces of blue fluorescence-labeled microspheres, 4000 pieces of green fluorescence-labeled microspheres and 4000 pieces of red fluorescence-labeled microspheres are sealed in the sheet which is placed in the cavity 4 and takes the polymethyl methacrylate as a carrier.

The diameters of the FITC marked substrate microspheres, the blue fluorescence marked microspheres, the green fluorescence marked microspheres and the red fluorescence marked microspheres are 80-120 micrometers; the microspheres marked with different color fluorescence are arranged in a monolayer in the polymethyl methacrylate sheet.

2. A preparation method of a counting calibration device for a droplet type digital PCR instrument comprises the following steps:

(1) 30000 regularly arranged pits are respectively etched on the surface of the polymethyl methacrylate material with the length of 18-26 mm and the width of 14-20 mm by using a micro-electro-mechanical system technology, wherein the diameter of each pit is 84-124 micrometers, and the height of each pit is 42-62 micrometers;

(2) under the field of a fluorescence microscope, a certain amount of microspheres marked with different colors of fluorescence are fully mixed by using a curing agent to prepare a microsphere suspension, and then the microsphere suspension is uniformly dripped on the surface of a polymethyl methacrylate material etched to form small pits with the diameter of 84-124 microns, and the state that the microspheres enter the small pits is observed under the fluorescence microscope after the microspheres are uniformly scraped;

(3) coating a curing agent on the boundary of the other polymethyl methacrylate material etched with the small pits and covering the other polymethyl methacrylate material with the microspheres, and irradiating by using 365nm UVA ultraviolet light to enable the two polymethyl methacrylate materials to be cured and bonded together to prepare a polymethyl methacrylate sheet containing a fixed number of fluorescent microspheres marked with different colors;

(4) carrying out full-view scanning on the prepared polymethyl methacrylate sheets required to be used in the cavities 1 to 4 on a fluorescence imaging scanner, carrying out accurate counting statistics on various fluorescent microspheres, and screening out the polymethyl methacrylate sheets which are closest to the polymethyl methacrylate sheets carrying the fluorescent microspheres with fixed quantity and different colors;

(5) respectively preparing 4 polymethyl methacrylate sheets containing fixed quantity and marked with fluorescent microspheres with different colors according to the steps (1) to (4), putting the 4 polymethyl methacrylate sheets into a mold from a cavity 1 to the cavity 4 according to a certain sequence, and sealing the mold to prepare the counting calibration device for the micro-drop digital PCR instrument.

3. A method for using a counting calibration device of a droplet type digital PCR instrument comprises the following steps:

(1) respectively placing the counting and calibrating devices of the droplet-type digital PCR instrument with the cavity height of 90-130 micrometers in the digital PCR instrument, and collecting and imaging the emitted fluorescence by an image sensor after excitation of an excitation light source;

(2) respectively imaging the fluorescent marked microspheres with different colors and the diameters of 80-120 micrometers on the surface of an image sensor;

(3) after data acquisition is carried out through a computer, the number of the fluorescent microspheres is compared and analyzed with the known fixed number in the cavities 1 to 4 of the counting and calibrating device of the digital PCR instrument and the fluorescent microspheres with different colors are marked;

(4) analyzing the comparison result, calibrating a counting system of the instrument and forming a calibration file;

(5) after the digital PCR detection of the sample is carried out, firstly, a calibration file is introduced to calibrate the counting system, and then, the microdroplets emitting fluorescence are counted to complete the subsequent data analysis work.

Technical Field

The invention relates to digital PCR detection, in particular to a counting calibration device for a droplet type digital PCR instrument, a preparation method and a use method.

Background

The digital PCR technique divides a sample into several tens to several tens of thousands and distributes them to different reaction units, one for each droplet generated by a droplet-type digital PCR instrument. Each reaction unit contains one or more copies of target molecules (DNA templates), then PCR amplification is carried out on the target molecules in each reaction unit, and after the amplification is finished, the fluorescence signals of each reaction unit are subjected to statistical analysis. So far, based on the difference of liquid separation methods, there are three main types of digital PCR technologies: the micro-reaction chamber/orifice plate, the large-scale integrated micro-fluidic chip and the micro-drop type digital PCR system, three types of digital PCR technologies realize liquid separation through a micro-porous plate, a micro-fluidic chip or micro-drops respectively, and each separated micro area can carry out independent PCR reaction, wherein the diameter of a micro-drop formed in the micro-drop type digital PCR is about 100 microns. Although the digital PCR technology has been proposed for only 20 years, the industrialization of the technology is rapidly developed due to the unique technical advantages and application prospects. At present, the technology is applied to the research fields of gene copy number variation, infection detection, gene sequencing, gene mutation detection and the like. The digital PCR technology belongs to absolute quantification of DNA molecules, and detection is realized by the real proportion of the number of droplets or the number of micro reaction chambers, so that the number of liquid separation is calibrated as much as possible, and the number of formed droplets is accurately calculated, so that the liquid separation error can be greatly reduced, and the detection accuracy and precision of a digital PCR instrument are greatly improved. The related technology has not been reported and published at present.

Disclosure of Invention

One of the purposes of the invention is to provide a counting calibration device for a droplet type digital PCR instrument and a preparation method thereof; another object of the present invention is to provide a method of using a counting calibration device for a digital PCR machine that can be used multiple times without additional devices.

The invention provides a counting and calibrating device for a micro-drop digital PCR (polymerase chain reaction) instrument, which comprises a cavity 1, a cavity 2, a cavity 3 and a cavity 4, wherein a polymethyl methacrylate sheet is placed in the cavity 1 to the cavity 4, and a fixed number of microspheres with different fluorescent marks are sealed in the polymethyl methacrylate sheet.

The cavities 1 to 4 are single-layer closed cavities, and the heights of the cavities are 90-130 micrometers;

30000 FITC-labeled substrate microspheres are sealed in the polymethyl methacrylate sheet; a sheet which takes polymethyl methacrylate as a carrier and is arranged in the cavity 2, 27000 substrate microspheres marked by FITC, 1000 microspheres marked by blue fluorescence, 1000 microspheres marked by green fluorescence and 1000 microspheres marked by red fluorescence are sealed in the polymethyl methacrylate sheet; a sheet which takes polymethyl methacrylate as a carrier and is arranged in the cavity 3, 24000 FITC-labeled substrate microspheres, 2000 blue fluorescence-labeled microspheres, 2000 green fluorescence-labeled microspheres and 2000 red fluorescence-labeled microspheres are sealed in the polymethyl methacrylate sheet; a sheet with polymethyl methacrylate as a carrier is placed in the cavity 4, 18000 FITC-labeled substrate microspheres, 4000 blue fluorescence-labeled microspheres, 4000 green fluorescence-labeled microspheres and 4000 red fluorescence-labeled microspheres are sealed in the polymethyl methacrylate sheet.

The diameters of the FITC marked substrate microspheres, the blue fluorescence marked microspheres, the green fluorescence marked microspheres and the red fluorescence marked microspheres are 80-120 micrometers; the microspheres marked with different color fluorescence are arranged in a monolayer in the polymethyl methacrylate sheet.

A preparation method of a counting calibration device for a droplet type digital PCR instrument comprises the following steps:

(1) using Micro-Electro-Mechanical System (MEMS) to etch 30000 regularly arranged pits on the surface of the polymethyl methacrylate material with the length of 18-26 mm and the width of 14-20 mm, wherein the diameter of each pit is 84-124 microns, and the height is 42-62 microns;

(2) under the field of a fluorescence microscope, a certain amount of microspheres marked with different colors of fluorescence are fully mixed by using a curing agent to prepare a microsphere suspension, and then the microsphere suspension is uniformly dripped on the surface of a polymethyl methacrylate material etched to form small pits with the diameter of 84-124 microns, and the state that the microspheres enter the small pits is observed under the fluorescence microscope after the microspheres are uniformly scraped;

(3) coating a curing agent on the boundary of the other polymethyl methacrylate material etched with the small pits and covering the other polymethyl methacrylate material with the microspheres, and irradiating by using 365nm UVA ultraviolet light to enable the two polymethyl methacrylate materials to be cured and bonded together to prepare a polymethyl methacrylate sheet containing a fixed number of fluorescent microspheres marked with different colors;

(4) carrying out full-view scanning on the prepared polymethyl methacrylate sheets required to be used in the cavities 1 to 4 on a fluorescence imaging scanner, carrying out accurate counting statistics on various fluorescent microspheres, and screening out the polymethyl methacrylate sheets which are closest to the polymethyl methacrylate sheets carrying the fluorescent microspheres with fixed quantity and different colors;

(5) and (3) respectively preparing 4 polymethyl methacrylate sheets containing different quantities of fluorescent microspheres marked with different colors according to the steps (1) to (4), putting the 4 polymethyl methacrylate sheets into a mold from the cavity 1 to the cavity 4 in a certain sequence, and sealing the mold to obtain the counting calibration device for the micro-drop digital PCR instrument.

A method for using a counting calibration device of a droplet type digital PCR instrument comprises the following steps:

(1) respectively placing the counting and calibrating devices of the droplet-type digital PCR instrument with the cavity height of 90-130 micrometers in the digital PCR instrument, and collecting and imaging the emitted fluorescence by an image sensor after excitation of an excitation light source;

(2) respectively imaging the fluorescent marked microspheres with different colors and the diameters of 80-120 micrometers on the surface of an image sensor;

(3) after data acquisition is carried out through a computer, the number of the fluorescent microspheres is compared and analyzed with the known fixed number in the cavities 1 to 4 of the counting and calibrating device of the digital PCR instrument and the fluorescent microspheres with different colors are marked;

(4) analyzing the comparison result, calibrating a counting system of the instrument and forming a calibration file;

(5) after the digital PCR detection of the sample is carried out, firstly, a calibration file is introduced to calibrate the counting system, and then, the microdroplets emitting fluorescence are counted to complete the subsequent data analysis work.

The invention has the following remarkable characteristics:

a fixed number of FITC marked substrate microspheres, red fluorescence marked microspheres, green fluorescence marked microspheres and blue fluorescence marked microspheres which imitate a micro-droplet structure in digital PCR are placed in a closed single-layer cavity, and a counting system of the digital PCR is counted and calibrated through the fixed number of microspheres marked with different colors of fluorescence in the single-layer cavity to form a calibration file, so that the accuracy and precision of detection on a sample are further improved.

Drawings

FIG. 1 shows the state of the microsphere solution under a fluorescent microscope.

FIG. 2 uses a fluorescence microscope (Sapphire)^TMBiomolecular Imager, Azure Biosystems, usa) to observe the state of the microspheres labeled with different color fluorescence and mixed.

FIG. 3 is a top view of the number and distribution of microspheres in a counting calibration device for a digital PCR machine. From left to right, 1000 microspheres carrying all the substrate microspheres and red, green and blue fluorescent markers, 2000 microspheres carrying the red, green and blue fluorescent markers and 4000 polymethyl methacrylate material sheets carrying the red, green and blue fluorescent markers are respectively arranged.

FIG. 4 is a schematic top view of a chamber structure of a counting calibration device for a digital micro-PCR apparatus.

FIG. 5 is a schematic diagram of a side view of a chamber structure of a counting calibration device for a digital micro-droplet PCR apparatus.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be noted that any combination of the non-conflicting embodiments or technical features may form a new embodiment.