CN111951294A - Micropore traversing method and device of PCR chip and storage medium - Google Patents
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Abstract
According to the micropore traversing method, the micropore traversing device and the storage medium of the PCR chip, the micropores of the digital PCR chip are traversed at high speed, high precision and high robustness to calculate the pixel sizes of the micropores and count the coordinates of the micropores; the method comprises the following steps: collecting an image of a micropore area of a PCR chip subjected to a detection reaction, and converting the image into a gray-scale image; traversing the micropores to extract all extreme value regions in the image, wherein the extreme value regions are one or more micropore communication regions with different gray levels; screening the extreme value region through the shape characteristics of the micropores, and obtaining a maximum stable extreme value region through the maximum extreme value region of the communication region; and acquiring the pixel area and the pixel coordinate of the micropore in the maximum stable extreme value area, and completing the inspection of the PCR chip.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a micropore traversing method and device of a PCR chip and a storage medium.
Background
Digital PCR is a recent quantitative technique, and nucleic acid quantification based on counting by a single-molecule PCR method is an absolute quantitative method. The method mainly adopts a micro-fluidic or micro-droplet method in the current analytical chemistry hot research field to disperse a large amount of diluted nucleic acid solution into micro-reactors or micro-droplets of a chip, wherein the number of nucleic acid templates in each reactor is less than or equal to 1. Thus, after PCR cycling, a reactor with a nucleic acid molecule template will give a fluorescent signal, and a reactor without a template will have no fluorescent signal. Based on the relative proportions and the volume of the reactor, the nucleic acid concentration of the original solution can be deduced.
Machine vision mainly uses a computer to simulate the visual function of a human, but not only is the simple extension of human eyes, but also has a part of functions of human brain, namely, information is extracted from the image of an objective object, processed and understood, and finally the information is used for actual detection, measurement and control.
After obtaining a reliable chip fluorescence photo, how to mark the negative and positive of the chip micropore in the photo by using a proper algorithm and count the position of each micropore is a key part of the algorithm of the digital PCR reader. The existing algorithm generally has the defects of low precision, poor robustness and long time consumption, so that a method for calculating the pixel size and the pixel coordinate position of the micropore of the chip at high speed, high precision and high robustness is needed.
Disclosure of Invention
The invention aims to provide a micropore traversing method, a micropore traversing device and a storage medium of a PCR chip, which can realize the calculation of the pixel size and the statistics of the pixel coordinate position of a micropore of the PCR chip in a high-speed, high-precision and high-robustness manner.
In order to achieve the above object, an aspect of the present invention provides a microwell traversal method for a PCR chip, comprising:
s100, collecting an image of a micropore area of a PCR chip subjected to detection reaction, and converting the image into a gray-scale image;
s200, traversing micropores to extract all extreme value regions in the image, wherein the extreme value regions are one or more micropore communication regions with different gray levels;
s300, screening the extreme value region through the shape characteristics of the micropores, and obtaining a maximum stable extreme value region through the maximum extreme value region of the communicated region;
s400, acquiring the pixel area and the pixel coordinate of the micropore in the maximum stable extreme value area, and completing the inspection of the PCR chip.
Further, the traversing process of the extremum region includes:
s201, initializing an image, and establishing a stack for storing a connected region and a stack of a region boundary;
s202, selecting any micropore in the image, taking the current micropore as a current communication area, putting the current micropore into a stack, and taking the gray value of the current micropore as the gray value of the current communication area;
s203, detecting the gray value of the adjacent micropore of the micropore, if the gray value of the adjacent micropore is not more than the gray value of the current communication area,
step S204 is executed, the hole site is added into the current communication area and is put into a stack, the gray value of the hole site is used as the gray value of the current communication area, and step S203 is continuously repeated;
if the gray value of the adjacent micropore is larger than that of the current connected region,
step S205 is performed to put the hole site in the stack as the boundary of the connected region.
Further, the traversing process of the extremum region further includes:
s206, acquiring the gray value of the boundary of the connected region in the pile, and judging as follows:
if the obtained boundary of the connected region in the heap is empty, traversing is finished;
if the gray value of the boundary of the connected region in the stack is equal to the gray value of the current connected region in the stack, executing step S203;
if the gray value of the boundary of the connected region in the stack is not less than the gray value of the current connected region in the stack, combining the connected regions in the stack until the pixel value of the current connected region is equal to the gray value of the boundary of the connected region;
s206, the merged current connected region is set as an extremum region.
S207, if the acquired gray value of the boundary of the connected region in the stack is smaller than the gray value of the current connected region in the stack, executing the step S205.
Further, in the traversal process of the extreme value region, the gray values of the adjacent micropores on the left side and the lower side of the micropore are detected by taking the micropore at the upper left corner of the image as a starting point.
Further, in the process of acquiring the maximum stable extremum region, the method further includes:
s301, screening the extreme value area through micropore shape characteristics, and deleting the area which does not accord with the micropore shape characteristics from the extreme value area, wherein the micropore shape characteristics comprise the shape, the size, the row spacing and the column spacing of micropores;
s302, adjusting the gray value of the extremum region after the result screening, and obtaining a maximum connected region to form a maximum stable extremum region.
In another aspect, the present invention further provides a microwell traversing apparatus for a PCR chip, comprising;
the acquisition unit is used for acquiring an image of a micropore area of the PCR chip subjected to the detection reaction and converting the image into a gray-scale image;
the acquisition unit traverses the micropores to extract all extreme value areas in the image, wherein the extreme value areas are one or more micropore communication areas with different gray levels;
the extraction unit screens the extreme value region through the shape characteristics of the micropores and obtains a maximum stable extreme value region through the maximum extreme value region of the communication region;
and the verification unit is used for acquiring the pixel area and the pixel coordinate of the micropore in the maximum stable extremum region and completing the detection of the PCR chip.
In another aspect, the present invention further provides a storage medium, where the storage medium stores a plurality of instructions, and the instructions are suitable for being loaded by a processor to perform the steps of the above-mentioned microwell traversal method for a PCR chip.
According to the micropore traversing method, device and storage medium of the PCR chip, provided by the invention, micropores of the digital PCR chip are traversed at high speed, high precision and high robustness to calculate the pixel size of the micropores and count the coordinates of each micropore, the calculation error of the pore size pixel size of the effective micropore is less than 10%, the pixel coordinate counting precision of the central point of the micropore is more than 95%, the misrecognition rate of the micropore is less than 0.1%, and the performance and efficiency of the algorithm of the digital PCR reader are improved.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a diagram of a micropore region to be identified in a digital PCR chip;
FIG. 2 is a flowchart of a method of micro-well traversal of a PCR chip according to the present invention;
FIG. 3 is a schematic diagram of the basic principle of the extremum region algorithm;
FIG. 4 is a flowchart of a method for extracting an extremum region according to the present invention;
FIG. 5 is a system block diagram of a microwell traversing apparatus for a PCR chip according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
FIG. 1 shows the microwell region to be identified in a digital PCR chip.
As shown in fig. 1, the digital PCR chip is a nucleic acid quantitative detection device based on a single molecule PCR method, which employs a microfluidic or microdroplet method to disperse a large amount of diluted nucleic acid solution into the micropore area of the chip, the micropores containing the nucleic acid molecular template give fluorescence signals, the micropore area is photographed and analyzed by a digital PCR reader, and the nucleic acid concentration of the original solution can be calculated according to the relative proportion of the fluorescence signals in the micropore area and the volume of the micropores.
FIG. 2 is a flowchart of a method for traversing a microwell of a PCR chip according to the present invention.
As shown in FIG. 2, the present invention employs a modified extreme area algorithm to traverse the microwells of a PCR chip, which comprises the following steps:
s100, collecting an image of a micropore area of the PCR chip subjected to the detection reaction, and converting the image into a gray-scale image.
S200, traversing the micropores to extract all extreme value regions in the image, wherein the extreme value regions are one or more micropore communication regions with different gray levels.
S300, screening the extreme value region through the shape characteristics of the micropores, and obtaining a maximum stable extreme value region through the extreme value region with the maximum communication region.
S400, acquiring the pixel area and the pixel coordinate of the micropore in the maximum stable extreme value area, and completing the inspection of the PCR chip.
Specifically, the invention adopts a digital PCR reader to finish the photographing of the micropore area of the PCR chip. After obtaining the image of the micropore area, the invention adopts an algorithm based on the extreme value area to mark, analyze and count the micropores of the chip in the image. The basic principle of the extremum region algorithm is shown in fig. 3, where 5 points represent 5 pixels and their heights represent pixel values. The ER region should be a region with two high sides and a low middle part, and the region can store water, and the pixel level of the ER region is the water surface height (the same height reference as the pixel). The left side is continuously injected with water. Looking at pixel 1 first, and at a higher degree than the pixel on the right, it is found that pixel 2 is lower than pixel 1, so water cannot be accumulated at pixel 1, and pixel 1 should be taken as a boundary. Looking now at pixel 2, pixel 3 is higher than pixel 2, so pixel 3 is also a border, as compared to the pixel on the right. Pixel 2 is a high on both sides and low in the middle, and can accumulate water, thus forming the first ER region. As water is injected, the water level rises until it reaches the lower boundary height, pixel 3. Pixel 3 is the right boundary and whether the water level can continue to increase depends on whether the pixel 3 on the right side is higher than pixel 3. Above pixel 3, the water level may continue to rise above pixel 3, thus obtaining a second ER region, consisting of pixel 2 and pixel 3, at the pixel level of pixel 3. But the pixel 4 to the right of the pixel 3 in the figure is lower than the pixel 3 and water will overflow. Looking now at pixel 4, pixel 5 to the right of pixel 4 is taller than pixel 4 so that water can accumulate at pixel 4, which will now form a second ER region at the pixel level of pixel 4. When the water level height of this region reaches the height of the lower border (pixel 3), the first ER region will merge with the second ER region to form a new ER region. When the water level reaches the pixel 5 height, pixel 1 height, two new ER regions will be obtained again.
The method for extracting the extreme region from the microwell region of the PCR chip of this embodiment will be described in detail with reference to FIG. 4.
Fig. 4 is a flowchart of a method of an extremum region extracting process of the present invention, as shown in fig. 4, the extremum region extracting process of the present invention is as follows:
s201, initializing an image, converting the micropore area image into a gray scale image, and establishing a stack for storing a communication area and a stack for storing an area boundary.
S202, any micropore in the image is selected, the current micropore is used as a current communication area and is placed in a stack, and the gray value of the current micropore is used as the gray value of the current communication area.
Specifically, in this embodiment, the micro-hole at the top left corner of the image can be selected as the starting point
S203, detecting the gray value of the adjacent micropore of the micropore, if the gray value of the adjacent micropore is not more than the gray value of the current communication area,
step S204 is executed, the hole site is added into the current communication area and is put into a stack, the gray value of the hole site is used as the gray value of the current communication area, and step S203 is continuously repeated;
if the gray value of the adjacent micropore is larger than that of the current connected region,
step S205 is performed to put the hole site in the stack as the boundary of the connected region.
S206, acquiring the gray value of the boundary of the connected region in the pile, and judging as follows:
if the obtained boundary of the connected region in the heap is empty, traversing is finished;
if the gray value of the boundary of the connected region in the stack is equal to the gray value of the current connected region in the stack, executing step S203;
if the obtained gray value of the boundary of the connected region in the stack is not less than the gray value of the current connected region in the stack, step S206 is executed to merge the connected regions in the stack until the pixel value of the current connected region is equal to the gray value of the boundary of the connected region, and the merged current connected region is set as the extremum region.
S207, if the acquired gray value of the boundary of the connected region in the stack is smaller than the gray value of the current connected region in the stack, executing the step S205.
Specifically, the heap in the algorithm is a container containing 256 elements. Each element corresponds to 0-255 per pixel value, each element is a real heap, and the corresponding size pixel is stored, and the pixel with the smallest pixel value is popped up each time. The pixels stored in the heap are boundary pixels. Each element in the stack is a connected domain, i.e. a connected set of pixels, which also has a pixel level and does not necessarily satisfy the gray level relation of the ER region. When the minimum boundary pixel popped from the stack is not equal to (i.e., larger than) the last popped pixel, the boundary pixel value of the connected component is larger than the top-of-stack connected component pixel level, and the pixel level of the connected component satisfies the pixel level relationship of the ER area, where the connected component is an ER area. At initialization, an empty area of 256 pixel level will be pushed onto the stack. When the algorithm is executed starting from the upper left corner of the image, only the right and lower neighborhood pixels are visited, all pixels can be traversed as well. It is worth noting that the pixels accessed in the algorithm are not accessed any more, the time complexity of the algorithm is in positive correlation with the number of the image pixels, and the execution efficiency is high. It only takes about 0.06s to extract all ER regions of a channel of a 480x640 size image.
Furthermore, after the extremum region is extracted, screening is performed to obtain a maximum stable extremum region. The screening process comprises the following steps:
s301, screening the extreme value area through micropore shape characteristics, and deleting the area which does not accord with the micropore shape characteristics from the extreme value area, wherein the micropore shape characteristics comprise the shape, the size, the row spacing and the column spacing of micropores;
s302, adjusting the gray value of the extremum region after the result screening, and obtaining a maximum connected region to form a maximum stable extremum region.
The size of the pixel of the micropore and the central coordinate of the micropore can be obtained through the returned maximum stable extreme value area of each screening condition, and a foundation is laid for the analysis of the negative and positive of the micropore of the digital PCR chip and the like.
FIG. 5 is a system block diagram of a microwell traversing apparatus for a PCR chip according to the present invention.
As shown in FIG. 5, the present invention provides a microwell traversing apparatus for PCR chip, comprising;
an acquisition unit 41 which acquires an image of a micro-pore region of the PCR chip subjected to the test reaction and converts the image into a gray scale image;
the acquiring unit 42 traverses the micropores to extract all extreme value regions in the image, wherein the extreme value regions are one or more micropore communication regions with different gray levels;
an extracting unit 43, which screens the extremum region according to the shape characteristics of the micropores and obtains a maximum stable extremum region according to the extremum region with the maximum communication region;
and the verification unit 44 is used for acquiring the pixel area and the pixel coordinate of the micropore in the maximum stable extremum region and completing the detection of the PCR chip.
The invention also provides a storage medium, wherein the storage medium stores a plurality of instructions, and the instructions are suitable for being loaded by a processor to execute the steps of the PCR chip micropore traversing method.
Computer storage media for embodiments of the invention may employ any combination of one or more computer-readable media. The computer readable medium may be a computer readable signal medium or a computer readable storage medium. A computer readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any combination of the foregoing. More specific examples (a non-exhaustive list) of the computer readable storage medium would include the following: an electrical connection having one or more wires, a portable computer diskette, a hard disk, a Random Access Memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing. In the context of this document, a computer readable storage medium may be any tangible medium that can contain, or store a program for use by or in connection with an instruction execution system, apparatus, or device.
A computer readable signal medium may include a propagated data signal with computer readable program code embodied therein, for example, in baseband or as part of a carrier wave. Such a propagated data signal may take many forms, including, but not limited to, electro-magnetic, optical, or any suitable combination thereof. A computer readable signal medium may also be any computer readable medium that is not a computer readable storage medium and that can communicate, propagate, or transport a program for use by or in connection with an instruction execution system, apparatus, or device.
Program code embodied on a computer readable medium may be transmitted using any appropriate medium, including but not limited to wireless, wireline, optical fiber cable, RF, etc., or any suitable combination of the foregoing.
Computer program code for carrying out operations for aspects of the present invention may be written in any combination of one or more programming languages, including an object oriented programming language such as Java, Smalltalk, C + +, and conventional procedural programming languages, such as the "C" programming language or similar programming languages. The program code may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or terminal. In the case of a remote computer, the remote computer may be connected to the user's computer through any type of network, including a Local Area Network (LAN) or a Wide Area Network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet service provider).
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A microwell traversal method of a PCR chip, comprising:
s100, collecting an image of a micropore area of a PCR chip subjected to detection reaction, and converting the image into a gray-scale image;
s200, traversing micropores to extract all extreme value regions in the image, wherein the extreme value regions are one or more micropore communication regions with different gray levels;
s300, screening the extreme value region through the shape characteristics of the micropores, and obtaining a maximum stable extreme value region through the maximum extreme value region of the communicated region;
s400, acquiring the pixel area and the pixel coordinate of the micropore in the maximum stable extreme value area, and completing the inspection of the PCR chip.
2. The microwell traversal method of a PCR chip as claimed in claim 1, wherein the traversal process of the extremum region comprises:
s201, initializing an image, and establishing a stack for storing a connected region and a stack of a region boundary;
s202, selecting any micropore in the image, taking the current micropore as a current communication area, putting the current micropore into a stack, and taking the gray value of the current micropore as the gray value of the current communication area;
s203, detecting the gray value of the adjacent micropore of the micropore, if the gray value of the adjacent micropore is not more than the gray value of the current communication area,
step S204 is executed, the hole site is added into the current communication area and is put into a stack, the gray value of the hole site is used as the gray value of the current communication area, and step S203 is continuously repeated;
if the gray value of the adjacent micropore is larger than that of the current connected region,
step S205 is performed to put the hole site in the stack as the boundary of the connected region.
3. The microwell traversal method of a PCR chip as claimed in claim 2, further comprising:
s206, acquiring the gray value of the boundary of the connected region in the pile, and judging as follows:
if the obtained boundary of the connected region in the heap is empty, traversing is finished;
if the gray value of the boundary of the connected region in the stack is equal to the gray value of the current connected region in the stack, executing step S203;
if the gray value of the boundary of the connected region in the stack is not less than the gray value of the current connected region in the stack, combining the connected regions in the stack until the pixel value of the current connected region is equal to the gray value of the boundary of the connected region;
s206, the merged current connected region is set as an extremum region.
S207, if the acquired gray value of the boundary of the connected region in the stack is smaller than the gray value of the current connected region in the stack, executing the step S205.
4. The microwell traversal method of PCR chip as claimed in claim 2, wherein the gray values of the adjacent microwells at left and lower sides of the microwell are detected from the microwell at the top left corner of the image as a starting point during the traversal of the extremum region.
5. The method for traversing the microwells of a PCR chip as claimed in claim 1, wherein the obtaining of the maximum stable extremum region further comprises:
s301, screening the extreme value area through micropore shape characteristics, and deleting the area which does not accord with the micropore shape characteristics from the extreme value area, wherein the micropore shape characteristics comprise the shape, the size, the row spacing and the column spacing of micropores;
s302, adjusting the gray value of the extremum region after the result screening, and obtaining a maximum connected region to form a maximum stable extremum region.
6. A microwell ergograph device for a PCR chip, comprising:
the acquisition unit is used for acquiring an image of a micropore area of the PCR chip subjected to the detection reaction and converting the image into a gray-scale image;
the acquisition unit traverses the micropores to extract all extreme value areas in the image, wherein the extreme value areas are one or more micropore communication areas with different gray levels;
the extraction unit screens the extreme value region through the shape characteristics of the micropores and obtains a maximum stable extreme value region through the maximum extreme value region of the communication region;
and the verification unit is used for acquiring the pixel area and the pixel coordinate of the micropore in the maximum stable extremum region and completing the detection of the PCR chip.
7. A storage medium storing a plurality of instructions adapted to be loaded by a processor to perform the steps of the PCR chip microwell traversal method of claims 1 to 5.
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