CN116879377A - Horizontal agarose gel electrophoresis detection analysis system and method thereof - Google Patents

Abstract

The invention relates to a horizontal agarose gel electrophoresis detection analysis system and a method thereof, wherein the horizontal agarose gel electrophoresis detection analysis system comprises: the device comprises a base for supporting, a supporting rod, an electrophoresis power connector, a light source fixing piece, a camera, a lens, an adjustable light source, a special electrophoresis tank and a precise moving platform; the optical imaging system is arranged in pairs by adopting side light sources, the illumination angle can be adjusted according to the requirement, the uniform irradiation of large-size plane gel is satisfied, the uniform excitation of fluorescent marked nucleic acid strips in the gel is realized, the light sources and the front of a camera are both provided with optical filters for reducing background light, and the camera is directly connected with a computer for real-time imaging and analysis. Compared with the prior art, the system can realize the analysis of the change of the gel electrophoresis strip along with time, and has the advantages of quick separation, on-line monitoring, imaging and analysis, high stability, high flux and the like.

Description

Horizontal agarose gel electrophoresis detection analysis system and method thereof

Technical Field

The invention relates to the technical field of agarose gel electrophoresis, in particular to a horizontal agarose gel electrophoresis detection analysis system and a method thereof.

Background

Agarose gel electrophoresis is widely used in the fields of biochemistry, molecular biology, medical diagnosis and the like as one of reliable separation technologies of biomacromolecules such as nucleic acid and the like. The information of purity, concentration, fragment size and the like of the nucleic acid in plasmid extraction verification, PCR amplification and gene sequencing can be obtained through gel electrophoresis separation of DNA and RNA. The development of the biomedical field provides higher and higher requirements for high throughput, high stability, usability and easy analysis of the detection of the agarose gel electrophoresis nucleic acid of the plate, and particularly, the online quantitative analysis of gel results can accelerate the whole biochemical analysis process, shorten the separation analysis period and improve the research and development efficiency.

First, traditional agarose gel electrophoresis adopts horizontal electrophoresis tank, realizes separation, identification and purification of different fragment nucleic acid, generally needs four steps of gel preparation, sample application, electrophoresis and result analysis, and whole experimental process separation, step are numerous, easily lead to experimental condition and electrophoresis process parameter to have the difference, are difficult to satisfy the repeatability and the accuracy of electrophoresis separation result.

Secondly, the existing agarose gel electrophoresis online observation technology has the problem of low sensitivity, and the existing nucleic acid agarose gel electrophoresis realizes electrophoresis observation (patent number: CN 1766581 A,CN211402238U,CN 203249894U), but the imaging mode is usually transmission imaging, and a light source is arranged below the gel, so that the material requirement on an electrophoresis tank is good in light transmittance on one hand, and the filter performance requirement on a filter plate is high on the other hand. When the excitation light source spectrum and the fluorescence spectrum are overlapped, the background interference is increased, the signal to noise ratio is increased, and the detection sensitivity is reduced.

Third, accurate quantification of agarose gel electrophoresis results is difficult, and information such as nucleic acid purity, concentration and fragment size is often required for nucleic acid gel electrophoresis. The migration distance and the band broadening of the same nucleic acid band are increased along with the increase of the electrophoresis time, and when the signal ratio of a plurality of nucleic acid bands is large in difference, particularly when background signal interference exists, the calculation of low signal intensity is easy to generate errors and cannot be accurately quantified.

The interference of these multifactorial factors greatly reduces the feasibility of quantitative analysis of the gel electrophoresis strip, and even leads to the failure of the electrophoresis result to realize quantitative analysis. The development of the integration of the electrophoresis device and the imaging detection has become a necessary trend. However, simply integrating the electrophoresis apparatus with the imaging apparatus may introduce some unexpected problems, such as uneven imaging surface, deflection, and the like, which may not accurately control parameters involved in the electrophoresis process, and the difficulty of implementing reliable quantitative analysis based on imaging is high. Therefore, intensive research and study on agarose gel electrophoresis real-time detection systems is necessary.

Disclosure of Invention

The invention aims to solve the problems and provide a horizontal agarose gel electrophoresis detection analysis system and a method thereof, wherein an electrophoresis device and an optical imaging device are integrated, the experimental process is flow-processed, the online monitoring and quantitative analysis of agarose gel electrophoresis are realized, the operation unification in the gel electrophoresis process is realized through the matching integration of the electrophoresis device and the optical imaging device, the determination of electrophoresis and imaging process parameters is increased, the accurate imaging of agarose gel electrophoresis results is realized, and the quantitative analysis of strips is realized. In order to realize the real-time detection and accurate quantitative analysis of gel electrophoresis strips, the uniform irradiation of a surface light source to the gel surface is the basis, and meanwhile, a special electrophoresis tank under uniform irradiation only generates low background, so that the accurate and sensitive detection of the strips in the gel is realized.

The aim of the invention is achieved by the following technical scheme:

a first object of the present invention is to provide a horizontal agarose gel electrophoresis detection analysis system, comprising a base, a support bar, an optical imaging device and an electrophoresis device; the base, the support rod and the side plates (the side plates and the support rod are integrated) form a closed box body (shading box), and the optical imaging device and the electrophoresis device are arranged in the box body; the optical imaging device comprises a light source fixing piece, a camera, a lens and an adjustable light source; the electrophoresis device comprises a special electrophoresis tank and a precise moving platform; the precise mobile platform is arranged on the base; the special electrophoresis tank is placed on a precise moving platform; the light source fixing piece is arranged above the side of the special electrophoresis tank, and the adjustable light source is arranged on the light source fixing piece; the camera fixing piece is arranged above the adjustable light source, and the camera is provided with a lens.

Further, the system also comprises an electrophoresis power connector; the upper electrode of the special electrophoresis tank is connected with an electrophoresis power connector, and the power connector is directly connected with an electrophoresis power.

Further, a track is arranged on the base, and the precise mobile platform is arranged on the track; the precise moving platform is controlled by a servo motor to move along the track in an oriented manner.

Further, the servo motor is in transmission connection with the screw rod, and the precise moving platform is moved along the track in a directional manner through the screw rod.

Further, the precise moving platform is controlled by a servo motor, and the precise moving platform is controlled to move a special electrophoresis tank into and out of a closed shading box, so that gel is placed, buffer solution is added and sample is loaded when the precise moving platform moves out of the box, and electrophoresis and imaging are performed in a closed environment after the precise moving platform moves into the box; the center shaft of the precise moving platform is aligned with the center positions of the adjustable light source and the lens, so that the special electrophoresis tank is ensured to be placed in the middle of the adjustable light source and aligned with the center position of the camera, and the image result of the imaging surface is ensured to be uniform.

Furthermore, the camera is directly connected with a computer, so that online imaging detection and analysis are realized.

Further, filters are arranged in front of the adjustable light source and the lens of the camera so as to reduce the overlapping area between the spectrum of the excitation light source and the spectrum of the fluorescent dye, reduce interference background and improve detection sensitivity.

Furthermore, the adjustable light source is an LED light source, the illumination angle can be adjusted according to the requirement, the uniform irradiation of the large-size planar gel is met, and the uniform excitation of the fluorescent marked nucleic acid strips in the gel is realized.

Further, the LED light source is a surface light source; the LED light sources are arranged above the sides of the special electrophoresis tank in pairs, the distance between the two light sources is not smaller than the width of the special electrophoresis tank, and the LED light sources are kept at a certain height above the special electrophoresis tank.

Further, the adjustable light source is an LED array area light source, a uniform filter is arranged in front of the light source, the filter cuts off the emission wavelength of the fluorescent dye (the short-pass filter can be selected to only transmit the light of the excitation wavelength of the fluorescent dye), so that background stray light is reduced, the background is reduced, and the signal to noise ratio is improved; a light-equalizing plate is arranged in front of the light source to ensure that the light source emits uniformly; the light source is a strip-shaped uniform light source, the length of the light source exceeds the length of the gel irradiation surface, the incident angle of the light source is set to be timely adjusted according to the imaging result, and the uniform illumination of the gel area is ensured.

Further, the camera is centrally arranged at the center of the adjustable light source, and the bottommost end of the lens connected with the camera is higher than the adjustable light source by a certain distance; and a light filter is arranged in front of the lens, and the background can be reduced by filtering out stray light only through light with the wavelength emitted by the fluorescent dye.

Further, the special electrophoresis tank is made of black light absorption, insulation and corrosion resistance materials (low reflection opaque materials); the special electrophoresis tank comprises a gel area and a non-gel area; and the special electrophoresis tank is covered with a special upper cover for shielding a non-gel area in the special electrophoresis tank.

Preferably, the special electrophoresis tank can be black Polycarbonate (PC), or transparent polymethyl methacrylate (PMMA) surface is coated with black light-absorbing and corrosion-resistant paint, and other materials and treatment methods meeting the conditions can also be adopted.

Further, the surface of the special electrophoresis tank is required to have a certain roughness, so that the background light is kept low under the illumination, and the special electrophoresis tank is designed to contain gel of 12X 16cm at maximum.

Further, the special upper cover is made of black light absorbing material and can be made of black light absorbing material; the gel is fixed at the center of the special electrophoresis tank, so that the gel area is completely exposed under the light source, the shape of the special upper cover can be changed, the gel loading operation is not influenced, and the background interference of the non-gel area is reduced.

Preferably, the special upper cover is made of a high polymer material such as black PC or PMMA.

The second object of the present invention is to provide a method for detecting and analyzing the horizontal agarose gel electrophoresis, which adopts the system to realize the real-time detection and accurate quantitative analysis of the gel electrophoresis strip, and the method comprises the following steps:

firstly, preparing blank gel by using a mould, placing the gel into a special electrophoresis tank after the gel is solidified, adding a proper amount of electrophoresis buffer solution, covering an upper cover of the electrophoresis tank, and adding a sample solution into a sample hole;

controlling a servo motor, moving a precise moving platform, moving a special electrophoresis tank to the most suitable imaging position, closing a box body, setting electrophoresis voltage and time, starting electrophoresis, setting parameters such as light source exposure time, photographing time step and the like, and photographing an electrophoresis photo by a camera according to a given time step in the electrophoresis process or observing the electrophoresis process in real time according to the need;

and thirdly, selecting a target area according to the recorded and stored electrophoresis pictures, comparing the separated bands in the analysis gel, and obtaining the time-varying parameters of the bands in the nucleic acid electrophoresis process to obtain the information of the relative concentration, peak width, peak area, intensity and the like of the nucleic acid bands.

Compared with the prior art, the invention has the following beneficial effects:

1) According to the horizontal agarose gel electrophoresis detection analysis system and the method thereof provided by the invention, the fluorescent dyed electrophoresis strip in the gel emits light to the sensor through the lens by excitation of the measuring light source, and the strip state in the gel can be recorded in real time in the electrophoresis process.

2) According to the horizontal agarose gel electrophoresis detection analysis system and the method thereof, which are provided by the invention, the migration of the strip can be tracked according to the recorded time sequence change of the strip in the gel, and the rapid analysis of the separated nucleic acid strip can be further performed based on the recorded time sequence strip.

3) The horizontal agarose gel electrophoresis detection analysis system and the method thereof can carry out quick separation analysis on samples based on fluorescence staining nucleic acid electrophoresis, are suitable for examining electrophoresis process changes of electrophoresis strips, and provide a quick separation analysis system and a strip analysis method based on an online imaging system for nucleic acid detection in the fields of biology, medicine, agriculture and the like.

Drawings

FIG. 1 is a schematic cross-sectional view of a horizontal agarose gel electrophoresis detection analysis system in an embodiment.

FIG. 2 is a run-time electrophoresis tank, light measuring source and camera center alignment of the horizontal agarose gel electrophoresis detection analysis system prepared in the example.

FIG. 3 shows a comparison of different imaging modes and the electrophoresis results of the present invention.

FIG. 4 shows the results (B) of the camera recording the distribution position (A) of the separated nucleic acid bands in the gel and the conversion of the nucleic acid bands into an electropherogram at the same time step when the present invention is operated.

FIG. 5 shows the variation of the peak area (A), peak value (B), peak position (C) and half-width (D) of 5 nucleic acid bands with separation time obtained by further analysis of the electrophoresis pattern after conversion of the electrophoresis bands according to the present invention at the same time step recorded by a camera.

In the figure:

1 base, 2 bracing pieces, 3 electrophoresis power connector, 4 light source mounting, 5 camera mounting, 6 camera, 7 camera lens, 8 adjustable light source, 9 special electrophoresis tank, 10 precision movement platform.

Detailed Description

The invention is described in detail below in connection with specific examples, but in no way limits the invention. In the technical scheme, the characteristics of preparation means, materials, structures or composition ratios and the like which are not explicitly described are regarded as common technical characteristics disclosed in the prior art.

A plate agarose gel electrophoresis detection analysis system mainly comprises: the device comprises a base for supporting, a supporting rod, an electrophoresis power connector, a light source fixing piece, a camera, a lens, an adjustable light source, a special electrophoresis tank and a precise moving platform; the optical imaging system is arranged in pairs by adopting side light sources, the illumination angle can be adjusted according to the requirement, the uniform irradiation of large-size plane gel is satisfied, the uniform excitation of fluorescent marked nucleic acid strips in the gel is realized, the light sources and the front of a camera are both provided with optical filters for reducing background light, and the camera is directly connected with a computer for real-time imaging and analysis. The special agarose gel electrophoresis tank for fluorescent imaging adopts a low-reflection opaque material, increases the surface roughness, reduces the background reflection, improves the detection sensitivity, meets the requirement of agarose gel high-sensitivity imaging, and is connected with a power supply for electrophoresis by an electrophoresis power supply connector. The electrophoresis tank is placed on a precise moving platform, and the front and back movement of the electrophoresis tank is controlled by a servo motor to realize accurate positioning of gel imaging. Based on the device, the analysis of the gel electrophoresis strip change with time can be realized.

Example 1

As shown in fig. 1, the horizontal agarose gel electrophoresis detection analysis system of the invention comprises a base 1 for supporting, a supporting rod 2, an optical imaging device and an electrophoresis device; the base 1, the support rods 2 and the side plates form a closed cavity box body together, and the optical imaging device and the electrophoresis device are arranged in the box body; the optical imaging device and the electrophoresis device are arranged in the box body; the optical imaging device comprises a light source fixing piece 4, a camera fixing piece 5, a camera 6, a lens 7 and an adjustable light source 8; the electrophoresis device comprises a special electrophoresis tank 9 and a precise mobile platform 10; the precise moving platform 10 is arranged on the base 1, and the special electrophoresis tank 9 is arranged on the precise moving platform 10 and can move along with the precise moving platform 10; the light source fixing piece 4 is arranged on the base 1, and the light source fixing piece 4 is higher than the electrophoresis tank 9 by a certain distance; the adjustable light source 8 is arranged on the light source fixing piece 4, and the arranged adjustable light source 8 can rotate by 90 degrees to ensure that the light source can uniformly irradiate the gel; the camera fixing piece 5 is arranged on the base 1, the camera 6 is arranged on the camera fixing piece 5, and the lens 7 is connected to the camera 6; the lowest end of the lens 7 is located a distance above the adjustable light source 8.

Filters are arranged in front of the adjustable light source 8 and the lens 7, and are used for reducing the background and improving the detection sensitivity.

The adjustable light source 8 is a led array area light source, a uniform filter is arranged in front of the light source, the emission wavelength of the filter cut-off fluorescent dye can select light with a short-pass filter which only penetrates through the excitation wavelength of the fluorescent dye, background stray light is reduced, the background is reduced, and the signal to noise ratio is improved; a light-equalizing plate is arranged in front of the light source to ensure that the light source emits uniformly; the light source is a strip uniform light source, the length of the light source exceeds the length of the gel irradiation surface, the incident angle of the light source is set with a reference imaging result, the rotation angle is adjusted timely, and the uniform illumination of the gel area is ensured; the light sources are placed above the sides of the special electrophoresis tank 9 in pairs, the distance between the two light sources is not smaller than the width of the special electrophoresis tank 9, and the light sources are kept at a certain height above the special electrophoresis tank 9.

As shown in fig. 2, under the working condition, the precise moving platform 10 can be adjusted to move the special electrophoresis tank 9 to a set position, at this time, the special electrophoresis tank 9 is aligned with the adjustable light source 8 symmetrically placed above the side, and at the same time, the lens 7 is positioned at the center of the gel imaging surface; the whole cavity is in a closed state under the working condition.

The camera 6 is directly connected with a computer, and can observe and collect fluorescent nucleic acid strip images in the electrophoresis process in real time and analyze the fluorescent nucleic acid strip images.

The electrophoresis power connector 3 is directly connected with an electrophoresis power supply to provide separation voltage for gel electrophoresis.

The special electrophoresis tank 9 is connected with the electrophoresis power connector 3 only at the working position through the metal electrode; the gel placing area in the special electrophoresis tank 9 is compatible with the size of a commercial gel making tool; the special electrophoresis tank 9 is made of opaque black material, and the light surface is further roughened to reduce reflection.

The invention can observe fluorescent stained nucleic acid bands in real time by a computer connected with the camera 6, and can record fluorescent nucleic acid electrophoresis bands directly based on given time steps.

After the recorded fluorescent nucleic acid electrophoresis strip chart is converted into an electrophoresis spectrogram, a series of parameters such as the peak area, the peak value, the peak position, the half-peak width and the like of the strip can be obtained; in the case of other condition determination, quantitative analysis can be realized according to the electrophoresis pattern.

The method of the horizontal agarose gel electrophoresis detection analysis system comprises the following steps:

(1) The special electrophoresis tank 9 is moved out by the precise moving platform 10, blank gel is prepared according to the requirement, electrophoresis is carried out after a proper amount of electrophoresis buffer is filled, the sample is subjected to fluorescent staining, the special electrophoresis tank 9 is moved to a working position by the precise moving platform 10, and the whole box body is sealed.

(2) Before the electrophoresis is started, setting optical imaging parameters and taking a picture as a background picture; after the electrophoresis is started, according to a set time step, the camera 6 automatically captures fluorescence-stained nucleic acid electrophoresis separation pictures (as shown in fig. 3) at different times, and the whole electrophoresis process can be seen on a computer at any time.

(3) And converting the recorded fluorescence-stained nucleic acid electrophoresis pictures into electrophoresis spectrograms at different times, and calculating the peak areas, peak values, peak positions and half-peak widths of corresponding bands according to the spectrograms (shown in figures 4 and 5).

(4) And carrying out subsequent analysis according to the calculation result, and further calculating the information such as the purity, concentration, fragment size and the like of the nucleic acid by tracking the time-varying condition of parameters.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Claims (10)

1. A horizontal agarose gel electrophoresis detection analysis system, which is characterized by comprising a base (1), a support rod (2), an optical imaging device and an electrophoresis device;

the base (1) and the support rod (2) form a closed box body, and the optical imaging device and the electrophoresis device are arranged in the box body;

the optical imaging device comprises a light source fixing piece (4), a camera fixing piece (5), a camera (6), a lens (7) and an adjustable light source (8);

the electrophoresis device comprises a special electrophoresis tank (9) and a precise moving platform (10);

the precise moving platform (10) is arranged on the base (1);

the special electrophoresis tank (9) is arranged on the precise moving platform (10);

the light source fixing piece (4) is arranged above the side of the special electrophoresis tank (9), and the adjustable light source (8) is arranged on the light source fixing piece (5);

the camera fixing piece (5) is arranged above the adjustable light source (8), and the camera (6) is provided with the lens (7).

2. A horizontal agarose gel electrophoresis detection analysis system according to claim 1, wherein the system further comprises an electrophoresis power connector (3);

the upper electrode of the special electrophoresis tank (9) is connected with the electrophoresis power connector (3), and the power connector (3) is directly connected with the electrophoresis power.

3. The horizontal agarose gel electrophoresis detection analysis system according to claim 1, wherein a rail is provided on the base (1), and the precision moving platform (10) is mounted on the rail.

4. The horizontal agarose gel electrophoresis detection and analysis system according to claim 1, wherein the camera (6) is directly connected with a computer, so that online imaging detection and analysis are realized.

5. The horizontal agarose gel electrophoresis detection analysis system according to claim 1, wherein filters are arranged in front of the lenses of the adjustable light source (8) and the camera (6) to reduce the overlapping area between the spectrum of the excitation light source and the spectrum of the fluorescent dye, and simultaneously reduce the interference background and improve the detection sensitivity.

6. The horizontal agarose gel electrophoresis detection analysis system according to claim 1, wherein the adjustable light source (8) is an LED light source.

7. The horizontal agarose gel electrophoresis detection and analysis system according to claim 6, wherein the LED light source is a surface light source;

the LED light sources are arranged above the sides of the special electrophoresis tank (9) in pairs, the distance between the two light sources is not smaller than the width of the special electrophoresis tank (9), and the LED light sources are kept at a certain height above the special electrophoresis tank (9).

8. The horizontal agarose gel electrophoresis detection analysis system according to claim 1, wherein the material of the special electrophoresis tank (9) is black light absorption, insulation and corrosion resistance material;

the special electrophoresis tank (9) comprises a gel area and a non-gel area;

the special electrophoresis tank (9) is covered with a special upper cover for shielding a non-gel area in the special electrophoresis tank (9).

9. The horizontal agarose gel electrophoresis detection and analysis system according to claim 8, wherein the material of the specially-made upper cover is black light-absorbing material;

the gel is fixed at the center of the special electrophoresis tank (9) to ensure that the gel area is completely exposed to the light source.

10. A method of a horizontal agarose gel electrophoresis detection assay system according to any of claims 1 to 9 comprising the steps of:

firstly, preparing blank gel by using a mould, placing the gel into a special electrophoresis tank (9) after the gel is solidified, adding a proper amount of electrophoresis buffer solution, and adding a sample solution into a sample hole;

moving the precise moving platform (10), moving the special electrophoresis tank (9) to the most suitable imaging position, closing the box body, setting electrophoresis voltage and time, starting electrophoresis, setting parameters such as light source exposure time, photographing time step and the like, and photographing an electrophoresis photo by the camera (6) according to a given time step in the electrophoresis process or observing the electrophoresis process in real time according to the requirement;

and thirdly, selecting a target area according to the recorded and stored electrophoresis pictures, comparing the separated bands in the analysis gel, and obtaining the time-varying parameters of the bands in the nucleic acid electrophoresis process to obtain the relative concentration, peak width, peak area and intensity of the nucleic acid bands.