CN110894462B

CN110894462B - PCR fluorescence detection device for eliminating aerosol interference

Info

Publication number: CN110894462B
Application number: CN201911229692.6A
Authority: CN
Inventors: 刘志华; 冯东平; 刘光明; 李社刚
Original assignee: EASTWIN SCIENTIFIC EQUIPMENT Inc
Current assignee: EASTWIN SCIENTIFIC EQUIPMENT Inc
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2023-08-01
Anticipated expiration: 2039-12-04
Also published as: CN110894462A

Abstract

The invention relates to a PCR fluorescence detection device for eliminating aerosol interference, which is used for detecting a PCR module and comprises the following components: a fluorescence detection assembly; and the ultraviolet radiation source is used for carrying out ultraviolet radiation on the PCR module. According to the PCR fluorescence detection device, the ultraviolet radiation source is used for carrying out ultraviolet radiation on the PCR module, the ultraviolet radiation can degrade aerosol in the air, on the surface of the PCR module and in the PCR module, interference of the aerosol is eliminated, and detection accuracy of the fluorescence detection assembly on the PCR module is improved.

Description

PCR fluorescence detection device for eliminating aerosol interference

Technical Field

The invention relates to the technical fields of molecular biology experiments, gene amplification technology and fluorescent quantitative detection and diagnosis, in particular to a PCR fluorescent detection device for eliminating aerosol interference.

Background

Along with the increasing expansion of research and application of molecular biology, the PCR (Polymerase Chain Reaction ) technology has gained wide acceptance because of its accuracy and reliability, and the DNA and RNA detection technology developed on the basis of this technology is endless and becomes one of the basic stones of molecular biology experiments, and also becomes a tool for human to recognize the microscopic world and understand itself.

The special laboratory provides clean environment, ventilation environment, professional operators and standard operation flow to ensure that the PCR reagent preparation and experimental process are not polluted. Since DNA amplification can amplify 1 copy to millions of times, on the one hand, detection is facilitated and on the other hand, a potential non-negligible risk is created. Aerosols are colloidal dispersions, also known as gas dispersions, formed by dispersing and suspending small particles of a solid or liquid in a gaseous medium. Aerosols in the air, on the surface of the test tube and on the surface of the module can affect the detection results and the subsequent experimental results can be completely erroneous.

Disclosure of Invention

In view of the above, it is necessary to provide a PCR fluorescence detection device. The method can eliminate the interference of aerosol in the PCR detection experiment, and improve the accuracy of the experiment.

A PCR fluorescence detection device for eliminating aerosol interference, for detecting a PCR module, comprising:

a fluorescence detection assembly;

and the ultraviolet radiation source is used for carrying out ultraviolet radiation on the PCR module.

According to the PCR fluorescence detection device, the ultraviolet radiation source is used for carrying out ultraviolet radiation on the PCR module, the ultraviolet radiation can degrade aerosols in the air, on the surface of the PCR module and in the PCR module, interference of the aerosols is eliminated, DNA chains in the aerosols are decomposed, so that after the aerosols enter a sample, DNA in the aerosols cannot participate in DNA amplification reaction, the aerosols cannot interfere with fluorescent signals, influence of the aerosols on PCR detection results is eliminated, and detection accuracy of the fluorescent detection assembly on the PCR module is improved.

In one embodiment, the fluorescence detection assembly includes: a bottom plate; the excitation component is arranged on the bottom plate and used for emitting excitation light to the PCR module; the detection component is used for carrying out fluorescence detection on the PCR module; the ultraviolet radiation source is arranged on the bottom plate or the excitation component.

In one embodiment, the two excitation components are formed and are arranged on the bottom plate, and the light paths of the two excitation components are parallel; the detection components are also formed with two, respectively corresponding to the two excitation components.

In one embodiment, the ultraviolet radiation source is disposed on the base plate and between the two excitation assemblies.

In one embodiment, the optical path of the detection component and the optical path of the corresponding excitation component are located on the same plane. The excitation component and the light path center axis of the detection component are coplanar to form a fluorescence detection channel.

In one embodiment, the included angle between the light path of the detection component and the light path of the corresponding excitation component is 90 °. The included angle between the light path of the detection component and the light path of the excitation component is 90 degrees, so that excitation light and ambient light passing through the top of the test tube cannot enter the detection part through refraction and reflection, and the light source background in the signal is reduced. Thereby improving the accuracy of fluorescence detection.

In one embodiment, the fluorescence detection device further comprises: the connecting frame is connected between the excitation assembly and the detection assembly; and the sliding component is used for driving the excitation component and the detection component to slide.

In one embodiment, the UV radiation source irradiates the PCR module with an intensity of 70uW/cm or more ² 。

In one embodiment, the PCR module has a plurality of light holes arranged at equal intervals, and a distance between the light paths of the two detection components is greater than a distance between the adjacent light holes.

In one embodiment, the distance between the light paths of the two detection assemblies is equal to 1.5 times the distance between adjacent light transmission holes.

Drawings

FIG. 1 is a front cross-sectional view of a PCR fluorescence detection apparatus for detecting a PCR module;

FIG. 2 is a side sectional view of a PCR fluorescence detection apparatus for detecting a PCR module;

FIG. 3 is an exploded view of a PCR fluorescence detection apparatus;

FIG. 4 is a plan view of the PCR fluorescence detection apparatus when detecting a PCR module.

Wherein: 1. a bracket; 11. a sample hole; 12. a first light hole; 13. a second light hole; 2. an excitation assembly; 21. an excitation light source; 22. an excitation light filter; 23. an excitation optical prism group; 3. a detection assembly; 31. detecting an optical prism group; 32. a fluorescence filter; 33. a channel synthesis prism group; 34. a large area photosensor; 4. a base; 5. an ultraviolet radiation source; 61. a motor; 62. a transmission shaft; 63. a fixed shaft; 64. a first belt; 65. a second belt; 7. a connecting frame; 8. a first fluorescent detection channel; 9. and a second fluorescent detection channel.

Detailed Description

The present invention will be further described in detail with reference to the drawings and the detailed description, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the detailed description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the invention.

It will be understood that when an element is referred to as being "fixed to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "left," "right," and the like are used herein for illustrative purposes only and are not meant to be the only embodiment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The first embodiment of the invention discloses a PCR fluorescence detection device for eliminating aerosol interference, which is used for detecting a PCR module and can eliminate the interference of aerosol on detection.

As shown in fig. 1, the PCR module includes: the PCR reaction test tube on support 1 with establish on support 1, a plurality of test tube sample holes 11 have been seted up to equidistant along the horizontal direction on the support 1, each PCR reaction test tube inserts respectively in each sample hole 11, combine fig. 1 and 2, set up two light trap with sample hole 11 intercommunication on the support 1, be first light trap 12 and second light trap 13 respectively, first light trap 11 is seted up in the bottom of support 1, second light trap 13 is seted up in the side of support 1, supplies PCR fluorescence detection device to detect.

The PCR fluorescence detection device comprises: fluorescence detection assembly 3, fluorescence detection assembly 3 includes: excitation subassembly 2 and detection subassembly 3, excitation subassembly 2 and detection subassembly 3 correspond with first light trap 12 and second light trap 13 on the support 1 respectively, and excitation subassembly 2 sends excitation light, shines the test tube through first light trap 12, and reagent in the test tube sends out the fluorescence of specific wavelength after being shone by excitation light, and detection subassembly 3 detects the fluorescence that reagent in the test tube sent through second light trap 13.

It should be noted that, during the detection, the detection result is easily interfered by aerosol, and the aerosol is a colloidal dispersion system formed by dispersing and suspending small solid or liquid particles in a gaseous medium, which is also called a gas dispersion system. Before and during the test, the test tube is sealed, but aerosol exists in the air, the surface of the test tube and in the light holes, and the detection result of fluorescent signals is interfered.

As shown in fig. 2 and 3, the PCR fluorescence detection device further includes: the ultraviolet radiation source 5 is arranged close to the excitation assembly 2, ultraviolet light is irradiated into the first light holes 12, aerosol in air can be degraded by ultraviolet light irradiation with certain intensity, interference of the aerosol is eliminated, and detection accuracy of the fluorescence detection assembly 3 on the PCR module is improved.

As shown in fig. 3, the fluorescence detection assembly 3 further includes: the base 4 and the excitation assembly 2 are arranged on the base 4, in this embodiment, two excitation assemblies 2 are arranged on the base 4, two detection assemblies 3 are also arranged and correspond to the excitation assemblies 2 one by one, wherein the corresponding excitation assemblies 2 are coplanar with the central axis of the optical path of the detection assembly 3, and a fluorescence detection channel is formed. The two detection modules 3 may share one photosensor 34, or may use separate photosensors 34. In the present embodiment, the two detection modules 3 share one photosensor 34, and each of the two detection modules 3 includes a channel synthesizing prism group 33, and the channel synthesizing prism group 33 guides light of two fluorescence detection channels into the photosensor 34. The ultraviolet radiation source 5 may be disposed on the excitation component 2, or may be disposed on the base 4 between the two excitation components 2, in this embodiment, the ultraviolet radiation source 5 is disposed on the base 4 and between the two excitation components 2, and may irradiate into the first light holes 12 corresponding to the two excitation components 2 on the support 1. Wherein the ultraviolet irradiation source 5 adopts a UV-C level deep ultraviolet LED lamp, and the irradiation intensity of the deep ultraviolet LED lamp irradiated to the bottom of the light hole reaches at least 70uW/cm ² Above, this intensity meets the requirements of medical ultraviolet disinfection lamps. Under ultraviolet irradiation, DNA chains in the aerosol are decomposed, and the aerosol in the air, the surface of the test tube and the inside of the light holes is eliminated, so that the aerosol is avoidedThe sol interferes with the fluorescent signal. In addition, the test tube PP material is difficult to pass through ultraviolet light, and the test tube is clung to the light hole, so that the ultraviolet light cannot penetrate through the test tube to enter the detection part, and the wavelength of the ultraviolet light is not in the passband range of the fluorescent filter 32, so that the ultraviolet light cannot influence the result of reagent fluorescence detection.

The excitation assembly 2 comprises: the excitation light source 21, the excitation optical prism group 23, and the excitation light filter 22, and the detection unit 3 includes: the detection optical prism group 31, the fluorescence filter 32 and the photoelectric sensor 34, wherein the excitation light filter 22 and the fluorescence filter 32 emit light dye or probe aiming at specific reagents, have corresponding excitation light wavelength and fluorescence wavelength, and the cross cut-off depth of the filters reaches over OD6, so that high-purity fluorescence signals are obtained. Simultaneously, the excitation component 2 emits excitation light from the bottom of the bracket 1, so that the interference of an external light source can be greatly avoided, the excitation optical prism group 23 and the detection optical prism group 31 are used for transmitting the excitation light or fluorescence on a light path, and the photoelectric sensor 34 is used for detecting the fluorescence.

In addition, it should be noted that the included angle between the light path of the detection component 3 and the light path of the excitation component 2 in this embodiment is 90 °, so that the excitation light and the ambient light passing through the top of the test tube cannot enter the detection portion through refraction and reflection, i.e. the light source background in the signal is reduced. Thereby improving the accuracy of fluorescence detection.

As shown in fig. 2, on the rack 1 of the PCR module, each sample hole 11 is arranged in the horizontal direction, each first light-transmitting hole 12 is arranged in the horizontal direction, each second light-transmitting hole 13 is arranged in the horizontal direction, and the connection line of each sample hole 11, the connection line of each first light-transmitting hole 12 and the connection line of each second light-transmitting hole 13 are parallel. The fluorescence detection assembly 3 further includes: the connecting frame 7 and the sliding component, the connecting frame 7 is connected between the excitation component 2 and the detection component 3, each excitation component 2 is connected with each detection component 3, the excitation component 2 and the detection component 3 are integrated, and the relative position and the relative included angle are always kept. The sliding component is connected with the connecting frame 7, and can drive the connecting frame 7, the excitation component 2 and the detection component 3 to move along the horizontal direction together, so as to carry out fluorescence detection on the reagent in each test tube on the PCR module.

The sliding component may be any mechanism for driving the connecting frame 7 to move, such as a rack and pinion driving mechanism, a turbine scroll driving mechanism, etc., and in this embodiment, the sliding component is a belt conveying mechanism, as shown in fig. 4, and the sliding component includes: the motor 61, the transmission shaft 62 parallel with the output shaft of the motor 61, the fixed axle 63 opposite to the transmission shaft 62, the output shaft of the motor 61 is connected with the transmission shaft 62 through the first belt 64, so that the transmission shaft 62 can rotate synchronously with the output shaft. Meanwhile, the transmission shaft 62 is connected with the fixed shaft 63 through a second belt 65, and the second belt 65 is arranged in the horizontal direction. When the motor 61 works, the second belt 65 between the transmission shaft 62 and the fixed shaft 63 can be driven to move, the connecting frame 7 is connected with the second belt 65, the detection assembly 3 and the excitation assembly 2 are driven to move under the driving of the second belt 65, the reagents in the test tubes sequentially pass through the test tubes in the sample holes 11, the excitation assembly 2 and the detection assembly 3 are not contacted with the support 1, and a smaller distance is reserved between the excitation assembly 2 and the support 1. Compared with the prior art that the fluorescence detection channel is switched through the motor 61 and the filter wheel, in the embodiment, the detection component 3 and the excitation component 2 respectively correspond to the second light hole 13 and the first light hole 12, the fluorescence filter 32 emits light to the specific reagent or the probe, has the corresponding fluorescence wavelength, reduces the switching difficulty of the fluorescence detection channel, ensures that the replacement of the fluorescence detection channel is more convenient when the detection object is replaced, and improves the experimental efficiency.

As shown in fig. 3, in the fluorescence detection device, the two detection components 3 and the excitation components 2 corresponding to the two detection components respectively form a first fluorescence detection channel 8 and a second fluorescence detection channel 9, and the slip component can complete double-color fluorescence detection by one action. The fluorescence detection device can simultaneously open two fluorescence detection channels to detect the reagent in each test tube, so that the detection efficiency is improved; the detection can also be carried out twice, and each time, one fluorescence detection channel is opened for the reagent in each test tube. The PCR detection assembly further includes: the main control module is electrically connected with the excitation light source 21, the photoelectric sensor 34 and the motor 61 and controls the opening and closing of the excitation assembly 2, the detection of the detection assembly 3 and the movement of the sliding assembly.

As shown in fig. 2, when one fluorescence detection channel is opened each time, one excitation component 2 is turned on and the other excitation component 2 is turned off in the first round of testing, and at this time, the signal obtained by the light point sensor of the detection component 3 corresponding to the turned-on excitation component 2 is the fluorescence signal of the fluorescence detection channel; and after detecting all the reagents in the test tubes, carrying out a second round of test, and sequentially detecting the reagents in each test tube by using the excitation assembly 2 which is closed in a first round, wherein the signal obtained by the light point sensor of the detection assembly 3 corresponding to the excitation assembly 2 is the fluorescent signal of the fluorescent detection channel. And (5) finishing the detection of the reagents in each test tube by the two detection channels.

As shown in fig. 1, when two fluorescence detection channels are simultaneously opened for detection, the fluorescence device simultaneously opens two excitation assemblies 2, wherein the distance between the light paths of the two excitation assemblies 2 is equal to 1.5 times the distance between the adjacent light holes. When the connecting frame 7 moves along the horizontal direction, only one excitation assembly 2 is aligned with one first light transmission hole 12 of the PCR module all the time, and the other excitation assembly 2 is staggered with the first light transmission hole 12. The two fluorescent detection channels sequentially detect each test tube, the two fluorescent channels alternately generate fluorescent signals, signal sequences of the respective channels can be obtained through time-sharing processing, detection is completed, and low background fluorescence and lower inter-channel crosstalk can be obtained during detection.

In fig. 3, the ultraviolet radiation source 5 needs to irradiate the air in the first light holes 12 and the surface of the test tube before the excitation channel excites the test tube, the ultraviolet radiation source 5 can operate independently, for example, before the test, the ultraviolet radiation source 5 is started independently, the two excitation components 2 are closed, the sliding component drives the ultraviolet radiation source 5 to irradiate each first light hole 12 and each surface of the test tube for one time, aerosol interference is eliminated, and then fluorescence test is performed, so that the accuracy of the fluorescence test can be greatly improved. The ultraviolet radiation source 5 can also synchronously operate along with fluorescence test, and can inhibit the tiny leakage aerosol pollution caused by consumable or sealing inaccuracy at any time by continuously switching on the ultraviolet radiation source 5. Thus, the PCR amplification experiment can obtain the correct analysis result.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A PCR fluorescence detection device for eliminating aerosol interference for detecting a PCR module, comprising:

a fluorescence detection assembly;

the ultraviolet radiation source is connected with the fluorescence detection assembly and used for carrying out ultraviolet radiation on the PCR module;

the PCR module comprises a bracket, wherein a plurality of test tube sample holes are formed in the bracket, a first light hole and a second light hole which are communicated with the same sample hole are also formed in the bracket, the first light hole is formed in the bottom of the bracket, and the second light hole is formed in the side face of the bracket;

the fluorescence detection assembly comprises an excitation assembly and a detection assembly, excitation light emitted by the excitation assembly irradiates the test tube through the first light holes, and the ultraviolet irradiation source is arranged close to the excitation assembly; the detection component carries out fluorescence detection on the reagent in the test tube through the second light hole.

2. The PCR fluorescence detection device of claim 1, wherein the fluorescence detection assembly includes a base plate;

the excitation component is arranged on the bottom plate;

the ultraviolet radiation source is arranged on the bottom plate or the excitation component.

3. The device for detecting PCR fluorescence with aerosol interference elimination according to claim 2, wherein two excitation components are formed and are both arranged on the bottom plate, and light paths of the two excitation components are parallel; the detection components are also formed with two, respectively corresponding to the two excitation components.

4. The aerosol-interference-free PCR fluorescence detection device as set forth in claim 3, wherein the ultraviolet radiation source is disposed on the base plate and between the two excitation assemblies.

5. A PCR fluorescence detection apparatus for eliminating aerosol interference according to claim 3, wherein the optical path of the detection assembly is in the same plane as the optical path of the corresponding excitation assembly.

6. The aerosol-interference-free PCR fluorescence detection device as set forth in claim 5, wherein the detection assembly has an optical path at an angle of 90 ° to the optical path of the corresponding excitation assembly.

7. The aerosol-interference-free PCR fluorescence detection apparatus according to claim 1, wherein the ultraviolet irradiation source irradiates the PCR module with an intensity of 70uW/cm or more ² 。

8. The apparatus of claim 3, wherein the PCR module has a plurality of light holes arranged in an equidistant manner, and a distance between light paths of the two detection components is greater than a distance between adjacent light holes.

9. The aerosol-interference-free PCR fluorescence detection device of claim 8, wherein a distance between optical paths of the two detection assemblies is equal to 1.5 times a distance between adjacent light-transmitting holes.

10. A PCR fluorescence detection apparatus for eliminating aerosol interference according to claim 2 or 3, further comprising:

the connecting frame is connected between the excitation assembly and the detection assembly;

and the sliding component is used for driving the excitation component and the detection component to slide.