CN113862143A

CN113862143A - Independent fluorescence channel detection system for real-time fluorescence quantitative PCR

Info

Publication number: CN113862143A
Application number: CN202111266723.2A
Authority: CN
Inventors: 谭文敏; 杨奇贤
Original assignee: Shenzhen Bode Zhiyuan Biotechnology Co ltd
Current assignee: Shenzhen Bode Zhiyuan Biotechnology Co ltd
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2021-12-31

Abstract

The invention discloses an independent fluorescence channel detection system for real-time fluorescence quantitative PCR, which belongs to the technical field of biological detection and comprises a light source, a light filter, a dichroic mirror and a photoelectric sensing module. The light source is used for emitting light to the detection sample, and the optical filter is used for screening light with specific wavelength in the light. The dichroic mirror is used for reflecting the light screened by the filter and detecting fluorescence excited by the light on the sample. The photoelectric sensing module is used for detecting the intensity of the emitted fluorescent signal. Through the scheme, a dynamic moving assembly is not required to be arranged, the fluorescence channel can detect the fluorescence emitted by different substances simultaneously, the integrated fluorescence detection module is adopted, the material quantity is reduced, the processing and assembling time is saved, the maintenance is also convenient, the cost is lower, the moving parts among modules are reduced, and the detection precision and efficiency are provided.

Description

Independent fluorescence channel detection system for real-time fluorescence quantitative PCR

Technical Field

The application relates to the technical field of detection, in particular to an independent fluorescence channel detection system for real-time fluorescence quantitative PCR.

Background

The digital PCR technology is an absolute nucleic acid molecule quantitative technology as a third-generation PCR technology. Digital PCR improves upon traditional PCR by dividing a PCR reaction into many smaller PCR reactions, each of which on average includes no more than one target nucleic acid molecule. Each small reaction contains approximately 1 or 0 target nucleic acid molecules, and at the end of PCR amplification, gives a positive or negative binary reading, and the initial copy number or concentration of the target nucleic acid molecule is determined based on the Poisson distribution principle and the number and ratio of positive droplets. Compared with the traditional fluorescent quantitative PCR, the digital PCR has more excellent sensitivity, specificity and accuracy, and is widely applied to the fields of tumor detection, prenatal diagnosis, single cell research and the like.

The current fluorescent detection implementation schemes of real-time fluorescent quantitative PCR in the market are three as follows: 1. a movable multi-fluorescence channel detection system and a movable multi-fluorescence channel detection method mainly adopt a moving mechanism to drive a detection module to scan above a sample pore plate in sequence, the detection module moves at high frequency, so that the test precision is reduced, the service life is shortened, the maintenance is inconvenient, one fluorescence detection channel is damaged, the whole fluorescence detection channel needs to be replaced, and the maintenance cost is high;

2. one is to adopt a plurality of detection heads of the full-coverage matrix type, light up scanning in turn during testing, this kind of mode probe is many, with high costs, and there are differences among every probe, too many probes will cause the detection error to be big, influence the experimental contrast effect;

3. the PCR temperature control module of movable many reaction tubes mainly adopts moving mechanism to drive PCR temperature control module at the below horizontal migration that detects the module, when removing to the detection module below, and integrated CCD module can shoot a plurality of reaction tubes, can cause the optical path difference like this, and the detection effect is insensitive, and is with high costs moreover.

Moreover, most importantly, the above schemes can only realize the fluorescence reaction detection of a single fluorescence excitation light signal by means of a single light source, but cannot realize multiple fluorescence reaction detections. Based on this, it is necessary to provide an independent fluorescence channel detection system for real-time fluorescence quantitative PCR, so as to solve the above problems.

Disclosure of Invention

It is an object of the embodiments of the present application to provide a real-time fluorescence quantitative PCR independent fluorescence channel detection system capable of solving the above-mentioned technical problems.

An independent fluorescence channel detection system for real-time fluorescence quantitative PCR, comprising:

the light source is used for emitting light to the detection sample;

the optical filter is used for screening light rays with specific wavelengths in the light rays;

a dichroic mirror for reflecting the light filtered by the filter and for reflecting fluorescence excited by the light passing through the detection sample;

the photoelectric sensing module for detecting and emitting the intensity of the fluorescence signal comprises:

a substrate;

the array is arranged on the plurality of photoelectric sensing elements on the substrate;

the light filter film is arranged in front of the photosensitive element and is configured to screen fluorescence of the detection sample excited by light rays by at least two wavelengths.

Further, the filter film is provided with different thicknesses along the direction of the filter film, so that the passing light passes through the filter film and is screened into fluorescence with different wavelengths.

Further, the filter film cross section is provided as a stepped structure having two or more stepped section planes.

Further, the dichroic mirror is arranged in an inclined manner at 45 degrees, the light source and the photoelectric sensing module are respectively arranged on one horizontal side and above the dichroic mirror, and the detection sample is arranged below the dichroic mirror.

Furthermore, a collimation optical module is arranged between the light source and the optical filter.

Further, still include the thermoblock that is used for supplying the detection sample to place, the thermoblock is equipped with temperature control device.

Furthermore, the temperature control device is a peltier, the peltier is arranged on the lower side of the temperature block, and a radiator is further arranged on the lower side of the peltier.

Further, the optical filter is an optical filter.

Furthermore, the filter is a rotatable filter wheel, and comprises at least two filters for filtering at least two wavelengths of light.

Furthermore, the optical filters are arranged in a fan shape, the optical filters jointly form a circular filter wheel, and the centers of the filter wheels are staggered with the center of the light source, so that the light rays do not pass through the centers of the filter wheels.

The invention has the beneficial effects that: through the above scheme, need not to set up dynamic removal subassembly, the light that the light source sent arouses the fluorescent material production fluorescence of reaction tube sample through light filter and dichroic mirror redirecting in proper order, the fluorescence of arousing crosses the filter membrane filtration of dichroic mirror final in integrated photoelectric sensing module in proper order again and crosses multiple pure fluorescence, and carry out photoelectric conversion, thereby single reaction tube independent multi-wavelength's PCR fluorescence channel detection has been realized, the fluorescence that a fluorescence channel can detect different fluorescent material simultaneously has been realized, adopt integrated fluorescence detection module simultaneously, reduce the material quantity, processing and assembly time have been saved, it is also convenient to maintain, the cost is lower, and the moving part between each module has been reduced, the precision and the efficiency of detection have been provided.

Drawings

In order to more clearly illustrate the solution of the present application, the drawings needed for describing the embodiments of the present application will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and that other drawings can be obtained by those skilled in the art without inventive effort.

FIG. 1 is a schematic cross-sectional view of an embodiment of the independent fluorescence channel detection system of the real-time fluorescence quantitative PCR of the present invention;

FIG. 2 is a schematic plan view of a photoelectric sensing module of the independent fluorescence channel detection system for real-time fluorescence quantitative PCR shown in FIG. 1;

FIG. 3 is a schematic cross-sectional plan view of the optoelectronic sensing module;

FIG. 4 is a schematic cross-sectional view of a filter of the optoelectronic sensor module shown in FIG. 3;

FIG. 5 is a schematic diagram of a partial cross-sectional structure of a filter in another embodiment;

FIG. 6 is a schematic plan view of a portion of the filter of the independent fluorescence channel detection system of the real-time quantitative PCR shown in FIG. 1;

FIG. 7 is a schematic plan view of a portion of an optical filter according to another embodiment.

Detailed Description

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 application belongs; the terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "including" and "having," and any variations thereof, in the description and claims of this application and the description of the above figures are intended to cover non-exclusive inclusions.

Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It is explicitly and implicitly understood by one skilled in the art that the embodiments described herein can be combined with other embodiments.

In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

As shown in fig. 1 to 7, the independent fluorescence channel detection system for real-time fluorescence quantitative PCR according to the embodiment described in the present application includes: light source 12, optical filter 14, dichroic mirror 4 and photoelectric sensing module 1. The light source 12 is used for emitting light to the test sample 11, and the filter 14 is used for screening light with a specific wavelength. The dichroic mirror 4 is used to reflect the light screened by the filter 14 and pass the fluorescence excited by the light on the detection sample 11. The photoelectric sensing module 1 is used for detecting the intensity of the emitted fluorescent signal.

Specifically, the light source 12 is an LED light source 12, and may be a tungsten halogen lamp or a laser light source, and the present invention does not limit the specific form of the light source 12. The photoelectric sensing module 1 includes: the photoelectric sensing device comprises a substrate 33, a plurality of photoelectric sensing elements 31 arranged on the substrate 33 in an array manner, and a filter film 32 arranged in front of the photosensitive elements. The filter 32 is used to filter the excitation light for exciting the sample 11 to pass only the fluorescence generated by the sample 11, so as to derive at least two fluorescence lights with different wavelengths. The photoelectric sensing elements 31 are used for respectively receiving the fluorescence with different wavelengths passing through the filter film 32, and converting the fluorescence into corresponding electric signals to be transmitted to an external receiving unit through the circuit interface 15 integrated on the substrate 33.

Through the above scheme, need not to set up dynamic removal subassembly, the light that light source 12 sent arouses the fluorescent material of reaction tube lens 11 through light filter 14 and dichroic mirror 4 in proper order and produces fluorescence, the fluorescence of arousing changes into parallel fluorescence through dichroic mirror 4 again in proper order, filter film 32 in integrated photoelectric sensing module 1 filters out multiple pure fluorescence at last, and carry out photoelectric conversion, thereby the independent multichannel PCR fluorescence passageway of single reaction tube detects, it can detect the fluorescence of different wavelength simultaneously to have realized a fluorescence passageway, adopt integrated fluorescence detection module simultaneously, reduce material quantity, processing and assemble duration have been saved, it is also convenient to maintain, the cost is lower, and the moving part between each module has been reduced, the precision and the efficiency of detecting have been improved. However, according to the same idea, the optical components such as the light source 12, the optical filter 14, the dichroic mirror 4, and the photoelectric sensing module 1 can be configured to be movable, so as to ensure that a plurality of reaction tube samples can be detected, and such structural changes still fall within the protection scope of the present invention.

Further, the filter 32 is configured to have different thicknesses in its own direction, so that the fluorescence passing through the filter 32 is screened for fluorescence of different wavelengths. Specifically, the filter film 32 is configured in a stepped structure having two or more stepped segment planes in cross section. In other embodiments, the filter 32 may be configured as a triangle or trapezoid, and such structural variations still fall within the scope of the present invention. Therefore, light rays with certain wavelengths can be filtered through the filter films 32 with different thicknesses, the required light ray wavelengths are reserved, and therefore the light rays are received by the corresponding photoelectric sensing elements 31, a system is formed for detecting multiple fluorescence reactions at the same time, and the integrated performance is better.

Preferably, the number of the photoelectric sensing elements 31 is 9, which respectively receive 9 different fluorescent lights and are uniformly arranged along the edge of the substrate 33. However, the present invention does not set an upper limit to the number of the specific photo-sensor elements 31, and more than 1 photo-sensor element 31 is within the protection scope of the present invention. Meanwhile, a baffle 34 is disposed around the substrate 33, the filter 32 is disposed at an end of the baffle 34, and the baffle 34 serves to fix the filter 32 and prevent the leakage light from affecting the photoelectric sensing element 31 to receive unnecessary light, thereby affecting the detection result. The PCB board 2 can be arranged on the back of the substrate 33, and the detection data can be exported through the interface 15 on the PCB board 2.

In this embodiment, the dichroic mirror 4 is disposed at an angle of 45 degrees, the light source 12 and the photoelectric sensing module 1 are respectively disposed on one horizontal side and above the dichroic mirror 4, and the detection sample 11 is disposed below the dichroic mirror 4. In another embodiment, the light source 12 may be disposed above the dichroic mirror 4, and the photoelectric sensing module 1 is disposed on the horizontal side of the dichroic mirror 4.

In another embodiment, the dichroic mirror 4 is disposed on one horizontal side of the detection sample 11, and the light source 12 and the photoelectric sensing module 1 are disposed on the other horizontal side and above the dichroic mirror 4, respectively, or the light source 12 may be disposed above the dichroic mirror 4, and the photoelectric sensing module 1 is disposed on the other horizontal side of the dichroic mirror 4.

In this embodiment, a collimating optical module 13 is further disposed between the light source 12 and the optical filter 14, and is used for collimating the light emitted from the light source 12, so as to more stably react on the detection sample 11. And a collimating optical module 3 may be disposed in front of the photodetecting module 1 to ensure the collimation of the light inputted into the photodetecting module 1.

In this embodiment, the detection sample 11 is placed in a reaction tube, and the reaction tube is placed in the temperature block 8, specifically, the temperature block 8 is provided with a counter bore for inserting the reaction tube. The temperature block 8 is provided with a temperature control device. The temperature control device is a Peltier 9, the Peltier 9 is arranged on the lower side of the temperature block 8, and a radiator 10 is further arranged on the lower side of the Peltier 9. In other embodiments, a resistance heater or an electromagnetic heating method may be used, and the present invention is not limited to the specific form of the temperature control device, and any conventional controllable heating method may be applied to this embodiment.

In one embodiment, the optical filter 14 is an optical filter 141, and the light source 12, the optical filter 141, and the dichroic mirror 4 are jointly fixed in the optical fixing frame 5 to form a stable connection relationship, so as to avoid detection errors caused by deviation due to movement or vibration.

In another embodiment, the filter 14 is a rotatable filter wheel, and includes at least two filters 141 for filtering at least two wavelengths of light. So, then can then set up the light of excitation before can following the excited of detection sample 11, possess the same function that detects sample 11 of different light excitations to improve and integrate the performance.

Specifically, the optical filters 141 are arranged in a fan shape, as shown in fig. 6, the optical filters 141 together form a circular filter wheel, and the center of the filter wheel is staggered from the center of the light source 12, so that the light does not pass through the center of the filter wheel. Different optical filters 141 are arranged in different fan-shaped areas, and the filter wheel can be driven to rotate by configuring a rotating mechanism, so that the effect of emitting light rays with different wavelengths by rotating the filter wheel is achieved.

In other embodiments, a circular or other shape filter 141 may be embedded on the filter wheel to set the light passing diameter, as shown in fig. 7.

It is to be understood that the above-described embodiments are merely illustrative of some, but not restrictive, of the broad invention, and that the appended drawings illustrate preferred embodiments of the invention and do not limit the scope of the invention. This application is capable of embodiments in many different forms and is provided for the purpose of enabling a thorough understanding of the disclosure of the application. Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that the present application may be practiced without modification or with equivalents of some of the features described in the foregoing embodiments. All equivalent structures made by using the contents of the specification and the drawings of the present application are directly or indirectly applied to other related technical fields and are within the protection scope of the present application.

Claims

1. Real-time fluorescence quantitative PCR's independent fluorescence channel detecting system, its characterized in that includes:

the light source is used for emitting light to the detection sample;

a dichroic mirror for reflecting the light filtered by the filter and detecting fluorescence excited by the light by the sample;

a substrate;

2. The independent fluorescence channel detection system for real-time quantitative PCR according to claim 1, wherein the filter film is configured to have different thicknesses along its own direction, so that the passing light passes through the filter film and is filtered into fluorescence of different wavelengths.

3. The independent fluorescence channel detection system for real-time fluorescent quantitative PCR of claim 1, wherein: the filter film section is arranged into a step structure, and the step structure is provided with two or more step section planes.

4. The independent fluorescence channel detection system for real-time fluorescent quantitative PCR of claim 1, wherein: the dichroic mirror is obliquely arranged at 45 degrees, the light source and the photoelectric sensing module are respectively arranged on one horizontal side and above the dichroic mirror, and the detection sample is arranged below the dichroic mirror.

5. The independent fluorescence channel detection system for real-time fluorescence quantitative PCR according to claim 4, wherein: and a collimating optical module is arranged between the light source and the optical filter.

6. The independent fluorescence channel detection system for real-time fluorescent quantitative PCR of claim 1, wherein: still include the thermoblock that is used for supplying the detection sample to place, the thermoblock is equipped with temperature control device.

7. The independent fluorescence channel detection system for real-time fluorescent quantitative PCR according to claim 6, wherein: the temperature control device is a Peltier, the Peltier is arranged on the lower side of the temperature block, and a radiator is further arranged on the lower side of the Peltier.

8. The independent fluorescence channel detection system for real-time fluorescent quantitative PCR of claim 1, wherein: the optical filter is an optical filter.

9. The independent fluorescence channel detection system for real-time quantitative PCR of claim 1, wherein the optical filter is a rotatable filter wheel comprising at least two optical filters for filtering at least two wavelengths of light.

10. The system of claim 9, wherein the filters are arranged in a sector shape, and the filters together form a circular filter wheel, and the center of the filter wheel is offset from the center of the light source, such that the light does not pass through the center of the filter wheel.