CN110935498A

CN110935498A - Fluorescence scanning system for PCR instrument

Info

Publication number: CN110935498A
Application number: CN201911229574.5A
Authority: CN
Inventors: 叶建新; 张伟; 徐辉; 常江波; 刘旭; 邓明辉; 王宪华
Original assignee: BEIJING BEITAI TECHNOLOGY Co Ltd
Current assignee: BEIJING BEITAI TECHNOLOGY Co Ltd
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2020-03-31

Abstract

The invention discloses a fluorescence scanning system which comprises a PCR tube temperature bath hole opening structure, a light path structure and a motion driving component, wherein the PCR tube temperature bath hole structure is arranged on a PCR temperature control module, the light path structure is arranged on the motion driving component, the light path structure is positioned on the side surface of the PCR tube temperature bath hole opening structure, the light path structure forms correlation or reflection relative to the PCR tube temperature bath hole opening structure, and simultaneously, the fluorescence scanning system can efficiently collect excited fluorescence at high speed for filtering and photoelectric detection. The invention not only ensures that the real-time fluorescence quantitative PCR system has simple structure and high fluorescence scanning efficiency, but also has good real-time performance and flexible application, and can easily realize simultaneous or time-sharing scanning of multiple light paths and simultaneous and independent work of multiple PCR.

Description

Fluorescence scanning system for PCR instrument

Technical Field

The invention relates to the field of real-time fluorescence quantitative PCR, in particular to a high-efficiency, high-speed and flexible PCR real-time fluorescence scanning system scheme.

Background

Briefly, PCR is the mass synthesis of In Vitro or In Vitro of a specific gene using DNA polymerase, which basically involves the specific linkage replication using DNA polymerase. The PCR instrument is an instrument for providing the whole PCR process. In order to better monitor the PCR process and simultaneously quantify the target DNA, a real-time fluorescence quantitative PCR instrument is also produced. The fluorescence quantitative technology of the real-time fluorescence quantitative PCR instrument also determines the main performance of the PCR instrument. There are many fluorescence detection techniques for PCR, and most of the commercially available instruments are imaging and scanning techniques. The optical path composition of the imaging mode adopts a high-power excitation light source, such as a halogen tungsten lamp, which can excite all reaction positions of 96 holes, emitted fluorescence is collected by an imaging device through a lens and judged, and the difference of detection data is caused by the optical path difference of different reaction positions in the mode. The other imaging mode is to use optical fiber as an optical transmission mechanism, so that the problem of optical path difference is solved, but the detection sensitivity is affected by transmission loss caused by the optical fiber. The scanning mode mostly adopts EP pipe top scanning or bottom scanning, and top scanning is bow-shaped scanning generally, carries out ranks scanning detection to 96 positions, nevertheless because the top cap generally has hot lid, and is more complicated to structural design, and in addition, the temperature of hot lid also can cause the influence to the detection device, and then influences the homogeneity of detection data. Bottom scanning mode, because there are heating device and heat dissipation device in the bottom, the light path is integrated again, and first structure complicacy problem, the influence of the rise and fall temperature of PCR temperature control module is bigger to detection device and light path moreover, can increase the standard deviation who detects data. The upper and bottom acquisition can both result in increased fluorescence acquisition distance due to the structural member of the thermal cover or heater, the optical acquisition efficiency is in a square relationship with the acquisition distance, and the optical acquisition efficiency of the upper and bottom acquisition can be greatly reduced. The existing fluorescence scanning technology is generally continuous scanning, and signal crosstalk among different fluorescence channels is easily caused due to simultaneous sampling of multiple different fluorescence channels. In order to solve the crosstalk problem of the signal, various structures and algorithms need to be additionally considered. The fluorescence scanning system provides a high-efficiency, high-speed and flexible PCR real-time fluorescence scanning system scheme, and can well solve the defects.

Disclosure of Invention

The invention provides a scheme of a PCR real-time fluorescence scanning system with high efficiency, high speed and flexibility. The invention can be flexibly applied to the fluorescence scanning of one path, two paths and multiple paths of light paths, and can scan fluorescence at high speed, high sensitivity and high dynamic range, thereby fundamentally improving the key performance of the real-time quantitative PCR instrument.

The utility model provides a fluorescence scanning system, includes PCR pipe temperature bath trompil structure, light path structure, motion drive assembly, PCR pipe temperature bath structure installs on PCR temperature control module, and the main characterized in that light path structure is located the motion drive assembly of PCR pipe temperature bath structure side, and excitation light and emission are aroused and fluorescence scanning in the side of PCR pipe temperature bath structure. The scanning is continuous moving line scanning, and the scanning process is not stopped. Such scanning motion can greatly improve the real-time performance of the acquisition system, improve the stability of signal acquisition and simultaneously reduce the motion noise during scanning.

Furthermore, the PCR tube temperature bath hole opening structure is provided with an opening on the side surface of the temperature bath hole, and is not limited to one-side or double-side opening. Typically, a single-sided aperture, with the application of excitation light forming a reflected light path with the emitted light; the holes are opened on two sides, and the excitation light and the emission light form a correlation light path.

Furthermore, because the light paths are arranged in parallel and parallel to the movement direction, all the light paths can pass through all the PCR tube warm bath holes, and therefore, the light path structure can be a single-light-path acquisition structure or a multi-light-path acquisition structure.

Further, the optical path structure, the photoelectric detection element, is not limited to a single detection element or multiple detection elements in general.

Further, the single detection element can be applied to single-light-path detection and multi-light-path detection. The excitation light source for single-light path detection can be constantly opened, and the excitation light source for multi-light path detection can perform multi-light path detection by a time-sharing flicker method.

Further, in the optical path structure, an optical device can be directly integrated on the moving structure, and the optical path can be guided to other stable structures through optical fibers for optical processing.

Further, the photodetection device in the optical path structure is not limited to a photocell, an avalanche diode, a photomultiplier tube, etc., nor is the light source limited to a light emitting diode LED, a laser, an inert gas lamp, etc. The optimal scheme is that the optical detection system with high sensitivity, high response rate, high stability and long service life can be conveniently realized by adding a Light Emitting Diode (LED) to a photoelectric detection device of a photomultiplier.

Furthermore, the motion driving component can be a motion structure with high-precision positioning, the motion mechanism is not limited to a stepping motor, a servo motor, a direct current motor and the like, and the positioning is not limited to pulse positioning of the stepping motor, positioning of a photoelectric encoder, positioning of a magnetic encoder and the like

Furthermore, the fluorescence scanning system is not limited to a single optical path structure corresponding to a single-PCR tube warm bath hole opening structure, but also can be a single optical path structure corresponding to a double-row PCR tube warm bath hole opening structure, so that the flux of PCR detection is improved, and the structure is more compact.

The invention also claims the application of the fluorescence scanning system, which is applied to the field of real-time fluorescence quantitative PCR.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below. It is appreciated that the following drawings depict only certain embodiments of the invention and are therefore not to be considered limiting of its scope. For a person skilled in the art, it is possible to derive other relevant figures from these figures without inventive effort.

FIG. 1 is a schematic diagram of a fluorescence scanning system according to the present invention;

fig. 2 is a schematic diagram of the optical path structure of the present invention.

Description of reference numerals:

PCR heating module 11, PCR hot bath 12, motion driving module 13, optical path structure 14, excitation light LED 21, excitation light filter 22, monochromatic excitation light 23, emission light filter 24, photodetector 25, single-wavelength emission light 26, dichroic mirror 27, and condenser objective 29.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, 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. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. 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.

It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

In the description of the present invention, it is to be understood that the terms "upper", "lower", "inside", "outside", "left", "right", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, or the orientations or positional relationships that the products of the present invention are conventionally placed in use, or the orientations or positional relationships that are conventionally understood by those skilled in the art, and are used for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.

Furthermore, the terms "first," "second," and the like are used merely to distinguish one description from another, and are not to be construed as indicating or implying relative importance.

In the description of the present invention, it is also to be noted that, unless otherwise explicitly stated or limited, the terms "disposed" and "connected" are to be interpreted broadly, and for example, "connected" may be a fixed connection, a detachable connection, or an integral connection; can be mechanically or electrically connected; the connection may be direct or indirect via an intermediate medium, and may be a communication between the two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

The following detailed description of embodiments of the invention refers to the accompanying drawings.

Example 1

The fluorescence scanning system of the present invention is further described by this embodiment with reference to fig. 1 and 2.

The fluorescence scanning system comprises a PCR heating module 11, a PCR warm bath hole 12, a motion driving component 13 and a light path structure 14.

The PCR heating module 11 controls the temperature through a semiconductor heater, so that the temperature of the PCR warm bath hole 12 is changed, the PCR warm bath hole 12 is a warm bath hole with a small hole on the side surface, and the PCR tube is just placed in the warm bath hole 12. The motion driving assembly 13 comprises a motor, a synchronous wheel and a synchronous belt. The optical path structure 14 is fixed to the timing belt and is driven by a motor so that the optical path structure 14 moves in a direction parallel to the arrangement of the small holes along the direction shown in fig. 1. The optical path component 14 includes an excitation light LED 21, an excitation light filter 22, a dichroic mirror 27, a condenser objective 29, an emission light filter 24, and a photodetector 25.

In this embodiment, the motion driving assembly 13 in fig. 1 employs a stepping motor with a step angle of 0.9 ° and a driver with a subdivision of 16, so that the positioning accuracy of the pulse reaches several um levels, and by using this high-accuracy position resolution to perform trigger sampling, 300 pieces of data of the measurement holes of each PCR hot bath hole 12 can be easily obtained, and by performing algorithms such as data integration and bubble sorting on the 300 pieces of data, the key performances such as sensitivity, signal stability, signal-to-noise ratio of the system can be effectively improved.

The optical path structure 14 in fig. 1 of this embodiment is fixed on the synchronous belt and mounted on a high-precision slide rail, so that the optical path has small jitter and is stable in the linear scanning process. The optical path structure 14 adopted in the present embodiment is based on the confocal principle, so that the optical path system has extremely high sensitivity and signal-to-noise ratio. The optical path structure 14 moves in the direction of motion shown in the figure parallel to the array of wells of the PCR hot bath 12, so that all wells can be scanned in one linear motion. Thus, theoretically, without being limited to size limitations, an infinite number of optical paths can be installed to scan independently and simultaneously to increase the throughput of test items for a PCR system.

In fig. 2 of this embodiment, the excitation light emitted from the light source 21 is filtered by the color filter 22 to become monochromatic excitation light 23 in a specific wavelength range, then totally reflected 28 by the dichroic mirror 27, finally converged by the focusing objective 29 to reach the measurement hole of the target PCR hot-bath 12, and the fluorescence is excited to form diffuse reflection, which is efficiently collected by the focusing objective 29, and then the scattered light is converged, transmitted through the dichroic mirror 27, filtered by the color filter 24 to form single-wavelength emission light 26, and then reaches the photodetection device 25. The photoelectric conversion by the photodetection device 25 is converted into an electric signal, which is introduced into the electric circuit. If a light path of a confocal principle is to be formed, a group of focusing eyepieces and small holes are added behind the color filter 24 to form confocal, so that most of background stray light can be filtered out due to the filtering of the small holes, the sensitivity of the system is greatly improved, the system is complex, and the debugging is difficult.

Claims

1. The fluorescence scanning system is characterized by comprising a PCR tube temperature bath hole opening structure, a light path structure and a motion driving assembly, wherein the PCR tube temperature bath hole structure is arranged on a PCR temperature control module, the light path structure is arranged on the motion driving assembly on the side surface of the PCR tube temperature bath hole structure, excitation light and emission light are excited and fluorescence scanned on the side surface of the PCR tube temperature bath hole structure, the scanning is continuous line scanning, and the scanning process is not stopped.

2. The fluorescence scanning system of claim 1, wherein the side of the hot well of the PCR tube hot well open structure is open, and the open hole is a single-sided or double-sided open hole.

3. The fluorescence scanning system of claim 1, wherein the excitation light and the emission light of the optical path structure are in a counter-incident or reflected optical path.

4. The fluorescence scanning system of claim 3, wherein the optical path structure is one or more optical collection structures.

5. The fluorescence scanning system of claim 4, wherein the optical path structure comprises a photoelectric detection element, and the photoelectric detection element is one path of detection element corresponding to one optical path or one path of detection element corresponding to multiple optical paths.

6. The fluorescence scanning system of claim 5, wherein the photodetector device is a photocell, avalanche diode, or photomultiplier tube, and the light source is a Light Emitting Diode (LED), laser, or inert gas lamp.

7. The fluorescence scanning system of claim 1, wherein the motion driving component is a high-precision positioning motion structure, and the motion mechanism is a stepping motor, a servo motor, or a dc motor, and the positioning is a stepping motor pulse positioning, a photoelectric encoder positioning, a grating ruler positioning, or a magnetic encoder positioning.

8. The fluorescence scanning system of claim 1, wherein the system is a single-piece optical path structure corresponding to a single-PCR tube hot-bath open-hole structure, or a single-piece optical path structure corresponding to a double-row PCR tube hot-bath open-hole structure.

9. The fluorescence scanning system of claim 1, wherein the excitation light of the line scan process is a continuous excitation or a scintillation excitation, and the emission light is collected continuously or scintillatively by the collection system, allowing simultaneous operation of multiple optical path systems.

10. Use of a fluorescence scanning system according to any of claims 1 to 9, in the field of real-time fluorescence quantitative PCR.