LAMP (loop-mediated isothermal amplification) rapid fluorescence detector based on multiple fluorescence detection reagents

Abstract

The invention belongs to the technical field of nucleic acid isothermal amplification detection, and discloses an LAMP rapid fluorescence detector based on multiple fluorescence detection reagents, the LAMP rapid fluorescence detector comprises a shell, a laser light source module, a reaction test tube and an optical beam splitter module are arranged in the shell, the optical beam splitter module is used for splitting an excitation light beam into at least two paths of independent sub light beams, at least two light path channels are formed, and a light path switch module and a light filter module are sequentially arranged on each light path channel; the propagation direction of the sub light beams deviating from the original propagation direction is changed through the reflective mirror module, the light beams are guided to the reaction test tube, and when detection is carried out, the light path switch module controls on-off of the light path channels, so that at any detection moment, only one light path channel is in a light beam access state, and the other light path channels are in a light beam blocking state. According to the invention, while the manufacturing cost is obviously reduced, the detection steps are simplified, the high-performance requirement of on-site rapid detection is met, and the use scene is expanded.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid isothermal amplification and detection technology, and relates to a LAMP rapid fluorescence detector, specifically a LAMP rapid fluorescence detector based on multiplex fluorescence detection reagents. Background Technology

[0002] Loop-mediated isothermal amplification (LAMP) technology, as a highly efficient isothermal amplification method for nucleic acids, has shown great application potential in fields such as pathogen diagnosis, food safety monitoring, and environmental microbial detection, especially in rapid on-site detection scenarios, due to its significant advantages such as fast reaction speed (usually completed within 15-60 minutes), simple operation (no need for complex thermal cycling equipment), high specificity (using 4-6 specific primers), and the ability to intuitively judge the results through the turbidity or fluorescence signal of the byproduct magnesium pyrophosphate precipitation.

[0003] Combining LAMP technology with fluorescence detection allows for real-time, quantitative monitoring of the amplification process by adding fluorescent dyes (such as SYBR Green I) that specifically bind to the amplified products or sequence-specific fluorescent probes (such as quenching fluorescent probes) to the reaction system. This significantly improves the sensitivity, accuracy, and objectivity of the results. In particular, multiplex fluorescent LAMP detection technology, by using probes labeled with fluorescent groups of different emission wavelengths targeting different targets, can simultaneously detect multiple target nucleic acid sequences in a single reaction tube, significantly increasing throughput and efficiency. This is of great significance for the typing of complex infectious pathogens and the development of multi-detection kits.

[0004] However, achieving efficient and reliable multiplex fluorescence LAMP detection is highly dependent on the corresponding detection instruments. Currently, existing real-time fluorescence detection devices typically use broadband light sources (such as high-pressure mercury lamps or pulsed xenon lamps) combined with filter wheels or monochromators to provide excitation light of different wavelengths. While these light sources can cover a wide excitation spectrum and meet the needs of various fluorescent dyes, the following inherent limitations severely restrict their application in rapid, portable detection, especially in the context of multiplex LAMP reagents: (1) High-pressure mercury lamps, xenon lamps and their associated power supply and heat dissipation systems are large in size and weight, and their power consumption is usually above tens of watts. This makes it difficult to miniaturize and lighten the whole machine, and it is heavily dependent on a stable mains power supply. It cannot meet the needs of use in environments without a stable power supply or in mobile environments such as the field, vehicle, and primary clinics. (2) To separate excitation light of a specific wavelength from a broadband light source, a complex filter wheel or grating system is required. This not only increases the complexity and cost of the mechanical structure, but also leads to energy loss in the optical path. Furthermore, the combination of excitation wavelengths is usually fixed or finitely adjustable, making it difficult to flexibly and accurately match them according to the subtle differences in the optimal excitation wavelengths of the fluorescent groups in different multiplex fluorescent detection reagents, which affects the detection sensitivity. Summary of the Invention

[0005] The present invention aims to provide a LAMP rapid fluorescence detector based on multiplex fluorescence detection reagents, so as to achieve rapid detection, applicability to various application scenarios, and improved detection sensitivity.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A rapid fluorescence detector for LAMP based on multiplex fluorescence detection reagents includes a housing, and the housing contains: The laser source module is used to provide the excitation beam; Reaction tubes are used to contain the LAMP reaction system and fluorescence detection reagents. An optical beam splitter module is used to split the excitation beam into at least two independent sub-beams, forming at least two optical path channels. Each optical path channel is provided with the following in sequence: Optical path switch module, used to control the on / off state of the corresponding optical path channel; The filter module is used to adjust the wavelength of the corresponding sub-beam to a preset value; Among them, the sub-beams that deviate from the original propagation direction are changed by the reflector module to guide the beam to the reaction tube to excite the fluorescence signal in the reaction tube. During detection, the optical path switch module controls the on / off state of the optical path channel so that at any detection time, only one optical path channel is in the beam pass state, and the other optical path channels are in the beam block state.

[0007] As a limitation of the present invention, the optical beam splitter module includes a primary optical beam splitter and a secondary optical beam splitter; The first-stage optical beam splitter splits the incident beam from the laser source module into a first transmitted beam and a first reflected beam according to a first preset ratio; the second-stage optical beam splitter splits the first transmitted beam into a second transmitted beam and a second reflected beam according to a second preset ratio.

[0008] As a further limitation of the present invention, the optical channel formed by the first reflected beam and the second reflected beam is finally converged into the reaction tube by the convex lens module.

[0009] As another limitation of the present invention, the first preset ratio is 70:30 and the second preset ratio is 50:50.

[0010] As a limitation of the present invention, the optical path switch module includes an electrically controlled dimming glass and a control switch.

[0011] As a further limitation of the present invention, the filter module is detachably disposed within the housing and can be replaced according to the multiplex fluorescence detection reagent used.

[0012] As a third limitation of the present invention, an observation window is provided on the shell at the position corresponding to the reaction tube, for observing the change of fluorescence color inside the reaction tube.

[0013] As a limitation of the present invention, the laser source module is a light-emitting diode.

[0014] By adopting the above-described technical solution, the beneficial effects achieved by this invention compared to the prior art are as follows: (1) This invention uses a low-power LED light source to replace the traditional bulky and high-energy-consuming broadband light source, and combines it with a precise miniaturized optical path design, which greatly reduces the size and weight of the whole machine and significantly reduces power consumption. This allows the instrument to get rid of its dependence on stable mains power and can be powered by a built-in battery or a mobile power source, thus adapting to environments such as the field, primary medical institutions, and mobile testing vehicles where there is no stable power source or where portable operation is required, thus broadening the application scenarios. (2) This invention combines multi-level optical beam splitting with independent channel optical path switching. Only one light source is needed to generate multiple independent excitation beams through physical optical path division. This not only simplifies the system from the source and reduces cost and power consumption, but also allows each optical path channel to be independently configured with filters of different wavelengths. The independent control of each optical path channel is achieved through electronically controlled dimming glass, realizing efficient, accurate and flexible matching of excitation wavelengths of different dyes in multiple fluorescent reagents. (3) The optical path of the present invention adopts a preset fixed structure. Only by simply switching the detection channel can the sequential excitation and detection of multiple target signals be completed. This eliminates the need for complex optical path calibration, wavelength adjustment and other professional operations, greatly reducing the threshold for use. The steps are extremely simplified and the detection efficiency is significantly improved. (4) The filter module of the present invention adopts a detachable structure, which can flexibly replace the corresponding wavelength filter group according to the multiple LAMP fluorescence reagent kits of different manufacturers and different detection items, so that one instrument can be adapted to multiple detection items, greatly enhancing the versatility and application range.

[0015] In summary, this invention significantly reduces manufacturing costs, simplifies testing procedures, meets the high-performance requirements of rapid on-site testing, and expands application scenarios. Attached Figure Description

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0017] Figure 1 This is a three-dimensional structural diagram of an embodiment of the present invention; Figure 2 This is a schematic diagram of the internal three-dimensional structure of an embodiment of the present invention; Figure 3 This is a top view of the structural arrangement of the embodiments of the present invention.

[0018] In the diagram: 1. Housing; 11. Observation window; 2. Laser source module; 3. Optical beam splitter module; 31. First-stage optical beam splitter; 32. Second-stage optical beam splitter; 4. Reflector module; 41. First reflector; 42. Second reflector; 5. Optical path switch module; 51. First electrically controlled dimming glass; 52. Second electrically controlled dimming glass; 53. Third electrically controlled dimming glass; 6. Filter module; 7. Convex lens module; 71. First convex lens; 72. Second convex lens; 8. Reaction tube. Detailed Implementation

[0019] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustrative and understanding purposes only and are not intended to limit the scope of the invention.

[0020] Example: A rapid fluorescence detector for LAMP based on multiplex fluorescence detection reagents like Figures 1-3 As shown, this embodiment includes a housing 1, the interior of which forms a darkroom environment to reduce ambient light interference. All the optical and electronic modules of this detector are integrated inside the housing 1. Within the housing 1, along the optical path transmission direction, are arranged a laser source module 2, an optical beam splitter module 3, a reflector module 4, an optical path switch module 5, a filter module 6, a convex lens module 7, and a reaction tube 8.

[0021] A support is installed inside the housing 1 to hold the reaction tube 8 containing the LAMP reaction system to be tested and the fluorescent detection reagent. The final emitted beams from all optical paths are precisely guided to the same detection area of ​​the reaction tube 8. An observation window 11 is provided on the housing 1, directly opposite the reaction tube 8. The observation window 11 can be made of transparent material, allowing the operator to directly observe the fluorescence color change caused by nucleic acid amplification inside the tube.

[0022] The laser light source module 2 serves as the excitation light source. In this embodiment, a high-brightness light-emitting diode (LED) is preferably used. The LED should have the characteristics of high stability, long life and low power consumption. The power supply for the LED can be a battery or a mobile power supply.

[0023] The optical beam splitter module 3 is used to precisely split the single excitation beam emitted by the laser source module 2 into multiple independent sub-beams. In this embodiment, the optical beam splitter module 3 includes a first-stage optical beam splitter 31 and a second-stage optical beam splitter 32, which split the single excitation beam into two sub-beams.

[0024] A primary optical beam splitter 31 is positioned along the optical path emitted from the laser source module 2, tilted at 45° along the direction of light propagation. The primary optical beam splitter 31 splits the incident beam from the laser source module 2 into beams at a first preset ratio of 70:30. The beam accounting for 70% of the total intensity continues to propagate along its original direction as the first transmitted beam, while the beam accounting for 30% of the total intensity propagates as the first reflected beam in a direction perpendicular to the first transmitted beam.

[0025] A secondary optical beam splitter 32 is positioned along the optical path of the first transmitted beam, tilted at 135° along the propagation direction of the first transmitted beam. The secondary beam splitter 32 splits the first transmitted beam a second time according to a second preset ratio of 50:50, generating two sub-beams, each with an intensity of 35% of the original beam. One sub-beam serves as the second transmitted beam, continuing to propagate along the initial direction, while the other serves as the second reflected beam, propagating in a direction perpendicular to the second transmitted beam. Since the tilt angles of the primary optical beam splitter 31 and the secondary optical beam splitter 32 are opposite, the propagation directions of the first and second reflected beams are opposite. In this embodiment, the distance from the center point of the primary optical beam splitter 31 to the center point of the secondary optical beam splitter 32 is 40 mm.

[0026] The reflector module 4 is used to guide the light path to a preset direction so that all light paths can eventually converge and illuminate the same reaction tube 8. In this embodiment, since both the first and second transmitted light beams propagate in a direction perpendicular to the transmitted light beam, two reflectors are needed to guide them to propagate in a direction parallel to the transmitted light beam. The reflector module 4 in this embodiment includes a first reflector 41 and a second reflector 42.

[0027] The first reflector 41 and the first-stage optical beam splitter 31 are arranged parallel to each other, with a parallel distance of 40mm between their center points. The first reflector 41 is tilted at a 45° angle to redirect the first reflected beam again, so that the first reflected beam propagates parallel to the initial beam, forming optical path channel A.

[0028] The second reflector 42 is set parallel to the second-stage optical beam splitter 32, with a parallel distance of 40mm between their center points. The second reflector 42 is tilted at a 135° angle to redirect the second reflected beam again, so that the second reflected beam propagates parallel to the initial beam, forming the optical path channel C.

[0029] The second projection beam, after passing through the secondary optical beam splitter 32, forms optical path channel B.

[0030] Optical path switch module 5 is disposed in each optical path channel and is used to control the on / off state of the light beam. In this embodiment, the optical path switch module 5 is specifically an electrically controlled dimming glass, the core of which is based on polymer dispersed liquid crystal (PDLC) technology. It can quickly switch between a transparent (light-transmitting) state and an opaque (light-blocking) state by applying or removing a driving voltage. Since this embodiment has three optical path channels, the optical path switch module 5 includes a first electrically controlled dimming glass 51, a second electrically controlled dimming glass 52, and a third electrically controlled dimming glass 53, which are respectively placed on the propagation paths corresponding to optical path channels A, B, and C. Each electrically controlled dimming glass is controlled by an independent control switch, which is disposed on the outer wall of the housing 1. During detection, the operator ensures that only one electrically controlled dimming glass in the optical path channel is in a transparent state (passage) at any given time, while the other two are in an opaque state (closed circuit), thereby avoiding interference caused by multiple stray lights entering the reaction tube 8 simultaneously. The center point distance between the first electrically controlled dimming glass 51 and the first reflector 41 is 70 mm. The distance between the center point of the second electronically controlled dimming glass 52 and the center point of the second-stage optical beam splitter 32 is 30mm, and the distance between the center point of the third electronically controlled dimming glass 53 and the center point of the second reflector 42 is 30mm.

[0031] The filter module 6, located after the optical path switch module 5, is used for spectral purification to match the precise excitation wavelength of different fluorescent dyes. The filter module 6 employs a pluggable modular design for easy replacement. Each channel is equipped with a bandpass filter. Optical path channel A is equipped with a filter with a center wavelength of 570nm for exciting dyes with emission spectra near 570nm (such as CY3 or HEX); optical path channel B is equipped with a filter 202 with a center wavelength of 520nm for exciting dyes with emission spectra near 520nm (such as FAM or SYBR Green I); and optical path channel C is equipped with a filter with a center wavelength of 670nm for exciting dyes with emission spectra near 670nm (such as Cy5). The distance from the center point of the filter module 6 to its electrically controlled dimming glass is 20mm.

[0032] A convex lens module 7 is disposed at the end of optical path channels A and C, used to converge parallel or slightly divergent light beams to the center of the reaction tube 8. In this embodiment, a first convex lens 71 is disposed 20 mm behind the filter in optical path channel A; a second convex lens 72 is disposed 20 mm behind the filter in optical path channel C. Since the propagation direction of optical path channel B remains unchanged, it can directly illuminate the center of the reaction tube 8.

[0033] The working principle and operation steps of this embodiment are as follows: 1. Preparation and sample addition: Dispense the premixed LAMP reaction solution into reaction tube 8, add the nucleic acid sample to be tested, and place it on the support inside the shell 1.

[0034] 2. Isothermal amplification: Start the external isothermal device to carry out the LAMP amplification reaction in reaction tube 8 under isothermal conditions of 60-65℃; 3. Sequential stimulation and observation: When the amplification reaction reaches a preset time point (e.g., 30 minutes) or when real-time monitoring is required, turn on the laser source module 6; The operator first turns on the control switch, making the first electrically controlled dimming glass 51 of the optical path channel A transparent. At this time, the light emitted by the light source passes sequentially through the first-stage optical beam splitter 31 (30% reflection), the first reflector 41, the first electrically controlled dimming glass 51, the 570nm filter, and the first convex lens 71, finally converging onto the reaction tube 8. The operator observes through the observation window 11 whether a specific fluorescent color corresponding to the 570nm excitation light appears inside the reaction tube 8.

[0035] Subsequently, the control switch for the first electrically controlled dimming glass 51 is closed, and the control switch for the second electrically controlled dimming glass 52 is opened. At this time, the second electrically controlled dimming glass 52 of the optical path channel B becomes transparent. The light beam passes through the first-stage optical beam splitter 31 (70% transmission), the second-stage optical beam splitter 32 (50% reflection), the second electrically controlled dimming glass 52, and the 520nm filter, directly illuminating the reaction tube 3. Observe whether a specific fluorescent color corresponding to the 520nm excitation light appears in the reaction tube 8.

[0036] Finally, close the second electrically controlled dimming glass 52 and open the control switch of the third electrically controlled dimming glass 53. At this time, optical path channel B is closed, and the third dimming glass 53 of optical path channel C becomes transparent. The light beam passes through the first-stage optical beam splitter 31 (70% transmission), the second-stage optical beam splitter 32 (50% reflection), the second reflector 42, the third electrically controlled dimming glass 53, the 670nm filter, and the second convex lens 72, and finally converges onto the reaction tube 8. Observe whether a specific fluorescent color corresponding to the 670nm excitation light appears in the reaction tube 8.

[0037] 4. Result Interpretation: The presence or absence of the corresponding three target nucleic acids in the sample can be qualitatively determined based on the fluorescence color observed in the three channels in sequence.

[0038] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid fluorescence detector for LAMP based on multiplex fluorescence detection reagents, characterized in that, Includes a housing, and inside the housing are: The laser source module is used to provide the excitation beam; Reaction tubes are used to contain the LAMP reaction system and fluorescence detection reagents. An optical beam splitter module is used to split the excitation beam into at least two independent sub-beams, forming at least two optical path channels. Each optical path channel is provided with the following in sequence: Optical path switch module, used to control the on / off state of the corresponding optical path channel; The filter module is used to adjust the wavelength of the corresponding sub-beam to a preset value; Among them, the sub-beams that deviate from the original propagation direction are changed by the reflector module to guide the beam to the reaction tube to excite the fluorescence signal in the reaction tube. During detection, the optical path switch module controls the on / off state of the optical path channel so that at any detection time, only one optical path channel is in the beam pass state, and the other optical path channels are in the beam block state.

2. The LAMP rapid fluorescence detector based on multiplex fluorescence detection reagents according to claim 1, characterized in that, The optical beam splitter module includes a primary optical beam splitter and a secondary optical beam splitter; The first-stage optical beam splitter splits the incident beam from the laser source module into a first transmitted beam and a first reflected beam according to a first preset ratio; the second-stage optical beam splitter splits the first transmitted beam into a second transmitted beam and a second reflected beam according to a second preset ratio.

3. The LAMP rapid fluorescence detector based on multiplex fluorescence detection reagents according to claim 2, characterized in that, The optical channel formed by the first and second reflected beams is finally converged into the reaction tube by the convex lens module.

4. A LAMP rapid fluorescence detector based on multiplex fluorescence detection reagents according to claim 2 or 3, characterized in that, The first preset ratio is 70:30, and the second preset ratio is 50:

50.

5. A LAMP rapid fluorescence detector based on multiplex fluorescence detection reagents according to claim 4, characterized in that, The optical path switch module includes an electronically controlled dimming glass and a control switch.

6. A LAMP rapid fluorescence detector based on multiplex fluorescence detection reagents according to claim 5, characterized in that, The filter module is detachably housed within the housing and can be replaced depending on the multiplex fluorescence detection reagent used.

7. A rapid fluorescence detector for LAMP based on multiplex fluorescence detection reagents according to any one of claims 1-3, 5, and 6, characterized in that, An observation window is provided on the shell at the position corresponding to the reaction tube, for observing the changes in fluorescence color inside the reaction tube.

8. A LAMP rapid fluorescence detector based on multiplex fluorescence detection reagents according to claim 7, characterized in that, The laser source module is a light-emitting diode.

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