CN220643115U - Real-time quantitative detection instrument for nucleic acid amplification products - Google Patents
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- CN220643115U CN220643115U CN202320899314.4U CN202320899314U CN220643115U CN 220643115 U CN220643115 U CN 220643115U CN 202320899314 U CN202320899314 U CN 202320899314U CN 220643115 U CN220643115 U CN 220643115U
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 41
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 41
- 238000010438 heat treatment Methods 0.000 claims abstract description 42
- 238000011897 real-time detection Methods 0.000 claims abstract description 35
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- AUIINJJXRXMPGT-UHFFFAOYSA-K trisodium 3-hydroxy-4-[(2-hydroxy-4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2,7-disulfonate Chemical compound [Na+].[Na+].[Na+].Oc1cc(c2ccccc2c1N=Nc1c(O)c(cc2cc(ccc12)S([O-])(=O)=O)S([O-])(=O)=O)S([O-])(=O)=O AUIINJJXRXMPGT-UHFFFAOYSA-K 0.000 description 2
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Abstract
The utility model provides a real-time quantitative detection instrument for a nucleic acid amplification product, which comprises a real-time detection module and a detection control module, wherein the real-time detection module comprises a real-time detection shell arranged in an instrument shell, a plurality of sample detection holes with upper openings are arranged in the real-time detection shell, heating chambers communicated with the sample detection holes are arranged at the bottoms of the sample detection holes, containing holes are respectively arranged at the positions, close to the bottom ends, of two sides of the sample detection holes, light passing holes are respectively arranged between the sample detection holes and the containing holes, two ends of the light passing holes are respectively communicated with the sample detection holes and the containing holes, the containing holes comprise a first containing hole and a second containing hole, and the detection control module comprises a heating driving element, an analog-to-digital conversion element, a data processing element, an input element, an output element and a power supply part.
Description
Technical Field
The utility model relates to the field of nucleic acid detection, in particular to a real-time quantitative detection instrument for a nucleic acid amplification product.
Background
The acquisition of the nucleic acid detection results depends on the analysis of the amplified products. If the amplification reaction generates an amplification product, the sample is positive, the detected target gene exists, and otherwise, the sample is negative. In addition, the yield of the amplified product is related to the initial template content, so that the content of the gene to be detected can be quantitatively determined based on the amount of the amplified reaction product. Common methods of product analysis are gel electrophoresis and real-time fluorescent quantitation. The gel electrophoresis method belongs to an end-point method, and after the amplification is completed, a sample is taken out, and the DNA content in the amplification solution is obtained through complex and time-consuming analysis steps.
The real-time fluorescence quantitative method can utilize fluorescent dye to detect the generation amount of DNA in real time in the amplification process, has simple operation and high analysis speed, but the required instrument and detection reagent are expensive, is mainly applied to large laboratories, is difficult to meet the field detection requirements in the fields of agricultural production, food safety detection and the like, and has high use cost which is not suitable for basic users; the structure of the real-time fluorescence quantitative detection instrument is complex, and because the principle of fluorescence detection is to use a light source with a certain wavelength as an excitation light source to enable a fluorescent reagent to generate an optical signal with another wavelength, the fluorescence detection instrument not only needs to detect the intensity of the optical signal, but also needs to consider the wavelength of the differential optical signal to avoid the problem of interference of residual excitation light, and therefore, the structure of the real-time fluorescence quantitative detection instrument is complex and expensive, and the current application is mainly limited to a large laboratory.
Because the amplification process needs to heat the nucleic acid amplification container to provide a proper amplification temperature, the existing detection device generally does not have an amplification function, and needs to take out a sample from an amplification instrument after amplification is completed in the amplification instrument, so that the sample can be analyzed, the use of other instruments is involved in the step, the steps comprise preparation of the instrument, sample placement, detection, sample taking out and the like, and furthermore, after the nucleic acid amplification reaction is completed, the enzyme needs to be kept at a high temperature for a period of time to inactivate, and then the sample can be taken out from the amplification instrument, so that the operation steps are more, and the result cannot be obtained immediately when the nucleic acid amplification reaction is completed; in addition, the conventional nucleic acid detection needs sample feeding detection, and a series of steps of sampling, transporting samples to a laboratory, detecting and feeding back detection results are carried out, so that extra sample feeding cost is needed, the detection period is long, the result is seriously lagged, and the method is difficult to adapt to the characteristics of high microbial pollution occurrence and diffusion speed.
Disclosure of Invention
The utility model aims to overcome the defects of the prior art, provide a real-time quantitative detection instrument for a nucleic acid amplification product, heat a nucleic acid amplification container, detect and record absorbance values in real time, and obtain a nucleic acid detection result according to the absorbance values, and solve the problems that the existing detection device depends on expensive amplification instruments and fluorescent reagents, is difficult to meet the field detection requirements of basic users, and has complicated manual operation steps.
For this purpose, the utility model adopts the following technical scheme:
the utility model provides a real-time quantitative determination instrument of nucleic acid amplification product, includes real-time detection module and the detection control module that sets up in the instrument shell, detection control module includes analog-to-digital conversion element, data processing element, input element, output element and power supply unit, real-time detection module includes real-time detection casing, be equipped with a plurality of sample detection holes of top open-ended in the real-time detection casing, the both sides of sample detection hole are close to bottom department and are equipped with the accommodation hole respectively, sample detection hole with be equipped with the light-passing hole between the accommodation hole, the both ends of light-passing hole communicate respectively the sample detection hole with the accommodation hole, its characterized in that, detection control module still includes heating drive element, the accommodation hole includes first accommodation hole and second accommodation hole, the bottom of sample detection hole is equipped with the heating chamber with it intercommunication.
On the basis of adopting the technical scheme, the utility model can also adopt the following further technical schemes or use the further technical schemes in combination:
the nucleic acid amplification container with the sample to be detected is placed in the sample detection hole, a heating element and a temperature sensor are arranged in the heating chamber, and a light source and a photoelectric sensor are respectively placed in the first accommodating hole and the second accommodating hole.
The color of the light emitted by the light source is red.
The nucleic acid amplification vessel is a transparent vessel that is transparent to light.
The input end of the data processing element is connected with the output end of the temperature sensor, the output end of the data processing element is connected with the input end of the heating driving element, the output end of the heating driving element is connected with the heating element, the input end of the analog-to-digital conversion element is connected with the output end of the photoelectric sensor, the output end of the analog-to-digital conversion element is connected with the input end of the data processing element, the input element is connected with the input end of the data processing element, and the output end of the data processing element is connected with the output element.
The hole shafts of the two light-passing holes on two sides of the sample detection hole are on the same axis, and the light-passing holes are perpendicularly intersected with the sample real-time detection hole.
The output element comprises a display screen and a sound generating device; the real-time detection module and the outside of detection control module are equipped with the instrument shell, the upper surface of instrument shell is equipped with the storehouse lid that can open and shut.
Compared with the prior art, the utility model has the following advantages:
(1) The utility model can monitor the product yield of the amplification reaction in real time, so that the detection result can be reported when the amplification reaction is finished, even in the process of the amplification reaction, the step and the time for taking out the sample for analysis after the amplification is finished are saved, and the utility model has the advantages of simplicity and speed.
(2) The utility model provides a low-price real-time quantitative detection instrument, which has the advantages of simple structure of a light detection module of the instrument, few types of required parts, capability of reducing the complexity and manufacturing cost of the instrument and low price.
(3) The utility model provides an instrument with high analysis speed, simple operation and high efficiency, which can synchronously complete detection while amplifying, can obtain results when amplifying is finished and even in the process of amplifying, and has the advantages of higher analysis speed, fewer operation steps and higher efficiency.
(4) The portable real-time quantitative detection instrument provided by the utility model better meets the actual detection requirements of a base layer, has a simple instrument structure, a small number of components and low energy consumption, can reduce the volume and the weight of the instrument, is powered by a battery, has good portability, can be applied to scenes such as field land, supermarkets, communities and families, and can realize field detection.
Drawings
FIG. 1 is a graph of real-time detection data for LAMP amplification of samples containing 0 and 10 copies/. Mu.L using the present utility model;
FIG. 2 is a graph of real-time detection data of LAMP amplification of samples containing different initial templates according to the present utility model;
FIG. 3 is a graph of real-time detection data of RPA amplification of samples containing different initial template concentrations in accordance with the present utility model;
FIG. 4 is a cross-sectional view of one embodiment of a real-time detection housing in accordance with the present utility model;
FIG. 5 is a three-dimensional schematic of an embodiment of a real-time detection housing of the present utility model;
FIG. 6 is a three-dimensional schematic of an eight-channel embodiment of the real-time detection housing of the present utility model.
Description of the embodiments
For a better understanding of the technical solutions of the present utility model by those skilled in the art, a preferred embodiment of the present utility model is described below with reference to specific examples, which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar functional elements throughout, but it is understood that the drawings are for illustrative purposes only and are not to be construed as limiting the utility model; for the purpose of better illustrating the embodiments, certain elements of the drawings may be omitted, enlarged or reduced and do not represent the actual product dimensions; it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted in order to illustrate the utility model only and not to limit the utility model.
The utility model is further illustrated by the following figures and examples, which are not intended to be limiting.
Reference is made to the accompanying drawings. In the utility model, LAMP amplification and RPA amplification are taken as examples, a conventional end-point method is used, a detection result obtained after amplification is finished, a negative sample is kept purple, and a positive sample is changed from original purple to blue. LAMP amplification was performed with a set of 10 copies/. Mu.L samples, and the result obtained by photographing one sample was taken every 5 minutes, and the reaction solution was initially dark purple in color, changed to light purple (or light blue) after about 25 minutes, and changed to dark blue after 30 minutes. It is clear that the change from violet to blue of hydroxynaphthol blue is not abrupt, but gradually changes with the progress of the amplification reaction, indicating that magnesium ions are continuously consumed by the byproducts during the amplification process, and the absorption of light in the red wavelength range by hydroxynaphthol blue is continuously enhanced. This means that, in addition to waiting for the analysis of the sample after the completion of the amplification, a new method can be employed in which the amplification result is judged from the change in absorbance of the nucleic acid amplification reaction solution to red light during the progress of the amplification reaction.
FIG. 1 is a graph of time-absorbance data obtained by taking the reaction time of the LAMP amplification process as the abscissa and the absorbance measured at intervals of 5 minutes as the ordinate. As can be seen from the graph, the absorbance value of the sample with the initial content of the detected gene of 10 copies/. Mu.L is continuously increased along with the progress of the amplification reaction, and the absorbance value of the sample with the initial content of the detected gene of 0 copies/. Mu.L is not obviously increased, so that whether the sample to be detected is a positive sample or not can be detected by judging the absorbance value. Fig. 2 and 3 are real-time detection results obtained by LAMP and RPA, respectively, using samples of different concentrations. The results shown in FIGS. 2 and 3 indicate that the amplification reaction occurred in the sample containing the tested gene, while the amplification reaction did not occur in the sample not containing the tested gene. Furthermore, the absorbance value of the positive sample remains a rapid rising trend over time, regardless of whether it is LAMP amplification or RPA amplification, and the rate of rise is related to the copy number of the initial template. Therefore, qualitative and quantitative results can be obtained in real time according to time-absorbance data of the nucleic acid amplification reaction system solution.
Compared with the conventional end-point method which requires waiting for amplification to be completed and then observing whether the color is purple (negative result) or blue (positive result), the method can distinguish negative and positive samples in real time according to whether the absorbance value increases with the increase of the amplification time in the amplification process, provide detection results earlier, and omit the steps of analyzing the result after the amplification is completed, such as taking out the sample from an instrument and observing the color of the sample. In addition, the LAMP of FIG. 2 and the RPA amplification result of FIG. 3 show that the application range of detecting whether the sample to be detected is a positive sample by judging the absorbance value is not limited to LAMP, and the LAMP can be applied to different types of nucleic acid amplification reaction systems and has good universality.
In order to heat a nucleic acid amplification container and detect and record absorbance values in real time in an amplification process, the utility model provides a nucleic acid amplification product real-time quantitative detection instrument which comprises a real-time detection module and a detection control module, wherein the real-time detection module and the detection control module are arranged in an instrument shell, the detection control module comprises an analog-to-digital conversion element, a data processing element, an input element, an output element and a power supply component, the real-time detection module comprises a real-time detection shell, a plurality of sample detection holes 2 with upper openings are arranged in the real-time detection shell, accommodating holes are respectively arranged at two sides of the sample detection holes 2 close to the bottom end, a light through hole 3 is arranged between the sample detection holes 2 and the accommodating holes, two ends of the light through hole 3 are respectively communicated with the sample detection holes 2 and the accommodating holes, the detection control module further comprises a heating driving element, the accommodating holes comprise a first accommodating hole 4 and a second accommodating hole 5, and a heating chamber 6 communicated with the sample detection holes 2 is arranged at the bottom of the sample detection holes 2.
A nucleic acid amplification container filled with a sample to be detected is placed in the sample detection hole 2, a heating element and a temperature sensor are arranged in the heating chamber 6, and a light source and a photoelectric sensor are respectively placed in the first accommodating hole 4 and the second accommodating hole 5; the heating element functions to provide the temperature required for the amplification reaction and the photosensor is capable of obtaining an optical sensing signal.
The light emitted by the light source is red in color.
The nucleic acid amplification vessel is a transparent vessel that is transparent to light.
The output end of the data processing element is connected with the input end of the heating driving element, the output end of the heating driving element is connected with the heating element, the input end of the analog-to-digital conversion element is connected with the output end of the photoelectric sensor, the output end of the analog-to-digital conversion element is connected with the input end of the data processing element, the input element is connected with the input end of the data processing element, and the output end of the data processing element is connected with the output element;
the user sets parameters such as heating temperature and time through the input element, the data processing element adjusts the heating power of the heating element according to the temperature detected by the temperature sensor through the heating driving element, the reaction temperature required by nucleic acid amplification is provided, the optical sensing signal obtained by the photoelectric sensor is converted, calculated and output by the analog-to-digital conversion element, the optical signal is converted into a digital signal, the digital signal is obtained by the data processing element from the analog-to-digital conversion element, the absorbance is calculated according to the lambert law, and the result is transmitted to the output element.
The hole axes of the two light-passing holes 3 on two sides of the sample detection hole 2 are on the same axis, and the light-passing holes 3 are vertically intersected with the sample real-time detection hole 2.
The output element comprises a display screen and a sound generating device;
the display screen is used for displaying detection data obtained from the data processing element, the sounding device is used for detecting absorbance values in the amplification process in real time, when the absorbance values reach a preset absorbance threshold value, the sounding device sends out prompt sounds, and when the amplification reaction is finished, the sounding device also sends out prompt sounds to indicate that the amplification is finished.
The power supply component provides energy for all electronic elements of the detection instrument.
The outside of the real-time detection module and the detection control module is provided with an instrument shell, and the upper surface of the instrument shell is provided with a bin cover which can be opened and closed so as to put in and take out a nucleic acid amplification container filled with a detected sample.
Fig. 4-6 are schematic diagrams of the structure of the real-time detection housing disclosed in the present utility model, as shown in fig. 4, the hole axes of two light-passing holes 3 on two sides of a sample detection hole 2 are on the same axis, and the light-passing holes 3 are perpendicular to the sample real-time detection hole 2, in this embodiment, a light source is placed in the first accommodation hole 4, a photoelectric sensor is placed in the second accommodation hole 5, the light source emits detection light, the detection light enters the sample detection hole 2 through the light-passing holes 3, and is absorbed by a detected sample part in a nucleic acid amplification container, the transmission light enters the light-passing hole 3 on the other side, and the laser sensor generates a response signal, i.e. an optical sensing signal.
The real-time detection shell structure disclosed by the utility model can comprise one or more groups of channels, such as a single channel, a double channel and a three-channel … … eight channels … …, the number of the channels is drawn according to practical application conditions, and each group of channels comprises a sample detection hole 2, a light transmission hole 3 and a containing hole correspondingly and is not exemplified here; FIG. 6 is a three-dimensional schematic diagram of an embodiment of an 8-channel real-time detection housing in a nucleic acid amplification product real-time quantitative detection instrument, which can be used for simultaneous detection of less than 8 samples.
A real-time quantitative detection instrument for nucleic acid amplification products comprises the following steps:
t1, instrument preparation: according to the requirements of different nucleic acid amplification reactions, setting the required temperature, amplification time and absorbance threshold through the data processing element, sending a heating instruction to the heating driving element by the data processing element, controlling the heating element to start heating by the heating driving element, and waiting for the instrument to reach the preset heating temperature.
T2, amplification and real-time monitoring: the method comprises the steps of placing a nucleic acid amplification container filled with a tested sample into a sample detection hole 2, enabling red light emitted by a light source in a first accommodating hole 4 to pass through a transparent nucleic acid amplification container in the sample detection hole 2, receiving the red light by a photoelectric sensor placed in a second accommodating hole 5, starting timing by an instrument, recording the first transmitted light intensity as initial light intensity, automatically measuring the intensity of the transmitted light once through the photoelectric sensor at regular intervals, converting and calculating the transmitted light intensity measured by the photoelectric sensor by an analog-digital conversion element, outputting a result, converting an optical signal into a digital signal, obtaining the digital signal by the data processing element, calculating the absorbance value (formula 1) of the sample liquid according to the lambert law, automatically storing the absorbance value by the data processing element, and displaying the detected absorbance value in real time in a display screen.
Absorbance value=lg (I 0 /I t ) (1)
Wherein I is 0 For the transmitted light intensity at the initial moment, i.e. the initial light intensity, I t The transmitted light intensity at time t.
When the absorbance value of the detected sample reaches or exceeds a preset absorbance threshold value for the first time, a sound generating device in the instrument generates prompt sound; when the timing reaches the preset amplification time, the sounding device sends out a prompt tone and the heating element stops heating, and a user can inquire the whole set of data detected in real time through the display screen.
The light source adopted in the embodiment is a light-emitting diode emitting red light, the photoelectric sensor is a photoresistor, the heating element is a PTC ceramic heating plate, and the temperature sensor is DS18B20; the heating driving element in the detection control module is a MOS tube field effect tube driving plate, the analog-to-digital conversion element is ADS1110, the input element is a 3-key keyboard, the data processing element is an ATmega328P singlechip, the output module is an LCD1602 liquid crystal display screen and a passive buzzer, and the power supply part is a 18650 type lithium battery and a 5V charging and discharging integrated module of Xintai micro-technology Co of Shenzhen city.
As shown in fig. 6, the real-time detection housing includes 8 channels, which can detect 1 to 8 samples simultaneously; the light source is 8 red light-emitting diodes, the photoelectric sensor is 8 photoresistors, the heating element is an MCH aluminum oxide ceramic heating sheet, and the temperature sensor is DS18B20; the heating driving element in the detection control module is a MOS tube field effect tube driving plate, the analog-to-digital conversion element is 2 ADS1115, the input element is a 3-key keyboard, the data processing element adopts an ATmega328P singlechip, the output module adopts an OLED display screen and a passive buzzer, and the power supply part is a 18650 type lithium battery and a 5V charging and discharging integrated module of Xintai micro-technology Co of Shenzhen city.
Based on the description of the present utility model and the accompanying drawings, one skilled in the art can easily manufacture or use a nucleic acid amplification product real-time quantitative detection apparatus of the present utility model, and can produce the positive effects described in the present utility model.
It is noted that the terms "comprising" and "having" and any variations thereof in the description of the utility model and the claims and the foregoing drawings are intended to cover non-exclusive inclusions. The terms "mounted," "configured," "provided," "connected," and "sleeved" are to be construed broadly. For example, it may be a fixed connection, a removable connection, or a unitary construction; may be a mechanical connection, or an electrical connection; may be directly connected, or indirectly connected through intervening media, or may be in internal communication between two mechanisms, elements, or components. The specific meaning of the above terms in the present utility model can be understood by those of ordinary skill in the art according to the specific circumstances.
In the description of the present utility model, it should be understood that the terms "one end," "another end," "outer side," "inner side," "horizontal," "end," "length," "outer end," "left," "right," and the like indicate orientations or positional relationships based on the orientations or positional relationships illustrated in the drawings, merely to facilitate description of the present utility model and simplify the description, and do not indicate or imply that the mechanisms or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and therefore should not be construed as limiting the present utility model. The terms "first," "second," and the like, are also used for simplicity of description only and are not indicative or implying relative importance.
Furthermore, variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed utility model, from a study of the drawings, the disclosure, and the appended claims. Furthermore, in the claims, the specification, the words "comprise", "comprising", and the like do not exclude other elements or steps, and the non-plural terms do not exclude a plurality.
The foregoing description is only illustrative of the preferred embodiments of the present utility model and is not intended to limit the scope of the utility model, i.e. all equivalent changes and modifications that may be made in accordance with the present utility model are covered by the appended claims, which are not intended to be construed as limiting.
Claims (7)
1. A nucleic acid amplification product real-time quantitative detection instrument is characterized in that: including instrument shell in real-time detection module and the detection control module that sets up, detection control module includes analog-to-digital conversion element, data processing element, input element, output element and power supply unit, real-time detection module includes real-time detection casing, be equipped with a plurality of sample detection holes of top open-ended in the real-time detection casing, the both sides of sample detection hole are close to bottom department and are equipped with the accommodation hole respectively, the sample detection hole with be equipped with the logical unthreaded hole between the accommodation hole, the both ends of logical unthreaded hole communicate respectively the sample detection hole with the accommodation hole, detection control module still includes heating drive element, the accommodation hole includes first accommodation hole and second accommodation hole, the bottom of sample detection hole is equipped with the heating chamber with it intercommunication.
2. The apparatus for real-time quantitative detection of a nucleic acid amplification product according to claim 1, wherein: the nucleic acid amplification container with the sample to be detected is placed in the sample detection hole, a heating element and a temperature sensor are arranged in the heating chamber, and a light source and a photoelectric sensor are respectively placed in the first accommodating hole and the second accommodating hole.
3. The apparatus for real-time quantitative detection of a nucleic acid amplification product according to claim 2, wherein: the color of the light emitted by the light source is red.
4. The apparatus for real-time quantitative detection of a nucleic acid amplification product according to claim 2, wherein: the nucleic acid amplification vessel is a transparent vessel that is transparent to light.
5. The apparatus for real-time quantitative detection of a nucleic acid amplification product according to claim 2, wherein: the input end of the data processing element is connected with the output end of the temperature sensor, the output end of the data processing element is connected with the input end of the heating driving element, the output end of the heating driving element is connected with the heating element, the input end of the analog-to-digital conversion element is connected with the output end of the photoelectric sensor, the output end of the analog-to-digital conversion element is connected with the input end of the data processing element, the input element is connected with the input end of the data processing element, and the output end of the data processing element is connected with the output element.
6. The apparatus for real-time quantitative detection of a nucleic acid amplification product according to claim 1, wherein: the hole shafts of the two light-passing holes on two sides of the sample detection hole are on the same axis, and the light-passing holes are perpendicularly intersected with the sample real-time detection hole.
7. The apparatus for real-time quantitative detection of a nucleic acid amplification product according to claim 1, wherein: the output element comprises a display screen and a sound generating device; the real-time detection module and the outside of detection control module are equipped with the instrument shell, the upper surface of instrument shell is equipped with the storehouse lid that can open and shut.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202320899314.4U CN220643115U (en) | 2023-04-20 | 2023-04-20 | Real-time quantitative detection instrument for nucleic acid amplification products |
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| CN202320899314.4U CN220643115U (en) | 2023-04-20 | 2023-04-20 | Real-time quantitative detection instrument for nucleic acid amplification products |
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| CN220643115U true CN220643115U (en) | 2024-03-22 |
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