WO2022126899A1 - 一种便携式荧光检测装置 - Google Patents

一种便携式荧光检测装置 Download PDF

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Publication number
WO2022126899A1
WO2022126899A1 PCT/CN2021/082668 CN2021082668W WO2022126899A1 WO 2022126899 A1 WO2022126899 A1 WO 2022126899A1 CN 2021082668 W CN2021082668 W CN 2021082668W WO 2022126899 A1 WO2022126899 A1 WO 2022126899A1
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Prior art keywords
sample
laser
fluorescence detection
fluorescence
detection
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PCT/CN2021/082668
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English (en)
French (fr)
Inventor
于继彬
戴军
杨晓虎
马攀攀
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苏州先达基因科技有限公司
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Priority claimed from CN202023009762.8U external-priority patent/CN214201190U/zh
Priority claimed from CN202011475002.8A external-priority patent/CN114624215A/zh
Application filed by 苏州先达基因科技有限公司 filed Critical 苏州先达基因科技有限公司
Priority to DE112021004082.0T priority Critical patent/DE112021004082T5/de
Publication of WO2022126899A1 publication Critical patent/WO2022126899A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/02—Mechanical
    • G01N2201/022—Casings
    • G01N2201/0221—Portable; cableless; compact; hand-held

Definitions

  • the present invention relates to a fluorescence detection device, in particular to a fluorescence detection device excited by a laser, and in particular to a fluorescence detection device excited by a laser for analyzing the nucleic acid content in a sample.
  • PCR instruments used for quantitative analysis of nucleic acids generally use LED lamps, halogen lamps, xenon lamps, etc. as the light source of the excitation device.
  • LED light has a large divergence angle, unstable brightness, weak brightness, inconsistent wavelengths, insufficient light purity, strong stray light interference, and poor signal-to-noise ratio, requiring multiple optical calibrations.
  • Various complex and demanding auxiliary accessories such as signal multiplication auxiliary that require expensive filters and light sensors.
  • the unstable brightness of the LED light source puts forward high requirements for optoelectronic engineers to choose the power supply method of the light source.
  • the weak LED brightness makes the excitation light emitted by the excitation device not strong enough, resulting in a complex PD receiving device and weak effective signals.
  • the electronic engineer also needs to extract and amplify this part of the effective signal, and the extracted effective amplified signal cannot be distorted.
  • the difficulty is greatly increased.
  • a PCR machine using an ordinary LED as the excitation device there are 1 plano-convex lens and 1 plano-convex lens, 2 bi-convex lenses and 1 filter lens in the excitation device.
  • their optical signal receiving device is also composed of a plurality of plano-convex, double-convex, and optical filters, and effective signals cannot be detected by wrong order and direction.
  • Halogen lamps, xenon lamps, etc. as the light source of the excitation device also have major disadvantages.
  • the present invention provides a portable fluorescence detection device, which uses a laser as an excitation light source, enlarges the volume of the sample, and allows the laser to irradiate the sample, so that the path of the laser penetrating the sample is extended to a certain range, so that more The fluorescent reagent is excited to generate fluorescence, thereby improving the detection sensitivity.
  • the distance of the sample path through which the laser light penetrates is not less than 3.7 mm, thereby increasing the optical path of the laser light in the sample.
  • the sample includes nucleic acid, which has been amplified, and at the same time, the sample also includes a fluorescent substance for indicating the amount of nucleic acid in the sample.
  • the laser generates more transmission and refraction in the process of penetrating the sample, thereby exciting more fluorescent substances to generate fluorescence, which greatly increases the optical signal intensity, greatly improves the detection sensitivity and accuracy, and realizes stable signal detection.
  • the lasers of different channels can be replaced to realize multi-channel detection of different samples.
  • the portable fluorescence detection device provided by the invention has the advantages of miniaturization, portability and low cost, can be used in public places and home detection, and can realize accurate detection of nucleic acid under limited professional knowledge and experimental equipment.
  • the research team found that when the laser penetrates the sample to a certain thickness or the path distance of the laser in the sample increases, the laser can generate more transmission and refraction during the process of penetrating the sample, making the laser in the The optical path in the sample is significantly increased, thereby exciting more fluorescent substances to generate fluorescence, which can greatly improve the sensitivity of nucleic acid fluorescent detection, expand the range, and realize the miniaturization and simplification of nucleic acid amplification equipment, such as PCR instruments.
  • Nucleic acid material is any similar nucleic acid, such as RNA, DNA or derivatives thereof, hybrid strands, and the like.
  • the portable fluorescence detection device reduces the volume of the device, improves the utilization rate of the internal space of the device, changes the LED excitation light source to laser excitation, and enlarges the reagent tube to 2.5-10 times of the general PCR instrument on the market.
  • the amount of reagents is also greatly increased by nearly 5-10 times, so that the optical signal intensity is greatly increased, and the device is truly portable and low-cost.
  • the present invention provides a portable fluorescence detection device, comprising a laser light source module and a sample cell module for loading a sample, the laser light source module is used for emitting laser light, and the emitted laser light is used to penetrate through the sample cell module. A sample that excites fluorescent substances contained in the sample within a distance of the penetrated path to fluoresce.
  • such a device does not necessarily contain a sample, but during detection, when the sample cell module contains a sample, the sample occupies a certain volume in the sample cell, so that the laser passes through the volume of the sample inside, Thus, the fluorescent substance in the sample is excited to emit fluorescence.
  • the dispersion angle of the laser is small, the light is emitted almost in parallel, the brightness is high, the monochromaticity is good, and no additional filter is required, the interference is small, and the optical signal-to-noise ratio is good, so it has the significant advantages of high sensitivity.
  • the laser as an exciter can make the fluorescence detection device simpler in structure, convenient in assembly and price advantage.
  • the sample here is generally in fluid form, or in a liquid state. These fluids in liquid state must occupy a certain volume in the container, and the form of the volume is determined by the shape of the container. So, when the shape of the container is determined, the way the laser enters determines the distance the laser travels through the sample.
  • the path distance of the laser penetrating the sample mentioned here refers to the length of the sample when the laser passes through. There are many cases. When the sample is placed vertically and the laser is injected from the side, the thickness it passes through is equivalent to the thickness of the sample container.
  • Diameter when the sample is placed vertically and the laser is injected from the bottom, the thickness it passes through is equivalent to the height of the sample in the container; when the sample is placed obliquely and the laser is injected from the side, the thickness it passes through is equivalent to the thickness of the sample.
  • the length of an oblique line of when the sample volume is very small and almost flattens the bottom of the container, when the laser is injected from the side of the sample, it can pass through the surface of the sample, and the length of the path it passes through is the thickness.
  • distance refers to the distance between the laser entering the liquid and the exit from the liquid or the distance of the path traveled. Here, it can be understood as the length of the route that the laser travels in the liquid.
  • the path distance that the sample in the sample cell module is penetrated by the laser is not less than 3.7mm
  • the research team also found that using the laser as the exciter, when the laser penetrates the sample to a thickness of more than 3.7mm, the laser can generate more transmission and refraction during the process of penetrating the sample, which can greatly improve the sensitivity of fluorescence detection of nucleic acid substances. Expand the range, so as to realize the miniaturization and simplification of the PCR instrument.
  • optical path distance through which the sample in the sample cell module is penetrated by the laser light is preferably 3.7mm-15mm.
  • the sample in the sample cell module is 3.7 mm or greater in thickness that is penetrated by the laser light. In some approaches, the length or distance of the path through the liquid is less than 15 mm.
  • the path distance of the sample in the sample cell module being penetrated by the laser light is 6mm-10mm.
  • sample in the sample cell module is contained in a laser penetrable sample container.
  • sample cell module may contain one or more sample containers.
  • sample container is a transparent centrifuge tube.
  • the size of the sample container is 0.5ml-5ml.
  • the portable fluorescence detection device provided by the present invention is mainly used to detect the content of target nucleic acid in a sample.
  • the sample to be detected needs to undergo constant temperature amplification, and the amplification system contains fluorescent probes. Therefore, the container for the sample is a container that can be easily purchased in the market. To the common specifications of transparent centrifuge tubes, including 0.5ml-5ml.
  • the laser emission power of the laser light source module is 5-500mw.
  • the emission power of the laser is related to the intensity of the emitted laser light, as well as the distance through the liquid to allow the fluorescence to be excited to emit fluorescence. It can be understood that when the power is fixed within a certain range, the distance that the laser penetrates the liquid has a direct relationship with the emitted fluorescence. Of course, if the power varies within a certain range, the laser intensity also varies.
  • the present invention provides a nucleic acid detection device, which includes a laser light source module, a fluorescence detection module, and a sample pool module; wherein, the sample pool module is used for containing samples containing nucleic acids, and the samples include nucleic acid indicators indicating the quantity of nucleic acids. Fluorescent substances, the laser light source module is used to emit laser light, so that the emitted laser light transmits and refracts in the process of penetrating the sample, thereby exciting more fluorescent substances to emit fluorescence.
  • the laser emission power of the laser light source module is 1-10000mw; 1-1000mw; 5-500mw; or 50-200mw. If the volume of the sample is determined, the excitation power can be changed to change the intensity of the laser, so as to achieve the intensity of the fluorescent substance to excite the fluorescent substance. In some approaches, when the excitation power is determined, the laser light is passed through the thickness distance of the liquid sample, thereby allowing more fluorescence to be excited to fluoresce.
  • the sample volume in the transparent centrifuge tube is 0.05ml-2.5ml
  • the laser emission power of the laser light source module is 100mw
  • the thickness of the laser penetrating the sample can be maintained in the range of 3.7mm-15mm , so as to achieve stable signal detection.
  • the size of the sample container is 0.5ml-2ml.
  • the size of the sample container is 0.5ml-1.5ml.
  • the number of the transparent centrifuge tubes is 1, the specification is 1.5ml, and the sample volume in the tube is 0.05ml-1.2ml.
  • test tube container is preferably a 1.5ml transparent centrifuge tube, and the sample volume in the tube is 0.05ml-1.2ml, the detection effect is better, and the accuracy and sensitivity are higher.
  • the portable fluorescence detection device only measures a small number of samples each time, for example, only one sample, two samples, three samples, and four samples are measured each time; Instead, it focuses on a smaller and more convenient design to realize on-site fluorescence detection at any time and anywhere.
  • the detection time of a single sample end-point method does not exceed 5s, and it can be tested multiple times by changing samples, without limiting the number of samples to be tested.
  • the sample contains target nucleic acid
  • the target nucleic acid is an amplified amplification product, or purified single-stranded or double-stranded DNA or single-stranded RNA, or artificially synthesized DNA or RNA.
  • the fluorescent substance is used as a labeling substance to indicate the quantity or content of the nucleic acid substance in the sample.
  • the fluorescent material is included on the nucleic acid sequence to indicate the quantity of the target nucleic acid.
  • the sample cell module includes a sample container and a constant temperature reaction block, and the sample container can be installed in the constant temperature reaction block.
  • the constant temperature reaction block is equipped with a base for fixing the transparent centrifuge tube. Bases of different specifications are used together with sample containers of different specifications. The use of sample containers of different specifications can be realized by replacing the base.
  • the isothermal reaction block can be used for isothermal amplification of nucleic acid contained in the sample.
  • the transparent centrifuge tube can be installed in a constant temperature reaction block, and the sample is controlled to maintain a constant temperature through the constant temperature reaction block.
  • heating rods can be installed inside the thermostatic reaction block for temperature control.
  • the laser light source module is replaceable, and is used to emit laser light of different wavelengths.
  • Lasers of different wavelengths can excite different fluorescent substances to emit fluorescence of different wavelengths, and by replacing lasers of different wavelengths, the detection of fluorescence of respective wavelengths can be realized, and the present invention can realize full-spectrum fluorescence detection.
  • the laser light source module may include one or more than one lasers, and different lasers are respectively used to emit lasers of different wavelengths to excite fluorescence of different wavelengths.
  • the laser includes a laser head and a laser head mounting barrel, wherein the laser head is mounted on the laser head mounting barrel.
  • Multiple lasers can be used in combination to further ensure the accuracy of detection.
  • the portable fluorescence detection device further includes a fluorescence detection module, the fluorescence detection module is used to receive fluorescence of different wavelengths, convert the optical signal into an electrical signal after receiving, and further convert it into a digital signal.
  • the fluorescence detection module includes one or more receptors, which are respectively used to receive fluorescence of different wavelengths; the receptors and the laser are arranged at 90 degrees.
  • the receiver includes a signal transfer board mounting block and a signal receiving board.
  • a filter can be installed in front of the light-transmitting hole of the signal receiving plate to help filter out interference light.
  • the laser light source module includes 1-4 lasers, respectively corresponding to the fluorescence detection module including 1-4 receivers; the 1-4 lasers can be turned on at the same time to use 1 or 2-4 combinations of them , and correspondingly open 1 or 2-4 combinations of receivers at the same time.
  • the laser is located on one side of the constant-temperature reaction block, and the light beam enters the transparent centrifuge tube directly from the light-transmitting hole of the constant-temperature reaction block, and the fluorescent reagent emits fluorescence after being excited by the light source;
  • the signal adapter board mounting block is connected to the signal receiving board, located on the other side of the constant temperature reaction block, and is placed at 90 degrees to the laser.
  • the signal adapter board mounting block and the signal receiving board are provided with light-transmitting holes.
  • the fluorescence detection device also includes a main control board, which can convert the optical signal into an electrical signal, and then transmit it to the display screen and output it as a digital signal.
  • the present invention provides a method for improving the sensitivity of a fluorescence detection device, which uses a laser as an excitation light source, and improves the sensitivity of the fluorescence detection device by increasing the distance path through which the sample is penetrated by the laser light.
  • a fluorescence detection device which uses a laser as an excitation light source, and improves the sensitivity of the fluorescence detection device by increasing the distance path through which the sample is penetrated by the laser light.
  • the volume is smaller, the weight is lighter, the shape is regular, and it is easy to carry.
  • the detection accuracy is high, the detection range is wide, and the number of detections is not limited, which improves the detection efficiency, increases the application environment range of fluorescence detection, and improves the portability of the fluorescence detection device.
  • the lasers with different excitation wavelengths can be replaced, and multiple lasers can be configured to be used in combination as needed.
  • the detection range is wide, and there are many fluorescent substances that can be detected, which can realize full-spectrum detection.
  • the reagent tube is large, the reagent tube is enlarged by 2.5-10 times, and the reagent volume is also greatly increased by nearly 5-10 times.
  • the detectable sample volume is large, the fluorescence detection signal is strong, the stability is good, and the detection effect is accurate.
  • the detection speed is fast.
  • the detection time of the single sample end-point method is as fast as 5s, and the number of detections is not limited.
  • test results can be read by directly placing the reagent tube into the sample pool.
  • Detection means assaying or testing for the presence or absence of a substance or material.
  • substances or materials are, for example, but not limited to, chemical substances, organic compounds, inorganic compounds, metabolites, drugs or drug metabolites, organic tissues or metabolites of organic tissues, nucleic acids, proteins or polymers.
  • detection can also represent the quantity of a test substance or material.
  • Assays also mean immunoassays, chemical assays, enzymatic assays, and the like.
  • the samples used by the detection device include biological fluids.
  • the initial state of the sample can be liquid, solid or semi-solid, and the solid or semi-solid sample can be transformed into a liquid sample by any suitable method, such as mixing, mashing, macerating, incubating, dissolving, enzymatic hydrolysis, etc., and then Poured into the collection chamber, the test element detects whether the sample contains the analyte.
  • Samples can be taken from humans, animals, plants, nature, etc. Samples taken from the human body, such as blood, serum, urine, cerebrospinal fluid, sweat, lymph, saliva, gastric juice and other liquid samples; feces, hair, keratin, tartar, finger/toenails and other solid or semi-solid samples .
  • Samples taken from plants can be solid samples such as roots, stems, and leaves; liquid or semi-solid samples such as tissue fluid and cell fluid prepared from roots, stems, and leaves.
  • Samples taken from nature can be liquid samples such as rainwater, river water, seawater, and groundwater; solid or semi-solid samples such as soil, rock, ores, and petroleum.
  • the samples of the present invention are liquid samples.
  • the sample of the present invention is a liquid sample containing a fluorescent substance.
  • the sample of the present invention is a nucleic acid-amplified liquid sample
  • the sample contains nucleic acid substances to be detected
  • the amplification system contains fluorescent probes
  • the fluorescent probes are used as labeling substances indicating the content of nucleic acid substances in the sample.
  • the fluorescent nucleic acid amplification mentioned here can be after the amplification reaction has been completed, or directly after the amplification reaction is performed in the portable fluorescence detection device provided by the present invention, and the detection can be performed directly after the amplification reaction is completed. Of course, it can also be a detection or test at any stage before nucleic acid amplification, during amplification, or after amplification.
  • the nucleic acid substance here can be the target nucleic acid to be detected, and the nucleic acid can be RNA or DNA, and the specific content of the nucleic acid substance, such as the copy number, is represented by a fluorescent labeling substance.
  • nucleic acid amplification in a variable temperature mode, such as PCR amplification, which is embodied in fluorescent PCR and digital PCR amplification.
  • PCR amplification which is embodied in fluorescent PCR and digital PCR amplification.
  • isothermal or isothermal nucleic acid amplification such as LAMP, RPA, TMA, RCA, NEAR, ERA, and amplification using cleavage enzymes, and the like.
  • a test device generally includes a test element, and the so-called test element refers to a component that can detect the analyte in the sample to be tested.
  • the detection of the analyte by the test element can be based on any technical principle, eg immunological, chemical, electrical, optical, molecular, physical, etc.
  • the test element of the present invention may be one kind or a combination of two or more test elements.
  • the test element has a detection area for displaying the detection result, and after the detection is performed, the detection area displays the detection result.
  • the detection device of the present invention is a fluorescence detection device that detects the fluorescence value.
  • the detection device is used to detect the intensity of fluorescence, and of course also includes components that convert optical signals into digital signals. Further, the detection device of the present invention is a fluorescence detection device for detecting the content of target nucleic acid in a sample.
  • Fluorescence detector is a kind of detector commonly used in high pressure liquid chromatograph.
  • the chromatographic fractions are irradiated with ultraviolet light, and when the sample components have fluorescent properties, they can be detected.
  • fluorescence refers to the transition of some electrons in the atom from the lowest vibrational energy level in the ground state to some higher vibrational energy levels after some substance absorbs light of the same frequency as its own. .
  • the electrons collide in the same molecule or other molecules, and consume a considerable amount of energy, thereby falling to the lowest vibrational energy level in the first electronic excited state. This form of energy transfer is called a nonradiative transition.
  • Fluorescence occurs when the vibrational energy level drops from the lowest vibrational energy level to some different energy levels in the ground state, and at the same time emits a light with a lower frequency and a longer wavelength than the original absorption.
  • the light absorbed by the compound is called excitation light, and the resulting fluorescence is called emission light.
  • the wavelength of fluorescence is always longer than the wavelength of ultraviolet light absorbed by the molecule, usually in the visible range.
  • the nature of fluorescence is closely related to the molecular structure, and not all molecules with different structures can emit fluorescence after being excited. Fluorescence involves two processes of light absorption and emission, so any fluorescent compound has two characteristic spectra: excitation spectrum and emission spectrum.
  • the portable fluorescence detection device provided by the present invention is a fluorescence detector that uses a laser to excite a fluorescent substance to emit fluorescence.
  • Fluorescence belongs to photoluminescence, and it is necessary to select an appropriate excitation light wavelength to facilitate detection.
  • the excitation wavelength can be determined by the excitation spectrum of the fluorescent compound.
  • the specific detection method of the excitation spectrum is to scan the excitation monochromator, so that the incident light of different wavelengths excites the fluorescent compound, and the generated fluorescence is detected by the light detection element through the emission monochromator of fixed wavelength.
  • the relationship between the fluorescence intensity and the excitation wavelength is obtained as the excitation spectrum.
  • the maximum wavelength of the excitation spectrum curve the number of molecules in the excited state is the largest, that is, the absorbed light energy is also the largest, which can produce the strongest fluorescence. When sensitivity is considered, the maximum excitation wavelength should be selected for the assay.
  • the portable fluorescence detection device of the present invention uses laser light as excitation light, and the excitation spectrum is the wavelength of the emitted laser light.
  • the fluorescence spectrum actually only refers to the fluorescence emission spectrum. It is a curve of the fluorescence intensity changing with the fluorescence wavelength obtained by the wavelength scanning of the emission monochromator when the wavelength of the excitation monochromator is fixed. Fluorescence spectrum can be used to identify fluorescent substances and be used as a basis for selecting appropriate measurement wavelengths in fluorescence measurement.
  • the energy distribution of the light source, the transmittance of the monochromator, and the response of the detector will vary with the wavelength, so the same compound will get different spectra on different instruments, and There is no analogy with each other, this spectrum is called apparent spectrum. If the same compound can obtain fluorescence spectra with the same characteristics on different instruments, the above characteristics of the instruments need to be corrected. The corrected spectrum is called the true fluorescence spectrum.
  • Excitation and emission wavelengths are necessary parameters for fluorescence detection.
  • the selection of the appropriate excitation wavelength and emission wavelength is important for the sensitivity and selectivity of detection, especially the detection sensitivity can be improved to a great extent.
  • Fluorescence quantitative PCR detector refers to an instrument that uses fluorescent dyes or fluorescently labeled specific probes to label and track PCR products and monitor the reaction process in real time. Combined with the corresponding software, the product can be qualitatively and quantitatively analyzed, and the initial concentration of the sample template to be tested can be calculated. Fluorescent groups are added to the PCR reaction system, and the entire PCR process is monitored in real time by the accumulation of fluorescent signals.
  • PCR can relatively quantify the abundance of a specific gene or species, compare the differences between different treatments or samples, such as gene copy number and expression level, to study the response of gene expression to treatment, and obtain information such as PCR amplification efficiency.
  • the portable fluorescence detection device provided by the present invention further includes a heating module for controlling the temperature of the sample, mainly for isothermal amplification of the nucleic acid contained in the sample, and the amplification system of the sample includes a fluorescent probe, and the fluorescent probe is on the fluorescent probe. Contains fluorophores and quenchers.
  • fluorescence in the ultraviolet-visible-near-infrared region and its fluorescence properties (excitation and emission wavelengths, intensity, lifetime, polarization, etc.) can be sensitively changed with the properties of the environment, such as polarity, refractive index, viscosity, etc. a class of fluorescent molecules.
  • fluorescent probes There are two types of fluorescent chemicals used in real-time PCR: fluorescent probes and fluorescent dyes.
  • a specific fluorescent probe is added along with a pair of primers.
  • the probe is an oligonucleotide, and the two ends are respectively labeled with a reporter fluorescent group and a quenching fluorescent group.
  • the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme cleaves and degrades the probe, making the reporter fluorophore and the quencher group.
  • the fluorescent group is separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, a fluorescent molecule is formed every time a DNA chain is amplified, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product.
  • the probe has a large molar absorption coefficient and a high fluorescence quantum yield; the fluorescence emission wavelength is at a long wavelength and has a large Stokes shift; when used in immunoassays, the binding to antigens or antibodies should not affect their activity. .
  • It can also be used to label undetermined nucleotide fragments, and to specifically and quantitatively detect the amount of nucleic acid.
  • fluorescent probes include fluorescein-based probes, inorganic ion fluorescent probes, fluorescent quantum dots, and molecular beacons.
  • fluorescent probes are widely used in nucleic acid staining, DNA electrophoresis, nucleic acid molecular hybridization, quantitative PCR technology and DNA sequencing.
  • the portable fluorescence detection device of the present invention uses a laser as the excitation light.
  • the excitation wavelength of the laser is 450-490 nm
  • the detected fluorescence emission wavelength is 515-530 nm
  • the detectable fluorescent substances include FAM, SYBR Green I, etc.
  • the excitation wavelength of the laser is 500-535nm
  • the detected fluorescence emission wavelength is 560-580nm
  • the detectable fluorescent substances include VIC, HEX, JOE, TAMRA, TET, Cy3, etc.
  • the excitation wavelength of the laser is 560-580nm 555-585nm
  • the detected fluorescence emission wavelength is 610-650nm
  • the detectable fluorescent substances include ROX, TEXAS-Red, etc.
  • the laser excitation wavelength is 620-650nm
  • the detected fluorescence emission wavelength is 675-730nm, which can be detected
  • the fluorescent substances include Cy5 and so on.
  • the invention can realize free conversion combination between instrument channels by replacing
  • Embodiment 1 is a schematic structural diagram of the portable fluorescence detection device provided in Embodiment 1
  • FIG. 2 is a schematic diagram of the structural disassembly of the portable fluorescence detection device provided in Example 1
  • FIG. 3 is a real photo of the portable fluorescence detection device provided in Example 1
  • Figure 4 is a comparison photo of the same 0.2ml samples in Example 3 being packed in 0.2ml, 0.5ml and 1.5ml transparent centrifuge tubes respectively
  • Fig. 5 is the real-time curve scanning result of using the portable fluorescence detection device in conjunction with the mycoplasma detection reagent in Example 7
  • Embodiment 1 The portable fluorescence detection device provided by the present invention
  • FIG. 1 is a schematic structural diagram of the portable fluorescence detection device
  • FIG. 2 is a schematic structural disassembly diagram of the portable fluorescence detection device
  • a portable fluorescence detection device includes a laser light source module 1 and a sample cell module 2.
  • the laser light source module 1 is used to emit laser light, and the laser acts on the sample in the sample cell module 2 to excite the samples contained in the sample.
  • the fluorescent substance emits fluorescence; the path distance of the sample in the sample cell module 2 being penetrated by the laser light is not less than 3.7 mm.
  • the path distance through which the sample in the sample cell module 2 is penetrated by the laser light is 3.7mm-15mm.
  • the laser is excited from the side, so the “distance” mentioned here refers to the maximum length of the sample that is penetrated by the laser when the sample is placed in the container, which is equivalent to the maximum diameter.
  • the “sample” mentioned here refers to a nucleic acid PCR amplification or isothermal amplification sample containing a fluorescent substance.
  • the sample in the sample pool module 2 is packed in a transparent centrifuge tube 3, and the size of the transparent centrifuge tube 3 is 0.5ml-5ml.
  • the laser emission power of the laser light source module 2 is 5-500mw.
  • the sample pool module 2 only contains one transparent centrifuge tube 3, and the sample volume in the transparent centrifuge tube 3 is 0.05ml-5ml.
  • the portable fluorescence detection device provided in this embodiment focuses on a smaller and more convenient design, and realizes on-site fluorescence detection at any time and anywhere.
  • the end point detection time of a single sample does not exceed 5s, and it can be detected multiple times by replacing the sample without limiting the detection sample. quantity.
  • the sample pool module 2 includes a transparent centrifuge tube 3, a transparent centrifuge tube cover 4 and a constant temperature reaction block 5, the transparent centrifuge tube 3 can be loaded into the constant temperature reaction block 5, and the transparent centrifuge tube cover 4 covers the transparent centrifuge tube 3 above.
  • a base matching the specifications of the transparent centrifuge tube 3 can be set in the constant temperature reaction block 5 to fix the transparent centrifuge tube 3, and the base specifications can be replaced, so that it can be respectively applied to transparent centrifuge tubes 3 of different specifications.
  • the isothermal reaction block 5 can be used for isothermal amplification of nucleic acid contained in the sample.
  • the transparent centrifuge tube 3 can be installed in the constant temperature reaction block 5, and the constant temperature of the sample is controlled by the constant temperature reaction block 5 to maintain a constant temperature.
  • a heating rod can be installed inside the constant temperature reaction block 5 for temperature control.
  • the laser light source module 1 in this embodiment can be only the laser 6 , and the laser 6 includes a laser head 7 and a laser head mounting barrel 8 , wherein the laser head 7 is mounted on the laser head mounting barrel 8 .
  • the laser 6 provided in this embodiment is replaceable and is used to emit lasers of different wavelengths, which can excite different fluorescent substances to emit fluorescence of different wavelengths. By replacing the lasers of different wavelengths, the detection of fluorescence of respective wavelengths can be realized. Spectral fluorescence detection.
  • the laser light source module 1 can also include one or more lasers 6, and different lasers 6 are respectively used to emit lasers of different wavelengths to excite fluorescence of different wavelengths; multiple lasers can be used in combination to further ensure detection By replacing the laser 6 or adding the laser 6, the free conversion combination between the device channels can be realized, which provides more convenience for accurate detection.
  • the fluorescence detection device further includes a fluorescence detection module 9, and the fluorescence detection module 9 includes one or more receivers 10, which are respectively used for receiving fluorescence of different wavelengths; put.
  • the receiver 10 includes a signal adapter board mounting block 11 and a signal receiving board 12 .
  • a filter 13 can be installed in front of the light-transmitting hole of the signal receiving plate 12 to help filter out interference light.
  • the laser light source module 1 may include 1-4 lasers 6, respectively corresponding to the detection module 9 including 1-4 receivers 10; the 1-4 lasers 6 may be turned on at the same time to use one or 2-4 combinations, and correspondingly open 1 or 2-4 combinations in the receiver 10 at the same time.
  • the position of the laser 6 is parallel to the receiver 10 on the right side of the sample cell module 2, which further increases the space utilization of the device and reduces the interference of light.
  • the laser 6 and the receiver 10 when the laser 6 and the receiver 10 are installed in two positions at the same time, the fluorescence detection channel is increased, and the detection range and types of samples are increased.
  • the four sides of the sample cell module 2 are installed at the same time
  • the laser light source module 1 can include 4 lasers 6 and 4 receivers 10 .
  • the excitation wavelength of the first laser 6 is 450-490 nm
  • the detection wavelength is 515-530 nm
  • the detectable fluorescent substances include FAM, Sybr Green I, etc.
  • the excitation wavelength of the second laser 6 is 500-535nm
  • the detection wavelength is 560-580nm
  • the detectable fluorescent substances include VIC, HEX, JOE, TAMRA, TET, Cy3, etc.
  • the excitation wavelength of the third laser 6 is 555-585nm, and the detection wavelength is 610-650nm.
  • the detected fluorescent substances include ROX, TEXAS-Red, etc.; the excitation wavelength of the fourth laser 6 is 620-650 nm, the detection wavelength is 675-730 nm, and the detectable fluorescent substances include Cy5 and the like.
  • the present invention can realize the free conversion combination between the instrument channels by replacing the laser 6 or adding the laser 6, and adding two exciters 6 and receivers 10 to the sample pool module 2 at the same time (Example 1: the sample pool module 2 simultaneously adds the first and the second Exciter 6 and receiver 10; Example 2: The first and third exciters 6 and receivers 10 are added to the sample cell module 2 at the same time, and so on, there can be various combinations) to achieve full-spectrum fluorescence detection.
  • the laser 6 is located on one side of the constant-temperature reaction block 5, and the light beam enters the transparent centrifuge tube 3 directly from the light-transmitting hole of the constant-temperature reaction block 5, and the fluorescent reagent is subjected to
  • the light source emits fluorescence after excitation
  • the signal adapter plate mounting block 11 is connected to the signal receiving plate 12, located on the other side of the constant temperature reaction block 5, and is placed at 90 degrees to the laser.
  • the signal adapter plate mounting block 11 and the signal receiving plate 12 Both are provided with light-transmitting holes 14 .
  • the fluorescence detection device further includes a main control board 15 , which can convert optical signals into electrical signals, which are then transmitted to the display screen and output as digital signals, and also includes a base 16 .
  • the portable fluorescence detection device provided in this embodiment is used for detection, since the laser is excited from the side, when the transparent centrifuge tube contains a normal volume of sample, the thickness of the laser penetrating the sample is equivalent to the diameter of the PCR tube.
  • the volumes of the samples to be tested that have been amplified are 0.025ml, 0.05ml, 0.1ml, 0.2ml, 0.4ml, 0.8ml and 1.2ml, respectively, and put them into 1.5ml reagent tubes.
  • the detection devices are respectively tested.
  • the laser emission power of the laser light source module is 100mw. When the laser is excited from the side, the influence of the same volume of samples in different specifications of reagent tubes on the detection results is investigated.
  • amplification reaction system 60mM Tris-acetic acid buffer (pH8.0), 100mM potassium acetate, 3mM dithiothreitol, 5% polyethylene glycol (20000), 2mM ATP, 20mM phosphocreatine acid, 420nM primer mix (upstream primer sequence, 5'-AATTTGTGCGAGTAAAACCTATGTAGCAGCAGAG-3'; downstream primer sequence, 5'-TTCCTTCTAAGCTCTGCAGCTTCATTCATCATC-3'), 200nM fluorescent probe (probe sequence 5'-AAAATGTCGGGAGCTGAACATATGGAAGG(FAM-dT) ( THF)T(BHQ1-dT)GAGCGAGTGCT-3'(C3-SPACER)), 100ng/ ⁇ l creatine kinase, 600ng/ ⁇ l bacteriophage gp32 protein, 150ng/ ⁇ l bacteriophage uvsX protein, 25ng/ ⁇ l bacteriophage uvsY
  • the above reaction was prepared into 1ml system, a total of 3 tubes were prepared, and the total volume was 3ml, which was fully shaken and mixed and centrifuged briefly. It was placed in a water bath, reacted at 37° C. for 30 minutes, and then reacted at 80° C. for 5 minutes to inactivate the enzyme components in the reaction components, and the reaction was fully completed.
  • the fluorescent probe is cleaved at THF and the fluorescent signal is released due to loss of quenching.
  • the laser is irradiated at the center of the reagent sample.
  • the average path distance penetrated by the laser increases from 3mm to 10.09mm.
  • the total amount of excited fluorescent substances It also increases in turn, so as to emit more fluorescence; when the sample volume is increased from 0.8ml to 1.2ml, especially when the sample is increased from 1.0ml to 1.2ml, the diameter of the centrifuge tube will not continue to increase because the upper end of the centrifuge tube is cylindrical.
  • the average path distance penetrated by the laser also slows down, and the growth rate of the optical path also slows down. Even when the sample volume continues to increase, the path distance penetrated by the laser does not increase because the diameter remains unchanged. , the optical path growth rate is no longer obvious. At this time, if the sensitivity needs to be further improved, a larger centrifuge tube needs to be replaced to further increase the distance of the path penetrated by the laser, thereby further increasing the optical path.
  • the sample volume is 0.05ml-0.4ml
  • the diameter gradually increases from bottom to top, and the path distance of the sample in the transparent centrifuge tube being penetrated by the laser increases from 5mm to 10mm.
  • the measured fluorescence value also increased rapidly from 6046 to 23663, an increase of nearly 4 times, and the detection sensitivity increased significantly.
  • the path distance of the sample penetrated by the laser is 10.09-10.22mm.
  • the path distance penetrated by the laser increases slowly, and the optical path length increases. The change trend of the measured fluorescence value is obviously slowed down, and the influence on the detection result is not obvious.
  • the thickness of the laser penetration also increases, which makes the transmission and refraction of the laser in the sample more sufficient, thereby exciting more fluorescent substances to emit fluorescence, and the measured fluorescence value increases significantly. high, thereby improving the detection sensitivity.
  • the "thickness" of the sample liquid penetrated by the laser refers to the length of the path of the light generated by the laser passing through the liquid sample.
  • the length of the path can be no less than 3 mm, no less than 4 mm, no less than 5 mm, no less than 6mm, not less than 8mm, not less than 9mm, not less than 10mm.
  • the intensity of the laser can be increased to improve the detection sensitivity.
  • the length of the path of the laser light through the liquid sample is adjusted together with the intensity of the laser light to achieve the detected result.
  • the path here can also be understood as such a way that after the laser passes through the liquid sample, the light generated by the laser is repeatedly refracted and reflected through the liquid sample.
  • the path can also be understood that one or more laser beams pass through the liquid sample at the same time, considerably extending the length of the path through the liquid.
  • Example 3 The influence of the same volume and different reagent penetration optical paths on the detection results
  • This implementation case examines the difference in fluorescence values of fluorescent reagents with the same concentration and volume under different penetration light paths. Take 0.2ml of the reagent in Example 2, put it into a 0.2ml PCR reagent tube, a 0.5ml transparent centrifuge tube, and a 1.5ml transparent centrifuge tube with a micropipette, respectively. For detection, the laser emission power of the laser light source module is set to 50mw.
  • the laser When the laser is excited from the side, adjust the position of the sample cell module 2 at the same time to ensure that the laser emission position and the detection position pass through the center of the reagent center, and the temperature is set to 55 °C, such as As shown in Figure 4, from left to right are 0.2ml PCR reagent tube, 0.5ml transparent centrifuge tube, 1.5ml transparent centrifuge tube, and 0.2ml sample is loaded at the same time, the distance of the path penetrated by the laser is equivalent to its diameter. The influence of the same volume of samples in different specifications of reagent tubes on the detection results.
  • the sample volume is 0.2ml, and they are placed in containers with volumes of 0.2ml, 0.5ml, and 1.5ml.
  • the path distance of the laser penetrating the sample is mainly determined by the shape of the container where the sample is located. Decide.
  • the paths penetrated by the laser are about 4.2mm, 5.1mm, and 7.8mm respectively, that is, as the volume of the container increases, the path distance of the laser penetrating the sample gradually increases, resulting in The transmission and refraction are also stronger, and the excitation of more fluorescent substances produces stronger fluorescence.
  • the purpose of the test in this example is to limit the dilution of the reagent to the limit concentration, so that the fluorescence intensity cannot be detected in a small volume, and by increasing the volume and the thickness of the reagent, the instrument can re-detect the fluorescence. In order to prove that the increase of the reagent volume or the reagent thickness is helpful to improve the sensitivity of the reagent to the sample.
  • the distance path of laser penetration is different, and the maximum path distance of the sample penetrated by the laser is equivalent to the diameter of the centrifuge tube where the highest liquid level of the sample is located.
  • the volume is increased from 0.025ml to 1.6ml, and the diameter of the centrifuge tube where the highest liquid level is located is increased from 3.9mm to 14.3mm, that is to say, the maximum path distance penetrated by the laser increases from 3.9mm to 14.3mm.
  • the resulting transmission and refraction also increase rapidly, and the optical path increases rapidly, so that the excited fluorescent substances in the sample also increase rapidly;
  • the sample volume is from 1.6ml to 3.2ml, the Because the upper end of the centrifuge tube is cylindrical, the diameter will not continue to increase, so the distance of the path penetrated by the laser will not increase, and the optical path will not continue to increase, so that the excited fluorescent substances in the sample will not continue to increase.
  • the reagent with small volume or small optical path cannot detect the fluorescence reading value.
  • the volume of the reagent is gradually increased, the path distance of the sample penetrated by the laser increases continuously, so that the rapid Increasing the optical path can make the transmission and refraction of the laser in the sample more sufficient, so as to excite more fluorescent substances to emit fluorescence, so that the measured fluorescence value can be gradually detected, and increases significantly with the increase of reagent volume or optical path. high, thereby improving the detection sensitivity.
  • the portable fluorescence detection device in Example 1 was used for the LAMP amplification reaction.
  • the final volume of reagents for each reaction was 0.25ml.
  • the reaction conditions were referred to the amplification in step 2 in Example 2 of ZL201310368890, and paraffin oil was used to seal the liquid to prevent gas Sol is produced.
  • the temperature was set to 61°C, and the reaction was carried out for 30 minutes.
  • Sybr Green I dye at a final concentration of 1x was added to perform end-point scanning to record the difference between negative and positive values, respectively.
  • the above case shows that the determined fluorescent device can be used to determine the negative and positive qualitative detection of samples after LAMP amplification.
  • AceQqPCR SYBR Green Master Mix (Low ROX Premixed) from Nanjing Novizan Biotechnology Co., Ltd. Cat. No.: Q131-02 dye-based fluorescence quantitative premixed PCR reagent was used for amplification, and the reaction was prepared according to the kit instructions.
  • the reaction system is 25ul per tube, and the amplified product is compared with the portable fluorescence detection device provided in Example 1, and the fluorescence intensity of the amplified product is compared and detected.
  • the minimum detection line of Q-PCR reagents is 5 copies/reaction, and the negative controls are all flat lines, and no fluorescence amplification curve appears.
  • Experiments were performed with a sample concentration of 5 copies/reaction. 16 reactions were tested on each instrument, with 8 positive control replicates and 8 negative control replicates. The experimental results show that the two instruments can detect all 8 positives, the melting curve is a single peak, and the results are normal; the negatives are also normal, and no amplification curve appears.
  • Upstream primer sequence mMPF 5'-TAATACTAATGAGTCGAGGA mCmUmU mA TT mG G mA mA mG-3';
  • Downstream primer sequence mMPR 5'-CAGCGACAGAGTCACCAAACAA mA mAAmCmG A mC mA-3';
  • Probe sequence mMPBP 5'-FAM-CTGCGTATmUmU C mC TACCA mA mAmGmGmC TACGCAG-BHQ1-3';
  • the final reaction concentration of the upstream primers contained in the reaction tube is 0.5uM, and the downstream is 0.2uM, and 500 copies of Mycoplasma pneumoniae are respectively added to the prepared reaction tube, and 1ul is added in each reaction.
  • the final buffer concentration of the reaction is shown in Table 6:
  • the final concentration of dNTPs was 0.4 mM; the final concentration of manganese ions was 2 mM; the final reaction volume was 0.3 ml, and the enzyme was added to the cap of the tube. , shake at 2000RPM/min for 30s; then transfer to the portable fluorescence detection device of Example 1, continue to react at 55°C for about 10min, collect the fluorescence signal value every 6s, the result is shown in Figure 5, when the end-point scan value When it is lower than 1000, it is judged as negative, and when it is higher than 2000, it is judged as positive.
  • Example 8 Using a portable fluorescence detection device with mycoplasma detection reagent to perform endpoint scanning to confirm the sensitivity
  • the probe was replaced with Rox fluorescently labeled reagent, mMPBP-R: 5'-Rox-CTGCGTATmUmU C mC TACCA mA mAmGmGmC TACGCAG-BHQ2-3' and other reaction conditions were consistent.
  • the samples were crudely lysed buccal swabs of Mycoplasma pneumoniae genomic DNA. After confirming the sample concentration by traditional fluorescence quantitative PCR, the positive samples were diluted with negative crudely lysed samples to a concentration of 400 copies in the milliliter sample, and 0.25ml was added to each reaction. Sample, a total of 100 copies, the total reaction system is still 0.3ml.
  • the experiment was repeated 20 times, and the end-point scanning method was used to confirm the end-point value.
  • the excitation wavelength of the fluorescence device was replaced with a laser of about 585 nm
  • the detection wavelength was replaced with the corresponding filter and optical signal detector of about 625 nm. According to the data obtained in a large number of experiments in the early stage, it is determined that when the end-point scan value is lower than 1000, it is judged as negative, and when it is higher than 1500, it is judged as positive.
  • the device of the present invention was used to determine the detection sensitivity of the mycoplasma detection reagent, and the results are shown in Table 7.
  • the nucleic acid content in the sample may be low during the detection of nucleic acid content, it is difficult to ensure accuracy when using a low sample volume for detection. Therefore, using a laser as the excitation light source and increasing the thickness of the sample penetrated by the laser can significantly improve the detection sensitivity and accuracy.
  • the laser In order to realize the miniaturization and convenience of the fluorescence detection device, the laser must be used as the excitation light source, and at the same time, the volume of the sample must be increased, so that the thickness of the sample penetrated by the laser increases, so as to achieve stable signal detection.

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Abstract

一种便携式荧光检测装置，包含激光光源模块(1)和用于装载样品的样品池模块(2)，采用激光作为激发光源，使激光穿透的样品的路径距离增大，从而增大光程，使光信号强度增加，提高检测灵敏度和准确性，实现了稳定的信号检测；并可更换不同通道的激光器，实现不同样品的多通道检测。该便携式荧光检测装置为小型化便携式、成本低、检测速度快、检测试剂管体积大，试剂量大、荧光检出信号值高、检出结果稳定、精确度高、检测范围广，可检测的荧光类物质多，无需专业人员操作，增大了荧光检测的实际应用领域。

Description

一种便携式荧光检测装置 技术领域
本发明涉及一种荧光检测装置，具体而言，涉及一种由激光激发的荧光检测装置，尤其涉及一种由激光激发的用于分析样本中核酸含量的荧光检测装置。
背景技术
目前用于定量分析核酸的PCR仪普遍使用LED灯和卤素、氙气灯等作为激发装置的光源。LED光的发散角大，亮度不稳定，亮度弱，波长不一致，光纯度不足，杂光干扰强，信噪比差，需要多次光校准，导致它在设计激发装置和接受装置的时候要添加各种复杂和苛刻的辅助配件，如需要昂贵的滤光片和光感应器信号倍增辅助。LED光源亮度不稳定对光电工程师选择光源供电方式提出了很高的要求。LED亮度弱致使激发装置发出的激发光不够强，导致PD接受装置复杂且有效信号弱，电子工程师还需对这部分有效信号进行提取和放大，且提取的有效放大信号不能失真导致电路设计时的难度大大增加。例如使用普通LED作为激发装置的PCR仪中，在激发装置中就有1个平凸透和1个平凸透镜，2个双凸透和1个滤光镜片组成，它们组装的时候是通过顺序和方向来增加光强的，它的光信号接受装置也是由多个平凸，双凸，滤光片组成，顺序和方向装错均无法检测到有效信号。卤素灯、氙气灯等作为激发装置的光源也有较大缺点，它自身发光体积大所需消耗的电功率增加，发光热量高要加散热装置，光的发散角度大也需要光的校准，这些缺点使得仪器不能小型化。虽然已有少量文献或专利报道采用激光作为激发光源来制备PCR仪，如ABI 7900HT荧光定量PCR仪，采用氩离子激光器，其价格昂贵，且波长单一，目前市场上已基本被淘汰。
普通的荧光定量PCR仪或恒温荧光仪器均需要非常复杂精密的光信号过滤和信号放大过程，并需要昂贵的光信号感应元件或装置，采用传统光学元件，复杂的加热元件等，导致现有PCR荧光检测设备存在集成度不足，光源和检测器数量多，多个光源和检测器之间的差异影响检测结果的一致性，需要离线设备和资源，设备大且昂贵。而且常规设备的精密性和复杂程度都要求复杂的参数设置，专业的检测人员操作，限制了其应用的普遍性，难以实现公共场合及家庭检测的常规检测。
这就需要对现有核酸扩增设备进行改变，提高检测的灵敏度，同时让设备仪器小型化或者微型化。
发明内容
为解决上述问题，本发明提供了一种便携式荧光检测装置，该装置采用激光作为激发光源，放大样品体积，让激光照射样品，从而让激光穿透样品的路径延长至一定的范围，使更多的荧光试剂被激发后产生荧光，从而提高检测的灵敏度。
在一些优选的方式中，使激光穿透的样品路径距离不小于3.7mm，从而增大激光在样品中的光程。在一些方式中，样品中包括核酸，该核酸是经过扩增过的，同时，样品中还包括荧光物质，用于指示样品中核酸数量。这样，激光在穿透样品过程中产生更多的透射与折射，从而激发更多的荧光物质产生荧光，使得光信号强度大大增加，大幅提高检测灵敏度和准确性，实现了稳定的信号检测。另外，并可更换不同通道的激光器，实现不同样品的多通道检测。本发明提供的便携式荧光检测装置具有小型化、便携式、成本低的优点，可用于公共场合及家庭检测，并能在有限的专业知识和实验设备下实现核酸的精确检测。
本研究小组令人惊喜地发现，使激光穿透样本达到一定厚度或者让激光在样品中的路径距离增大的时候，激光在穿透样品过程中可产生更多的透射与折射，使激光在样品中的光程显著增大，从而激发更多的荧光物质产生荧光，可大幅提高核酸物质荧光检测的灵敏度，扩大量程，并能实现核酸扩增设备，例如PCR仪的小型化简单化设计。换句话说，就是让激光穿过一定厚度的溶液或者让激光进入液体中并在液体中穿过的距离增大，该溶液中含有被测试的样品和荧光物质，这样可以达到高灵敏度的检测荧光的含量，从而可以高灵敏度检测对应的核酸物质。核酸物质是任何类似的核酸，例如RNA、DNA或其衍生物、杂交链等。
本发明提供的便携式荧光检测装置，减小了装置的体积，提高了装置内部空间利用率，将LED激发光源改为激光器激发，将试剂管放大为市面上的一般PCR仪的2.5-10倍，试剂量也大大提高了近5-10倍，使得光信号强度大大增加，真正实现了装置的便携式和低成本。
一方面，本发明提供了一种便携式荧光检测装置，包含激光光源模块和用于装载样品的样品池模块，所述激光光源模块用于发射激光，发射的激光用于穿透样品池模块中的样品，激发被穿透路径距离内的样品中包含的荧光物质发出荧光。
可以理解，这样的设备并不必然含有样品，而是在检测的时候，当样品池模块中含有样品的时候，样品在样品池里占据一定的体积，从而让激光穿过该体积的样品内部，从而让样品中的荧光物质被激发而发出荧光。
激光的分散角小，光几乎是平行发射的，亮度高、单色性好、甚至可以不需要额外配备滤光片、干扰小，光信噪比好，因此具有敏感度高的显著优点，选用激光作为激发器，可使荧光检测装置结构更简单，组装方便并具价格优势。
这里的样品一般是流体形式，或者液体状态。这些液体状态的流体在容器里，一定占据一定的体积，该体积的形式是由容器的形状决定的。所以，当容器的形状确定的情况下，激光的进入方式就确定激光穿过样品的距离。这里所说的激光穿透样本的路径距离是指样本被激光穿过时的长度，具体有多种情况，当样本竖直放置，激光从侧面射入时，其穿过的厚度相当于样本容器的直径；当样本竖直放置，激光从底部射入时，其穿过的厚度相当于样本在容器内的高度；当样本倾斜放置，激光从侧面射入时，其穿过的厚度相当于贯穿样本的一条斜线的长度；当样本体积量很小，近乎平铺容器底部时，激光从样本侧面射入时，可从样本表层穿过，其穿过的路径长度即为所述厚度。所谓的“距离”是指激光进入液体到从液体出来之间的长度或者走过的路径的距离，这里可以理解为激光在液体中经过的路线的长度。
进一步地，所述样品池模块中的样品被激光穿透的路径距离为不小于3.7mm
同时研究小组还发现，采用激光作为激发器，当激光穿透样本达到3.7mm以上厚度时，激光在穿透样品过程中能产生更多的透射与折射，可大幅提高核酸物质荧光检测的灵敏度，扩大量程，从而真正实现PCR仪的小型化简单化设计。
进一步地，所述样品池模块中的样品被激光穿透的光程距离优选为3.7mm-15mm。
在一些方式中，所述样品池模块中的样品被激光穿透的厚度为3.7mm或者大于3.7mm。在一些方式中，穿过液体的路径的长度或者距离小于15mm。
进一步地，所述样品池模块中的样品被激光穿透的路径距离为6mm-10mm。
进一步地，所述样品池模块中的样品被装在激光可穿透的样品容器内。
进一步地，所述样品池模块可含有1个或1个以上的样品容器内。
进一步地，所述样品容器内为透明离心管。
进一步地，所述样品容器的规格为0.5ml-5ml。
本发明提供的便携式荧光检测装置主要用于检测样品中的靶核酸含量，待检测的样品需要经过恒温扩增，扩增体系中包含荧光探针，因此装样品的容器采用的是市面上容易购买到的普通规格的透明离心管，包括0.5ml-5ml。
进一步地，激光光源模块的激光发射功率为5-500mw。激光的发射功率与发出的激光强度有关，也与穿透液体的距离从而让荧光激发发出荧光的强度相关。可以理解，当功率固定在一定范围内的时候，激光穿透液体的距离与发出的荧光具有直接关系。当然，如果功率在一定范围内变化，激光强度也发生变化。
所以，另一方面，本发明提供一种核酸检测设备，该设备包括激光光源模块、荧光检测模块和样品池模块；其中，样品池模块用于装含有核酸的样品，样品中包括指示核酸数量的荧光物质，所述激光光源模块用于发射激光，让发射的激光在穿透样品过程中产生透射与折射，从而激发更多的荧光物质发出荧光。
在一些方式中，激光光源模块的激光发射功率为1-10000mw；1-1000mw；5-500mw；或者50-200mw。如果样品的体积确定，可以改变激发功率来改变激光的强度，从而实现荧光物质激发荧光物质的强度。在一些方式中，当激发功率确定的情况下，让激光穿过液体样品的厚度距离，从而让更多的荧光被激发而发出荧光。
当采用0.5ml-5ml的透明离心管，透明离心管内的样品体积为0.05ml-2.5ml，激光光源模块的激光发射功率为100mw时，可使激光穿透样本的厚度保持在3.7mm-15mm范围内，从而实现稳定的信号检测。
进一步地，所述样品容器的规格为0.5ml-2ml。
进一步地，所述样品容器的规格为0.5ml-1.5ml。
进一步地，所述透明离心管的个数为1根，规格为1.5ml，管内样品体积为0.05ml-1.2ml。
当所述试管容器优选为1根1.5ml规格的透明离心管，管内的样品体积为0.05ml-1.2ml时，检测效果更好，精确度和灵敏度更高。
本发明提供的便携式荧光检测装置，每次只测少量几个样品，如，每次只测一个样本，两个样本，三个样本，四个样本；不同于目前市面追求的高通量检测，而是着眼于更小型、更便捷的设计，实现随时随处现场荧光检测，单个样品终点法检测时间不超过5s，且可以通过更换样品，多次检测，不限制检测样品数量。
进一步地，所述的样品中含有目的核酸，所述目的核酸是经过扩增过的扩增产物，或者经过提纯过的单链或双链DNA或单链RNA，或者人工合成的DNA或RNA。
进一步地，所述荧光物质作为标记物质指示样品中核酸物质的数量的多少或者含量的高低。
进一步地，所述荧光物资被包括在核酸序列上用来指示目的核酸的数量。
进一步地，所述样品池模块包括样品容器和恒温反应块，所述样品容器可装入恒温反应块内。恒温反应块内装有用于固定透明离心管的底座，不同规格的底座与不同规格的样品容器配套使用，通过更换底座可实现不同规格样品容器的使用。
进一步地，所述恒温反应块可用于使样品中含有的核酸进等温扩增。
进一步地，透明离心管可安装在恒温反应块内，通过恒温反应块控制样品保持恒定温度。在一些 实施方式中，可以采用加热棒安装于恒温反应块内部用于温度的控制。
进一步地，所述激光光源模块可更换，用于发射不同波长的激光。
不同波长的激光，可以激发不同的荧光物质发出不同波长的荧光，通过更换不同波长的激光，可以实现各自波长荧光的检测，本发明可实现全光谱荧光检测。
进一步地，所述激光光源模块可包含一个或一个以上的激光器，不同的激光器分别用于发射不同波长的激光，用于激发不同波长的荧光。
进一步地，所述激光器包括激光头和激光头安装桶，其中激光头安装在激光头安装桶上。
多个激光器可以进行组合使用，进一步保证检测的精确度。
通过更换激光器或增加激光器，实现装置通道间的自由转换组合，为精准检测提供更多的便利。
进一步地，所述便携式荧光检测装置还包括荧光检测模块，所述荧光检测模块用于接受不同波长的荧光，并将光学信号接收后转化为电信号，再进一步转换为数字信号。
所述荧光检测模块包含一个或一个以上的接受器，分别用于接受不同波长的荧光；接受器与激光器呈90度摆放。
进一步地，所述接受器包括信号转接板安装块和信号接收板。
进一步地，在信号接收板透光孔前可安装滤光片，帮助过滤掉干扰光。
进一步地，所述激光光源模块包含1-4个激光器，分别对应于荧光检测模块包含1-4个接受器；所述1-4个激光器可同时开启使用其中的1个或2-4个组合，并对应同时开启接受器中的1个或2-4个组合。
进一步地，恒温反应块的后方和右侧有透光孔；激光器位于恒温反应块的一侧，光束从恒温反应块的透光孔直射进入透明离心管内部，荧光试剂受光源激发后发出荧光；信号转接板安装块和信号接收板连接，位于恒温反应块的另一侧，与激光器呈90度摆放，信号转接板安装块和信号接收板上都设有透光孔。
进一步地，所述荧光检测装置还包括主控板，可将光信号转变为电信号，然后传输到显示屏上输出为数字信号。
再一方面，本发明提供一种提高荧光检测装置灵敏度的方法，所述荧光检测装置采用激光作为激发光源，通过增加样品被激光穿透的距离路径来提高荧光检测装置的灵敏度，所述样品中含有目的核酸，以及用于标记目的核酸数量的荧光物质。
本发明提供的便携式荧光检测装置，具有如下的有益效果：
1、比市场上常见的荧光检测仪体积小，质量轻，外形规整，便于携带。
2、检测准确度高，检测范围广，不限数量检测，提高了检测效率，增加了荧光检测的应用环境范围，提高了荧光检测装置的便携性。
3、可以更换不同激发波长的激光器，也可配置多个激光器根据需要组合使用，检测范围广，可检测的荧光类物质多，可实现全光谱检测。
4、试剂管大，试剂管放大2.5-10倍，试剂量也大大提高了近5-10倍，可检测样品体积大，荧光检出信号强，稳定性好，检出效果精确。
5、检测速度快，单个样品终点法检测时间最快至需要5s，且不限检测数量。
6、不需要复杂的参数设置，直接将试剂管放入样品池就可读出检测结果。
7、因为激光功率大，试剂内的光程增长，带来的有益效果，让我们可以不需要精密昂贵的光学感应器，光电放大和精密的计算，而同样达到高灵敏度效果。这样，成本大大降低，仪器体积可以缩 小至苹果大小，稳定性也大大提高。
详细说明
下面对本发明涉及的结构或技术术语做进一步的说明，如果没有特别指明，按照本领域的通用的一般术语进行理解和解释。这些说明仅仅是采用举例的方式进行说明本发明的方式是如何实现的，并不能对本发明构成任何的限制，本发明的范围由权利要求进行限定和表达。
检测
检测表示化验或测试一种物质或材料是否存在。所述的物质或材料例如、但并不限于化学物质、有机化合物、无机化合物、新陈代谢产物、药物或者药物代谢物、有机组织或有机组织的代谢物、核酸、蛋白质或聚合物。另外，检测还可以表示测试物质或材料的数量。化验还表示免疫检测、化学检测、酶检测等。
样品
检测装置所使用的样品包括生物液体。样本的初始状态可以是液态、固态或半固态的，固态或半固态的样本可以通过任何适当的方法转化成液态样本，例如混合、捣碎、浸软、孵育、溶解、酶解等等，然后倒入收集腔中，通过测试元件检测样本是否含有被分析物。样本可以取自人体、动物、植物、自然界等。取自人体的样本，例如可以是血液、血清、尿液、脑脊髓液、汗液、淋巴液、唾液、胃液等液态样本；粪便、毛发、角质、牙垢、指/趾甲等固态或半固态的样本。取自植物的样本，例如可以是根、茎、叶等固态样本；由根、茎、叶制备的组织液、细胞液等液态或半固态样本。取自自然界的样本，例如可以是雨水、河水、海水、地下水等液态样本；土壤、岩石、矿石、石油等固态或半固态样本。
在一些方式中，本发明所述的样品为液态样本。
在一些方式中，本发明所述样品为包含荧光物质的液态样本。
进一步地，本发明所述样品为经核酸扩增的液态样本，样本中包含需检测的核酸物质，扩增体系中包含荧光探针，荧光探针作为指示样品中核酸物质多少含量的标记物质。这里所说的荧光核酸扩增，可以是已经完成扩增反应后，也可以直接在本发明所提供的便携式荧光检测装置进行扩增反应后，扩增反应结束后直接进行检测。当然，也可以是处于核酸扩增前，扩增中，扩增后的任何阶段的检测或者测试。这里的核酸物质可以是被检测的目标核酸，核酸可以是RNA，也可以是DNA，利用荧光标记物质来表示核酸物质的具体含量，例如拷贝数。
这里的任何能够让核酸扩增的技术都是作为本发明的一个具体的方式，例如变温模式的核酸扩增，例如PCR扩增，具体体现为荧光PCR、数字PCR扩增。还比如等温或者恒温核酸扩增，例如LAMP，RPA，TMA，RCA，NEAR，ERA，以及利用切割酶的扩增等等。
测试装置
测试装置中一般包括测试元件，所谓的测试元件指的是能够对待测样本中的被分析物质进行检测的部件。测试元件对被分析物质的检测可以基于任何技术原理，例如免疫学、化学、电学、光学，分子学、物理学等。本发明的测试元件可以是一种，也可以是两种以上测试元件的组合。测试元件具有 用于显示检测结果的检测区，进行检测之后、检测区显示检测结果。
本发明所述的检测装置是通过检测荧光值的荧光检测装置。该检测装置来检测荧光的强度的设备，当然也包括把光学信号转化为数字信号的元器件。进一步地，本发明所述的检测装置是用于检测样本中的靶核酸含量的荧光检测装置。
荧光检测仪
荧光检测器是高压液相色谱仪常用的一种检测器。用紫外线照射色谱馏分，当试样组分具有荧光性能时，即可检出。
从电子跃迁的角度来讲，荧光是指某些物质吸收了与它本身特征频率相同的光线以后，原子中的某些电子从基态中的最低振动能级跃迁到较高的某些振动能级。电子在同类分子或其他分子中撞击，消耗了相当的能量，从而下降到第一电子激发态中的最低振动能级，能量的这种转移形式称为无辐射跃迁。由最低振动能级下降到基态中的某些不同能级，同时发出比原来吸收的频率低、波长长的一种光，就是荧光。被化合物吸收的光称为激发光，产生的荧光称为发射光。荧光的波长总要长于分子吸收的紫外光波长，通常在可见光范围内。荧光的性质与分子结构有密切关系，不同结构的分子被激发后，并不是都能发射荧光。荧光涉及光的吸收和发射两个过程，因此任何荧光化合物，都有两种特征的光谱：激发光谱和发射光谱。
本发明提供的便携式荧光检测装置为采用激光激发荧光物质发出荧光的荧光检测仪。
激发光谱
荧光属于光致发光，需选择合适的激发光波长以利于检测。激发波长可通过荧光化合物的激发光谱来确定。激发光谱的具体检测办法是通过扫描激发单色器，使不同波长的入射光激发荧光化合物，产生的荧光通过固定波长的发射单色器，由光检测元件检测。最终得到荧光强度对激发波长的关系曲线就是激发光谱。在激发光谱曲线的最大波长处，处于激发态的分子数目最多，即所吸收的光能量也最多，能产生最强的荧光。当考虑灵敏度时，测定应选择最大激发波长。
本发明所述的便携式荧光检测装置采用激光为激发光，激发光谱即为发射的激光波长。
发射光谱
一般所说的荧光光谱，实际上仅指荧光发射光谱。它是在激发单色器波长固定时，发射单色器进行波长扫描所得的荧光强度随荧光波长变化的曲线。荧光光谱可供鉴别荧光物质，并作为荧光测定时选择合适的测定波长的依据。
另外，由于荧光测量仪器的特性，使光源的能量分布、单色器的透射率和检测器的响应等性能会随波长而变，所以同一化合物在不同的仪器上会得到不同的光谱图，且彼此间无类比性，这种光谱称为表观光谱。要使同一化合物在不同的仪器上能得到具有相同特性的荧光光谱，则需要对仪器的上述特性进行校正。经过校正的光谱称为真正的荧光光谱。
激发波长和发射波长是荧光检测的必要参数。选择合适的激发波长和发射波长，对检测的灵敏度和选择性都很重要，尤其是可以较大程度地提高检测灵敏度。
荧光PCR定量检测仪
荧光定量PCR检测仪，是指通过荧光染料或荧光标记的特异性探针，对PCR产物进行标记跟踪，实时监测反应过程的仪器。结合相应的软件可以对产物进行定性和定量分析，计算待测样品模板的初始浓度。在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程。
最后通过标准曲线对未知模板进行定量分析，包括TaqMan荧光探针或SYBR Green I等荧光染料。PCR能对特定基因或种的丰度进行相对定量，比较不同处理或样品间的差异，如基因拷贝数、表达量来研究基因表达对处理的响应，同时可以获得PCR扩增效率等信息。
本发明提供的便携式荧光检测装置还包括加热模块，用于控制样品的温度，主要用于使样品中含有的核酸进行等温扩增，同时样品的扩增体系中包含荧光探针，荧光探针上包含荧光基团和淬灭集团。
荧光探针
在紫外-可见-近红外区有特征荧光，并且其荧光性质(激发和发射波长、强度、寿命、偏振等)可随所处环境的性质，如极性、折射率、粘度等改变而灵敏地改变的一类荧光性分子。与核酸(DNA或RNA)、蛋白质或其他大分子结构非共价相互作用而使一种或几种荧光性质发生改变的小分子物质。可用于研究大分子物质的性质和行为。
荧光定量PCR所使用的荧光化学制剂可分为两种：荧光探针和荧光染料。PCR扩增时在加入一对引物的同时加入一个特异性的荧光探针，该探针为一寡核苷酸，两端分别标记一个报告荧光基团和一个淬灭荧光基团。探针完整时，报告基团发射的荧光信号被淬灭基团吸收；PCR扩增时，Taq酶的5’-3’外切酶活性将探针酶切降解，使报告荧光基团和淬灭荧光基团分离，从而荧光监测系统可接收到荧光信号，即每扩增一条DNA链，就有一个荧光分子形成，实现了荧光信号的累积与PCR产物形成完全同步。
最常用于荧光免疫法中标记抗原或抗体，亦可用于微环境，如表面活性剂胶束、双分子膜、蛋白质活性位点等处微观特性的探测。通常要求探针的摩尔吸光系数大，荧光量子产率高；荧光发射波长处于长波且有较大的斯托克斯位移；用于免疫分析时，与抗原或抗体的结合不应影响它们的活性。
也可用于标记待定的核苷酸片断，用与特异性地、定量地检测核酸的量。
常用的荧光探针有荧光素类探针、无机离子荧光探针、荧光量子点、分子信标等。荧光探针除应用于核酸和蛋白质的定量分析外，在核酸染色、DNA电泳、核酸分子杂交、定量PCR技术以及DNA测序上都有着广泛的应用。
本发明所述的便携式荧光检测装置采用激光为激发光，有些实施例中，当激光的激发波长为450-490nm，检测的荧光发射波长为515-530nm，可检测的荧光类物质包括FAM、SYBR Green I等；当激光的激发波长为500-535nm，检测的荧光发射波长为560-580nm，可检测的荧光类物质包括VIC、HEX、JOE、TAMRA、TET、Cy3等；当激光的激发波长为555-585nm，检测的荧光发射波长为610-650nm，可检测的荧光类物质包括ROX、TEXAS-Red等；当激光的激发波长为620-650nm，检测的荧光发射波长为675-730nm，可检测的荧光类物质包括Cy5等。本发明可通过更换激光器或增加激光器实现仪器通道间的自由转换组合，可以实现全光谱荧光检测。
附图说明
图1为实施例1提供的便携式荧光检测装置的结构示意图
图2为实施例1提供的便携式荧光检测装置的结构拆分示意图
图3为实施例1提供的便携式荧光检测装置的实物照片
图4为实施例3中同为0.2ml的样品分别装在0.2ml、0.5ml和1.5ml透明离心管中的对比照片
图5为实施例7中利用便携式荧光检测装置配合支原体检测试剂的实时曲线扫描结果
具体实施方式
下面结合附图对本发明的优选实施例作进一步详细描述，需要指出的是，以下所述实施例旨在便于对本发明的理解，而对其不起任何限定作用。本发明具体实施例中使用的原料、设备均为已知产品，通过购买市售产品获得。
实施例1 本发明提供的便携式荧光检测装置
本实施例提供的便携式荧光检测装置如图1、图2和图3所示，其中图1为便携式荧光检测装置结构示意图，图2为便携式荧光检测装置的结构拆分示意图，图3为制得的便携式荧光检测装置的实物照片。
本实施例提供的一种便携式荧光检测装置，包含激光光源模块1和样品池模块2，所述激光光源模块1用于发射激光，激光作用于样品池模块2中的样品，激发样品中包含的荧光物质发出荧光；所述样品池模块2中的样品被激光穿透的路径距离不小于3.7mm。
优选地，所述样品池模块2中的样品被激光穿透的路径距离为3.7mm-15mm。
本实施例提供的便携式荧光检测装置，激光从侧面激发，因此这里所说的“距离”指样品装在容器时，被激光穿透的最大长度，相当于最大直径处。这里所说的“样品”是指含有荧光物质的核酸PCR扩增或等温扩增样本。
优选地，所述样品池模块2中的样品装在透明离心管3内，所述透明离心管3的规格为0.5ml-5ml。
优选地，所述激光光源模块2的激光发射功率为5-500mw。
优选地，所述样品池模块2只含有1根透明离心管3，透明离心管3内的样品体积为0.05ml-5ml。本实施例提供的便携式荧光检测装置，着眼于更小型、更便捷的设计，实现随时随处现场荧光检测，单个样品终点检测时间不超过5s，且可以通过更换样品，多次检测，不限制检测样品数量。
优选地，所述样品池模块2包括透明离心管3、透明离心管盖4和恒温反应块5，所述透明离心管3可装入恒温反应块5，透明离心管盖4盖在透明离心管3上方。恒温反应块5内可以通过设置与透明离心管3的规格相配套的底座，用于固定透明离心管3，底座规格可以更换，从而可以分别适用于不同规格的透明离心管3。
优选地，所述恒温反应块5可用于使样品中含有的核酸进等温扩增。
优选地，透明离心管3可安装在恒温反应块5内，通过恒温反应块5控制样品保持恒定温度。在一些实施方式中，可以采用加热棒安装于恒温反应块5内部用于温度的控制。
优选地，本实施例中激光光源模块1可以仅为激光器6，激光器6包括激光头7和激光头安装桶8，其中激光头7安装在激光头安装桶8上。本实施例提供的激光器6可更换，用于发射不同波长的激光，可以激发不同的荧光物质发出不同波长的荧光，通过更换不同波长的激光，可以实现各自波长荧光的检测，本发明可实现全光谱荧光检测。
优选地，激光光源模块1还可包含一个或一个以上的激光器6，不同的激光器6分别用于发射不同波长的激光，用于激发不同波长的荧光；多个激光器可以进行组合使用，进一步保证检测的精确度；通过更换激光器6或增加激光器6，实现装置通道间的自由转换组合，为精准检测提供更多的便利。
优选地，所述荧光检测装置还包括荧光检测模块9，所述荧光检测模块9包含一个或一个以上的接受器10，分别用于接受不同波长的荧光；接受器10与激光器6呈90度摆放。
优选地，所述接受器10包括信号转接板安装块11和信号接收板12。
优选地，在信号接收板12透光孔前可安装滤光片13，帮助过滤掉干扰光。
优选地，所述激光光源模块1可以包含1-4个激光器6，分别对应于检测模块9包含1-4个接受器10；所述1-4个激光器6可同时开启使用其中的1个或2-4个组合，并对应同时开启接受器10中的1个或2-4个组合。
一些方式中，激光器6的位置在样品池模块2的右侧平行于接受器10，进一步增大了装置的空间利用率，降低了光的干扰。
还有一些方式中，当在两个位置同时装有激光器6和接受器10，增加了荧光检测通道，增多了样品的检测范围和种类，如在样品池模块2的四个面都分别同时装有激光器6和接受器10，则可实现激光光源模块1可以包含4个激光器6和4个接受器10。
优选地，当包含四个激光器6时，第一激光器6激发波长为450-490nm，检测波长为515-530nm，可检测的荧光类物质包括FAM、Sybr Green I等；第二激光器6激发波长为500-535nm，检测波长为560-580nm，可检测的荧光类物质包括VIC、HEX、JOE、TAMRA、TET、Cy3等；第三激光器6激发波长为555-585nm，检测波长为610-650nm，可检测的荧光类物质包括ROX、TEXAS-Red等；第四激光器6激发波长为620-650nm，检测波长为675-730nm，可检测的荧光类物质包括Cy5等。本发明可通过更换激光器6或增加激光器6实现仪器通道间的自由转换组合，样本池模块2上同时加两个激发器6和接受器10(例1：样本池模块2同时加第一，二的激发器6和接受器10；例2：样本池模块2同时加第一、三的激发器6和接受器10以此类推可以有多种组合)则可以实现全光谱荧光检测。
优选地，恒温反应块5的后方和右侧有透光孔14；激光器6位于恒温反应块5的一侧，光束从恒温反应块5的透光孔直射进入透明离心管3内部，荧光试剂受光源激发后发出荧光；信号转接板安装块11和信号接收板12连接，位于恒温反应块5的另一侧，与激光器呈90度摆放，信号转接板安装块11和信号接收板12上都设有透光孔14。
优选地，所述荧光检测装置还包括主控板15，可将光信号转变为电信号，然后传输到显示屏上输出为数字信号，同时还包括底座16。
采用本实施例提供的便携式荧光检测装置进行检测时，由于激光是从侧面激发的，当透明离心管装有正常体积的样品时，激光穿透样品的厚度则与PCR管的直径相当。
实施例2 不同体积样品对检测结果的影响
分别采用已经完成扩增的待测样品的体积为0.025ml、0.05ml、0.1ml、0.2ml、0.4ml，0.8ml，1.2ml装入1.5ml的试剂管中，通过实施例1提供的便携式荧光检测装置分别进行检测，激光光源模块的激光发射功率为100mw，激光从侧面激发时，考察相同体积的样品装在不同规格试剂管内对检测结果的影响。
具体实验过程如下：
配制扩增反应体系：60mM三羟甲基氨基甲烷-醋酸缓冲液(pH8.0)、100mM醋酸钾、3mM二硫苏糖醇、5％聚乙二醇(20000)、2mM ATP、20mM磷酸肌酸、420nM引物混合物(上游引物序列，5’-AATTTGTGCGAGTAAACCTATGTAGCAGCAGAG-3’；下游引物序列，5’-TTCCTTCTAAGCTCTGCAGCTTCATTCATCATC-3’)、200nM荧光探针(探针序列为5'-AAAATGTCGGGAGCTGAACATATGGAAGG(FAM-dT)(THF)T(BHQ1-dT)GAGCGAGTGCT-3'(C3-SPACER))、100ng/μl肌酸激酶、600ng/μl噬菌体gp32蛋白、150ng/μl噬菌体uvsX蛋白、25ng/μl噬菌体uvsY蛋白、80ng/μl klenow聚合酶大片段(exo-)、50ng/μl核酸外切酶III、450μM dNTP引物混合物、14mM醋酸镁、1000拷贝阳性质粒模板。
将上述反应配制1ml体系，共计配制3管，总体积为3ml，充分振荡混匀并短暂离心。放置在水浴锅内，37℃反应30分钟后，在80℃反应5分钟灭活反应组分中酶成分，反应充分结束。荧光探针在THF处被切割，荧光信号因失去淬灭作用而被释放。为保证荧光试剂的均一性，将上述反应液全部混合至5ml离心管并充分混匀后，分别取0.025ml、0.05ml、0.1ml、0.2ml、0.4ml，0.8ml，1.2ml反应液装入1.5ml规格的透明离心管中。将分装好的透明离心管放置在实施例1提供的便携式荧光检测装置，同时调节样本池模块2位置，以确保激光发射位置和检测位置通过试剂中心的中心位置，温度设定为37℃，进行荧光数值读取。
其中不同体积样品在1.5ml透明离心管中时，激光照射于试剂样品中心位置。随着样品体积由0.025ml增大到0.8ml，被激光穿透的平均路径距离由3mm增大到10.09mm，此时随着样品被穿透路径距离的增大，所激发的荧光物质总量也依次增多，从而发出更多的荧光；当样品体积由0.8ml-1.2ml时，尤其是当样品由1.0ml增大到1.2ml时，因离心管上端为圆柱形，直径不再继续增大，因此被激光穿透的平均路径距离也增速放缓，光程增速也放缓，甚至当样品体积继续增大时，因直径不变，被激光穿透的路径距离也不再增大，光程增速也不再明显，此时若需进一步提高灵敏度，则需更换更大的离心管，使被激光穿透的路径距离进一步增大，从而进一步增大光程。
仪器实验数据如表1所示。
表1不同体积样品在1.5ml规格透明离心管的检测结果
Figure PCTCN2021082668-appb-000001
由表1的检测结果可以看出：
采用1.5ml规格的透明离心管时，当样品体积为0.025ml时，被激光穿透的样品体积小，厚度也较小，被激光穿透的路径距离为3mm，此时激光穿透时产生透射与折射非常少，光程较小，测得的荧光值也是偏小。如果采用此体积反应时容易导致检测结果出现误差。
而当样品体积为0.05ml-0.4ml时，由于透明离心管底部类似圆锥形，由下向上直径逐渐变大，透明离心管内的样品被激光穿透的路径距离从5mm增大到10mm，光程逐步增大，测得的荧光值也由6046迅速增大到23663，增长了近4倍，检测灵敏度明显升高。
当样品体积为0.8ml-1.2ml时，样品被激光穿透的路径距离为10.09-10.22mm，此时由于离心管上端形状的限制，被激光穿透的路径距离增长变缓，光程增长变缓，使测得的荧光值变化趋势明显变缓，对检测结果影响不明显。
可见，随样品体积的增大，其激光穿透的厚度也在增加，使得激光在样品中产生的透射与折射更充分，从而激发更多的荧光物质发出荧光，使测得的荧光值明显升高，从而提高检测灵敏度。
这里的样品液体被激光穿透的“厚度”是指激光发生的光穿过液体样本的路径的长度，比如路径的长度可以是不小于3毫米，不小于4毫米，不小于5毫米，不小于6毫米，不小于8毫米，不小于9毫米，不小于10毫米。在一些方式中，当液体的厚度一定的时候，可以增加激光的强度来提高检测的灵敏度。在一些方式中，激光穿过液体样本的路径的长度与激光的强度共同调节来实现检测的结果。这里的路径还可以理解为这样的方式，激光穿过液体样品后，激光发生的光多次反复折与反射穿过液体样本。当然，还可以理解是一束或者多束激光同时穿过液体的样本，相当远延长了穿过液体的路径的长度。
实施例3 相同体积不同试剂穿透光程对检测结果的影响
本次实施案例考察在相同浓度相同体积的荧光试剂，在不同的穿透光程下，其荧光值的差异。将实施例2中的试剂取0.2ml，用微量移液器分别放入0.2ml PCR试剂管、0.5ml透明离心管、1.5ml透明离心管中，通过实施例1提供的便携式荧光检测装置分别进行检测，激光光源模块的激光发射功率设置为50mw，激光从侧面激发时，同时调节样本池模块2位置，以确保激光发射位置和检测位置通过试剂中心的中心位置，温度设定为55℃，如图4所示，其中从左到右分别为0.2ml PCR试剂管、0.5ml透明离心管、1.5ml透明离心管，同时装了0.2ml样品，被激光穿透的路径距离相当于其直径，考察相同体积的样品装在不同规格试剂管内对检测结果的影响。
相同浓度的样品，样品体积都为0.2ml，分别装在体积为0.2ml、0.5ml、1.5ml的容器内，激光从侧面照射时，激光穿透样品的路径距离，主要由样品所在的容器形状决定。如图4可见，由于容器体积都为0.2ml，被激光穿透的路径分别约为4.2、5.1、7.8mm，即随着容器体积增大，激光穿透样品的路径距离也逐渐增大，产生的透射和折射也较强，对更多荧光物质的激发产生了更强的荧光。
仪器所测得实验数据如表2所示。
表2不同体积样品在1.5ml规格透明离心管的检测结果
Figure PCTCN2021082668-appb-000002
Figure PCTCN2021082668-appb-000003
由表2的检测结果可以看出，在相同浓度相同体积下，而激光穿透的试剂光程越长，则会产生更多的折射，激发的荧光物质会越多，所检测到的荧光强度也越强。因此荧光强度的变化于样品体积相关性较小，而与样品被激光穿透的路径距离相关性更大。
实施例4荧光试剂稀释后对检测结果的影响
在进行核酸荧光定量PCR过程中，经常会遇到低浓度目标样本的情况。对于低浓度模板样本扩增后所产生的荧光强度也较低。在特定的反应试剂体积下，当样本浓度低至一定极限后，所产生的荧光强度不能再被检测到，这也决定了试剂和仪器组合的扩增灵敏度。也就是说，当仪器对于荧光信号变化敏感度越高，检测试剂和仪器的对于样本的灵敏度也越高。本实施案例的测试目的是通过有限稀释试剂至极限浓度，使其在小体积下无法检测的荧光强度，而通过增加体积和试剂厚度，使仪器能够重新检测到荧光。以印证对于试剂体积或试剂厚度增加后，有助于提高试剂对于样本的灵敏度的提高。
取实施例2实验中的反应后试剂0.5ml，用水分别稀释10倍后，按照表2装入5ml的透明离心管中，共分装8管，通过实施例1提供的便携式荧光检测装置，采用5ml透明离心管，激光光源模块的激光发射功率为300mw，激光从侧面激发时，同时调节样本池模块2位置，以确保激光发射位置和检测位置通过试剂中心的中心位置，分别进行检测，考察低浓度的荧光在不同体积及试剂光程下对检测结果的影响。温度设定为37℃。
其中，不同体积的相同样品，在5ml透明离心管中时，激光穿透的距离路径各不同，样品被激光穿透的最大路径距离相当于样品最高液位所处的离心管直径，随着样品体积由0.025ml增大到1.6ml，最高液位所处的离心管直径从3.9mm增大到14.3mm，也就是说被激光穿透的最大路径距离由3.9mm增大到14.3mm，此时随着样品被穿透路径距离的增大，产生的透射和折射也迅速增多，光程迅速增加，从而使样品中被激发的荧光物质也迅速增多；当样品体积由1.6ml-3.2ml时，因离心管上端为圆柱形，直径不再继续增大，因此被激光穿透的路径距离不再变大，光程不再继续增加，从而使样品中被激发的荧光物质也不再继续增多。
仪器检测实验数据如表3所示。
表3样品稀释不同倍数在5ml规格透明离心管的检测结果
Figure PCTCN2021082668-appb-000004
Figure PCTCN2021082668-appb-000005
由表3的检测结果可以看出：
当荧光试剂的荧光物质大大降低后，小体积或小光程的试剂，无法被检测荧光读值，而当逐步加大试剂体积时，使样品被激光穿透的路径距离不断增大，从而迅速增大光程，能使激光在样品中产生的透射与折射更充分，从而激发更多的荧光物质发出荧光，使测得的荧光值逐步被检测到，并随试剂体积或光程增加明显升高，从而提高检测灵敏度。
实施例5 便携式荧光检测装置用于LAMP扩增
采用实施例1中的便携式荧光检测装置，用于LAMP扩增反应，每个反应的试剂终体积为0.25ml，反应条件参考ZL201310368890实施例2中步骤2扩增，使用石蜡油封住液体，防止气溶胶产生。温度设定61℃，反应30分钟。反应结束后，增加终浓度为1x的Sybr Green I染料进行终点扫描法分别记录阴性与阳性值的差异。
表4 LAMP扩增实验中阴性与阳性样本终点检测结果
Figure PCTCN2021082668-appb-000006
从表4中可以看出，阴性样本与阳性样本扩增后产物的荧光值差异迥异，可以明显区分阴、阳性，同时用参考专利实施例2步骤3所述的紫外观察对比，发现阴性与阳性结果与本仪器测试结果均一致，即：阳性样本的LAMP扩增产物稀释液的颜色由橙色变为绿色，而阴性结果保持橙色。
上述案例表明所确定荧光装置可以用于判断LAMP扩增后样本的阴性与阳性定性检测。
实施例6 采用便携式荧光检测装置与PCR荧光检测仪器检测结果比较
本实施例选择市面上的两种传统荧光定量PCR仪器(苏州雅睿科技有限公司，型号：MA-6000，采用LED配合滤光片和透镜组合作为激发光源；美国Bio-Rad，型号：CFX MiniOpticon System)，退火温度设置为54℃，每个循环30s，总反应设定为40个循环，其它仪器设置也均为常规设置，并通过熔解曲线辅助判断特异性产物扩增情况。采用双向引物为MPP-1QF:5'ACAAATAAGTGGAGGTAAAGC 3'；MPP-1QR:5'TGTCTGACTGCGAGAATAA 3'。采用南京诺唯赞生物科技有限公司AceQqPCR SYBR Green Master Mix(Low ROX Premixed)货号：Q131-02染料法荧光定量预混PCR试剂进行扩增，配制反应参考试剂盒说明书。反应体系为每管25ul，扩增后产物与实施例1提供的便携式荧光检测装置进行比较，比较检测扩增后产物的荧光强度。
经前期实验优化验证，Q-PCR的试剂最低检测线为5拷贝/反应，且阴性对照均为平线，无荧光扩增曲线出现。采用5拷贝/反应的样本浓度进行实验。每个仪器上均测试16个反应，8个阳性对照重复和8个阴性对照重复。实验结果表明，两台仪器针对8个阳性均能够检测出，熔解曲线为单一峰，结果正常；阴性也均正常，无扩增曲线出现。
便携式荧光装置对比实验：由于本发明荧光装置采用的体积较大，故将以上扩增后的产物，同一个仪器扩增后的阳性和阴性分别混合到同一1.5ml透明离心管后，再进行大体系的荧光值测量。检测结果如表5所示。
表5相同样品经不同PCR荧光检测仪器检测结果
Figure PCTCN2021082668-appb-000007
由表5的检测结果可知，通过传统荧光定量PCR扩增后的产物，经过样本体积放大后采用本发明提供的便携式荧光检测装置进行检测均可以检测到荧光信号值的明显差异。
同时，为进一步验证结果，将荧光定量PCR仪器作为恒温扩增仪使用时，采用等温扩增试剂，能够检测到的阳性扩增产物经混合后采用本便携式荧光检测装置也均检测到阴阳性扩增产物荧光差异。表明在作为恒温扩增使用时，通过放大扩增试剂体积，可以达到荧光定量PCR仪器的荧光检测效果。
实施例7利用便携式荧光检测装置配合支原体检测试剂的实时曲线扫描
参考专利申请202010160768.0，其中使用的引物、探针为经过2’-O-methyl-修饰，且探针序列5端加FAM、3端加BHQ1。
上游引物序列mMPF：5‘-TAATACTAATGAGTCGAGGA mCmUmU mA TT mG G mA mA mG-3’；
下游引物序列mMPR：5‘-CAGCGACAGAGTCACCAAACAA mA mAAmCmG A mC mA-3’；
探针序列mMPBP：5'-FAM-CTGCGTATmUmU C mC TACCA mA mAmGmGmC TACGCAG-BHQ1-3'；
其中，反应管中包含上游引物终反应浓度为0.5uM，下游0.2uM，并向制备好的反应管中分别加 入肺炎支原体500拷贝，每个反应中添加1ul。反应的缓冲液终浓度如表6所示：
表6反应的缓冲液终浓度
Figure PCTCN2021082668-appb-000008
另添加dNTPs终浓度0.4mM；锰离子终浓度，2mM；终反应体积为0.3ml，并将酶加至管盖，拧上盖子后颠倒摇动混匀后放入恒温震荡仪，温度设置为55℃，2000RPM/分钟震荡30s；之后转移至本实施例1的便携式荧光检测装置上，在55℃条件下继续反应10min左右，每6s采集一次荧光信号值，结果如图5所示，当终点扫描值低于1000时判断为阴性，高于2000时判断为阳性。
实施例8 利用便携式荧光检测装置配合支原体检测试剂的进行终点扫描法确认灵敏度
根据实施例7的实验，将探针更换为Rox荧光标记的试剂，mMPBP-R：5'-Rox-CTGCGTATmUmU C mC TACCA mA mAmGmGmC TACGCAG-BHQ2-3'其它反应条件均一致。样本采用粗裂解的口腔拭子肺炎支原体基因组DNA，经过传统荧光定量PCR法确认样本浓度后，采用阴性粗裂的样本稀释阳性样本至毫升样本中含400拷贝浓度，并向每反应中添加0.25ml样本，总100拷贝，总反应体系仍采用0.3ml。分别重复20次实验，采用终点扫描法确认终点值，对应的，将荧光装置的激发波长更换为585nm左右的激光器，检测波长更换为对应的625nm左右的滤光片和光信号检测器。根据前期大量实验时所获得数据，确定当终点扫描值低于1000时判断为阴性，高于1500时判断为阳性。应用本发明装置确定支原体检测试剂检测的灵敏度，结果如表7所示。
表7支原体检测试剂的荧光扫描结果
Figure PCTCN2021082668-appb-000009
Figure PCTCN2021082668-appb-000010
以上实验结果表明，在预设的阴阳性判断标准前提下，采用本专利的发明装置，所有20个阳性样本均能检测为阳性，在放大反应体积的情况下，配合检测试剂检测灵敏度可以达到传统荧光定量PCR的灵敏度一般行业检测标准(400-1000拷贝/毫升)。而且采用本解决方案时，不需要进行样本抽提。使用场景变得极为灵活，且操作非常简便。为今后核酸的POCT现场化检测提供了有效的解决方案。
由于在进行核酸含量检测过程中，样品中的核酸含量有可能较低，此时采用低样品体积进行检测时，难以保证精确性。因此采用激光作为激发光源，并使样品被激光穿透的厚度增大，才能显著提升检测灵敏度和精确性。为了实现荧光检测装置的小型化和便捷化，必须采用激光作为激发光源，同时务必需要保证样品体积增大，使样品被激光穿透的厚度增大，从而实现稳定的信号检测。
虽然本发明披露如上，但本发明并非限定于此。如根据其在医学上的应用范围均可做扩展。任何本领域技术人员，在不脱离本发明的精神和范围内，均可作各种更动与修改，因此本发明的保护范围应当以权利要求所限定的范围为准。

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  1. 一种便携式荧光检测装置，其特征在于，包含激光光源模块和用于装载样品的样品池模块，所述激光光源模块用于发射激光，发射的激光用于穿透样品池模块中的样品，激发被穿透路径距离内的样品中包含的荧光物质发出荧光。
  2. 根据权利要求1所述的便携式荧光检测装置，其特征在于，所述的样品中含有目的核酸，所述目的核酸是经过扩增过的扩增产物，或者经过提纯过的单链或双链DNA或单链RNA，或者人工合成的DNA或RNA。
  3. 根据权利要求1或2所述的便携式荧光检测装置，其中，所述荧光物质作为标记物质指示样品中核酸物质的数量的多少或者含量的高低。
  4. 根据权利要求1-3任一项所述的便携式荧光检测装置，所述的荧光物资被包括在核酸序列上用来指示目的核酸的数量。
  5. 根据权利要求1-4任一项所述的便携式荧光检测装置，其特征在于，所述样品池模块中的样品被激光穿透的路径距离为不小于3.7mm，优选3.7mm-15mm，最优选为6-10mm。
  6. 根据权利要求1-5任一项所述的便携式荧光检测装置，其特征在于，所述样品池模块中的样品被装在激光可穿透的样品容器内。
  7. 根据权利要求1-6任一项所述的便携式荧光检测装置，其特征在于，所述样品池模块可含有1个或1个以上的样品容器内。
  8. 根据权利要求1-7任一项所述的便携式荧光检测装置，其特征在于，所述样品容器的规格为0.5ml-5ml，优选为0.5ml-2ml，最优选为0.5ml-1.5ml。
  9. 根据权利要求1-8任一项所述的便携式荧光检测装置，其特征在于，所述激光光源模块可更换，用于发射不同波长的激光；所述便携式荧光检测装置还包括荧光检测模块，所述荧光检测模块用于接受不同波长的荧光，并将光学信号接收后转化为电信号，再进一步转换为数字信号。
  10. 根据权利要求1所述的便携式荧光检测装置,还包括用于控制样品温度的恒温反应块和检测模块；激光光源模块和检测模块分别位于样品池模块的两侧。
  11. 根据权利要求10所述的便携式荧光检测装置，其特征在于，所述样品池模块还包括样品容器，所述样品容器安装在恒温反应块内，样品容器包含反应杯和反应杯盖。
  12. 如权利要求11所述的装置，其特征在于，所述恒温反应块内设有与样品容器配套的底座；所述恒温反应块开口向上，样品容器由上向下插入恒温反应块内的底座。
  13. 如权利要求10-12任一项所述的装置，其特征在于，所述恒温反应块上设有透光孔，用于激光或荧光穿过。
  14. 如权利要求10所述的装置，其特征在于，所述激光光源模块和检测模块分别位于样品池模块的两侧并呈90度摆放。
  15. 如权利要求1所述的装置，其特征在于，所述激光器包括激光头和激光头安装桶。
  16. 如权利要求10所述的装置，其特征在于，所述检测模块包含接受器，用于接受荧光；所述接受器包括信号转接板安装块和信号接收板。
  17. 如权利要求10所述的装置，其特征在于，所述激光光源模块包含1-4个激光器，分别对应于检测模块包含1-4个接受器。
  18. 如权利要10所述的便携式荧光检测装置，其特征在于，所述荧光检测装置还包括主控板和底座，所述主控板可将光信号转变为电信号，然后传输到显示屏上输出为数字信号。
  19. 一种提高荧光检测装置灵敏度的方法，其特征在于，所述荧光检测装置采用激光作为激发光源，通过增加样品被激光穿透的距离路径来提高荧光检测装置的灵敏度，所述样品中含有目的核酸，以及用于标记目的核酸数量的荧光物质。
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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003021212A1 (en) * 2001-08-28 2003-03-13 Baylor College Of Medicine Pulsed-multiline excitation for color-blind fluorescence detection
WO2006119277A2 (en) * 2005-05-04 2006-11-09 Stratagene California System and method for a pulsed light source used in fluorescence detection
CN101542273A (zh) * 2006-08-24 2009-09-23 新加坡科技研究局 紧凑式光学检测系统
CN106092895A (zh) * 2016-08-23 2016-11-09 中国电子科技集团公司第四十九研究所 一种水体叶绿素浓度原位检测装置及其检测方法
CN107389644A (zh) * 2017-08-11 2017-11-24 长春理工大学 一种快速荧光定量装置
CN107817227A (zh) * 2016-09-12 2018-03-20 台达电子国际（新加坡）私人有限公司 荧光检测装置
CN109883969A (zh) * 2019-03-18 2019-06-14 长春美泰仪器有限公司 一种饮用水中溴酸盐含量检测方法
CN110132909A (zh) * 2018-02-08 2019-08-16 台达电子国际（新加坡）私人有限公司 荧光检测装置
CN110220872A (zh) * 2018-03-02 2019-09-10 台达电子国际（新加坡）私人有限公司 可携式多色荧光检测装置
CN210090300U (zh) * 2019-03-18 2020-02-18 长春美泰仪器有限公司 一种饮用水中溴酸盐含量检测装置

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003021212A1 (en) * 2001-08-28 2003-03-13 Baylor College Of Medicine Pulsed-multiline excitation for color-blind fluorescence detection
WO2006119277A2 (en) * 2005-05-04 2006-11-09 Stratagene California System and method for a pulsed light source used in fluorescence detection
CN101542273A (zh) * 2006-08-24 2009-09-23 新加坡科技研究局 紧凑式光学检测系统
CN106092895A (zh) * 2016-08-23 2016-11-09 中国电子科技集团公司第四十九研究所 一种水体叶绿素浓度原位检测装置及其检测方法
CN107817227A (zh) * 2016-09-12 2018-03-20 台达电子国际（新加坡）私人有限公司 荧光检测装置
CN107389644A (zh) * 2017-08-11 2017-11-24 长春理工大学 一种快速荧光定量装置
CN110132909A (zh) * 2018-02-08 2019-08-16 台达电子国际（新加坡）私人有限公司 荧光检测装置
CN110220872A (zh) * 2018-03-02 2019-09-10 台达电子国际（新加坡）私人有限公司 可携式多色荧光检测装置
CN109883969A (zh) * 2019-03-18 2019-06-14 长春美泰仪器有限公司 一种饮用水中溴酸盐含量检测方法
CN210090300U (zh) * 2019-03-18 2020-02-18 长春美泰仪器有限公司 一种饮用水中溴酸盐含量检测装置

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