CN214201190U - Portable fluorescence detection device - Google Patents

Abstract

The utility model provides a portable fluorescence detection device, which comprises a laser light source module, a sample cell module and a detection module, wherein the sample cell module comprises a constant temperature reaction block, the laser light source module and the detection module are respectively positioned at two sides of the sample cell module, the device adopts laser as an excitation light source, the path distance of a sample penetrated by the laser is increased, the fluorescence signal intensity is enhanced, the detection sensitivity and accuracy are greatly improved, and stable signal detection is realized; and can realize multi-channel detection of different samples. The utility model provides a portable fluorescence detection device has miniaturization, portable, with low costs, detection speed is fast, detect reagent pipe is bulky, and the reagent volume is big, fluorescence detection signal value is high, it is stable, the accuracy is high, detection range is wide to detect the result, detectable fluorescence class material is many, has increased fluorescence detection's practical application field and place, need not professional's operation, has improved fluorescence detection device's portability.

Description

Portable fluorescence detection device

Technical Field

The utility model relates to a fluorescence detection device particularly, relates to a fluorescence detection device by laser excitation, especially relates to a fluorescence detection device by laser excitation that is arranged in analyzing nucleic acid content in the sample.

Background

At present, an LED lamp, a halogen lamp, a xenon lamp, and the like are commonly used as a light source of an excitation device in a PCR instrument for quantitatively analyzing nucleic acid. The divergence angle of LED light is large, the brightness is unstable, the brightness is weak, the wavelength is inconsistent, the light purity is insufficient, the stray light interference is strong, the signal-to-noise ratio is poor, and multiple light calibrations are needed, so that various complex and harsh auxiliary accessories are added when an excitation device and a receiving device are designed, for example, expensive optical filters and light sensor signal multiplication assistance are needed. The unstable brightness of the LED light source puts high requirements on the selection of a light source power supply mode by a photoelectric engineer. The weak LED brightness causes the excitation light emitted by the excitation device to be not strong enough, so that the PD receiving device is complex and effective signals are weak, an electronic engineer needs to extract and amplify the effective signals, and the difficulty in circuit design is greatly increased because the extracted effective amplified signals cannot be distorted. For example, in a PCR instrument using a common LED as an excitation device, the excitation device includes 1 plano-convex lens and 1 plano-convex lens, 2 biconvex lenses and 1 filter lens, which are assembled in sequence and direction to increase the light intensity, and its optical signal receiving device includes a plurality of plano-convex, biconvex, and filter lenses, which are assembled in sequence and direction, and no effective signal can be detected. Halogen lamps, xenon lamps and the like also have great disadvantages as light sources of the excitation devices, the electric power consumption is increased due to large self-luminous volume, a heat dissipation device is required due to high luminous heat, and the light calibration is required due to large light diffusion angle, so that the instrument cannot be miniaturized. Although there are a few documents or patents that use laser as excitation light source to prepare PCR instrument, such as ABI 7900HT fluorescence quantitative PCR instrument, and argon ion laser, it is expensive and has single wavelength, and it is almost eliminated in the market.

The conventional fluorescent quantitative PCR instrument or constant-temperature fluorescent instrument needs very complicated and precise optical signal filtering and signal amplifying processes, needs expensive optical signal sensing elements or devices, adopts traditional optical elements, complicated heating elements and the like, and causes the existing PCR fluorescent detection equipment to have insufficient integration level, a large number of light sources and detectors, the consistency of detection results is influenced by the difference between a plurality of light sources and detectors, and the equipment needs offline equipment and resources and is large and expensive. And the precision and complexity of the conventional equipment require complex parameter setting, and professional detection personnel operate the equipment, so that the universality of the application of the equipment is limited, and the conventional detection of public and household detection is difficult to realize.

It is necessary to modify the existing nucleic acid amplification apparatus to improve the sensitivity of detection and to miniaturize or miniaturize the apparatus.

SUMMERY OF THE UTILITY MODEL

In order to solve the problem, the utility model provides a portable fluorescence detection device, the device adopt laser as excitation light source, enlarge the sample volume, let laser irradiation sample to let laser pierce through the path extension to certain scope of sample, make more fluorescence reagent arouse the back and produce fluorescence, thereby improve the sensitivity that detects.

In some preferred modes, the sample path distance penetrated by the laser is not less than 3.7mm, so that the optical path of the laser in the sample is increased. In some embodiments, the sample includes nucleic acids that have been amplified, and the sample further includes a fluorescent substance that indicates the amount of nucleic acids in the sample. Therefore, laser generates more transmission and refraction in the process of penetrating through a sample, so that more fluorescent substances are excited to generate fluorescence, the intensity of an optical signal is greatly increased, the detection sensitivity and accuracy are greatly improved, and stable signal detection is realized. In addition, the laser devices with different channels can be replaced, and multi-channel detection of different samples is achieved. The utility model provides a portable fluorescence detection device has miniaturization, portable, advantage with low costs, can be used to public occasion and family and detect to can realize the accurate detection of nucleic acid under limited professional knowledge and experimental facilities.

The research group finds that when the laser penetrates a sample to reach a certain thickness or the path distance of the laser in the sample is increased, the laser can generate more transmission and refraction in the process of penetrating the sample, so that the optical path of the laser in the sample is obviously increased, more fluorescent substances are excited to generate fluorescence, the sensitivity of nucleic acid substance fluorescence detection can be greatly improved, the range is expanded, and the miniaturization and simplification design of nucleic acid amplification equipment, such as a PCR instrument, can be realized. In other words, the distance that the laser beam passes through a solution having a certain thickness or enters the liquid and passes through the liquid is increased, and the solution contains the sample to be tested and the fluorescent substance, so that the content of the fluorescence can be detected with high sensitivity, and the corresponding nucleic acid substance can be detected with high sensitivity. The nucleic acid material is any similar nucleic acid, such as RNA, DNA or derivatives thereof, hybrid strand, etc.

The utility model provides a portable fluorescence detection device has reduced the volume of device, has improved device inner space utilization, changes LED excitation light source into the laser instrument and arouses, for 2.5-10 times of the general PCR appearance on the market with the reagent pipe amplification, the reagent volume has also improved nearly 5-10 times greatly for light signal intensity greatly increased has really realized the portable and low-cost of device.

In one aspect, the utility model provides a portable fluorescence detection device contains laser light source module and is used for loading the sample cell module of sample, laser light source module is used for transmitting laser, and the laser of transmission is arranged in piercing through the sample in the sample cell module, excites the fluorescent material that contains in the sample that is penetrated in the path distance and sends fluorescence.

It will be appreciated that such an apparatus does not necessarily contain a sample, but that, when a sample is contained in the cell module at the time of detection, the sample occupies a volume in the cell so that the laser light passes through the interior of the sample in that volume, thereby causing a fluorescent substance in the sample to be excited to fluoresce.

The dispersion angle of the laser is small, the light is almost emitted in parallel, the brightness is high, the monochromaticity is good, even the light filter does not need to be additionally arranged, the interference is small, the optical signal to noise ratio is good, and therefore the fluorescence detection device has the remarkable advantage of high sensitivity, the laser is selected as the exciter, the structure of the fluorescence detection device is simpler, the assembly is convenient, and the price advantage is achieved.

The sample is generally in a fluid form, or liquid state. These fluids in the liquid state must occupy a volume in the container, the form of which is determined by the shape of the container. Therefore, when the shape of the container is determined, the laser is entered in a manner that determines the distance the laser has traveled through the sample. The path distance of the laser penetrating the sample is the length of the sample penetrated by the laser, and particularly, when the sample is vertically placed and the laser is injected from the side, the penetrating thickness of the sample is equivalent to the diameter of a sample container; when the sample is vertically placed and the laser is injected from the bottom, the penetrating thickness of the sample corresponds to the height of the sample in the container; when the sample is placed obliquely and the laser is injected from the side, the penetrating thickness of the sample is equivalent to the length of an oblique line penetrating through the sample; when the sample volume is small and is approximately flat at the bottom of the container, the laser can penetrate through the surface layer of the sample when being injected from the side of the sample, and the length of the path through which the laser penetrates is the thickness. By "distance" is meant the distance between the laser light entering the liquid and the liquid exiting it or the path travelled, which is herein understood to be the length of the path the laser light has travelled in the liquid.

Further, the path distance of the sample in the sample cell module penetrated by the laser is not less than 3.7mm

Meanwhile, research groups also find that laser is used as an exciter, when the laser penetrates through a sample to reach the thickness of more than 3.7mm, the laser can generate more transmission and refraction in the process of penetrating through the sample, the sensitivity of fluorescence detection of nucleic acid substances can be greatly improved, the measuring range is expanded, and therefore the miniaturized and simplified design of the PCR instrument is really realized.

Further, the optical path distance of the sample in the sample cell module penetrated by the laser is preferably 3.7mm-15 mm.

In some embodiments, the sample in the sample cell module is penetrated by the laser to a thickness of 3.7mm or more than 3.7 mm. In some approaches, the length or distance of the path through the liquid is less than 15 mm.

Further, the path distance of the sample in the sample pool module penetrated by the laser is 6mm-10 mm.

Further, the sample in the sample cell module is contained in a sample container that is transparent to the laser.

Further, the sample container comprises a reaction cup and a reaction cup cover.

Further, the sample cell module may contain 1 or more than 1 sample container.

Further, a transparent centrifuge tube is arranged in the sample container.

Further, the sample container has a size of 0.5ml to 5 ml.

The utility model provides a portable fluorescence detection device mainly used detects the target nucleic acid content in the sample, and the sample that awaits measuring needs to be through constant temperature amplification, contains fluorescence probe in the amplification system, and what consequently adorn the container of sample adopted is the transparent centrifuging tube of the ordinary specification of purchasing easily on the market, including 0.5ml-5 ml.

Further, the laser emission power of the laser light source module is 5-500 mw. The emission power of the laser is related to the intensity of the emitted laser light and also to the distance of penetration through the liquid, which in turn causes the fluorescence excitation to emit fluorescence. It will be appreciated that the distance the laser penetrates the liquid has a direct relationship to the emitted fluorescence light when the power is fixed within a certain range. Of course, if the power is varied within a certain range, the laser intensity is also varied.

Therefore, in another aspect, the present invention provides a nucleic acid detecting apparatus, which includes a laser light source module, a fluorescence detecting module, and a sample cell module; the sample cell module is used for containing a sample containing nucleic acid, the sample comprises fluorescent substances indicating the quantity of the nucleic acid, and the laser source module is used for emitting laser, so that the emitted laser generates transmission and refraction in the process of penetrating through the sample, and more fluorescent substances are excited to emit fluorescence.

In some modes, the laser emission power of the laser light source module is 1-10000 mw; 1-1000 mw; 5-500 mw; or 50-200 mw. If the volume of the sample is determined, the excitation power can be varied to vary the intensity of the laser, thereby achieving an intensity at which the fluorescent substance excites the fluorescent substance. In some embodiments, the laser is directed through the thickness of the liquid sample when the excitation power is determined, thereby allowing more fluorescence to be excited to fluoresce.

When a transparent centrifugal tube of 0.5ml-5ml is adopted, the volume of a sample in the transparent centrifugal tube is 0.05ml-2.5ml, and the laser emission power of the laser source module is 100mw, the thickness of a laser penetrating sample can be kept within the range of 3.7mm-15mm, and therefore stable signal detection is achieved.

Further, the sample container has a specification of 0.5ml to 2 ml.

Further, the sample container has a specification of 0.5ml to 1.5 ml.

Furthermore, the number of the transparent centrifuge tubes is 1, the specification is 1.5ml, and the volume of the sample in the tube is 0.05ml-1.2 ml.

When the test tube container is 1 transparent centrifuge tube with the specification of 1.5ml, and the sample volume in the tube is 0.05ml-1.2ml, the detection effect is better, and the accuracy and the sensitivity are higher.

The portable fluorescence detection device provided by the utility model only detects a small number of samples at a time, for example, only detects one sample, two samples, three samples and four samples at a time; the method is different from the high-throughput detection pursued in the market at present, focuses on the design of being smaller and more convenient, realizes the fluorescence detection anywhere at any time, has the detection time of a single sample endpoint method not exceeding 5s, can detect for multiple times by replacing the sample, and does not limit the number of the detected samples.

Further, the sample contains a target nucleic acid, and the target nucleic acid is an amplified product, or purified single-stranded or double-stranded DNA or single-stranded RNA, or artificially synthesized DNA or RNA.

Further, the fluorescent substance as a labeling substance indicates the amount of the nucleic acid substance in the sample or the level of the content.

Further, the fluorescent material is included on the nucleic acid sequence to indicate the amount of the nucleic acid of interest.

Further, the sample cell module comprises a sample container and a constant temperature reaction block, wherein the sample container can be arranged in the constant temperature reaction block. The base that is used for fixed transparent centrifuging tube is equipped with in the constant temperature reaction piece, and the base of different specifications uses with the transparent centrifuging tube of different specifications is supporting, can realize the use of different specification sample containers through changing the base.

Furthermore, the opening of the constant temperature reaction block is upward, and the sample container is inserted into the base in the constant temperature reaction block from top to bottom.

Further, the isothermal reaction block can be used to subject nucleic acids contained in a sample to isothermal amplification.

Further, the transparent centrifuge tube can be installed in a constant temperature reaction block, and the sample is controlled by the constant temperature reaction block to keep a constant temperature. In some embodiments, can be used with heating rod installed in the constant temperature reaction block for temperature control.

Further, the laser light source module is replaceable and used for emitting laser with different wavelengths.

The laser of different wavelengths can arouse different fluorescent substances to send the fluorescence of different wavelengths, through the laser of changing different wavelengths, can realize the detection of wavelength fluorescence separately, the utility model discloses can realize full gloss register for easy reference fluorescence detection.

Further, the laser light source module may include one or more lasers, and different lasers are respectively used for emitting laser light with different wavelengths and exciting fluorescence with different wavelengths.

Further, the laser instrument includes laser head and laser head installation bucket, and wherein the laser head is installed on the laser head installation bucket.

Multiple lasers can be combined for use, and the detection accuracy is further guaranteed.

By replacing the laser or adding the laser, free conversion combination among the channels of the device is realized, and more convenience is provided for accurate detection.

Furthermore, the portable fluorescence detection device also comprises a fluorescence detection module, wherein the fluorescence detection module is used for receiving fluorescence with different wavelengths, converting the received optical signals into electric signals and further converting the electric signals into digital signals.

The fluorescence detection module comprises one or more than one receiver, and the receivers are respectively used for receiving fluorescence with different wavelengths; the receiver is placed at 90 degrees to the laser.

Further, the receiver comprises a signal adapter plate mounting block and a signal receiving plate.

Further, an optical filter can be arranged in front of the light holes of the signal receiving plate to help filter out interference light.

Further, the laser light source module comprises 1-4 lasers, and 1-4 receivers are respectively arranged corresponding to the fluorescence detection module; the 1-4 lasers may be simultaneously turned on using 1 or 2-4 combinations thereof, and corresponding to simultaneously turning on 1 or 2-4 combinations in the receiver.

Furthermore, light holes are arranged at the rear and the right side of the constant-temperature reaction block; the laser is positioned at one side of the constant-temperature reaction block, light beams directly enter the transparent centrifugal tube from a 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 plate mounting block is connected with the signal receiving plate, is positioned on the other side of the constant-temperature reaction block and is placed at 90 degrees with the laser, and light holes are formed in the signal adapter plate mounting block and the signal receiving plate.

Furthermore, the fluorescence detection device also comprises a main control panel which can convert the optical signal into an electric signal and then transmit the electric signal to a display screen to output the electric signal as a digital signal.

In still another aspect, the present invention provides a method for improving the sensitivity of a fluorescence detection apparatus, which uses laser as an excitation light source, by increasing a distance path through which a sample containing a target nucleic acid and a fluorescent substance for labeling the amount of the target nucleic acid is penetrated by the laser, thereby improving the sensitivity of the fluorescence detection apparatus.

The utility model provides a portable fluorescence detection device has following beneficial effect:

1. compared with common fluorescence detectors in the market, the fluorescence detector has the advantages of small volume, light weight, regular appearance and portability.

2. The detection accuracy is high, the detection range is wide, the quantity detection is not limited, the detection efficiency is improved, the application environment range of the fluorescence detection is enlarged, and the portability of the fluorescence detection device is improved.

3. The laser with different excitation wavelengths can be replaced, a plurality of lasers can be configured to be combined according to needs, the detection range is wide, the detectable fluorescent substances are more, and full spectrum detection can be realized.

4. The reagent tube is large, the amplification of the reagent tube is 2.5-10 times, the reagent amount is greatly improved by 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.

5. The detection speed is high, the detection time of a single sample endpoint method is as fast as 5s, and the detection quantity is not limited.

6. The detection result can be read by directly placing the reagent tube into the sample cell without complex parameter setting.

7. Because the laser power is high, the optical path in the reagent is increased, and the beneficial effect is brought, the high-sensitivity effect can be achieved without a precise and expensive optical sensor, photoelectric amplification and precise calculation. Therefore, the cost is greatly reduced, the size of the instrument can be reduced to the size of an apple, and the stability is also greatly improved.

Detailed Description

Further description of the present invention will be made with respect to structural or technical terms, which are used to explain the present invention, if not otherwise indicated, in accordance with the common general terms used in the art. These descriptions are given by way of example only to illustrate how the invention may be embodied and not to limit the invention in any way, the scope of which is defined and expressed by the claims.

Detection of

Detection means assaying or testing for the presence or absence of a substance or material. Such as, but not limited to, chemicals, organic compounds, inorganic compounds, metabolic products, drugs or drug metabolites, organic tissues or metabolites of organic tissues, nucleic acids, proteins or polymers. In addition, detection may also indicate the amount of the test substance or material. Assays also refer to immunodetection, chemical detection, enzymatic detection, and the like.

Sample (I)

The sample used in the test device comprises a biological fluid. The initial state of the sample may be liquid, solid or semi-solid, and the solid or semi-solid sample may be converted to a liquid sample by any suitable method, such as mixing, triturating, macerating, incubating, dissolving, enzymatically digesting, etc., and then poured into the collection chamber, and the sample may be tested for the presence of the analyte by the test element. The sample can be obtained from human body, animal, plant, nature, etc. The sample taken from human body can be, for example, blood, serum, urine, cerebrospinal fluid, sweat, lymph, saliva, gastric juice, etc.; solid or semi-solid samples of feces, hair, cutin, tartar, nails, and the like. Samples taken from plants, which may be, for example, solid samples of roots, stems, leaves, etc.; liquid or semisolid samples such as tissue fluid and cell fluid prepared from root, stem and leaf. The sample taken from nature may be, for example, a liquid sample such as rainwater, river water, seawater, or groundwater; solid or semi-solid samples of soil, rock, ore, petroleum, etc.

In some embodiments, the sample of the present invention is a liquid sample.

In some embodiments, the sample is a liquid sample comprising a fluorescent substance.

Furthermore, the sample of the present invention is a liquid sample obtained by nucleic acid amplification, the sample contains nucleic acid substances to be detected, the amplification system contains a fluorescent probe, and the fluorescent probe is used as a marker substance for indicating the content of the nucleic acid substances in the sample. The fluorescent nucleic acid amplification may be performed after the amplification reaction is completed, or may be performed directly after the amplification reaction is completed in the portable fluorescence detection apparatus of the present invention. Of course, detection or testing may be performed at any stage prior to, during, or after nucleic acid amplification. The nucleic acid substance may be a target nucleic acid to be detected, the nucleic acid may be RNA or DNA, and the specific content of the nucleic acid substance, for example, the copy number, is represented by a fluorescent labeling substance.

Any technique that allows nucleic acid amplification, such as temperature-variable mode nucleic acid amplification, e.g., PCR amplification, embodied as fluorescence PCR, digital PCR amplification, is a specific embodiment of the present invention. Also such as isothermal or isothermal nucleic acid amplification, e.g., LAMP, RPA, TMA, RCA, NEAR, ERA, and amplification using a cleaving enzyme, and the like.

Testing device

The test device generally includes a test element, which is a component capable of detecting an analyte in a sample to be tested. The detection of the analyte by the test element can be based on any technical principle, such as immunology, chemistry, electricity, optics, molecular, physics, etc. The test element of the utility model can be one type, and can also be a combination of more than two types of test elements. The test element is provided with a detection area for displaying a detection result, and the detection area displays the detection result after the detection is carried out.

The detection device of the utility model is a fluorescence detection device for detecting fluorescence value. The device for detecting the intensity of the fluorescence by the detection device also comprises a component for converting an optical signal into a digital signal. Further, the detection device of the present invention is a fluorescence detection device for detecting the content of a target nucleic acid in a sample.

Fluorescence detector

The fluorescence detector is one commonly used detector for high pressure liquid chromatography. The chromatographic fraction is irradiated with ultraviolet light and detected when the sample component has fluorescent properties.

In terms of electronic transition, fluorescence means that some electrons in an atom transition from the lowest vibrational level in the ground state to some higher vibrational level after some substance absorbs light of the same characteristic frequency as itself. Electrons collide in the same or other molecules and consume considerable energy, thereby dropping to the lowest vibrational level in the excited state of the first electron, this form of transfer of energy being referred to as a radiationless transition. From the lowest vibrational level down to some different level in the ground state, while emitting a light of lower frequency and longer wavelength than the original absorption, i.e. fluorescence. 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 molecules with different structures cannot emit fluorescence after being excited. Fluorescence involves both absorption and emission of light, and therefore any fluorescent compound has two characteristic spectra: excitation spectra and emission spectra.

The utility model provides a portable fluorescence detection device is for adopting laser excitation fluorescent material to send the fluorescence detector of fluorescence.

Excitation spectroscopy

Fluorescence is photoluminescence, and the wavelength of excitation light needs to be selected to be suitable for detection. The excitation wavelength can be determined by the excitation spectrum of the fluorescent compound. The specific detection method of excitation spectrum is to excite fluorescent compound with incident light of different wavelengths by scanning excitation monochromator, and the generated fluorescence is detected by light detecting element through emission monochromator of fixed wavelength. Finally, the obtained curve of the fluorescence intensity to the excitation wavelength is the excitation spectrum. At the maximum wavelength of the excitation spectrum curve, the number of molecules in the excited state is the largest, i.e., the absorbed light energy is the largest, and the strongest fluorescence can be generated. When considering sensitivity, the assay should select the maximum excitation wavelength.

Portable fluorescence detection device adopt laser to be the exciting light, the excitation spectrum is the laser wavelength of transmission promptly.

Emission spectrum

The fluorescence spectrum is generally referred to as a fluorescence emission spectrum. It is a curve of fluorescence intensity changing with fluorescence wavelength obtained by wavelength scanning of an emission monochromator when the wavelength of an excitation monochromator is fixed. The fluorescence spectrum can be used for identifying fluorescent substances and can be used as a basis for selecting proper measurement wavelength in fluorescence measurement.

In addition, due to the characteristics of fluorescence measuring instruments, the energy distribution of a light source, the transmittance of a monochromator, the response of a detector and other properties vary with the wavelength, so that the same compound can obtain different spectrograms on different instruments without analogy, and the spectrums are called apparent spectrums. In order to obtain fluorescence spectra of the same compound on different instruments with the same characteristics, the above characteristics of the instrument need to be corrected. The corrected spectrum is referred to as the true fluorescence spectrum.

The excitation wavelength and emission wavelength are essential parameters for fluorescence detection. The selection of appropriate excitation and emission wavelengths is important to the sensitivity and selectivity of the detection, and in particular, the detection sensitivity can be improved to a greater extent.

Fluorescent PCR quantitative detector

The fluorescent quantitative PCR detector is an instrument for labeling and tracking a PCR product and monitoring a reaction process in real time through a fluorescent dye or a fluorescent labeled specific probe. And the product can be qualitatively and quantitatively analyzed by combining corresponding software, and the initial concentration of the sample template to be detected is calculated. And adding a fluorescent group into a PCR reaction system, and utilizing fluorescent signal accumulation to monitor the whole PCR process in real time.

And finally, carrying out quantitative analysis on the unknown template through a standard curve, wherein the quantitative analysis comprises fluorescent dyes such as TaqMan fluorescent probes or SYBR Green I and the like. PCR can relatively quantify the abundance of a specific gene or species, compare differences among different treatments or samples, such as gene copy number and expression amount to study the response of gene expression to treatment, and simultaneously obtain information such as PCR amplification efficiency.

The utility model provides a portable fluorescence detection device still includes heating module for the temperature of control sample, the nucleic acid that mainly used contained in making the sample carries out isothermal amplification, contains fluorescence probe in the amplification system of sample simultaneously, contains fluorophore and quenching group on the fluorescence probe.

Fluorescent probe

A class of fluorescent molecules that fluoresce characteristically in the uv-vis-nir and whose fluorescent properties (excitation and emission wavelengths, intensity, lifetime, polarization, etc.) can change sensitively with changes in the properties of the environment, such as polarity, refractive index, viscosity, etc. Small molecule substances that interact non-covalently with nucleic acids (DNA or RNA), proteins or other macromolecular structures to alter one or more fluorescent properties. Can be used for researching the properties and behaviors of macromolecular substances.

The fluorescent chemicals used for fluorescent quantitative PCR can be divided into two categories: fluorescent probes and fluorescent dyes. During PCR amplification, a pair of primers is added, and a specific fluorescent probe is added at the same time, wherein the probe is an oligonucleotide, and two ends of the probe are respectively marked with a reporter fluorescent group and a quenching fluorescent group. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, the 5 '-3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous.

The fluorescent probe is most commonly used for labeling antigens or antibodies in a fluorescence immunoassay method, and can also be used for detecting micro characteristics of micro environments, such as surfactant micelles, bimolecular membranes, protein active sites and the like. The molar absorptivity of the probe is generally required to be large, and the fluorescence quantum yield is required to be high; the fluorescence emission wavelength is in long wave and has larger Stokes shift; for use in immunoassays, binding to an antigen or antibody should not affect their activity.

It can also be used to label a pending nucleotide fragment for specific, quantitative detection of the amount of nucleic acid.

Common fluorescent probes include fluorescein probes, inorganic ion fluorescent probes, fluorescent quantum dots, molecular beacons, and the like. Fluorescent probes are widely used in nucleic acid staining, DNA electrophoresis, nucleic acid molecular hybridization, quantitative PCR techniques, and DNA sequencing, in addition to quantitative analysis of nucleic acids and proteins.

The portable fluorescence detection device of the utility model adopts laser as excitation light, in some embodiments, when the excitation wavelength of the laser is 450-doped 490nm, the fluorescence emission wavelength of the detection is 515-doped 530nm, and the detectable fluorescence substances include FAM, SYBR Green I and the like; when the excitation wavelength of the laser is 500-535nm and the detected fluorescence emission wavelength is 560-580nm, the detectable fluorescent substances comprise VIC, HEX, JOE, TAMRA, TET, Cy3 and the like; when the excitation wavelength of the laser is 555-585nm and the detected fluorescence emission wavelength is 610-650nm, the detectable fluorescent substances comprise ROX, TEXAS-Red and the like; when the excitation wavelength of the laser is 620-650nm and the detected fluorescence emission wavelength is 675-730nm, the detectable fluorescent substance comprises Cy5 and the like. The utility model discloses the accessible is changed the laser instrument or is increased the laser instrument and realize the free conversion combination between the instrument passageway, can realize full gloss register for easy reference fluorescence detection.

Drawings

FIG. 1 is a schematic structural diagram of a portable fluorescence detection device provided in embodiment 1

FIG. 2 is a schematic view of the portable fluorescence detection device according to embodiment 1, illustrating the structure thereof being disassembled

Detailed Description

In the following, preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings, it being noted that the embodiments described below are intended to facilitate understanding of the present invention without any limiting effect. The raw materials and the equipment used in the embodiment of the utility model are known products and are obtained by purchasing products sold in the market.

Embodiment 1 the utility model provides a portable fluorescence detection device

Fig. 1 and fig. 2 show a portable fluorescence detection device provided in this embodiment, where fig. 1 is a schematic structural diagram of the portable fluorescence detection device, and fig. 2 is a schematic structural diagram of the portable fluorescence detection device.

The portable fluorescence detection device provided by the embodiment comprises a laser light source module 1 and a sample cell module 2, wherein the laser light source module 1 is used for emitting laser, the laser acts on a sample in the sample cell module 2, and a fluorescent substance contained in the sample is excited to emit fluorescence; the path distance of the sample in the sample cell module 2 penetrated by the laser is not less than 3.7 mm.

Preferably, the distance of the path of the sample in the sample cell module 2 penetrated by the laser is 3.7mm-15 mm.

In the portable fluorescence detection device provided in this embodiment, the laser is excited from the side, and therefore the "distance" as used herein refers to the maximum length of penetration of the laser when the sample is loaded in the container, which corresponds to the maximum diameter. The term "sample" as used herein refers to a sample obtained by PCR amplification or isothermal amplification of nucleic acids containing a fluorescent substance.

Preferably, the sample in the sample cell module 2 is contained in a transparent centrifuge tube 3, and the transparent centrifuge tube 3 has a size of 0.5ml to 5 ml.

Preferably, the laser emission power of the laser light source module 2 is 5-500 mw.

Preferably, the sample cell module 2 contains only 1 transparent centrifuge tube 3, and the sample volume in the transparent centrifuge tube 3 is 0.05ml-5 ml. The portable fluorescence detection device that this embodiment provided, look at more small-size, more convenient design, realize at any time and everywhere on-the-spot fluorescence detection, single sample terminal point check-out time is no longer than 5s, and can be through changing the sample, detect many times, do not restrict the detection sample quantity.

Preferably, the sample cell module 2 comprises a transparent centrifuge tube 3, a transparent centrifuge tube cover 4 and an isothermal reaction block 5, the transparent centrifuge tube 3 can be loaded with the isothermal reaction block 5, and the transparent centrifuge tube cover 4 is covered above the transparent centrifuge tube 3. Can be through setting up the base that matches with transparent centrifuging tube 3's specification in the constant temperature reaction piece 5 for fixed transparent centrifuging tube 3, the base specification can be changed, thereby can be applicable to the transparent centrifuging tube 3 of different specifications respectively.

Preferably, the isothermal reaction block 5 can be used to subject nucleic acids contained in a sample to isothermal amplification.

Preferably, the transparent centrifuge tube 3 can be installed in the isothermal reaction block 5, and the sample is controlled to maintain a constant temperature by the isothermal reaction block 5. In some embodiments, a heating rod may be installed inside the isothermal reaction block 5 for temperature control.

Preferably, the laser source module 1 in this embodiment may 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 instrument 6 that this embodiment provided is removable for the laser of launching different wavelengths can arouse different fluorescent substances to send the fluorescence of different wavelengths, through the laser of changing different wavelengths, can realize the detection of wavelength fluorescence separately, the utility model discloses can realize full spectrum fluorescence detection.

Preferably, the laser light source module 1 may further include one or more lasers 6, and the different lasers 6 are respectively used for emitting laser light with different wavelengths for exciting fluorescence with different wavelengths; a plurality of lasers can be combined for use, so that the detection accuracy is further ensured; by replacing the laser 6 or adding the laser 6, free conversion combination among device channels is realized, and more convenience is provided for accurate detection.

Preferably, the fluorescence detection device further comprises a fluorescence detection module 9, wherein the fluorescence detection module 9 comprises one or more than one receptor 10 for respectively receiving fluorescence with different wavelengths; the receiver 10 is positioned at 90 degrees to the laser 6.

Preferably, the receiver 10 includes a signal adapter plate mounting block 11 and a signal receiving plate 12.

Preferably, an optical filter 13 may be installed in front of the light transmission holes of the signal receiving plate 12 to help filter out the interference light.

Preferably, the laser light source module 1 may comprise 1-4 lasers 6, and 1-4 receptors 10 corresponding to the detection module 9; the 1-4 lasers 6 may be simultaneously activated for use in 1 or 2-4 combinations thereof, and correspondingly simultaneously activated for 1 or 2-4 combinations of the receivers 10.

In some ways, the laser 6 is positioned parallel to the receptor 10 on the right side of the cuvette module 2, further increasing the space utilization of the apparatus and reducing light interference.

In some other ways, when the laser 6 and the receiver 10 are installed at two positions simultaneously, the fluorescence detection channel is added, and the detection range and the type of the sample are increased, for example, when the laser 6 and the receiver 10 are installed at four sides of the sample cell module 2 simultaneously, the laser source module 1 can contain 4 lasers 6 and 4 receivers 10.

Preferably, when four lasers 6 are included, the excitation wavelength of the first laser 6 is 450-490nm, the detection wavelength is 515-530nm, and 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, and the detectable fluorescent substances comprise VIC, HEX, JOE, TAMRA, TET, Cy3 and the like; the excitation wavelength of the third laser 6 is 555-585nm, the detection wavelength is 610-650nm, and the detectable fluorescent substances comprise ROX, TEXAS-Red and the like; the excitation wavelength of the fourth laser 6 is 650nm and the detection wavelength is 675 nm and 730nm, and the detectable fluorescent substances comprise Cy5 and the like. The utility model discloses the free conversion combination between the instrument passageway can be realized to the accessible change laser instrument 6 or increase laser instrument 6, adds two exciters 6 and acceptors 10 simultaneously on the sample cell module 2 (example 1: the sample cell module 2 adds first simultaneously, the exciters 6 and acceptors 10 of second; example 2: the sample cell module 2 adds first, third exciters 6 and acceptors 10 simultaneously and so on can have multiple combination) then can realize full spectrum fluorescence detection.

Preferably, light holes 14 are formed at the rear and right sides of the thermostatic reaction block 5; the laser 6 is positioned at one side of the constant temperature reaction block 5, light beams directly enter the transparent centrifuge tube 3 from a light-transmitting hole of the constant temperature reaction block 5, and the fluorescent reagent emits fluorescence after being excited by a light source; the signal adapter plate mounting block 11 is connected with the signal receiving plate 12, is positioned on the other side of the constant temperature reaction block 5, is arranged at 90 degrees with the laser, and is provided with light holes 14 on both the signal adapter plate mounting block 11 and the signal receiving plate 12.

Preferably, the fluorescence detection device further comprises a main control panel 15, which can convert the optical signal into an electrical signal, and then transmit the electrical signal to a display screen to output the signal as a digital signal, and a base 16.

When the portable fluorescence detection device provided by the embodiment is used for detection, because laser is excited from the side surface, when a transparent centrifugal tube is filled with a sample with a normal volume, the thickness of the sample penetrated by the laser is equivalent to the diameter of a PCR tube.

EXAMPLE 2 Effect of different volumes of sample on test results

The amplified samples to be detected with the volumes of 0.025ml, 0.05ml, 0.1ml, 0.2ml, 0.4ml, 0.8ml and 1.2ml are respectively filled into reagent tubes with the volumes of 1.5ml, the portable fluorescence detection device provided by the embodiment 1 is used for respectively detecting, the laser emission power of the laser light source module is 100mw, and when laser is excited from the side, the influence of the samples with the same volume filled in the reagent tubes with different specifications on the detection result is examined.

The specific experimental process is as follows:

preparing an amplification reaction system: 60mM Tris-acetate buffer (pH8.0), 100mM potassium acetate, 3mM dithiothreitol, 5% polyethylene glycol (20000), 2mM ATP, 20mM creatine phosphate, 420nM primer mix (upstream primer sequence, 5'-AATTTGTGCGAGTAAACCTATGTAGCAGCAGAG-3'; downstream primer sequence, 5'-TTCCTTCTAAGCTCTGCAGCTTCATTCATCATC-3'), 200nM fluorescent probe (probe sequence 5'-AAAATGTCGGGAGCTGAACATATGGAAGG (FAM-dT) (THF) T (BHQ1-dT) GAGCGAGTGCT-3' (C3-SPAC ER)), 100 ng/. mu.l creatine kinase, 600 ng/. mu.l phage gp32 protein, 150 ng/. mu.l phage uvsX protein, 25 ng/. mu.l phage uvsY protein, 80 ng/. mu.l klenow polymerase large fragment (exo-), 50 ng/. mu.l exonuclease TP III, 450. mu.M dNTP primer mix, 14mM magnesium acetate, 1000 copies of positive plasmid template.

Preparing 1ml system by the reaction, preparing 3 tubes in total with the total volume of 3ml, fully oscillating, uniformly mixing and centrifuging for a short time. Placing the mixture in a water bath kettle, reacting for 30 minutes at 37 ℃, then reacting for 5 minutes at 80 ℃ to inactivate enzyme components in the reaction components, and fully finishing the reaction. The fluorescent probe is cleaved at THF and the fluorescent signal is released due to loss of quenching. In order to ensure the uniformity of the fluorescent reagent, the reaction solution is completely mixed into a 5ml centrifuge tube and is fully and uniformly mixed, and then 0.025ml, 0.05ml, 0.1ml, 0.2ml, 0.4ml, 0.8ml and 1.2ml of the reaction solution are respectively filled into a transparent centrifuge tube with the specification of 1.5 ml. The transparent centrifugal tube that will divide well is placed at the portable fluorescence detection device that embodiment 1 provided, adjusts sample cell module 2 positions simultaneously to ensure that laser emission position and detection position pass through the central point of reagent center and put, the temperature sets for 37 ℃, carries out fluorescence numerical value and reads.

Wherein, when samples with different volumes are in a 1.5ml transparent centrifuge tube, laser irradiates the center of the reagent sample. As the sample volume is increased from 0.025ml to 0.8ml, the average path distance penetrated by the laser is increased from 3mm to 10.09mm, and the total amount of the excited fluorescent substances is increased sequentially as the path distance penetrated by the sample is increased, so that more fluorescence is emitted; when the sample volume is from 0.8ml to 1.2ml, especially when the sample is increased from 1.0ml to 1.2ml, the diameter is not increased continuously because the upper end of the centrifuge tube is cylindrical, so the average path distance penetrated by the laser is increased and slowed down, and the optical path is increased and slowed down, even when the sample volume is increased continuously, the path distance penetrated by the laser is not increased and the optical path is increased obviously because the diameter is unchanged, and at the moment, if the sensitivity is required to be further improved, the centrifuge tube with a larger size is required to be replaced, so the path distance penetrated by the laser is further increased, and the optical path is further increased.

The instrument experimental data are shown in table 1.

TABLE 1 detection results of samples of different volumes in a 1.5ml transparent centrifuge tube

As can be seen from the test results of table 1:

when a transparent centrifugal tube with the specification of 1.5ml is adopted, when the volume of a sample is 0.025ml, the sample penetrated by laser is small in volume and small in thickness, the path distance penetrated by the laser is 3mm, the transmission and refraction generated during the penetration of the laser are very small, the optical path is small, and the measured fluorescence value is also small. If the volume reaction is adopted, the detection result is easy to cause error.

When the sample volume is 0.05ml-0.4ml, the diameter of the transparent centrifugal tube is gradually increased from bottom to top due to the fact that the bottom of the transparent centrifugal tube is similar to a cone, the path distance of the sample in the transparent centrifugal tube penetrated by laser is increased from 5mm to 10mm, the optical path is gradually increased, the measured fluorescence value is also rapidly increased from 6046 to 23663, the increase is nearly 4 times, and the detection sensitivity is obviously improved.

When the volume of the sample is 0.8ml-1.2ml, the path distance of the sample penetrated by the laser is 10.09-10.22mm, and at the moment, due to the limitation of the shape of the upper end of the centrifugal tube, the path distance penetrated by the laser is gradually increased, the optical path is gradually increased, so that the change trend of the measured fluorescence value is obviously gradually reduced, and the influence on the detection result is not obvious.

Therefore, as the volume of the sample is increased, the thickness of the laser penetration is increased, so that the transmission and refraction of the laser in the sample are more sufficient, more fluorescent substances are excited to emit fluorescence, the measured fluorescence value is obviously increased, and the detection sensitivity is improved.

The "thickness" of the sample liquid penetrated by the laser refers to the length of the path of the laser-generated light through the liquid sample, and for example, the length of the path may be not less than 3mm, not less than 4mm, not less than 5mm, not less than 6mm, not less than 8mm, not less than 9mm, and not less than 10 mm. In some embodiments, when the thickness of the liquid is constant, the intensity of the laser can be increased to improve the detection sensitivity. In some approaches, the length of the path of the laser through the liquid sample is adjusted in conjunction with the intensity of the laser to achieve the results of the detection. The path is also understood to mean the manner in which the light generated by the laser is repeatedly refracted and reflected through the liquid sample after the laser has passed through the liquid sample. Of course, it is also understood that one or more lasers may be simultaneously passed through a sample of liquid, substantially extending the length of the path through the liquid.

EXAMPLE 3 Effect of the same volume of different reagents on the detection results

The present embodiment examines the difference of fluorescence values of the fluorescent reagents with the same concentration and the same volume under different penetration light paths. Taking 0.2ml of the reagent in the embodiment 2, respectively placing the reagent into a 0.2ml PCR reagent tube, a 0.5ml transparent centrifuge tube and a 1.5ml transparent centrifuge tube by using a micropipette, respectively detecting by using the portable fluorescence detection device provided by the embodiment 1, setting the laser emission power of a laser source module to be 50mw, simultaneously adjusting the position of a sample cell module 2 when laser is excited from the side surface so as to ensure that the laser emission position and the detection position pass through the center position of the center of the reagent, setting the temperature to be 55 ℃, respectively penetrating through the 0.2ml PCR reagent tube, the 0.5ml transparent centrifuge tube and the 1.5ml transparent centrifuge tube, simultaneously containing 0.2ml of the sample, enabling the path distance penetrated by the laser to be equivalent to the diameter of the sample, and inspecting the influence of the sample with the same volume on the detection result when the sample is contained in the reagent tubes with different specifications.

The samples with the same concentration and the sample volume of 0.2ml are respectively arranged in containers with the volumes of 0.2ml, 0.5ml and 1.5ml, and the distance of the laser penetrating through the samples when the laser is irradiated from the side is mainly determined by the shape of the container in which the samples are arranged. Since the container volume is 0.2ml, the paths penetrated by the laser are about 4.2 mm, 5.1 mm and 7.8mm respectively, namely, as the container volume is increased, the path distance of the laser penetrating the sample is gradually increased, the generated transmission and refraction are stronger, and stronger fluorescence is generated for excitation of more fluorescent substances.

The experimental data obtained by the instrument are shown in table 2.

TABLE 2 detection results of samples of different volumes in a 1.5ml transparent centrifuge tube

As can be seen from the results of the measurements in Table 2, the longer the optical path of the reagent is penetrated by the laser light, the more refraction occurs, the more fluorescent substance is excited, and the stronger the fluorescence intensity is detected. The change in fluorescence intensity is therefore less correlated with the sample volume and more correlated with the path distance traversed by the laser.

Example 4 Effect of fluorescent reagent diluted on assay results

In the fluorescent quantitative PCR of nucleic acid, the low concentration of target sample is often encountered. The fluorescence intensity generated after amplification is also lower for low concentration template samples. The resulting fluorescence intensity is no longer detectable at a particular reaction reagent volume when the sample concentration is low to a certain limit, which also determines the amplification sensitivity of the reagent and instrument combination. That is, as the instrument becomes more sensitive to changes in the fluorescent signal, the detection reagent and the instrument become more sensitive to the sample. The test objective of this embodiment is to enable the instrument to re-detect fluorescence by limiting dilution of the reagent to a limiting concentration, making it undetectable in a small volume, and by increasing the volume and reagent thickness. To verify that the sensitivity of the reagent to the sample is increased with respect to the increase in reagent volume or reagent thickness.

Taking 0.5ml of the reacted reagent in the experiment of example 2, diluting the reacted reagent by 10 times with water respectively, then loading the diluted reagent into 5ml transparent centrifuge tubes according to table 2, and loading 8 tubes in total, using the 5ml transparent centrifuge tube through the portable fluorescence detection device provided by example 1, wherein the laser emission power of the laser source module is 300mw, and when the laser is excited from the side, adjusting the position of the sample cell module 2 simultaneously to ensure that the laser emission position and the detection position pass through the center position of the center of the reagent, respectively detecting, and inspecting the influence of the low-concentration fluorescence on the detection result under different volumes and reagent optical distances. The temperature was set at 37 ℃.

When the sample with different volumes is in a 5ml transparent centrifuge tube, the distance and the path penetrated by the laser are different, the maximum path distance penetrated by the laser of the sample is equivalent to the diameter of the centrifuge tube at the highest liquid level of the sample, and as the sample volume is increased from 0.025ml to 1.6ml, the diameter of the centrifuge tube at the highest liquid level is increased from 3.9mm to 14.3mm, namely the maximum path distance penetrated by the laser is increased from 3.9mm to 14.3mm, at the moment, as the path distance penetrated by the sample is increased, the generated transmission and refraction are also increased rapidly, the optical path is increased rapidly, and therefore, the excited fluorescent substances in the sample are also increased rapidly; when the sample volume is from 1.6ml to 3.2ml, the diameter is not increased continuously because the upper end of the centrifugal tube is cylindrical, so the path distance penetrated by the laser is not increased, the optical path is not increased continuously, and the excited fluorescent substances in the sample are not increased continuously.

The experimental data of the instrumental tests are shown in Table 3.

TABLE 3 detection results of samples diluted by different times in a 5ml transparent centrifuge tube

As can be seen from the test results of table 3:

when the volume of the reagent is gradually increased, the path distance of the sample penetrated by the laser is continuously increased, so that the optical path is rapidly increased, the transmission and refraction of the laser generated in the sample are more sufficient, more fluorescent substances are excited to emit fluorescence, the measured fluorescence value is gradually detected, and the fluorescence value is obviously increased along with the increase of the volume of the reagent or the optical path, so that the detection sensitivity is improved.

Example 5 use of a Portable fluorescence detection device for LAMP amplification

The portable fluorescence detection device in example 1 is used for LAMP amplification reaction, the reagent final volume of each reaction is 0.25ml, the reaction conditions refer to ZL201310368890, step 2 in example 2 is used for amplification, and paraffin oil is used for sealing liquid to prevent aerosol generation. The temperature was set at 61 ℃ and the reaction was carried out for 30 minutes. After the reaction is finished, adding Sybr Green I dye with the final concentration of 1x to carry out an end point scanning method to record the difference between negative and positive values respectively.

TABLE 4 detection results of the end-points of negative and positive samples in LAMP amplification experiments

As can be seen from Table 4, the fluorescence values of the amplified products of the negative sample and the positive sample are different, and can be distinguished from negative and positive, and meanwhile, by using the ultraviolet observation comparison described in step 3 of reference patent example 2, the negative and positive results are consistent with the test results of the instrument, namely: the color of the LAMP amplification product dilution of the positive sample changes from orange to green, while the negative result remains orange.

The above cases show that the determined fluorescence device can be used for determining the negative and positive qualitative detection of the sample after LAMP amplification.

Example 6 comparison of the results of detection with a PCR fluorescence detector using a Portable fluorescence detection device

In this example, two conventional fluorescence quantitative PCR instruments (model: MA-6000, Acui technology Limited, Suzhou, using a combination of LED and a filter and a lens as an excitation light source; U.S. Bio-Rad, model: CFX miniOpticon System) were selected, the annealing temperature was set at 54 ℃, 30 seconds per cycle, the total reaction was set at 40 cycles, the other instrument settings were all normal settings, and the specific product amplification condition was judged by the aid of the melting curve. Adopting a bidirectional primer MPP-1QF:5'ACAAATAAGTGGAGGTAAAGC 3'; MPP-1QR:5'TGTCTGACTGCGAGAATAA 3'. The method adopts AceQ qPCR SYBR Green Master Mix (Low ROX premix) of Nanjing Novozam Biotechnology limited with the cargo number: and (3) carrying out amplification by using the Q131-02 dye method fluorescent quantitative premixed PCR reagent, and preparing a reaction reference kit instruction. 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 detected by comparison.

Through preliminary experiment optimization verification, the lowest detection line of the Q-PCR reagent is 5 copies/reaction, and negative controls are all flat lines without a fluorescent amplification curve. The experiment was performed with a sample concentration of 5 copies/reaction. 16 reactions, 8 positive control replicates and 8 negative control replicates were tested on each instrument. The experimental result shows that the two instruments can detect 8 positive, the melting curve is a single peak, and the result is normal; the negatives were all normal, and no amplification curve appeared.

Portable fluorescent device comparative experiment: because the utility model discloses the volume that fluorescence device adopted is great, so will be above the result after the amplification, positive and negative after same instrument amplification mix respectively to same 1.5ml transparent centrifuging tube after, carry out the fluorescence value measurement of big system again. The results are shown in Table 5.

TABLE 5 detection results of the same samples by different PCR fluorescence detectors

Can know by the testing result of table 5, through the product after traditional fluorescence quantitative PCR amplifys, adopt after sample volume enlargies the utility model provides a portable fluorescence detection device detects the obvious difference that all can detect fluorescence signal value.

Meanwhile, in order to further verify the result, when the fluorescence quantitative PCR instrument is used as a constant temperature amplification instrument, isothermal amplification reagents are adopted, and the fluorescence difference of the positive and negative amplification products can be detected by adopting the portable fluorescence detection device after the positive amplification products are mixed. The method shows that when the PCR kit is used for isothermal amplification, the fluorescence detection effect of a fluorescence quantitative PCR instrument can be achieved by amplifying the volume of an amplification reagent.

Since the nucleic acid content in the sample may be low during the detection of the nucleic acid content, it is difficult to ensure the accuracy when the detection is performed with a low sample volume. Therefore, the detection sensitivity and accuracy can be significantly improved by using laser as an excitation light source and increasing the thickness of the sample penetrated by the laser. In order to realize the miniaturization and the convenience of the fluorescence detection device, laser is used as an excitation light source, and meanwhile, the volume of a sample is required to be increased, so that the thickness of the sample penetrated by the laser is increased, and stable signal detection is realized.

Although the present invention is disclosed above, the present invention is not limited thereto. For example, the application range of the medicine can be expanded. Various changes and modifications may be effected therein by one of ordinary skill in the pertinent art without departing from the scope or spirit of the present invention, and the scope of the present invention is defined by the appended claims.

Claims (10)

1. A portable fluorescence detection device is characterized by comprising a laser light source module, a sample cell module and a detection module, wherein the sample cell module comprises a constant temperature reaction block for controlling the temperature of a sample; the laser light source module and the detection module are respectively positioned at two sides of the sample cell module.

2. The apparatus of claim 1, wherein the sample cell module further comprises a sample container mounted within the isothermal reaction block, the sample container comprising a reaction cup and a reaction cup lid.

3. The device as claimed in claim 2, wherein a base matched with the sample container is arranged in the constant temperature reaction block; the opening of the constant temperature reaction block is upward, and the sample container is inserted into the base in the constant temperature reaction block from top to bottom.

4. The device according to any one of claims 1 to 3, wherein the isothermal reaction block is provided with a light-transmitting hole for the passage of laser light or fluorescence.

5. The apparatus of claim 1, wherein the laser source module and the detection module are respectively located at two sides of the sample cell module and are disposed at 90 degrees.

6. The apparatus of claim 1 or 5, wherein the laser source module comprises a laser for emitting laser light for penetrating the sample in the sample cell module to excite fluorescent substances contained in the sample within a distance of the penetrated path to emit fluorescence.

7. The apparatus of claim 6, wherein the laser comprises a laser head and a laser head mounting barrel.

8. The apparatus of claim 7, wherein the detection module comprises a receptor for receiving fluorescence; the receiver comprises a signal adapter plate mounting block and a signal receiving plate.

9. The apparatus of claim 8, wherein the laser source module comprises 1-4 lasers and 1-4 receivers corresponding to the detection module, respectively.

10. The device of claim 9, further comprising a main control board and a base, wherein the main control board can convert the optical signal into an electrical signal and then transmit the electrical signal to the display screen to output the digital signal.

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