KR101802460B1

KR101802460B1 - Gene Diagnostic Apparatus

Info

Publication number: KR101802460B1
Application number: KR1020150184209A
Authority: KR
Inventors: 조원창
Original assignee: 조원창
Priority date: 2015-12-22
Filing date: 2015-12-22
Publication date: 2017-11-28
Also published as: KR20170074662A

Abstract

According to the present invention, there is provided a plasma display panel comprising: a first heating element; A heating block disposed above the first heating element and having insertion holes into which one or more sample tubes can be inserted; And an optical system for introducing light into a sample in the sample tube through a through hole formed in one side of the heating block and detecting a fluorescence signal of the sample.

Description

[0001] GENE DIAGNOSTIC APPARATUS [0002]

The present invention relates to a gene diagnosis apparatus, and more particularly, to a gene diagnosis apparatus for detecting a fluorescence signal from a gene sample contained in a sample tube using a fluorescence optical system.

Just as the discovery of restriction enzymes has opened the beginnings of genetic engineering, the Polymerase Chain Reaction (PCR) technology has helped to advance the life sciences and helped to achieve its full potential. The PCR method is a technique for mass-amplifying a specific region of DNA or RNA in a reaction vessel, and its principle is very simple and easy to apply. In addition to the field of pure molecular biology, the PCR method is applied to a variety of fields such as medicine, science, agriculture, Furthermore, it is expanding its application range from archeology to anthropology.

Gene amplification technology has been extensively used for R & D and diagnosis purposes in life sciences genetic engineering and medical fields. In particular, gene amplification technology by polymerase chain reaction (PCR) has been widely used.

PCR is an abbreviation for polymerase chain reaction. It uses DNA polymerase to amplify a specific DNA sequence in the genome as necessary. Polymerase chain reaction (PCR) is widely used to amplify a large number of genes using only a small amount of genes with the use of gene cloning enzymes in order to carry out researches using genes, experiments, and detection of microorganisms. In order to amplify these genes, PCR is necessary to control the temperature of the gene sample. PCR generally consists of three steps. Denaturation to separate the two strands of DNA, annealing to bind the primer to the end of the nucleotide sequence, extension to synthesize DNA using DNA polymerase, ). Since different temperatures are required, the reaction is performed by repeatedly performing the temperature cycle, and this process can be amplified exponentially by cyclic repetition.

The thermal denaturation process of PCR is usually carried out at around 95 ° C. Since the binding reaction and the polymerization reaction of PCR proceed at a temperature of about 55 ° C to 75 ° C lower than that of the PCR, It is necessary to repeatedly raise and lower the temperature of the contained PCR container.

As such, a thermal cycler is widely used as a device for lifting the temperature of the sample periodically.

A method for detecting PCR reaction in real time, the fluorescence detection method is used in most of the devices currently on sale. This fluorescence detection method can be combined with a method of using a fluorescent dye such as SYBR Green I by binding to a double strand DNA generated by a PCR reaction and a method of combining between two primers used for a PCR reaction (DNA polymerase) is used as a probe, and a fluorophore and a fluorescence inhibitor (Quencher) are bonded to both ends of the probe. The exonuclease And TagMan (R) method, which analyzes fluorescence when a probe is cleaved by using an activity (exonuclease activity) to separate a fluorescent chromophore and a fluorescence inhibition terminal. In most commercialized devices, various devices for detecting fluorescence in containers using PCR containers are on sale. The fluorescence detector is mainly composed of a light source, a detection sensor such as a photodiode (PD), a PMT, and a CCD sensor, an optical system including a lens including a mirror, a lens and a filter, and a fluorescence signal processing board.

LED, halogen lamp, white light, etc. are used for the light source part, CCD sensor, PD, PMT and the like are used for the detection sensor part. A collimating lens for condensing the excitation light irradiated from the light source, a dichroic mirror for selectively transmitting or reflecting the light according to the wavelength, an excitation light selected by the dichroic mirror to be condensed and irradiated onto the sample in the tube, An objective lens for condensing fluorescence generated in the tube by irradiation of excitation light, and a focusing lens system for condensing the fluorescence selected by the dichroic mirror. The fluorescence detector has an imaging type in which an optical device is fixed and a scanning type in which all or a part of the optical device moves while moving around the PCR container. This is determined by the number of containers to be inspected and the intended use of the product, each with its advantages and disadvantages. In the case of the scanning type, since the sample is inspected using the same fluorescent detector in order to inspect the samples of plural PCR containers, the calibration process of the separate fluorescent detector is not required unlike the image type, .

Peltier devices are used as heating devices in PCR. The Peltier device performs heating and cooling in the same device while switching the current direction, and serves as a cooling function on the lower surface of the Peltier device and performs heating on the upper surface. The upper surface of the Peltier element is in contact with a block of aluminum, and the aluminum block transfers heat of the heater to a sample tube mounted on the aluminum block. When the sample tube is cooled, a cooling fin is attached to the lower surface of the Peltier element so that cooling can be performed quickly.

In most cases, the fluorescence measuring part is located on the upper surface of the sample tube in order to measure the fluorescent amplification amount of the sample tube, irrespective of the image type or the scanning type. In order to prevent this phenomenon, the tube lid corresponding to the upper part of the sample tube is set at a temperature higher than the PCR cycle temperature by about 100 ° C. It keeps the temperature to prevent evaporation, and a separate heating device is installed here. Therefore, the fluorescence measurement unit and the evaporation-prevention heating apparatus are placed at the same position, so that interference between each other is required and the structure becomes large.

PCR In detail, PCR proceeds in three reaction steps. The first step is a denaturation step. In this step, the double stranded DNA is treated at 90 ° C or higher to separate each strand of DNA. The second step is an annealing step. In this step, two kinds of primers (primer) to each single strand of complementary DNA. In this case, the condition is usually from 55 to 60 ° C for 30 seconds to several minutes. The third is an extension step, in which a DNA polymerase is activated to extend the primer. The time required for the elongation reaction depends on the concentration of the template DNA, the size of the amplified fragment, and the reaction temperature. When using Thermusaquaticus (Taq) polymerase, which is commonly used, it takes about 30 seconds to several minutes at 72 ℃.

It is an object of the present invention to provide an improved genetic diagnostic device capable of solving the problems of the prior art.

It is another object of the present invention to provide a gene diagnostic apparatus capable of performing rapid and accurate gene diagnosis on a large number of samples.

Another object of the present invention is to provide a genetic diagnostic device which can be operated without interfering with a Peltier element, a heater and an optical system necessary for gene amplification.

In order to achieve the above object, according to the present invention,

A first heating element;

A heating block disposed above the first heating element and having insertion holes into which one or more sample tubes can be inserted; And

And an optical system for introducing light into the sample in the sample tube through a through hole formed in one side of the heating block and detecting a fluorescence signal of the sample.

According to an aspect of the present invention, the optical system is installed to be reciprocally movable along one side of the heating block.

According to another aspect of the present invention,

Light source;

A collimating lens for focusing light from the light source;

A first filter for filtering light in a used wavelength range of the light;

A dichroic mirror for refracting the filtered light;

An objective lens for passing the light refracted by the dichroic mirror to enter the sample;

A second filter for filtering the fluorescence signal of the sample transmitted through the dichroic mirror; And

And a detector for detecting light having passed through the second filter.

According to another aspect of the present invention, there is further provided a fin and a fan disposed below the first heating element.

According to another aspect of the present invention, the heating block has at least one through-hole formed in the first side surface and at least one through-hole formed in the second side surface opposite to the first side surface,

The optical system includes a first optical system moving along the first side and a second optical system moving along the second side.

According to another aspect of the present invention, the optical system includes two or more optical systems fixed to each other so as to correspond to cases of detecting different types of fluorescence when two or more kinds of fluorescent dyes used in a sample tube are detected, .

According to another aspect of the present invention, there is further provided a second heating element disposed on the heating block.

According to another aspect of the present invention, the first heating element is a Peltier element, and the second heating element is a heater for heating.

According to another aspect of the present invention, the light source and the detector of the optical system are located in the same direction or opposite to each other about the sample tube.

According to another aspect of the present invention, the light source of the optical system simultaneously irradiates the excitation light source with a plurality of fixed number of samples, while the detector detects the fluorescent signal while moving along one side of the sample tube.

In the present invention, a fluorescent detector is placed on the side of a heating block on which a PCR tube is mounted, and the amount of fluorescence as a result of PCR is measured. In order to measure the gene amplification result using a plurality of sample tubes, it is possible to linearly move the fluorescence detector by applying a motor and a moving mechanism. That is, a structure of a fluorescence measuring part is taken to irradiate the excitation light source on the side of the sample tube mounted vertically to the heating block, and to measure the amount of fluorescent light returned to the excitation light. In this case, interference with the evaporation-preventing heating device located on the upper surface of the sample tube is avoided, and it is isolated from the high-temperature heat generated in the heating device, so that deterioration of the performance of the detection sensor of the fluorescence measurement part can be prevented. The fluorescence measurement section of the present invention is constituted by a light source section, a lens system, and a detection sensor section. Since the fluorescence measuring unit can be scanned while being moved by a driving unit and a guide unit for moving the fluorescence measuring unit with the same structure, a plurality of sample tubes can be inspected by the same fluorescence measuring apparatus.

In the gene diagnosis apparatus according to the present invention, the cost of the fluorescence measuring unit, which occupies a large portion of the cost, can be greatly reduced, and since the fluorescence measuring unit measures the fluorescence amount of a plurality of sample tubes, As a difference in brightness changes, it is necessary to perform an optical calibration that makes the light amount of the light source the same, since a difference in measured values may occur even for the same sample. However, if a fluorescence measurement unit is constructed as in the present invention, Since the fluorescence optical system measures a plurality of sample tubes, a separate calibration process is unnecessary or simple.

The fluorescence measuring unit of the present invention has a structure in which light is irradiated from the side of the tube and the amount of fluorescence can be measured on the same line (the incident hole and the exit hole are the same) in which the light is irradiated. In addition, it is possible to combine a plurality of different fluorescence measuring units having different measurement wavelength ranges, and unlike a complicated multi-channel fluorescence measuring unit that can be seen in other products, excitation and emission wavelengths When the other filters are manufactured in the same manner and are combined and mounted, it is possible to easily construct a multi-channel optical system.

In another embodiment of the present invention, when a plurality of sample tubes are arranged in two rows, the fluorescence measuring unit can be measured while scanning the left and right sides of each row of the tubes. An upper heater is separately provided on the upper surface of the sample tube of the present invention to prevent water vapor from being generated in the sample tube when the temperature rises and falls. The incidence holes / exit holes made on the side of the heating block that transfers heat to the sample tube can be made perpendicular to the center line of the vertically standing sample tube or perpendicular to the outer surface of the sample tube have.

In the present invention, there is provided a structure in which an optical system is placed on a side surface of a sample tube vertically mounted on a heating block, and an excitation light is irradiated, and the amount of the fluorescent light returned to the excitation light is measured. In the present invention, a Peltier device is disposed below the heating block, and a heating source for preventing evaporation is provided on the upper part of the heating block to have a complicated structure for measuring fluorescence in the upper part or the lower part. Therefore, if the optical system is provided on the side surface of the sample tube as in the present invention, the optical signal can be detected by avoiding the interference between the Peltier element and the upper heat source, thereby simplifying the product structure and reducing the cost.

On the other hand, since the optical system can be moved and scanned by using the driving device, a plurality of sample tubes can be measured with the same optical system, and the calibration process for each optical system, which is indispensably required when using several optical systems, The use of a fluorescence detector (including an optical system) improves the reproducibility of the test results. Even if a large number of sample tubes are inspected, the number of fluorescence detectors can be minimized.

The sample tube can be arranged in one or two or more rows, making it possible to easily configure the optical system for the number of rows of sample tubes. It is easy to measure multiple sample tubes.

In order to detect a large number of fluorescence signals, a complicated multi-channel optical system was required. However, in the present invention, a plurality of optical systems applying a filter corresponding to a fluorescence signal to be detected are attached to one guide and scanned to detect a plurality of fluorescence signals can do. This is advantageous in that a necessary optical system can be easily applied in accordance with the type and number of fluorescent signals to be measured.

1 is a schematic perspective view of an embodiment of a gene diagnosis apparatus according to the present invention.
2 is a schematic perspective view of a second embodiment of the gene diagnosis apparatus according to the present invention.
3 is a schematic perspective view of a third embodiment of the gene diagnosis apparatus according to the present invention.
4 is a side view showing an enlarged view of a part of the heating block and the optical system.
5 is an enlarged perspective view of a heating block and a part of an optical system.
Fig. 6 is an explanatory diagram showing the configuration of the optical system described with reference to Figs. 1 to 5. Fig.
7A and 7B are schematic perspective views showing a fourth embodiment of the present invention.
8A and 8B are schematic perspective views showing a fifth embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

1 is a schematic perspective view of an embodiment of a gene diagnosis apparatus according to the present invention.

Referring to FIG. 1, a first heating element 13 made of a Peltier element and insertion holes, which are disposed on the first heating element 13 and into which one or more sample tubes T can be inserted, are formed A second heating element 17 such as a heating heater disposed on the heating block 15 and a through hole 15a formed in the side surface of the heating block 15, And an optical system (OS) that receives light through the sample in the sample tube (T) through a light source (not shown) and detects the fluorescence signal of the sample.

The heating block 15 is composed of a top surface and a bottom surface opposed to each other and a hexahedron having two pairs of side surfaces facing each other as shown in the figure. The inlet of the insertion hole into which the sample tube T is inserted is formed on the upper surface of the heating block 15 and the insertion hole extends along the height of the heating block 15. [ 4, the through hole 15a is formed on one side of the heating block 15, and a sample contained in the sample tube T inserted into the heating block 15 through the through hole 15a Light can be incident, and the fluorescence signal of the sample can be detected.

A radiating fin 12 is disposed below the first heating element 13 which can be manufactured by a Peltier element and a fan 11 is installed under the radiating fin 12. [ The fan 11 is fixed to the base B and a side wall 27 is provided on one side of the base B.

In order to detect the optical signal of the sample contained in one or more sample tubes T, the optical system OS can reciprocate parallel to the side of the heating block 15. The movement of the optical system OS is controlled by a drive motor M fixed to the side wall 27 and a motor pulley 16 coupled to the rotation shaft of the drive motor M and a belt 24, and a moving part 22 fixed to the belt 24. The optical system OS is connected to the moving part 22 through the bracket 21 and the moving part 22 is guided horizontally along the guide 25. [ Therefore, when the belt 24 travels by the rotation of the motor M, the moving part 22 moves horizontally, whereby the optical system OS can also be moved. The optical system 24 measures the amount of fluorescence in the sample tube through the through-hole 15a of the heating block 15.

The Peltier element, which can be used as the first heating element 13, is heated at the upper surface and cooled at the lower surface according to the direction dial of the current as described above. Therefore, the sample contained in the sample tube T can be heated when necessary, and the once heated Peltier element can be quickly cooled by the cooling operation at the lower surface. It is also preferable to dispose the radiating fin 12 and the fan 11 under the first heating element 13 to assist cooling. On the other hand, the upper heater, which is the second heating element 17 disposed on the top of the heating block 15, may be a general heater, which prevents the evaporation phenomenon in the sample tube due to temperature rise and drop. The second heating element 17 is held at the top of the heating block 15 by a support connected to the base B and the side wall 27.

As a modification of the embodiment shown in Fig. 1, the optical system OS is provided with only a detector,

2 is a schematic perspective view of a second embodiment of the gene diagnosis apparatus according to the present invention.

Referring to the drawings, it can be understood that the overall configuration is similar to the embodiment of FIG. 1, and a description of similar portions will be omitted.

In FIG. 2, an insertion hole is formed in the heating block 15 'so that the sample tubes T can be inserted in two rows, so that when the sample tubes T are inserted into the heating block 15', two of them are overlapped with each other State. First and second optical systems OS1 and OS2 are disposed on both sides of the heating block 15 ', respectively, for incident light and fluorescence signal detection for each of the two overlapping sample tubes T. In FIG. 2, denoted by 15a 'are through holes formed in the second side of the heating block 15', and through holes are also formed in the first side opposite to the second side. Light is incident from the optical system through the through hole 15a ', and the fluorescence signal of the sample is also detected. That is, the heating block has at least one through-hole formed in the first side face and at least one through-hole formed in the second side face opposite to the first side face, and the optical system includes a first optical system moving along the first side face, And a second optical system moving along the side surface.

The moving part 22 driven by the motor M is coupled to the first optical system OS1 through the first bracket 32a. The first optical system OS1 and the second optical system OS2 are connected to each other through a connection part 31. [ The connection part 31 connects the optical systems O1 and O2 above the upper heater 17 so as not to interfere with the upper heater 17. [ The second optical system OS2 is connected to the guide 35a through the second bracket 32b and the guide 35a is guided along the rail 35b fixed to the base B. Thus, the second optical system OS2 can perform work on the sample tube T disposed in the heating block 15 'concurrently with the first optical system OS1.

3 is a schematic perspective view of a third embodiment of the gene diagnosis apparatus according to the present invention.

Referring to the drawings, the moving unit 22 is connected to three optical systems OS1, OS2, OS3 through a bracket 21. [ The three optical systems OS1, OS2, OS3 are arranged so as to face the same side of the heating block 15. Therefore, when there is only one fluorescent dye used in the sample tubes T, it is possible to measure the fluorescence signal of the sample tube simultaneously with the quantity of the optical system by using a plurality of optical systems, have. That is, by providing two or more optical systems OS1, OS2, OS3 fixed to each other so as to correspond to two or more through-holes 15a formed on one side of the heating block 15, faster fluorescence signal detection is performed .

On the other hand, when a plurality of types of fluorescent dyes used in the sample tube are different from each other and a plurality of fluorescence filters of two or more optical systems are appropriately used in different kinds thereof, And measurements can be performed. That is, even when a multiplex test is required, it can be handled easily.

Fig. 4 is a side view showing an enlarged view of a part of the heating block and the optical system, and Fig. 5 is an enlarged perspective view of a heating block and a part of the optical system.

Referring to FIG. 4, a through hole 15a is formed in a side surface of the heating block 15. An optical system OS may be arranged to correspond to the through hole 15a. 5, a heating block 15 is formed to accommodate a plurality of sample tubes T, and the optical system OS is moved along the heating block 15 to a position corresponding to the through hole 15a . &Lt; / RTI > When the detection of the fluorescence signal from the sample of the sample tube T is completed through any one of the through holes 15a, it moves to the position of the aperture corresponding to the next adjacent sample tube T and repeats detection of the same fluorescence signal.

The heating blocks shown in Fig. 5 are formed in a cylindrical shape rather than a hexahedron, and are connected to each other. 5, it is understood that through holes are formed in the respective circumferential surfaces corresponding to the side surfaces of the cylindrical heating block, light is incident from the optical system OS through the through holes, and fluorescence signals from the sample can be detected .

Fig. 6 is an explanatory diagram showing the configuration of the optical system described with reference to Figs. 1 to 5. Fig.

An optical system OS includes a light source 51, a collimating lens 52 for focusing the light from the light source 51, a first filter 53 for filtering light in the used wavelength range of the light, A dichroic mirror 54 for refracting the filtered light, an objective lens 55 for passing the light reflected by the dichroic mirror 54 and entering the sample, and a dichroic mirror 54 A second filter 57 for filtering the fluorescence signal of the sample transmitted through the second filter 57 and a detector 60 for detecting the light passing through the second filter 57.

As the light source, an LED can be mainly used, and a photodiode can be used as a detector.

In the optical system having the above-described configuration, the light emitted from the light source 51 is focused on a point through the collimating lens 52, and then passes through the first filter 53. At this time, 1 filter 53 as shown in Fig. The first filter used depends on the type of fluorescence of the sample to be measured, and one optical system can measure only the fluorescence signal of a specific wavelength.

The filtered light reaches the dichroic mirror 54, and the light refracted by 90 degrees by the dichroic mirror is irradiated through the objective lens 55 to the sample to be measured. The emitted light causes the fluorescence of the sample to be emitted and the emitted fluorescence signal passes through the objective lens 55, the dichroic mirror 54, the second filter 57, the focusing lens 58, .

In the optical system of the present invention, one optical system can detect one fluorescence signal. In order to detect a plurality of fluorescent signals, which are generally used, a fluorescent filter is replaced, but a different kind of filter is applied to the same optical system structure, and a pre-manufactured optical system is selected and used adjacent to each other. (I.e., three optical summer systems OS1, OS2, OS3 are fixed adjacently to each other as shown in Fig. 3). That is, the number of optical systems equal to the number of required fluorescence signals is used. Therefore, a fluorescent detector can be easily manufactured and applied. A plurality of optical systems used at this time are combined and moved together on one moving axis, and horizontally move on the side of the sample tube to detect the fluorescent signal.

Figures 7A and 7B show another embodiment of the present invention.

Referring to the drawing, a detector OS-D of the optical system is installed on one side of the bridge 71 and faces the first side of the heating block 75 and a light source OS-L of the optical system is installed on the other side, Towards the second side of block 75. A through hole 75a is formed in the second side surface of the heating block 75 and a through hole (not shown) corresponding to the second side surface of the heating block 75 is formed. The bridge 71 can reciprocate along the heating block 75 by the operation of the drive motor and the belt or the like described with reference to Fig.

The light from the light source OS-L of the optical system is incident on the sample tube T through the through hole 75a of the heating block 75 and the fluorescent signal from the sample tube T (OS-D) through a through hole formed in one side surface of the substrate. In this case, it will be understood that the optical system can be constructed without using the dichroic mirror described with reference to Fig. That is, it can be understood that the light source and the detector of the optical system are located in opposite directions with respect to the sample tube.

8A and 8B show another embodiment of the present invention.

The OS-D detector of the optical system is movably installed on the first side of the heating block 85 while the light source OS-L of the optical system is disposed on the second side of the heating block 85, Is fixed to the base (B) by the light source fixing portion (86). In the heating block 85, through holes corresponding to the first side and the second side are formed. A plurality of light sources are provided in the light source unit OS-L, so that light can be simultaneously incident through all the holes formed in the second side surface of the heating block 85. [ The detector OS-D can move along the heating block 85 by a motor, a belt, or the like shown in Fig. 1, and thus can detect a fluorescent signal in accordance with the movement of the detector OS-D. That is, the light source of the optical system is provided for a plurality of fixed number of samples to simultaneously irradiate the excitation light source to each sample, while the detector can detect the fluorescent signal while moving along one side of the sample tube.

Claims

A first heating element;
A heating block disposed above the first heating element and capable of being inserted in two rows of sample tubes, the heating block comprising: at least one first through-holes formed in the first side; and at least one through- A heating block having second through holes;
A second heating element disposed on top of the heating block;
A first optical system having a light source for emitting light to a sample in the sample tube through each of the first holes and a detector for detecting a fluorescence signal of the sample;
A second optical system having a light source for emitting light to the sample in the sample tube through each of the second holes and a detector for detecting the fluorescence signal of the sample;
A connecting portion connecting the first optical system and the second optical system to each other; And
And a moving part coupled to the first optical system and driven by the motor such that the first optical system and the second optical system can reciprocate simultaneously along the first side and the second side of the heating block, .

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The method according to claim 1,
Wherein each of the first optical system and the second optical system comprises:
A collimating lens for focusing light from the light source;
A first filter for filtering light in a used wavelength range of the light;
A dichroic mirror for refracting the filtered light;
An objective lens for passing the light refracted by the dichroic mirror to enter the sample; And
And a second filter for filtering the fluorescence signal of the sample transmitted through the dichroic mirror,
And the light having passed through the second filter is detected by the detector.

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