KR20130056408A - Device for detecting nucleic acid amplification reaction product - Google Patents

Device for detecting nucleic acid amplification reaction product Download PDF

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Publication number
KR20130056408A
KR20130056408A KR1020110122005A KR20110122005A KR20130056408A KR 20130056408 A KR20130056408 A KR 20130056408A KR 1020110122005 A KR1020110122005 A KR 1020110122005A KR 20110122005 A KR20110122005 A KR 20110122005A KR 20130056408 A KR20130056408 A KR 20130056408A
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South Korea
Prior art keywords
light
fluorescence
polarization
beam splitter
polarizer
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KR1020110122005A
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Korean (ko)
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이현영
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이현영
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/119—Fluorescence polarisation

Abstract

The present invention relates to a real-time monitoring device for nucleic acid amplification reaction products for monitoring in real time the production of the reaction product generated during the reaction while performing a nucleic acid amplification reaction such as a polymerase chain reaction of a plurality of trace samples. The present invention relates to a real-time monitoring device for a biochemical reaction including a polarizer, a polarizing beam splitter, a polarization converting system, and the like to effectively separate the interference of excitation light and fluorescence.

Description

Device for detecting nucleic acid amplification reaction product

The present invention relates to a device for detecting a nucleic acid amplification reaction product, and more particularly, a plurality of trace samples of a reaction product generated during the reaction while performing a nucleic acid amplification reaction such as polymerase chain reaction (PCR). A device for detecting nucleic acid amplification products for monitoring production in real time.

Recently, a real-time PCR method has been developed to monitor a reaction product in real time while performing a polymerase chain reaction. This method does not require electrophoresis on the gel, can identify amplification products during the reaction cycle, and has the advantage of obtaining quantitative results. The apparatus used for the real-time PCR is a device incorporating a thermal cycler (PCR) for a PCR reaction and a fluorometer for real-time detection of the reactants.

In general, the monitoring of real-time PCR is used for fluorescence detection using a fluorescent reagent, and the following methods are representative.

1) Interchelating method is a method of detecting fluorescence that is developed by amplification by adding a reagent (Interchelator; for example, SYBRGreen I, EtBr, etc.) that binds to double-stranded DNA to the reaction system and amplifies it. When the interchelator binds to the stranded DNA, it displays fluorescence. The fluorescence intensity can be detected to measure the melting temperature of the amplified DNA as well as quantitatively.

2) TaqManTM

 As a probe method, an oligonucleotide in which the 5 'end is modified with a fluorescent material (FAM, etc.) and the 3' end with a quencher material (TAMRA, etc.) is added. Specific annealing with TaqMan ™ probe template DNA under annealing conditions but fluorescence is inhibited by the quencher. In the expansion reaction, the template is decomposed by 5 '-> 3' exonuclease activity possessed by Taq DNA polymerase, and the inhibition by capture is eliminated.

 3) As a molecular beacon method, an oligonucleotide (Molecular Beacon probe) that forms a hairpin secondary structure in which both ends are modified with a fluorescent material (FAM, TAMRA, etc.) and a quencher material (DABCYL, etc.) is added to the reaction. Molecular Beaconprobe hybridizes specifically to regions complementary to the template under annealing conditions. At this time, the distance between the fluorescent material and the quencher material is far away and the suppression by the quencher material is eliminated. In addition, Molecular Beacon probe, which did not hybridize, has a secondary structure and is suppressed by the quencher material and does not exhibit fluorescence.

 The conventional apparatus for real-time PCR is a thermoelectric element 1c, a block 1 for transferring heat to the reaction tube (2a) containing the sample and the inside of the reaction tube as shown in FIG. It consists of an irradiation light source 11 for irradiating light to a sample and a light receiving portion 78 for receiving fluorescence emitted from the sample (US Patent No. 6,818,437). The principle of operation of the above technique is to operate the irradiation light source 11 and the light receiving unit 78 at the end of each cycle while repeatedly performing the cooling or heating cycle using the thermoelectric element 1c to react the nucleic acid sample solution in the tube. The amount of fluorescence emitted from the sample is measured to display the degree of reaction in real time. The irradiation light source 11 is a white light source, and uses a band pass filter 7 to generate excitation light having a wavelength that matches the fluorescent probe used. The dichroic beam splitter 6 is a device for separating excitation light and fluorescence. In FIG. 1, a dichroic beam splitter 6 reflects a wavelength lower than a specific wavelength and passes a high wavelength. Another band pass filter 8 is a filter for selectively transmitting only the fluorescence from the sample to the light receiving element 78. In addition, Fresnel lens 3 was used to make the excitation light parallel.

 Recently, real-time PCR experiments using multiple colored fluorescent probes at the same time have been introduced. However, when the wavelength of the fluorescent probe used in the prior art is different, it is necessary to use different dichroic beam splitters 6 according to the fluorescent probes. Therefore, in the prior art, the band pass filters 7 and 8 and the dichroic beam splitter 6 are configured in a module form to simultaneously acquire data while simultaneously changing to match the fluorescent probe. In this case, for example, when five kinds of band pass filters are used, five kinds of dichroic beam splitters must be used for the band pass filters.

In addition, the dichroic beam splitter used in the prior art has a problem in that the excitation light is about 105 times brighter than the fluorescence emitted from the sample, so that the excitation light and the fluorescence cannot be completely separated. Reflection light of the excitation light by the optical component is incident on the light-receiving unit, causing interference with the fluorescence generated in the sample.

Factors that reflect the excitation light include: 1) Fresnel lens (3) used to make the excitation light parallel, and 2) lid (2b) used to prevent evaporation of the sample in the reaction tube (2a). Or transparent tape and 3) elements that occur in the reaction tube 2a itself.

The present invention has been proposed to solve the above problems, by using the polarizing characteristics of light (polarizing) to have the excitation light and the fluorescence in different directions to separate the excitation light and fluorescence in the prior art It aims to effectively separate excitation light and fluorescence irrespective of wavelength and remove incident light reflected from the excitation light to the light receiving unit without using a dichroic beam splitter used as a tool.

In order to achieve the above object, a real-time monitoring apparatus for a nucleic acid amplification reaction product according to the present invention includes a polarization converter (102) for polarizing excitation light excited from a light source (100); A condenser lens 103 for condensing the polarized light converted from the polarization converter 102; An optical waveguide 104 which converts and transmits the polarized light collected by the condenser lens 103 into a uniform surface light source; A first band pass filter 105 for passing only the polarized light of a specific wavelength according to the excitation characteristic of the fluorescent probe through the polarization of the surface light source transmitted from the optical waveguide 104; A polarization beam splitter (108) for separating the polarized light having a specific wavelength passing through the first band pass filter (105) according to polarization characteristics; A surface mirror 109 which transmits the polarized excitation light separated from the polarization beam splitter 108 in a specific direction to the sample and reflects the fluorescence generated from the sample to the polarization beam splitter 108; The fluorescence reflected by the surface mirror 109 is transmitted to the polarization beam splitter 108, and only the polarization component in a direction different from the excitation light is reflected and transmitted to the light receiving unit 113, and by the polarization converter 102. A first polarizer 111 for polarizing to have a component such as converted polarized light; A light receiving unit 113 for receiving fluorescence generated from a sample; And a control unit.

 In addition, the present invention includes a second polarizer 107 for polarizing so as to have a component opposite to the polarizer 102 and the first polarizer 111 in order to prevent the polarized excitation light is reflected to the light receiving unit; And further comprising:

 In addition, the present invention further includes a second band pass filter 112 for passing fluorescence generated by the polarized excitation light passing through the first polarizer 111 according to the fluorescence characteristics of the fluorescent probe. It is characterized by comprising.

 In addition, the present invention is provided between the light source 100 and the polarization converter 102, the ultraviolet and infrared cut filter 101 to block ultraviolet rays and infrared rays; And further comprising:

 In addition, the present invention is characterized in that the sample container is further provided with the sample is accommodated.

The sample vessel is a tube or well plate having 1 to 1536 wells, a Petri dish (bacterial dish), a slide, a filter, and a Terasaki plate. It is preferable that it is any one selected from a PCR plate (PCRplate).

As described above, the real-time monitoring device of the nucleic acid amplification reaction product according to the present invention is an optical component (for example, Fresnel lens, glass heater, etc.) in which the excitation light is located on the optical path in the conventional real-time PCR device. ) And the cap or transparent tape used to prevent evaporation of the sample and reflected by the reaction tube, etc. is incident to the light-receiving unit and thus cannot collect only the fluorescence generated in the sample. In case of processing a large amount of sample, the capacity of the sample is reduced, and the amount of fluorescence generated in the sample itself is further reduced.

By improving the problem of the interference by the excitation light, the present invention has an advantage that the excitation light and the fluorescence have different polarities by using the polarization characteristic of the light so that the interference of the excitation light cannot affect the fluorescence. In addition, the present invention uses only one polarizing beam splitter to separate fluorescence and excitation light regardless of the wavelength and number of band pass filters without using a dichroic beam splitter as in the prior art. Since it can receive fluorescence, there is no need to install a plurality of dichroic beam splitters, and there is no need for replacement work, and there is no need for a mechanism for setting the dichroic beam splitter for each wavelength.

1 is a view showing a real-time monitoring device of the nucleic acid amplification reaction products of the prior art.
Figure 2 is a perspective view showing a real-time monitoring device of the nucleic acid amplification reaction product of the present invention.
3 shows polarization characteristics of light;
4 is a view illustrating polarization characteristics of a polarizing beam splitter.
Figure 5 is a photograph of the reaction tube (plate) in the light receiving unit when using a non-polarization optical system.
Figure 6 is a photograph of the reaction tube (plate) in the light receiving unit when using the optical system of the present invention.

With reference to the accompanying drawings, a preferred embodiment of a real-time monitoring device of the nucleic acid amplification reaction product according to the present invention configured as described above will be described in detail.

The present invention provides an apparatus for effectively separating the excitation light and the fluorescence emitted from the sample using the polarization property of light.

2 is a perspective view showing a real-time monitoring device of the nucleic acid amplification reaction product of the present invention, Figure 3 is a view showing the polarization characteristics of light, Figure 4 is a view showing the polarization characteristics of a polarizing beam splitter (Polarizing Beam Splitter), 5 is a photograph of a reaction tube (plate) in the light receiving unit when using a non-polarization optical system, Figure 6 is a photograph of a reaction tube (plate) in the light receiving unit when using the optical system of the present invention.

As shown, the real-time monitoring device of the nucleic acid amplification reaction product according to the present invention comprises a polarization converter 102 for polarizing the excitation light excited from the light source 100; A condenser lens 103 for condensing the polarized light converted from the polarization converter 102; An optical waveguide 104 for making a uniform surface light source of polarized light collected by the condenser lens 103; A first band pass filter (105) for passing the polarized light transmitted from the optical waveguide (104); A polarization beam splitter (108) for separating the polarization passing through the first band pass filter (105); A surface mirror 109 which transmits polarized light in a specific direction separated from the polarization beam splitter 108 to a sample and reflects fluorescence generated from the sample; A second polarizer 107 that polarizes the fluorescence reflected by the surface mirror 109; a material that passes only fluorescence of a specific wavelength in accordance with the light emission characteristics of the fluorescent probe among the polarized fluorescence transmitted from the second polarizer 107; Two band pass filters 112; A light receiving unit 113 for receiving fluorescence passing through the second band pass filter 112; It consists of

Monitoring of real-time PCR according to the present invention is a fluorescence detection using a fluorescent reagent disclosed in the Patent Application Publication No. 10- <42> 2006-0009246 (Applicant's name: real-time monitoring device of the biochemical reaction) Use

 The light source 100 may be a white light source such as a tungsten halogen lamp, a xenon discharge lamp, and a monochromatic light source such as a laser that are generally used as a light source. have.

Light emitted from the light source 100 generating the excitation light has a property of parallel light and has a non-polarization characteristic. This light has both S and P wave components as shown in FIG. That is, it is composed of S waves and P waves perpendicular to each other in the advancing direction.

In addition, the light emitted from the light source 100 has not only a visible light region but also components of normal ultraviolet rays and infrared rays. Therefore, in order to remove components of the ultraviolet and infrared rays, which are not necessary in the optical system, it is preferable to provide the ultraviolet and infrared cut filter 101 to remove the components of the ultraviolet and infrared rays.

 The polarizing converting system 102 converts the excitation light to have the same polarity in a specific direction. That is, it serves to convert the incident non-polarization beam to have one polarity. The reason for using the polarization converter 102 is that the light is composed of S wave and P wave, but if one polarity is removed to obtain only one polarity, the total amount of light is reduced to 50% or less. This is because there is an advantage that can be converted to a polarization component to increase the use efficiency of light.

The condenser lens 103 collects the polarized light converted from the polarization converter 102 to inject the converted polarized light into a small lighttunnel 104.

The optical waveguide 104 is used as a means for producing a uniform surface light source as disclosed in International Patent Publication No. WO 2004/088291.

The first band pass filter 105 serves to pass only the polarized excitation light having a specific wavelength according to the excitation characteristic of the fluorescent probe through the polarized excitation light transmitted from the optical waveguide 104.

According to the present invention, when using various types of fluorescent probes, different types of bandpass filters should be used according to respective fluorescent probes, and accordingly, different types of dichroic beam splitters (reference numeral 6 of FIG. 1). The problem that must be used (if there are 5 sets of band pass filters, 5 dichroic beam splitters are also required) can be used to separate fluorescence and excitation light without using a dichroic beam splitter.

The polarization beam splitter 108 is an optical component having a characteristic of separating light having one polarization component and light having the remaining polarization component to each other by 90 degrees when unpolarized light is incident.

The polarization beam splitter 108 separates excitation light having a specific wavelength passing through the first band pass filter 105 into respective polarization components.

The polarized light separated by the polarization beam splitter 108 is transmitted to the surface mirror 109, and the remaining polarized light is separated in the opposite direction of the light receiver 113.

In the present invention, for the sake of explanation, it is assumed that the polarization converter 102 converts a non-polarization beam into an S wave. Of course, even when converted to P-wave, the same effect can be obtained by adjusting only the angles of the polarizer and the polarizing beam splitter according to the situation.

 The sample vessel is a tube or well plate having 1 to 1536 wells, a Petri dish (bacterial dish), a slide, a filter, and a Terasaki plate. It is preferable that it is any one selected from a PCR plate (PCRplate), and any sample container which can receive and measure a sample is possible.

In addition, it is preferable that an imaging lens 106 is provided between the first band pass filter 105 and the polarization beam splitter 108 to spread out as a uniform surface light source according to the size of the sample container in which the sample is accommodated. Do. The imaging lens 106 spreads the excitation light so that the intensity of the excitation light becomes uniform when the excitation light reaches the tube (plate) containing the sample to detect fluorescence.

The surface mirror 109 transmits the polarized light split from the polarization beam splitter 108 to the sample and reflects the fluorescence generated from the sample to the polarization beam splitter 108. The surface mirror 109 ) Only serves to convert the light path.

Fresnel lens 110 is used to make the excitation light reflected by the surface mirror 109 into parallel light to reach the tube containing the sample well. The excitation light incident in this way excites the fluorescent probe to emit fluorescence. The fluorescence generated from the sample is incident to the light receiving unit again through a Fresnel lens and a polarization beam splitter.

 The first polarizer 111 has a polarization characteristic when the excitation light polarized by the polarization converter 102 passes through an optical component (a condenser lens 103, an optical waveguide 104, an imaging lens 106, etc.). Since this may be disturbed, the polarization beam splitter 108 serves to remove the polarization components in the unnecessary direction as much as possible before the excitation light is incident on the polarization beam splitter 108. That is, as the excitation light of the polarized S-wave component passes through the optical path, it serves to remove a small amount of P-wave component that may be caused by the polarization is disturbed.

 The fluorescence generated from the sample also has non-polarization characteristics and thus has both S-wave and P-wave components. When the fluorescence of this characteristic is incident on the polarization beam splitter 108 as shown in FIG. 4, the P wave is reflected and enters the light receiving unit 113, and the S wave passes through the polarization beam splitter 108 and is incident toward the excitation light source. The second polarizer 107 is rotated by 90 degrees in the same kind or direction as the first polarizer 111, and this time, it is provided so that only P wave may pass. Accordingly, the second polarizer 107 serves to remove light of the S-wave component that may not be completely separated from the polarization beam splitter 108.

 Here, even though the excitation light is reflected on the optical path and incident toward the light receiving part 113, the excitation light has an S-wave characteristic and thus is incident again toward the excitation light source through the polarization beam splitter 108 and blocked by the second polarizer 107. The light receiving unit 113 may not be incident.

The fluorescence reflected by the surface mirror 109 is transmitted to the polarization beam splitter 108 and has a component opposite to the polarization converted by the first polarizer 111 and the polarization converter 102, that is, excitation light. It is possible to effectively separate the fluorescence by having different polarities and fluorescence so that the interference of excitation light does not affect the fluorescence.

As described above, the present invention has one polarization component for the excitation light and removes one polarization component from the light receiving unit collecting fluorescence from the sample, and passes only the remaining polarization component to the light receiving sensor, thereby effectively removing the excitation light. Will be.

The second band pass filter 112 serves to pass only the fluorescence of a specific wavelength in accordance with the light emission characteristics of the fluorescent probe of the polarized fluorescence transmitted from the second polarizer 107.

 The light receiving unit 113 serves to receive fluorescence that has passed through the second band pass filter 112, and is provided with a fluorescent probe (not shown) to collect fluorescence emitted from a sample. The light receiving lens 114 of the light receiving unit 113 serves to form an image fluorescence generated in the sample on the image sensor.

In addition, the present invention is a first polarizer 111 for polarizing the excitation light is not converted by the polarization converter 102; It is desirable to be further equipped. Since the polarized light may be broken while the polarized light passes through the optical component, the first polarizer 111 may be used before passing the polarized beam splitter 108 to increase the beam separation efficiency of the polarized beam splitter 108. -As a polarizer, it plays a role to make use of polarization characteristics.

 In addition, the present invention is provided between the light source 100 and the polarization converter 102, the ultraviolet and infrared cut filter 101 to block ultraviolet rays and infrared rays; And further comprising:

  The present invention utilizes the polarization characteristics of the light, the excitation light is used to prevent evaporation of a particular optical component or sample on the optical path (see reference numeral 2b in FIG. 1) or the transparent tape and reaction tube (reference 2a in FIG. 1). In order to prevent the incident light from being reflected to the light-receiving part and the like, the excitation light and the fluorescence have different polarization characteristics to minimize interference of the fluorescence by the excitation light. Since the reflected polarized light still has the same polarization property, it is possible to efficiently separate the excitation light and the fluorescence in the present invention.

(Example)

5 is a photograph of a reaction tube (plate) in a light receiving unit in an optical system using an unpolarized excitation light source and non-polarized fluorescence. In this picture, the left and right sides of the center are wells containing fluorescent probes, and the reflected light is visible through several wells in the center.

In the case of Figure 6 is an image of the excitation light source and the polarized fluorescence polarized as in the present invention photographed from the light receiving unit. As shown in the figure, the reflected light at the bottom center disappeared completely.

100: light source 101: UV and infrared cut filter
102: Polarizing converting system
103: condenser lens
104: light tunnel
105: first band pass filter
106: imaging lens 107: second polarizer
108: Polarizing Beam Splitter
109: Surface Mirror
110: Fresnel lens
111: first polarizer
112: second band pass filter 113: light receiving unit
114: light receiving lens

Claims (1)

In the device for detecting a nucleic acid amplification reaction product,
Condensing lens 103 for collecting the excitation light generated from the light source 100
An optical waveguide 104 converting the excitation light collected by the condenser lens 103 into a uniform surface light source and transmitting the converted light;
A first band pass filter (105) for passing the polarization of the surface light source transmitted from the optical waveguide (104) to pass the polarization of a specific wavelength in accordance with the excitation characteristic of the fluorescent probe;
A first polarizer 111 for polarizing the excitation light having a specific wavelength passing through the first band pass filter 105;
A polarization beam splitter (108) for separating polarization of a specific wavelength passing through the first polarizer (111);
A surface mirror 109 which transmits the polarized light separated from the polarization beam splitter 108 to the sample and reflects the fluorescence generated from the sample to the polarization beam splitter 108;
A second polarizer 107 which fluorescence reflected by the surface mirror 109 is transmitted to the polarization beam splitter 108 to polarize the fluorescence so as to have a component opposite to the polarization converted by the first polarizer 111;
A second band pass filter (112) for passing only fluorescence of a specific wavelength in accordance with light emission characteristics of the fluorescent probe among the polarized fluorescence transmitted from the second polarizer (107);
And a light receiving unit (113) for receiving fluorescence that has passed through the second band pass filter (112).
KR1020110122005A 2011-11-22 2011-11-22 Device for detecting nucleic acid amplification reaction product KR20130056408A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220015018A (en) * 2020-07-30 2022-02-08 한림대학교 산학협력단 Apparatus for detecting PCR fluorescence with fluorescence concentrating and apparatus, method and program for concentrating fluorescence

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220015018A (en) * 2020-07-30 2022-02-08 한림대학교 산학협력단 Apparatus for detecting PCR fluorescence with fluorescence concentrating and apparatus, method and program for concentrating fluorescence

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