WO2024186194A1 - Gene amplification inspection device and inspection method using same - Google Patents

Abstract

The present invention relates to a gene amplification inspection device and an inspection method using same, and comprises: a sample container in which a PCR sample including a nanostructure is stored; a first light source for irradiating the sample container with near-infrared rays; a second light source for irradiating the sample container with visible rays; and an optical detector for measuring a fluorescence signal from the sample container, wherein the near-infrared rays heat the nanostructure and the PCR sample, and the visible rays generate a fluorescence signal in the nanostructure and the PCR sample.

Description

Gene amplification test device and test method using the same

The present invention relates to a gene amplification testing device and a testing method using the same, and more specifically, to a gene amplification testing device capable of testing for diseases, etc. by amplifying genes from a small amount of sample, and a testing method using the same.

Infectious diseases are a serious threat, accounting for 25% of global mortality. In addition, the continued overuse of antibiotics has led to the emergence and spread of multidrug-resistant bacteria, which is expected to cause up to 10 million deaths annually in the coming decades.

Therefore, in resource-limited settings, simple, inexpensive tests that can rapidly identify bacterial pathogens and resistance to them could provide significant benefits in preventing the spread of infections and enabling prompt, targeted treatment.

However, the standard method for detecting bacteria in clinical settings is based on microbial culture, which is time-consuming and takes at least several days to several weeks to identify the species and resistance type of bacteria. In addition, molecular detection methods based on RT-PCR (real-time polymerase chain reaction) are highly sensitive and can shorten the analysis time to within a few hours, but they require expensive equipment including thermal cycling and real-time fluorescence detection modules, which limits their application to field diagnosis.

Real-time PCR is a device used to detect trace amounts of specific proteins and genes. In order to amplify the genes contained in the measurement sample, the temperature of the sample is repeatedly raised and lowered between preset low (~60 degrees Celsius) and high (~90 degrees Celsius) points.

A single round trip between low and high temperature points is defined as a temperature cycle, and during this process, a specific gene is exponentially amplified through processes such as denaturation of the genetic strand, annealing with a primer, and extension by a polymerase.

Real-time gene amplification devices can measure the process of amplifying specific genes in real time using templates and markers, so they do not require electrophoresis required in conventional PCR, allowing for quick and easy interpretation of results, and have the advantage of allowing accurate quantification of DNA and RNA.

Existing real-time gene amplification devices use a thermoelectric heating block to control the temperature of the sample, but they have the disadvantages of limited temperature change speed, high power consumption, and large volume. To supplement this, plasmonic gene amplification devices that increase the temperature of the sample by utilizing the surface plasmon resonance effect of metal nanoparticles have been proposed.

However, the device configuration according to this proposal has a limitation that it uses LED lighting in the visible range as a light source that induces the plasmonic resonance effect, and the LED lighting that provides a heating effect during fluorescence measurement must be blinked. This limits the ability to actively control the sample temperature, and as a result, the gene amplification efficiency is lower than that of existing real-time gene amplification devices.

The present invention provides a genetic amplification test device capable of promoting heating and generation of a fluorescent signal by including a nanostructure in a PCR sample and enabling simple and convenient precise diagnosis by direct measurement of the fluorescent signal, and a test method using the same.

According to one embodiment of the present invention, the present invention comprises: a sample container storing a PCR sample including a nanostructure; a first light source irradiating near-infrared light to the sample container; a second light source irradiating visible light to the sample container; and an optical detector measuring a fluorescence signal from the sample container, wherein the near-infrared light heats the nanostructure and the PCR sample, and the visible light generates a fluorescence signal in the nanostructure and the PCR sample.

In addition, the invention further includes a control unit that controls the output of the first light source so as to maintain the temperature of the PCR sample at a set temperature value.

In addition, it further includes a temperature sensing member that measures the temperature of the PCR sample and transmits the measured value to the control unit.

Additionally, the temperature sensing member includes a thermocouple.

Additionally, the control unit includes a PID controller that generates a control signal by performing PID feedback through the measurement value transmitted by the temperature sensing member.

Additionally, the optical detector includes a filter that blocks near-infrared rays and transmits only wavelengths corresponding to the fluorescence signal.

According to another embodiment of the present invention, the present invention comprises a step of irradiating near-infrared light to a sample container in which a PCR sample including a nanostructure is stored by a first light source; a step of irradiating visible light to the sample container by a second light source; and a step of measuring a fluorescence signal from the sample container, wherein the nanostructure and the PCR sample are heated by the near-infrared light and generate a fluorescence signal by the visible light.

In addition, prior to the step of irradiating the visible light, the method further includes a step of maintaining the temperature of the sample container at isotherm.

Additionally, the step of maintaining the temperature isothermally measures the temperature of the sample container, and the control unit performs PID feedback based on the temperature of the sample container to control the output of the first light source.

Additionally, in the step of measuring the fluorescence signal, the fluorescence signal is measured using an optical detector.

Additionally, the optical detector includes a filter that blocks near-infrared rays and transmits only wavelengths corresponding to the fluorescence signal.

Specific details of other embodiments are included in the detailed description and drawings.

The genetic amplification test device according to the present invention and the test method using the same have the following effects.

First, it has the advantage of including nanostructures as well as PCR samples, and the nanostructures promote heating of the PCR sample and emission of fluorescent signals, allowing for rapid cycle testing.

Second, it has the advantage of being able to miniaturize the device by eliminating the heating block previously used to heat PCR samples and instead using a light source to heat PCR samples.

FIG. 1 is a block diagram illustrating the configuration of a genetic amplification test device according to one embodiment of the present invention.

FIG. 2 is a graph showing the state in which the temperature is maintained when the fluorescence signal of a PCR sample is excited in the genetic amplification test device according to FIG. 1.

FIG. 3 is a flowchart illustrating a testing method using a genetic amplification testing device according to one embodiment of the present invention.

Hereinafter, with reference to the attached drawings, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. The present invention may be implemented in various different forms and is not limited to the embodiments described herein.

It is noted that the drawings are schematic and not drawn to scale. The relative dimensions and proportions of parts in the drawings are exaggerated or reduced in size for clarity and convenience in the drawings, and any dimensions are for illustration only and not limiting. In addition, the same structural elements or parts appearing in more than one drawing are denoted by the same reference numerals to indicate similar features.

The embodiments of the present invention specifically illustrate ideal embodiments of the present invention. As a result, various modifications of the diagrams are expected. Accordingly, the embodiments are not limited to the specific form of the illustrated area, and also include modifications of the form by manufacturing, for example.

Hereinafter, a genetic amplification test device according to the present invention will be specifically described with reference to FIGS. 1 and 2.

A genetic amplification test device according to one embodiment of the present invention includes a sample container (110), a first light source (120), a second light source (130), an optical detector (140), and a control unit (150).

The above sample container (110) stores a PCR sample (111) for genetic amplification testing. The PCR sample (111) includes a nanostructure (112).

The first light source (120) is provided to irradiate light to the sample container (110). Specifically, the first light source (120) irradiates near-infrared rays to the sample container (110). The nanostructure (112) stored in the sample container (110) is heated by the near-infrared rays irradiated by the first light source (120).

The above nanostructure (112) has a resonance wavelength in the near-infrared. Therefore, the nanostructure (112) is heated by causing a plasmonic resonance effect by the near-infrared ray irradiated by the first light source (120), thereby promoting heating and temperature increase of the PCR sample (111).

In this embodiment, the resonance wavelength of the nanostructure (112) that responds to near-infrared light is 750 nm to 850 nm. The first light source (120) has a wavelength of 785 nm to 808 nm and includes one of a laser and an LED.

The second light source (130) is provided to irradiate light to the sample container (110) like the first light source (120). The second light source (130) irradiates visible light to the sample container (110).

The above PCR sample (111) generates a fluorescence signal by a visible light source irradiated from the second light source (130). Specifically, the PCR sample (111) absorbs visible light and re-emits it, generating a fluorescence signal.

In this embodiment, the second light source (130) is a laser having a wavelength of 480 nm to 490 nm, but is not limited thereto.

The optical detector (140) measures a fluorescence signal from the sample container (110). As described above, when the PCR sample (111) generates a fluorescence signal by visible light irradiated from the second light source (130), the optical detector (140) measures it.

The above optical detector (140) detects a fluorescence signal by measuring the intensity of light emitted from the PCR sample (111).

The optical detector (140) includes a filter (141). The filter (141) blocks near-infrared light irradiated to the sample container (110) and transmits only the wavelength of light emitted from the PCR sample (111), that is, the wavelength corresponding to the fluorescence signal, so that the fluorescence signal can be measured by measuring the intensity of the light.

The above control unit (150) controls the output of the first light source (120) and the second light source (130). As described above, the PCR sample (111) and the nanostructure (112) of the sample container (110) are heated by the near-infrared rays irradiated from the first light source (120) and when the temperature rises, the temperature must be maintained at a set temperature value.

That is, since it is difficult to maintain the temperature of the sample container (110) constant if the first light source (120) continuously outputs near-infrared rays, the output of the first light source (120) is adjusted. Meanwhile, the output control of the first light source (120) is performed based on the temperature of the sample container (110), which will be described in more detail later.

The output of the second light source (130) is also controlled by the control unit (150) like the first light source (120). The optical detector (150) measures the fluorescence signal from the sample container (110), and the measurement of the fluorescence signal is performed while the sample container (110) maintains a set temperature value. Therefore, the control unit (150) controls the output of the second light source (130) so that the second light source (130) irradiates or does not irradiate visible light to the sample container (110).

The above-described genetic amplification test device further includes a temperature sensing member (160). The temperature sensing member (160) measures the temperature of the sample container (110). Specifically, the temperature sensing member (160) measures the temperature of the PCR sample (111) in the sample container (110). As described above, the control unit (150) controls the output of the first light source (120) based on the temperature of the PCR sample (111), so the temperature of the PCR sample (111) is measured by the temperature sensing member (160).

The above control unit (150) generates a control signal (output value) by PID feedback based on the temperature measurement value of the PCR sample (111) obtained by the temperature sensing member (160) and controls the output of the first light source (120).

The above control unit (150) includes a PID controller (151). The temperature sensing member (160) transmits the temperature measurement value measured in the sample container (110) to the PID controller (151).

The above PID controller (151) calculates the error between the input temperature measurement value and the set temperature value, and performs PID control to generate an output value to be output by the first light source (120). The control unit (150) transmits this output value to the first light source (120), and the first light source (120) controls the output by the control unit (150).

The above temperature sensing member (160) includes a thermocouple, and in the present embodiment, the thermocouple is provided as the temperature sensing member (160).

FIG. 2 is a graph showing a cycle of increasing and decreasing temperature measured in the sample container (110) in the genetic amplification test device (100) according to one embodiment of the present invention.

Referring to Fig. 2, it can be confirmed that the temperature of the sample container (110) is maintained for a certain period of time when the temperature is 60° and 95°. This is because the temperature is maintained for a set period of time at a set temperature value by controlling the output of the first light source (120) by the temperature sensing member (160), the PID controller (151), and the control unit (150) as described above.

The above optical detector (140) measures the fluorescence signal of the sample container (110) when the sample container (110) maintains a set temperature value.

In this embodiment, since amplification occurs when the temperature of the sample container (110) is 60°, the fluorescence signal is measured while maintaining the temperature at 60°. The annealing temperature of the primer used for producing the PCR sample is set to 5° to 10° below the melting temperature. In addition, each polymerase has an appropriate amplification temperature.

In the case of a PCR sample in which a cycle is performed in a two-step manner as in one embodiment of the present invention, heat treatment and amplification are performed in one step, and the appropriate temperature range is presented differently depending on the specifications of each PCR sample. In this embodiment, since heat treatment and amplification are performed in the appropriate temperature range of 60° to 65°, the fluorescence signal is measured while maintaining 60° as described above.

And whether amplification actually occurs can be confirmed by measuring the fluorescence signal and seeing that the fluorescence signal increases exponentially after a certain number of cycles.

Below, a test method using a genetic amplification test device according to the present invention will be described with reference to FIG. 3.

First, a first light source (120) irradiates near-infrared rays to a sample container (110) in which a PCR sample (111) including a nanostructure (112) is stored. (Step S110) The nanostructure (112) and the PCR sample (111) stored in the sample container (110) are heated by the near-infrared rays. In particular, the nanostructure (112) promotes heating and amplification of the PCR sample (111), thereby improving the amplification efficiency of the PCR sample (111).

Next, a second light source (130) irradiates visible light to the sample container (110). (Step S130) The PCR sample (111) and the nanostructure (112) stored in the sample container (110) generate a fluorescence signal by the visible light irradiated by the second light source (130).

The above PCR sample (111) generates a fluorescence signal by the visible light, and the nanostructure (112) promotes the amplification of the fluorescence signal of the PCR sample (111), thereby improving efficiency.

When the PCR sample (111) and the nanostructure (112) are heated by the first light source (120) in the above step S110, the output of the first light source (120) is controlled so that the temperature of the PCR sample (111) is maintained at a set temperature value. (Step S120)

In the above step S120, the temperature sensing member (160) measures the temperature of the PCR sample (111). The temperature sensing member (160) measures the temperature of the PCR sample (111) in a non-contact manner. The temperature sensing member (160) measures the temperature of the sample container (110) 10 to 30 times per second in a non-contact manner.

The temperature sensing member (160) equipped with a thermocouple measures the temperature of the PCR sample (111) and transmits the measured value to the PID controller (151). The PID controller (151) generates a control signal through PID control, and the control unit (150) controls the output of the first light source (120) according to the output value generated by the PID controller (151) to maintain the temperature of the PCR sample (111) at a set temperature value.

Step S120 of maintaining the temperature of the above PCR sample (11) at a set temperature value is performed before step S130 of irradiating visible light.

When visible light is irradiated to the above sample container (110), the fluorescence signal of the PCR sample (111) is measured. (Step S140)

The fluorescence signal of the above PCR sample (111) is measured through an optical detector (140). Meanwhile, the sample container (110) is simultaneously irradiated with near-infrared light from the first light source (120) and visible light from the second light source (130).

The optical detector (140) includes a filter (141). The filter (141) blocks near-infrared rays irradiated from the first light source (120) and passes only wavelengths corresponding to fluorescence signals generated by visible light, so that the optical detector (140) measures the fluorescence signals.

And the examination of the PCR sample is completed through the measured fluorescence signal.

The gene amplification test device according to the present invention and the gene amplification test method using the same have the advantage of being able to operate at a faster cycle than conventional gene amplification test devices. Specifically, the nanostructure stored in the sample container can shorten the time required for gene amplification of a PCR sample, that is, the time required for gene amplification of a PCR sample.

In particular, in conventional genetic amplification testing devices, a thermoelectric element type heating block was used to heat a PCR sample, but in the present invention, a light source is used to heat the PCR sample, so the genetic amplification testing device can be miniaturized and power consumption can be reduced.

Accordingly, it has the advantage of being able to quickly perform precise diagnosis on site.

Although the embodiments of the present invention have been described with reference to the attached drawings, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical idea or essential features thereof.

Therefore, the embodiments described above should be understood as illustrative and not restrictive in all respects, and the scope of the present invention is indicated by the claims described below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (11)

A sample container storing a PCR sample containing a nanostructure; A first light source for irradiating near-infrared rays onto the sample container; a second light source for irradiating visible light onto the sample container; and An optical detector for measuring a fluorescence signal from the sample container, A genetic amplification test device in which the near-infrared light heats the nanostructure and the PCR sample, and the visible light generates a fluorescent signal in the nanostructure and the PCR sample. In the first paragraph, A genetic amplification test device further comprising a control unit that controls the output of the first light source so as to maintain the temperature of the PCR sample at a set temperature value. In the second paragraph, A genetic amplification test device further comprising a temperature sensing member that measures the temperature of the PCR sample and transmits the measured value to the control unit. In the third paragraph, The above temperature sensing member is a genetic amplification test device including a thermocouple. In the third paragraph, The above control unit, A genetic amplification test device including a PID controller that generates a control signal by performing PID feedback through the measurement value transmitted by the temperature sensing member. In the first paragraph, The above optical detector, A genetic amplification test device including a filter that blocks the near-infrared rays and transmits only the wavelength corresponding to the fluorescence signal. A step of irradiating a sample container containing a PCR sample including a nanostructure with near-infrared light by a first light source; a step of a second light source irradiating visible light onto the sample container; and Comprising a step of measuring a fluorescence signal from the above sample container, A genetic amplification test method in which the nanostructure and the PCR sample are heated by the near-infrared light and generate a fluorescent signal by the visible light. In Article 7, Before the step of irradiating the above visible light, A genetic amplification test method further comprising a step of maintaining the temperature of the sample container at isotherm. In Article 8, The step of maintaining the above isotherm is as follows: A genetic amplification test method comprising: measuring the temperature of the sample container; and controlling the output of the first light source by a control unit performing PID feedback based on the temperature of the sample container. In Article 7, In the step of measuring the above fluorescence signal, A genetic amplification test method for measuring the above fluorescence signal using an optical detector. In Article 9, A genetic amplification test method wherein the optical detector includes a filter that blocks near-infrared rays and transmits only the wavelength corresponding to the fluorescence signal.

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