KR20230163236A - Nucleic Acid Amplification Test System - Google Patents

Abstract

In the nucleic acid amplification test system,
A power supply unit 100 that converts commercial AC power supplied from the outside into DC power and supplies it; a central processing unit 200 that controls heating and cooling of the reaction tube, and analyzes and stores optically measured data in the reaction tube; a temperature circulation unit 300 that heats and cools the reaction tube to amplify the genes of the material contained in the reaction tube; An optical inspection unit 400 that optically analyzes the material contained in the reaction tube; An optical drive unit 500 that drives the optical inspection unit 400 by operation control of the central operation processing unit 200; And an interface unit 600 that inputs operation commands for the test system and outputs test results. A nucleic acid amplification test system comprising a.

Description

Nucleic Acid Amplification Test System}

The present invention amplifies and tests DNA in large quantities in a reaction tube.

This relates to a nucleic acid amplification testing system.

PCR stands for Polymerase Chain Reaction and is a technology that amplifies the amount of DNA using DNA polymerase.

According to PCR, even if there is a very small amount of DNA, we can greatly increase the amount of DNA we want with enormous quantity and high accuracy. Additionally, the amplification time is very short, making it the most important basic technology in modern biology.

PCR mixes the template DNA to be amplified, a primer that binds to a specific position to be amplified, DNA polymerase, and dNTP (Deoxynucleotide triphosphate), a material for making new DNA, 94 Heat is applied to ~96℃ to separate one DNA strand into two strands. Afterwards, when the temperature is cooled to about 50-64°C, the primer and DNA polymerase bind to a specific site on the single-stranded DNA, and are then synthesized to form two DNAs. If this process is repeated, the desired amount of DNA increases through an explosive chain reaction, such as 4, 8, or 16.

Meanwhile, according to the high-speed nucleic acid amplification device (domestic registration patent 10-2081480), which is a prior patent previously applied for and registered by the present applicant,

In the device used for high-speed nucleic acid amplification reaction,

A holder that fixes the position of the tube in which the reaction occurs;

a heating module including a heater that generates heat to heat the tube and a heating block connected to the heater;

a driving module that moves the heating module up and down to vary the distance between the heating module and the holder; And a cooling module that cools the tube fixed to the holder,

The cooling module is,

It includes a blowing fan that generates cooling wind, and a blowing nozzle that delivers the cooling wind generated by the blowing fan to a tube fixed to the holder,

There is a high-speed nucleic acid amplification device in which the blowing nozzle is closed when the heating module is adjacent to the holder and is opened when the heating module is spaced away from the holder.

Republic of Korea Patent Publication No. 10-2081480 (announced on April 24, 2020)

As disclosed in <Figure 1>, the existing high-speed nucleic acid amplification device is

The heating block 220 is coupled to the module base and has a mounting wall so that the light trapping part and the emission filter 330 are each mounted, and the mounting wall corresponds to the position of the light transmitting part 224 of the heating block 220. Thus, a through hole communicating with the light transmitting unit 224 is formed to allow light to pass through.

And the light capture unit is composed of a member in which a predetermined sensor unit 324 is mounted on a predetermined PCB 322, and the sensor unit 324 has a number corresponding to the number of light transmitting units 224 and the arrangement of the tubes, and It has an array.

That is, a plurality of light transmitting units 224 are formed according to the number of the plurality of depressions 222 of the heating block 220, and a plurality of light capturing units are provided corresponding to each light transmitting unit 224.

However, the existing nucleic acid amplification device using a fixed single-wavelength optical unit requires a cumbersome calibration setting process to maintain the same performance of a plurality of optical units, and all reaction tubes must be set to one standard.

In addition, since only a single wavelength can be applied to a fixed optical unit due to its structure, there are limitations to multiplex PCR testing and the number of detectable wavelengths is also limited.

In order to solve the above problems, this patented invention proposes an inspection system that is equipped with a scanning type and a reflective optical module, and can measure a plurality of optical units, each with different detection wavelengths, while moving to the position of the reaction tube.

In the nucleic acid amplification test system according to the present invention, an optical system that detects a plurality of different wavelengths moves linearly back and forth to the position of the reaction tube, and the same optical system for each wavelength measures the fluorescent substance of the sample in the reaction tube, thereby providing the same optical system performance. This is guaranteed, and by adopting a simple combination structure that allows linear movement of multiple optical systems, it is very advantageous in various aspects of production, use, and maintenance.

Figure 1 is an example of an existing inspection device.
Figure 2 shows the main configuration of the nucleic acid amplification testing system according to the present invention.
Figure 3 is a temperature circulation unit according to the present invention.
Figure 4 shows an optical inspection unit according to the present invention.
Figure 5 is a diagram showing the appearance of the nucleic acid amplification testing system according to the present invention.
Figure 6 is a diagram showing the operation of the optical inspection unit according to the present invention.
7(a) to 7(d) are examples of optical measurement according to the present invention.
Figure 8 is an exploded view of the optical module according to the present invention.
Figure 9 is a configuration diagram of the first body of the optical module according to the present invention.
Figure 10 is a coupling relationship diagram of two optical modules according to the present invention.
Figure 11 is an analysis algorithm of the central operation processing unit according to the present invention.
Figure 12 is a data correction diagram according to the present invention.
Figure 13 is a diagram showing the amplification start point determination and Ct value calculation according to the present invention.
Figure 14 is a diagram of baseline adjustment and graph output according to the present invention.
Figure 15 is a quantitative value calculation algorithm according to the present invention.

Figure 2 is a block diagram showing the main configuration of the nucleic acid amplification testing system according to the present patented invention.

As disclosed in Figure 2, the present patented invention,

In the nucleic acid amplification test system,

A power supply unit 100 that receives commercial alternating current power from the outside, converts it into direct current power, and supplies it;

a central processing unit 200 that controls heating and cooling of the reaction tube, and analyzes and stores data measured in the reaction tube;

A temperature circulation unit 300 that amplifies the genetic sample in the reaction tube by heating and cooling the reaction tube;

An optical inspection unit 400 that optically analyzes the reaction solution in the reaction tube;

An optical drive unit 500 that drives the optical inspection unit 400 by operation control of the central operation processing unit 200;

It includes an interface unit 600 that inputs operation commands for the inspection system and outputs inspection results.

In the reaction tube, in addition to the sample to be analyzed, a high-concentration control sample, a low-concentration control sample, and an internal control material whose quantitative value is already known are added to the reaction solution.

When commercial AC power is supplied to this system, the power supply unit 100 converts it into direct current power and supplies it to the central arithmetic processing unit 200, and the central arithmetic processing unit 200 delivers direct current power to each connected component device.

As shown in the drawing described later, after inserting a reaction tube containing a mixed solution of the analysis target and control sample into the nucleic acid amplification test system of the present patent, an operation control command is input through the interface unit 600, and the nucleic acid amplification test system is input. The inspection process is carried out by amplification and optical measurement.

The process of nucleic acid amplification proceeds in three stages, with DNA denaturing, annealing, and DNA synthesis steps maintained at a specific temperature for a certain period of time.

During the nucleic acid amplification process, the central processing unit 200 drives the temperature circulation unit 300 to heat and cool the reaction tube to a specific temperature.

The temperature circulation unit 300 includes a peltier 310, a thermistor 320, and a fan 330, as shown in FIG. 3.

In the DNA denaturing step, the Peltier 310 in the temperature circulation unit 300 heats the reaction tube.

The central processing unit 200 detects the temperature of the heated reaction tube through the thermistor 320.

By compensating for the difference between the temperature sensed by the thermistor 320 and following the control input and applying a driving current to the Peltier 310, the temperature of the Peltier 310 is kept constant.

In the DNA denaturing step, the reaction tube is heated to 90-95°C.

In the annealing step, the fan 330 in the temperature circulation unit 300 is driven to cool the heated reaction tube to lower the temperature to 50-60°C.

Additionally, in the DNA synthesis step, the temperature of the reaction tube is maintained at 60-70°C, and the amount of fluorescence expression of the gene sample in the reaction tube amplified as a result of DNA synthesis is measured using the optical inspection unit 400. .

The optical inspection unit 400 irradiates light from the LED light source 420 to the reaction tube through the optical module 410, and the light expressed by fluorescence by the fluorescent material in the reaction tube again passes through the optical module 410 to the detection sensor. Receive light at (430).

The amount of light detected by the detection sensor 430, such as a CCD or photodiode, is proportional to the amount of fluorescent material in the reaction tube, that is, the amount of synthesized DNA.

The detection sensor 430 outputs an electrical signal proportional to the intensity of the detected light, and is transmitted to the central processing unit 200.

The central processing unit 200 records and stores the received data for each reaction tube.

Preferably, the above process of nucleic acid amplification and optical measurement of fluorescent material is repeated several times, and the number of repetitions may vary depending on the type of amplification target nucleic acid.

The central processing unit 200 analyzes and processes data recorded and stored as the result of optical measurement of fluorescent substances using analysis algorithm software, outputs and displays the analysis results on the interface unit 600, and performs operation control of the system by user input. Runs the software (GUI) that

Now let's look at an embodiment of the nucleic acid amplification test system according to the present invention disclosed in Figure 5.

The temperature circulation unit 300 is equipped with a Peltier 310 for heating and a fan 330 for cooling, and the reaction tube alternately blows wind generated from the Peltier 310 and the fan 330 to maintain high temperature. Contact induces a temperature change in the reaction tube.

The optimal temperature change range according to the nucleic acid to be amplified is adjusted by varying the contact time.

Accordingly, the temperature of the sample is driven to rapidly repeat a specific section within the range of 50 to 100°C to amplify the target nucleic acid.

As shown in FIG. 6, under the operational control of the central operation processing unit 200, the optical drive unit 500 moves the optical inspection unit 400 in a straight line back and forth to a position corresponding to the reaction tube in the direction of the arrow, and DNA is measured optically.

The optical inspection unit 400 according to the present invention is provided with a plurality of optical modules 410 of different wavelength bands, and according to the embodiment disclosed in FIGS. 6 and 7, there are four optical modules 410 (1) of different wavelength bands. ~(4) is included in the optical inspection unit 400.

The optical inspection unit 400 according to an embodiment of the present invention includes optical modules 410 (1) to (4) that detect four different wavelengths, and each optical module moves to the position of a plurality of reaction tubes. It operates by using the same optical system for each wavelength to measure the fluorescent material of the test sample in the reaction tube, and the optical inspection unit 400, which is a combination of a plurality of optical modules, can move back and forth in a straight line along the position of the reaction tube. Adopting a structure is advantageous in terms of production, use and maintenance.

In the reaction tube according to the above embodiment, there is a reaction solution in which four things are mixed: a high-concentration control sample whose quantitative value is already known, a low-concentration control sample, an internal control material, and a sample to be analyzed.

7 (a) to 7 (d), four optical modules 410 (1) to (4) sequentially irradiate LED light source light in four different wavelength bands and emit light emitted from the mixed reaction solution. sense each.

In Figure 7 (a), the optical module 410 (1) irradiates LED optical light in the 450nm to 490nm wavelength band to reaction tube No. 1 as shown in the figure.

And the light in the 520nm to 540nm band emitted from the mixed reaction solution in reaction tube 1 is received by a detection sensor.

When light reception by the detection sensor is completed for reaction tube No. 1, the optical module 410 (1) moves to reaction tube No. 2 by the operation of the optical drive unit 500, and the 450 nm to 490 nm band for reaction tube No. 2 irradiates LED optical light.

And the light in the 520nm to 540nm band emitted from the mixed reaction solution in reaction tube No. 2 is received by a detection sensor.

As shown in the drawing, the optical module 410 (1) sequentially moves from reaction tube No. 1 to reaction tube No. 8 to conduct optical analysis of the sample in the reaction tube.

When the optical measurement by the optical module 410 (1) is completed, it returns to the original position as shown in Figure 7 (b), and sequentially moves from reaction tube 1 to reaction tube 8, and the optical module ( 410) Perform optical analysis of the sample in the reaction tube according to (2).

At this time, the optical module 410 (2) radiates LED optical light in the 515 nm to 535 nm wavelength band and receives light in the 560 nm to 590 nm band emitted from the fluorescent material of the sample in the reaction tube with a detection sensor.

When the optical measurement by the optical module 410 (2) is completed, the optical module 410 (3) moves sequentially from reaction tube 1 to reaction tube 8, as shown in FIG. 7 (c). Perform optical analysis of the sample in the reaction tube.

At this time, the optical module 410 (3) irradiates LED optical light in the 560nm to 590nm wavelength band and receives light in the 610nm to 650nm band emitted from the fluorescent material of the sample in the reaction tube with a detection sensor.

When the optical measurement by the optical module 410 (3) is completed, it sequentially moves from reaction tube 1 to reaction tube 8, as shown in FIG. 7 (d), to the optical module 410 (4). Perform optical analysis of the sample in the reaction tube.

At this time, the optical module 410 (4) radiates LED optical light in the 620nm to 650nm wavelength band and receives light in the 660nm to 690nm band emitted from the fluorescent material of the sample in the reaction tube with a detection sensor.

The present invention is not limited to the excitation wavelength bands for each channel for the four channels from the optical module 410 (1) to the optical module 410 (4), and allows excitation wavelengths different from the four wavelength bands. Channels can be added, and depending on the number of channels added, the number of optical modules increases from 4 to the number added.

When an optical module is added, the types of genetic samples that can be optically inspected within the reaction tube can be added.

As disclosed in the above embodiment, the electrical signal received by the detection sensor from the optical module 410 (1) to the optical module 410 (4) is transmitted to the central processing unit.

Since the optical system that detects a plurality of different wavelengths moves to individual positions in the eight reaction tubes and measures them, the same optical system measures each wavelength. Therefore, the condition that the performance of the optical system must be the same is always satisfied.

Additionally, the amplification efficiency of the control and sample becomes the same.

Depending on the sample, PCR inhibitors may be present, and even if the same amplification reagent is used, the amplification efficiency of the reaction solution with the control added and the reaction solution with the sample added may be different.

To improve this, high-concentration control samples, low-concentration control samples, and internal controls whose values are already known are added to the reaction solution of the sample to be analyzed.

In this case, multiple signals (high-concentration control sample, low-concentration control sample, internal control material, sample to be analyzed, etc.) can be generated in one reaction tube.

Since the reaction solution environment in the reaction tube is the same, the control sample and the analysis target satisfy the same amplification efficiency conditions, and since the control sample is added to the reaction solution, a separate control material reaction tube is not required.

Therefore, all eight reaction tubes are exposed to the light of the sample to be analyzed. Can be used for measurements.

When the optical measurement process from the optical module 410 (1) to the optical module 410 (4) is completed as disclosed in the above embodiment,

Again, the central processing unit 200 drives the temperature circulation unit 300 to heat and cool the reaction tube.

Then, the light from the LED light source 420 is irradiated to the reaction tube through the optical module 410, and the light emitted by the fluorescent substance in the reaction tube passes through the optical module 410 again to the detection sensor 430. Light is collected and the second cycle of optical analysis proceeds.

Afterwards, the cycle of temperature cycling and optical analysis is repeated, and the amplified nucleic acid is tested.

The number of times the cycle of temperature cycling and optical analysis is repeated may vary depending on the type of nucleic acid being targeted.

Figure 8 is an exploded view of the optical module 410.

The optical module 410 consists of a first body 410(a) and a second body 410(b), and the first body 410(a) on the left is equipped with devices necessary for optical measurement. It is a frame, and the second body 410(b) on the right is a frame cover that fixes and supports the devices mounted on the first body 410(a).

As shown in Figure 9, the first body of the optical module includes a module incident lens 411, an input optical filter 412, a dichroic mirror 413 that reflects only light of a specific wavelength, a reaction tube incident lens 414, and an output side. An optical filter 415 and a sensor output lens 416 are installed.

Figure 10 is a diagram showing the combination of two optical modules.

A pair of coupling protrusions 417 provided on the second body 410(b) of the left optical module and a pair of coupling grooves 418 provided on the first body 410(a) of the right optical module. The two optical modules are joined by snapping at corresponding positions.

A plurality of optical modules 410 are coupled to the optical inspection unit 400 using the coupling protrusion-coupling groove fitting method described above.

As shown in FIG. 11, the electrical signal measured by the detection sensor is analyzed and processed by software built into the central processing unit.

<Database creation steps>

For each reaction tube, a database of raw data of electrical signals is recorded and stored for each fluorescence channel.

<Smoothing step>

The raw data of each reaction tube is corrected using a 5-section moving average (see Figure 12).

<Amplification starting point determination and Ct value calculation steps>

The data is rotated based on the straight line connecting the start point and the end point, the X value with the minimum Y value is determined as the amplification start point, and the Ct value (Cycle Threshold Value) is calculated (see Figure 13).

< Baseline adjustment and graph output steps>

The range from the starting point to before the amplification start point is set as the baseline section, and the slope and Y-intercept of this section are adjusted to ‘0’ to output a graph (see Figure 14).

Figure 15 is an algorithm for calculating quantitative values for each reaction through quantitative lines created with control values.

<Control Ct value calling step>

Specify the reaction tube and fluorescence channel containing high- and low-concentration control samples, and call the Ct value of the specified signal, respectively.

Specify reaction tubes and fluorescence channels containing high and low control samples (if high and low control samples are used in separate tubes), or specify fluorescence channels for high and low control samples in a single tube (if high and low control samples are used in separate tubes). (when applying the included reaction tube) and call the Ct value of the specified signal, respectively.

<Control concentration call step>

Enter the concentration values of high and low concentration control samples or recall the saved values.

< Quantitative line creation stage>

A quantitative line is created using the concentration and Ct values of the high- and low-concentration controls.

< Quantitative value calculation step>

Calculate the concentration value of the sample by substituting the Ct value of the reaction sample into the quantitative line.

Claims (11)

In the nucleic acid amplification test system,
A power supply unit 100 that converts commercial AC power supplied from the outside into DC power and supplies it;
a central processing unit 200 that controls heating and cooling of the reaction tube, and analyzes and stores optically measured data in the reaction tube;
a temperature circulation unit 300 that heats and cools the reaction tube to amplify the genes of the material contained in the reaction tube;
An optical inspection unit 400 that optically analyzes the material contained in the reaction tube;
An optical drive unit 500 that drives the optical inspection unit 400 by operation control of the central operation processing unit 200; and
A nucleic acid amplification test system comprising an interface unit 600 that inputs operation commands for the test system and outputs test results.
In claim 1,
The temperature circulation unit 300,
DNA denaturing step of heating the reaction tube in the range of 90 to 95°C;
An annealing step of cooling the reaction tube down to the range of 50-60°C; and
Including a DNA synthesis step of maintaining the temperature of the reaction tube in the range of 60-70°C,
Nucleic acid amplification test system characterized by amplifying the genes of the material contained in the reaction tube
In claim 1,
The temperature circulation unit 300 is a nucleic acid amplification test system characterized in that it includes a peltier 310 as a reaction tube heating means and a fan 330 as a cooling means.
In claim 1,
The temperature circulation unit 300 detects the temperature of the heated reaction tube and includes a thermistor 320 to which a driving current is applied so that the detected temperature follows the control input.
In claim 1,
The optical inspection unit 400 is
An LED light source 420 that irradiates light to the solution contained in the reaction tube; and
A nucleic acid amplification test system comprising a detection sensor 430 that detects light expressed by a fluorescent substance in a solution contained in a reaction tube.
In claim 1,
The optical inspection unit 400 is a nucleic acid amplification inspection system characterized in that it is provided with a plurality of optical modules 410 having different excitation wavelength bands for a plurality of channels.
In claim 6,
A nucleic acid amplification test system characterized in that the excitation wavelength band includes 450 nm to 490 nm, 515 nm to 535 nm, 560 nm to 590 nm, and 620 nm to 650 nm.
In claim 6,
The optical driving unit 500 sequentially moves the optical inspection unit 400 linearly to the corresponding position of the reaction tube so that the optical module 410 can perform optical measurements on the solutions contained in all reaction tubes, and one optical module ( When the optical measurement by 410 is completed, the optical inspection unit 400 returns to the position before moving so that the reaction tube can be optically measured by another optical module 410, and then sequentially moves straight back to the corresponding position of the reaction tube. A nucleic acid amplification inspection system characterized in that the performance of the optical system can always satisfy the same conditions by measuring the solutions contained in all reaction tubes by the same optical system.
In claim 6,
The optical module 410 consists of a first body 410(a) and a second body 410(b), and the first body 410(a) is equipped with devices necessary for optical measurement. A nucleic acid amplification test system, wherein the second body (410(b)) is a frame cover that fixes and supports the devices mounted on the first body (410(a)).
In claim 1,
The analysis algorithm of the central operation processing unit 200 is,
(i) recording and storing raw data of electrical signals for each reaction tube and each fluorescence channel;
(ii) correcting the raw data of each reaction tube using a 5-section moving average;
(iii) rotating the data based on a straight line connecting the start point and the end point, determining the X value with the minimum Y value as the amplification start point, and calculating the Ct value (Cycle Threshold Value);
(iv) setting the range from the start point to before the amplification start point as a baseline section, and adjusting the overall value of the slope and Y-intercept of this section to '0' to output a graph; a nucleic acid amplification test comprising: system
In claim 1,
The algorithm for calculating the quantitative value of the central processing unit 200 is:
(i) specifying reaction tubes and fluorescence channels containing high-concentration and low-concentration control samples and recalling the Ct values of the designated signals, respectively;
(ii) recalling input or stored values as concentration values of high-concentration and low-concentration control samples;
(iii) creating a quantitative line using the concentration values and Ct values of the high-concentration and low-concentration controls;
(iv) calculating the concentration value of the sample by substituting the Ct value of the reaction sample into the quantitative line; a nucleic acid amplification test system comprising: