U.S. patent application number 17/614827 was filed with the patent office on 2022-08-04 for method for detecting target nucleic acid in sample.
The applicant listed for this patent is SEEGENE, INC.. Invention is credited to Jun Hyeok JEONG, Young Wook KIM, Hye Jin LEE.
Application Number | 20220244181 17/614827 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-04 |
United States Patent
Application |
20220244181 |
Kind Code |
A1 |
KIM; Young Wook ; et
al. |
August 4, 2022 |
METHOD FOR DETECTING TARGET NUCLEIC ACID IN SAMPLE
Abstract
The disclosure relates to a method for detecting a target
nucleic acid in a sample using a nucleic acid reaction detection
device comprising a plurality of reaction areas independent from
each other. According to the disclosure, the method may
simultaneously perform two or more nucleic acid reactions which are
carried out with different protocols and independently measure
emitted optical signals, thereby detecting the target nucleic
acid.
Inventors: |
KIM; Young Wook; (Seoul,
KR) ; LEE; Hye Jin; (Anyang-si, KR) ; JEONG;
Jun Hyeok; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEEGENE, INC. |
Seoul |
|
KR |
|
|
Appl. No.: |
17/614827 |
Filed: |
May 29, 2020 |
PCT Filed: |
May 29, 2020 |
PCT NO: |
PCT/KR2020/007040 |
371 Date: |
November 29, 2021 |
International
Class: |
G01N 21/64 20060101
G01N021/64; B01L 3/00 20060101 B01L003/00; C12Q 1/686 20060101
C12Q001/686 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2019 |
KR |
10-2019-0064472 |
Jun 28, 2019 |
KR |
10-2019-0078284 |
Dec 6, 2019 |
KR |
10-2019-0162094 |
Claims
1. A method for detecting a target nucleic acid in a sample, the
method comprising: (a) positioning a first sample set in a first
reaction area of a nucleic acid reaction detection device and a
second sample set in a second reaction area of the nucleic acid
reaction detection device and performing a nucleic acid reaction;
wherein the nucleic acid reaction detection device comprising a
sample reaction module comprising the first reaction area and the
second reaction area thermally independent from each other, an
optical module for detecting nucleic acid reaction of samples in
the first reaction area and the second reaction area, and a
controller; wherein temperatures of the first reaction area and the
second reaction area are controlled according to different
protocols; (b) measuring optical signals from the first sample set
and the second sample set; wherein the optical signals from the
first sample set and the second sample set are measured
synchronously; wherein the wavelength bands of the optical signals
synchronously measured from the first sample set and the second
sample set are different from each other; and (c) detecting a
target nucleic acid in a sample included in the first sample set
and the second sample set by analyzing the measured optical
signals.
2. The method according to claim 1, wherein the step of measuring
the optical signals comprises (i) a step of synchronously measuring
the optical signals from the first sample set and the second sample
set, wherein the wavelength bands of the optical signals
synchronously measured from the first sample set and the second
sample set are different from each other, and (ii) a step of
measuring an optical signal from one of the first sample set and
the second sample set without measuring an optical signal from the
other of the first sample set and the second sample set.
3. The method according to claim 1, wherein the measurement of an
optical signal for the first sample set is performed by
sequentially measuring optical signals of two or more different
wavelength bands for the first sample set.
4. The method according to claim 1, wherein the first sample set
comprises a plurality of samples, and wherein measuring an optical
signal from the first sample set positioned in the first reaction
area is simultaneously measuring optical signals from two or more
samples included in the first sample set.
5. The method according to claim 1, wherein the controller operates
the nucleic acid reaction detection device to perform an operation
comprising at least one of the steps of: (i) measuring an optical
signal for one sample set of the first sample set and the second
sample set without measuring an optical signal for the other sample
set: and (ii) synchronously measuring the optical signals from the
first sample set and the second sample set, wherein the wavelength
bands of the optical signals synchronously measured from the first
sample set and the second sample set are different from each
other.
6. The method according to claim 1, wherein the optical module
comprises a plurality of light sources, and wherein at least two of
the plurality of light sources produce excitation lights of
different wavelength bands from each other.
7. The method according to claim 1, wherein the optical module
comprises a plurality of detectors, and wherein each of the
plurality of detectors detects light emitted from a sample in a
predetermined and fixed area of the sample reaction module.
8. The method according to claim 1, wherein the optical module
comprises a plurality of detectors independently controlled,
wherein one or more different detectors among the plurality of
detectors are assigned to each of the first reaction area and the
second reaction area, and wherein optical signals are detected from
the first sample set and the second sample set by the one or more
different detectors assigned respectively.
9. A nucleic acid reaction detection device, comprising: a sample
reaction module comprising a first reaction area and a second
reaction area thermally independent from each other; an optical
module for detecting nucleic acid reaction of samples in the sample
reaction module; and one or more controllers electrically connected
with the sample reaction module and the optical module, wherein the
optical module is configured to synchronously measure an optical
signal of a first wavelength band from a sample in the first
reaction area and an optical signal of a second wavelength band
from a sample in the second reaction area, wherein the second
wavelength band is different from the first wavelength band, and
wherein the controller is configured to control temperatures of the
first reaction area and the second reaction area independently and
control optical signal measurement of a sample in the first
reaction area and optical signal measurement of a sample in the
second reaction area independently.
10. The nucleic acid reaction detection device according to claim
9, wherein the controller receives distinct protocols for the first
reaction area and the second reaction area; controls temperatures
of the first reaction area and the second reaction area
independently according to the received protocols; and operates the
nucleic acid reaction detection device to perform an operation
comprising at least one of the steps of, based on the received
protocols for all the reaction areas: (i) measuring an optical
signal for a sample in one reaction area of the first reaction area
and the second reaction area without measuring an optical signal
for a sample in the other reaction area; and (ii) synchronously
measuring an optical signal of a first wavelength band from a
sample in the first reaction area and an optical signal of a second
wavelength band from a sample in the second reaction area, wherein
the second wavelength band is different from the first wavelength
band.
11. The nucleic acid reaction detection device according to claim
9, wherein the optical module comprises a light source for
illuminating a sample in a reaction area with an excitation light
and a detector for sensing an emission light received from the
sample in the reaction area.
12. The nucleic acid reaction detection device according to claim
9, wherein the optical module comprises a plurality of detectors,
and wherein each of the plurality of detectors detects light
emitted from a sample in a predetermined and fixed area of the
sample reaction module.
13. The nucleic acid reaction detection device according to claim
9, wherein the optical module simultaneously illuminates a
plurality of samples in the same reaction area with an excitation
light.
14. The nucleic acid reaction detection device according to claim
9, wherein the optical module comprises a plurality of light
sources, and wherein at least two of the plurality of light sources
produce excitation lights of different wavelength bands from each
other.
15. The nucleic acid reaction detection device according to claim
9, wherein the sample reaction module is configured so that each of
the first reaction area and the second reaction area is capable of
receiving at least two or more samples.
Description
TECHNICAL FIELD
[0001] The disclosure relates to a method for detecting a target
nucleic acid in a sample using a nucleic acid reaction detection
device.
BACKGROUND ART
[0002] Nucleic acid amplification reaction well known as
polynucleotide chain reaction (PCR) includes repeated cycles of
double-stranded DNA denaturation, annealing of the oligonucleotide
primers to DNA templates, and extension/elongation of the primers
with the DNA polymerase (Mullis et al., U.S. Pat. Nos. 4,683,195,
4,683,202, and 4,800,159; Saiki et al., (1985) Science 230,
1350-1354).
[0003] A typical PCR device (or machine) causes amplification in
the reaction vessel positioned in the thermal block by varying the
temperature of the thermal block according to a predetermined
sequence. DNA denaturation is performed at about 95.degree. C., and
primer annealing and elongation are performed at a lower
temperature ranging from 55.degree. C. to 75.degree. C. The
temperature and reaction time for each step of DNA denaturation,
annealing, and elongation need to be set to differ depending on,
e.g., the sequence of oligonucleotides, e.g., the primers and probe
used for analysis, the target nucleic acid for analysis, and each
sample.
[0004] Real-time PCR is a PCR-based technique for detecting a
target nucleic acid from a sample in real-time. To detect a
particular target nucleic acid, a signal generator is used which
radiates detectable fluorescent signals in proportion to the amount
of the target nucleic acid upon PCR. To that end, an intercalator
may be used which emits a fluorescent signal when combined with the
dual-helix DNA, or oligonucleotides including a fluorophore and
quencher molecules suppressing fluorescent emissions therefrom may
be used. The fluorescent signal proportional to the amount of the
target nucleic acid is detected in real-time at each cycle and is
measured to obtain an amplification curve or amplification profile
curve which displays the strength of the fluorescent signal
detected per cycle.
[0005] The real-time PCR device is able to measure, in real-time,
the signals while the nucleic acid reaction proceeds. Typically,
the real-time PCR device illuminates the substrate with light and
measures the resultant fluorescence, thereby detecting the nucleic
acid in the reaction vessel. To that end, the real-time PCR device
includes a light source for radiating an excitation light to the
sample and a detector for receiving the light emitted from the
sample.
[0006] Conventional nucleic acid reaction detection devices may
analyze the nucleic acid reaction on multiple samples using a
single device. However, the reaction proceeds under the same
condition for all the samples and, since it is configured to
radiate the same wavelength of light at the same time, only
simultaneous analysis on the same mark for the plurality of samples
is possible. Thus, analysis of various kinds of samples in small
quantities takes an increased overall processing time and higher
costs because each kind of sample needs to be analyzed
separately.
[0007] To address such issues, a device has been developed which is
equipped with a plurality of thermally independent thermal blocks
and is able to perform nucleic acid reaction with different thermal
protocols. However, this nucleic acid reaction detection device may
simply amplify the nucleic acid without real-time optical
measurement or, although performing nucleic acid reaction based on
a different thermal protocol for each thermal block thermally
independent, analyzes the plurality of thermal blocks with one
optical analyzing module. This device is unable to simultaneously
measure optical signals of different wavelength bands from the
samples individually received in the thermally-independent thermal
blocks. Thus, the device cannot simultaneously analyze two or more
nucleic acid reactions, for which the optical signal measurement
time is independently determined, because the reaction is used
based on the different thermal protocols.
[0008] Therefore, a need exists for a device capable of
simultaneously performing different nucleic acid reactions in a
single device and performing independent and real-time optical
analysis and a method for detecting a nucleic acid using the
same.
DISCLOSURE OF INVENTION
Technical Problem
[0009] The inventors have tried to develop a new method for
detecting a target nucleic acid in a sample, for use in PCR devices
comprising two or more reaction areas thermally independent from
each other such that two or more nucleic acid reactions based on
distinct protocols are capable of being performed simultaneously in
a single device. As a result, the inventors have found that the
target nucleic acid in the sample included in each sample set may
be detected by positioning a first sample set in a first reaction
area and a second sample set in a second reaction area in a nucleic
acid reaction detection device, which comprises a sample reaction
module comprising the first reaction area and the second reaction
area thermally independent from each other, an optical module
capable of detecting nucleic acid reaction in samples in the first
reaction area and the second reaction area, and a controller, and
in which the temperatures of the first reaction area and the second
reaction area are controlled according to protocols independent
from each other, performing nucleic acid reaction, synchronously
measuring optical signals from the first sample set and the second
sample set, wherein the wavelength bands of the optical signals
from the first sample set and the second sample set are different
from each other and analyzing the measured optical signals.
[0010] Thus, a purpose of the present disclosure is to provide a
method for detecting a target nucleic acid in a sample.
[0011] Another purpose of the present disclosure is to provide a
nucleic acid reaction detection device that may synchronously
measure an optical signals of a first wavelength band generated
from the sample in the first reaction area and an optical signal of
a second wavelength band (which differs from the first wavelength
band) generated from the sample in the second reaction area.
[0012] The other objectives and advantages of the disclosure will
be apparent from the embodiments and claims described below in
detail with reference to the accompanying drawings.
Solution to Problem
[0013] To achieve the foregoing objects, according to one aspect of
the disclosure, there is provided a method for detecting a target
nucleic acid in a sample, comprising:
[0014] (a) positioning a first sample set in a first reaction area
of a nucleic acid reaction detection device and a second sample set
in a second reaction area of the nucleic acid reaction detection
device and performing a nucleic acid reaction, the nucleic acid
reaction detection device comprising a sample reaction module
comprising the first reaction area and the second reaction area
thermally independent from each other and an optical module for
detecting nucleic acid reaction of samples in the first reaction
area and the second reaction area, and a controller; wherein
temperatures of the first reaction area and the second reaction
area are controlled according to different protocols; (b) measuring
optical signals from the first sample set and the second sample
set; wherein the optical signals from the first sample set and the
second sample set are measured synchronously; wherein the
wavelength bands of the optical signals synchronously measured from
the first sample set and the second sample set are different from
each other; and (c) detecting a target nucleic acid in a sample
included in the first sample set and the second sample set by
analyzing the measured optical signals.
[0015] To achieve the foregoing objects, according to one aspect of
the disclosure, there is provided a nucleic acid reaction detection
device, comprising: a sample reaction module comprising a first
reaction area and a second reaction area thermally independent from
each other; an optical module for detecting nucleic acid reaction
in samples in the sample reaction module; and one or more
controllers electrically connected with the sample reaction module
and the optical module, wherein the optical module is configured to
synchronously measure an optical signal of a first wavelength band
from a sample in the first reaction area and an optical signal of a
second wavelength band from a sample in the second reaction area,
wherein the second wavelength band is different from the first
wavelength band, and wherein the controller is configured to
control temperatures of the first reaction area and the second
reaction area independently and control optical signal measurement
of a sample in the first reaction area and optical signal
measurement of a sample in the second reaction area
independently.
Advantageous Effects of Invention
[0016] According to the disclosure, the method and device may
simultaneously perform two or more nucleic acid reactions which are
carried out with different protocols and independently measure
emitted optical signals, thereby detecting the target nucleic acid.
The method and device may thus analyze various kinds of samples in
small quantities, providing advantages in light of sample analysis
time and costs.
[0017] Where the optical module measures optical signals while
being moved by a moving means, the measurement takes long and the
measurement time is varied between the individual samples. However,
the device and method according to the disclosure are able to
synchronously measure optical signals of different wavelength bands
generated from a plurality of samples included in the sample set
where nucleic acid reaction has been performed in different
reaction areas and may thus detect the target nucleic acid in a
precise and efficient manner as compared with conventional
analyzers which may perform optical signal measurement only
sequentially.
[0018] The method and device according to the disclosure may detect
two or more target nucleic acids in one sample. Therefore, the
method and device according to the disclosure enable multiplex
target nucleic acid detection from two or more sample sets for
detecting different target nucleic acids.
BRIEF DESCRIPTION OF DRAWINGS
[0019] FIG. 1 is a view illustrating a nucleic acid reaction
detection device according to an embodiment of the disclosure;
[0020] FIG. 2 is a view illustrating a structure of a nucleic acid
reaction detection device with a separate light source for each
reaction area, according to an embodiment of the disclosure;
[0021] FIG. 3 is a view illustrating a structure of a nucleic acid
reaction detection device with a light source shared by a plurality
of reaction areas, according to an embodiment of the
disclosure;
[0022] FIG. 4 is a view illustrating a structure in which a
separate light source is provided for each reaction area according
to an embodiment of the disclosure;
[0023] FIG. 5 is a view illustrating a structure in which a light
source is shared by two or more reaction areas according to an
embodiment of the disclosure;
[0024] FIG. 6 is a view illustrating a sample reaction module and
an optical module using a light source comprising a plurality of
light emitting elements in a nucleic acid reaction detection device
according to an embodiment of the disclosure;
[0025] FIG. 7 is a view illustrating an optical path of a nucleic
acid reaction detection device according to an embodiment of the
disclosure; and
[0026] FIG. 8 is a view illustrating a structure in which a
controller is electrically connected with another component of a
nucleic acid reaction detection device according to an embodiment
of the disclosure.
MODE FOR THE INVENTION
[0027] The configuration and effects of the present invention are
now described in further detail in connection with embodiments
thereof. The embodiments are provided merely to specifically
describe the present invention, and it is obvious to one of
ordinary skill in the art that the scope of the present invention
is not limited to the embodiments.
[0028] I. Detection of Target Nucleic Acid in Sample
[0029] According to an embodiment of the disclosure, there is
provided a method for detecting a target nucleic acid in a sample,
comprising:
[0030] (a) positioning a first sample set in a first reaction area
of a nucleic acid reaction detection device and a second sample set
in a second reaction area of the nucleic acid reaction detection
device and performing a nucleic acid reaction; wherein the nucleic
acid reaction detection device comprising a sample reaction module
comprising the first reaction area and the second reaction area
thermally independent from each other and an optical module and a
controller for detecting nucleic acid reaction in the first
reaction area and the second reaction area, and a controller;
wherein temperatures of the first reaction area and the second
reaction area are controlled according to different protocols;
[0031] (b) measuring optical signals from the first sample set and
the second sample set; wherein the optical signals from the first
sample set and the second sample set are measured synchronously;
wherein the wavelength bands of the optical signals synchronously
measured from the first sample set and the second sample set are
different from each other; and
[0032] (c) detecting a target nucleic acid in a sample included in
the first sample set and the second sample set by analyzing the
measured optical signals.
[0033] The inventors have tried to develop a new method for
detecting a target nucleic acid in a sample, for use in PCR devices
comprising two or more reaction areas thermally independent from
each other such that two or more nucleic acid reactions based on
distinct protocols are capable of being performed simultaneously in
a single device. As a result, the inventors have found that the
target nucleic acid in the sample included in each sample set may
be detected by positioning a first sample set in a first reaction
area and a second sample set in a second reaction area in a nucleic
acid reaction detection device, which comprises a sample reaction
module comprising the first reaction area and the second reaction
area thermally independent from each other, an optical module
capable of detecting nucleic acid reaction in samples in the first
reaction area and the second reaction area, and a controller, and
in which the temperatures of the first reaction area and the second
reaction area are controlled according to protocols independent
from each other, performing nucleic acid reaction, synchronously
measuring optical signals from the first sample set and the second
sample set, and analyzing the measured optical signals.
[0034] Each step of the method is described below in detail:
[0035] Step (a): Nucleic Acid Reaction
[0036] According to the disclosure, the first sample set and the
second sample set are positioned in the first reaction area and the
second reaction area, respectively, of the nucleic acid reaction
detection device, and nucleic acid reaction is performed.
[0037] According to an embodiment, the nucleic acid reaction
detection device refers to a device that detects the target nucleic
acid using nucleic acid reaction. In particular, the nucleic acid
reaction detection device is a device that comprises a sample
reaction module comprising the first reaction area and the second
reaction area thermally independent from each other, an optical
module for detecting nucleic acid reaction of samples in the first
reaction area and the second reaction area, and a controller, and
in which the temperatures of the first reaction area and the second
reaction area may be controlled according to different protocols.
The nucleic acid reaction detection device is described in more
detail below in Section II.
[0038] As used herein, the term "sample" may encompass biological
samples (e.g., cells, tissues, or fluids from biological sources)
and non-biological samples (e.g., foods, water, and soil). The
biological samples include virus, germs, tissues, cells, blood
(e.g., whole blood, plasma, and serum), lymph, bone marrow fluid,
saliva, sputum, swab, aspiration, milk, urine, stool, ocular humor,
semen, brain extracts, spinal fluid, joint fluid, thymus fluid,
bronchoalveolar lavage fluid, ascites, and amniotic fluid. The
sample may undergo a nucleic acid extraction process. Where the
extracted nucleic acid is RNA, reverse transcription may be added
to synthesize cDNA from the extracted RNA (Joseph Sambrook, et al.,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (2001)).
[0039] The nucleic acid reaction means a series of physical or
chemical reactions that generate a signal dependent upon the
presence or absence of a nucleic acid with a specific sequence in
the sample or the amount thereof. The nucleic acid reaction may be
a reaction that includes a hybridization of the nucleic acid with
the specific sequence in the sample and other nucleic acid or
material, copying, cutting, or decomposition of the nucleic acid
with the specific sequence in the sample.
[0040] According to an embodiment of the disclosure, the nucleic
acid reaction may be a signal-generating process that may generate
a signal, dependent upon the presence or absence of target nucleic
acid in the sample or the amount thereof. The signal-generating
process may be a genetic analysis process, such as PCR, real-time
PCR, or microarray. According to an embodiment of the disclosure,
the nucleic acid reaction may be PCR or real-time PCR.
[0041] According to an embodiment of the disclosure, the
signal-generating process may be an amplification reaction of
signal. The amplification reaction means a reaction to increase or
decrease signals. According to an embodiment of the disclosure, the
amplification reaction means increasing (or amplifying) signals,
relying on the presence of the target nucleic acid using a
signal-generation means. The amplification reaction may, or may
not, be accompanied by amplification of the target nucleic acid.
Thus, according to an embodiment of the disclosure, the nucleic
acid reaction may be performed along with, or without,
amplification of target nucleic acid molecules. Specifically,
according to the disclosure, the nucleic acid reaction may be a
signal amplification reaction that is performed accompanied by
amplification of the target analyte.
[0042] The signal-generating process is accompanied by a signal
change. As used herein, the term "signal" means a measurable
output.
[0043] The signal change may serve as an indicator qualitatively or
quantitatively indicating the presence/absence of the target
nucleic acid or the amount thereof. Examples of the indicator
include the strength of fluorescence, the strength of luminescence,
the strength of chemiluminescence, the strength of bioluminescence,
and the strength of phosphorescence. The indicator most widely used
is the strength of fluorescence. The signal change may include a
signal decrease as well as a signal increase.
[0044] As used herein, `signal` refers to an optical signal. The
optical signal may be luminescence, phosphorescence,
chemiluminescence, fluorescence, polarized fluorescence, or other
colored signal.
[0045] To generate the optical signal, a signal-generating means
may be included in the sample. A wide variety of the
signal-generating means have been known to one of skill in the art.
The signal-generating means comprises a label itself or a
label-attached oligonucleotide. The label may include a fluorescent
label, a luminescent label, a chemiluminescent label, an
electrochemical label, and a metal label. The label per se like an
intercalating dye may serve as signal-generating means.
Alternatively, a single label or interactive dual label including a
donor molecule and an acceptor molecule may be used as
signal-generating means in the form of linkage to at least one
oligonucleotide. The signal-generating means may comprise
additional components for generating signals such as an enzyme with
nucleolytic activity (e.g., 5'-nucleases and 3'-nucleases).
[0046] According to an embodiment of the disclosure, more
specifically, the label used for the signal-generating means may
include a single fluorescent label or interactive dual label
(including, e.g., a fluorescence reporter molecule and a quencher
molecule) including a donor molecule and an acceptor molecule. The
fluorescent label or fluorescent reporter molecule comprises, but
is not limited to, FAM.TM., TET.TM., VIC.TM. JOE.TM., HEX.TM., CY3,
TAMRA.TM., ROX.TM., Texas Red, CY5, CY5.5, and Quasar 705.
[0047] There are known various methods of generating an optical
signal to indicate the presence of the target nucleic acid using
nucleic acid reaction. Representative examples include: TaqMan.TM.
probe method (U.S. Pat. No. 5,210,015), molecule beacon method
(Tyagi et al., Nature Biotechnology v. 14 Mar. 1996), Scorpion
method (Whitcombe et al., Nature Biotechnology 17:804-807(1999)),
Sunrise or Amplifluor method (Nazarenko et al., 2516-2521 Nucleic
Acids Research, 25(12):2516-2521(1997), and U.S. Pat. No.
6,117,635), Lux method (U.S. Pat. No. 7,537,886), CPT (Duck P, et
al. Biotechniques, 9:142-148(1990)), LNA method (U.S. Pat. No.
6,977,295), Plexor method (Sherrill C B, et al., Journal of the
American Chemical Society, 126:4550-4556(2004)), Hybeacons.TM. (D.
J. French, et al., Molecular and Cellular Probes (2001) 13, 363-374
and U.S. Pat. No. 7,348,141), Dual-labeled, self-quenched probe;
U.S. Pat. No. 5,876,930), hybridization probe (Bernard P S, et al.,
Clin Chem 2000, 46, 147-148), PTOCE (PTO cleavage and extension)
method (WO 2012/096523), PCE-SH (PTO Cleavage and
Extension-Dependent Signaling Oligonucleotide Hybridization) method
(WO 2013/115442), PCE-NH (PTO Cleavage and Extension-Dependent
Non-Hybridization) method (PCT/KR2013/012312) and CER method (WO
2011/037306).
[0048] According to an embodiment, the nucleic acid reaction
detection device may be a device that performs nucleic acid
amplification reaction to amplify the nucleic acid with a specific
nucleic sequence. The target nucleic acid may be amplified by
various methods. For example, methods for amplifying the target
nucleic acid include the polymerase chain reaction (PCR), ligase
chain reaction (LCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR
Protocols: A Guide to Methods and Applications (Innis et al., eds,
1990)), strand displacement amplification (SDA)) (Walker, et al.
Nucleic Acids Res. 20(7):1691-6 (1992); Walker PCR Methods Appl
3(1):1-6 (1993)), transcription-mediated amplification (Phyffer, et
al., J. Clin. Microbiol. 34:834-841 (1996); Vuorinen, et al., J.
Clin. Microbiol. 33:1856-1859 (1995)), nucleic acid sequence-based
amplification (NASBA) (Compton, Nature 350(6313):91-2 (1991)),
rolling circle amplification (RCA) (Lisby, Mol. Biotechnol.
12(1):75-99 (1999); Hatch et al., Genet. Anal. 15(2):35-40 (1999))
and Q-beta Replicase (Lizardi et al., Biol Technology 6:1197
(1988)).
[0049] In particular, according to an embodiment, the nucleic acid
reaction detection device may be a device that performs nucleic
acid amplification reaction, accompanied by a temperature change.
For example, to amplify deoxyribonucleic acid (DNA) with a specific
nucleic sequence, the nucleic acid amplification reaction may
include a denaturing step, an annealing step, and an extension (or
amplification) step.
[0050] According to an embodiment, the nucleic acid reaction
detection device may be a device that performs the target nucleic
acid amplification reaction, accompanied by a temperature change,
and the reaction of generating an optical signal, dependent on the
presence of the target nucleic acid and detects a generated optical
signal.
[0051] The nucleic acid reaction detection device comprises a
sample reaction module. The sample reaction module is a module that
receives a sample, the target for analysis, and performs nucleic
acid reaction. The sample reaction module comprises a reaction area
for receiving the sample or a reaction vessel containing the
sample. A plurality of samples may be received in the sample
reaction module. The sample reaction module according to the
disclosure comprises a first reaction area and a second reaction
area thermally independent from each other.
[0052] According to an embodiment of the disclosure, the sample
reaction module may comprise other additional reaction area(s) than
the first reaction area and the second reaction area. For example,
the sample reaction module may further comprise a third reaction
area and a fourth reaction area. The reaction areas each may be
controlled thermally independently. The reaction areas being
controlled thermally independently may mean that the temperature of
each reaction area may be individually controlled. For example,
while the first reaction area remains at 94.degree. C. at a first
time, the second reaction area may be cooled down from 94.degree.
C. to 60.degree. C. or heated up from 60.degree. C. to 75.degree.
C. or may remain at a specific temperature. By so doing, a separate
nucleic acid reaction may be performed in each reaction area of the
nucleic acid reaction detection device. The number of reaction
areas included in the sample reaction module may range from 2 to
24, but is not limited thereto. For example, the number of reaction
areas comprised in the sample reaction module may be 2, 3, 4, 5, 6,
8, 10, 12, 16, 20, or 24. According to an embodiment of the
disclosure, the reaction areas are thermally independent from each
other. In other words, heat does not transfer from one reaction
area to another. For example, a thermal insulation material or an
air gap may be provided between the reaction areas.
[0053] In the nucleic acid reaction step, the temperatures of the
first reaction area and the second reaction area are controlled
according to protocols independent from each other.
[0054] As used herein, the protocol means a set of instructions to
perform a unit operation. The unit operation includes the operation
of controlling temperature over time and the operation of measuring
signal. The protocol comprises information regarding the order and
timings of performing various unit operations in the protocol.
[0055] The method according to the disclosure is characterized by
allowing the first sample set and the second sample set to react
according to protocols independent from each other. Sample set
refers to a set of samples including one or more samples. For the
samples included in one sample set, nucleic acid reaction and
detection are performed according to the same protocol. According
to an embodiment of the disclosure, the first sample set may
comprise a plurality of samples.
[0056] The first sample set and the second sample set are samples
for detecting different target nucleic acids from each other. Thus,
the optimal protocols for the first sample set and second sample
set differ from each other for efficient nucleic acid reaction and
detection. The sample reaction module of the present disclosure may
control temperature independently for each reaction area and, thus,
for the first sample set and second sample set, reaction may be
performed according to protocols independent from each other.
[0057] Step (b): Measurement of Optical Signal
[0058] In the step of optical signal measurement, optical signals
from the first sample set and the second sample set are measured.
Optical signal measurement means measuring an optical signal that
is generated depending on the presence of the target nucleic acid
in the sample or the amount thereof.
[0059] In the method according to the disclosure, the nucleic acid
reaction may be repeated several times. According to an embodiment
of the disclosure, optical signal measurement may be performed once
after the plurality of nucleic acid reactions all have been
finished. Selectively, according to an embodiment of the
disclosure, the optical signal measurement may be performed several
times between the plurality of nucleic acid reactions. Preferably,
the optical signal measurement may be performed for each nucleic
acid reactions of the plurality of nucleic acid reactions.
[0060] Further, since nucleic acid reaction is performed on the
first sample set and the second sample set according to independent
protocols, the times for measuring the optical signals for the
first sample set and the second sample set are determined
independently from each other.
[0061] Specifically, according to the method of the disclosure, the
measurement step of the disclosure may comprise the step of
synchronously measuring optical signals from the first sample set
and the second sample set. Or, the measurement step of the
disclosure may comprise the step of measuring an optical signal
from one sample set of the first sample set and the second sample
set without measuring an optical signal for the other sample set of
the first sample set and the second sample set. The measurement
step of the disclosure, which comprises a number of measurements of
optical signals, generally comprises both of the measurement steps
disclose above. The wavelength bands of the optical signals
measured from the first sample set and the second sample set may be
identical to or different from each other.
[0062] The conventional target nucleic acid detector, which has
thermal blocks thermally independent from each other and may thus
simultaneously perform different nucleic acid reactions, lacks an
optical module capable of real-time optical measurement or is
configured to simultaneously measure the thermal blocks at the same
wavelength. The device with such a structure may only adopt the
method of collecting the sample after nucleic acid reaction is
finished and performing analysis separately or the method of
simultaneously measuring optical signals of the same wavelength
band on all the samples where nucleic acid reaction proceeds in the
device. Such a measurement method is not suitable for real-time
analysis of different nucleic acid reactions which are performed by
independent protocols in a single device.
[0063] The target nucleic acid detection method according to the
disclosure comprises the step of synchronously measuring an optical
signal measured from the first sample set and an optical signal
measured from the second sample set, which are of different
wavelength bands. According to an embodiment of the disclosure, the
target nucleic acid detection method according to the disclosure
comprises the measurement step of synchronously measuring optical
signals from the first sample set and the second sample set. The
optical signal measured from the first sample set and the optical
signal measured from the second sample set have different
wavelength bands. The optical signals of different wavelength bands
mean optical signals generated from different labels. Thus, the
target nucleic acid detection method according to the disclosure
may comprise the step of synchronously detecting different labels
from the first sample set and the second sample set.
[0064] The synchronous measurement may encompass the case where
optical signals for two reaction areas are measured at the same
time. The synchronous measurement may also encompass the case where
although the times of measurement of optical signals for two
reaction areas do not simply overlap each other, the optical signal
for one reaction area is measured and, then, the optical signal for
the other reaction area is measured without any physical component,
e.g., a light source or filter for the device. The synchronous
measurement may also encompass the case where measurement data of
the optical signals for two reaction areas is treated as obtained
in substantially the same time period.
[0065] The target nucleic acid detection method of the disclosure,
which comprises (i) the step of performing a nucleic acid reaction
on a first sample set and a second sample set positioned in two
reaction areas thermally independent from each other, according to
independent protocols and (ii) the step of measuring an optical
signal from each sample set, wherein the measurement step comprises
the sub-step of synchronously measuring an optical signal from the
first sample set and an optical signal from the second sample set,
which are of different wavelength bands, is a brand-new method that
is not performed by conventional nucleic acid reaction detection
devices which lack an optical module or are configured to
simultaneously measure all the samples in the thermal block in the
device, at the same wavelength.
[0066] According to an embodiment of the disclosure, the measuring
step may comprise the step of synchronously measuring the optical
signals from the first sample set and the second sample set,
wherein the wavelength bands of the optical signals synchronously
measured from the first sample set and the second sample set are
different from each other and the step of measuring an optical
signal from one of the first sample set and the second sample set
without measuring an optical signal from the other of the first
sample set and the second sample set.
[0067] According to an embodiment of the disclosure, in the target
nucleic acid detection method according to the disclosure, the
measuring step may further comprise the step of measuring the
optical signal from the first sample set without measuring the
optical signal for the second sample set. According to an
embodiment of the disclosure, in the target nucleic acid detection
method according to the disclosure, the measuring step may further
comprise the step of measuring the optical signal from the second
sample set without measuring the optical signal for the first
sample set.
[0068] When real-time PCR is performed on the first sample set and
the second sample set according to separate protocols in the single
device, the periods of measurement of the optical signals for the
two sample sets differ from each other. In this case, the single
device may synchronously measure optical signals of different
wavelength bands from the first sample set and the second sample
set in some cycle and, in other cycle, may measure the optical
signal from one sample set of the first sample set and the second
sample set without measuring the optical signal for the other
sample set of the first sample set and the second sample set.
[0069] The wavelength band of the measured optical signal may
include the whole or part of the wavelength band of the emission
light generated from the used label. The wavelength band of the
measured optical signal may include the whole or part of the
wavelength band of the emission light generated from one or more
label selected from the group consisting of, but not limited to,
FAM.TM., TET.TM., VIC.TM., JOE.TM., HEX.TM. CY3, TAMRA.TM.,
ROX.TM., Texas Red, CY5, CY5.5, and Quasar 705.
[0070] The method according to the disclosure may apply to
real-time multiplex nucleic acid detection methods. Multiplex
nucleic acid detection means distinctively detecting, in real-time,
two or more target nucleic acids in one sample. To assay optically
real-time multiplex nucleic acid detection, there is needed the
step of measuring optical signals of two or more wavelength bands
respectively corresponding to the target nucleic acids in one
sample. Thus, according to an embodiment of the disclosure, the
measurement of an optical signal for the first sample set may be
performed by sequentially measuring optical signals of two or more
different wavelength bands for the first sample set. The number of
the different wavelength bands may be two or more or may be three
or more, or may be seven or less or may be six or less.
[0071] According to an embodiment of the disclosure, the first
sample set may comprise a plurality of samples. Measuring an
optical signal from the first sample set positioned in the first
reaction area may be simultaneously measuring optical signals from
two or more samples included in the first sample set. The number of
the samples included in the first sample set, although not
specifically limited, may be, e.g., 1 or 2 or more or may be 1563,
1000, 800, 500, 400, 384, 100, 96, 32 or less.
[0072] According to an embodiment of the disclosure, the second
sample set may comprise a plurality of samples. Measuring an
optical signal from the second sample set positioned in the second
reaction area may be simultaneously measuring optical signals from
two or more samples included in the second sample set. The number
of the samples included in the second sample set, although not
specifically limited, may be, e.g., 1 or 2 or more or may be 1563,
1000, 800, 500, 400, 384, 100, 96, 32 or less.
[0073] According to an embodiment of the disclosure, the nucleic
acid reaction detection device comprises an optical module capable
of detecting nucleic acid reaction in the samples in the first
reaction area and second reaction area. According to an embodiment
of the disclosure, the optical signal measurement is performed by
the optical module of the nucleic acid reaction detection device.
The optical module comprises a light source for illuminating the
sample in the reaction area with an excitation light and a detector
for sensing an emission light received from the sample in the
reaction area. The detector may be, e.g., a CCD (Charge Coupled
device), CMOS (Complementary Metal Oxide Semiconductor field effect
transistor) or photodiode. The light radiated from the light source
may also be referred to as an excitation light, and the light
emitted from the label excited by the excitation light may also be
referred to as an emission light.
[0074] To synchronously measure the optical signal from the first
sample set and the optical signal from the second sample set, which
are of different wavelength bands, different wavelength bands of
light need to be synchronously radiated to the first reaction area
where the first sample set is positioned and the second reaction
area where the second sample set is positioned, and the emission
lights generated from the first sample set and the second sample
set need to be synchronously sensed.
[0075] Thus, the light source of the optical module is
characterized by being able to synchronously radiate different
lights to the first reaction area where the first sample set is
positioned and the second reaction area where the second sample set
is positioned. Further, the detector of the optical module is
characterized by being able to synchronously sense the emission
lights generated from the first sample set and the second sample
set.
[0076] According to an embodiment of the disclosure, the optical
module comprises a plurality of light sources. At least two of the
plurality of light sources may produce excitation lights of
different wavelength bands from each other. For example, the
optical module may comprise a first light source and a second light
source. The wavelength bands of the lights radiated from the first
light source and the second light source may differ from each
other. Alternatively, the optical module may comprise a first light
source to a fourth light source. The wavelength bands of the lights
radiated from the first light source and the second light source
may differ from each other. Alternatively, the optical module may
comprise a first light source to a fourth light source. The
wavelength bands of the lights radiated from the first light source
to the fourth light source may differ from each other. The number
of the light sources comprised in the optical module is not
particularly limited but, as an example, may be two or more or may
be 100, 50, 40, 30, 20, 10 or less.
[0077] As such, when the optical module of the nucleic acid
reaction detection device used for the method according to the
disclosure comprises a plurality of light sources, and at least two
of the plurality of light sources are configured to produce
excitation lights of different wavelength bands from each other, it
may be possible to synchronously measure optical signals of
different wavelength bands from the first sample set and the second
sample set.
[0078] Meanwhile, according to an embodiment of the disclosure, the
optical module may comprise a plurality of detectors. Each of the
plurality of detectors may detect light emitted from a sample in a
predetermined and fixed area of the sample reaction module.
[0079] The detector senses the emission light generated from the
sample. The detector may be, e.g., a CCD (Charge Coupled device),
CMOS (Complementary Metal Oxide Semiconductor field effect
transistor) or photodiode.
[0080] In the method according to the disclosure, there may be
provided a plurality of detectors. When a plurality of detectors
are used, the number of samples sensed by each detector may be
reduced, so that more accurate detection is possible. The number of
the detectors, although not specifically limited, may be, e.g., 2,
3, 4, or more or may be 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9,
8, 7, 6 or less.
[0081] According to an embodiment of the disclosure, the optical
module comprises a plurality of detectors independently controlled,
and one or more different detectors among the plurality of
detectors may be assigned to each of the first reaction area and
the second reaction area. Optical signals may be measured from the
first sample set and second sample set by the one or more different
detectors assigned.
[0082] The plurality of detectors each may sense an emission light
generated from one reaction area. In the method according to the
disclosure, one or more detectors may be assigned to each reaction
area, and the two or more detectors may sense emission lights, with
each reaction area segmented. For example, where the nucleic acid
reaction detection device used for the method according to the
disclosure comprises two reaction areas thermally independent from
each other and comprises four detectors, a first detector and a
second detector may sense the emission light generated from the
sample positioned in the first reaction area, and a third detector
and a fourth detector may sense the emission light generated from
the sample positioned in the second reaction area.
[0083] In the method according to the disclosure, each detector may
be a detector that senses the emission light generated from the
sample in a predetermined and fixed detection area of the sample
reaction module. The detection area means an area where one
detector detects an emission light in the sample reaction module.
When the plurality of detectors each sense the emission light
generated from the sample positioned in the predetermined detection
area, this means that the detection area where each detector
performs detection in the sample reaction module is previously
determined. The samples positioned in the same detection area in
the sample reaction module may be detected by the same detector,
and the samples positioned in different detection areas may be
detected by different detectors. For example, a first detector may
detect only emission lights generated from a first detection area,
which is a fixed area in the sample reaction module but may not
detect emission lights generated from other area.
[0084] The nucleic acid reaction detection device comprises a
controller. The controller may be electrically connected with the
sample reaction module and the optical module. The controller
controls the temperatures of the first reaction area and the second
reaction area independently and controls optical signal measurement
of the sample in the first reaction area and optical signal
measurement of the sample in the second reaction area
independently. There may be provided one or more controllers.
[0085] According to an embodiment of the disclosure, the controller
is characterized by operating the nucleic acid reaction detection
device to perform an operation comprising at least one of the steps
of:
[0086] (i) measuring an optical signal for one sample set of the
first sample set and the second sample set without measuring an
optical signal for the other sample set and (ii) synchronously
measuring the optical signals from the first sample set and the
second sample set, wherein the wavelength bands of the optical
signals synchronously measured from the first sample set and the
second sample set are different from each other.
[0087] Reaction is performed on the sample positioned in the first
reaction area and the sample positioned in the second reaction area
by protocols independent from each other, and the first reaction
area and the second reaction area may individually or
simultaneously reach the light detection time. When it becomes time
to detecting optical signal only for one of the first reaction area
and the second reaction area, the controller operates to perform
the operation of measuring the optical signal for the sample in one
reaction area of the first reaction area and the second reaction
area without measuring the optical signal for the sample in the
other reaction area. Further, when it becomes time to detecting
optical signals for both the first reaction area and the second
reaction area, the controller operates to perform the operation of
measuring the optical signal for the sample in each reaction area.
At this time, the wavelength bands of the optical signals measured
from the first reaction area and the second reaction area may
differ from each other.
[0088] Step (c): Detection of Target Nucleic Acid
[0089] According to the disclosure, the measured optical signals
are analyzed, detecting the target nucleic acid in the sample
included in the first sample set and the second sample set.
[0090] The detection of the target nucleic acid means
quantitatively or qualitatively measuring the target nucleic acid
in the sample using the optical signals measured from the sample.
The quantitative or qualitative detection may be the detection of
the presence or absence of the target nucleic acid in the sample or
the detection of the amount of the target nucleic acid in the
sample.
[0091] The detection may be performed by a known detection
method.
[0092] The detection may be performed by a method of determining
that the target nucleic acid corresponding to the measured optical
signal is present in the sample when the value of the measured
optical signal is a predetermined threshold or more. Alternatively,
the detection may be performed by a method of calculating the
variation in the fluorescent signal, which is increased as the
nucleic acid reaction is repeated and determining whether there is
the target nucleic acid based on the calculated variation.
Alternatively, the detection may be performed by a method of
obtaining a standard curve using a reference sample including the
target nucleic acid in various concentrations, comparing the
standard curve with the optical signal measured from each sample,
and calculating the initial amount of the target nucleic acid
included in each sample. Alternatively, the detection may be the
detection of the target nucleic acid based on the optical signals
obtained by melting analysis between the target nucleic acid and
the signal-generating means in each of the nucleic acid reaction
repeated.
[0093] II. Nucleic Acid Reaction Detection Device
[0094] According to another embodiment of the disclosure, there is
provided a nucleic acid reaction detection device.
[0095] FIG. 1 is a view illustrating a nucleic acid reaction
detection device 10 according to an embodiment of the
disclosure.
[0096] Referring to FIG. 1, the nucleic acid reaction detection
device 10 according to the disclosure comprises a sample reaction
module 100 comprising a first reaction area and a second reaction
area thermally independent from each other, an optical module 200
capable of detecting nucleic acid reaction of all samples in the
sample reaction module, and one or more controllers 300
electrically connected with the sample reaction module and the
optical module. In the nucleic acid reaction detection device
according to the disclosure, the optical module is configured to be
able to synchronously measure an optical signal of a first
wavelength band from a sample in the first reaction area and an
optical signal of a second wavelength band from a sample in the
second reaction area, wherein the second wavelength band differs
from the first wavelength band. The controller is configured to
control the temperatures of the first reaction area and the second
reaction area independently and control optical signal measurement
of the sample in the first reaction area and optical signal
measurement of the sample in the second reaction area
independently.
[0097] According to an embodiment of the disclosure, the nucleic
acid reaction detection device 10 comprises the sample reaction
module 100. The sample reaction module 100 is a module that
receives samples which are to be analyzed and performs nucleic acid
reaction.
[0098] Referring to FIG. 2, the sample reaction module 100
comprises a first reaction area 111 and a second reaction area 112.
Each reaction area is thermally independent from another. A thermal
insulation material or air gap may be provided between the first
reaction area and the second reaction area to prevent heat
transfer. According to an embodiment of the disclosure, the sample
reaction module 100 comprises the first reaction area 111 and the
second reaction area 112 thermally independent from each other. The
sample reaction module 100 may comprise reaction areas 110 and heat
transfer modules 120.
[0099] According to an embodiment of the disclosure, the sample
reaction module 100 may be configured so that each of the first
reaction area 111 and the second reaction area 112 is able to
receive at least two or more samples. To that end, the sample
reaction module 100 comprises the reaction area 110 for receiving
the sample or a reaction vessel including the sample. The reaction
area 110 may comprise a plurality of receiving spaces 115. The
receiving space 115 for receiving the sample may be a well for
receiving the reaction vessel. Each reaction area may receive at
least two or more samples using the plurality of receiving spaces
115. Although not particularly limited, the number of the receiving
spaces 115 included in each reaction area may be 2 to 96, 2 to 84,
2 to 72, 2 to 64, 2 to 56, 2 to 48, 2 to 40, or 2 to 32. The sample
reaction module 100 adjusts the temperature of the received samples
and performs nucleic acid reaction. Thus, the reaction area 110 may
be formed of a metal or metal alloy with superior thermal
conductivity. The reaction area 110 may be, e.g., a thermal block.
The thermal block has a volume and is formed of a metal with
superior thermal conductivity. The thermal block may have a well
for receiving samples. A heat transfer module, e.g., a Peltier
element, may contact one surface of the thermal block to adjust the
temperature.
[0100] Another example of the reaction area 110 is a heating plate.
The heating plate may be formed of a plate for receiving samples
and a thin metal sheet attached to the plate. The heating plate may
be operated in such a manner that the plate is heated by applying
electric current to the thin metal sheet.
[0101] Another example of the reaction area 110 may be formed of
one or more chips or cartridges. An example of the cartridge is a
fluid cartridge comprising a flow channel.
[0102] The sample reaction module may further comprise the heat
transfer module 120 for supplying heat to the reaction area 110 or
absorbing heat from the reaction area 110. The heat transfer module
120 may switch the reaction area 110 into two or more temperatures.
The heat transfer module 120 may be a device capable of heating and
cooling both. The heat transfer module 120 may separately comprise
a heat supplying means for supplying heat and a heat absorbing
means for absorbing heat. According to an embodiment of the
disclosure, the heat transfer module 120 may be a Peltier element
capable of both heat supply and heat absorption to/from the
reaction area 110 and positioned in the form of a plate on the
bottom of the reaction area 110.
[0103] As shown in FIG. 2, in the nucleic acid reaction detection
device according to the disclosure, the first reaction area and the
second reaction area may be reaction areas 110 physically separated
from each other. The nucleic acid reaction detection device
according to the disclosure may comprise heat transfer modules 120
physically separated from each other to change the temperatures of
the first reaction area and the second reaction area independently.
Thus, in the nucleic acid reaction detection device according to
the disclosure, the first reaction area 111 and the second reaction
area 112 may be controlled thermally independently from each
other.
[0104] The reaction areas 111 and 112 may be controlled thermally
independently from each other. The reaction areas being controlled
thermally independently may mean that the temperature of each
reaction area may be individually controlled. For example, while
the first reaction area remains at 94.degree. C. at a first time,
the second reaction area may be cooled down from 94.degree. C. to
60.degree. C. or heated up from 60.degree. C. to 75.degree. C. or
may remain at a specific temperature. By so doing, a separate
nucleic acid reaction may be performed in each reaction area of the
nucleic acid reaction detection device. The number of reaction
areas included in the sample reaction module may range from 2 to
24, but is not limited thereto. For example, the number of reaction
areas included in the sample reaction module may be 2, 3, 4, 5, 6,
8, 10, 12, 16, 20, or 24. According to an embodiment, the nucleic
acid reaction detection device may comprise two or more reaction
areas, four or more reaction areas, six or more reaction areas, or
eight or more reaction areas. According to an embodiment, the
nucleic acid reaction detection device may comprise 24 or less
reaction areas, 20 or less reaction areas, 16 or less reaction
areas, or 12 or less reaction areas. According to an embodiment of
the disclosure, the sample reaction module may comprise other
additional reaction area(s) than the first reaction area and the
second reaction area. For example, the sample reaction module may
further comprise a third reaction area and a fourth reaction area.
The reaction areas each may be controlled thermally
independently.
[0105] Meanwhile, the sample reaction module 100 according to the
disclosure may further comprise a pressure lid 130. Referring to
FIG. 7, the pressure lid 130 provides pressure to the reaction
vessels containing samples positioned on the reaction area 110. The
pressure lid 130 may contact the covers of the reaction vessels and
press the covers of the reaction vessels, providing pressure to the
reaction vessels. The pressure lid 130 may maintain a high
temperature. For example, the pressure lid 130 may comprise a heat
plate (not shown) to maintain a temperature of 105.degree. C. The
pressure lid 130 comprises a plurality of holes 131. The holes 131
of the pressure lid 130 are formed in the positions corresponding
to the reaction vessels containing samples, positioned on the
reaction area 110.
[0106] Referring to FIG. 1, the nucleic acid reaction detection
device according to the disclosure comprises the optical module 200
capable of detecting nucleic acid reaction of the samples in the
sample reaction module.
[0107] The optical module 200 radiates excitation lights to the
samples in the sample reaction module 100 and senses the emission
lights generated by the excitation lights, thereby detecting the
nucleic acid reaction in the sample.
[0108] According to an embodiment of the disclosure, the optical
module 200 may comprise a light source 210 for illuminating the
sample in the reaction area with an excitation light and a detector
220 for sensing an emission light received from the sample in the
reaction area.
[0109] The light source 210 illuminates the sample in the reaction
area with an excitation light. The excitation light refers to the
light generated from the light source 210 and reaching the sample
positioned in the sample receiving space 115. The label in the
sample may be excited by the excitation light. The excited label
may emit an optical signal, e.g., fluorescent light. The light
radiated from the light source 210 may also be referred to as an
excitation light, and the light emitted from the label excited by
the excitation light may also be referred to as an emission
light.
[0110] Referring to FIG. 3, there may be provided a plurality of
light sources 210. In such a case, the optical module 200 is
configured so that, when a first light source 210a radiates light
to the first reaction area 111, a second light source 210b may
radiate light of a different wavelength band from that of the first
light source 210a to the second reaction area 112. In this case,
the optical module 200 may synchronously measure the optical signal
of the first wavelength band generated from the sample in the first
reaction area and the optical signal of the second wavelength band
(which differs from the first wavelength band) generated from the
sample in the second reaction area 112. According to an embodiment
of the disclosure, at least two of the plurality of light sources
210 may radiate excitation lights of different wavelength bands
from each other. The excitation lights of different wavelength
bands may excite different labels. The excited label may emit an
emission light, which is an optical signal, and the different
labels emit emission lights of different wavelength band. For
example, emission light of a first wavelength band may excite a
first label, and emission light of a second wavelength band may
excite a second label. The number of the light sources 210 included
in the optical module 200 is not particularly limited but, as an
example, may be two or more or may be 100, 50, 40, 30, 20, 10 or
less.
[0111] According to the disclosure, the optical module 200 may
comprise a light source wheel 213. The light source wheel 213
receives the plurality of light sources 210. The light source wheel
213 receives the light sources 210 may mean that the light sources
210 are attached or fixed to the light source wheel 213. The light
source wheel 213 may be connected to a power transmission device,
e.g., a motor, to rotate and may thus switch the light sources to
illuminate each reaction area with light.
[0112] According to an embodiment of the disclosure, the wavelength
band of the excitation light radiated to the first reaction area
111 and the wavelength band of the excitation light radiated to the
second reaction area, each, may be independently determined. For
example, referring to FIGS. 2 and 4, a plurality of light sources
may be configured to radiate excitation lights of different
wavelength bands to the first reaction area, and another plurality
of light sources may be separately configured to radiate excitation
lights of different wavelength bands to the second reaction area.
Specifically, as shown in FIG. 4A, the sample reaction module 100
according to the disclosure may comprise two reaction areas 111 and
112, and the optical module 200 comprises two light source wheels
213 each comprising a plurality of light sources 210 to allow each
wavelength band of excitation light radiated to each reaction area
to be independently determined. As another example, as shown in
FIG. 4B, the sample reaction module 100 according to the disclosure
may comprise four reaction areas 111, 112, 113, and 114. In this
case, the optical module 200 may comprise four light source wheels
213 each comprising a plurality of light sources 210 for the four
reaction areas.
[0113] The sample reaction module according to the disclosure may
comprise four reaction areas. In this case, the optical module may
comprise four light source wheels each comprising a plurality of
light sources for the four reaction areas.
[0114] According to an embodiment of the disclosure, the wavelength
band of the excitation light radiated to the first reaction area
and the wavelength band of the excitation light radiated to the
second reaction area may be synchronously changed. For example, as
shown in FIG. 5, a plurality of light sources capable of radiating
excitation lights of different wavelength bands may be connected to
a moving member, e.g., the light source wheel 213 to be
individually positioned on two or more reaction areas. For example,
where the light source wheel 213 comprises two light sources for
radiating light of different wavelength bands, the light sources
may be positioned on the first reaction area 111 and the second
reaction area 112 and may switch their positions (refer to FIG.
5A). Besides, as shown in FIG. 5B, the light source wheel 213 may
comprise four light sources for radiating light of different
wavelength bands and may synchronously radiate light of different
wavelength bands to the first reaction area 111 and the second
reaction area 112. In such a case, as the light source wheel 213
rotates, the light source 210a may be repositioned from the first
reaction area 111 to the second reaction area 112, and another
light source 210c may be positioned on the first reaction area
111.
[0115] As shown in FIG. 5C, the light source wheel 213 may comprise
four light sources for radiating light of different wavelength
bands And the four light sources may be respectively positioned on
different reaction areas 111, 112, 113, and 114 to synchronously
radiate light of different wavelength bands to the four reaction
areas. In this case, as the light source wheel 213 rotates, the
light source 210a may be moved from the first reaction area 111 to
the second reaction area 112, the light source 210b may be moved
from the second reaction area 112 to the third reaction area 113,
the light source 210c may be moved from the third reaction area 113
to the fourth reaction area 114, and the light source 210d may be
moved from the fourth reaction area 114 to the first reaction area
111. In the disclosure, such repositioning may be referred to as
"cyclic movement." Such configuration may economically adjust the
number of light sources used as compared with the number of the
reaction areas.
[0116] Referring to FIG. 6, the light source 210 comprises a light
emitting element 211. There may be provided a plurality of light
emitting elements 211. The light emitting element may be a light
emitting diode (LED). The light emitting element may be a light
emitting element that selectively radiates light of a wavelength
band that is to be emitted from the light source 210.
[0117] The light source 210 may comprise a filter 212. The filter
212 allows the reaction area to be illuminated with a light of a
wavelength band intended by the light source 210. The filter 212
may be a filter that selectively transmits the light of the
intended wavelength band among the lights generated from the light
emitting element. According to an embodiment of the disclosure, the
light source may comprise a plurality of filters, and the filters
may be switched and used depending on the label used in the sample
in the reaction area.
[0118] According to an embodiment of the disclosure, the optical
module 200 comprises a plurality of light sources. At least two
light sources of the plurality of light sources may produce
excitation lights of different wavelength bands. According to an
embodiment of the disclosure, the optical module 200 may
selectively radiate excitation lights of two or more wavelength
bands. The optical module 200 may comprise a plurality of light
sources 210. The optical module 200 may comprise two or more light
sources 210 that radiate light of different wavelength bands. In
the optical module 200, each of light sources may be configured by
selecting a light emitting element 211 that generates light of a
specific wavelength band for the each of light sources. In the
optical module 200, the filter 212 of each light source may be
selected and configured as a filter that selectively transmits
light of a different wavelength band for each light source. The
plurality of light sources 210 that radiate light of different
wavelength bands may be fastened to the light source wheel 213, and
the light source wheel 213 may move the light sources 210 to allow
light of different wavelength bands to be sequentially radiated to
the reaction areas 111 and 112. By such a configuration, the
optical module may selectively radiate excitation lights of
different wavelength bands to one reaction area. Thus, the device
according to the disclosure may detect a plurality of target
nucleic acids that are detected by different labels in one
sample.
[0119] According to an embodiment of the disclosure, the optical
module may simultaneously radiate excitation lights to the
plurality of samples in the same reaction area. According to an
embodiment of the disclosure, the optical module may comprise a
plurality of light sources, and one of the plurality of light
sources may simultaneously radiate excitation lights to the
plurality of samples in one reaction area. Thus, the optical module
may detect optical signals from a plurality of samples more quickly
than when one light source detects optical signals while
sequentially radiating excitation lights to a plurality of
samples.
[0120] Referring to FIG. 2, the optical module 200 according to the
disclosure comprises a detector 220. The detector senses light.
Specifically, the detector senses emission lights generated from
samples. The detector may be, e.g., a CCD (Charge Coupled device),
CMOS (Complementary Metal Oxide Semiconductor field effect
transistor) or photodiode.
[0121] According to an embodiment of the disclosure, one detector
may sense all of the emission lights generated from all the
reaction areas. In this case, one detector is configured to
separately detect different wavelength bands of emission lights
generated from the plurality of reaction areas.
[0122] According to an embodiment of the disclosure, the optical
module may comprise two or more detectors 220a and 220b as shown in
FIG. 2. In such a case, each of the lights generated from the first
reaction area 111 and the second reaction area 112 may be detected
using an independent detector. According to an embodiment of the
disclosure, the optical module comprises a plurality of detectors
independently controlled, and one or more different detectors among
the plurality of detectors may be assigned to each of the first
reaction area and the second reaction area. Optical signals may be
measured from the first sample set and second sample set by the one
or more different detectors assigned.
[0123] According to an embodiment of the disclosure, two or more
detectors may be used in one reaction area. In this case, two or
more detectors may sense emission lights from one reaction area,
with the reaction area separated.
[0124] The optical module is configured to be able to synchronously
measure an optical signal of a first wavelength band from the
sample in the first reaction area and an optical signal of a second
wavelength band from the sample in the second reaction area, the
second wavelength band different from the first wavelength
band.
[0125] As shown in FIG. 7, according to an embodiment, the optical
module may comprise a plurality of detectors 220a and 220b, and
each of the plurality of detectors may be configured to sense the
emission light generated from the sample positioned in a fixed and
predetermined detection area 111 or 112 in the sample receiving
module. The emission light refers to light that is generated from
the sample and reaches the detector. Specifically, the emission
light is light that is generated by a signal-generating means,
e.g., the label in the sample. In the disclosure, the emission
light corresponds to a signal or an optical signal.
[0126] The optical module 200 may be configured so that each of the
plurality of detectors senses the emission light generated from the
sample positioned in the respectively predetermined detection area.
The detection area means an area where one detector detects an
emission light in the sample reaction module. When the plurality of
detectors each sense the emission light generated from the sample
positioned in the predetermined detection area, this means that the
detection area where each detector performs detection in the sample
reaction module is previously determined. The samples positioned in
the same detection area in the sample reaction module may be
detected by the same detector, and the samples positioned in
different detection areas may be detected by different detectors.
In each detector, the detection area for sensing emission lights
may be fixed. For example, a first detector may detect only
emission lights generated from a first detection area, which is a
fixed area in the sample reaction module but may not detect
emission lights generated from other area.
[0127] According to an embodiment of the disclosure, one detection
area may be included in one reaction area. In other words, the
detection area may not be present over two or more reaction areas.
It is preferable to measure the emission lights generated from
different reaction areas by different detectors rather than
measuring all of the lights synchronously generated from two or
more reaction areas by one detector. For example, a first detector
220a may be configured to detect only emission lights generated
from the first reaction area 111, but not emission lights generated
from other reaction area, e.g., the second reaction area 112.
Further, a second detector 220b may be configured to detect only
emission lights generated from the second reaction area 112, but
not emission lights generated from other area, e.g., the first
reaction area 111.
[0128] According to an embodiment of the disclosure, one reaction
area may comprise one or more detection areas. The number of
detectors in charge of one reaction area may be determined
depending on the shape and size of the reaction area and detection
schemes, and the number of detectors may be one or two or more.
[0129] According to an embodiment of the disclosure, the optical
module 200 may additionally comprise a filter 230 which may be
positioned on an emission light path 270 to the detector. The
filter may be a filter that selectively transmits the wavelength
band of light, as the target for measurement, among the emission
lights. According to an embodiment of the disclosure, the optical
module may comprise a plurality of filters 230, and the filter
positioned on the emission light path 270 may be replaced depending
on the wavelength of the emission light to be measured. The
replacement of the filter may be performed in such a manner that a
plurality of filters 230 are attached to a filter wheel 235, and
the filter wheel 235 is rotated as necessary.
[0130] Referring to FIG. 7, the optical module 200 may comprise a
beam splitter 240. The beam splitter 240 reflects part of the
excitation lights or emission lights, establishing an intended
optical path. For example, as shown in FIG. 7, the excitation light
generated from the light source 210 passes through the beam
splitter 240 and reaches the sample received in the sample
receiving space 115 of the reaction area 110, forming an excitation
light path 260. The emission light emitted from the sample is
reflected by the beam splitter 240 to the detector 220, forming an
emission light path 270.
[0131] The optical module 200 may comprise a light blocking means
250. The light blocking means 250 may be configured so that the
excitation light radiated from, e.g., the first reaction area 111
is not radiated to the second reaction area 112 or other reaction
area. Further, the light blocking means 250 may be configured so
that, when the excitation light is radiated from the light source
210a to the first reaction area 111, other lights than the radiated
excitation light are not radiated to the first reaction area 111.
The light blocking means 250 may be disposed between the adjacent
light sources. Alternatively, the light blocking means 250 may be
disposed between the adjacent detectors. According to an
embodiment, the light blocking means 250 may be shaped as a
rectangular or circular tube. According to an embodiment, the light
blocking means 250 may be shaped as a combination of a plurality of
tubes.
[0132] Referring to FIG. 8, the nucleic acid reaction detection
device according to the disclosure comprises the controller 300.
There may be provided one or more controllers 300 electrically
connected with the sample reaction module 100 and the optical
module 200. The controller may be, e.g., a computer, a
micro-processor, or a programmable logic device.
[0133] The controller 300 controls each of the temperatures of the
first reaction area 111 and the second reaction area 112
independently. Specifically, the controller 300 may be electrically
connected with the heat transfer module 120a of the first reaction
area and control the direction and magnitude of the current applied
to the heat transfer module 120a of the first reaction area 111,
thereby controlling the temperature of the first reaction area 111.
Further, the controller 300 is electrically connected with the heat
transfer module 120b of the second reaction area 112 and control
the direction, magnitude, and time of the current applied to the
heat transfer module 120b of the second reaction area 112
independently from the control of the temperature of the first
reaction area 111, thereby controlling the temperature of the
second reaction area 112. The controller 300 receives the protocol
for the first sample set positioned in the first reaction area,
controls the temperature of the first reaction area 111 according
to the protocol for the first sample set, receives the protocol for
the second sample set positioned in the second reaction area, and
controls the temperature of the second reaction area 112 according
to the protocol for the second sample set.
[0134] Further, according to an embodiment of the disclosure, the
controller 300 is configured to control the optical signal
measurement on the sample in the first reaction area and the
optical signal measurement on the sample in the second reaction
area independently. Specifically, the controller 300 may move the
light source wheel 213 to move the first light source 210a or
second light source 210b to a predetermined position so that the
light of wavelength band capable of generating an optical signal
from the sample in the first reaction area may be radiated to the
first reaction area. Further, the controller 300 may supply current
to the light source 210a so that the light source 210a is able to
radiate the excitation light 260a to the sample. Further, the
controller 300 may move the filter wheel 235a to position the
filter 230a, which is able to selectively transmit the emission
light 270a, on the emission light path 270a and supply current to
the detector 220a so that the detector 220a is able to sense the
emission light 270a generated from the sample by the excitation
light 260a radiated from the light source 210a. To measure the
optical signal from the sample in the second reaction area, the
controller may move the light source wheel 213 and the filter wheel
230b and supply current to the light source 210b and the detector
220b as described above.
[0135] The controller 300 may operate the sample reaction module
100 and the optical module 200 of the nucleic acid reaction
detection device so that the temperature control and optical signal
measurement on each reaction area are performed in the order
determined by the protocol.
[0136] The controller 300 may receive the protocol for each sample
and according to the received protocol independently control the
temperature of each reaction area of the sample reaction module and
independently control the optical signal measurement on each
reaction area of the optical module. For example, the controller
300 may operate the device to selectively perform optical signal
measurement only on the sample in the first reaction area without
measuring the optical signal for the sample in the second reaction
area. Alternatively, the controller 300 may operate the device to
selectively perform optical signal measurement only on the sample
in the second reaction area without measuring the optical signal
for the sample in the first reaction area. Or, the controller 300
may operate the device to synchronously perform optical signal
measurement on the samples in the first reaction area and the
second reaction area.
[0137] According to an embodiment of the disclosure, the controller
may receive distinct protocols for the first reaction area and the
second reaction area, control temperatures of the first reaction
area and the second reaction area according to the received
protocols independently, and operate the nucleic acid reaction
detection device to perform an operation comprising at least one of
the steps of, based on the received protocols for all the reaction
areas:
[0138] (i) measuring an optical signal for a sample in one reaction
area of the first reaction area and the second reaction area
without measuring an optical signal for a sample in the other
reaction area; and (ii) synchronously measuring an optical signal
of a first wavelength band from a sample in the first reaction area
and an optical signal of a second wavelength band from a sample in
the second reaction area, wherein the second wavelength band is
different from the first wavelength band.
[0139] Reaction is performed on the sample positioned in the first
reaction area and the sample positioned in the second reaction area
by protocols independent from each other, and the first reaction
area and the second reaction area may individually or
simultaneously reach the light detection time. When it becomes time
to detecting optical signal only for one of the first reaction area
and the second reaction area, the controller 300 operates to
perform the operation of measuring the optical signal for the
sample in one reaction area of the first reaction area and the
second reaction area without measuring the optical signal for the
sample in the other reaction area. Further, when it becomes time to
detecting optical signals for both the first reaction area and the
second reaction area, the controller 300 operates to perform the
operation of measuring the optical signal for the sample in each
reaction area. At this time, the wavelength bands of the optical
signals measured from the first reaction area and the second
reaction area may differ from each other.
[0140] According to an embodiment of the disclosure, the controller
may be configured to control the sample reaction module and optical
module to be able to selectively perform optical signal measurement
on one area of the first reaction area and the second reaction area
under the condition where the temperatures of the first reaction
area and the second reaction area differ from each other. For
example, referring to FIG. 8, the controller 300 may control the
light source 260a, filter wheel 230a, and detector 220a for the
first reaction area to measure the optical signal for the first
reaction area while simultaneously controlling the heat transfer
module 120 of the second reaction area 112 to allow a nucleic acid
reaction to occur in the second reaction area.
[0141] Further, the controller may operate the device to be able to
detect a plurality of target nucleic acids in one sample.
Specifically, the controller may allow optical signals of two or
more different wavelength bands to be measured sequentially for one
reaction area. According to an embodiment of the disclosure, the
controller may be configured to control the sample reaction module
and optical module to be able to sequentially measure optical
signals of two or more different wavelength bands for one area of
the first reaction area and the second reaction area under the
condition where the temperatures of the first reaction area and the
second reaction area differ from each other.
[0142] According to an embodiment, referring to FIG. 8, at a first
time, the controller may move the first light source 210a to the
position where the excitation light may be radiated to the first
reaction area 111 and applies current to allow the first light
source 210a to radiate an excitation light. Then, the controller
may drive the filter wheel 235a to position the filter, which is
able to selectively transmit the emission light generated from the
sample by the excitation light from the first light source 210a, on
the excitation light path 270a and apply current to the detector
220a, thereby measuring the optical signal from the first reaction
area 111. After the optical signal measurement using the first
light source 210a is finished, the controller drives the light
source wheel 213 to move the second light source 210b to the
position where it may radiate the excitation light to the first
reaction area 111 at a second time and applies current to allow the
second light source 210b to radiate the excitation light. Then, the
controller may drive the filter wheel 235a to position the filter,
which is able to measure the emission light generated from the
sample by the excitation light from the second light source 210b,
on the excitation light path 270a and apply current to the detector
220a, thereby measuring the optical signal from the first reaction
area 111. While the above-described optical signal measurement is
carried out on the first reaction area 111, the controller 300 may
change the temperature of the second reaction area 112 and perform
nucleic acid reaction.
[0143] By the above-described operations of the controller 300, the
nucleic acid reaction detection device 10 may detect each of a
plurality of targets for each of a plurality of sample sets where
optical signal detection on the plurality of targets and nucleic
acid reaction are performed according to different protocols.
[0144] While embodiments of the disclosure have been described
above, it will be easily appreciated by one of ordinary skill in
the art that the disclosure is not limited thereto. Thus, the scope
of the invention is defined by the appended claims and equivalents
thereof.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0145] This application claims priority from Korean Patent
Application No. 10-2019-0078284, filed on Jun. 28, 2019, and Korean
Patent Application No. 10-2019-0064472, filed on May 31, 2019,
Korean Patent Application No. 10-2019-0162094, filed on Dec. 6,
2019, in the Korean Intellectual Property Office, the disclosure of
which is incorporated herein by reference in its entirety.
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