KR102559634B1 - Apparatus and method for pcr diagnosis based on multiwavelength light source - Google Patents

Abstract

An apparatus for PCR diagnosis according to an embodiment of the present disclosure includes a multi-wavelength light source outputting a first light source signal and a second light source signal having different wavelengths, a transmitter for applying the first light source signal and the second light source signal to a DNA sample, a code generator for generating a first code signal and a second code signal corresponding to the first and second light source signals, respectively, and a first delay signal and a second code signal respectively corresponding to the first and second code signals. A delay controller that generates a delay signal, and a receiver that compares first and second fluorescence signals emitted from the DNA sample with the first and second delay signals, and cumulatively integrates the first fluorescence signal or the second fluorescence signal based on the comparison result, wherein the first fluorescence signal is emitted from the DNA sample in response to the first light source signal, and the second fluorescence signal is emitted from the DNA sample in response to the second light source signal.

Description

Apparatus and method for PCR diagnosis based on multi-wavelength light source {APPARATUS AND METHOD FOR PCR DIAGNOSIS BASED ON MULTIWAVELENGTH LIGHT SOURCE}

The present disclosure relates to a diagnostic device, and more particularly, to a device and method for PCR diagnosis based on a multi-wavelength light source.

Molecular diagnosis technology is a technology that analyzes molecules that cause diseases in the body, such as diseases caused by viruses and other genetic diseases. Molecular diagnostic techniques allow DNA amplification to analyze with high accuracy whether or not disease-causing DNA is present. Molecular diagnosis technology pre-processes a bio sample to be measured to extract DNA, and then replicates and amplifies a desired portion of the extracted DNA using a polymerase chain reaction (PCR). After attaching a fluorescent substance (eg, SYBR green) to the amplified DNA, the intensity of the fluorescence signal emitted by an optical method is measured. When there is emission of fluorescence corresponding to the attached phosphor, it is determined that the sample contains DNA causing disease.

The more DNA is amplified, the more fluorescent substances can be attached and the intensity of fluorescence can increase. In the case of general PCR, the amplification process proceeds in 30 cycles, and 2 ³⁰ DNA chains are copied. When using conventional PCR, it takes about 4 hours to prepare a bio sample and proceed with 30 cycles of amplification. In addition, it is also possible to detect multiple DNAs in one sample at once. For this purpose, it is common to configure two or more phosphors and use multi-wavelength light sources. A multi-wavelength light source used in conventional PCR is continuous light (white light).

However, when continuous light is used to detect a plurality of DNAs at once, there is a problem in that signals generated from multiple phosphors as well as signals generated from phosphors not attached to DNA chains are mixed to generate noise. In addition, there is a problem in that fluorescence intensity of continuous light of the phosphor decreases over time (photo bleaching). Therefore, there is a need for a technique capable of accurately and efficiently detecting a signal generated from a phosphor while minimizing a PCR amplification cycle.

US Patent Publication US 2019/0383739 A1 (Jonathan M. Rothberg), "Optical system and assay chip for probing, detecting and analyzing molecules", 2019.12.19 US Patent Publication US 2018/0031476 A1 (Reuven Duer), "Waveguide-based detection system with scanning light source", 2018.02.01

The present disclosure provides an apparatus and method for PCR diagnosis capable of minimizing a DNA amplification cycle and improving detection accuracy in order to simultaneously analyze a plurality of DNAs.

An apparatus for PCR diagnosis according to an embodiment of the present disclosure includes a multi-wavelength light source outputting a first light source signal and a second light source signal having different wavelengths, a transmitter for applying the first light source signal and the second light source signal to a DNA sample, a code generator for generating a first code signal and a second code signal corresponding to the first and second light source signals, respectively, a first fluorescence signal and a second fluorescence signal emitted from the DNA sample, and a first delay signal. And a receiver that calculates a correlation function between the second delay signals, determines whether each of the first and second fluorescence signals, and each of the first and second delay signals corresponds to the same code signal based on the value of the calculated correlation function, and cumulatively integrates the first and second fluorescence signals over a period in which the level of the delay signal corresponding to the same code signal is maintained at a logic high value, based on the determination result; Is emitted from the DNA sample in response to the first light source signal, and the second fluorescence signal is emitted from the DNA sample in response to the second light source signal.

A method for PCR diagnosis according to an embodiment of the present disclosure includes generating a plurality of code signals, applying a plurality of light source signals having different wavelengths to a DNA sample at different times based on the plurality of code signals, generating a plurality of delay signals respectively corresponding to the plurality of code signals, calculating a correlation function between a fluorescence signal emitted from the DNA sample and the plurality of delay signals, and based on the value of the calculated correlation function, the fluorescence signal and each of the plurality of signals are identical to each other. Determining whether the fluorescent signal corresponds to a signal, and cumulatively integrating the fluorescence signal over a period in which a level of a delay signal corresponding to the same code signal is maintained at a logic high value based on the determination result.

According to an embodiment of the present disclosure, a problem in which fluorescence intensity of continuous light of a phosphor decreases over time may be improved.

In addition, according to an embodiment of the present disclosure, it is possible to detect only desired fluorescence without a separate fluorescence filter, so detection accuracy of PCR diagnosis can be improved.

1 shows a device for PCR diagnosis according to an embodiment of the present disclosure.
FIG. 2 shows an example of a code signal generated by the code generator of FIG. 1 .
Figure 3 shows the structure of the optical fiber included in the device of Figure 1.
4 conceptually illustrates the operation of a device for PCR diagnosis according to an embodiment of the present disclosure.
5 conceptually illustrates the operation of a device for PCR diagnosis according to another embodiment of the present disclosure.

In the following, embodiments of the present disclosure will be described clearly and in detail so that those skilled in the art can easily practice the present disclosure.

Components described with reference to terms such as unit, unit, module, block, ~or, ~er, etc. used in the detailed description and functional blocks shown in the drawings may be implemented in the form of software, hardware, or a combination thereof. Illustratively, the software may be machine code, firmware, embedded code, and application software. For example, the hardware may include an electrical circuit, an electronic circuit, a processor, a computer, an integrated circuit, integrated circuit cores, a pressure sensor, an inertial sensor, a microelectromechanical system (MEMS), a passive component, or a combination thereof.

1 shows an apparatus 100 for PCR diagnosis according to an embodiment of the present disclosure. The apparatus 100 may apply the light source signal LS to the DNA sample 10 including a plurality of DNAs to which a phosphor is attached. The phosphor attached to each DNA of the DNA sample 10 may emit a fluorescence signal FS in response to the input light source signal LS. The device 100 may receive a fluorescence signal (FS) emitted from the DNA sample 10 and may detect DNA to be analyzed. Apparatus 100 may include a transmitter 110 , a code generator 120 , a delay controller 130 , and a receiver 140 .

The transmitter 110 may include a plurality of multi-wavelength light sources for applying to the DNA sample 10 . For example, each of the plurality of light sources may be any one of a laser diode (LD) and a light emitting diode (LED) having a limited wavelength range. Hereinafter, for a clear description, it is assumed that a plurality of light sources in the present disclosure are LD, but the present disclosure is not limited thereto. In addition, the light source of the present disclosure hereinafter means a multi-wavelength light source. The number of the plurality of light sources may be equal to the number of different phosphors attached to the plurality of DNAs of the DNA sample 10 .

Signals output from the plurality of light sources of the transmitter 110 may be modulated based on the code signal CODE received from the code generator 120 . The transmitter 110 may include at least one of circuitry, software, and firmware for modulating signals output from the plurality of light sources based on the code signal CODE.

For example, the level of a signal output from the plurality of light sources may be modulated so that the level of the code signal CODE is the same as the original level while maintaining the logic high value and becomes 0 while the level of the code signal CODE maintains the logic low value. For example, signals output from a plurality of light sources may be modulated by a pulse amplitude modulation (PAM) method based on a code signal (CODE).

The transmitter 110 may include a plurality of optical fiber bundles for applying the modulated light source signals LS to the DNA sample 10 . Each light source signal LS having a different wavelength may be transferred through a corresponding fiber bundle and combined in one output fiber using wavelength division multiplexing (WDM). The output optical fiber may include a splitter at an end thereof, and each light source signal LS having a different wavelength may be applied to a plurality of DNAs through the splitter.

The code generator 120 may generate a code signal CODE for modulating signals output from a plurality of light sources of the transmitter 110 . For example, the code signal CODE may be a pulse signal having a logic high value or a logic low value. The code generator 120 may modulate signals output from the light sources by applying a code signal CODE to each light source having a different wavelength.

Specifically, the level of the modulated light source signal LS may be the same as the level of the signal originally output from the light source while the level of the corresponding code signal CODE is maintained at the logic high value, and may be 0 while the level of the corresponding code signal CODE is maintained at the logic low value. In other words, each modulated light source signal LS may be applied to the DNA sample 10 while the level of the code signal CODE is maintained at a logic high value.

For example, a time period during which the level of the code signal CODE is maintained at a logic high value may be equal to a length of a predetermined reference time period. For example, the length of the predetermined reference time may be equal to or longer than the lifetime of a phosphor attached to DNA to be applied. In addition, the time at which the signal level starts to rise from the logic low value to the logic high value may be different for each code signal CODE applied to each light source having a different wavelength.

As a result, each light source signal LS having a different wavelength may be temporally dispersed and applied to the DNA sample 10 . Furthermore, the code generator 120 may transmit the generated code signal CODE to the delay controller 130 . The code signal CODE will be described in more detail with reference to FIG. 2 .

Different phosphors may be attached to each of the plurality of DNAs of the DNA sample 10 . Different phosphors may absorb light source signals LS having different wavelengths. A phosphor attached to each DNA may absorb a related light source signal (LS) and emit a fluorescence signal (FS). For example, the phosphor may be a cyber green (SYBR green) phosphor that absorbs a light source having a wavelength of 530 nm or less and emits a fluorescence signal having a wavelength of 550 nm or more.

Therefore, the phosphor attached to each DNA of the present disclosure can absorb light source signals LS having different wavelengths at different times determined based on the code signal CODE, and can emit a fluorescence signal FS corresponding to the wavelength of the absorbed light source signal LS. Fluorescence signals FS emitted from each phosphor may be provided to the receiver 140 .

The delay controller 130 may receive the code signal CODE generated by the code generator 120 and generate a delay signal DELAY corresponding to the code signal CODE. For example, the delay signal DELAY may be a signal obtained by delaying the code signal CODE by a reference time. The delay controller 130 may transmit the delay signal DELAY to the receiver 140 .

The receiving unit 140 may receive fluorescence signals FS emitted from phosphors attached to each DNA of the DNA sample 10 . The receiver 140 may include a plurality of optical fiber bundles to receive the fluorescence signal FS. A detection array (eg, a silicon detection array) for converting the fluorescence signal FS into an electrical signal may be positioned at the ends of the plurality of optical fiber bundles. Accordingly, the fluorescence signal provided from the DNA sample 10 may be converted into an electrical signal in the detection array through the plurality of optical fiber bundles. Also, the receiver 140 may receive the delay signal DELAY from the delay controller 130 .

The receiver 140 may perform correlation integral on the fluorescence signal FS and the delay signal DELAY. First, the receiver 140 may calculate a correlation function between the fluorescence signal FS and the delay signal DELAY. By calculating the correlation function between the fluorescence signal FS and the delay signal DELAY, the receiving unit 140 can determine whether or not the coding of the input fluorescence signal FS and the delay signal DELAY match (that is, the fluorescence signal FS and the delay signal DELAY correspond to the same code signal CODE).

Next, when the coding of the input fluorescence signal FS and the delay signal DELAY match, the receiving unit 140 may accumulate and integrate the fluorescence signal FS for sections in which the level of the delay signal DELAY is maintained at a logic high value. Accordingly, while the level of the delay signal DELAY is maintained at the logic high value, the corresponding level of the fluorescence signal FS may be accumulated. The receiver 140 may include at least one of a circuit, software, or firmware for calculating a correlation function between the fluorescence signal FS and the delay signal DELAY and cumulatively integrating the fluorescence signal FS.

Since the fluorescence signal FS is cumulatively integrated, a gain can be increased and a DNA replication cycle can be minimized. Also, since the delay signal DELAY is a signal in which the code signal CODE is delayed by a predetermined reference time, a signal obtained by accumulative integration of the fluorescence signal FS may be a signal from which the light source signal LS applied to the corresponding phosphor is removed. Therefore, the receiver 140 of the present disclosure may not include a filter for removing the light source signal LS that was separately applied to the phosphor, and the signal-to-noise ratio (SNR) may be improved.

FIG. 2 shows an example of a code signal generated by the code generator 120 of FIG. 1 . As described with reference to FIG. 1 , the code signal CODE of the present disclosure may be a pulse signal having a logic high value (indicated by “1” in FIG. 2 ) or a logic low value (indicated by “0” in FIG. 2 ). In FIG. 2 , a horizontal axis may represent time, and a vertical axis may represent a signal level. Hereinafter, it will be described with reference to FIG. 1 together with FIG. 2 .

For clarity, it is assumed that the transmitter 110 includes six light sources LD1 to LD6 having different wavelengths, and each of the six light sources LD1 to LD6 outputs first to sixth light source signals LS1 to LS6. Also, it is assumed that the code generator 120 generates six code signals CODE1 to CODE6 respectively corresponding to the six light source signals LS1 to LS6. However, the present disclosure is not limited thereto, and the number of light sources or code signals may vary.

For convenience of description, the first code signal CODE1 will be described as an example. The first code signal CODE1 may be divided into three sections T1 to T3. The first period T1 is a period in which the signal level is a logic high (“1”) value, and the length of the first period T1 may be determined according to the length of the reference time described with reference to FIG. 1 . The first code signal CODE1 is applied to the first light source LD1 so that the first light source signal LS1 is applied to the corresponding phosphor attached to the DNA during the first period T1.

As described with reference to FIG. 1 , the length of the reference time during which the signal level is maintained at the logic high (“1”) value may be equal to or longer than the fluorescence lifetime of the phosphor corresponding to the first light source signal LS1. Also, the period in which the signal level is a logic high (“1”) value cannot be continued for a time longer than the reference time. Accordingly, after the signal level of the first code signal CODE1 is maintained at the logic high (“1”) value during the reference, a period in which the signal level is the logic low (“0”) value may follow. During the first period T1, the signal levels of the second to sixth code signals CODE2 to CODE6 may be maintained at a logic low (“0”) value. Thus, only the first light source signal LS1 may be applied to the DNA sample 10, and the second to sixth light source signals LS2 to LS6 may not be applied to the DNA sample 10.

As shown in FIG. 2, the second period T2 is a period in which the signal level is a logic low (“0”) value, and the length of the second period T2 is the length of the first period T1 (ie, the length of the reference time). In response to the first light source signal LS1 applied during the first period T1, the phosphor attached to the DNA may emit a first fluorescence signal FS1 during the second period T2. A part of the first fluorescence signal FS1 is shown in the hatched area in FIG. 2 .

Furthermore, the signal level of the first delay signal (not shown) output from the delay controller 130 based on the first code signal CODE1 may be logic high (“1”) in the second period T2. In the second period T2, the first delay signal and the first fluorescence signal FS1 may be compared by the receiver 140 (e.g., a correlation function may be calculated), and the first fluorescence signal FS1 may be cumulatively integrated for a period in which the level of the first delay signal is maintained at a logic high level.

Since the third period T3 is a period in which the signal level of any one of the second to sixth code signals CODE2 to CODE6 is maintained at a logic high (“1”) value, the signal level of the first code signal CODE1 may be maintained at a logic low (“0”) value so that the first light source signal LS1 is not applied. Changes in signal levels of the second to sixth code signals CODE2 to CODE6 over time are also the same as the description for the first code signal CODE1.

However, the present disclosure is not limited to the above, and the code signal generated by the code generator 120 allows light source signals having different wavelengths to be applied at different times, but may be a pulse signal in which a logic high (“1”) and a logic low (“0”) value alternate, unlike the first to sixth code signals CODE1 to CODE6 shown in FIG.

FIG. 3 shows the structure of an optical fiber included in the device 100 of FIG. 1 . The light source signal LS output from the light source of the transmitter 110 may be applied to the DNA sample 10 through the central optical fiber of the optical fiber bundle. The fluorescence signal FS emitted from the phosphor attached to the DNA of the DNA sample 10 in response to the light source signal LS may be provided to the detection array of the receiver 140 through the outer fibers of the fiber bundle, and the fluorescence signal FS may be converted into an electrical signal in the detection array.

Therefore, according to an embodiment of the present disclosure, a fluorescence signal FS having a weak intensity emitted from a phosphor attached to DNA can be transmitted through a plurality of optical fiber bundles, and thus loss of the fluorescence signal FS can be minimized.

4 conceptually illustrates the operation of the apparatus 100 for PCR diagnosis according to an embodiment of the present disclosure.

The DNA sample 10 shown in FIG. 4 may include a plurality of DNAs s1 to s19, and a phosphor may be attached to each of the DNAs s1 to s19. The phosphor attached to each of the DNAs s1 to s19 may emit a fluorescence signal by absorbing a light source signal having a corresponding wavelength. It is assumed that the number of different phosphors attached to each of the DNAs s1 to s19 in FIG. 4 is 6, and the transmitter 110 includes first to sixth light sources LD1 to LD6 respectively corresponding to the 6 different phosphors. The first to sixth light sources LD1 to LD6 may have first to sixth wavelengths λ1 to λ6 different from each other. However, the present disclosure is not limited thereto, and the number of DNAs, the number of phosphors, and the number of light sources included in the DNA sample 10 may be set differently from those shown in FIG. 4 .

The first to sixth light sources LD1 to LD6 are connected to the optical fiber bundle, respectively, and the first to sixth light source signals LS1 to LS6 may be output through the corresponding optical fiber bundle. As described with reference to FIGS. 1 to 2 , the first to sixth light source signals LS1 to LS6 may be modulated by the first to sixth code signals CODE1 to CODE6 output from the code generator 120 .

As described with reference to FIG. 1 , the first to sixth light source signals LS1 to LS6 may be combined in one output optical fiber using wavelength division multiplexing (WDM). The output optical fiber may include a splitter at an end, and the first to sixth light source signals LS1 to LS6 having different wavelengths (λ1 to λ6) may be applied to the plurality of DNAs s1 to s19 at different times through the splitter.

The phosphor attached to the plurality of DNAs s1 to s19 may receive the first to sixth light source signals LS1 to LS6 at different times through a plurality of optical fiber bundles, and emit corresponding first to sixth fluorescence signals FS1 to FS6. The first to sixth fluorescence signals FS1 to FS6 emitted from the DNA sample 10 may be converted into electrical signals in a detection array of the receiver 140 through a plurality of optical fiber bundles. Also, the delay controller 130 may generate the first to sixth delay signals DELAY1 to DELAY6 based on the first to sixth code signals CODE1 to CODE6 and transmit them to the receiver 140 .

The receiving unit 140 may compare the first to sixth fluorescence signals FS1 to FS6 and the corresponding first to sixth delay signals DELAY1 to DELAY6, and may cumulatively integrate each fluorescence signal FS1 to FS6. Thus, the device 100 may detect a plurality of DNAs s1 to s19.

5 conceptually illustrates the operation of the apparatus 100 for PCR diagnosis according to another embodiment of the present disclosure.

4, the first to sixth light source signals LS1 to LS6 are provided to the DNA sample 10 through a plurality of optical fiber bundles, and the first to sixth fluorescence signals FS1 to FS6 are provided to the receiver 140 through a plurality of optical fiber bundles. In contrast, in FIG. It is shown in (10) that the first to sixth fluorescence signals FS1 to FS6 are provided to the receiving unit 140 through one multi-mode fiber having a large size. Hereinafter, a description overlapping with that of FIG. 4 will be omitted.

As shown in FIG. 5, a single mode optical fiber and a multimode optical fiber can be attached to each other. The end of the single-mode optical fiber is inclined at an arbitrary angle to provide the first to sixth light source signals LS1 to LS6 to the center of the DNA sample 10 . As a result, the incident first to sixth light source signals LS1 to LS6 may be bent and applied to the center of the DNA sample 10 . The first to sixth fluorescence signals FS1 to FS6 may be received through a multimode optical fiber, provided to a detection array of the receiver 140, and converted into electrical signals.

The foregoing are specific embodiments for carrying out the present disclosure. The present disclosure will include not only the above-described embodiments, but also embodiments that can be simply or easily changed in design. In addition, the present disclosure will also include techniques that can be easily modified and implemented using the embodiments. Therefore, the scope of the present disclosure should not be limited to the above-described embodiments and should be defined by not only the claims to be described later but also those equivalent to the claims of the present disclosure.

100: device for PCR diagnosis 110: transmission unit
120: code generator 130: delay controller
140: receiver

Claims (11)

a transmitter including a multi-wavelength light source outputting a first light source signal and a second light source signal having different wavelengths, and applying the first light source signal and the second light source signal to a DNA sample;
a code generator configured to generate a first code signal and a second code signal respectively corresponding to the first light source signal and the second light source signal;
a delay controller configured to generate a first delay signal and a second delay signal respectively corresponding to the first code signal and the second code signal; and
Calculate a correlation function between the first and second fluorescence signals emitted from the DNA sample, and the first delay signal and the second delay signal, determine whether the first and second fluorescence signals, respectively, and the first and second delay signals correspond to the same code signal based on the value of the calculated correlation function, and determine whether the first and second fluorescence signals correspond to the same code signal based on the determination result A receiver that cumulatively integrates for a period in which the level is maintained at a logic high value,
The first fluorescence signal is emitted from the DNA sample in response to the first light source signal, and the second fluorescence signal is emitted from the DNA sample in response to the second light source signal. According to claim 1,
The first light source signal and the second light source signal are applied to the DNA sample at different times determined based on the first code signal and the second code signal, respectively. According to claim 1,
The first code signal and the second code signal are composed of a first section having a logic high value, a second section having a logic low value, and a third section having a logic low value, wherein the first section is not continuous, and the length of the first section and the second section are the same. According to claim 3,
The first light source signal and the second light source signal are applied to the DNA sample during a period in which the first code signal has a logic high value and a period in which the second code signal has a logic high value, respectively;
The length of the first section of the first code signal is set to be equal to or longer than the fluorescence lifetime of the phosphor emitting the first fluorescence signal, and the length of the first section of the second code signal is set to be equal to or longer than the fluorescence lifetime of the phosphor emitting the second fluorescence signal;
The fluorescence lifetime is a PCR diagnostic device indicating a period during which a phosphor can emit a fluorescence signal. According to claim 3,
A period in which the first code signal has a logic high value and a period in which the second code signal has a logic high value are different from each other. According to claim 1,
The first delay signal is a signal delayed by a time when the level of the first code signal has a logic high value compared to the first code signal, and
The second delay signal is a signal delayed by a time when the level of the second code signal has a logic high value compared to the second code signal. delete According to claim 1,
The first light source signal and the second light source signal are applied to the DNA sample through a central bundle of a plurality of optical fiber bundles, and the first fluorescence signal and the second fluorescence signal are applied to the receiving unit through outer bundles of the plurality of optical fiber bundles. Apparatus for diagnosing PCR. According to claim 1,
The first light source signal and the second light source signal are applied to the DNA sample through a single-mode optical fiber, and the first fluorescence signal and the second fluorescence signal are provided to the receiver through a multi-mode optical fiber. Apparatus for diagnosis. According to claim 9,
The single-mode optical fiber is tilted at a predetermined angle so that the first light source signal and the second light source signal can be applied to the center of the DNA sample. generating a plurality of code signals;
applying a plurality of light source signals having different wavelengths to a DNA sample at different times based on the plurality of code signals;
generating a plurality of delay signals respectively corresponding to the plurality of code signals;
calculating a correlation function between a fluorescent signal emitted from the DNA sample and the plurality of delay signals;
determining whether the fluorescent signal and each of the plurality of signals correspond to the same code signal based on the calculated value of the correlation function; and
Accumulatively integrating the fluorescence signal over a period in which a level of a delay signal corresponding to the same code signal is maintained at a logic high value based on the determination result.