METHOD FOR DETECTING AND QUANTIFYING TARGET NUCLEIC ACID IN REAL TIME USING SINGLE SIGNAL FLUORESCENT MATERIAL

Abstract

Provided is a method for detecting and quantifying a nucleic acid in real time and at high speed. The present disclosure provides a real-time high-speed PCR method in which fluorescent signals can be measured from a single-wavelength light source by using a single signal fluorescent material under continuous temperature control. Thus, the PCR method can be performed with a compact lightweight device with a simplified structure.

Claims

1. A method for detecting and quantifying a target nucleic acid in real time, the method comprising: (a) repeatedly performing DNA denaturation, annealing, and DNA extension of a target sequence and an internal control sequence, using a PCR reaction solution containing a single-signal fluorescent material and a primer pair that specifically bind to the target sequence under continuous temperature control; and (b) measuring a fluorescence signal at regular intervals from the start of heating for DNA denaturation to the completion of DNA denaturation.

2. The method of claim 1, wherein the single-signal fluorescent material in step (a) is an intercalating dye.

3. The method of claim 1, wherein the internal control sequence in step (a) is used to remove a false negative of the PCR reaction.

4. The method of claim 1, wherein the continuous temperature control in step (a) comprises heating a reaction vessel to a first temperature by bringing the reaction vessel containing the PCR reaction solution into contact with a heating block, and then cooling the vessel to a second temperature by separating the heated reaction vessel from the heating block and exposing the separated reaction vessel to an artificial air flow for a predetermined period of time, and the first temperature is a temperature at which the DNA denaturation is performed, and the second temperature is a temperature at which the annealing and/or the DNA extension is performed.

5. The method of claim 1, wherein the continuous temperature control in step (a) comprises: heating the reaction vessel to a first temperature by bringing the reaction vessel containing the PCR reaction solution into contact with the heating block; cooling the reaction vessel to a third temperature by separating the heated reaction vessel from the heating block and then exposing the separated vessel to an artificial air flow for a predetermined period of time; and heating the cooled reaction vessel to a second temperature by bringing the cooled reaction vessel into contact with the heating block and then separating the reaction vessel from the heating block, wherein the first temperature is a temperature at which the DNA denaturation is performed, the third temperature is a temperature at which the annealing is performed, and the second temperature is a temperature at which the DNA extension is performed.

6. The method according to claim 4, wherein, in the cooling of the reaction vessel, the heating block is fixed at a position, and the reaction vessel is moved upward to a predetermined position from the heating bock and so that the reaction vessel and the heating block are separated from each other.

7. The method of claim 6, wherein, in the cooling of the reaction vessel, the artificial air flow is continuously supplied.

8. The method according to claim 4, wherein, in the cooling of the reaction vessel, the reaction vessel is fixed at a position, and the heating block is moved downward to a predetermined position under the reaction vessel so that the reaction vessel and the heating block are separated from each other.

9. The method of claim 8, wherein, in the cooling of the reaction vessel, the artificial air flow is supplied only in a state in which the heating block is separated from the vessel.

10. The method according to claim 4, wherein, in the cooling of the reaction vessel, a predetermined time period is determined by the following general Formula 1.
t=4+2*e.sup.−(v−7.4)/6.2  [General Formula 1] In the above general Formula 1, t means a predetermined time, and v is the speed of the artificial air flow.

11. The method according to claim 4, wherein the reaction vessel and the heating block are spaced by a distance of 0.5 to 2 cm in a state in which the reaction vessel and the heating block are separated from each other.

12. The method of claim 4, wherein when only the annealing is performed at the second temperature, the method further comprises separating the reaction vessel from the heating block again after bringing the reaction vessel that is cooled to a second temperature into contact with the heating block so that the reaction vessel is heated to a fourth temperature, wherein the fourth temperature is a temperature at which the DNA extension is performed.

13. The method of claim 1, wherein, in step (b), the fluorescence signal is measured at regular time intervals from the start of the heating to the completion of the DNA denaturation, the fluorescence signal measured at a time point Tx among the measured fluorescence signals is selected, and the fluorescence signal measured at the time point Tx is a fluorescence signal measured at a time point at which a temperature higher than the melting point of an amplified product of the internal control sequence is reached.

14. The method of claim 13, wherein, in step (b), the fluorescence signal (signal B) measured at the start of the heating comprises a fluorescence signal of an amplified product of the target sequence and a fluorescence signal of an amplified product of the internal control sequence, and the fluorescence signal (signal A) measured at the time point Tx comprises only the fluorescence signal of the amplified product of the target sequence.

15. The method of claim 1, wherein the interval in step (b) is 0.5 to 1 second.

16. The method of claim 1, wherein the method further comprises checking whether there is a false negative by checking whether the internal control sequence is amplified or not.

17. The method according to claim 5, wherein, in the cooling of the reaction vessel, the heating block is fixed at a position, and the reaction vessel is moved upward to a predetermined position from the heating bock and so that the reaction vessel and the heating block are separated from each other.

18. The method according to claim 5, wherein, in the cooling of the reaction vessel, the reaction vessel is fixed at a position, and the heating block is moved downward to a predetermined position under the reaction vessel so that the reaction vessel and the heating block are separated from each other.

19. The method according to claim 5, wherein, in the cooling of the reaction vessel, a predetermined time period is determined by the following general Formula 1.
t=4+2*e.sup.−(v−7.4)/6.2  [General Formula 1] In the above general Formula 1, t means a predetermined time, and v is the speed of the artificial air flow.

20. The method according to claim 5, wherein the reaction vessel and the heating block are spaced by a distance of 0.5 to 2 cm in a state in which the reaction vessel and the heating block are separated from each other.

Description

DESCRIPTION OF DRAWINGS

[0066] FIGS. 1A and 1B schematically show the appearance of a PCR device to which the method according to the present disclosure is applied, and the PCR device is composed of a reaction vessel 1, a heating block 2, and a blowing fan 3, and the reaction vessel 1 contained reaction solution is moved so as to be in contact with the heating block 2 and the wind 5 generated from the blowing fan, respectively;

[0067] FIGS. 2A and 2B schematically show a method in which heating and cooling are achieved in a PCR device to which the method according to the present disclosure is applied, the reaction vessel 1 is moved to the heating block 2 and brought into contact with the reaction solution is heated, and then the reaction vessel 1 move in front of the blowing fan 3 and comes into contact with the wind 5 generated from the blowing fan to cool the reaction solution;

[0068] FIG. 3 is a schematic diagram of a method for measuring the fluorescence of the PCR reaction and the amplification product using the Bio-rad CFX 96 device;

[0069] FIG. 4 is an optical curve measured at 60° C. using a Bio-rad CFX96 device and is an amplification curve in which the amounts of the fluorescence signal derived from the amplification product of the internal control and the fluorescence signal derived from the amplification product of the target sequence are combined;

[0070] FIG. 5 is an optical curve measured at 84° C. using a Bio-rad CFX96 device. The amplification product of the internal control is denatured at a temperature of 79° C. or higher and thus does not exhibit fluorescence; FIG. 5 is an optical amplification curve in which a fluorescence signal derived from an amplification product of an internal control sequence is removed from the amplification curve of FIG. 4, and is an amplification curve is composed only of a fluorescence signal derived from an amplification product of a target sequence;

[0071] FIG. 6 is a schematic diagram showing a method of measuring the PCR reaction and fluorescence of an amplification product using a device to which the present disclosure is applied;

[0072] FIG. 7 is an optical curve is composed of data of an optical measurement sequence 1 using a device to which the present disclosure is applied and is an amplification curve that is the sum of the fluorescence signal derived from the amplification product of the internal control sequence and the amount of fluorescence derived from the amplification product of the target sequence; and

[0073] FIG. 8 is an optical curve is composed of data of sequence 11 optically measured using the device to which the present disclosure is applied and is an amplification curve of the signal excluding the fluorescence signal derived from the amplification product of the internal control sequence in the amplification curve of FIG. 7 and is an amplification curve is composed only of the fluorescence signal derived from the amplification product of the target sequence.

BEST MODE

Mode for Disclosure

[0074] Hereinafter, the present disclosure will be described in detail through examples. The following examples only illustrate the present disclosure, but the scope of the present disclosure is not limited to the following examples.

EXAMPLE

Example 1: Confirmation of Target Nucleic Acid Detection

[0075] 1) HCV Viral RNA Detection

[0076] A control material of the Quanti-HCV v1.0 kit was extracted and mixed with a PCR reaction solution to detect HCV viral RNA. 20 μl of a PCR reaction solution containing different concentrations of control WT RNA was added to each of the reaction vessel strips to which several PCR tubes were connected, and after closing the lid of the vessel, pre-denaturation for 1 minute in a heating block heated to 98° C. was performed. The denaturation step was performed by contact with the heating block for 8 seconds, and an artificial air flow was provided by a blowing fan to cool for 9 seconds to perform annealing and DNA extension steps. PCR reaction was performed by repeating this process 45 times, and the specific PCR reaction solution, PCR device, and reaction conditions are shown in Table 1 below:

TABLE-US-00001 TABLE 1 Condition The present disclosure Related art RNA Internal control gene and HCV RNA extraction: extraction Viral RNA extraction kit (SEF-016, Ugenecell, KR) PCR reaction Bioplastic 0.1 mL tube strip(low profile) vessel PCR ExAmplar (Ugenecell, KR) CFX 96 (Bio-rad, US) amplification instrument PCR reaction PCR mixture: Primer (forward: 5′-GTTCTGCGGAACCGGTGAGTACA-3′/ solution reverse: 5′-CGCRACCCAACRCTACTCGGCTA-3′; R is A or G) Real MOD 2X RT-PCR enzyme mix (iNtRon, KR) PCR A. Reverse transcription (48° C., 15 min) A. Reverse transcription (48° C., amplification B. Initial denaturation (98° C., 5 min) 15 min) B. Initial denaturation conditions C. Heating (98° C. (contact heating block), (98° C., 5 min) C. Heating (95° C., 15 seconds)-Measure signals at 0.8 seconds 20 seconds) D. Amplification, intervals for 15 seconds of heating first optical measurement (60° C., (represented by sequence) 10 seconds) E. Secondary optical D. Cooling (9 seconds)  measurement (84° C., 10 seconds) E. Standing (12 seconds) C-E: Repeat 45 times C-E: Repeat 45 times

[0077] 13 IU and 300 IU of HCV target RNA were added to the reaction tube, respectively, and 10 IU of internal control RNA was added to all tubes. NCs without the addition of HCV target RNA (negative control) were also tested. The fluorescence of the amplification products was measured through the optics of each amplifier. In the above experiment, in order to compare with the conventional PCR method, a commercially available real time PCR device (CFX 96 of Bio-rad Corporation) was used. The PCR reaction using Bio-rad's CFX 96 was performed as shown in FIG. 3, the optical curve measured at 60° C. is shown in FIG. 4, and the optical curve measured at 84° C. is shown in FIG. 5.

[0078] The PCR reaction using the device of the present disclosure was performed as shown in FIG. 6, and the optical curves accordingly are shown in FIGS. 7 and 8, respectively, in which FIG. 7 is composed of the signal of sequence 1, and FIG. 8 is composed of the signal of sequence 11.

[0079] As a result of the experiment, as can be compared in FIGS. 4, 5, and 7, 8, in the amplification curve (FIG. 4) obtained through the general signal measurement method used in the conventional real time PCR device, since the signal by target sequence amplification and the signal by internal gene sequence amplification were overlapped, it was difficult to distinguish only the signal of the target sequence of interest. Therefore, in order to distinguish only the signal of the target sequence, the measurement temperature had to be adjusted to remove the signal by amplifying the internal gene sequence (FIG. 5). On the other hand, in the PCR device to which the present disclosure is applied, it was confirmed that it is possible to detect only the fluorescence signal of the amplification product for the target sequence because the fluorescence signal of the internal control group sequence may be excluded according to the measurement time point of the fluorescence signal (FIGS. 7 and 8).