WO2016122198A1

WO2016122198A1 - Method for solving problem of nucleic acid detection due to threshold cycle (ct) of real-time polymerase chain reaction using pna probe

Info

Publication number: WO2016122198A1
Application number: PCT/KR2016/000842
Authority: WO
Inventors: 박재신; 박희경; 정진욱
Original assignee: 주식회사 시선바이오머티리얼스
Priority date: 2015-01-30
Filing date: 2016-01-27
Publication date: 2016-08-04

Abstract

The present invention relates to a method for solving the problem of nucleic acid detection due to a threshold cycle (Ct) of the real-time polymerase chain reaction using a PNA probe and, more specifically, to a method for analyzing a melting curve using a PNA probe to which a reporter and a quencher for detecting a target nucleic acid are bound, to a method for detecting and quantifying a target nucleic acid to solve the problem of the detection of the target nucleic acid due to a threshold cycle (Ct) using the same, and to a kit for detecting and quantifying a target nucleic acid, using the method and comprising a PNA probe. The use of the method for detecting and quantifying a target nucleic acid using a PNA probe, to which a reporter and a quencher are bound, according to the present invention leads to the solving of a technical problem caused by performing the determination on the basis of the threshold cycle (Ct) value of the real-time polymerase chain reaction, which is used in the method for the diagnosis and the identification and analysis of individuals, thereby improving the accuracy in the detection and quantification result read-out of a target nucleic acid or a target gene and reducing economic, social, and medical costs.

Description

Method for solving the problem of nucleic acid detection according to the critical cycle (Ct) of real time polymerase chain reaction using PNA probe

The present invention relates to a method for solving the problem of nucleic acid detection according to the critical cycle (Ct) of the real-time polymerase chain reaction using a PNA probe, and more specifically, a reporter and a quencher for detecting a target nucleic acid ( Fusion curve analysis method using a PNA probe coupled to a quencher, a method for detecting and quantifying a target nucleic acid for solving a detection problem of a target nucleic acid according to a threshold cycle (Ct) using the same, a PNA probe using the method It relates to a kit for detecting and quantifying a target nucleic acid comprising a.

Since Watson and Creek (James D. Watson & Francis Crick) identified the double helix structure of DNA in the 1950s, large-scale automated DNA synthesis and analysis techniques were developed and deployed in the 2000s to achieve rapid advances in molecular biology. It is used in various fields of application. Some examples of applications include DNA hybridization probes, primers for DNA sequencing, DNA amplification, and oligos. for gene synthesis). In particular, real-time polymerase chain reaction (Real-Time PCR) of the DNA amplification technology is used in the field of diagnosing diseases or identifying individuals (eg, sex) in real time using a synthetic probe (probe).

Representative probes used in the real-time polymerase chain reaction are hydrolysis probes, which are prepared by complementary base sequences for a specific gene (specific sequence) to be analyzed and include the genes. Used for amplification and fusion analysis of sequences.

TaqMan probe structurally has a structure in which a fluorescent (fluorescent) at the 5 'end, and a quencher at the 3' end is coupled, and the probe is subjected to an annealing step during a polymerase chain reaction (PCR) process. In the (annealing step) hybridization to the complementary DNA sequence (hybridize), but the development of fluorescence is suppressed by the quencher. However, the 5 'end of fluorescence is cut off by the activity of the 5' exonuclease of Taq DNA polymerase, which results in color development.

The technique applying the real-time polymerase chain reaction using the hydrolysis-type probe is used for reading such as disease diagnosis and individual identification. If the reading is incorrect, it may threaten human life and cause economic loss. Accurate and fast reading is required.

In the case of target genes to be used for the above-described methods of diagnosis and identification of individuals, analysis is performed in single or mixed individuals. In particular, when a small amount of the target gene is present in a mixed individual, it is difficult to detect the gene. The quantitative detection of the target gene as described above is a result of the threshold cycle (Ct) value using a commercially available hydrolysis probe, which causes a lot of confusion during reading. In most cases, the target gene is detected based on a fixed Ct value. Read whether it is (Stephen A. Bustin et al, Clin Chem , 55 (4): 611-22, 2009; Carr, AC et al., PLoS One , 7 (5): e37640, 2012; Schefe JH et al, J Mol Med , 84 (11): 901-10, 2006; Holland PM et al., Proc Natl Acad Sci USA , 88 (16): 7276-7280, 1991).

In real-time PCR, the amount of PCR amplification product can be detected by fluorescence signal. Intercalating method using an intercalator that binds to double-stranded DNA as a detection method, and uses an oligonucleotide labeled with a fluorescent substance at the 5 'end and a quencher at the 3' end. How to do it. When the above methods are used, the intensity of the fluorescence signal increases with increasing amount of polynucleotides as the Real-Time PCR progresses, and the user generates an amplification profile curve indicating the intensity of the fluorescence signal according to the number of amplification cycles. You get

Amplification profile curves generally include a baseline region where the fluorescence signal of the background does not reflect the actual amount of polynucleotides, an exponential region where the fluorescence signal increases with the increase in the amount of polynucleotide products, and PCR. When the reaction reaches saturation, it is divided into a plateau region where no increase in fluorescence signal intensity is observed. The baseline region appears early in the PCR reaction because the amount of PCR amplification products has not yet reached a detectable amount.

In general, the intensity of the fluorescence signal when the point of transition from the baseline region to the exponential region, that is, when the amount of PCR amplification products reaches a detectable amount by fluorescence is called a threshold and corresponds to a threshold in the amplification profile curve. The number of amplification cycles is called the threshold cycle (Ct) value. When Real-Time PCR is performed at different initial polynucleotide concentrations, the larger the initial polynucleotide amount, the smaller the number of amplification cycles in which the amount of amplification products reaches a detectable amount. Thus, the log value of the initial polynucleotide amount and the threshold cycle value are in inversely strong relations, and typically the desired polynucleotide quantification is performed using the threshold cycle value. Polynucleotide quantification using real-time polymerase chain reaction has large absolute and relative quantification. Absolute quantification is a method of quantitating a standard curve of critical cycle values for a polynucleotide amount by performing a real-time polymerase chain reaction with a sample having a known polynucleotide amount. This method compares the relative amount with other samples even if the nucleotide amount is not known. For relative quantification, standard curves may or may not be used. In all these cases, in order to quantify, the threshold value must first be determined to calculate the threshold cycle value.

The number of cells in a reliable sample of a general real-time polymerase chain reaction is about 10 ² to 10 ³ . If the quantitative value of the target gene in the mixed population is less than the number of reliable cells, it will have a higher Ct value than the DNA sample with the large amount of the target gene. However, the method of determining the result through the Ct value ignores fluorescence signals below the Ct value in most cases, even though the actual target gene exists because of the quantitative limitation of the target gene. For example, the identification of microorganisms that cause disease in the blood in emergency patients is a very high risk factor and incurs time costs for further verification.

One of the disadvantages of all PCR reactions is that there is always a non-specific band even when amplification is performed by preparing an oligo primer corresponding to the target nucleic acid. In general polymerase chain reaction, it is possible to confirm the gene length whether the target nucleic acid amplified by electrophoresis is accurate.However, in real time polymerase chain reaction, the authenticity of the gene (the presence of genes) is shown as the Ct value displayed on the machine according to the amplification. Or base mutations). Therefore, the method of diagnosis of Ct value criteria and analysis of individual identification carries the risk of making a mistake in reading.

Peptide Nucleic Acid (PNA), which is used for the analysis of diagnosis and individual identification, is an artificial nucleotide and has excellent complementary binding ability with DNA or RNA. In addition, it has excellent biological stability against nuclease and protease, and chemical stability and water solubility also have advantages over other artificially synthesized oligos (Nielsen PE et al, Science, 254 (5037): 1497-500, 1991). Santosh B et al, Biomed Res Int, 2014 (Article ID 540451), 2014).

One characteristic of the PNA probe is that it hybridizes in the presence of complementary gene sequences, regardless of the activity of the 5 'exonuclease of Taq DNA polymerase. Is to send. In particular, the PNA probe, like the TaqMan probe, may detect a target gene through an additional melting step (melting step) after the amplification is completed, including a signal during the nucleic acid amplification process. In particular, when identifying the presence of a small amount of the target gene, the additional amplification step can solve the shortcomings of the determination method using most of the Ct value.

Accordingly, the present inventors minimize the discrimination error of the presence of the target nucleic acid due to the false positive and false negative signals according to the critical cycle (Ct) value shown in the existing Real-Time PCR process, and the target nucleic acid. Efforts have been made to effectively detect and quantify the activity of PNA probes in combination with a reporter and a quencher to enable rapid and accurate detection or quantification of target nucleic acids or target genes through lysis curve analysis. It confirmed and completed this invention.

발명의 요약Summary of the Invention

Disclosure of Invention An object of the present invention is to minimize the detection error of a target nucleic acid by false positive and false negative signals according to the threshold cycle (Ct) value shown in Real-Time PCR, and to detect a target nucleic acid. Or to provide a target nucleic acid fusion curve analysis method using a PNA probe combined with a reporter (quenching) and a reporter (quenching) to quantify, the kit for the detection or quantification of the target nucleic acid through the melting curve analysis.

In order to achieve the above object, the present invention is (a) adding a sample suspected to contain the target nucleic acid and a PNA probe and TaqMan probe to which the nucleic acid polymerase, primer, reporter and quencher are bound, hybridize, and then asymmetric polymerization Amplifying using an asymmetric polymerase chain reaction; (b) melting the hybridized product with varying temperature to obtain a melting curve; And (c) analyzing the obtained fusion curves to detect or quantify the target nucleic acid, thereby solving the problem of detecting the target nucleic acid according to the threshold cycle (Ct) of the real-time polymerase chain reaction. A method for detecting or quantifying target nucleic acid through target nucleic acid fusion curve analysis using a PNA probe in which a reporter and a quencher are combined is provided.

The present invention also provides a kit for detecting or quantifying target nucleic acid by using a target nucleic acid fusion curve analysis using the above method and including a reporter and a quencher-coupled PNA probe.

The present invention also relates to (a) adding a sample suspected of containing a target nucleic acid and a nucleic acid polymerase, a primer and a TaqMan probe, hybridizing and amplifying using an asymmetric polymerase chain reaction. step; (b) adding and hybridizing the probe with the reporter and quencher to the amplification product; (c) melting the hybridized product of step (b) while varying the temperature to obtain a melting curve; And (d) analyzing the obtained fusion curves to solve the problem of detecting target nucleic acids according to a threshold cycle (Ct) of a real-time polymerase chain reaction, comprising detecting or quantifying target nucleic acids. A method for detecting or quantifying target nucleic acid through target nucleic acid fusion curve analysis using a PNA probe in which a reporter and a quencher are combined is provided.

1 is a schematic diagram showing an improved detection method of a target nucleic acid according to Ct value using a PNA probe.

Figure 2 shows the amplification curve and melting curve temperature conditions for confirming the detection of the target nucleic acid according to the Ct value.

Figure 3 compares the amplification curves according to symmetric or asymmetric gene amplification of real-time polymerase chain reaction to read the results in Ct value.

4 compares the peaks of the melting curve of the hydrolysis probe and the PNA probe.

5 shows an amplification curve using only the hydrolysis probe.

Figure 6 shows the peak of the melting curve using the PNA probe.

7 shows an amplification curve for identifying low concentration of nonspecific amplification products using PNA probe.

Figure 8 shows the melting curve peaks for distinguishing actual target nucleic acids from low concentrations of nonspecific amplification products.

9 shows the melting curve peaks obtained by an improved assay to distinguish between actual target nucleic acids and low concentrations of nonspecific amplification products.

발명의 상세한 설명 및 바람직한 Detailed description of the invention and preferred 구현예Embodiment

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In an embodiment of the present invention, the amplification curve and the melting curve were analyzed to solve the problem of detecting the target nucleic acid according to the Ct value by mixing the artificial synthetic oligo and PNA probe. WSD (White Spot) as a target nucleic acid in order to minimize the error of discrimination of the presence or absence of a target nucleic acid by false positive and false negative signals, which is a problem of nucleic acid detection according to the threshold cycle (Ct), and to effectively detect the target nucleic acid. Disease) Synthetic oligo was used, and primers and probes (hydrolysis probe and PNA probe) for amplifying the nucleic acid were used. As a result, as shown in Figure 3, the actual starting point of the asymmetric and symmetric gene amplification corresponds to 25 cycles, but slightly less than the set Ct and relative fluorescence units (RFU) value to confirm the presence of the nucleic acid It was difficult to judge the existence. Difficulty in determining the presence or absence of the target nucleic acid identified above, as shown in Figure 4, through the fusion step of the asymmetric gene amplification product was able to clearly distinguish the authenticity of the result.

Therefore, in one aspect, the present invention provides a method for preparing a hybrid target comprising: (a) adding a PNA probe and a TaqMan probe to which a sample suspected of containing a target nucleic acid and a nucleic acid polymerase, a primer, a reporter, and a quencher are bound, hybridized, and then asymmetric polymerase. Amplifying using an asymmetric polymerase chain reaction; (b) melting the hybridized product with varying temperature to obtain a melting curve; And (c) analyzing the obtained fusion curves to detect or quantify the target nucleic acid, thereby solving the problem of detecting the target nucleic acid according to the threshold cycle (Ct) of the real-time polymerase chain reaction. Method of detecting or quantifying target nucleic acid by target nucleic acid fusion curve analysis using PNA probe combined with reporter and quencher, and using reporter and reporter and quencher The present invention relates to a kit for detecting or quantifying target nucleic acid through target nucleic acid fusion curve analysis using a PNA probe.

In real-time PCR, an amplification cycle in which the intensity of a fluorescence signal when the amount of PCR amplification products reaches a fluorescence detectable amount is called a threshold and corresponds to a threshold in an amplification profile curve. The number of times is called the threshold cycle (Ct) value. When Real-Time PCR is performed at different initial polynucleotide concentrations, the larger the initial polynucleotide amount, the smaller the number of amplification cycles in which the amount of amplification products reaches a detectable amount. Thus, the log value of the initial polynucleotide amount and the threshold cycle value are in inversely strong relations, and typically the desired polynucleotide quantification is performed using the threshold cycle value.

In the present invention, "solve the problem of detecting the target nucleic acid according to the threshold cycle (Ct) of real-time polymerase chain reaction" is the critical cycle (Ct) appearing in the conventional real-time PCR (Real-Time PCR) process Means to minimize the error of discrimination of the presence of the target nucleic acid by false positive and false negative signal according to the value, and to effectively detect and quantify the target nucleic acid, but is not limited thereto.

In the present invention, "sample" includes various samples, and preferably, a biosample is analyzed using the method of the present invention. Biosamples of plant, animal, human, fungus, bacterial and viral origin can be analyzed. When analyzing a sample of mammalian or human origin, the sample may be derived from a specific tissue or organ. Representative examples of tissues include connective, skin, muscle or nerve tissue. Representative examples of organs include eyes, brain, lungs, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testes, ovaries, uterus, rectum, nervous system, Glands and internal vessels are included. The biosample to be analyzed includes any cell, tissue, fluid from a biological source, or any other medium that can be well analyzed by the present invention, which is the consumption of humans, animals, humans or animals. Samples obtained from food prepared for use are included. In addition, the biological sample to be analyzed includes a bodily fluid sample, which includes blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain grinds), spinal fluid, appendix, spleen And tonsil tissue extracts, but is not limited thereto.

In the present invention, the PNA probe completely hybridizes with the base sequence of the target nucleic acid (perfect match) to show the expected melting temperature (Tm) value, and incomplete hybridization (mismatch) with the target nucleic acid having a base sequence difference It may be characterized by showing a lower melting temperature (Tm) value than expected.

In the present invention, the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and It may be characterized in that at least one selected from the group consisting of CY5, the quencher may be characterized in that at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl. have.

In the present invention, the false-positive and false-negative signals can be distinguished using the difference in the melting curve according to the target nucleic acid sequence, the PNA probe further comprises a locked nucleic acid (LNA) It can be characterized.

Locked nucleic acid (LNA) that can be contained in the PNA probe in the present invention is a modified RNA nucleotide, modified in the form of combining the 2 'oxygen atoms and 4' carbon atoms of the ribose moiety (ribose moiety) It is. LNAs can form oligonucleotides in a mixed form with DNA or RNA, and the locked ribose conformation of the blocked nucleic acids allows stacking between bases and pre-organization of the backbone. enhances the hybridization performance of oligonucleotides by promoting -organization. Oligonucleotides used in molecular biological assays, such as DNA microarrays, FISH probes, and Quantitative PCR (Q-PCR) probes, use LNA nucleotides to increase sensitivity and specificity. In addition, nucleotides containing LNA are easy to detect short-length RNA or DNA targets, and have high resistance to nucleases, and thus are easy to use in vivo / in vitro .

'Target nucleic acid' or 'synthetic oligo' of the present invention means a nucleic acid sequence to be detected or not, and includes a specific site of the nucleic acid sequence of the 'target gene' encoding a protein having physiological and biochemical functions. , Annealed or hybridized with primers or probes under hybridization, annealing or amplification conditions.

By 'hybridization' of the present invention is meant that complementary single stranded nucleic acids form a double-stranded nucleic acid. Hybridization can occur when the complementarity between two nucleic acid strands is perfect or even when some mismatch base is present. The degree of complementarity required for hybridization may vary depending on the hybridization conditions, and may be particularly controlled by temperature.

The PNA probe including the reporter and the quencher of the present invention hybridizes with the target nucleic acid and generates a fluorescence signal. As the temperature increases, the PNA probe rapidly melts with the target artificial oligo oligomer at an appropriate melting temperature of the probe, thereby extinguishing the fluorescent signal. The presence or absence of base denaturation of the target synthetic oligo may be detected through high resolution fluorescence melting curve analysis (FMCA) obtained from the fluorescence signal according to the temperature change.

The probe of the present invention may combine a reporter and a fluorescent material of a quencher capable of quenching reporter fluorescence at both ends. The reporter is reported as FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5. At least one selected from the group consisting of, the quencher may be one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, but is not limited thereto, preferably Dabcyl You can use (FAM-labeled).

The kits of the present invention may optionally include reagents necessary to conduct target nucleic acid amplification reactions (eg, PCR reactions) such as buffers, DNA polymerase cofactors and deoxyribonucleotide-5-triphosphates. Optionally, the kits of the present invention may also include various polynucleotide molecules, reverse transcriptases, various buffers and reagents, and antibodies that inhibit DNA polymerase activity. In addition, the optimum amount of reagent used in a particular reaction of the kit can be easily determined by those skilled in the art having learned the disclosure herein. Typically, kits of the present invention can be made in a separate package or compartment containing the aforementioned components.

In another embodiment of the present invention, the synthetic oligo oligonucleotide and the PNA probe were separated to analyze the amplification curve and the melting curve for solving the detection problem of the target nucleic acid according to the Ct value. To determine whether white spot disease (WSD) was detected, target nucleic acids were amplified symmetrically and asymmetrically using a PNA probe suitable for high temperature. As a result, as shown in Figure 5, the amplification curve was difficult to distinguish the presence or absence of the nucleic acid according to the set Ct Cutoff value. Therefore, an additional PNA probe was added to the amplification product and a fusion step was performed to determine the authenticity of the correct target nucleic acid. As a result, as shown in Figure 6, when the target nucleic acid was added it was confirmed whether the detection by showing the peak of the melting on the melting curve.

In another embodiment of the present invention, a method of distinguishing an amplification product of a real target nucleic acid from a low concentration of nonspecific amplification product using a PNA probe was made. First, in the analysis of the target nucleic acid, as a result of confirming the aspect of the amplification curve according to the presence of a low concentration of non-specific amplification products, as shown in Figure 7, the positive control when identifying the amplification curve using PROBE-3 (PNA probe) When the target nucleic acid of 0.01 ng was used, the Ct value was 36 when the RFU (light intensity or the concentration of the product of the amplified target nucleic acid) was 1000, but the Ct value was used when the low concentration of the target nucleic acid (0.001 ng / sample) was used. Couldn't decide. However, low concentrations of the target nucleic acid and negative control showed a slight amplification curve.

In addition, in order to confirm the authenticity of the low concentration of non-specific amplification product, the dissolution curve analysis was performed using PROBE-4 (PNA probe), as shown in Figure 8, in the melting curve analysis 0.01ng target nucleic acid By checking the intrinsic temperature (Tm) 64.0 ℃ corresponding to PROBE-4 (PNA probe) it was confirmed that the target nucleic acid is real. However, in the target nucleic acid of 0.001ng, the Tm value was 60 ° C., and the negative control group did not show the Tm value, so it was not possible to distinguish the amplification product from the non-specific amplification product. Therefore, it was confirmed that the concentration of the minimum amplification product is required to confirm whether the amplification product is the actual target nucleic acid amplification product or the nonspecific amplification product at the intrinsic temperature using the PNA probe.

In addition, as a result of confirming the improved melting curve analysis method that can distinguish the authenticity of the low concentration of the non-specific amplification product using the PNA probe, as shown in Figure 9, the target nucleic acid (0.001 ng / sample lower than the negative control) ), And further amplification and increasing the binding capacity of the PNA probe was able to distinguish the actual target nucleic acid and the non-specific product negative control.

Therefore, in another aspect, the present invention provides a method for preparing an asymmetric polymerase chain reaction, comprising: (a) adding a sample suspected of containing a target nucleic acid, a nucleic acid polymerase, a primer, and a TaqMan probe, hybridizing, and then performing an asymmetric polymerase chain reaction. Amplifying using; (b) adding and hybridizing the probe with the reporter and quencher to the amplification product; (c) melting the hybridized product of step (b) while varying the temperature to obtain a melting curve; And (d) analyzing the obtained fusion curves to solve the problem of detecting target nucleic acids according to a threshold cycle (Ct) of a real-time polymerase chain reaction, comprising detecting or quantifying target nucleic acids. Method of detecting or quantifying target nucleic acid by target nucleic acid fusion curve analysis using PNA probe combined with reporter and quencher, and using reporter and reporter and quencher The present invention relates to a kit for detecting or quantifying a target nucleic acid by analyzing a target nucleic acid fusion curve including a PNA probe.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: Preparation of primer and probe for detection of target nucleic acid

Based on the WSD (White Spot Disease) sequence as the target nucleic acid to minimize the detection error of the presence of the target nucleic acid by false positive and false negative signals, which is a problem of nucleic acid detection according to the threshold cycle (Ct) The WSD artificial oligo oligonucleotide was used, and primers and probes (hydrolysis probe and PNA probe) for amplifying the nucleic acid were prepared and used based on the nucleotide sequence shown in the OIE standard.

The PNA probe used in the present invention may be prepared to additionally contain locked nucleic acid (LNA). Contained nucleic acids are modified RNA nucleotides that are modified in the form of a combination of 2 'oxygen atoms and 4' carbon atoms of a ribose moiety. LNAs can form probes (oligonucleotides) in mixed form with DNA or RNA, and the locked ribose conformation of blocked nucleic acids allows stacking between bases and pre-backing of backbones. Promoting pre-organization increases the hybridization performance of the probe.

In order to solve the problem of detecting a target nucleic acid according to a non-specific band and a Ct value, an artificial synthetic oligo having an OIE primer sequence but having one base sequence complementary to the probe was prepared. Complementary artificial synthetic oligos capable of confirming abnormalities of the PNA probe were prepared and used (see Table 1; O in Table 1 represents a linker and K represents lysine).

All PNA probes (FAM-labeled, Dabcyl) used in the present invention were synthesized by HPLC purification method in Panagene (Panagene, Korea), and the purity of all synthesized probes was confirmed by mass spectrometry, and further compared with the target nucleic acid. Unnecessary secondary structures of the probes were avoided for effective binding. In addition, in order to avoid crossing with the fluorescent probes marked on the OIE, fluorescence of different wavelengths was produced.

PCR (Polymerase Chain Reaction) was performed using the synthesized synthetic oligo oligo and PNA probes using a CFX96 ™ Real-Time system (Bio-Rad, USA), followed by an amplification curve and a melting curve analysis ( 1).

Table 1 below shows probes and oligo sequences for the detection of artificial synthetic oligos.

Example 2: Analysis method of amplification curve and melting curve for solving Ct value by mixing artificial synthetic oligo and PNA probe

The conditions for the analysis of the amplification curve and the melting curve for minimizing the discrimination error of the presence of the target nucleic acid by false-positive and false-negative signals, the detection problems of the target nucleic acid according to the Ct value, and the effective detection of the target nucleic acid were as follows. ; 12.5 μl 2X TaqMan Universal PCR Master Mix (PE Applied Biosystems, Foster City, Calif., USA) so that the total volume is 25 μl, TaqMan probe (PROBE-3, SEQ ID NO: 3) (neoprov, Korea) 1.5 μl / 1 pmol, Primer-1-F (SEQ ID NO: 5): Primer-1-R (SEQ ID NO: 7) = (3 μl / 1 pmol: 3 μl / 10 pmol), PROBE-1 (SEQ ID NO: 1) 0.5 μl / 10 pmol, sterile distilled water 3.5 μl was added to prepare a reaction for PCR amplification. 20ng was used for the target nucleic acid added to the reaction, and the NCT-1 artificial oligos for the comparative experiment were also added to have the same quantitative value and Ct value as the actual target nucleic acid.

For the amplification curve analysis, the reaction was performed 40 times for 2 minutes at 50 ° C, 10 minutes at 95 ° C, 15 seconds at 95 ° C, and 1 minute at 60 ° C. The process for the calcite is performed in a denaturation step of 3 minutes at 90 ° C., 1 minute at 75 ° C., and 1 minute at 55 ° C., followed by melting curve analysis to measure fluorescence by increasing the temperature from 20 ° C. to 90 ° C. in 0.5 ° C. steps. It was. It was kept stationary for 5 seconds between each step (Figure 2). In order to solve the detection problem of the target nucleic acid according to the Ct value by real time polymerase chain reaction under the above conditions, the amplification curve and the melting curve analysis were performed by varying the concentration of the sample.

Non-specific amplification (non-specific) that can occur by real-time polymerase chain reaction using NTC-1 artificial oligos that are not affected by gene amplification and artificially amplified by actual samples containing target nucleic acid. band)) and below the Ct value (cycle to determine that no target nucleic acid is present). In the experiment, the Ct value was set to 30 and the RFU (relative fluorescence units; light intensity) was set to cutoff, and the target nucleic acid was not present when the cycle was 30 or more and less than RFU 1000. In addition, in order to specifically use a PNA probe, asymmetric gene amplification must be performed for single-strand production. Thus, an amplification curve experiment was performed by comparing symmetric and asymmetric.

As a result, as shown in Figure 3, it was confirmed that it is very difficult to determine the presence of the target nucleic acid of the actual sample. In other words, the starting point of the actual asymmetric and symmetric gene amplification corresponds to 25 cycles, but it was difficult to determine the presence of the target nucleic acid to the extent that it is slightly below the set Ct value and RFU value to confirm the presence of the nucleic acid of the actual sample. Here, the blue line of FIG. 3 is an actual sample containing a target nucleic acid, and the red line corresponds to a negative control group (D.W). This result is seen in most real-time polymerase reactors that determine the presence or absence of a target nucleic acid by Ct value. Difficulty in determining the presence or absence of the target nucleic acid identified above, as shown in Figure 4, through the fusion step of the asymmetric gene amplification product was able to clearly distinguish the authenticity of the result.

Example 3: Analysis method of amplification curve and melting curve for solving Ct value by separating artificial synthetic oligo and PNA probe

Experimental conditions for determining the WSD of the OIE regulations should be amplified target symmetrically using a primer for amplifying the target nucleic acid. On the other hand, in order to analyze the melting curve using the PNA probe, it is necessary to amplify the target nucleic acid asymmetrically (Asymmetric). In order to solve the disadvantages caused by these conflicting experimental conditions, a PNA probe suitable for high temperature was manufactured.

Target nucleic acid amplification according to OIE criteria is performed by using a hydrolysis probe only, and when a result of the amplification curve of Ct value or non-specific band (non-specific band) is generated, the PNA probe is additionally added. It is attached and confirmed. In this manner, the target nucleic acid was single-stranded to a high temperature while complying with the OIE standard, and then the experiment was performed to bind the PNA probe.

The conditions of the amplification curve experiment to solve the detection problem of the target nucleic acid according to the Ct value are as follows; 12.5 μl 2X TaqMan Universal PCR Master Mix (PE Applied Biosystems, Foster City, Calif., USA) so that the total volume is 25 μl, TaqMan probe (PROBE-3, SEQ ID NO: 3) (neoprov, Korea) 1.5 μl / 1 pmol, Primer-1-F (SEQ ID NO: 5): Primer-1-R (SEQ ID NO: 7) = (3 μl / 1 pmol: 3 μl / 10 pmol) and 5 μl of sterilized distilled water were added to prepare a reaction for PCR amplification.

For the amplification curve analysis, the reaction was performed 40 times for 2 minutes at 50 ° C, 10 minutes at 95 ° C, 15 seconds at 95 ° C, and 1 minute at 60 ° C.

As a result, as shown in Figure 5, the amplification curve was difficult to distinguish the presence or absence of the nucleic acid according to the set Ct Cut off value. Here, the blue line of Fig. 5 is a sample containing the actual target nucleic acid, and the red line corresponds to the negative control group (DW), if the Ct value is 30 or more and the RFU value is 1000 or less, it is assumed that the target nucleic acid is substantially present. In the case of the above experiment, it may be difficult to determine the result, or it may be determined that the target nucleic acid does not exist.

Therefore, when the above results are obtained, the addition of the PNA probe to the amplification product may further determine the authenticity of the target nucleic acid. In the case of making a single strand to which the PNA probe can bind, a general target nucleic acid can be identified by preparing a PNA probe having a binding force at a temperature higher than a denatured temperature (about 75 ° C. or more).

The experimental conditions for the melting curve analysis using the PNA probe were as follows; 25 [mu] l of the amplification product used for amplification curve analysis and 0.5 [mu] l (10 pmol) of PROBE-2 (SEQ ID NO: 2) were added.

For the amplification curve analysis, the reaction was performed 40 times for 2 minutes at 50 ° C, 10 minutes at 95 ° C, 15 seconds at 95 ° C, and 1 minute at 60 ° C. The process for the calcite is performed in a denaturation step of 3 minutes at 90 ° C., 1 minute at 75 ° C., and 1 minute at 55 ° C., followed by melting curve analysis to measure fluorescence by increasing the temperature from 20 ° C. to 90 ° C. in 0.5 ° C. steps. It was. The suspension was held for 5 seconds between each step.

As a result, as shown in Figure 6, it was confirmed that the melting peak in the melting curve of the sample to which the target nucleic acid is added. Here, the blue line of FIG. 6 is an amplification product of a sample containing a target nucleic acid, and the red line is a negative control (D.W).

Example 4 Method of Distinguishing Amplification Products of Real Target Nucleic Acids from Low Concentration Non-Specific Amplification Products Using PNA Probes

In the analysis of the target nucleic acid, in order to verify the method of effectively detecting the target nucleic acid and to minimize the error of discrimination of the presence or absence of the target nucleic acid by false-positive and false-negative signals, which is a problem with low concentration of nonspecific amplification products, The base sequence of WSD (White Spot Disease) virus was used, and a primer and a probe for amplification were prepared by using a phosphate tagging primer having a nucleotide sequence as described in OIE guidelines.

PROBE-3 (SEQ ID NO: 3) and PROBE-4 (SEQ ID NO: 4) were used as probes to confirm the presence of an amplification curve due to the appearance of low concentration of nonspecific amplification products. WSD-derived target nucleic acid and distilled water as a negative control, to obtain an amplification curve. The experimental method is as follows; 2X TaqMan Universal PCR Master Mix (PE Applied Biosystems, Foster City, CA, USA) 12.5 μl, PROBE-3 (SEQ ID NO: 3) (neoprov, Korea) 1.5 μl / 1 pmol, Primer-2 so that the total volume is 25 μl. -F (SEQ ID NO: 6), Primer-1-R (SEQ ID NO: 7) (3 μl / 1 pmol: 3 μl / 10 pmol) and 5 μl of sterilized distilled water were added to prepare a reaction for PCR amplification. The target nucleic acid in the experiment was 0.01 ng (positive control) and 0.001 ng (low concentration of target nucleic acid) per sample, and distilled water was used as a negative control.

As a result, as shown in Figure 7, when using a target control of 0.01ng as a positive control when confirming the amplification curve using PROBE-3 (SEQ ID NO: 3) RFU (light intensity or the concentration of the product of the amplified target nucleic acid) The Ct value was 36 when the) value was 1000, but the Ct value could not be determined when using a low concentration of the target nucleic acid (0.001 ng / sample). However, low concentrations of the target nucleic acid and negative control showed a slight amplification curve.

On the other hand, to verify the authenticity of the low concentration of non-specific amplification product, the dissolution curve analysis was performed using PROBE-4 (SEQ ID NO: 4).

In order to identify low concentrations of nonspecific amplification products, the concentration of primers was added symmetrically, and experiments were performed using the method of Korean Patent Application No. (10-2013-0106692) as a method for increasing the binding force of the PNA probe. The experimental method is as follows; Experiments were performed by adding 0.5 μl / unit of Lambda exonulease and 0.5 μl / 10 pmol of PROBE-4 (SEQ ID NO: 4) to 0.01 ng target nucleic acid, 0.001 ng target nucleic acid, or negative control, which were amplification products per sample, respectively.

The melting curve analysis using the PNA probe was first performed by removing the strand bound to the phosphate group at the 5 'end of the product generated through amplification to make a single strand. The melting curve analysis was performed after 5 minutes at 95 ° C, 30 seconds at 75 ° C, and 30 seconds at 55 ° C, followed by melting curve analysis at 1 ° C from 45 ° C to 80 ° C. Was performed.

As a result, as shown in Figure 8, in the analysis of the melting curve of the target nucleic acid of 0.01ng intrinsic temperature (Tm) 64.0 ℃ corresponding to PROBE-4 (SEQ ID NO: 4) it was confirmed that the target nucleic acid is real . However, in the target nucleic acid of 0.001ng, the Tm value was 60 ° C., and the negative control group did not show the Tm value, so it was not possible to distinguish the amplification product from the non-specific amplification product. Therefore, using a PNA probe (PROBE-4, SEQ ID NO: 4) it was confirmed that the minimum amplification product concentration is necessary to confirm whether the amplification product is a real target nucleic acid amplification product or a non-specific amplification product at a unique temperature.

On the other hand, the PNA probe was used to identify an improved melting curve analysis method that can distinguish the authenticity of low concentration nonspecific amplification products.

The method for confirming the presence or absence of an amplification curve of a low concentration of nonspecific amplification products was the same as the amplification method used in FIG. 7. The experimental method is as follows; 5 μl of target product amplified to a total volume of 20 μl, 10 μl of 2X qPCR PreMix (Eye Biomaterials, Korea), 0.5 μl / 10 pmol of PROBE-4 (SEQ ID NO: 4), Primer-1-R (SEQ ID NO: 7) (0.5 μl / 100 pmol) and 4 μl of sterilized distilled water were added. The target nucleic acid in the experiment was 0.01 ng (positive control) and 0.001 ng (low concentration of target nucleic acid) per sample, and distilled water was used as a negative control.

The process for melting curve analysis was performed 15 minutes at 95 ° C. for 5 minutes, 95 ° C. for 3 seconds, and 56 ° C. for 30 seconds, at 76 ° C. for 30 seconds, for 5 minutes at 95 ° C., at 75 ° C. for 30 seconds, and at 55 ° C. After 30 seconds in the denaturation step, the melting curve analysis to measure the fluorescence was carried out in 1 ℃ increments from 45 ℃ to 80 ℃.

As a result, as shown in FIG. 9, a lower concentration of the target nucleic acid (0.001 ng / sample) and the additional amplification and the binding force of the PNA probe were distinguished from the actual target nucleic acid and the non-specific product negative control as compared to the negative control. .

Critical Cycle (Ct) of Real-Time Polymerase Chain Reaction Used in Diagnostic and Individual Identification Methods When Using Targeted Nucleic Acid Detection and Quantitative Methods Using a PNA Probe Associated with a Reporter and a Quencher According to the Present Invention By solving the technical problem of discriminating by reference value, the accuracy of detection and reading of quantitative results of target nucleic acid or target gene can be improved, and economic, social and medical cost can be reduced.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims

Target nucleic acid using a reporter and quencher coupled PNA probe that solves the problem of detection of target nucleic acid along a threshold cycle (Ct) of real-time polymerase chain reaction, including the following steps: Methods for detecting or quantifying target nucleic acids by melting curve analysis:

(a) adding a PNA probe and TaqMan probe bound to a sample suspected of containing a target nucleic acid and a nucleic acid polymerase, primer, reporter and quencher, hybridizing, and then performing an asymmetric polymerase chain reaction. Amplifying using;

(b) melting the hybridized product with varying temperature to obtain a melting curve; And

(c) analyzing the resulting melting curve to detect or quantify the target nucleic acid.
The method of claim 1, wherein the PNA probe has a perfect match with the base sequence of the target nucleic acid (perfect match) to show the expected melting temperature (Tm) value, and incomplete hybridization (mismatch) with the target nucleic acid having a base sequence difference Method for detecting or quantifying target nucleic acid through a target nucleic acid fusion curve analysis, characterized in that the lower than expected temperature (Tm) value.
According to claim 1, wherein the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 And one or more selected from the group consisting of CY5. A method for detecting or quantifying a target nucleic acid through a target nucleic acid fusion curve analysis.
The method of claim 1, wherein the quencher is one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, detection or quantification of the target nucleic acid by target nucleic acid fusion curve analysis Way.
The method of claim 1, wherein the false-positive and false-negative signals can be distinguished using the difference in the fusion curve according to the target nucleic acid sequence.
The method of claim 1, wherein the PNA probe further comprises a locked nucleic acid (LNA).
A kit for detecting or quantifying target nucleic acid by using a method according to any one of claims 1 to 6, comprising a PNA probe coupled with a reporter and a quencher.
The method of claim 7, wherein the PNA probe has a perfect match with the base sequence of the target nucleic acid (perfect match) shows the expected melting temperature (Tm) value, and incomplete hybridization (mismatch) with the target nucleic acid having a base sequence difference A kit for the detection or quantification of a target nucleic acid through a target nucleic acid fusion curve analysis, characterized in that the lower than expected fusion temperature (Tm) value.
The method of claim 7, wherein the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 And a target nucleic acid fusion curve analysis kit for detecting or quantifying a target nucleic acid, characterized in that at least one selected from the group consisting of CY5.
The method according to claim 7, wherein the quencher is one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl detection or quantification of target nucleic acid by target nucleic acid fusion curve analysis Kit.
8. The kit according to claim 7, wherein the false positive and false negative signals can be distinguished using the difference in the fusion curve according to the target nucleic acid sequence.
Target nucleic acid using a reporter and quencher coupled PNA probe that solves the problem of detection of target nucleic acid along a threshold cycle (Ct) of real-time polymerase chain reaction, including the following steps: Methods for detecting or quantifying target nucleic acids by melting curve analysis:

(a) adding and hybridizing a sample suspected of containing the target nucleic acid with a nucleic acid polymerase, a primer and a TaqMan probe, followed by amplification using an asymmetric Polymerase Chain Reaction;

(b) adding and hybridizing the probe with the reporter and quencher to the amplification product;

(c) melting the hybridized product of step (b) while varying the temperature to obtain a melting curve; And

(d) analyzing the resulting melting curve to detect or quantify the target nucleic acid.
The method of claim 12, wherein the PNA probe is completely hybridized to the base sequence of the target nucleic acid (perfect match) shows the expected melting temperature (Tm) value, and incomplete hybridization (mismatch) with the target nucleic acid having a base sequence difference Method for detecting or quantifying target nucleic acid through a target nucleic acid fusion curve analysis, characterized in that the lower than expected temperature (Tm) value.
The method of claim 12, wherein the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 And one or more selected from the group consisting of CY5. A method for detecting or quantifying a target nucleic acid through a target nucleic acid fusion curve analysis.
The method of claim 12, wherein the quencher is one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl detection or quantification of the target nucleic acid by target nucleic acid fusion curve analysis Way.
The method of claim 12, wherein the false-positive and false-negative signals can be distinguished using the difference in the melting curve according to the target nucleic acid sequence.
The method of claim 12, wherein the PNA probe further comprises Locked Nucleic Acid (LNA).
18. A kit for detecting or quantifying target nucleic acid using a target nucleic acid fusion curve analysis using a method according to any one of claims 12 to 17, and comprising a PNA probe coupled with a reporter and a quencher.
19. The method of claim 18, wherein the PNA probe is in perfect hybridization with the base sequence of the target nucleic acid (perfect match) shows the expected melting temperature (Tm) value, and incomplete hybridization (mismatch) with the target nucleic acid having a base sequence difference A kit for the detection or quantification of a target nucleic acid through a target nucleic acid fusion curve analysis, characterized in that the lower than expected fusion temperature (Tm) value.
19. The method according to claim 18, wherein the quencher is one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, detection or quantification of target nucleic acid by target nucleic acid fusion curve analysis Kit.
19. The method of claim 18, wherein the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 And a target nucleic acid fusion curve analysis kit for detecting or quantifying a target nucleic acid, characterized in that at least one selected from the group consisting of CY5.
19. The method according to claim 18, wherein the quencher is one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, detection or quantification of target nucleic acid by target nucleic acid fusion curve analysis Kit.
19. The kit for detecting or quantifying a target nucleic acid through a target nucleic acid fusion curve analysis according to claim 18, wherein false-positive and false-negative signals can be distinguished using the difference in the fusion curve according to the target nucleic acid sequence.