KR20140091944A - Melting Curve Analysis Using Self Internal Control and PNA Probe Comprising Reporter and Quenching, Method and Kit for Analyzing Target DNA Detection Using Melting Curve Analysis - Google Patents

Melting Curve Analysis Using Self Internal Control and PNA Probe Comprising Reporter and Quenching, Method and Kit for Analyzing Target DNA Detection Using Melting Curve Analysis Download PDF

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KR20140091944A
KR20140091944A KR1020130004022A KR20130004022A KR20140091944A KR 20140091944 A KR20140091944 A KR 20140091944A KR 1020130004022 A KR1020130004022 A KR 1020130004022A KR 20130004022 A KR20130004022 A KR 20130004022A KR 20140091944 A KR20140091944 A KR 20140091944A
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정진욱
송민식
허덕회
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주식회사 시선바이오머티리얼스
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Abstract

The present invention relates to a method for analyzing a melting curve using a PNA probe with which an internal control, a reporter, and a quencher are combined, a method for detecting a target nucleic acid, and a kit for detecting a target nucleic acid, and more specifically, to a method for analyzing a melting curve using the melting temperature difference between a target nucleic acid and an internal control, which is characterized by substituting, deleting, or inserting one to four bases of a target nucleic acid base sequence for effectively detecting a target nucleic acid, and for distinguishing a false negative signal from a false positive signal of a PCR, and a method for detecting a target nucleic acid and a kit for detecting a target nucleic acid using the same. The present invention has an effect of distinguishing a false negative signal from a false positive signal, and effectively detecting a target nucleic acid through the melting curve analysis by using a PNA probe with which an internal control, which is characterized by substituting, deleting, or inserting one to four bases of a target nucleic acid base sequence, a reporter, and a quencher are combined.

Description

[TECHNICAL FIELD] The present invention relates to a method for analyzing a melt curve using a PNA probe coupled with an internal control and a reporter and a quencher, a method for detecting a target nucleic acid using the probe, and a method for detecting a target nucleic acid Target DNA Detection Using Melting Curve Analysis}

The present invention relates to a melting curve analysis method using a PNA probe coupled with an internal control and a reporter and a quencher, a target nucleic acid detection method and a target nucleic acid detection kit using the same, more particularly, to a false positive and false negative false negative), and 1 to 4 bases of the target nucleic acid sequence are substituted, deleted or inserted so as to effectively detect the target nucleic acid, and a fusion curve analysis method using the difference between the fusion temperatures of the target nucleic acid A target nucleic acid detection method using the same, and a target nucleic acid detection kit.

PCR technology, a nucleic acid amplification technology developed by Kary Mullis in 1983, is regarded as one of the greatest technological and economic value biotechnologies of the 20th century and has revolutionized whole molecular biology. It can be used as basic core technology in all fields of biotechnology such as gene cloning, sequencing, genetic diagnosis, gene fingerprint analysis and infectious disease diagnosis because it can amplify a very small amount of nucleic acid sample in a simple and convenient manner. (Bartlett, Stirling, Methods Mol Biol . 3 (6): 226, 2003).

In addition, the nucleic acid amplification technology has developed into a quantitative amplification technology of nucleic acid since the 1990s, and the market has been rapidly increasing. Overcoming the fact that the existing PCR technique can not predict the initial concentration before amplification of the target nucleic acid, Technologies that can accurately diagnose are widely used. Typically a non-specific fluorescent substances in SYBR the green and sequencing quantitative polymerase chain reaction (qPCR) the real-time PCR technique using specific TaqMan probe and so on are widely used and have been reported by many research results (yichangmuk, BioWave, 9 (8) , 2007). This real-time PCR technique has recently been recognized as the most powerful technique in the field of gene analysis and has been widely used in the field of nucleic acid analysis and has proved its value as it is being used to confirm the H1N1 virus infection, .

A representative DNA-binding dye, SYBR Green, is inserted between double-stranded DNAs and displays fluorescence. However, since SYBR Green is likely to be inserted non-specifically between DNA strands, it is not suitable for specific target detection. When SYBR Green technology is applied to real-time multiplex detection, melting curve analysis ), It is considered unsuitable in terms of temporal and efficient analysis.

In addition, primers or probes of conventional real-time multiplex PCR methods are more likely to generate non-specific hybridization (RN Gunson, et al ., Journal of Clinical In the case of label primer-based real-time multiplex PCR, dimer formation of the labeled primer is considered to be a major problem because it can generate a false positive signal, and PCR amplification is not performed even though the target nucleic acid is present ( Virology , 43: 372, There is a problem that it is difficult to distinguish the false signal caused by the probe problem.

A method for distinguishing false-negative signals using internal control has been studied (Burggraf S. et al ., Clin Chem . , 50 (5): 819, 2004), there is a problem that the length of the DNA probe must be increased in order to produce the specificity and proper melting temperature (Tm) for producing the internal control using the base mutation. In the above paper, PCR was performed using a binary probe method to increase the length. However, it is difficult to design and manufacture the probe, and since the binary probe technique has difficulties in using various fluorescence than the general fluorescence-photon photon method, Which is a major limitation in multiplex detection.

On the other hand, PNA is more thermally and biologically stable than DNA, has excellent recognition and binding ability for target nucleic acid, and can be used as a probe of a real-time PCR technique for detecting a target nucleic acid. However, since the real-time PCR method uses a fluorescent substance in detecting a target nucleic acid in a sample, there is a problem in that a probe having two different wavelengths of fluorescence is required to detect two or more target nucleic acids, detection is a large limitation.

Accordingly, the present inventors have made intensive efforts to minimize the error caused by the false positive and false negative signals appearing in the conventional PCR process and to effectively detect the target nucleic acid. As a result, it has been found that one to four bases of the target nucleic acid sequence are substituted, deleted or inserted Using a PNA probe with internal control and reporter and quenching features, it is possible to effectively detect the target nucleic acid through dissolution curve analysis and to identify the false positive and false negative signals Thereby completing the present invention.

It is an object of the present invention to provide an internal control and a PNA coupled with a reporter and a quenching, characterized in that one to four bases of the target nucleic acid sequence are substituted, deleted or inserted to detect a target nucleic acid. A method for analyzing a fusion curve for detecting a target nucleic acid using a probe, a method for detecting a target nucleic acid using the probe, and a kit.

In order to achieve the above object, the present invention provides a PNA probe having internal control and reporter and quenching combined, characterized in that 1 to 4 bases of the target nucleic acid sequence are substituted, deleted or inserted. A method for analyzing a melting curve for detecting a target nucleic acid using the method.

The present invention also provides a method for detecting a nucleic acid comprising: (a) separating a target nucleic acid from a sample of a sample; (b) 1 to 4 bases of the target nucleic acid sequence are substituted, deleted, or inserted, characterized in that the PNA probe and the primer are combined with a target nucleic acid and a PNA probe and a target nucleic acid Hybridizing internal control; (c) melting the hybridized product while changing the temperature to obtain a melting curve; And (d) analyzing the obtained melting curve to detect the presence or absence of a target nucleic acid.

The present invention also provides a fusion curve analysis of a PNA probe coupled with an internal control and a reporter and a quenching, characterized in that one to four bases of the target nucleic acid sequence are substituted, deleted or inserted A kit for detecting a target nucleic acid is provided.

Using a PNA probe coupled with an internal control and reporter and quenching, characterized in that 1 to 4 bases of the target nucleic acid sequence of the present invention are substituted, deleted or inserted, analysis of the dissolution curve The target nucleic acid can be effectively detected and the false positive and false negative signals can be discriminated.

Figure 1 shows the sequence of a PNA capable of complementary binding to a target nucleic acid and internal control with a sequence of a target nucleic acid and an internal control with one base substituted for one base corresponding to the target nucleic acid.
FIG. 2 is a graph showing a change in melting curve and a false negative signal according to presence or absence of target nucleic acid using the internal control system of the present invention. FIG.
FIG. 3 is a graph showing the melting curve according to measurement of fluorescence at two temperatures in the detection of a target nucleic acid using the internal control system of the present invention. FIG.
FIG. 4 is a graph showing an amplification plot and a melting peak according to the presence or absence of a target nucleic acid in the detection of a target nucleic acid using the internal control system of the present invention.
Figure 5 shows the detection and quantification of target nucleic acid using real-time PCR. (A) PCR amplification curve according to target nucleic acid concentration, (B) Melting curve according to target nucleic acid concentration, (C) (NTC: negative control added with internal control only). Fig. 2 (b) is a graph showing the dissociation curve according to the target nucleic acid concentration.
6 is a graph showing the melting curves of a number of target nucleic acids using PNA labeled with FAM, HEX, texas Red and Cy5 reporter, respectively.
FIG. 7 is a diagram illustrating the degree of hybridization of PNA according to the position of the base mutation in the base sequence region and the number of mutation bases, which are combined with the internal control and the PNA probe.
FIG. 8 is a graph showing the change of the melting curve according to the number of mutation bases of the (A) end position deletion and (B) middle position deletion in the nucleotide sequence region where the internal control and the PNA probe are combined .
Figure 9 shows the position of PNA probe hybridization according to the base displacement position of the internal control sequence.
FIG. 10 is a comparison between the internal control according to the base displacement position of the internal control sequence and the melting curve of the target nucleic acid.

Unless otherwise defined, 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.

The present invention, in one aspect, is directed to a target using a PNA probe coupled with an internal control and a reporter and a quenching, characterized in that one to four bases of the target nucleic acid sequence are substituted, deleted or inserted To a method of analyzing a melting curve for nucleic acid detection.

The 'target nucleic acid' of the present invention means a nucleic acid sequence to be detected, and is annealed or hybridized with a primer or a probe under hybridization, annealing or amplification conditions. The 'target nucleic acid' is not different from the term 'target nucleic acid', 'target nucleic acid sequence' or 'target sequence' as used herein, and is used in this specification.

The 'hybridization' of the present invention means that complementary single-stranded nucleic acids form a double-stranded nucleic acid. Hybridization can occur either in perfect match between two nucleic acid strands, or even in the presence of some mismatching bases. The degree of complementarity required for hybridization can vary depending on the hybridization conditions, and can be controlled, in particular, by temperature.

The PNA probe comprising the reporter and the quencher of the present invention hybridizes with the target nucleic acid and generates a fluorescence signal. As the temperature rises, the fluorescence signal is rapidly extinguished with the target nucleic acid at the optimal melting temperature of the probe, The presence or absence of the target nucleic acid can be detected through the analysis of the melting curve of the high resolution obtained from the fluorescence signal according to the present invention.

The probe of the present invention may be combined with a quencher fluorescent material capable of quenching reporter and reporter fluorescence at both ends thereof. The reporter may be selected from the group consisting of 6-carboxyfluorescein, Texas red, HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy-4,7-dichlorofluorescein) And the quencher may be at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, but is not limited thereto. Preferably, Dabcyl is used .

In the present invention, the PNA probe is characterized by a perfect match with the target nucleic acid sequence, and is characterized by incomplete hybridization (mismatch) with the internal control.

The internal control of the present invention is designed to have a difference in dissolution temperature (Tm) from a target nucleic acid which is completely hybridized with a PNA probe due to incompatibility with the PNA probe. As shown in FIG. 1, the internal control has a target nucleic acid sequence And the PNA probe binding sequence is prepared so that one or more bases are substituted for mismatching. In addition to the mutation method by substitution, the internal control can be designed to be mutated through deletion or insertion of a base have.

In the present invention, a PNA probe binds preferentially to a target nucleic acid in the presence of a target nucleic acid and exhibits a predicted melting temperature (Tm) value. When the target nucleic acid is absent, it binds to internal control, And the difference between the internal control and the target nucleic acid dissolution curve can be used to distinguish between false positive and false negative signals.

In general, when performing real-time PCR techniques using a labeled probe or a primer to simultaneously detect a plurality of target nucleic acid sequences, it is very difficult to determine the proper sequence of the oligonucleotide as a primer and a probe, and a dimer of the oligonucleotide there is a high possibility that a false positive signal generated by dimer formation or non-specific hybridization occurs. If the target nucleic acid is present in very small amounts or does not exist, the discrimination or determination of whether the signal from the real-time multiplex PCR is a positive signal due to the presence of the target nucleic acid sequence or an error signal due to the dimer formation of the label primer There is a problem that the PCR is not amplified even though the target nucleic acid is present or a false negative signal is generated due to the probe problem.

In order to discriminate the false negative signal, a method of using a binary probe for the difference of the fusion temperature between the internal control and the target nucleic acid has been studied (Burggraf S. et al ., Clin Chem . , 50 (5): 819, 2004). When a binary probe is used for the difference in melting temperature with the target nucleic acid, a false negative signal is generated when the concentration of the target nucleic acid is high or low. There is a possibility to affect the number. However, in the case of the PNA probe of the present invention, since it is specifically prepared only for the target nucleic acid, it binds to the target nucleic acid competitively in the presence of the target nucleic acid and does not affect the copy number.

As shown in FIG. 2, when the target nucleic acid is present, the PNA probe binds to the target nucleic acid and the internal control to show two peaks. The internal control and the internal control of the PNA probe Is measured at a low melting temperature (Tm). In the absence of the target nucleic acid, only one peak is visible because the PNA probes bind only to the internal control.

The melting curve analysis method of the present invention is based on the fact that a PNA probe combined with a reporter and a quenching is attached to a complementary target nucleic acid instead of labeling the primer as in real-time multiplex PCR, (Tm) as shown in FIG. 2C, the false positive signal due to the primer dimer can be excluded. If the dissolution curve is not present as shown in FIG. 2C, the PCR reaction can not be performed, It is possible to identify the false-negative signal that appears as a combination abnormality.

In one embodiment of the present invention, PNA probes, target nucleic acids, and internal control DNA oligomers and primers were prepared (Table 1) for the detection of target nucleic acids by dissolution curve analysis. Asymmetric PCR ) Method was used to perform real-time PCR and fusion curve analysis. As a result, since the binding temperature of the probe binding to the target nucleic acid and the internal control is different according to the sequence variation of the internal control, the presence or absence of the target nucleic acid can be confirmed in real time by measuring the fluorescence at the two temperatures in the real- 3). In addition to the dissolution curve peak analysis, as shown in FIG. 4, the amplification plot can be measured at different temperatures to confirm whether there is a target nucleic acid in real time.

In addition, the present invention, as shown in the above Examples and FIGS. 2 to 4, shows that even when the internal control giving a single base change is used, the PNA probe preferentially binds to the target nucleic acid in the presence of the target nucleic acid (Tm), the presence or absence of the target nucleic acid can be easily determined using the melting curve analysis method.

In the present invention, negative control (NTC) with internal control alone and various concentrations of target nucleic acid and internal control were mixed and analyzed using real-time PCR. As a result, NTC showed no amplification in the amplification curve (FIG. 5C) (FIG. 5B and FIG. 5D), and it was confirmed that the presence or absence of a false negative signal can be clearly distinguished. Further, when the fluorescence measurement temperature is measured at a temperature at which the melting curve peak of the internal control does not appear (more than 50 ° C in FIG. 3) during the real-time PCR process based on the melting curve peak shown in FIG. 3, the amplification curve of the internal control appears And only the amplification curve of the target nucleic acid is displayed. Therefore, it has been confirmed that the method of detecting the target nucleic acid using the internal control of the present invention is capable of quantitative analysis and real-time analysis using the Ct (Cycle Threshold) value.

The internal control of the present invention may include, but is not limited to, continuous or discontinuous substitution, deletion or insertion of bases at the middle or end of the sequence for differences in melting temperature (Tm) with the target nucleic acid, Can be substituted, deleted or inserted.

When the internal control of the present invention and the deletion of 1 to 4 bases corresponding to the end of the site that is associated with the PNA probe are continuously deleted, the ends of the PNA probe undergo internal control and incomplete hybridization (mismatch) When the difference in temperature (Tm) occurs and successive deletion of one to four bases corresponding to the middle part of the internal control and the binding site of the PNA probe, the middle part of the PNA probe has internal control and incomplete Hybridization is performed to form a loop, and thus a difference in melting temperature Tm occurs (FIG. 7).

In one embodiment of the present invention, when one or more bases corresponding to the center position and the end position of the base sequence which binds to the internal control and the PNA probe are successively deleted , A difference in the target nucleic acid (perfect match) and the melting temperature (Tm) occurred. As the number of deleted bases increased, the difference in melting temperature (Tm) with the target nucleic acid became larger. As the number of mutated bases increases, the difference in melting temperature becomes larger. However, when the number of base mutations continuously attached increases, there arises a problem that PNA can not bind to the base. Therefore, it is preferable to mutate 1 to 4 bases .

In the present invention, as shown in FIG. 9 and Table 3, a fusion curve analysis with the target nucleic acid was performed using an internal control or an internal control in which a base was inserted or deleted according to the hybridization position of the target nucleic acid and the PNA probe. As a result, it was confirmed that the internal control in which the base was deleted or inserted regardless of the base mutation position of the entire base sequence of the internal control had a difference in melting temperature (Tm) with the target nucleic acid, and the target nucleic acid, And the internal control in which the base was inserted (Fig. 10).

In another aspect, the present invention provides a method for detecting a target nucleic acid, comprising: (a) separating a target nucleic acid from a sample of a sample; (b) 1 to 4 bases of the target nucleic acid sequence are substituted, deleted, or inserted, characterized in that the PNA probe and the primer are combined with a target nucleic acid and a PNA probe and a target nucleic acid Hybridizing internal control; (c) melting the hybridized product while changing the temperature to obtain a melting curve; And (d) analyzing the obtained melting curve to detect the presence or absence of a target nucleic acid.

In the present invention, the 'sample' includes various samples, and preferably the biosample is analyzed using the method of the present invention. Biological samples of plant, animal, human, fungal, bacterial and viral origin can be analyzed. When analyzing a sample of mammalian or human origin, the sample may be from a particular tissue or organ. Representative examples of tissues include binding, skin, muscle or nervous tissue. Representative examples of organs include, but are not limited to, eyes, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testes, Line and inner blood vessels. The biological sample to be analyzed includes any cells, tissues, fluids from the biological source, or any other medium that can be well analyzed by the present invention, including human, animal, human or animal Lt; RTI ID = 0.0 > a < / RTI > In addition, the biological sample to be analyzed includes a body fluid sample, which may be a blood sample, serum, plasma, lymph, milk, urine, feces, eye milk, saliva, semen, brain extract And tonsil tissue extracts.

The target nucleic acid of the sample is DNA or RNA, and the molecule may be double-stranded or single-stranded. When the nucleic acid as the starting material is a double strand, it is preferable to make the two strands into a single strand, or a partial single strand form. Methods known to separate strands include, but are not limited to, heat, alkaline, formamide, urea and glycoconjal treatment, enzymatic methods (e.g., helicase action) and binding proteins. For example, the strand separation can be achieved by heat treatment at a temperature of 80 to 105 ° C. A common method of processing as described above is by Joseph Sambrook meat al ,, Molecular Cloning , < / RTI > 2001.

The target nucleic acid detection method of the present invention is characterized in that the PNA probe binds preferentially to the target nucleic acid in the presence of the target nucleic acid and exhibits the expected fusion temperature (Tm) value, and binds to the internal control in the absence of the target nucleic acid, (Tm) value. The difference between the internal control and the target nucleic acid dissolution curve can be used to identify the presence or absence of the target nucleic acid and the false positive / false negative signal.

In the present invention, the target nucleic acid detection method of the present invention may be characterized in that two or more target nucleic acids are used, and the reporter labeled on the PNA probe is different for each target nucleic acid, thereby detecting two or more target nucleic acids.

In one embodiment of the invention, four sets of PNA probes (FAM-, HEX-, texas Red-, Cy5-labeled), primers, target nucleic acids and internal controls shown in Table 1 were added to one tube As a result, real-time PCR and melting curve analysis were performed. As a result, it was confirmed that a target nucleic acid detection method using the internal control system of the present invention can simultaneously detect a plurality of target nucleic acids (FIG. 6).

In another aspect, the present invention includes a PNA probe coupled with an internal control and a reporter and a quenching, characterized in that one to four bases of the target nucleic acid sequence are substituted, deleted or inserted And a kit for detecting a target nucleic acid using a fusion curve analysis method.

The kit of the present invention can optionally include reagents necessary for conducting a target amplification PCR reaction (e. G., PCR reaction) such as a buffer, a DNA polymerase joiner and deoxyribonucleotide-5-triphosphate. Alternatively, the kit of the present invention may also include various polynucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.

Also, the optimal amount of reagent used in a particular reaction in a kit can be readily determined by those skilled in the art having the teachings herein. Typically, the kit of the present invention is fabricated as a separate package or compartment comprising the aforementioned components.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Detection of target nucleic acid using internal control

(1) Production of internal controls and target nucleic acid oligomers, primers and PNA probes used in the melting curve assay

PNA probes, target nucleic acids and internal control DNA oligomers and primers were prepared for target nucleic acid detection through the dissolution curve analysis of the present invention.

First, PNA probes were designed using a PNA probe designer (Applied Biosystems, USA). All PNA probes (FAM-, HEX-, texas Red-, and Cy5-labeled) used in the present invention were synthesized by HPLC purification method from Panagene (Korea) and the target nucleic acid and oligonucleotide (Neoprobe, Korea) using PAGE purification method. The purity of all synthesized probes was verified using mass spectrometry and unnecessary secondary structure of the probe was avoided for more effective binding with the target nucleic acid.

4 sets of oligomers corresponding to the internal control and the target nucleic acid shown in Table 1 were arbitrarily designed and synthesized (Neoprobe, Korea), and the internal control was changed to one sequence when compared with the target nucleic acid sequence (mismatch). In addition, the PNA probe was designed to be complementary to the internal control and the target nucleic acid. For multiple detection of the target nucleic acid, a different reporter was labeled with FAM, HEX, texas Red and Cy5 on the PNA probe for each target nucleic acid, Dabcyl was used.

The PNA, DNA oligomer sequence used in the Self-Internal Control system (SICS) Set Target sequence (5'-3 ') Primer sequence (5'-3 ') Probe sequence (5'-3 ') FAM
Target TCGTCAATGGAGCGTCGGTATTGCCATCAGCCGGGGCCGGAACAGGTTAT GAACTGTGTACCC CCCTTGGCAGGAAATATAGCCCACGCTGGGGCCTCGATCAAGCACGATCATTGCCATGAGACG
(SEQ ID NO: 1) ATGGAGCGTCGGTATTGCCATC
(SEQ ID NO: 3) Dabcyl-GAACTGTGTACCC-OK-FAM
(SEQ ID NO: 5)
SIC-target TCGTCAATGGAGCGTCGGTATTGCCATCGAATTAGTACTATGGCGAGTAT GAACTGT A TACCC CACAAGCCTGCGCGGTGGGGTATTATGATAAATTCGATCAAGCACGATCATTGCCATGAGACG
(SEQ ID NO: 2) ATGGCAATGATCGTGCTTGATCG
(SEQ ID NO: 4) Cy5 Target ≪ RTI ID =
(SEQ ID NO: 6) GGCAAGCCACGTTTGGTG
(SEQ ID NO: 8) Dabcyl-CTTCTTATGGCCC-OK-Cy5
(SEQ ID NO: 10) SIC-target GGCAAGCCACGTTTGGTGGTTACAACTGTCTTGCTT CTTAT A GCCC TCCCAGTCCTAGCACCTCTGACACATGCAGCTCC
(SEQ ID NO: 7) GGAGCTGCATGTGTCAGAGG
(SEQ ID NO: 9) HEX Target ATGCCATAGCATTTTTATCCAAAGTTTTTGACTTC TACCTCCCTCTTT CCTCCTCCTTTTAGCCTGATACAGATTAAATC
(SEQ ID NO: 11) ATGCCATAGCATTTTTATCCA
(SEQ ID NO: 13) Dabcyl-TACCTCCCTCTTT-OK-HEX
(SEQ ID NO: 15) SIC-target ATGCCATAGCATTTTTTCCAAAGTTTTTGACTTC TACC C CCCTCTTT CCTCCTCCTTTTAGCCTGATACAGATTAAATC
(SEQ ID NO: 12) GATTTAATCTGTATCAGG
(SEQ ID NO: 14) Texas Red Target AGCACCCAGTCCGCCCTGAGCATAAAACCACCAGCA ATCTCTCAATACC AAACACCTTTATTCCACTCTCGGCATGGACG
(SEQ ID NO: 16) AGCACCCAGTCCGCCCTGAGC
(SEQ ID NO: 18) Dabcyl-ATCTCTCAATACC-OK-Texas Red
(SEQ ID NO: 20) SIC-target AGCACCCAGTCCGCCCTGAGCATAAAACCACCAGCA ATCTC C CAATACC AAACACCTTTATTCCACTCTCGGCATGGACG
(SEQ ID NO: 17) CGTCCATGCCGAGAGTG
(SEQ ID NO: 19)

In Table 1, underlined letters indicate hybridization with PNA probes, internal control (SIC-target) nucleotide sequences in boldface indicate mutated bases, O means linker, and K means lysine.

(2) Melting curve analysis for target nucleic acid detection

The PCR between the probe and the synthesized target oligonucleotide and internal control was performed using a CFX96 ™ Real-Time system (BIO-RAD, USA). All the experimental conditions were asymmetric PCR (asymmetric PCR) was used. The conditions of asymmetric PCR are as follows; 0.5 μl of internal control and 0.5 μl of internal control were added to 1 × PNAqPCR ™ PreMix (PANAGENE, Korea), 2.5 mM MgCl 2 , 200 μM dNTPs, 1.0 U Taq polymerase, 0.05 μM forward primer and 0.5 μM reverse primer (asymmetric PCR) Mu] l of target DNA (1 x 10 5 copies of synthetic DNA) and then real-time PCR. Real-time PCR was performed at 95 ° C for 7 minutes, followed by 95 ° C for 10 seconds, 50 ° C for 15 seconds, and 74 ° C for 30 seconds, followed by 50 cycles and fluorescence at 50 ° C. Melting curve analysis was performed by denaturation at 95 ° C for 3 minutes, followed by hybridization at 35 ° C for 5 minutes, followed by a solubility curve analysis in which fluorescence was measured by increasing the temperature from 35 ° C to 85 ° C by 1 ° C. The stationary state was maintained for 5 seconds between each step.

As a result, since the binding temperature of the probe binding to the target nucleic acid and the internal control is different according to the sequence variation of the internal control, the presence or absence of the target nucleic acid can be confirmed in real time by measuring the fluorescence at the two temperatures in the real- 3). In addition to the dissolution curve peak analysis, as shown in FIG. 4, the amplification plot can be measured at different temperatures to confirm whether there is a target nucleic acid in real time.

In addition, negative control (NTC) with internal control alone and various concentrations of target nucleic acid and internal control were mixed and analyzed using real-time PCR. As a result, NTC did not show amplification in the amplification curve (FIG. 5C) (FIG. 5B and FIG. 5D), it was confirmed that quantitative analysis and real-time analysis using a Ct (Cycle Threshold) value were possible as well as discrimination of presence or absence of a false negative signal.

With internal controls PNA The probe  Analysis of the melting curve according to the position of the base mutation and the number of mutation bases in the base sequence

(1) internal control and production of target nucleic acid oligomers, primers and PNA probes

The internal control DNA and the target nucleic acid DNA were prepared as follows to measure the change of the dissolution curve according to the base mutation position and the number of mutation bases in the base sequence region where the internal control of the present invention and the PNA probe bind.

First, PNA probes were designed using a PNA probe designer (Applied Biosystems, USA). All the PNA probes (FAM-labeled) used in the present invention were synthesized by HPLC purification method in Panagene (Korea). The target nucleic acid and the oligonucleotide of the internal control were purified from the neoprobe (Neoprobe, Korea) . The purity of all synthesized probes was verified using mass spectrometry and unnecessary secondary structure of the probe was avoided for more effective binding with the target nucleic acid.

The internal control corresponds to the center position and the end position of the base sequence to which the internal control and the PNA probe bind, as shown in Table 2, for the difference in melting temperature (Tm) with the target nucleic acid The primers and the PNA probes (FAM-labeled) were designed so that complementary binding with the target nucleic acid and internal control with 1 to 4 bases deleted Respectively.

Internal control and target nucleic acid, primer and PNA probe oligomer sequence Application Target sequence (5'-3 ') SEQ ID NO: end position deletion Perfect match
(target DNA) TCGTCAATGGAGCGTCGGTATTGCCATCAGCCGGGGCCGGAACAGGTTAT GAACTGTGTACCC CCCTTGGCAGGAAATATAGCCCACGCTGGGGCCTCGATCAAGCACGATCATTGCCATGAGACG SEQ ID NO: 1 single del TCGTCAATGGAGCGTCGGTATTGCCATCAGCCGGGGCCGGAACAGGTTAT GAACTGTGTACC CCCTTGGCAGGAAATATAGCCCACGCTGGGGCCTCGATCAAGCACGATCATTGCCATGAGACG SEQ ID NO: 21 double del ≪ RTI ID = 0.0 & SEQ ID NO: 22 triple del ≪ RTI ID = 0.0 & SEQ ID NO: 23 quadruple del ≪ RTI ID = 0.0 & SEQ ID NO: 24 평점 위치
deletion Perfect match
(target DNA) TCGTCAATGGAGCGTCGGTATTGCCATCAGCCGGGGCCGGAACAGGTTAT GAACTGTGTACCC CCCTTGGCAGGAAATATAGCCCACGCTGGGGCCTCGATCAAGCACGATCATTGCCATGAGACG SEQ ID NO: 1 single del ≪ RTI ID = 0.0 & SEQ ID NO: 25 double del TCGTCAATGGAGCGTCGGTATTGCCATCAGCCGGGGCCGGAACAGGTTAT GAACTGTACCC CCCTTGGCAGGAAATATAGCCCACGCTGGGGCCTCGATCAAGCACGATCATTGCCATGAGACG SEQ ID NO: 26 triple del TCGTCAATGGAGCGTCGGTATTGCCATCAGCCGGGGCCGGAACAGGTTAT GAACTTACCC CCCTTGGCAGGAAATATAGCCCACGCTGGGGCCTCGATCAAGCACGATCATTGCCATGAGACG SEQ ID NO: 27 quadruple del TCGTCAATGGAGCGTCGGTATTGCCATCAGCCGGGGCCGGAACAGGTTAT GAACTACCC CCCTTGGCAGGAAATATAGCCCACGCTGGGGCCTCGATCAAGCACGATCATTGCCATGAGACG SEQ ID NO: 28 Prime Forward ATGGAGCGTCGGTATTGCCATC SEQ ID NO: 3 everse ATGGCAATGATCGTGCTTGATCG SEQ ID NO: 4 Probe AM Dabcyl-GAACTGTGTACCC-O-K-FAM SEQ ID NO: 5

In Table 2, O means linker, K means lysine, underlined letter and boldface means hybridization with PNA probe and deleted base.

As shown in Table 2, when one to four bases corresponding to the ends of the internal control and the PNA probe are continuously deleted, the ends of the PNA probe are subjected to internal control and incomplete hybridization (mismatch) When the melting temperature (Tm) is varied, and one to four bases corresponding to the middle part of the internal control and the binding site of the PNA probe are successively deleted, the middle part of the PNA probe is inside Control is incompletely hybridized with each other to form a loop, which causes a difference in melting temperature (Tm) (FIG. 7).

(2) Analysis of the melting curve according to the position of the base mutation and the number of mutation bases in the base sequence region where the internal control and the PNA probe are combined

PCR was performed using the CFX96 ™ Real-Time system (BIO-RAD, USA) for the analysis of the fusion curve using the target nucleic acid, internal control, and PNA probe synthesized above. Asymmetric PCR (asymmetric PCR) was used to generate. The conditions of asymmetric PCR are as follows; 0.5 μl of internal control and 0.5 μl of internal control were added to 1 × PNAqPCR ™ PreMix (PANAGENE, Korea), 2.5 mM MgCl 2 , 200 μM dNTPs, 1.0 U Taq polymerase, 0.05 μM forward primer and 0.5 μM reverse primer (asymmetric PCR) Mu] l of target DNA (1 x 10 5 copies of synthetic DNA) and then real-time PCR. Real-time PCR was performed at 95 ° C for 7 minutes, followed by 95 ° C for 10 seconds, 50 ° C for 15 seconds, and 74 ° C for 30 seconds, followed by 50 cycles and fluorescence at 50 ° C. Melting curve analysis was performed by denaturation at 95 ° C for 3 minutes, followed by hybridization at 35 ° C for 5 minutes, followed by a solubility curve analysis in which fluorescence was measured by increasing the temperature from 35 ° C to 85 ° C by 1 ° C. The stationary state was maintained for 5 seconds between each step.

As a result, when 1 to 4 bases corresponding to the center position and the end position of the base sequence binding to the internal control and the PNA probe were successively deleted, the target nucleic acid (perfect match and the melting temperature (Tm) were found to be different from each other. As the number of deleted bases increased, the difference of the melting temperature (Tm) with the target nucleic acid became larger (FIG. 8).

Analysis of melting curve according to the base position of internal control

(1) internal control and production of target nucleic acid oligomers, primers and PNA probes

In order to measure the change of the dissolution curve according to the position and length of the base mutation position of the internal control of the present invention, internal control DNA and target nucleic acid DNA were prepared as follows.

First, PNA probes were designed using a PNA probe designer (Applied Biosystems, USA). All PNA probes (FAM-, HEX-, texas Red-, and Cy5-labeled) used in the present invention were synthesized by HPLC purification method from Panagene (Korea) and the target nucleic acid and oligonucleotide (Neoprobe, Korea) using PAGE purification method. The purity of all synthesized probes was verified using mass spectrometry and unnecessary secondary structure of the probe was avoided for more effective binding with the target nucleic acid.

In the present invention, an internal control in which one base is inserted or deleted (insertion and deletion detection) is prepared in each of four parts of the internal control whole nucleotide sequence. The four PNA probes (FAM-, HEX-, texas Red-, and Cy5-labeled) used in the experiments were designed to complement each other in four parts of the target nucleic acid sequence (149 bp) (149 bp) and internal control (153 bp), each of which contained one base, were used. The mutated bases were assigned a 10 bp difference between the primers or PNA probes and complementary Bonding was made. In addition, the mutation sites were basically located at the center of each probe so as to obtain a change in melting temperature (Tm) of 5 or more (Tables 3 and 9).

Internal control and target nucleic acid, primer and PNA probe oligomer sequence Application Target sequence (5'-3 ') SEQ ID NO: Target DNA Perfect match GGCTCAGCCATCTTACCTGTGGCACAGGTTGAACT GTG TACCCCCCCTTCGTTGGGGCCT CCG TAGACTTAACAACTTTATTCGT ATG ATCAATTCTTGTGTCTTGCTTCT TAT GGCCCTCCCAGTTTCTGATTCTTCGGACACCCGGC SEQ ID NO: 29 All Deletion GGCTCAGCCATCTTACCTGTGGCACAGGTTGAACT GG TACCCCCCCTTCGTTGGGGCCT CG TAGACTTAACAACTTTATTCGT AG ATCAATTCTTGTGTCTTGCTTCT TT GGCCCTCCCAGTTTCTGATTCTTCGGACACCCGGC SEQ ID NO: 30 All Insertion GGCTCAGCCATCTTACCTGTGGCACAGGTTGAACT G C C G CG TG TACCCCCCCTTCGTTGGGGCCT TAGACTTAACAACTTTATTCGT AT G T A T TA ATCAATTCTTGTGTCTTGCTTCT GGCCCTCCCAGTTTCTGATTCTTCGGACACCCGGC SEQ ID NO: 31 Primer Forward TCAGCCATCTTACCTGTGGC SEQ ID NO: 32 Reverse GGGTGTCCGAAGAATCAGAA SEQ ID NO: 33 Probe FAM Dabcyl-GTTGAACTGTGTACCC-O-K (FAM) SEQ ID NO: 34 HEX Dabcyl-GCCTCCGTAGACTTA-O-K (HEX) SEQ ID NO: 35 Texas Red Dabcyl-TTCGTATGATCAATTCTT-O-K (TexasRed) SEQ ID NO: 36 Cy5 Dabcyl-CTTCTTATGGCCCTCCC-O-K (Cy5) SEQ ID NO: 37

In Table 3, O means linker and K means lysine, and underlined letters and bold text means portions hybridized with PNA probes and portions deleted.

(2) Dissolution curve analysis according to internal control length

PCR was performed using the CFX96 ™ Real-Time system (BIO-RAD, USA) for the analysis of the fusion curve using the target nucleic acid, internal control, and PNA probe synthesized above. Asymmetric PCR (asymmetric PCR) was used to generate. The conditions of asymmetric PCR are as follows; 0.5 μl of internal control and 0.5 μl of internal control were added to 1 × PNAqPCR ™ PreMix (PANAGENE, Korea), 2.5 mM MgCl 2 , 200 μM dNTPs, 1.0 U Taq polymerase, 0.05 μM forward primer and 0.5 μM reverse primer (asymmetric PCR) Mu] l of target DNA (1 x 10 5 copies of synthetic DNA) and then real-time PCR. Real-time PCR was carried out by denaturing at 95 ° C for 7 minutes, then reacting at 95 ° C for 10 seconds, at 50 ° C for 15 seconds, and at 74 ° C for 30 seconds, followed by 50 cycles, and fluorescence was measured in real time. Melting curve analysis was performed by denaturation at 95 ° C for 3 minutes, followed by hybridization at 35 ° C for 5 minutes, followed by a solubility curve analysis in which fluorescence was measured by increasing the temperature from 35 ° C to 85 ° C by 1 ° C. The stationary state was maintained for 5 seconds between each step.

As a result, it was confirmed that the internal control in which the base was deleted or inserted regardless of the base mutation position of the entire base sequence of the internal control had a difference in melting temperature (Tm) with the target nucleic acid, and the target nucleic acid, And the internal control in which the base was inserted (Fig. 10).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Seasunbiomaterials <120> Melting curve analysis using Self Internal Control and PNA probe          Comprising Reporter and Quenching, Method and Kit for Analyzing          Target DNA Detection Using Melting Curve Analysis <130> P12-B278 <160> 37 <170> Kopatentin 2.0 <210> 1 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> artificial target DNA <400> 1 tcgtcaatgg agcgtcggta ttgccatcag ccggggccgg aacaggttat gaactgtgta 60 ccccccttgg caggaaatat agcccacgct ggggcctcga tcaagcacga tcattgccat 120 gagacg 126 <210> 2 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> artificial SIC-target DNA <400> 2 tcgtcaatgg agcgtcggta ttgccatcga attagtacta tggcgagtat gaactgtata 60 ccccacaagc ctgcgcggtg gggtattatg ataaattcga tcaagcacga tcattgccat 120 gagacg 126 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> target DNA forward primer <400> 3 atggagcgtc ggtattgcca tc 22 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> target DNA reverse primer <400> 4 atggcaatga tcgtgcttga tcg 23 <210> 5 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <400> 5 gaactgtgta ccc 13 <210> 6 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> artificial target DNA <400> 6 ggcaagccac gtttggtggt tacaactgtc ttgcttctta tggccctccc agtcctagca 60 cctctgacac atgcagctcc 80 <210> 7 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> artificial SIC-target DNA <400> 7 ggcaagccac gtttggtggt tacaactgtc ttgcttctta tagccctccc agtcctagca 60 cctctgacac atgcagctcc 80 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> target DNA forward primer <400> 8 ggcaagccac gtttggtg 18 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> target DNA reverse primer <400> 9 ggagctgcat gtgtcagagg 20 <210> 10 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <400> 10 cttcttatgg ccc 13 <210> 11 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> artificial target DNA <400> 11 atgccatagc atttttatcc aaagtttttg acttctacct ccctctttcc tcctcctttt 60 agcctgatac agattaaatc 80 <210> 12 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> artificial SIC-target DNA <400> 12 atgccatagc atttttatcc aaagtttttg acttctaccc ccctctttcc tcctcctttt 60 agcctgatac agattaaatc 80 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> target DNA forward primer <400> 13 atgccatagc atttttatcc a 21 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> target DNA reverse primer <400> 14 gatttaatct gtatcagg 18 <210> 15 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <400> 15 tacctccctc ttt 13 <210> 16 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> artificial target DNA <400> 16 agcacccagt ccgccctgag cataaaacca ccagcaatct ctcaatacca aacaccttta 60 ttccactctc ggcatggacg 80 <210> 17 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> artificial SIC-target DNA <400> 17 agcacccagt ccgccctgag cataaaacca ccagcaatct cccaatacca aacaccttta 60 ttccactctc ggcatggacg 80 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> target DNA forward primer <400> 18 agcacccagt ccgccctgag c 21 <210> 19 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> target DNA reverse primer <400> 19 cgtccatgcc gagagt 16 <210> 20 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <400> 20 atctctcaat acc 13 <210> 21 <211> 125 <212> DNA <213> Artificial Sequence <220> <223> SIC-target DNA sigle deletion <400> 21 tcgtcaatgg agcgtcggta ttgccatcag ccggggccgg aacaggttat gaactgtgta 60 cccccttggc aggaaatata gcccacgctg gggcctcgat caagcacgat cattgccatg 120 agacg 125 <210> 22 <211> 124 <212> DNA <213> Artificial Sequence <220> <223> SIC-target DNA double deletion <400> 22 tcgtcaatgg agcgtcggta ttgccatcag ccggggccgg aacaggttat gaactgtgta 60 ccccttggca ggaaatatag cccacgctgg ggcctcgatc aagcacgatc attgccatga 120 gacg 124 <210> 23 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> SIC-target DNA triple deletion <400> 23 tcgtcaatgg agcgtcggta ttgccatcag ccggggccgg aacaggttat gaactgtgta 60 cccttggcag gaaatatagc ccacgctggg gcctcgatca agcacgatca ttgccatgag 120 acg 123 <210> 24 <211> 122 <212> DNA <213> Artificial Sequence <220> <223> SIC-target DNA quadruple deletion <400> 24 tcgtcaatgg agcgtcggta ttgccatcag ccggggccgg aacaggttat gaactgtgtc 60 ccttggcagg aaatatagcc cacgctgggg cctcgatcaa gcacgatcat tgccatgaga 120 cg 122 <210> 25 <211> 125 <212> DNA <213> Artificial Sequence <220> <223> SIC-target DNA single deletion <400> 25 tcgtcaatgg agcgtcggta ttgccatcag ccggggccgg aacaggttat gaactggtac 60 cccccttggc aggaaatata gcccacgctg gggcctcgat caagcacgat cattgccatg 120 agacg 125 <210> 26 <211> 124 <212> DNA <213> Artificial Sequence <220> <223> SIC-target DNA double deletion <400> 26 tcgtcaatgg agcgtcggta ttgccatcag ccggggccgg aacaggttat gaactgtacc 60 ccccttggca ggaaatatag cccacgctgg ggcctcgatc aagcacgatc attgccatga 120 gacg 124 <210> 27 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> SIC-target DNA triple deletion <400> 27 tcgtcaatgg agcgtcggta ttgccatcag ccggggccgg aacaggttat gaacttaccc 60 cccttggcag gaaatatagc ccacgctggg gcctcgatca agcacgatca ttgccatgag 120 acg 123 <210> 28 <211> 122 <212> DNA <213> Artificial Sequence <220> <223> SIC-target DNA quadruple deletion <400> 28 tcgtcaatgg agcgtcggta ttgccatcag ccggggccgg aacaggttat gaactacccc 60 ccttggcagg aaatatagcc cacgctgggg cctcgatcaa gcacgatcat tgccatgaga 120 cg 122 <210> 29 <211> 149 <212> DNA <213> Artificial Sequence <220> <223> artificial target DNA <400> 29 ggctcagcca tcttacctgt ggcacaggtt gaactgtgta cccccccttc gttggggcct 60 ccgtagactt aacaacttta ttcgtatgat caattcttgt gtcttgcttc ttatggccct 120 cccagtttct gattcttcgg acacccggc 149 <210> 30 <211> 145 <212> DNA <213> Artificial Sequence <220> <223> artificial SIC-target DNA <400> 30 ggctcagcca tcttacctgt ggcacaggtt gaactggtac ccccccttcg ttggggcctc 60 gtagacttaa caactttatt cgtagatcaa ttcttgtgtc ttgcttcttt ggccctccca 120 gtttctgatt cttcggacac ccggc 145 <210> 31 <211> 153 <212> DNA <213> Artificial Sequence <220> <223> artificial SIC-target DNA <400> 31 ggctcagcca tcttacctgt ggcacaggtt gaactgctgt accccccctt cgttggggcc 60 tcgcgtagac ttaacaactt tattcgtatt gatcaattct tgtgtcttgc ttcttaatgg 120 ccctcccagt ttctgattct tcggacaccc ggc 153 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> target DNA forward primer <400> 32 tcagccatct tacctgtggc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> target DNA reverse primer <400> 33 gggtgtccga agaatcagaa 20 <210> 34 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <400> 34 gttgaactgt gtaccc 16 <210> 35 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <400> 35 gcctccgtag actta 15 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <400> 36 ttcgtatgat caattctt 18 <210> 37 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <400> 37 cttcttatgg ccctccc 17

Claims (16)

Characterized in that one to four bases of the target nucleic acid sequence are substituted, deleted or inserted, and a melting curve analysis for detection of the target nucleic acid using a PNA probe coupled with reporter and quenching Way.
2. The method of claim 1, wherein the PNA probe is in perfect match with the target nucleic acid sequence and incompletely hybridizes with the internal control.
2. The method of claim 1, wherein the PNA probe binds preferentially to the target nucleic acid in the presence of the target nucleic acid and exhibits an expected fusion temperature (Tm) value, and binds to the internal control in the absence of the target nucleic acid, ) Value of the melting curve.
The method according to claim 1, wherein false positive and false negative signals can be distinguished by using the difference between the internal control and the target nucleic acid dissolution curve.
4. The method of claim 1 wherein the reporter is selected from the group consisting of 6-carboxyfluorescein, Texas red, HEX (2 ', 4', 5 ', 7', - tetrachloro-6-carboxy-4,7-dichlorofluorescein) CY &lt; 5 &gt;.
2. The method according to claim 1, wherein the quenching is one or more selected from the group consisting of 6-carboxytetramethyl-rhodamine (TAMRA), BHQ1, BHQ2 and Dabcyl.
A method for detecting a target nucleic acid comprising the steps of:
(a) separating a target nucleic acid from a specimen sample;
(b) 1 to 4 bases of the target nucleic acid sequence are substituted, deleted, or inserted, characterized in that the PNA probe and the primer are combined with a target nucleic acid and a PNA probe and a target nucleic acid Hybridizing internal control;
(c) melting the hybridized product while changing the temperature to obtain a melting curve; And
(d) analyzing the obtained melting curve to detect the presence or absence of a target nucleic acid.
8. The method of claim 7, wherein the PNA probe is characterized by complete hybridization with the target nucleic acid sequence and incomplete hybridization with the internal control.
8. The method of claim 7, wherein the PNA probe binds preferentially to the target nucleic acid in the presence of the target nucleic acid and exhibits an expected fusion temperature (Tm) value, and binds to the internal control in the absence of the target nucleic acid, ) Of the target nucleic acid.
The method according to claim 7, wherein the false positive and false negative signals can be distinguished by using the difference between the internal control and the target nucleic acid dissolution curve.
The method of detecting a target nucleic acid according to claim 7, wherein two or more target nucleic acids are used and the reporter labeled with the PNA probe is different for each target nucleic acid to detect two or more target nucleic acids.
8. The method of claim 7 wherein the reporter is selected from the group consisting of FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7', - tetrachloro-6-carboxy-4,7-dichlorofluorescein) CY5. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
8. The method according to claim 7, wherein the quenching is one or more selected from the group consisting of 6-carboxytetramethyl-rhodamine (TAMRA), BHQ1, BHQ2 and Dabcyl.
Characterized in that one to four bases of the target nucleic acid sequence are substituted, deleted or inserted, and wherein the PNA probe comprises a reporter and a quenching combined PNA probe, Kits for detecting nucleic acids.
15. The method of claim 14, wherein the PNA probe binds preferentially to the target nucleic acid in the presence of the target nucleic acid and exhibits a predicted melting temperature (Tm) value, and binds to the internal control in the absence of the target nucleic acid, ) Of the target nucleic acid.
The kit for detecting a target nucleic acid according to claim 15, wherein the false positive and false negative signals can be distinguished by using the difference between the internal control and the target nucleic acid dissolution curve.
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KR101642784B1 (en) * 2016-01-15 2016-07-27 대한민국 Genetic Markers for Discrimination of Viral Hemorrhagic Septicemia Virus, and Method for Discrimination of Causative Virus Using the Same
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