Method for detecting target nucleic acid sequence using cleaved complementary tag fragment and a composition therefor

Abstract

The present invention relates to a method and a composition for detecting a target nucleic acid sequence using a cleaved complementary tag fragment. Specifically, the present invention relates to a method for linking a complementary tag sequence to a PCR primer so that a tagging can be produced by a restriction enzyme during a PCR reaction, diversifying the complementary tag sequence to be linked to each primer by utilizing factors such as length and nucleic acid combination, etc., and distinguishing the target sequence using the same. According to the present invention, a cleaved complementary tag fragment (CCTF) under stringent conditions is a complementary sequence to any sequence at the 5′ end linked to the primer and cannot be formed unless a PCR reaction and a restriction enzyme reaction occur, and the cleaved single strand is formed only when hybridization to the target sequence occurs and a primer extension product complementary to the target sequence is formed, so as to have a higher degree of accuracy secured by reading the cleaved single strand. In addition, the CCTF can be used to identify a plurality of target nucleic acid sequences by selecting various analytical techniques and analysis equipment according to a user's intention. For example, a result can be confirmed rapidly and accurately in genetic testing, identification of organisms in a sample, diagnosis of microbial or viral infection, etc.

Claims

1. A primer comprising: a random nucleic acid sequence non-complementary to a target sequence, having 5 to 50 nucleotides in length, wherein the random sequence is located at the 5′end of the primer; a restriction enzyme recognition sequence; and a nucleic acid sequence complementary to the target sequence, wherein the primer comprises one sequence selected from the group consisting of SEO ID NOs: 1, 3, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 74, 76, 78, 80, 82, 84, 86, 115, 117, 119, 121, 123, 125, 127, 129, 131, 151, 153, 155, 156, 159, 161, 163, 164, 166, 168, 170, 204, 205, 207, 218, 220 and 222.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a representative diagram illustrating the formation process of CTPO and CCTF used in a PCR reaction, and an example for the analysis of CCTF, as a schematic diagram of CCTF formation.

(2) FIG. 2 shows the results of the formation of CCTF and MALDI analysis in dual target PCR. CTPO was designed to form different CCTFs for each target sequence, amplified, and analyzed by MALDI, and as a results, a peak corresponding to the masses of CCTF 1 obtained by amplifying Neisseria gonorrhoeae (NG) and cleaving it and CCTF2 obtained by amplifying Mycoplasma hominis (MH) and cleaving it, were observed.

(3) FIG. 3 shows the results of Real-time PCR Melting Peak analysis for causative organisms of sexually transmitted diseases. As the results representing the multiple target dissociation temperature measurements to each target of Chlamydia trachomatis(CT), Neisseri gonorrhea (NG) Mycoplasma hominis(MH), Mycoplasma genitalium(MG), Trichomonas vaginalis(TV), Ureaplasma urealyticum(UU), Ureaplasma parvum(UP), Candida albicans(CA), Gardnerella vaginalis(GV), Herpes simplex virus 1(HSV 1), Herpes simplex virus 2(HSV 2), Treponema pallidum(TP) and Internal Control (IC), the peak was observed at the inherent dissociation temperature that each SCO has (CT: FAM 80° C., NG: HEX 76.5° C., MH: HEX 68° C., MG: CalRed610 67.5° C., TV: Quasar670 71.5° C., UU: CalRed610 77° C., UP: FAM 77° C., CA: FAM 65° C., GV: Quasar670 78.5° C., HSV 1: Quasar705 73.5° C., HSV 2: Quasar705 79° C., TP: Quasar705 66° C., IC: Quasar670 63.5° C.) (a)(b)(c)(d)(e)(f), and no peak of SCO that visualized CCTF was observed when the target sequence was not added in the same composition (g).

(4) FIG. 4 shows the results of Real-time PCR Melting Peak analysis for the causative organism of the gastrointestinal diseases. As the results representing the multiple inherent dissociation temperature measurements to each target of Rotavirus A(RVA), Astrovirus(AstV), Adenovirus F40(AdV 40), Adenovirus F41(AdV 41). Norovirus GI(NoV GI), Norovirus GII(NoV GII) and External Control, the peak was observed at the inherent dissociation temperature that each SCO has (RVA: HEX 78° C., AstV: CalRed610 78° C., AdV 40: CalRed610 67° C., AdV 41: CalRed610 67° C., NoV GI: FAM 68° C., NoV Gil: FAM 84° C., EC: HEX 69° C.) (a)(b)(c)(d), and no peak of SCO that visualizes CCTF was observed when the target sequence was not added in the same composition (e).

(5) FIG. 5 shows the results of Real-time PCR Melting Peak analysis for Human Papilloma Virus (HPV) detection. As a result of multiple inherent dissociation temperature measurements of each target of type 16, type 18, type 33, type 35, type 51, type 53, type 59, type 68a, type 82 and IC, the peak was observed at the inherent dissociation temperature that each SCO has (type 16: HEX 76.5° C., type 18: FAM 78° C., type 33: Quasar670 71° C., type 35: Quasar670 710° C., type 51: Quasar670 71° C. type 53: Quasar670 710° C., type 59: Quasar670 71° C., type 68a: Quasar670 71° C., type 82: Quasar670 71° C., IC: Quasar670 67.5° C.) (a)(b)(c)(d), and no peak of SCO that visualizes CCTF was observed when the target sequence was not added in the same composition (e).

(6) FIG. 6 shows the result of Real-time PCR Melting Peak analysis for detection of respiratory disease-induced virus. As a result of multiple inherent dissociation temperature measurements of each target of Influenza A/H1N1(H1), Influenza A/H3N2(H3), Influenza A/H1N1/2009pdm(2009pdm), Influenza B(Flu B), Parainfluenza 1(PIV 1), Parainfluenza 3(PIV 3), Respiratory syncytial virus A(RSV A), Respiratory syncytial virus B(RSV B), Human metapneumovirus(MPV), Adenovirus(AdV), External control (EC), the peak was observed at the inherent dissociation temperature that each SCO has (H1: FAM 67.5° C., H3: FAM 76.5° C., 2009pdm: FAM 86.5° C., Flu B: CalRed610 83.5° C., PIV 1: Quasar670 66° C., PIV 3: Quasar670 74° C., RSV A: HEX 63.5° C., RSV B: CalRed610 72° C., MPV: HEX 86° C., ADV: Quasar670 85° C., EC: CalRed610 68.5° C.) (a)(b)(c)(d)(e), and no peak of SCO that visualizes CCTF was observed when the target sequence was not added in the same composition (f).

(7) FIG. 7 shows the results of Real-time PCR Melting Peak analysis to analyze the genotype of rs6265, a single nucleotide polymorphism of BDNF gene. As a result representing the multiple inherent dissociation temperature measurements of each target of mutant A/A, wild type G/G and heterozygote A/G, the peak was observed at the inherent dissociation temperature that each SCO has (A/A: 76.5° C., A/G: 76.5° C. 75° C., G/G 75° C., IC: 66° C.) (a)(b)(c)(d), and no peak of SCO that visualizes CCTF was observed when the target sequence was not added in the same composition (e).

(8) FIG. 8 shows the results of real-time PCR Ct graph. As a result, representing fluorescent amplification curves and standard curves of SCO under the experimental condition of a multi-real-time polymerization chain reaction experiment of Neisseria. gonorrhea (NG), Mycoplasma. hominis (MH), Ureaplasma. parvum (UP) in which genomic DNA of each of the above causative organism was diluted by 10-folds from 100 pg/ul concentration, (a) graph shows the results of fluorescence amplification curves plotted when three target sequences are present at each concentration simultaneously, (b) graph shows a negative result plotted when all three target sequences are not included. When the standard curve is represented by a single fluorescence amplification curve of the graph corresponding NG of (a) graphs, it can be represented as (c) and (d), and the graph corresponding to MG graph can be represented as (e) and (f), and the curve corresponding to UP can be represented as (g) and (h).

MODE FOR INVENTION

(9) Hereinafter, the present invention will be described in detail with reference to Examples. These examples are for illustrative purposes only and thus, are not interpreted to limit the scope of the present invention.

Example 1. Formation of CCTF and MALDI Analysis in Dual Target PCR

(10) This experiment was conducted to prove that the CCTF formed during the PCR reaction for the detection of multiple target sequences can be detected in a target-specific manner by analyzing the mass using MALDI-TOF MS. In Example 1, the causative organism of sexually transmitted diseases, DNAs of Neisseria. gonorrhoeae (NG) and Mycoplasma. Hominis (MH) were used as the targets.

(11) 1. Target Template DNA and Primers Manufactured by Sequence Specific Manner

(12) The forward primers of NG and MH targeting in this example were manufactured based on the method described in the Detailed Description of the Invention as CTPO. The 5′end of the forward primer was an arbitrary nucleotide sequence consisting of a sequence non-complementary to the DNA of NG and MH so that it could be used as a template of CCTF, and a restriction enzyme recognition sequence was consecutively located thereon. The sequence after the restriction enzyme recognition sequence up to the 3′end is composed of a sequence complementary to the target region of the DNA of NG and MH, and plays a role as a primer. In addition, the 5′end of forward primer is composed of a different number of nucleotides with each other and has a different mass value for each CCTF generated, in order to design that the amplification products can be distinguished from each other as the mass when CCTF is formed. The reverse primer is consisted of a sequence complementary to the target site of the DNA of NG and MH.

(13) Primer information and target sequence information being amplified and generated are as follows.

(14) TABLE-US-00001 Primer 1: (SEQ ID. NO: 1) 5′-TGAACTAT *  TCCGACGTTTCGGTTGTGTTGAAACACCGCCCGG-3′ Primer 2: (SEQ ID. NO: 2) 5′-GCTCCTTATTCGGTTTGACCGG-3′ Primer 3: (SEQ ID. NO: 3) 5′-ATCTATGATA*  TTTAGCTCCTATTGCCAACGTATTGG-3′ Primer 4: (SEQ ID. NO: 4) 5′-TGTGTGGAGCATCTTGTAATCTTTGGTC-3′
Amplified product 1: GenBank: CP012028.1/Position (start-end): 251416-251506

(15) TABLE-US-00002 (SEQ ID. NO: 5) 5′ TGAACTAT *  TCCGACGTTTCGGTTGTGTTGAAACACCGC CCGGAACCCGATATAATCCGCCCTTCAACATCAGTGAAAATCTTTTTTT TAACCGGTCAAACCGAATAAGGAGC-3′
Amplified product 2: GenBank: AJ243692.1/Position (start-end): 835-944

(16) TABLE-US-00003 (SEQ ID. NO: 6) 5′ ATCTATGATA*  TTTAGCTCCTATTGCCAACGTATTGGAA AAAAACTTTGGTATTGAAAAAGGATTTATGACAACAGTCCACTCATAT ACAGCAGACCAAAGATTACAAGATGCTCCACACA-3′

(17) The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. In the examples of the present invention, the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme.

(18) The sequence and mass of the CCTF produced in the amplified product are as follows.

(19) TABLE-US-00004 CCTF 1: (SEQ ID. NO: 7) 5′-CCAGGATAGTTCA-3′/4038.6 Da CCTF 2: (SEQ ID. NO: 8) 5′-CCAGGTATCATAGAT-3′/4351.8 Da

(20) 2. PCR Amplification

(21) Primer 1 and Primer 3 as forward primers, and Primer 2 and Primer 4 as reverse primers were subjected and PCR reaction was performed simultaneously, and then, the formation of CCTF was determined.

(22) 20 μl Of the total reaction solution comprising each Primer 3 μM, PspGI (NEB, USA) 2U, PCR buffer (1×), MgSO.sub.4 3 mM, dNTP 400 μM, Vent Polymerase (NEB, USA) 1 U and NG, MH template DNA 100 pg/ul was subjected to PCR reaction using C1000 PCR (Bio-Rad, USA) under the following conditions:

(23) 94° C. 10 mins,

(24) 94° C. 30 secs, 62° C. 30 secs 72° C. 30 secs (35 cycles),

(25) 85° C. 2.5 hours

(26) 3. Purification and Desalting of the Cleaved Fragments During the PCR Reaction

(27) Oasis (Waters) C18 reverse phase column chromatography was used to isolate the DNA fragments cleaved by treatment with a restriction enzyme during the PCR reaction from the above solution. To the solution treated with the restriction enzyme, 70 μl of 0.15 M triethylammonium acetate (TEAA, pH 7.6) was added and allowed to stand for 1 minute. Resin was activated by passing 1 ml of 100% acetonitrile (ACN; Sigma. USA) and 0.1 M TEAA to the column, and then, 100 μl of a mixed solution of the solution treated with the restriction enzyme and 0.15M TEAA, 2 ml of 0.1M TEAA and 1 ml of the third distilled water were passed through in this order. The column was placed on a Collection Plate and 100 μl of 70% ACN was passed. When the eluate was collected on the collection plate, the collection plate was dried at 120° C. for 60 minutes.

(28) 4. MALDI-TOF MS Analysis

(29) 4 μl of MALDI matrix [22.8 mg ammonium citrate, 148.5 mg hydroxypicolinic acid, 1.12 ml acetonitrile, 7.8 ml H.sub.2O] was previously dotting on Anchor chip plate of MALDI-TOF mass spectrometry (Biflex IV, Bruker), and then, was dried at 37° C. for 30 minutes. 10 μl of the third distilled water was dissolved in a sample of the collection plate after the purification and desalting procedure, and 2 μl of the solution was dropped onto the dried MALDI Matrix, the Maldin Matrix was dried again at 37° C. for 30 minutes, and then was analyzed by MALDI-TOF mass spectrometry. The analysis method follows the manual of the MALDI-TOF mass spectrometry.

(30) The result of analyzing the CCTF produced by the above reaction using a mass spectrometer is as shown in FIG. 2. From the result of FIG. 2, it can be confirmed the peaks of 4083 Da, the mass of CCTF 1 which can be formed when performing PCT with the combination of Primer 1 and Primer 2, and 4351 Da. the mass of CCTF 2 which can be formed when performing PCT with the combination of Primer 3 and Primer 4 (a). These results demonstrated that the PCR amplification product can be analyzed using CCTF formed by CTPO, and that CCTF can be used to accurately amplify and differentiate the target sequence in the reaction product comprising various primers.

(31) Therefore, it was demonstrated that the target nucleic acid sequence can be detected more precisely than the conventional PCR method by performing the PCR using the CCTF marking technique and distinguishing the tag fragments of various lengths through mass analysis using MALDI-TOF MS after performing PCR.

Example 2. Formation of CCTF and Analysis of Inherent Dissociation Temperature Peak of CCTF in Multiple Target PCR

(32) The CCTF generated during the PCR reaction is combined with the SCO capable of generating a fluorescence signal at the inherent dissociation temperature to form an intrinsic dissociation temperature peak, which can be observed directly after the PCR process using a real-time PCR instrument. During the PCR reaction, CCTF is formed, and at the same time it is hybridized with the CCTF complementary sequence region of SCO to form a double strand. By measuring the inherent dissociation temperature of SCO seen when the double strand is dissociated into a single strand, the kinds of CCTF can be discriminated and analyzed simultaneously with PCR through a real-time PCR instrument. The SCO used in this example used different fluorescent reporters, respectively, and the inherent dissociation temperature was adjusted to enable discrimination of CCTF.

(33) In this example, CCTF analysis was performed using a real-time PCR instrument using 12 kinds of the causative organisms of the sexually transmitted diseases, 5 types of the causative organisms of gastrointestinal diseases, 9 types of HPV subtypes, 10 types of the causative organisms of the respiratory disease and single base mutation rs6265 nucleic acid of BDNF gene, respectively.

(34) 1. Formation of CCTF in multi-target PCR of the causative organisms of the sexually transmitted diseases and analysis of the inherent dissociation temperature peak of CCTF CCTF analysis for Chlamydia trachomatis(CT), Neisseria. gonorrhea (NG) Mycoplasma hominis(MH), Mycoplasma genitalium(MG), Trichomonas vaginalis(TV), Ureaplasma urealyticum(UU), Ureaplasma parvum(UP), Candida albicans(CA), Gardnerella vaginalis(GV), Herpes simplex virus 1(HSV 1), Herpes simplex virus 2(HSV 2), Treponema pallidum(TP), the causatives agents of sexually transmitted diseases and Internal control (IC) DNA was performed using Real-time PCR instrumentation.

(35) 1) Primer for Target Sequence Template DNA Constructed by the Sequence-Specific Manner

(36) The forward primer used in this example was CTPO and was constructed on the same principle as in Example 1 above. The 5′end of CTPO was composed of 19-20 mers of nucleotide sequences, and was composed of a sequence non-complementary to DNA of the target sequence to form CCTF. The restriction enzyme recognition sequence was then located, and from this up to the 3′ end, it was composed of the sequence complementary to each target site was composed to play a role as a primer. The reverse primer was composed of sequence complementary to the target site to be amplified.

(37) In addition, SCO, which forms a complementary bond with CCTF to be a double-stranded template, was positioned by positioning fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the fluorescent reporter molecular was positioned so as to have a certain distance.

(38) Primer information and target sequence information which is amplified and generated are as follows

(39) TABLE-US-00005 Primer 5: (SEQ ID. NO: 9) 5′-CCACTCCAGCCGGCTGACA*CCAGGACTTGGTGTGACGCTATC AGCAT-3′ Primer 6: (SEQ ID. NO: 10) 5′-GTTTTCAAAACACGGTCGAAAACAAAGTC-3′ Primer 7: (SEQ ID. NO: 11) 5′-CATCGCCACGAGCCGGTTAA*CCAGGTTGAAACACCGCCCGGA ACCC-3′ Primer 8: (SEQ ID. NO: 12) 5′-GCTCCTTATTCGGTTTGACCGGT-3′ Primer 9: (SEQ ID. NO: 13) 5′-ACTCACGCTAATGGAGCGCA*CCAGGTTTAGCTCCTATTGCCA ACGTATTGG-3′ Primer 10: (SEQ ID. NO: 14) 5′-TGTGTGGAGCATCTTGTAATCTTTGGTC-3′ Primer 11: (SEQ ID. NO: 15) 5′-GCTACCCAGCCGGCTACAAG*CCAGGCTTTTATGGTGCTTATA TTGGTGGCATG-3′ Primer 12: (SEQ ID. NO: 16) 5′-CTGTATAACGTTGTGCAGCAGGTC-3′ Primer 13: (SEQ ID. NO: 17) TGCCGCGTGATTCGATCCCA*CCAGGTATGTCCGGCACAACATGCG CT-3′ Primer 14: (SEQ ID. NO: 18) 5′-GAGCTTACGAAGGTCGGAGTTGA-3′ Primer 15: (SEQ ID. NO: 19) 5′-TCTCATAGCTGGGCCGCTG*CCAGGAAGTAGCATATGATGAAG CACACAACA-3′ Primer 16: (SEQ ID. NO: 20) 5′-TAATGCAACGTGCATTTGCTTCAAC-3′ Primer 17: (SEQ ID. NO: 21) 5′-CAGATCGTTGGCACTCTGCGA*CCAGGTTAAAGTAGCATATGA TCAAGCTCATTCA-3′ Primer 18: (SEQ ID. NO: 22) 5′-TTGTAATGATACAACGAGCATCATCATTAAT-3′ Primer 19: (SEQ ID. NO: 23) 5′-GCTCGTATGCCGCTCCATATA*CCAGGCCAAATCTGGATCTTC CTCTGCATC-3′ Primer 20: (SEQ ID. NO: 24) 5′-GAGCTTGAGCTGGACCCAGAG-3′ Primer 21: (SEQ ID. NO: 25) 5′-ACGTGCCGTGCATCGTTGCA*CCAGGCAACCGGCTCCATTTTG GTGGAG-3′ Primer 22: (SEQ ID. NO: 26) 5′-CGTCACGTCCTTCATCGGTCC-3 Primer 23: (SEQ ID. NO: 27) 5′-TCGCAGTCCCGTCGAGGAA*CCAGGAGGCCTGGCTATCCGGAG AAAC-3′ Primer 24: (SEQ ID. NO: 28) 5′-CGTTGTGTTGGCCGCAGGTC-3′ Primer 25: (SEQ ID. NO: 29) 5′-CTCATAGCTAGGCGCCTG*CCAGGGCTGCACGTGGGTCTGTTG TG-3′ Primer 26: (SEQ ID. NO: 30) 5′-GGAAACGCAGGCCACGAAACC-3′ Primer 27: (SEQ ID. NO: 31) 5′-GCTTCGCGTCTCAGGCCTGT*CCAGGGGGCATTACAGTTTTGC GTCATGAC-3′ Primer 28: (SEQ ID. NO: 32) 5′-CAAGTCTGAGCACTTGCACCG-3′ Primer 29: (SEQ ID. NO: 33) 5′-CTGTTAGCTCTGCGAGCT*CCAGGGGAGCGACACTTGTTGGTG TTGAC-3′ Primer 30: (SEQ ID. NO: 34) 5′-TGATGAAATGAAGCCACCCGTGC-3′ SCO 1: (SEQ ID. NO: 35) TCGGAGCCAGCGCGGCGTAAAC[T(FAM)]CCACTCCAGCCGGCTG ACA[BHQI] SCO 2: (SEQ ID. NO: 36) TACAACAGCAGTACGGAGACGAC[T(HEX)]CATCGCCACGAGCCG GTTAA[BHQI] SCO 3: (SEQ ID. NO: 37) ATTTATTCTTACTCGATGTTAAA[T(HEX)]ACTCACGCTAATGGA GCGCA[BHQI] SCO 4: (SEQ ID. NO: 38) TATATATATATATTATTATAAA[T(CalRed610)]GCTACCCAGCC GGCTACAAG[BHQ2] SCO 5: (SEQ ID. NO: 39) AAGAATAACTACTACAATCTACT[T(Quasar670)]TGCCGCGTGA TTCGATCCCA[BHQ2] SCO 6: (SEQ ID. NO: 40) TTATTATTATTATTATTATATA[T(CalRed610)]TCTCATAGCTG GGCCGCTG[BHQ2] SCO 7: (SEQ ID. NO: 41) AATCTTCAATGCTTACCGTA[T(FAM)]CAGATCGTTGGCACTCTGC GA[BHQ1] SCO 8: (SEQ ID. NO: 42) AAAATAAATAATATAATATA[T(FAM)]GCTCGTATGCCGCTCCATA TA[BHQ1] SCO 9: (SEQ ID. NO: 43) TCGGAGCCAGCGCGGCGTAACG[T(Quasar670)]ACGTGCCGTGC ATCGTTGCA[BHQ2] SCO 10: (SEQ ID. NO: 44) AAGAATAACTACTACAATCTAC[T(Quasar705)]TTCGCAGTCCC GTCGAGGAA[BHQ2] SCO 11: (SEQ ID. NO: 45) TCGGAGCCAGCGCGGCGTAA[T(Quasar705)]CTCTCATAGCTAG GCGCCTG[BHQ2] SCO 12: (SEQ ID. NO: 46) AAAATAAATAATATAATATAG[T(Quasar705)]CTTCGCGTCTCA GGCCTGT[BHQ2] SCO 13: (SEQ ID. NO: 47) AAAATAAATAATATAATATA[T(Quasar670)]TCTGTTAGCTCTG CGAGCT[BHQ2]
Amplified product 3: GenBank: X52557.1/Position (start-end): 157-227

(40) TABLE-US-00006 (SEQ ID. NO: 48) CCACTCCAGCCGGCTGACA* ACTTGGTGTGACGCTATCAGCAT GCGTATGGGTTACTATGGTGACTTTGTTTTCGACCGTGTTTTGAAAAC
Amplified product 4: GenBank: X52364.1/Position (start-end): 375-459

(41) TABLE-US-00007 (SEQ ID. NO: 49) CGCCCACCGCATCCCGCGCCCCTCCCTCAGCA* TTGAAACACCG CCCGGAACCCGATATAATCCGCCCTTCAACATCAGTGAAAATCTTTTTT TAACCGGTCAAACCGAATAAGGAGC
Amplified product 5: GenBank: AJ243692.1/Position (start-end): 835-944

(42) TABLE-US-00008 (SEQ ID. NO: 50) ACTCACGCTAATGGAGCGCA* TTTAGCTCCTATTGCCAACGTA TTGGAAAAAAACTTTGGTATTGAAAAAGGATTTATGACAACAGTCCACT CATATACAGCAGACCAAAGATTACAAGATGCTCCACACA
Amplified product 6: GenBank: U09251.1/Position (start-end): 3462-3687

(43) TABLE-US-00009 (SEQ ID. NO: 51) GCTACCCAGCCGGCTACAAG* CTTTATGGTGCTTATATTGGTGG CATGCACCATGATCGTCCTTTTAAAAAGTCTGCGAGGATTGTTGGTGAT GTAATGAGTAAATTCCACCCTCATGGTGATATGGCAATATATGACACCA TGTCAAGAATGGCTCAAGACTTTTCATTAAGATACCTTTTAATTGATGG TCATGGTAATTTTGGTTCTATAGATGGTGATAGACCTGCTGCACAACGT TATACAG
Amplified product 7: GenBank: XM_001582993.1/Position (start-end): 705-768

(44) TABLE-US-00010 (SEQ ID. NO: 52) TGCCGCGTGATTCGATCCCA* TATGTCCGGCACAACATGCGCTT ATGTCCGGCACAACATGCGCTCTCCGCTTCCCAGGTCAGCTCAACTCCG ACCTTCGTAAGCTC
Amplified product 8: GenBank: AF085700.2/Position (start-end): 4673-4873

(45) TABLE-US-00011 (SEQ ID. NO: 53) TCTCATAGCTGGGCCGCTG* AAGTAGCATATGATGAAGCACACA ACAAAATGGCGCATACTGTGTATTTCACTAATTTCTATCGTTCATCAAAA CCACTATTTTTAGATGAAGAAGACCCAATTAATCCCTGTTTTCAAACTAT TAGTATGGGTGGGGGTTATGTATCTGGTGAAGTGTATCGTTCTGATTTTG AAGTTGAAGCAAATGCACGTTGCATTA
Amplified product 9: GenBank: AF085733.2/Position (start-end): 4677-4886

(46) TABLE-US-00012 (SEQ ID. NO: 54) CAGATCGTTGGCACTCTGCGA* TTAAAGTAGCATATGATCAAGC TCATTCAAAAATGGCACATACTGTCTATTTTACGAATTTTTATCGTTCA TCTAAACCTTTATTTTTAGATGAAGAAGATCCAATCAACCCCTGTTTTC AAACAATTAGTATGGGTGGTGGATATGTTTCAGGTGAAATTTATCGTTC TGATTTTGAAATTAATGATGATGCTCGTTGTATCATTACAA
Amplified product 10: GenBank: M90812.1/Position (start-end): 1736-1811

(47) TABLE-US-00013 (SEQ ID. NO: 55) GCTCGTATGCCGCTCCATATA* CCAAATCTGGATCTTCCTCTG CATCTGCTTCTGGATCATCAAGCAGCAGCACCAGCTCTGGGTCCAGCTCA AGCTC
Amplified product 11: GenBank: L08167.1/Position (start-end): 273-434

(48) TABLE-US-00014 (SEQ ID. NO: 56) ACGTGCCGTGCATCGTTGCA* CAACCGGCTCCATTTTGGTGGA GTCGCTTGATCGTTTTGTGATCGTTTAGTGTGATGATTTATTATGTCTAG AGAGTTAAGCGATAGGCTTTTACTGGTGTATCACTGTAAGGGCGTATTGG TTGGATGCCTTGGTAGACAGGACCGATGAAGGACGTGACG
Amplified product 12: DQ889502.1/Position (start-end): 123860-124007

(49) TABLE-US-00015 (SEQ ID. NO: 57) TCGCAGTCCCGTCGAGGAA* AGGCCTGGCTATCCGGAGAAACA GCACACGACTTGGCGTTCTGTGTGTCGCGATGTCTCTGCGCGCAGTCTG GCATCTGGGGCTTTTGGGAAGCCTCGTGGGGGCTGTTCTTGCCGCCACC CATCGGGGACCTGCGGCCAACACAACG
Amplified product 13: GenBank: EU018100.1/Position (start-end): 561-746

(50) TABLE-US-00016 (SEQ ID. NO: 58) CTCATAGCTAGGCGCCTG* GCTGCACGTGGGTCTGTTGTGGGT AGAGGTGGGCGGGGAGGGCCCCGGCCCCACCGCCCCCCCCACAGGCGGC GCGTGCGGAGGGCGGCCCGTGCGTCCCCCCGGTCCCCGCGGGCCGCCCG TGGCGCTTCGGTGCCCCCGGTATGGTATTCCGCCCCCAACCCCGGGTTT CGTGGCCTGCGTTTCC
Amplified product 14: GenBank: U57757.1/Position (start-end): 910-1067

(51) TABLE-US-00017 (SEQ ID. NO: 59) GCTTCGCGTCTCAGGCCTGT* GGCATTACAGTTTTGCGTCATG ACGGCTTTGAAGCTGACGACCTCATTGCAACCCTAGCAAAACGAGTTGC GGCTGAGCACTGTCATGTTGTGATTATCTCCTCAGATAAAGATGTACTT CAGCTTGTGTGTGATACGGTGCAAGTGCTCAGACTTG
Amplified product 15: GenBank: NM_001035551.2/Position (start-end): 214-369

(52) TABLE-US-00018 (SEQ ID. NO: 60) CTGTTAGCTCTGCGAGCT* GGAGCGACACTTGTTGGTGTTGAC AAGTTCGGTAACAAATACTACCAGAAGCTAGGCGATACTCAATACGGTAT GCACAGATGGGTAGAGTATGCTTCAAAGGATCGTTACAACGCATCTCAA GTACCAGCTGAATGGCACGGGTGGCTTCATTTCATCA

(53) The bold and slanted font of the Primer sequences means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. The sequence of the CCTF produced from the amplified product is as follows.

(54) TABLE-US-00019 CCTF 3: (SEQ ID. NO: 61) 5′-CCTGGTGTCAGCCGGCTGGAGTGG-3′ CCTF 4: (SEQ ID. NO: 62) 5′-CCTGGTTAACCGGCTCGTGGCGATG-3′ CCTF 5: (SEQ ID. NO: 63) 5′-CCTGGTGCGCTCCATTAGCGTGAGT-3′ CCTF 6: (SEQ ID. NO: 64) 5′-CCTGGCTTGTAGCCGGCTGGGTAGC-3′ CCTF 7: (SEQ ID. NO: 65) 5′-CCTGGTGGGATCGAATCACGCGGCA-3′ CCTF 8: (SEQ ID. NO: 66) 5′-CCTGGCAGCGGCCCAGCTATGAGA-3′ CCTF 9: (SEQ ID. NO: 67) 5′-CCTGGTCGCAGAGTGCCAACGATCTG-3′ CCTF 10: (SEQ ID. NO: 68) 5′-CCTGGTATATGGAGCGGCATACGAGC-3′ CCTF 11: (SEQ ID. NO: 69) 5′-CCTGGTGCAACGATGCACGGCACGT-3′ CCTF 12: (SEQ ID. NO: 70) 5′-CCTGGTTCCTCGACGGGACTGCGA-3′ CCTF 13: (SEQ ID. NO: 71) 5′-CCTGGCAGGCGCCTAGCTATGAG-3′ CCTF 14: (SEQ ID. NO: 72) 5′-CCTGGACAGGCCTGAGACGCGAAGC-3′ CCTF 15: (SEQ ID. NO: 73) 5′-CCTGGAGCTCGCAGAGCTAACAG-3′

(55) 2) PCR Amplification and Determination of SCO Inherent Dissociation Temperature PCR reaction was performed using the following CFX96 Real-time PCR (Bio-Rad, USA) with 20 μl of total reaction solution of each of Primer 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and SCO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 prepared by adding 0.15 μM, PspGI (NEB, USA) 5U, PCR buffer (1×), MgCl.sub.2 2.5 mM, dNTP 200 μM, h-Taq DNA polymerase (Solgent, Korea) 1.6 U, template DNA of genomic DNA of CT, NO, MH, MG, TV, UU, UP, CA, GV, HSV1, HSV2, TP and IC 100 pg/rxn, respectively.

(56) 95° C. 15 mins,

(57) 95° C. 30 secs, 63° C. 1 min (50 cycles).

(58) A reaction was performed using a cycle at the denaturation temperature of 95° C. for 15 minutes once, and with a cycle at the denaturation temperature of 95° C. for 30 seconds, and an annealing temperature of 63° C. for 1 minute 50 times. After the reaction, the reaction mixture was cooled to 50° C. in the same apparatus, held at 50° C. for 30 seconds, and then slowly heated from 50° C. to 95° C. to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager 1.6.

(59) FIG. 3(a) shows the results of multiple inherent dissociation temperature measurements for causative organisms of CT, NO, MH, MG, TV, UU, UP, CA, GV, HSV1, HSV2, TP, IC. The peak was observed at the inherent dissociation temperature that each SCO has (CT: FAM 80° C., NO: HEX 76.5° C., MH: HEX 68° C., MG: CalRed610 67.5° C., TV: Quasar670 71.5° C., UU: CalRed610 77° C., UP: FAM 77° C., CA: FAM 65° C., GV: Quasar670 78.5° C., HSV 1: Quasar705 73.5° C., HSV 2: Quasar705 79° C., TP: Quasar705 66° C., IC: Quasar670 63.5° C.) (a)(b)(c)(d)(e)(t), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (g).

(60) Therefore, it was proved that the target nucleic acid sequence can be detected more quickly and simply than the conventional PCR method by analyzing the fluorescence of the SCO using the real-time PCR instrument, simultaneously with the PCR using the marking technique of CCTF.

(61) 2. Formation of CCTF in Multi-Target PCR of the Causative Organism for the Gastrointestinal Diseases and Analysis for the Inherent Dissociation Temperature Peak of CCTF

(62) CCTF analysis was performed with Real-time PCR instrument for DNA of the causative organisms of the gastrointestinal diseases, Rotavirus A(RVA), Astrovirus(AstV), Adenovirus F40(AdV 40), Adenovirus F41(AdV 41), Norovirus GI(NoV GI), Norovirus GII(NoV GII), and External control (EC).

(63) 1) Primer for the Target Sequence of Template DNA Constructed in the Sequence-Specific Manner

(64) The forward primer used in this example was CTPO and was constructed on the same principle as in Example 1 above. The 5′end of CTPO was composed of 19-20 mers of nucleotide sequences, and was composed of a sequence non-complementary to DNA of the target sequence so as to form CCTF. The restriction enzyme recognition sequence was consecutively located, and after this up to the 3′ end, it was composed of the sequence complementary to each target site to play a role as a primer. The reverse primer was composed of sequence complementary to the target site to be amplified.

(65) In addition, SCO, which forms a complementary bond with CCTF to be a double-stranded template, was positioned by positioning fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the fluorescent reporter molecular was positioned so as to have a certain distance.

(66) Primer information and target sequence information which is amplified and generated are as follows.

(67) TABLE-US-00020 Primer 31: (SEQ ID. NO: 74) 5′-GCAGGAGCCTCTCATCTCG*CCAGGCTCATTTATAGACARCTT CTCACTAATTC-3′ Primer 32: (SEQ ID. NO: 75) 5′-AGTTTTTTCTGATCCAATYTGYTCTATTTC-3′ Primer 33: (SEQ ID. NO: 76) 5′-TCAGACGGTTCGAGGCTCC*CCAGGARGATYAAGCGTGGAGTA TAYATGG-3′ Primer 34: (SEQ ID. NO: 77) 5′-TTTGCGTGCYTCTTCACACGC-3′ Primer 35: (SEQ ID. NO: 78) 5′-AACGCGAATCGACCGGAT*CCAGGCGCGATGTGTTTGCCGATA AAAC-3′ Primer 37: (SEQ ID. NO: 79) 5′-CATTGCGTCTGCCCCACTTG-3′ Primer 38: (SEQ ID. NO: 80) 5′-AACGCGAATCGACCGGAT*CCAGGAAACAAGAACACCTATGCCT ACATGAAC-3′ Primer 39: (SEQ ID. NO: 81) 5′-ATGTTAACGTCCTTCCTGAAGTTCCAC-3 Primer 40: (SEQ ID. NO: 82) 5′-TAGATCGGACTGCGAATCG*CCAGGGAGATCGCRATCTYCTGC CCGA-3 Primer 41: (SEQ ID. NO: 83) 5′-RGCGTCCTTAGACGCCATCATC-3 Primer 42: (SEQ ID. NO: 84) 5′-ATCTACAGCGTCGCATCACG*CCAGGCGCAATCTGGCTCCCART TTTGTG-3 Primer 43: (SEQ ID. NO: 85) 5′-GCGTCAYTCGACGCCATCYTCA-3 Primer 44: (SEQ ID. NO: 86) 5′-CATAGGTCGAGGTCCTCAC*CCAGGGCAAACTCCGGCATCTACTA ATAGACG-3 Primer 45: (SEQ ID. NO: 87) 5′-AAGCGGTGATCCGCACAGTG-3 SCO 14: (SEQ ID. NO: 88) TCGGCCGATCGTCCATAGAGTCAAGC[T(HEX)]CGCAGGAGCCTCTCA TCTCG[BHQ1] SCO 15: (SEQ ID. NO: 89) TCACGATGAGCGAGTTGAGCTACG[T(Calred 610]ATCAGACGGTTCGAGGCTCC[BHQ2] SCO 16: (SEQ ID. NO: 90) TGTTCAATATATAATGATAATATG[T(Calred610)]AACGCGAAT CGACCGGAT[BHQ2] SCO 17: (SEQ ID. NO: 91) TGTTCAATATATAATGATAATATG[T(Calred610)]AACGCGAA TCGACCGGAT[BHQ2] SCO 18: (SEQ ID. NO: 92) ACATTTATAATACAGTATTTTA[T(FAM)]TAGATCGGACTGCGAAT CG[BHQ1] SCO 19: (SEQ ID. NO: 93) AGCTCCTGCCAGTACTGCCATCCA[T(FAM)]ATCTACAGCGTC GCATCACG[BHQ1] SCO 20: (SEQ ID. NO: 94) TAGTTATAATGAATAACTATTAT[T(HEX)]CATAGGTCGAGGTC CTCAC[BHQ1]
Amplified product 16: GenBank: KT694942.1/Position (start-end): 19-99

(68) TABLE-US-00021 (SEQ ID. NO: 95) GCAGGAGCCTCTCATCTCG * CTCATTTATAGACARCTTCTCAC TAATTCATATTCAGTAGATTTACATGATGAAATAGARCARATTGGATCAG AAAAAACT
Amplified product 17: GenBank: AB000287.1/Position (start-end): 2232-2321

(69) TABLE-US-00022 (SEQ ID. NO: 96) TCAGACGGTTCGAGGCTCC * ARGATYAAGCGTGGAGTATAYAT GGACCTGCTTGTCTCGGGGGCAAGCCCAGGCAATGCATGGTCCCATGCGT GTGAAGARGCACGCAAA
Amplified product 18: GenBank: KM274923.1/Position (start-end): 121-179

(70) TABLE-US-00023 (SEQ ID. NO: 97) AACGCGAATCGACCGGAT* CGCGATGTGTTTGCCGATAAAACGT ACCAACCGGAGCCCCAAGTGGGGCAGACGCAATG
Amplified product 19: GenBank: AB330122.1/Position (start-end): 1407-1691

(71) TABLE-US-00024 (SEQ ID. NO: 98) AACGCGAATCGACCGGAT * AAACAAGAACACCTATGCCTACAT GAACGGTCGGGTGGCGGTTCCTAGCGCCCTCGATACCTACGTAAACATCG GGGCACGGTGGTCTCCAGATCCCATGGACAATGTTAACCCCTTCAATCAC CACCGTAACGCCGGTCTGCGCTATCGATCCATGCTCTTGGGCAACGGGCG TTACGTACCCTTCCACATTCAAGTCCCCCAGAAGTTTTTTGCCATTAAAA ATCTCCTCCTCTTACCGGGTTCCTACACCTACGAGTGGAACTTCAGGAAG GACGTTAACAT
Amplified product 20: GenBank: LN854564.1/Position (start-end): 5325-5378

(72) TABLE-US-00025 (SEQ ID. NO: 99) TAGATCGGACTGCGAATCG * GAGATCGCRATCTYCTGCCCGAA TTCGTAAATGATGATGGCGTCTAAGGACGCY
Amplified product 21: GenBank: KT202798.1/Position (start-end): 5060-5107

(73) TABLE-US-00026 (SEQ ID. NO: 100) ATCTACAGCGTCGCATCACG * CGCAATCTGGCTCCCARTTTTG TGAATGARGATGGCGTCGARTGACGC
Amplified product 22: GenBank: EF204940.1/Position (start-end): 1707-1878

(74) TABLE-US-00027 (SEQ ID. NO: 101) CATAGGTCGAGGTCCTCAC* GCAAACTCCGGCATCTACTAATAG ACGCCGGCCATTCAAACATGAGGATTACCCATGTCGAAGACAACAAAGAA GTTCAACTCTTTATGTATTGATCTTCCTCGCGATCTTTCTCTCGAAATTT ACCAATCAATTGCTTCTGTCGCTACTGGAAGCGGTGATCCGCACAGTG

(75) The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. The sequence of the CCTF produced from the amplified product is as follows.

(76) TABLE-US-00028 CCTF 16: (SEQ ID. NO: 102) 5′-CCTGGTGTCAGCCGGCTGGAGTGG-3′ CCTF 17: (SEQ ID. NO: 103) 5′-CCTGGTTAACCGGCTCGTGGCGATG-3′ CCTF 18: (SEQ ID. NO: 104) 5′-CCTGGTGCGCTCCATTAGCGTGAGT-3′ CCTF 19: (SEQ ID. NO: 105) 5′-CCTGGCTTGTAGCCGGCTGGGTAGC-3′ CCTF 20: (SEQ ID. NO: 106) 5′-CCTGGTGGGATCGAATCACGCGGCA-3′ CCTF 21: (SEQ ID. NO: 107) 5′-CCTGGCAGCGGCCCAGCTATGAGA-3′ CCTF 22: (SEQ ID. NO: 108) 5′-CCTGGTCGCAGAGTGCCAACGATCTG-3′ CCTF 23: (SEQ ID. NO: 109) 5′-CCTGGTATATGGAGCGGCATACGAGC-3′ CCTF 24: (SEQ ID. NO: 110) 5′-CCTGGTGCAACGATGCACGGCACGT-3′ CCTF 25: (SEQ ID. NO: 111) 5′-CCTGGTTCCTCGACGGGACTGCGA-3′ CCTF 26: (SEQ ID. NO: 112) 5′-CCTGGCAGGCGCCTAGCTATGAG-3′ CCTF 27: (SEQ ID. NO: 113) 5′-CCTGGACAGGCCTGAGACGCGAAGC-3′ CCTF 28: (SEQ ID. NO: 114) 5′-CCTGGAGCTCGCAGAGCTAACAG-3′

(77) 2) PCR Amplification and Determination of the Inherent Dissociation Temperature of SCO

(78) The following PCR reaction was performed using CFX96 Real-time PCR (Bio-Rad, USA) with 20 μl of total reaction solution of each of Primer 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and SCO 14, 15, 16, 17, 18, 19, 20 prepared by adding 0.15 μM, PspGI (NEB, USA) 1U, PCR buffer (1×), MgCl.sub.2 2.5 mM, dNTP 200 μM, DTT 0.1 mM, RNase Inhibitor 1U, SuperiorScript III (Enzynomics, Korea) 1 U and the nucleic acid of the genomic RNA of RVA, AstV, AdV 40, AdV41, NoV GI, NoV GII and EC(MS2 phage) 1×10{circumflex over ( )}.sup.4 pg/rxn, respectively.

(79) 55° C. 20 mins, 95° C. 10 mins

(80) 95° C. 30 secs, 63° C. 1 mins (50 cycles).

(81) A reverse transcription reaction was performed using a cycle at the denaturation temperature of 55° C. for 20 minutes once, and with a cycle at the denaturation temperature of 95° C. for 10 minutes 1 time, and with a cycle at an annealing temperature of 63° C. for 1 minute 50 times repeatedly. After the reaction, the reaction mixture was cooled to 50° C. in the same apparatus, held at 50° C. for 30 seconds, and then slowly heated from 50° C. to 95° C. to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager 1.6.

(82) FIG. 4 shows the results of multiple inherent dissociation temperature measurements for causative organisms of RVA, AstV, AdV 40, AdV 41, NoV GI, NoV GII. It was identified that the peak was observed at the inherent dissociation temperature that each SCO has: RVA: HEX 78° C., AstV: CalRed610 78° C., AdV 40: CalRed610 67° C., AdV 41: CalRed610 67° C., NoV GI: FAM 68° C., NoV Gil: FAM 84° C., EC: HEX 69° C. (a)(b)(c)(d), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (e).

(83) Therefore, it was proved that the target nucleic acid sequence can be detected more quickly and simply than the conventional PCR method by analyzing the fluorescence of the SCO using the real-time PCR instrument, simultaneously with the PCR using the marking technique of CCTF.

(84) 3. Formation of CCTF and Analysis for the Inherent Dissociation Temperature Peak of CCTF in Multi-Target PCR for Detecting the Human Papillomavirus

(85) CCTF analysis was performed with Real-time PCR instrument for DNA of subtypes of Human Papillomavirus (HPV), 16 type, 18 type, 33 type, 35 type, 51 type, 53 type, 59 type, 68a type, 82 type and Internal control (1C).

(86) 1) Primer of the Target Sequence Template DNA, Constructed in the Sequence-Specific Manner

(87) The forward primer used in this example was CTPO and was constructed on the same principle as in Example 1 above. The 5′end of CTPO was composed of 19˜20 mers of nucleotide sequences, and was composed of a sequence non-complementary to DNA of the target sequence to form CCTF. The restriction enzyme recognition sequence was consecutively located, and after this up to the 3′ end, a sequence complementary to each target site was composed to play a role as a primer. The reverse primer was composed of sequence complementary to the target site to be amplified.

(88) In addition, SCO, which forms a complementary bond with CCTF to be a double-stranded template, was positioned by positioning fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the fluorescent reporter molecular was positioned so as to have a certain distance.

(89) Primer information and target sequence information which is amplified and generated are as follows.

(90) TABLE-US-00029 Primer 46: (SEQ ID. NO: 115) 5′-CTCTGATAGCGACTGCTCGCA*CCAGGATAATATAAGGGGTCGG TGGACCGG-3′ Primer 47: (SEQ ID. NO: 116) 5′-CTCCATGCATGATTACAGCTGGGTT-3′ Primer 48: (SEQ ID. NO: 117) 5′-ATCGGTCTCCTGAAAGCTGCG*CCAGGCAGAAGGTACAGACGGG GAGGGC-3′ Primer 49: (SEQ ID. NO: 118) 5′-CACCTCCAGCCGCTCCCCTAAT-3′ Primer 50: (SEQ ID. NO: 119) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGCAACGATAACCGACCAC CACAAGCA-3′ Primer 51: (SEQ ID. NO: 120) 5′-CGGGGTCTGCACAGAACAGCTTT-3′ Primer 52: (SEQ ID. NO: 121) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGAGGACCCAGCTGAACGA CCTTACAA-3′ Primer 53: (SEQ ID. NO: 122) 5′-CTGTCCACCGTCCACCGATGTTATG-3′ Primer 54: (SEQ ID. NO: 123) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGGCTGGCAACGTACACGACAA CG-3′ Primer 55: (SEQ ID. NO: 124) 5′-GCTGTACAACGCGAAGGGTGTC-3′ Primer 56: (SEQ ID. NO: 125) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGTCCACCTATGCACCGAA ACCTCCAA-3′ Primer 57: (SEQ ID. NO: 126) 5′TGCAGTGACGAGTCCCCGTGTAGTA-3′ Primer 58: (SEQ ID. NO: 127) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGGACTGTACACCGTATGC AGCGTG-3′ Primer 59: (SEQ ID. NO: 128) 5′-GCGTATCAGCAGCTCATGTAA-3′ Primer 60: (SEQ ID. NO: 129) 5′-CTGGCGTAGAGCACTTACGCT*CCAGGACAAACTCGACGTCGTC TCGGAA-3′ Primer 61: (SEQ ID. NO: 130) 5′-CAGGTCACCACAACAAAGGCTCCGT-3′ Primer 62: (SEQ ID. NO: 131) 5′-ATCAGGACGCAGCCGGTTCT*CCAGGCCAAGGACAGGTACGGCT GTCATC-3′ Primer 63: (SEQ ID. NO: 132) 5′-GGTGCCCTTGAGGTTGTCCAGGTG-3′ SCO 21: (SEQ ID. NO: 133) GAGACGTTTAAGTCCGCGACCGCTC[T(HEX)]CTGATAGCGACTGC TCGCA[BHQ1] SCO 22: (SEQ ID. NO: 134) CAGGCGACGTCCATATGGTGCGCTA[T(FAM)]CGGTCTCCTGAAAG CTGCG[BHQ1] SCO 23: (SEQ ID. NO: 135) CCCTTAGGTAACGTCTGGC[T(Qusar 670)]GGCGTAGAGCACTTAC GCT[BHQ 2] SCO 24: (SEQ ID. NO: 136) AAACTTTAATTATTGTATA[T(FAM)]CAGGACGCAGCCGGTTCT [BHQ 1]
Amplified product 23: GenBank: LC193821.1/Position (start-end): 480-571

(91) TABLE-US-00030 (SEQ ID. NO: 137) CTCTGATAGCGACTGCTCGCA * ATAATATAAGGGGTCGGTGGA CCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACC CAGCTGTAATCATGCATGGAG
Amplified product 24: GenBank: KC470209.1/Position (start-end): 538-747

(92) TABLE-US-00031 (SEQ ID. NO: 138) ATCGGTCTCCTGAAAGCTGCG * CACGACAGGAACGACTCCAAC GACGCAGAGAAACACAAGTATAATATTAAGTATGCATGGACCTAAGGCAA CATTGCAAGACATTGTATTGCATTTAGAGCCCCAAAATGAAATTCCGGTT GACCTTCTATGTCACGAGCAATTAAGCGACTCAGAGGAAGAAAACGATGA AATAGATGGAGTTAATCATCAACATTTACCAGCCCGACG
Amplified product 25: GenBank: KU298894.1/Position (start-end): 535-860

(93) TABLE-US-00032 (SEQ ID. NO: 139) CTGGCGTAGAGCACTTACGCT * ACGCCATGAGAGGACACAAGC CAACGTTAAAGGAATATGTTTTAGATTTATATCCTGAACCAACTGACCTA TACTGCTATGAGCAATTAAGTGACAGCTCAGATGAGGATGAAGGCTTGGA CCGGCCAGATGGACAAGCACAACCAGCCACAGCTGATTACTACATTGTAA CCTGTTGTCACACTTGTAACACCACAGTTCGTTTATGTGTCAACAGTACA GCAAGTGACCTACGAACCATACAGCAACTACTTATGGGCACAGTGAATAT TGTGTGCCCTACCTGTGCACAACAATAAACATCATCTACAATGGCCGATC CTGAA
Amplified product 26: GenBank: M74117.1/Position (start-end): 117-509

(94) TABLE-US-00033 (SEQ ID. NO: 140) CTGGCGTAGAGCACTTACGCT * AGGAGGACCCAGCTGAACGAC CTTACAAACTGCATGATTTGTGCAACGAGGTAGAAGAAAGCATCCATGAA ATTTGTTTGAATTGTGTATACTGCAAACAAGAATTACAGCGGAGTGAGGT ATATGACTTTGCATGCTATGATTTGTGTATAGTATATAGAGAAGGCCAGC CATATGGAGTATGCATGAAATGTTTAAAATTTTATTCAAAAATAAGTGAA TATAGATGGTATAGATATAGTGTGTATGGAGAAACGTTAGAAAAACAATG CAACAAACAGTTATGTCATTTATTAATTAGGTGTATTACATGTCAAAAAC CGCTGTGTCCAGTTGAAAAGCAAAGACATTTAGAAGAAAAAAAACGATTC CATAACATCGGTGGACGGTGGACAG
Amplified product 27: GenBank: KU298905.1/Position (start-end): 512-812

(95) TABLE-US-00034 (SEQ ID. NO: 141) CTGGCGTAGAGCACTTACGCT * GCTGGCAACGTACACGACAAC GTAACGAAACCCAAGTGTAATAAAGCCATGCGTGGTAATGTACCACAATT AAAAGATGTAGTATTGCATTTAACACCACAGACTGAAATTGACTTGCAAT GCTACGAGCAATTTGACAGCTCAGAGGAGGAGGATGAAGTAGATAATATG CGTGACCAGCTACCAGAAAGACGGGCTGGACAGGCTACGTGTTACAGAAT TGAAGCTCCGTGTTGCAGGTGTTCAAGTGTAGTACAACTGGCAGTGGAAA GCAGTGGAGACACCCTTCGCGTTGTACAGC
Amplified product 28: GenBank: KU298906.1/Position (start-end): 3374-3558

(96) TABLE-US-00035 (SEQ ID. NO: 142) CTGGCGTAGAGCACTTACGCT * TCCACCTATGCACCGAAACCT CCAAGACCTCCGCATTGTCCGTGGGTGCCAAAGACACACACCTACAACCA CCACAGAAACGACGACGACCAGACGTCACAGACTCCAGAAACACCAAGTA CCCCAACAACCTTTTGCGGGGACAACAATCCGTGGACAGTACTACACGGG GACTCGTCACTGCA
Amplified product 29: GenBank: KU298922.1/Position (start-end): 226-366

(97) TABLE-US-00036 (SEQ ID. NO: 143) CTGGCGTAGAGCACTTACGCT * GTTAAGACCGAAAACGGTGCA TATAAAGGTAGTTAGAAAGAAAAGGGCAACGGCATGGCACGCTTTGAGGA TCCTACACAACGACCATACAAACTGCCTGACTTGAGCACAACATTGAATA TTCCTCTGCATGATATTCGC
Amplified product 30: GenBank: KC470271.1/Position (start-end): 3389-3541

(98) TABLE-US-00037 (SEQ ID. NO: 144) CTGGCGTAGAGCACTTACGCT * ATGGCGCTATTTCACAACCCT GAGGAACGGCCATACAAATTGCCAGACCTGTGCAGGACATTGGACACTAC ATTGCATGACGTTACAATAGAGTGTGTCTATTGCAGAAGGCAACTACAAC GGACAGAGGTATATGAATTTGCCTTTAGTGAC
Amplified product 31: GenBank: EF450778.1/Position (start-end): 431-681

(99) TABLE-US-00038 (SEQ ID. NO: 145) GCTCATATGCGGCGCCATTTA* GCAGGTTGCTATCAAGGTTACA AGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAG ACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTT TCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTG AGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAG GTGAAGGCTCATGGCAAGAAAGTGCTCGG

(100) The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. The sequence of the CCTF produced from the amplified product is as follows.

(101) TABLE-US-00039 CCTF 29: (SEQ ID. NO: 146) 5′-TGCGAGCAGTCGCTATCAGAG-3′ CCTF 30: (SEQ ID. NO: 147) 5′-CGCAGCTTTCAGGAGACCGAT-3′ CCTF 31: (SEQ ID. NO: 148) 5′-AGCGTAAGTGCTCTACGCCAG-3′ CCTF 32: (SEQ ID. NO: 149) 5′-AGAACCGGCTGCGTCCTGAT-3′

(102) 2) PCR Amplification and Determination of the Inherent Dissociation Temperature of SCO

(103) The following PCR reaction was performed using CFX96 Real-time PCR (Bio-Rad, USA) with 20 μl of total reaction solution of each of Primer 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 and SCO 21, 22, 23, 24 prepared by adding 0.15 μM, PspGI (NEB, USA) 5U, PCR buffer (1×), MgCl 2 2.5 mM, dNTP 200 μM, h-Taq DNA polymerase (Solgent, Korea) 1.6 U and HPV type 16, type 18, type 33, type 35, type 51, type 53, type 59, type 68a, type 82 and template DNA of genomic DNA of IC 100 pg/rxn, respectively.

(104) 95° C. 15 mins,

(105) 95° C. 30 secs, 63° C. 1 mins (50 cycles).

(106) A reaction was performed using a cycle at the denaturation temperature of 95° C. for 15 minutes once, and with a cycle at the denaturation temperature of 95° C. for 30 second and at an annealing temperature of 63° C. for 1 minute 50 times repeatedly. After the reaction, the reaction mixture was cooled to 50° C. in the same apparatus, held at 50° C. for 30 seconds, and then slowly heated from 50° C. to 95° C. to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager 1.6.

(107) FIG. 5 shows the results of multiple inherent dissociation temperature measurements for each target of type 16, type 18, type 33, type 35, type 51, type 53, type 59, type 68a, type 82, IC. It was identified that the peak was observed at the inherent dissociation temperature that each SCO has (type 16: HEX 76.5° C., type 18: FAM 78° C., type 33: Quasar670 71° C., type 35: Quasar670 71° C., type 51: Quasar670 71° C., type 53: Quasar670 71° C., type 59: Quasar670 71° C., type 68 a: Quasar670 71° C., type 82: Quasar670 71° C., IC: Quasar670 67.5° C.) (a)(b)(c)(d), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (e).

(108) Therefore, it was proved that the target nucleic acid sequence can be detected more quickly and simply than the conventional PCR method by analyzing the fluorescence of the SCO using the real-time PCR instrument, simultaneously with the PCR using the marking technique of CCTF.

(109) 4. Formation of CCTF and Analysis for the Inherent Dissociation Temperature Peak of CCTF in Multi-Target PCR for Detecting the Causative Organism of the Respiratory Diseases

(110) CCTF analysis was performed using Real-time PCR instrument of nucleic acids of the causative organisms of the respiratory diseases, Influenza A/H1N1(Flu A/H1N1), Influenza A/H3N2(Flu A/H3N2), Influenza A/H1N1/2009pdm(Flu A/H1N1/2009pdm), Influenza B(Flu B), Parainfluenza 1(PIV1), Parainfluenza 3(PIV3), Respiratory syncytial virus A(RSV A), Respiratory syncytial virus B(RSV B), Human metapneumovirus(MPV), Adenovirus(AdV) and External control (EC).

(111) 1) Primer for the Target Sequence of Template DNA, Constructed in the Sequence-Specific Manner

(112) The forward primer used in this example was CTPO and was constructed on the same principle as in Example 1 above. The 5′end of CTPO was composed of 19-20 mers of nucleotide sequences, and was composed of a sequence non-complementary to DNA of the target sequence so as to form CCTF. The restriction enzyme recognition sequence was then consecutively located, and after this up to the 3′ end, a sequence complementary to each target site was composed to play a role as a primer. The reverse primer was composed of sequence complementary to the target site to be amplified.

(113) In addition, SCO, which forms a complementary bond with CCTF to be a double-stranded template, was positioned by positioning fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the fluorescent reporter molecular was positioned so as to have a certain distance.

(114) Primer information and target sequence information which is amplified and generated are as follows.

(115) TABLE-US-00040 Primer 64: (SEQ ID. NO: 150) 5′-TTGCTATGGCTGACGGGGAAGAATGG-3′ Primer 65: (SEQ ID. NO: 151) 5′-GCCCCGTTGAGAGCACGAAT*CCAGGGGGGTGAATCTTCTGCTT AATGTGAAGACA C-3′ Primer 66: (SEQ ID. NO: 152) 5′-GGGCACCATGCAGTACCAAACGGAAC-3′ Primer 67: (SEQ ID. NO: 153) 5′-CCGTGGCGCGAACTTATCGA*CCAGGATCACACTGAGGGTCTCC CAATAGAGC-3′ Primer 68: (SEQ ID. NO: 154) 5′-TCAAAGACTAAGTGGTGCCATGGATGAAC-3′ Primer 69: (SEQ ID. NO: 155) 5′-AAGTGACCTGCCATTGCGCG*CCAGGTATGTCTACAGCAGAGGG ACCCAGC-3′ Primer 70: (SEQ ID. NO: 156) 5′-GGCTTAGAGCACCGCGTCATT*CCAGGTGTCGCTACTGGAAGCG GTGATC-3′ Primer 71: (SEQ ID. NO: 157) 5′-GCGATAGCTAAGGTACGACGGGTC-3′ Primer 72: (SEQ ID. NO: 158) 5′-GTAGATTCGATCCATGCTCCTCTACTACC-3′ Primer 73: (SEQ ID. NO: 159) 5′-CGTCTTACATGCGCAAGCGG*CCAGGTGATATTGAGTTCGGTAA TGCAAGATCTGC-3′ Primer 74: (SEQ ID. NO: 160) 5′-CCATAGAGATGGCAATAGATGAAGAGC-3′ Primer 75: (SEQ ID. NO: 161) 5′-AGGCGTTCCGCTTCAACGAG*CCAGGTTGTCAGATTCTGTAGC TTGCTCAGTC-3′ Primer 76: (SEQ ID. NO: 162) 5′-GGTGGTGATCCCAACTTGTTATATCGAAG-3′ Primer 77: (SEQ ID. NO: 163) 5′-TCCGTCTGCGAAGATCTGAGC*CCAGGTTCAATCTATCRTCTG ACAGATCTTGAAGT-3′ Primer 78: (SEQ ID. NO: 164) 5′-GTGTCACGACGCGCGAATCT*CCAGGAGATCGTGACCAGTAT AATAGCTCAACAC-3′ Primer 79: (SEQ ID. NO: 165) 5′-TTTCAGACAATGCAGGGATAACACCAGC-3′ Primer 80: (SEQ ID. NO: 166) 5′-CCCAGAACGATTTGCGGCGT*CCAGGCTTGGTCCTCTCTTAGG AGGCAAGC-3′ Primer 81: (SEQ ID. NO: 167) 5′-AGGATGCTTCGGAGTACCTGAG-3′ Primer 82: (SEQ ID. NO: 168) 5′-TGCATTGCCGTCGCAGAGAC*CCAGGCAACGGGCACGAAG CGCATC-3′ Primer 83: (SEQ ID. NO: 169) GCCCTAATGATAAGACAGGCAGTTGTGG Primer 84: (SEQ ID. NO: 170) 5′-ATGCGCTTGGATTGCCGATG*CCAGGAGCCCTGTTAGTTCTGG ATGCTGAACA-3′ SCO 33: (SEQ ID. NO: 171) CTTATAGATTATA[T(FAM)]TGCCCCGTTGAGAGCACGAAT[BHQ1] SCO 34: (SEQ ID. NO: 172) CTAAGTAAGCCTATATCGAAT[T(FAM)]CCGTGGCGCGAACTTAT CGA[BHQ1] SCO 35: (SEQ ID. NO: 173) CGTACTGCACTCGCCTACGAC[T(Cal Fluor Red 610)] AAGTGACCTGCCATTGCGCG[BHQ2] SCO 36: (SEQ ID. NO: 174) CTTATAAGTTACA[T(Cal Fluor Red 610)]GGCTTAGAGCACCG CGTCATT[BHQ2] SCO 37: (SEQ ID. NO: 175) CTAATTGTAATAC[T(Quasar 670)]CGTCTTACATGCGCAAGCGG [BHQ2] SCO 38: (SEQ ID. NO: 176) CTAATCGTATGAGATCTATGA[T(Quasar 670)] AGGCGTTCCGCTTCAACGAG[BHQ2] SCO 39: (SEQ ID. NO: 177) TCATAGACATTTA[T(Cal Fluor Gold 540)]TCCGTCTGCGAAG ATCTGAGC[BHQ1] SCO 40: (SEQ ID. NO: 178) TACGAATCTGACCTAGTAAGA[T(Cal Fluor Gold 540)] GTGTCACGACGCGCGAATCT[BHQ1] SCO 41: (SEQ ID. NO: 179) TGCCACTAACAGGCCGCTAGA[T(Cal Fluor Gold 540)]CC CAGAACGATTTGCGGCGT[BHQ1] SCO 42: (SEQ ID. NO: 180) TCGAGCGTGCGCCAGATCCA[T(Quasar 670)] TGCATTGCCGTCGCAGAGAC[BHQ2] SCO 43: (SEQ ID. NO: 181) TCGACTGTGCCTGCGTCCGTA[T(FAM)]ATGCGCTTGGATTGCCGATG [BHQ1]
Amplified product 32: GenBank: KU558787.1/Position (start-end): 428-621

(116) TABLE-US-00041 (SEQ ID. NO: 182) TTGCTATGGCTGACGGGGAAGAATGGTTTGTACCCAAACCTGAGCATGTC CTATGTAAACAACAAAGAGAAAGAAGTCCTTGTGCTATGGGGTGTTCATC ACCCACCTAACATAGGGAACCAAAGGGCCCTCTACCATACAGAAAATGCT TATGTCTCTGTAGTGTCTTCACATTATAGCAGAAGATTCACCCC* ATTCGTGCTCTCAACGGGGC
Amplified product 33: GenBank: CY221934.1/Position (start-end): 111-296

(117) TABLE-US-00042 (SEQ ID. NO: 183) GGGCACCATGCAGTACCAAACGGAACGATAGTGAAAACAATCACAAATGA CCAAATTGAAGTTACTAATGCTACTGAGTTGGTTCAGAATTCCTCAATAG GTGAAATATGCGACAGTCCTCATCAGATCCTTGATGGAGAGAACTGCACA CTAATAGATGCTCTATTGGGAGACCCTCAGTGTGAT* TCGATA AGTTCGCGCCACGG
Amplified product 34: GenBank: CY221750.1/Position (start-end): 1291-1501

(118) TABLE-US-00043 (SEQ ID. NO: 184) TCAAAGACTAAGTGGTGCCATGGATGAACTCCACAACGAAATACTCGAGC TGGATGAAAAAGTGGATGACCTCAGAGCTGACACTATAAGCTCACAAATA GAACTTGCAGTCTTGCTTTCCAACGAAGGAATAATAAACAGTGAAGATGA GCATCTATTGGCACTTGAGAGAAAACTAAAGAAAATGCTGGGTCCCTCTG CTGTAGACATA* CGCGCAATGGCAGGTCACTT
Amplified product 35: GenBank: JF719743.1/Position (start-end): 1816-1950

(119) TABLE-US-00044 (SEQ ID. NO: 185) GGCTTAGAGCACCGCGTCATT* TGTCGCTACTGGAAGCGGTGA TCCGCACAGTGACGACTTTACAGCAATTGCTTACTTAAGGGACGAATTGC TCGCAAAGCATCCGACCTTAGGTTCTGGTAATGACGAGGCGACCCGTCGT ACCTTAGCTATCGC
Amplified product 36: GenBank: KX639498.1 z/Position (start-end): 4035-4253

(120) TABLE-US-00045 (SEQ ID. NO: 186) GTAGATTCGATCCATGCTCCTCTACTACCATGGTCCAGCCGACTGAGACA AGGGATGATATATAATGCCAATAAAGTAGCTCTGGCACCCCAATGTCTCC CAGTCGACAAAGATATCAGATTCAGAGTTGTATTTGTCAACGGAACATCA CTGGGTACAATCACAATTGCCAAGGTCCCAAAAACTCTTGCAGATCTTGC ATTACCGAACTCAATATCA* CCGCTTGCGCATGTAAGACG
Amplified product 37: GenBank: KY369876.1/Position (start-end): 1310-1463

(121) TABLE-US-00046 (SEQ ID. NO: 187) CCATAGAGATGGCAATAGATGAAGAGCCAGAACAATTCGAACATAGAGCA GACCAAGAACAAGATGGGGAACCTCAATCATCTATAATCCAATATGCTTG GGCAGAAGGAAACAGAAGCGATGACCGGACTGAGCAAGCTACAGAATCTG ACAA* CTCGTTGAAGCGGAACGCCT
Amplified product 38: GenBank: KX894800.1/Position (start-end): 11378-11529

(122) TABLE-US-00047 (SEQ ID. NO: 188) GGTGGTGATCCCAACTTGTTATATCGAAGTTTCTATAGAAGAACTCCTGA TTTCCTCACAGAGGCTATAGTTCACTCTGTGTTCATACTTAGTTATTATA CAAACCATGATTTAAAGGATAAACTTCAAGATCTGTCAGAYGATAGATTG AA* GCTCAGATCTTCGCAGACGGA
Amplified product 39: GenBank: KY249683.1/Position (start-end): 11465-11577

(123) TABLE-US-00048 (SEQ ID. NO: 189) GGTGGTGATCCTAATTTGTTATATCGAAGCTTTTATAGGAGAACTCCAG ACTTCCTTACAGAAGCTATAGTACATTCAGTGTTCGTGTTGAGCTATTA TACTGGTCACGATCT*  AGATTCGCGCGTCGTGACAC
Amplified product 40: GenBank: KJ627391.1/Position (start-end): 3631-3933

(124) TABLE-US-00049 (SEQ ID. NO: 190) TTTCAGACAATGCAGGGATAACACCAGCAATATCATTGGACCTAATGAC TGATGCTGAACTGGCCAGAGCTGTATCATACATGCCAACATCTGCAGGG CAGATAAAGCTGATGTTGGAGAACCGCGCAATGGTAAGGAGAAAAGGAT TTGGAATCCTAATAGGGGTCTACGGAAGCTCTGTGATTTACATGGTTCA ATTGCCGATCTTTGGTGTCATAGATACACCTTGTTGGATAATCAAGGCA GCTCCCTCTTGCTCAGAAAAAAACGGGAATTATGCTTGCCTCCTAAGAG AGGACCAAG*  ACGCCGCAAATCGTTCTGGG
Amplified product 41: GenBank: KT963081.1/Position (start-end): 18437-18598

(125) TABLE-US-00050 (SEQ ID. NO: 191) AGGATGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGTTCGCCCGTGC AACAGACACCTACTTCAGTATGGGGAACAAGTTTAGAAACCCCACAGTG GCGCCCACCCACGATGTGACCACCGACCGTAGCCAGCGACTGATGCTGC GCTTCGTGCCCGITG*  GTCTCTGCGACGGCAATGCA
Amplified product 42: GenBank: CY221624.1/Position (start-end): 988-1252

(126) TABLE-US-00051 (SEQ ID. NO: 192) GCCCTAATGATAAGACAGGCAGTTGTGGTCCAGTATCGTCTAATGGAGC AAATGGAGTAAAAGGATTTTCATTCAAATACGGCAATGGTGTTTGGATA GGGAGAACTAAAAGCATTAGTTCAAGAAAAGGTTTTGAGATGATTTGGG ATCCGAATGGATGGACTGGGACTGACAATAAATTCTCAATAAAGCAAGA TATCGTAGGAATAAATGAGTGGTCAGGGTATAGCGGGAGTTTTGTTCAG CATCCAGAACTAACAGGGCT*  CATCGGCAATCCAAGCGCAT

(127) The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. The sequence of the CCTF produced from the amplified product is as follows.

(128) TABLE-US-00052 CCTF 33: (SEQ ID. NO: 193) 5′-ATTCGTGCTCTCAACGGGGC-3′ CCTF 34: (SEQ ID. NO: 194) 5′-TCGATAAGTTCGCGCCACGG-3′ CCTF 35: (SEQ ID. NO: 195) 5′-CGCGCAATGGCAGGTCACTT-3′ CCTF 36: (SEQ ID. NO: 196) 5′-AATGACGCGGTGCTCTAAGCC-3′ CCTF 37: (SEQ ID. NO: 197) 5′-CCGCTTGCGCATGTAAGACG-3′ CCTF 38: (SEQ ID. NO: 198) 5′-CTCGTTGAAGCGGAACGCCT-3′ CCTF 39: (SEQ ID. NO: 199) 5′-GCTCAGATCTTCGCAGACGGA-3′ CCTF 40: (SEQ ID. NO: 200) 5′-AGATTCGCGCGTCGTGACAC-3′ CCTF 41: (SEQ ID. NO: 201) 5′-ACGCCGCAAATCGTTCTGGG-3′ CCTF 42: (SEQ ID. NO: 202) 5′-GTCTCTGCGACGGCAATGCA-3′ CCTF 43: (SEQ ID. NO: 203) 5′-CATCGGCAATCCAAGCGCAT-3′

(129) 2) PCR Amplification and Determination of SCO Inherent Dissociation Temperature

(130) The following PCR reaction was performed using CFX96 Real-time PCR (Bio-Rad, USA) with 20 μl of total reaction solution of each of 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 and SCO 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 prepared by adding 0.15 μM, PspGI (NEB, USA) 1U, PCR buffer (1×), MgCl.sub.2 2.5 mM, dNTP 200 μM, DTT 0.1 mM, RNase Inhibitor IU. SuperiorScript III (Enzynomics, Korea) 1U Flu A/H1N1, Flu A/H3N2, Flu A/H1N1/2009pdm, the template nucleic acid of the genomic RNA of Flu B, PIV1, PIV3, RSV A, RSV B, hMPV, ADV and MS2 phage 1×10{circumflex over ( )}.sup.4 copies/rx, respectively.

(131) 55° C. 20 mins, 95° C. 10 mins

(132) 95° C. 30 secs, 63° C. 1 min (50 cycles).

(133) A reaction was repeatedly performed with a cycle at the reverse transcription reaction temperature of 55° C. for 20 minutes once, and with a cycle at the denaturation temperature of 95° C. for 30 seconds, and an annealing temperature of 63° C. for 1 minute 50 times repeatedly. After the reaction, the reaction mixture was cooled to 50° C. in the same apparatus, held at 50° C. for 30 seconds, and then slowly heated from 50° C. to 95° C. to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager 1.6.

(134) FIG. 6 shows the results of multiple inherent dissociation temperature measurements for causative organisms of Flu A/H1N1, Flu A/H3N2, Flu A/H1N1/2009pdm, Flu B, PIV1, PIV3, RSV A, RSV B, hMPV, ADV, EC(Ms2 phage). It was confirmed that the peak was observed at the inherent dissociation temperature that each SCO has (Flu A/H1N1: 67.5° C. Flu A/H3N2: 76.5° C., Flu A/H1N1/2009pdm: 86.5° C., Flu B: 83.5° C., PIV1: 66° C., PIV3: 74° C., RSV A: 63.5° C., RSV B: 72° C., hMPV: 86° C., ADV: 85° C.) (a)(b)(c)(d)(e), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (I).

(135) Therefore, it was proved that the target nucleic acid sequence can be detected more quickly and simply than the conventional PCR method by analyzing the fluorescence of the SCO using the real-time PCR instrument, simultaneously with the PCR using the marking technique of CCTF.

(136) 5. Formation of CCTF and Analysis for the Inherent Dissociation Temperature Peak of CCTF in Multi-Target PCR for Analyzing the Single Nucleotide Polymorphism Genotype of BDNF Gene

(137) CCTF analysis was performed with Real-time PCR instrument for analyzing the genotype of rs6265, single nucleotide polymorphism of BDNF gene.

(138) 1) Primer for the Target Sequence of Template DNA, Constructed in the Sequence-Specific Manner

(139) The forward primer used in this example was CTPO and was constructed on the same principle as in Example 1 above. The 5′end of CTPO was composed of 19-20 mers of nucleotide sequences, and was composed of a sequence non-complementary to DNA of the target sequence so as to form CCTF. The restriction enzyme recognition sequence was then located, and from this up to the 3′ end, a sequence complementary to each target site was composed to play a role as a primer. The reverse primer was composed of sequence complementary to the target site to be amplified.

(140) In addition, SCO, which forms a complementary bond with CCTF to be a double-stranded template, was positioned by positioning fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the fluorescent reporter molecular was positioned so as to have a certain distance.

(141) Primer information and target sequence information which is amplified and generated are as follows.

(142) TABLE-US-00053 Primer 85: (SEQ ID. NO: 204) 5′-ACGAGGCCTGTCCGCTTACTAG*CCAGGCTGGTCCTCATCCAACAG CTCTTCTATCGC-3′ Primer86: (SEQ ID. NO: 205) 5′-CCGGGTACGCTAAGTCCGCTAT*CCAGGTTCTGGTCCTCATCCAAC AGCTCTTCTATCGT-3′ Primer 87: (SEQ ID. NO: 206) 5′-GACCCATGGGACTCIGGAGAGCGTGAA-3′ Primer 88: (SEQ ID. NO: 207) 5′-GCTCATATGCGGCGCCATTTA*CCAGGGCAGGTTGCTATCAAGGTT ACAAGACAG-3′ Primer 89: (SEQ ID. NO: 208) 5′-CCGAGCACTTTCTTGCCATGAGCC-3′ SCO 44: (SEQ ID. NO: 209) GTAGCACGCTTCGAATGGC[T(HEX)]ATACGAGGCCTGTCCGCTTACT AG[BHQ1] SCO 45: (SEQ ID. NO: 210) GATACGGAGGTCCGAAGGCAG[T(FAM)]GTTGGTTACCCTAACGCGCC GGA[BHQ1] SCO 46: (SEQ ID. NO: 211) ATTAGTTTAACTATTATATT[T(FAM)]TATGCTCATATGCGGCGCCAT TTA[BHQ1]
Amplified product 43: GenBank: NT_009237.19/Position (start-end): 27598340-27598451

(143) TABLE-US-00054 (SEQ ID. NO: 212) ACGAGGCCTGTCCGCTTACTAG*  CTGGTCCTCATCCAACAGC TCTTCTATCACGTGTTCGAAAGTGTCAGCCAATGATGTCAAGCCTCTTG AACCTGCCTTGGGCCCATTCACGCTCTCCAGAGTCCCATGGGTC
Amplified product 44: GenBank: NT_009237.19/Position (start-end): 27598338-27598451

(144) TABLE-US-00055 (SEQ ID. NO: 213) CCGGGTACGCTAAGTCCGCTAT*  TTCTGGTCCTCATCCAAC AGCTCTTCTATCACGTGTTCGAAAGTGTCAGCCAATGATGTCAAGCCTC TTGAACCTGCCTTGGGCCCATTCACGCTCTCCAGAGTCCCATGGGTC
Amplified product 45: GenBank: EF450778.1/Position (start-end): 431-681

(145) TABLE-US-00056 (SEQ ID. NO: 214) GCTCATATGCGGCGCCATTTA*  GCAGGTTGCTATCAAGGTTA CAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAG AAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCT ATTTTCCCACCCTTAGGCTGCTGGTGGTGGTCTACCCTTGGACCCAGAG GTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGC AACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGG

(146) The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. The sequence of the CCTF produced from the amplified product is as follows.

(147) TABLE-US-00057 CCTF 44: (SEQ ID. NO: 215) 5′-CTAGTAAGCGGACAGGCCTCGT-3′ CCTF 45: (SEQ ID. NO: 216) 5′-ATAGCGGACTTAGCGTACCCGG-3′ CCTF 46: (SEQ ID. NO: 217) 5′-TAAATGGCGCCGCATATGAG-3′

(148) 2) PCR Amplification and Determination of SCO Inherent Dissociation

(149) PCR reaction was performed using the following CFX96 Real-time PCR (Bio-Rad, USA) with 20 μl of total reaction solution of each of Primer 85, 86, 87, 88, 89 and SCO 44, 45,46 prepared by adding 0.15 μM, PspGI (NEB, USA) 5U, PCR buffer (1×), MgCl.sub.2 2.5 mM, dNTP 200 μM, h-Taq DNA polymerase (Solgent, Korea) 1.6 U and Flu A/H1N1. Flu A/H3N2, Flu A/H1N1/2009pdm, the template nucleic acids of the genomic RNA of Flu B, PIV1, PIV3, RSV A, RSV B, hMPV, ADV and MS2 phage 1×10{circumflex over ( )}.sup.4 copies/rxn, respectively. 95° C. 15 mins, 95° C. 30 secs, 63° C. 1 min (50 cycles).

(150) A reaction was performed using a cycle at the denaturation temperature of 95° C. for 15 minutes once, and with a cycle at the denaturation temperature of 95° C. for 30 seconds, and an annealing temperature of 63° C. for 1 minute 50 times repeatedly. After the reaction, the reaction mixture was cooled to 50° C. in the same apparatus, held at 50° C. for 30 seconds, and then slowly heated from 50° C. to 95° C. to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager 1.6.

(151) FIG. 7 (a) shows the results of multiple inherent dissociation temperature measurements for the genotype of mutant type A/A, wild type G/G and heterozygote A/G of rs6265 and IC. It was identified that the peak was observed at the inherent dissociation temperature that each SCO has (A/A: 76.5° C., A/G: 76.5° C. 75° C., G/G 75° C., IC: 66° C.) (a)(b)(c)(d), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (e).

(152) Therefore, it was proved that the target nucleic acid sequence can be detected more quickly and simply than the conventional PCR method by analyzing the fluorescence of the SCO using the real-time PCR instrument, simultaneously with the PCR using the marking technique of CCTF.

Example 3. Formation of CCTF and Analysis for Ct Graph of CCTF in Multiple Target PCR

(153) It has been proved in Example 2 that SCO can be used to confirm whether CCTF is generated with a real-time PCR device. The SCO used in the above method is simultaneously formed during the reaction in which the target sequence is generated during the PCR amplification process, and it is possible to identify CCTF generated by real-time fluorescence analysis. Based on this, the present example demonstrated that a standard curve formation is possible when analyzing the formation of CCTF using SCO in the case of PCR with multiple target sequences.

(154) In order to perform this experiment, the causative organisms of sexually transmitted infections (STI), Neisseria. gonorrhea (NG), Mycoplasma. hominis (MH), Ureaplasma. parvum (UP) were selected.

(155) 1. Construction of Specific Primer of Target Template DNA

(156) The forward primer used in this example was constructed based on the method described in the detailed description of the invention above as CPTO. The 5′end of the forward primer was composed of a 19-mer or 21-mer nucleotide sequence, and was composed of non-complementary sequences to DNA of each causative organism so as to form CCTF. The restriction enzyme recognition sequence was then consecutively located. After this up to the 3′ end, a sequence complementary to DNA of each causative organism was composed to play a role as a primer. The reverse primer was composed of sequence complementary to the target site of DNA by each causative organism.

(157) In addition, SCO, which forms a dimer with CCTF, was designed to have a double tag, and was separately designed for each causative organism. SCO was designed by positioning quencher (BHQ-1 or BHQ-2) at 3 end, with reporter molecular (each FAM, HEX, CAL Fluor Red 610) positioned at a certain distance, and its sequence was complementary to CCTF sequence to be analyzed.

(158) Primer information and target sequence information which is amplified and generated are as follows.

(159) TABLE-US-00058 Primer 90: (SEQ ID. NO: 218) 5′-CTCATCGCCACGAGCCGGTTAA * TTGAAACACCGCCCGGAA CCC-3′ Primer 91: (SEQ ID. NO: 219) 5′-GCTCCTTATTCGGTTTGACCGGT-3′ Primer 92: (SEQ ID. NO: 220) 5′-GCTCGCAGGTACGGCACCATTCA * CAGAAGGTATGATAACA ACGGTAGAGC-3′ Primer 93: (SEQ ID. NO: 221) 5′-CCCCTTTGCACCGTTGAGGGG-3′ Primer 94: (SEQ ID. NO: 222) 5′-AGTCGATTATGTCTGAGGCCGCG * TTAAAGTAGCATATGA TCAAGCTCATTCA-3′ Primer 95: (SEQ ID. NO: 223) 5′-GATCCTGACATATAATCATTATCTCCTTTTATAAA-3′ SCO 47: (SEQ ID. NO: 224) TC[T(HEX)]CATCGCCACGAGCCGGTTAA[BHQ] SCO 48: (SEQ ID. NO: 225) TG[T(CAL Fluor Red 610)]CGCAGGTACGGCACCATTCA[BHQ2] SCO 49: (SEQ ID. NO: 226) TAG[T(FAM)]CGATTATGTCTGAGGCCGCG[BHQ]
Amplified product 46: GenBank: X52364.1/Position (start-end): 375-459

(160) TABLE-US-00059 (SEQ ID. NO: 227) CTCATCGCCACGAGCCGGTTAA * TTGAAACACCGCCCGGAAC CCGATATAATCCGCCCTTCAACATCAGTGAAAATCTTTTTTTAACCGGT CAAACCGAATAAGGAGC
Amplified product 47: GenBank: M31431.1/Position (start-end): 1455-1535

(161) TABLE-US-00060 (SEQ ID. NO: 228) GCTCGCAGGTACGGCACCATTCA * CAGAAGGTATGATAACAA CGGTAGAGCTTTATATGATATTAACTTAGCAAAAATGGAAAACCCCTCA ACGGTGCAAAGGGG
Amplified product 48: GenBank: AF085733.2/Position (start-end): 416-502

(162) TABLE-US-00061 (SEQ ID. NO: 229) AGTCGATTATGTCTGAGGCCGCG * GTTTCTGTACACGATCC AATT[T/c]ACAAATAACATTTACAATTCGTAAAATTTTTTTATAAAAG GAGATAATGATTATATGTCAGGATC

(163) The bold and slanted font of the Primer sequence means the restriction enzyme recognition sequence, and the underline is the complementary sequence of the CCTF produced thereby. the part represented by * is a tag that modified dCTP was inserted into C in the recognition sequence to block the site cleaved by the PspGI restriction enzyme. In SCO, the parentheses mean the position of the nucleotide sequence in which the fluorescent offsetting molecule and the fluorescent reporter are located. Primer and primer corresponding to NG in SCO is the same as those used in Example 2. The sequence of the CCTF produced from the amplified product is as follows.

(164) TABLE-US-00062 CCTF 47: (SEQ ID. NO: 230) 5′-CCTGGTTAACCGGCTCGTGGCGATGAG-3′ CCTF 48: (SEQ ID. NO: 231) 5′-CCTGGTGAATGGTGCCGTACCTGCGAGC-3′ CCTF 49: (SEQ ID. NO: 232) 5′-CCTGGCGCGGCCTCAGACATAATCGACT-3′

(165) 2. PCR Amplification and Determination of SCO Inherent Dissociation

(166) PCR reaction was performed using the following CFX96 Real-time PCR (Bio-Rad, USA) with 20 μl of total reaction solution obtained by adding three kinds of the specific forward primers and three kinds of reverse primers of each target sequence, as mentioned in the above primer design, and three kinds of SCO to be 0.15 μM, respectively, and adding PspGI (NEB, USA) 2 U, PCR buffer (1×), MgCl.sub.2 2.5 mM, dNTP 200 μM, h-Taq DNA polymerase (Solgent, Korea) 1.6 U, and contained the template DNA diluted by 10-folds with 100 pg/μl genomic DNA proven by the conventional quantitation method for each causative organism.

(167) 95° C. 15 mins,

(168) 95° C. 30 secs, 63° C. 1 min (50 cycles).

(169) A reaction was repeatedly performed with a cycle at the denaturation temperature of 95° C. for 15 minutes once, and with a cycle at the denaturation temperature of 95° C. for 30 seconds, and an annealing temperature of 63° C. for 1 minute 50 times. In addition, fluorescence signals were collected at the annealing stage, and the data analysis was performed with Bio-Rad CFX Manager 1.6. Cycle threshold (Ct) was started with an algebraic amplifier using a known number of DNA concentrations to create a standard curve for the strain.

(170) As shown in (a) of FIG. 8, the expected fluorescence amplification curves of SCO could be observed with each of different graphs depending on the concentration of the template. Also, any peak was observed when the template DNA was not added (b). As the results showing fluorescence amplification curves and standards of SCO represented by the experimental condition of Polymerase Chain Reaction of NO (solid line), MG (dotted line), and UP (circle), dilutions for genomic DNA of each causative organism diluted by 10-folds starting from the concentration of 100 pg, graph (a) indicates the fluorescence amplification curve drawn when the three target sequences are present at the same time by the concentration, graph (b) is the negative result drawn when all three target sequences are not included. When the graph corresponding to NO in graph (a) is represented by the single fluorescence amplification curve and thus the standard curve, it can be represented by (c) and (d), respectively. The graph corresponding to MG can be expressed by (e) and (f), respectively, and the curve corresponding to UP can be represented by (g) and (h), respectively.

(171) Regression coefficient (r.sup.2) in the linear regression analysis of the standard curve was represented by NO 0.9982, MG 0.999, UP 0.9992, respectively. The slope of the regression plot was NG −3.85, MG −3.89, and UP −3.66, respectively. It could be identified that the respective amplification efficiency (E=10.sup.[−1/slope]−1) was 81.8% for NO, 80.7% for MG and 87.6% for UP, respectively, and thus, they were listed in the proper range of between 80 and 120%.

(172) From this Example, when reading the different CCTFs by each of causal organisms using the real-time PCR instrument, it was demonstrated that the relative amount of CCTF to be generated by measuring a degree of the real-time fluorescence of SCO is grasped, and by using this, the Ct value is confirmed, and therefore, the identifying of the target sequence is possible.