Patent Description:
Polymerase Chain Reaction (PCR) is one of techniques very usefully utilized in detecting and analyzing low concentration nucleic acids. The detection of the nucleic acid is based on the complementarity of the double strand oligonucleotide sequences and the extension reaction of each DNA polymerase, and the target nucleic acid sequence can be detected using this (<NPL>).

Multiple PCR is a method that can simultaneously amplify nucleic acids of multiple target sequences, and is relatively fast and simple compared to other methods, and thus plays a very large role in diagnosis field such as genetic test, identification of organisms in samples, and microbial or viral infection, etc..

The most common method for confirming the results of such multiplex PCR is to design primers by varying an amplification product size of the target sequence as desired in PCR, and to analyze the size of the amplified product by electrophoresis of the PCR result, and then to confirm as to whether amplification of the target sequence is made. In this case, the number of genes that can be amplified at one time is limited to <NUM> to <NUM> experimentally because there is a restriction that the size of the amplification product should be limited within a narrow range, due to that the efficiency of amplification depends on the size of the amplification product that can be generated during the PCR reaction and thus a uniform amplification efficiency cannot be guaranteed. In this case, it also occurs the case that the size of the desired gene amplification product may overlap. Therefore, there is a limit to the interpretation of the detection method when the multiple PCR is analyzed depending on the size.

Real-time PCR guarantees a confirmation of a rapid PCR result in confirming the PCR results, and it can identify as to whether the amplification is made by marking fluorescent material regardless of the size of the amplified product. The methods performing and detecting Real-time PCR can be divided into intercalating method and probe method, wherein the intercalating method is referred to a method of confirming fluorescence intensity by inserting fluorescent substance between double-stranded base sequences. Since this method cannot distinguish the amplification products forming the double strands, and can observe all of them as the fluorescence of the same wavelength. Therefore, it has a limit on identifying the amplification product by each target sequence to detect and identify at least one amplified product simultaneously. The probe method is a method of detecting the amplified product by reading the fluorescence value of the probe designated for each target sequence and, in the case of using this method, since the amplification product can be detected only in the number of analyzable fluorescence channels of a device to be used, the multiple analysis over the number of fluorescent channels is not suitable for this.

Therefore, studies were continuously carried out to insert the tag during PCR to enable the maximum number of multiple analysis.

In the case of Luminex's xTAG technology, a constant base sequence comprised of a random array of thymine (T), adenine (A), and guanine (G), which constitutes the nucleic acid, was set and named xTAG. It is a method comprising inserting xTAG sequence into the primer to be located the xTAG sequence at the end in the amplification of the target sequence to be observed, so that the xTAG was inserted into the amplification product during the PCR procedure, and secondarily joining the xTAG with a bead to which the complementary sequence to xTAG attached to form a complementary bond between the two base sequences, detecting the target using the same, and analyzing the target sequence with fluorescence of the bead. In this method, even though the xTAG does not participate in the amplification, if the primer is not completely removed after the amplification, it has problems that there is a possibility that it binds to the complementary xTAG of the bead to recognize the mark, and an error occurs that the complementary sequence of xTAG forms non-specific reaction by PCR and thus non-specific target is detected (<CIT> and <CIT>).

In order to solve this problem, studies has been continuously performed that a tag is constructed during the PCR reaction, the tag does not affect the PCR reaction, the maximum numbers of multiple detections are possible.

<CIT> discloses the detection of a target nucleic acid sequence by a PCEC (PTO Cleavage and Extension-Dependent Cleavage) assay. The method is characterized by generating a cleavage site for a nucleolytic enzyme on the extended duplex of which the formation is dependent on the presence of a target nucleic acid sequence. The method detects the occurrence of the cleavage of the extended duplex, thereby determining the presence of the target nucleic acid sequence.

The present disclosure is derived to solve the above problems and to meet the above needs and the object of the present disclosure is to provide a method for solving the uncertainty which can be occurred when the results are determined depending on the length of the generated product in amplifying and analyzing a target sequence using an amplification reaction such as PCR, and for solving the restriction to the maximum numbers of amplification that can be identified in multiple detection.

The another object of the present disclosure is to provide a method for improving accuracy by solving errors due to non-specific amplification which can be caused by the use of artificial sequence as a tag itself in identifying a target sequence amplification by forming the tag.

In order to accomplish the above object, the present invention provides a primer comprising:.

wherein the restriction enzyme recognition sequence comprises a modified dNTP in the restriction enzyme recognition sequence and the primer comprises one sequence selected from the group consisting of SEQ ID. NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

In one embodiment of the present invention, the restriction enzyme recognition sequence is preferably one selected from the group consisting of Pho I, PspGI, BstNI, TfiI, ApeKI, TspMI, BstBI, BstEII, BstNI, BstUI, BssKI, BstYI, TaqI, MwoI, TseI, Tsp451, Tsp509I, TspRI, Tth111I, Nb. BstNBI restriction enzymes and Nick restriction enzymes, but is not limited thereto.

The modified dNTP is inserted at the cleavage site of the restriction enzyme recognition sequence of the primer, for the purpose of that a cleaved by-product other than the cleaved complementary tag fragment allow not to participate in the reaction, and the modified dNTP to be inserted into the cleavage site is phosphorothioated dNTP, dNTP containing <NUM>-deazapurine, or a <NUM>'-O-methyl nucleotide (<NUM>'-OmeN) in a DNA template, but is not limited thereto.

Furthermore, the present disclosure provides a method for forming a tag to be used in classifying and analyzing the kinds of the target sequences amplified in the Polymerase Chain Reaction, and identifying it, comprising:.

In one embodiment of the present disclosure, it is preferable to analyze the mass of the cleaved complementary tag fragment to identify the cleaved complementary tag fragment in the above method, and the instrument used for the mass spectrometry is preferably a matrix-assisted laser desorption-ionization-time-of-flight mass spectrometer ((MALDI -TOF MS), a Liquid Chromatography Mass Spectrometer, or a High Performance Liquid Chromatography Mass Spectrometer, but is not limited thereto.

In another embodiment of the present disclosure, the mass per unit electric charge (m / z) of the cleaved tag fragment to be used for mass spectrometry is preferably from greater than <NUM> to <NUM> Da or less, but is not limited thereto.

In another embodiment of the present disclosure, in order to preserve the mass of a cleaved complementary tag fragment to be used in mass analysis during the amplification process, it is preferable to use DNA polymerase that the function of adenine-addition elongation effect (A tailing) at the <NUM> 'end, which is an intrinsic property of the nucleic acid polymerase, is inhibited, but is not limited thereto.

In another embodiment of the present disclosure, it is preferable to analyze the fluorescence signal using the oligonucleotide that is tagged by fluorescence and Quencher and has the complementary sequence of the cleaved complementary tag fragment as the identification method of the cleaved complementary tag fragment, but it is not limited thereto.

In another embodiment of the disclosure, it is preferable to analyze the dissociation temperature and melting peak by varying the inherent dissociation temperature at which the double strand of the oligonucleotide and the cleaved complementary tag fragment are dissociated into a single strand, and to identify the presence of the target sequence by identifying the cleaved complementary tag fragment in the method, but is not limited thereto.

In yet another embodiment of the present disclosure, the oligonucleotide is preferably <NUM> or more in length, but is not limited thereto.

In another embodiment of the present disclosure, it is preferable to attach a quencher to the nucleotide at the <NUM> 'end of the oligonucleotide in order to prevent elongation of the base sequence from the oligonucleotide in the method, but is not limited thereto.

In another embodiment of the present disclosure, it is preferable to identify the complementary tag fragment cleaved by analyzing the cycle threshold (Ct) value of the fluorescence signal of the oligonucleotide, but not limited thereto.

In a preferred embodiment of the present disclosure, it is preferable to identify causative organisms of a sexually transmitted disease in the said method, and the sexually transmitted disease causative organism is preferable one selected from the group consisting of Chlamydia trachomatis, Neisseria. Gonorrhea, Mycoplasma hominis, Mycoplasma genitalium, Trichomonas vaginalis, Ureaplasma urealyticum, Ureaplasma parvum, Candida albicans, Gardnerella vaginalis, Herpes simplex virus <NUM>, Herpes simplex virus <NUM>, Treponema pallidum, but is not limited thereto.

The present disclosure also provides a composition for diagnosing sexually-transmitted diseases, comprising the primer of the present invention as an effective component.

In another embodiment of the present disclosure, it is preferable to identify the causative organism of gastrointestinal tract disease, wherein the causative organism of gastrointestinal tract disease is selected from the group consisting of Rotavirus A, Astrovirus, Adenovirus F40, Adenovirus F41, Norovirus GI and Norovirus GII, but is not limited thereto.

The present disclosure also provides a composition for diagnosing a gastrointestinal disease agent comprising the primer of the present invention as an effective component.

In another example of the present disclosure, it is preferable to identify a human papilloma virus in the method, and the subpopulations of the human papilloma virus is preferably selected from the group consisting of types <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, 68a, and <NUM>, but is not limited thereto.

The present disclosure also provides a composition for diagnosing HPV comprising the primer of the present invention as an effective component.

In another preferred example of the present disclosure, it is preferable to identify a causative organism of the respiratory disease in the method, and the causative organism of the respiratory disease is one being selected from the group consisting of Influenza A /H1N1, Influenza A / H3N2, Influenza A / H1N1 / 2009pdm, Influenza B, Parainfluenza <NUM>, Parainfluenza <NUM>, Respiratory syncytial virus A, Respiratory syncytial virus B, Human metapneumovirus, Adenovirus, but is not limited thereto.

The present disclosure also provides a composition for the diagnosis of respiratory diseases, comprising the primer of the present invention as an effective component.

In another example of the present disclosure, the method is preferably a single nucleotide polymorphism (SNP), wherein the single base mutation is preferably one selected from the group consisting of r6265 of the Brain-derived neurotrophic factor gene (BDNF gene), but is not limited thereto.

The present disclosure also provides a composition for analyzing the BDNF gene rs6265 gene comprising the primer of the present invention as an effective component.

Hereinafter, the present invention will be described.

The present inventors have tried our best to develop the method that can perform a multiplex amplification reaction on a large number of targets at one time by clearly distinguishing each amplification product through an easier, faster and more efficient method in preforming the amplification reaction and can analyze the results.

As a result, so as to be able to generate a nucleic acid sequence which can be used as a tag in an amplification reaction, when a sequence serving as a template for a tag was inserted into a primer, and only tag was cleaved by a restriction enzyme, we confirmed that the generated tag can play a role as the tag for detecting the target sequence and also identified that it can identify the amplification efficiently and rapidly than other existing methods in the multiplex amplification reaction analysis by applying it to various analysis methods, and thus, has been completed the present invention.

The present disclosure relates to a method of forming tags to be used for sorting and analyzing kinds of amplified target sequences during a PCR reaction.

In particular, the present disclosure is characterized in comprising the steps of: (<NUM>) hybridizing a target sequence with a primer comprising a template of a tag for generating the tag, (<NUM>) generating the tag from the template of the tag using a restriction enzyme during the PCR reaction, and (<NUM>) analyzing the generated tag with various analysis equipment to identify the tag.

Hereinafter, a method for making the primer of the present invention will be described in detail.

In step (<NUM>), prior to hybridizing the CTPO and the target sequence, the structure of CTPO is divided into a template portion of the CCTF, a restriction enzyme recognition sequence, and a sequence complementary to the target as shown in the following Formula <NUM>.

The A site in the structural formula <NUM> is comprised of a random sequence to be a template of the CCTF, and the complementary sequence of the CCTF template, that is, the CCTF site, is elongated by amplifying it after annealing with the target sequence and then the CCTF site is released by the restriction enzyme during the amplification. The released CCTF is characterized by being a random sequence having <NUM> or more oligonucleotides in length so that it can be specifically analyzed as a tag. Random sequences can be used in any sequence that does not create a by-product during the PCR reaction. The nucleotide sequence to be used as a template for CCTF is free from any sequence that does not cause a hybridization reaction during the amplification reaction.

B is a restriction enzyme recognition sequence, which means a specific recognition sequence of restriction enzymes and Nick restriction enzymes having thermal stability that can be used during amplification. For example, it includes Pho I, PspGI, BstNI, TfiI, ApeKI, TspMI, BstBI, BstEII, BstNI, BstUI, BssKI, BstYI, TaqI, MwoI, TseI, Tsp45I, Tsp509I, TspRI, Tth1111, Nb. BstNBI, etc..

Most preferably, among them, PspGI can be used, and the restriction enzyme used in Example of the present invention is PspGI.

The modified dNTP is inserted into a site cleaved by the restriction enzyme in the restriction enzyme recognition sequence of CTPO so as not to exist and participate the cleaved by-products other than CCTF in the reaction. Examples thereof include phosphorothioated dNTPs, dNTPs containing <NUM>-deazapurine, or <NUM>'-O-methyl nucleotides (<NUM>'-OMeN) in DNA templates, etc. The prior art, <NPL> and <NPL> can be applied to the present disclosure. Most preferably, a phosphothiolated bond is inserted into the cleavage site among the recognition sequence to prevent the cleavage of the template of CCTF by a restriction enzyme, thereby securing a template capable of generating CCTF and to prevent a by-product which can be generated by releasing the template of CCTF into the reaction solution, thereby increasing the efficiency of the reaction. It represents the effects of the invention different from the prior art, SDA (Strand Displacement Amplification) method (<CIT>) in view of that it generates CCTF and prevents the template to inflow to the reaction solution.

The C site shown in the structural formula <NUM> means a part after the restriction enzyme recognition sequence up to the <NUM> 'end, and is composed of a target specific sequence so that it binds specifically to the target during amplification so as to maintain its role as a primer.

In step (<NUM>), when the amplification product is formed by CTPO, and the amplified product present in the double strand is cleaved to CCTF by the restriction enzyme and released into the reaction solution, the appropriate concentration of the restriction enzyme to be used can be varied depending on the purpose of use. In addition, the results are different depending on the type of polymerase to be used, which can be also varied depending on the purpose of use. For example, when CCTF is formed for the purpose of mass spectrometry, it is preferable that the weight of CCTF should be kept constant regardless of the amplification process and should not reflect the intrinsic property of the nucleic acid polymerase. Therefore, a nucleic acid polymerase having no adenine addition extension effect (A tailing) at the <NUM> 'end, which is an intrinsic property of the nucleic acid polymerase, should be selected and used. Among the nucleic acid polymerase enzymes that do not make A tailing, Phusion polymerase, Vent polymerase, Deep Vent polymerase, Bst polymerase, etc. are present.

However, when CCTF analysis method using other techniques than mass analysis is applied, there is no variation in the results due to the A tailing effect, and thus, any polymerase can be used.

In order to increase the efficiency of the restriction enzyme to generate CCTF and to maximize the effect by promoting the influx into the reaction solution, a restriction enzyme reaction time can be further added during the PCR process. Reaction time, reaction temperature, etc. can be applied differently depending on the kind of the specific restriction enzyme and the reaction intention.

In step (<NUM>), as the step that the generated CCTF is analyzed through various analysis equipment to identify the target nucleic acid sequence, when the mass of the generated CCTF is directly analyzed, the kinds of CCTF are diversified through recombination of length and sequence, Mass spectrometry such as MALDI-TOF MS, LC MS and HPLC MS can be used to observe the intrinsic mass of the generated CCTF, and the amplified target sequence can be identified and identified using the said mass. It is preferable to observe it through MALDI-TOF MS, the range of mass of CCTF which is easy to observe is <NUM> Da or more. The amplification products can be observed by forming various CCTFs in the mass range as above.

The amplified target sequence can be identified by observing the fluorescence signal of CCTF, and this is the method which comprises hybridizing CCTF with SCO which is tagged with the fluorescence and the quencher so that the generated CCTF can provide the fluorescence signal at the inherent dissociation temperature, and is the sequence complementary to the CCTF having the inherent dissociation temperature, analyzing the fluorescence signal at the inherent dissociation temperature, and confirming the generation of CCTF, thereby identifying the presence of the target nucleic acid sequence.

For the release of CCTF, as described above, the use concentration of the restriction enzyme is designated according to the purpose of use, and the kind of the polymerase is not related to the A tailing unlike the mass analysis. The CCTF released from the amplification product and introduced into the reaction solution reacts with the SCO present in the reaction solution, wherein the component of the SCO is as follows.

The complementary sequence of CCTF exists to enable hybridization with CCTF from the <NUM> 'end to the <NUM>' end and the sequence of SCO is determined by CCTF length, sequence recombination depending on CCTF. In order to diversify the kinds of tags in step (<NUM>), the combination of the length and the sequence may be designed differently to give the inherent dissociation temperature of CCTF and SCO, such as in the case using the method such the length of CCTF and the method of sequence recombination, etc. In this case, the SCO is composed of a complementary sequence of CCTF, and the fluorescent substance is contained in the sequence, and the position of the fluorescent substance is possible in anywhere at least a certain length apart from the quencher. At the <NUM> 'end of the SCO, a blocker is positioned so that SCO serves as a primer during the reaction to prevent the nucleotide sequence from elongation. Spacer C3, Phosphat, ddC, Inverted END and Quencher, etc. may be used as the blocker, but not limited thereto. In particular, when the quencher is located at the <NUM> 'end of SCO, the SCO is served as a primer during the reaction to prevent the nucleotide sequence from elongation, and simultaneously hybridizes with CCTF to suppress the emission of the fluorescent material by the FRET phenomenon, before it forms a double strand with CCTF. By using a quencher in combination with a substance preventing nucleotide sequence elongation, an unnecessary modification reaction can be shortened in the production of SCO, thereby increasing the yield of the production reaction and further reducing the manufacturing cost. By using the hybridization of CCTF generated during the reaction with SCO contained in the reaction, it can be identified as to whether CCTF is generated by identifying the dissociation of the double strand with the fluorescence and analyzing it to confirm whether CCTF is generated due to the target sequence, and then the target sequence can be identified. The range of temperature that can be defined by the inherent dissociation temperature of the SCO is ~ <NUM>, and if there is no interference of the dissociation temperature of each double strand, there is no limitation in defining the inherent dissociation temperature for each fluorescent substance.

The combination of SCO's fluorophore and quencher can be exemplified as Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, Alexa Fluor <NUM>, ATTO <NUM>, ATTO <NUM>, ATTO <NUM>, ATTO <NUM>, ATTO <NUM>, ATTO <NUM>, ATTO <NUM>, ATTO <NUM>, ATTO Rho6G, ATTO 540Q, ATTO <NUM>, ATTO <NUM>, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO 580Q, ATTO Rho101, ATTO <NUM>, ATTO Rho13, ATTO <NUM>, ATTO <NUM>, ATTO 612Q, ATTO <NUM>, ATTO Rho14, ATTO <NUM>, ATTO <NUM>, ATTO 647N, ATTO <NUM>, ATTO Oxa12, ATTO <NUM>, ATTO <NUM>, ATTO <NUM>, ATTO <NUM>, ATTO <NUM>, ATTO MB2, AMCA, AMCA-S, BODIPY FL, BODIPY R6G, BODIPY <NUM>/<NUM>, BODIPY TMR, BODIPY <NUM>/<NUM>, BODIPY <NUM>/<NUM>, BODIPY <NUM>/<NUM>, BODIPY <NUM>/<NUM>, BODIPY TR, BODIPY <NUM>/<NUM>, BODIPY <NUM>/<NUM>, Biosearch Blue, CAL Fluor Gold <NUM>, CAL Fluor Orange <NUM>, CAL Fluor Red <NUM>, CAL Fluor Red <NUM>, CAL Fluor Red <NUM>, Pulsar <NUM>, Quasar <NUM>, Quasar <NUM>, Quasar <NUM>. FAM, Fluorescein, Fluorescein-C3, Calcein, Carboxyrhodamine <NUM>, Carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Cy2, Cy3, Cy5, Cy3. <NUM>, Cy5. <NUM>, Cy7, Dansyl, Dapoxyl, Dialkylaminocoumarin, <NUM>',<NUM>'- Dichloro-<NUM>',<NUM>'-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin, HEX, Hydroxycoumarin, IRD40, IRD <NUM>, IRD <NUM>, JOE, Lissamine rhodamine B, LC Red <NUM>, LC Red <NUM>, Marina Blue, Methoxycoumarin, Naphthofluorescein, NED, Oregon Green <NUM>, Oregon Green <NUM>, Oregon Green <NUM>, Pacific Blue, PyMPO, Pyrene, Phycoerythrin, Rhodamine <NUM>, Rhodamine Green, Rhodamine Red, Rhodol Green, <NUM>',<NUM>',<NUM>',<NUM>'-Tetra-bromosulfonefluorescein, Tetramethyl-rhodamine (TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X. TET, VIC, Yakima Yellow, BMN-Q460, DDQ-I, Dabcyl, BMN-Q530, BMN-Q535, Eclipse, Iowa Black FQ, BHQ-<NUM>, TQ2, IQ4, QSY-<NUM>, BHQ-<NUM>, TQ3, DDQ-II, BBQ-<NUM>, Iowa Black RQ, QSY-<NUM>, BHQ-<NUM>, etc., and may also include any fluorescent material and quencher.

In addition, the reaction between SCO and CCTF occurs simultaneously with the amplification reaction and the CCTF formation reaction, and in this case, by utilizing the fact that the double strand formation ratio of SCO represents a similar efficiency to the amplification amount of the target sequence, the Ct graph of the SCO having the inherent dissociation temperature can be made, and by using this, it is possible to identify the target sequence in a different manner from the inherent dissociation temperature analysis method.

The above content made by the solving means of the present invention will be described in more detail as the most preferable embodiment through the Examples of the present invention.

In the method of the present disclosure, since an arbitrary tag (CCTF) is generated and cleaved by restriction enzymes during the amplification reaction, the double strand of the restriction enzyme recognition sequence is not formed before the amplification reaction of the artificial sequence (CTPO) added to form the tag and thus, there is no possibility that it is randomly cleaved; since the tags are generated only by the reaction products specifically generated to the target sequence during PCR, the method of the present disclosure has the high accuracy for forming CCTF, and can obtain more delicate analysis results than the existing PCR result analysis depending on the length of the PCR amplification product or the specificity of the specific sequence; and the method of the present disclosure can distinguish and interpret amplification products specifically even if various kinds of amplification products are produced in the same length. In addition, since the analysis of the resultant CCTF can be applied to most of the analysis using base sequence, the device for interpretation can be selected and applied ordinarily. In particular, the method of the present disclosure can be used in the fields of diagnosis, etc., which require rapid multiple analysis using an amplification reaction.

Hereinafter, the present invention will be described in detail with reference to Examples.

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 <NUM>, the causative organism of sexually transmitted diseases, DNAs of Neisseria. gonorrhoeae (NG) and Mycoplasma. Hominis (MH) were used as the targets.

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 <NUM> '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 <NUM> '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 <NUM> '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.

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

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.

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

Primer <NUM> and Primer <NUM> as forward primers, and Primer <NUM> and Primer <NUM> as reverse primers were subjected and PCR reaction was performed simultaneously, and then, the formation of CCTF was determined.

<NUM>µℓ Of the total reaction solution comprising each Primer <NUM>, PspGI (NEB, USA) 2U, PCR buffer (1x), MgSO <NUM> <NUM>, dNTP <NUM>, Vent Polymerase (NEB, USA) <NUM> U and NG, MH template DNA <NUM> pg/ul was subjected to PCR reaction using C1000 PCR (Bio-Rad, USA) under the following conditions:.

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, <NUM>µL of <NUM> triethylammonium acetate (TEAA, pH <NUM>) was added and allowed to stand for <NUM> minute. Resin was activated by passing <NUM> of <NUM>% acetonitrile (ACN; Sigma, USA) and <NUM> TEAA to the column, and then, <NUM>µL of a mixed solution of the solution treated with the restriction enzyme and <NUM> TEAA, <NUM> of <NUM> TEAA and <NUM> of the third distilled water were passed through in this order. The column was placed on a Collection Plate and <NUM>µL of <NUM>% ACN was passed. When the eluate was collected on the collection plate, the collection plate was dried at <NUM> for <NUM> minutes.

<NUM>µL of MALDI matrix [<NUM> ammonium citrate, <NUM> hydroxypicolinic acid, <NUM> acetonitrile, <NUM> H <NUM>O] was previously dotting on Anchor chip plate of MALDI-TOF mass spectrometry (Biflex IV, Bruker), and then, was dried at <NUM> for <NUM> minutes. <NUM>µL of the third distilled water was dissolved in a sample of the collection plate after the purification and desalting procedure, and <NUM>µL of the solution was dropped onto the dried MALDI Matrix, the Maldin Matrix was dried again at <NUM> for <NUM> minutes, and then was analyzed by MALDI-TOF mass spectrometry. The analysis method follows the manual of the MALDI-TOF mass spectrometry.

The result of analyzing the CCTF produced by the above reaction using a mass spectrometer is as shown in <FIG>. From the result of <FIG>, it can be confirmed the peaks of <NUM> Da, the mass of CCTF <NUM> which can be formed when performing PCT with the combination of Primer <NUM> and Primer <NUM>, and <NUM> Da, the mass of CCTF <NUM> which can be formed when performing PCT with the combination of Primer <NUM> and Primer <NUM> (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.

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.

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.

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

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 <NUM>(HSV <NUM>), Herpes simplex virus <NUM>(HSV <NUM>), Treponema pallidum(TP), the causatives agents of sexually transmitted diseases and Internal control (IC) DNA was performed using Real-time PCR instrumentation.

The forward primer used in this example was CTPO and was constructed on the same principle as in Example <NUM> above. The <NUM> 'end of CTPO was composed of <NUM><NUM> 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 <NUM>' 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.

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

The primer of the invention comprises one sequence selected from the group consisting of SEQ ID. NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. The remaining SEQ ID. NOs are reference examples only.

Primer information and target sequence information which is amplified and generated are as follows
<IMG>.

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.

PCR reaction was performed using the following CFX96 Real-time PCR (Bio-Rad, USA) with <NUM>µℓ of total reaction solution of each of Primer <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and SCO <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> prepared by adding <NUM>, PspGI (NEB, USA) 5U, PCR buffer (1x), MgCl<NUM> <NUM>, dNTP <NUM>, h-Taq DNA polymerase (Solgent, Korea) <NUM> U, template DNA of genomic DNA of CT, NG, MH, MG, TV, UU, UP, CA, GV, HSV1, HSV2, TP and IC <NUM> pg/rxn, respectively.

A reaction was performed using a cycle at the denaturation temperature of <NUM> for <NUM> minutes once, and with a cycle at the denaturation temperature of <NUM> for <NUM> seconds, and an annealing temperature of <NUM> for <NUM> minute <NUM> times. After the reaction, the reaction mixture was cooled to <NUM> in the same apparatus, held at <NUM> for <NUM> seconds, and then slowly heated from <NUM> to <NUM> to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager <NUM>.

<FIG>, (a) shows the results of multiple inherent dissociation temperature measurements for causative organisms of CT, NG, 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 <NUM>, NG : HEX <NUM>, MH : HEX <NUM>, MG : CalRed610 <NUM>, TV : Quasar670 <NUM> UU : CalRed610 <NUM>, UP : FAM <NUM>, CA : FAM <NUM>, GV : Quasar670 <NUM>, HSV <NUM> : Quasar705 <NUM>, HSV <NUM> : Quasar705 <NUM>, TP : Quasar705 <NUM>, IC : Quasar670 <NUM>) (a)(b)(c)(d)(e)(f), and no peak of SCO visualizing CCTF was observed when the target sequence was not added in the same composition (g).

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.

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.

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 <NUM>), Adenovirus F41(AdV <NUM>), Norovirus G (NoV G ), Norovirus G (NoV G ), and External control (EC).

The forward primer used in this example was CTPO and was constructed on the same principle as in Example <NUM> above. The <NUM> 'end of CTPO was composed of <NUM><NUM> 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 <NUM>' 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.

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

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

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.

The following PCR reaction was performed using CFX96 Real-time PCR (Bio-Rad, USA) with <NUM>µL of total reaction solution of each of Primer <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and SCO <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> prepared by adding <NUM>, PspGI (NEB, USA) 1U, PCR buffer (1x), MgCl <NUM> <NUM>, dNTP <NUM>, DTT <NUM>, RNase Inhibitor 1U, SuperiorScript III (Enzynomics, Korea) 1U and the nucleic acid of the genomic RNA of RVA, AstV, AdV <NUM>, AdV41, NoV G , NoV G and EC(MS2 phage) 1x10 ^ <NUM> pg/rxn, respectively.

A reverse transcription reaction was performed using a cycle at the denaturation temperature of <NUM> for <NUM> minutes once, and with a cycle at the denaturation temperature of <NUM> for <NUM> minutes <NUM> time, and with a cycle at an annealing temperature of <NUM> for <NUM> minute <NUM> times repeatedly. After the reaction, the reaction mixture was cooled to <NUM> in the same apparatus, held at <NUM> for <NUM> seconds, and then slowly heated from <NUM> to <NUM> to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager <NUM>.

<FIG> shows the results of multiple inherent dissociation temperature measurements for causative organisms of RVA, AstV, AdV <NUM>, AdV <NUM>, NoV G , NoV G. It was identified that the peak was observed at the inherent dissociation temperature that each SCO has : RVA: HEX <NUM>, AstV: CalRed610 <NUM>, AdV <NUM>: CalRed610 <NUM>, AdV <NUM>: CalRed610 <NUM>, NoV G : FAM <NUM>, NoV G : FAM <NUM>, EC: HEX <NUM> (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).

CCTF analysis was performed with Real-time PCR instrument for DNA of subtypes of Human Papillomavirus (HPV), <NUM> type, <NUM> type, <NUM> type, <NUM> type, <NUM> type, <NUM> type, <NUM> type, 68a type, <NUM> type and Internal control (IC).

The forward primer used in this example was CTPO and was constructed on the same principle as in Example <NUM> above. The <NUM> 'end of CTPO was composed of <NUM><NUM> 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 <NUM>' 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.

The following PCR reaction was performed using CFX96 Real-time PCR (Bio-Rad, USA) with <NUM>µL of total reaction solution of each of Primer <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and SCO <NUM>, <NUM>, <NUM>, <NUM> prepared by adding <NUM>, PspGI (NEB, USA) 5U, PCR buffer (1x), MgCl<NUM> <NUM>, dNTP <NUM>, h-Taq DNA polymerase (Solgent, Korea) <NUM> U and HPV type <NUM>, type <NUM>, type <NUM>, type <NUM>, type <NUM>, type <NUM>, type <NUM>, type 68a, type <NUM> and template DNA of genomic DNA of IC <NUM> pg/rxn, respectively.

A reaction was performed using a cycle at the denaturation temperature of <NUM> for <NUM> minutes once, and with a cycle at the denaturation temperature of <NUM> for <NUM> second and at an annealing temperature of <NUM> for <NUM> minute <NUM> times repeatedly. After the reaction, the reaction mixture was cooled to <NUM> in the same apparatus, held at <NUM> for <NUM> seconds, and then slowly heated from <NUM> to <NUM> to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager <NUM>.

<FIG> shows the results of multiple inherent dissociation temperature measurements for each target of type <NUM>, type <NUM>, type <NUM>, type <NUM>, type <NUM>, type <NUM>, type <NUM>, type 68a, type <NUM>, IC. It was identified that the peak was observed at the inherent dissociation temperature that each SCO has (type <NUM>: HEX <NUM>, type <NUM>: FAM <NUM>, type <NUM>: Quasar670 <NUM>, type <NUM>: Quasar670 <NUM>, type <NUM>: Quasar670 <NUM>, type <NUM>: Quasar670 <NUM>, type <NUM>: Quasar670 <NUM>, type <NUM> a: Quasar670 <NUM>, type <NUM>: Quasar670 <NUM>, IC: Quasar670 <NUM>) (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).

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 <NUM>(PIV1), Parainfluenza <NUM>(PIV3), Respiratory syncytial virus A(RSV A), Respiratory syncytial virus B(RSV B), Human metapneumovirus(MPV), Adenovirus(AdV) and External control (EC).

The forward primer used in this example was CTPO and was constructed on the same principle as in Example <NUM> above. The <NUM> 'end of CTPO was composed of <NUM><NUM> 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 <NUM>' 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.

Primer information and target sequence information which is amplified and generated are as follows. The primer of the invention comprises one sequence selected from the group consisting of SEQ ID. NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. The remaining SEQ ID. NOs are reference examples only.

The following PCR reaction was performed using CFX96 Real-time PCR (Bio-Rad, USA) with <NUM>µL of total reaction solution of each of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and SCO <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> prepared by adding <NUM>, PspGI (NEB, USA) 1U, PCR buffer (1x), MgCl<NUM> <NUM>, dNTP <NUM>, DTT <NUM>, RNase Inhibitor 1U, 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 1x10 ^<NUM> copies/rx, respectively.

A reaction was repeatedly performed with a cycle at the reverse transcription reaction temperature of <NUM> for <NUM> minutes once, and with a cycle at the denaturation temperature of <NUM> for <NUM> seconds, and an annealing temperature of <NUM> for <NUM> minute <NUM> times repeatedly. After the reaction, the reaction mixture was cooled to <NUM> in the same apparatus, held at <NUM> for <NUM> seconds, and then slowly heated from <NUM> to <NUM> to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager <NUM>.

<FIG> 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: <NUM>, Flu A/H3N2: <NUM>, Flu A/H1N1/2009pdm: <NUM>, Flu B: <NUM>, PIV1: <NUM>, PIV3: <NUM>, RSV A: <NUM>, RSV B: <NUM>, hMPV: <NUM>, ADV: <NUM>) (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 (f).

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

The forward primer used in this example was CTPO and was constructed on the same principle as in Example <NUM> above. The <NUM> 'end of CTPO was composed of <NUM><NUM> 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 <NUM>' 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.

Primer information and target sequence information which is amplified and generated are as follows. <IMG>
<IMG>.

PCR reaction was performed using the following CFX96 Real-time PCR (Bio-Rad, USA) with <NUM>µL of total reaction solution of each of Primer <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and SCO <NUM>, <NUM>, <NUM> prepared by adding <NUM>, PspGI (NEB, USA) 5U, PCR buffer (1x), MgCl <NUM> <NUM>, dNTP <NUM>, h-Taq DNA polymerase (Solgent, Korea) <NUM> 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 1x10 ^<NUM> copies/rxn, respectively.

A reaction was performed using a cycle at the denaturation temperature of <NUM> for <NUM> minutes once, and with a cycle at the denaturation temperature of <NUM> for <NUM> seconds, and an annealing temperature of <NUM> for <NUM> minute <NUM> times repeatedly. After the reaction, the reaction mixture was cooled to <NUM> in the same apparatus, held at <NUM> for <NUM> seconds, and then slowly heated from <NUM> to <NUM> to obtain an inherent dissociation temperature analysis curve. Data analysis was performed with Bio-Rad CFX Manager <NUM>.

<FIG>, (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: <NUM>, A/G: <NUM> <NUM>, G/G <NUM>, IC: <NUM>) (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).

It has been proved in Example <NUM> 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.

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.

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 <NUM> 'end of the forward primer was composed of a <NUM>-mer or <NUM>-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 <NUM>' 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.

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-<NUM> or BHQ-<NUM>) at <NUM>' end, with reporter molecular (each FAM, HEX, CAL Fluor Red <NUM>) positioned at a certain distance, and its sequence was complementary to CCTF sequence to be analyzed.

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

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 <NUM>. The sequence of the CCTF produced from the amplified product is as follows.

PCR reaction was performed using the following CFX96 Real-time PCR (Bio-Rad, USA) with <NUM>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 <NUM>, respectively, and adding PspGI (NEB, USA) <NUM> U, PCR buffer (1x), MgCl<NUM> <NUM>, dNTP <NUM>, h-Taq DNA polymerase (Solgent, Korea) <NUM> U, and contained the template DNA diluted by <NUM>-folds with <NUM> pg/µL genomic DNA proven by the conventional quantitation method for each causative organism.

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

As shown in (a) of <FIG>, 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 NG (solid line), MG (dotted line), and UP (circle), dilutions for genomic DNA of each causative organism diluted by <NUM>-folds starting from the concentration of <NUM> 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 NG 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.

Regression coefficient (r <NUM>) in the linear regression analysis of the standard curve was represented by NG <NUM>, MG <NUM>, UP <NUM>, respectively. The slope of the regression plot was NG -<NUM>, MG -<NUM>, and UP -<NUM>, respectively. It could be identified that the respective amplification efficiency (E = <NUM> [-<NUM>/ slope] -<NUM>) was <NUM>% for NG, <NUM>% for MG and <NUM>% for UP, respectively, and thus, they were listed in the proper range of between <NUM> and <NUM>%.

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.

Claim 1:
A primer comprising from <NUM>' to <NUM>':
a random nucleic acid non-complementary to a target sequence, having <NUM> to <NUM> nucleotides in length, wherein the random sequence is located at the <NUM>' end of the primer;
a restriction enzyme recognition sequence; and
a nucleic acid sequence complementary to the target sequence,
wherein the restriction enzyme recognition sequence comprises a modified dNTP in the restriction enzyme recognition sequence and the primer comprises one sequence selected from the group consisting of SEQ ID. NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.