KR20180033911A - Method for multiplex detection of target nucleic acid or genetic variation using multiple probes - Google Patents

Abstract

The present invention relates to a multi-detection method for a target nucleic acid or genetic variation by using a multi-probe. More specifically, the present invention relates to a multi-detection method for a target nucleic acid or genetic variation, comprising a step of mixing a nucleic acid, a primer set having a base sequence complementary to a nucleic acid, and an excision reagent, and through a lengthening reaction, amplifying a nucleic acid-probe complex, and measuring an amount of isolated fragments, wherein the nucleic acid has two or more nucleic acid probes obtained from a sample, the nucleic acid probes having a structure of R_1-X-R_2; having two identical or different detectable markers attached at both terminals of the probe or inside thereof; and showing a complementary binding to a target nucleic acid or genetic variation to be detected. According to the present invention, the multi-detection method for a target nucleic acid is an instrument capable of detecting N fluorescent materials by using at least five pairs of nucleic acid probes prepared using one common primer set and at least two fluorescent materials, and can simultaneously detect (2^N-1+1) different target nucleic acids. According to the present invention, the multi-detection method for a genetic variation according to the present invention uses a nucleic acid probe with fluorescent materials different from each other attached thereto, and therefore can confirm the region of a genetic variation or the genotype of a genetic variation without a separate analysis of a base sequence, and thus may be beneficially used in making diagnosis and prognosis of various diseases.

Description

[0001] The present invention relates to a method for multiplex detection of a target nucleic acid or a genetic variation,

The present invention, more particularly R ₁ base sequence complementary to the base sequence of having the structure of the -XR _2, the target nucleic acid to be detected, or genetic variation relates to a method for detecting multiple target nucleic acid or the genetic variation And more particularly to a method for multiplex detection of a target nucleic acid or a genetic variation using two or more nucleic acid probes with the same or at least two different fluorescent substances attached thereto.

The diagnostic field of particular nucleic acids is being used to distinguish single nucleotide polymorphism (SNP), to detect and identify pathogenic bacteria or viruses, and to diagnose genetic diseases. Thus, many methods have been proposed for the rapid and accurate detection of a specific nucleic acid, and many studies related thereto have been carried out (W. Shen et al. , 2013, Biosen and Bioele., 42: 165-172; ML Ermini et al. , 2014, Biosen and Bioele., 61: 28-37, K. Chang et al. , 2015, Biosen and Bioele., 66: 297-307.).

Specifically, the most commonly used methods for detecting a specific nucleic acid include a polymerase chain reaction (PCR) method and a multiplex polymerase chain reaction (Multiplex PCR) method.

The polymerase chain reaction (PCR) can bind to the template DNA, and a primer or a probe having a fluorescent substance and a small molecule bonded thereto can be arbitrarily designed, so that only a desired site of a gene to be detected can be amplified accurately. However, when one nucleic acid can be amplified in a single reaction and the number of nucleic acids to be amplified is large, it is troublesome to repeatedly perform the same operation.

Multiplex PCR is an advantage of simultaneous analysis of a large number of nucleic acids by performing several polymerase chain reaction in a tube. However, when a large number of primers or probes are used in a single tube, a cross reaction occurs between the probe and the primer or the primer. Therefore, there is a limit to the number of nucleic acids that can be amplified at one time. (Hardenbol et al. , 2003, Nat. Biotechnol., 21: 673). However, there is a disadvantage in that it does not require good results in sensitivity and specificity.

In addition, since only one fluorescent substance can be labeled for one nucleic acid to be detected and the number of fluorescent channels that can be simultaneously analyzed at one time is usually limited to 4 to 7 types of equipment used for detecting a fluorescent substance at present, In order to analyze the above nucleic acid, there is a problem that the same operation of two or more times must be repeated.

Therefore, in recent years, researches have been actively conducted to enable mass analysis by simultaneously amplifying a plurality of nucleic acids using a common primer without using a multiple polymerase chain reaction. Representative techniques include SNPlex, Goldengate assay, and molecular inversion probes (MIPs).

The SNPlex performs purification using an exonuclease after the oligonucleotide ligation assay (OLA), amplifies the PCR using a common primer sequence at both ends of the probe, (Tobler et al. , J. Biomol. Tech., 16: 398, 2005) by using the ZipCode nucleotide sequence contained in the probe.

The Goldengate assay performs an allele-specific primer extension reaction with an upstream probe in a genomic DNA immobilized on a solid surface and then performs a DNA-binding reaction with a downstream probe The probe is removed by a washing procedure, and then the amplified polymerase chain reaction product is amplified by a common primer sequence included in a probe such as SNPlex, (Illumina BeadChip) (Shen et al. , Mutat. Res., 573: 70, 2005).

Molecular inversion probes (MIPs) can be prepared by Gap-ligation using a padlock probe, and then probes and genomic DNA that have not been DNA-ligated can be isolated using exonuclease After the padlock probe was linearized with uracil-N-glycosylase, the polymerase chain reaction was performed using the common primer base sequence included in the probe, and the GenFlex Tag Array (Hardenbol et al. , Nat. Biotechnol., 21: 673, 2003).

However, these methods require contamination between different samples due to the necessity of carrying out reaction by transferring part of the product reacted in the first tube to the second tube or using various kinds of enzymes, There is a problem.

Accordingly, the present inventors have made efforts to develop a method for rapidly and accurately detecting a nucleic acid or a genetic variation more than the number of fluorescent substances while overcoming the disadvantages as described above, and have a structure of R ₁ -XR ₂ , A nucleic acid probe comprising a base sequence capable of complementary binding to a base sequence of a genetic mutation is developed, and two or more nucleic acid probes having the same or at least two or more different fluorescent substances attached to the nucleic acid probe are hybridized with a biological sample And then the inside of the probe is cut with a cleavage reagent to measure the amount of the separated probe fragments, thereby confirming that the target nucleic acid or the genetic variation can be detected more rapidly than the number of the used fluorescent material, and the present invention has been completed .

It is an object of the present invention to provide a method for detecting multiple target nucleic acids more than the number of fluorescent materials used by using multiple probes.

It is another object of the present invention to provide a method for detecting a genetic mutation site present on a chromosome using multiple probes.

In one embodiment, the present invention provides a method for detecting a nucleic acid comprising: a) obtaining a sample comprising a target nucleic acid; b) preparing a nucleic acid probe having a structure of R ₁ -XR ₂ and consisting of a base sequence capable of complementary binding with a base sequence of a target nucleic acid to be detected; c) mixing the sample obtained in step a), two or more nucleic acid probes prepared in step b), a primer set having a base sequence complementary to the sample obtained in step a), and a cleavage reagent Amplifying the target nucleic acid-probe complex through the reaction; And d) measuring the amount of the probe fragment isolated from the target nucleic acid-probe complex amplified in the step c).

The method of multiplex detection of the target nucleic acid according to the present invention will be described in detail in each step as follows.

a) obtaining a sample containing the target nucleic acid.

In the present invention, the target nucleic acid refers to RNA or DNA to be detected from a sample, or cDNA obtained by amplifying the RNA with a reverse transcription polymerase.

In the present invention, the sample may be a biological sample, DNA, RNA, or a fragment thereof separated from the biological sample. Specifically, the sample may be at least one selected from the group consisting of blood, saliva, urine, feces, tissues, cells, and biopsy specimens, or DMA, RNA, or fragments thereof separated from the stored biological sample, But is not limited thereto.

The stored biological sample may be stored for a period of one year or more, for example, one to ten years, by a conventional method known in the art.

The DNA or RNA may be DNA or RNA derived from tissues frozen or stored in formalin at room temperature. Separation of DNA, RNA, or fragments thereof from the biological sample can be accomplished using methods commonly known in the art.

b) preparing a nucleic acid probe.

Specifically, the nucleic acid probe of the present invention has a structure of R ₁ -XR ₂ and is synthesized by synthesizing a base sequence which is capable of complementary binding with all or a part of base sequences of a target nucleic acid to be detected.

Wherein R ₁ and R ₂ are DNA or RNA, preferably DNA, consisting of 1 to 20, preferably 1 to 15 nucleotide sequences. Wherein X is an RNA or DNA, preferably RNA, which is composed of 1 to 10, preferably 1 to 6, more preferably 4 bases and can be cleaved by a cleavage reagent. Further, R ₁ , R ₂ and X may be synthesized so as to be totally methylated or partially methylated to prevent non-specific cleavage.

It is preferable that the nucleic acid probe of the present invention has the same or at least two different detectable markers on both ends or inside of the probe. The detectable marker may be a fluorescent substance capable of covalent or non-covalent binding to the probe, or a fluorescent pair (fluorescent substance and small molecule substance).

The fluorescent materials include Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Rhodamine, TAMRA, FAM, VIC, FITC, , Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor Orange 546, Calfluor red 610, Quasar 670 and Biotin 1, BHQ-2, BHQ-3, lowa Black RQ-Sp, QSY-7, QSY-2, and the like, and the minerals may be selected from the group consisting of DDQ-1, Dabcyl, Eclipase, 6-TAMRA, MGBNFQ. ≪ / RTI > However, the present invention is not limited thereto.

When a fluorescent pair is used as a detectable marker in the present invention, the fluorescent substance and the minerals are added to the ends or internals of X of the nucleic acid probe at 1 to 20, preferably 1 to 7, more preferably 2 to 6 More preferably at a position spaced apart by a distance of four base pairs.

In the present invention, more target nucleic acids can be detected than the number of fluorescent substances used through the combination of nucleic acid probes.

As one specific example, five target nucleic acids can be detected through the combination of two kinds of nucleic acid probes having different base sequences using two fluorescent substances (FAM and HEX).

Target nucleic acid The first probe The second probe One FAM - 2 HEX - 3 FAM FAM 4 HEX HEX 5 FAM HEX

As another specific example, 19 different target nucleic acids can be detected through the combination of three kinds of nucleic acid probes having different base sequences using three fluorescent materials (FAM, HEX or VIC).

Target nucleic acid The first probe The second probe The third probe One FAM - - 2 HEX - - 3 VIC - - 4 FAM FAM - 5 HEX HEX - 6 VIC VIC - 7 FAM VIC - 8 FAM HEX - 9 VIC HEX - 10 FAM FAM FAM 11 HEX HEX HEX 12 VIC VIC VIC 13 FAM VIC VIC 14 FAM HEX HEX 15 FAM FAM VIC 16 FAM FAM HEX 17 VIC HEX HEX 18 VIC VIC HEX 19 FAM VIC HEX

In another embodiment, 69 pairs of target nucleic acids are detected through the combination of four kinds of nucleic acid probes having different base sequences using four (FAM, HEX, VIC, or TET) fluorescent materials .

Target nucleic acid The first probe The second probe The third probe The fourth probe One FAM - - - 2 HEX - - - 3 VIC - - - 4 TET - - - 5 FAM FMA - - 6 HEX HEX - - 7 VIC VIC - - 8 TET TET - - 9 FAM HEX - - 10 FAM VIC - - 11 FAM TET - - 12 HEX VIC - - 13 HEX TET - - 14 VIC TET - - 15 FAM FMA FMA - 16 HEX HEX HEX - 17 VIC VIC VIC - 18 TET TET TET - 19 FAM FMA HEX - 20 FAM FMA VIC - 21 FAM FMA TET - 22 HEX HEX FAM - 23 HEX HEX VIC - 24 HEX HEX TET - 25 TET TET FAM - 26 TET TET HEX - 27 TET TET VIC - 28 VIC VIC FMA - 29 VIC VIC HEX - 30 VIC VIC TET - 31 FAM HEX TET - 32 FAM HEX VIC - 33 FAM VIC TET - 34 HEX VIC TET - 35 FAM FMA FMA FMA 36 HEX HEX HEX HEX 37 VIC VIC VIC VIC 38 TET TET TET TET 39 FAM FMA FMA HEX 40 FAM FMA FMA VIC 41 FAM FMA FMA TET 42 HEX HEX HEX FAM 43 HEX HEX HEX VIC 44 HEX HEX HEX TET 45 VIC VIC VIC FAM 46 VIC VIC VIC HEX 47 VIC VIC VIC TET 48 TET TET TET FAM 49 TET TET TET HEX 50 TET TET TET VIC 51 FAM FMA HEX HEX 52 FAM FMA HEX VIC 53 FAM FMA HEX TET 54 FAM FMA VIC VIC 55 FAM FMA TET TET 56 HEX HEX VIC VIC 57 HEX HEX VIC TET 58 HEX HEX VIC FAM 59 HEX HEX TET TET 60 HEX HEX FAM TET 61 VIC VIC TET TET 62 VIC VIC TET FAM 63 VIC VIC TET HEX 64 VIC VIC FAM HEX 65 VIC VIC HEX FAM 66 TET TET FAM VIC 67 TET TET FAM HEX 68 TET TET HEX VIC 69 FAM HEX VIC TET

That is, the nucleic acid probe prepared according to the present invention can be hybridized with the target nucleic acid without a separate reaction process, and even when the sequence or complementary sequence used to amplify the sample containing the target nucleic acid is overlapped with the probe, Hybridization with, and cleavage is possible. In addition, more than five pairs of nucleic acid probes prepared using at least two fluorescent materials can be used to analyze more target nucleic acids than the number of fluorescent materials used. This simplifies the design of the probe and is only cleaved by the cleavage reagent. Therefore, the amount of the nucleic acid to be detected can be more accurately measured than that of the probe degraded by the conventional DNA polymerase, and more target nucleic acids And the analysis cost can be reduced.

c) amplifying the complex with the target nucleic acid-probe.

Specifically, the step c) includes the steps of: preparing a sample containing the target nucleic acid obtained in step a), two or more nucleic acid probes prepared in step b), and a primer set having a base sequence complementary to a sample containing the target nucleic acid , And cleavage reagent, followed by amplification of the target nucleic acid-probe complex through an elongation reaction.

In the present invention, the primer set is complementary to the sequence of the target nucleic acid, and is preferably composed of a forward primer having 15 to 30 bases and a reverse primer.

The sequence of the target nucleic acid complementary to the forward primer in the present invention indicates the distance between the target nucleic acid sequence complementary to the nucleic acid probe of the present invention and the target nucleic acid sequence overlapping with 1 to 18 bases or complementary to the nucleic acid probe of the present invention .

In the present invention, the amplification of the target nucleic acid-probe complex is preferably performed at an isothermal temperature at which the nucleic acid probe of the present invention and the target nucleic acid can be annealed and the activity of the enzyme used is not substantially inhibited. Here, the isothermal temperature means that there is no thermal cycling and does not necessarily mean a physically identical temperature.

As one specific example, the isothermal temperature at which the amplification reaction is carried out in the present invention may be 30 to 75 캜, preferably 37 to 70 캜, more preferably 50 to 65 캜.

In the present invention, the amplification reaction of the target nucleic acid-probe complex may be carried out by polymerase chain reaction, rolling circle amplification, strand displacement amplification and nucleic acid sequence-based amplification acid sequence based amplification).

In the present invention, the amplification reaction of the target nucleic acid-probe complex may be performed by using a reagent which requires amplification, such as a suitable amount of DNA polymerase (for example, Thermus aquatiucs (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis or Phyrococcus may include furiosis thermostable DNA polymerase), DNA polymerase joinja (Mg ^{2 +),} buffer, dNTPs (dATP, dCTP, dGTP and dTTP) and water (dH ₂ O) was obtained from (Pfu). The buffer solution includes, but is not limited to, Triton X-100, dimethylsufoxide (DMSO), Tween 20, nonidet P40, PEG 6000, formamide and bovine serum albumin (BSA) And may be further used.

In the present invention, the cleavage reagent is an enzyme-mediated material that specifically cleaves only the X site of the nucleic acid probe in the amplified target nucleic acid-nucleic acid probe complex. The cleavage reagent can be used as long as it can specifically cleave only the X site of the nucleic acid probe. For example, one selected from the group consisting of deoxyribonuclease (DNase), ribonuclease (RNase), helicase, exonuclease, and endonuclease Ribonucleases (RNase) can be used.

d) measuring the amount of the cleaved nucleic acid probe fragment using the cleavage reagent.

In the present invention, measurement of the amount of nucleic acid probe fragments can be performed using various detection methods. Specifically, the fragments of the probe separated according to the present invention are preferably measured in real time or after the completion of the reaction, and can be obtained by measuring changes in fluorescence intensity or chemiluminescence.

The measurement of the fluorescence intensity or the chemiluminescence may be performed using any measuring device capable of detecting fluorescent markers known in the art, for example, a TRIAD Multimode Detector, a Wallac / Victor fluorescence) or a Perkin-Elmer LB50B luminescence spectrometer, but the present invention is not limited thereto.

The measurement and detection of the amount of the separated probe may vary depending on the type of the marker or the detection marker introduced into the probe or the reaction solution.

Specifically, the fluorescence value detected is proportional to the number of probe fragments separated from the target nucleic acid-probe complex amplified according to the present invention or the number of fluorescent substances attached to the probe, and the same kind of probe or fluorescent substance has the same level Lt; / RTI >

As a specific example, two nucleic acid probes each showing a complementary bond to a different target nucleic acid and having a FAM attached thereto were mixed with a sample containing the target nucleic acid. As a result, one nucleic acid probe with FAM attached thereto was used At the same time, it was confirmed that the amplification curve was about 2 times as much as that of the product obtained by amplification. On the other hand, when two nucleic acid probes showing complementary binding to the same target nucleic acid and two FAM and HEX-attached nucleic acid probes were mixed with a sample containing the target nucleic acid, it was confirmed that a double amplification curve was obtained.

As another specific example, when a nucleic acid probe prepared using three different fluorescent materials is used, 10 different target nucleic acids are detected according to a curve measured according to the type of fluorescent substance attached to each nucleic acid probe .

Therefore, the method of multiplex detection of a target nucleic acid according to the present invention is a method for hybridizing a target nucleic acid with a target nucleic acid without crossing reaction between the probe and the primer even if there is no difference in the gap between the base or the complementary sequence and the base, And a cleavable nucleic acid probe are used, it is possible to amplify the target nucleic acid to be detected accurately. In addition, a device capable of detecting N fluorescent materials using one common primer set and five or more pairs of nucleic acid probes prepared using at least two fluorescent substances, has (2 ^N -1 + N) different targets Nucleic acid can be detected at the same time.

In another aspect, the present invention provides a method for multiple detection of a genetic variation present on a chromosome using two or more nucleic acid probes each labeled with at least two different fluorescent substances.

In particular, a method for multiple detection of a genetic variation of the present invention is characterized by comprising the steps of:

a) obtaining a sample to be tested for the presence or absence of a nucleic acid comprising a genetic variation; b) preparing a nucleic acid probe having a structure of R ₁ -XR ₂ and consisting of a base sequence capable of binding complementary to a base sequence of a nucleic acid including a genetic mutation to be detected; c) mixing the sample obtained in step a), two or more nucleic acid probes prepared in step b), a primer set having a base sequence complementary to the sample obtained in step a), and a cleavage reagent Amplifying a nucleic acid-probe complex comprising a genetic mutation through a reaction; And d) measuring the amount of the probe fragment separated from the nucleic acid-probe complex comprising the genetic mutation amplified in step c).

A method for detecting multiple genetic mutations according to the present invention will be described in detail with reference to the following steps.

a) obtaining a sample to be tested for the presence of a nucleic acid comprising a genetic variation;

In the present invention, the genetic variation refers to a phenomenon caused by an allele, a single nucleotide polymorphism (SNP), a mutation, or a combination thereof. At this time, the allele refers to a gene having a different locus in the same locus on one chromosome or a gene having a different base sequence located in the same gene position on a homologous chromosome. The mutation can be a point mutation, a transition mutation, a transversion mutation, a missense mutation, nonsense mutation, duplication, deletion, insertion, translocation, inversion inversion), and combinations thereof. The single base polymorphism refers to mutation of one or several dozens of bases in a human chromosome.

In the present invention, the sample to be tested for the presence or absence of the nucleic acid including the genetic mutation may be a biological sample, DNA isolated from the biological sample, cDNA obtained by amplifying RNA, reverse transcription polymerase, or fragments thereof , Preferably cDNA obtained by amplifying DNA, RNA, RNA with a reverse transcription polymerase, or a fragment thereof.

The biological sample may be any one or more selected from the group consisting of various types of samples obtained from an individual, specifically, blood, saliva, urine, feces, tissue, cells, and biopsy specimens.

The DNA or RNA may be DNA or RNA derived from tissues frozen or stored in formalin at room temperature. Separation of the cDNA obtained by amplifying DNA, RNA, RNA from the biological sample with a reverse transcription polymerase, or a fragment thereof can be performed using a method commonly known in the art.

b) preparing a nucleic acid probe.

Specifically, the step b) is a step of preparing a nucleic acid probe comprising a nucleotide sequence capable of binding complementary to all or a part of the nucleotide sequence including the genetic mutation site after determining a genetic mutation site.

In the present invention, the determination of the genetic mutation site includes a SNP database provided from a bioinformation database for genetic variation, for example, a national center for biotechnology information (NCBI), a wiki- A wiki-style database, an OMIM database, and the like.

In the present invention, the nucleic acid probe has a structure of R ₁ -XR ₂ and is prepared by synthesizing a nucleotide sequence comprising a nucleotide sequence complementary to all or a part of the nucleotide sequence including the determined genetic mutation site .

In the present invention, R ₁ and R ₂ of the nucleic acid probe are DNA or RNA, preferably DNA, consisting of 1 to 20, preferably 1 to 15 nucleotide sequences. X is composed of 1 to 10, preferably 1 to 6, more preferably 4 nucleotide sequences capable of complementary binding with a base sequence comprising at least one genetic mutation site, Lt; / RTI > or RNA, preferably RNA. Further, R ₁ , R ₂ and X may be synthesized so as to be totally methylated or partially methylated to prevent non-specific cleavage.

It is preferable that the nucleic acid probe of the present invention has a form in which at least two detectable markers are attached to both ends or inside of the probe. The detectable marker may be a fluorescent substance capable of covalent or non-covalent binding to the probe, or a fluorescent pair (fluorescent substance and small molecule substance).

The fluorescent materials include Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Rhodamine, TAMRA, FAM, VIC, FITC, , Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor Orange 546, Calfluor red 610, Quasar 670 and Biotin 1, BHQ-2, BHQ-3, lowa Black RQ-Sp, QSY-7, QSY-2, and the like, and the minerals may be selected from the group consisting of DDQ-1, Dabcyl, Eclipase, 6-TAMRA, MGBNFQ. ≪ / RTI > However, the present invention is not limited thereto.

When a fluorescent pair is used as a detectable marker in the present invention, the fluorescent substance and the minerals are added to the ends or internals of X of the nucleic acid probe at 1 to 20, preferably 1 to 7, more preferably 2 to 6 More preferably at a position spaced apart by a distance of four base pairs.

In the present invention, the nucleic acid probe is produced using at least two or more different fluorescent materials.

As one specific example, when three fluorescent substances (FAM, VIC, or HEX) are used to detect six genetic mutations present in a nucleic acid: i) a complementary sequence with a normal base at a different genetic mutation site Or a nucleic acid probe having a different fluorescent substance attached thereto, or (ii) a nucleic acid probe having complementary binding with a normal base at the same genetic mutation site but having a different fluorescent substance attached thereto.

As another concrete example, a genotype in which the genetic mutation site "G" is mutated in the nucleotide sequence of "... CTGT ..." existing on the chromosome is detected using three fluorescent substances (FAM, VIC, or HEX) A nucleic acid probe comprising a base sequence including a base to which different fluorescent substances are attached and G can be mutated can be prepared and used.

As described above, the nucleic acid probe prepared according to the present invention can be hybridized with a nucleic acid containing a genetic mutation without a separate reaction process, and the sequence of a primer used for amplifying a nucleic acid containing a genetic mutation or complement It is possible to hybridize and cleave the probe and the nucleic acid. This facilitates the design of the probe, and since it is cleaved only by the cleavage reagent, the amount of the nucleic acid to be detected can be measured more accurately than the probe that is degraded by the conventional DNA polymerase. In addition, since a nucleic acid probe having a different fluorescent material is used, it is possible to identify a genetic mutation site or a genotype of a genetic mutation without analyzing the base sequence, thereby reducing the analysis cost.

c) amplifying the complex with a nucleic acid-probe containing a genetic mutation.

Specifically, the step c) comprises: a step of obtaining a sample obtained in step a), two or more nucleic acid probes prepared in step b), a primer set having a base sequence complementary to the sample obtained in step a) Amplifying a nucleic acid-probe complex containing a genetic mutation through a renal reaction after mixing the reagents.

In the present invention, it is preferable that the primer set is complementary to a sequence of a nucleic acid including a genetic mutation, and is composed of a forward primer having 15 to 30 bases and a reverse primer.

In the present invention, the sequence of the nucleic acid comprising the genetic mutation complementary to the forward primer may be a nucleic acid sequence having a complementary binding to the nucleic acid probe of the present invention and a nucleic acid sequence complementary to the nucleic acid probe of the present invention And does not show the distance between the nucleic acid sequence and the sequence.

In the present invention, amplification of a nucleic acid-probe complex containing a genetic mutation can be achieved by amplifying the nucleic acid comprising the nucleic acid probe of the present invention and the genetic mutation at an isothermal temperature that does not substantially inhibit the activity of the enzyme used . Here, the isothermal temperature means that there is no thermal cycling and does not necessarily mean a physically identical temperature.

As one specific example, the isothermal temperature at which the amplification reaction is carried out in the present invention may be 30 to 75 캜, preferably 37 to 70 캜, more preferably 50 to 65 캜.

In the present invention, the amplification reaction of the nucleic acid-probe complex containing the genetic mutation can be carried out by polymerase chain reaction, rolling circle amplification, strand displacement amplification, And a nucleic acid sequence based amplification.

In the present invention, the amplification reaction of the target nucleic acid-probe complex may be performed by using a reagent which requires amplification, such as a suitable amount of DNA polymerase (for example, Thermus aquatiucs (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis or Phyrococcus may include furiosis thermostable DNA polymerase), DNA polymerase joinja (Mg ^{2 +),} buffer, dNTPs (dATP, dCTP, dGTP and dTTP) and water (dH ₂ O) was obtained from (Pfu). The buffer solution includes, but is not limited to, Triton X-100, dimethylsufoxide (DMSO), Tween 20, nonidet P40, PEG 6000, formamide and bovine serum albumin (BSA) And may be further used.

In the present invention, the cleavage reagent refers to a substance that specifically cleaves only the X site of a nucleic acid probe in a nucleic acid-probe complex containing the amplified genetic mutation mediated by an enzyme. The cleavage reagent can be used as long as it can specifically cleave only the X site of the nucleic acid probe. For example, one selected from the group consisting of deoxyribonuclease (DNase), ribonuclease (RNase), helicase, exonuclease, and endonuclease Ribonucleases (RNase) can be used.

d) measuring the amount of the cleaved nucleic acid probe fragment using the cleavage reagent.

In the present invention, measurement of the amount of nucleic acid probe fragments can be performed using various detection methods. Specifically, the fragments of the probe separated according to the present invention are preferably measured in real time or after the completion of the reaction, and can be obtained by measuring changes in fluorescence intensity or chemiluminescence.

The change in fluorescence intensity or the method of measuring chemiluminescence, the apparatus, the measurement of the amount of the separated probe, and the detection method are specifically described in the step d) of the method for multiplex detection of the target nucleic acid.

The measurement and detection of the amount of the separated probe may vary depending on the type of the marker or the detection marker introduced into the probe or the reaction solution.

Specifically, the fluorescence value detected is proportional to the number of probe fragments separated from the nucleic acid-probe complex containing the amplified genetic mutation according to the present invention or the number of fluorescent substances attached to the probe, The fluorescent substance shows the same level of fluorescence amplification curve.

As a specific example, the amplification curve of 9 nucleic acid probes prepared in Table 5 and the samples to be tested for the presence of nucleic acid having a genetic mutation were amplified only in the fluorescence channel of HEX and the third genetic It was found that the mutation occurred at the mutation site.

Further, in order to detect a genotype in which the "G" mutation of the genetic mutation site in the nucleotide sequence of "... CTGT ..." existing on the chromosome was detected, the four nucleic acid probes prepared in the following Table 7 and the nucleic acid having the genetic mutation As a result of amplification by mixing the sample to be tested for presence, an amplification curve appeared only in the fluorescence channel of FAM, and it was found that the wild type base "G" was changed to "T".

Therefore, the method for detecting a genetic mutation according to the present invention is a method for detecting a genetic mutation, in which the sequence of the primer used for amplifying a nucleic acid containing a genetic mutation or the complementary sequence and the nucleotide sequence A nucleic acid probe capable of hybridization and cleavage with a target nucleic acid is used, so that it is possible to amplify the genetic mutation site to be detected accurately. In addition, amplification using one common primer set and a nucleic acid probe having a different fluorescent substance can advantageously detect the genotype of genetic mutation site or genetic variation without an additional base sequence analysis.

As described above, the multiplex detection method of a target nucleic acid according to the present invention can accurately amplify a target nucleic acid to be detected and use five or more pairs of nucleic acid probes prepared by using one common primer set and at least two or more fluorescent substances ( ^2N- 1 + N) different target nucleic acids can be simultaneously detected by using a device capable of detecting N fluorescent materials.

In addition, the method of multiplex detection of a genetic variation according to the present invention can accurately amplify a nucleic acid containing a genetic mutation to be detected. Since nucleic acid probes with different fluorescent materials are used, It is possible to identify the genetic variation site or the genotype of the genetic variation, thereby reducing the analysis cost.

Therefore, the multiplex detection method of a target nucleic acid or a genetic variation of the present invention can be usefully used for diagnosing various diseases and for diagnosing a prognosis.

Hereinafter, the present invention will be described in more detail by way of examples and the like. It will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples and the like according to the gist of the present invention. will be.

Manufacturing example  1: for detection of target nucleic acid Probe  And primer  making

1-1. Probe making

After extracting RNA from mouse blood, the elongated cDNA was prepared by a reverse transcription polymerase chain reaction. Then, the cDN was transformed into a microbacterium strain, which is a respiratory bacterium, using a perseptive ciosystem expedite nucleic acid synthesis system as an A template Microbacterium lacticum ) and the genomic DNA of Microbacterium oxydans ) were synthesized. The nucleotide probes were designed to be complementary to specific nucleic acids showing genetic differences between the genomic DNAs of P. oxydans .

At this time, the bases 1 to 9 and the bases 14 to 15 were deoxyribonucleotides (DNA), and the four bases between 10 and 13 were synthesized to be ribonucleotides (RNAs).

During the synthesis of the nucleic acid probe, a fluorescent probe, FAM, VIC or HEX, was inserted into the ninth base and the 14th base of the nucleic acid probe to prepare a nucleic acid probe capable of detecting the target nucleic acid.

Then, the probe was treated overnight in a 1 M tetrabutylammonium fluoride solution to remove sialyl protecting groups bound to RNA, and then 1 M TEAA (triethylammonium acetate) Solution. The oligonucleotides were then desalted using Sephadex G-25 column (Pharmacia) using distilled water. The final samples were then collected by fraction and electrophoresed on a 20% polyacrylamide gel for denaturation (7 M urea added), then the appropriate oligonucleotide bands were cut out of the gel and analyzed by S & S ELUTRAP electro- Separation System; Schleicher & Schuell) to elute the probe electrically.

1-2. Primer production

Microbacterium species extracted from the blood of patients with pneumonia ( Bacrobacterium sp.) was made into cDNA through a reverse transcription polymerase chain reaction, and then the cDNA was used as a template and a primer capable of complementary binding using a perseptive ciosystem expedite nucleic acid synthesis system .

Manufacturing example  2: Single nucleotide polymorphism (SNP) detection Probe  And primer  making

2-1. Probe making

Based on the information on the casein gene of bovine b, we searched for a SNP related sequence existing in the gene using the NCBI database. As a result, 6 SNPs were identified and 6 nucleic acid probes were synthesized based on the SNPs.

At this time, the four bases including the SNP were ribonucleotides (RNA), and the SNPs were synthesized so that the bases from -1 to -9 bases and the bases 1 to 3 were deoxyribonucleotides (DNA).

During the synthesis of the nucleic acid probe, a nucleic acid probe capable of detecting single base polymorphism was prepared by inserting the fluorescent material FAM, VIC or HEX into four bases including the SNP of the nucleic acid probe.

The first genetic mutation site Second genetic mutation site Third genetic mutation site Fourth genetic mutation site Fifth genetic mutation site 6th genetic mutation site Attached to the probe
Fluorescent material FAM VIC HEX FAM FAM HEX VIC HEX VIC

Then, the probe was eluted by the same method as in Production Example 1-1.

2-2. Primer production

The b-casein RNA extracted from bovine blood is made into cDNA through a reverse transcription polymerase chain reaction, and then the cDNA can be used as a template for complementary binding using a perseptive ciosystem expedite nucleic acid synthesis system Primer set was prepared.

Example  1: nucleic acid according to the present invention The probe  Target nucleic acid detection experiment using

In the presence of 10 pmol of the probe prepared in Preparation Example 1-1 and 5 units of heat-resistant RNase H, the genomic DNA of the pneumonia patient, the primer set prepared in Preparation Example 1-2, 50 Ta Taq Polymerase Buffer, 0.2 mM dNTP and 2.5 units of Taq polymerase were subjected to polymerase chain reaction (PCR) and fluorescence intensity was monitored. The results are shown in Table 6.

Single nucleotide polymorphism Target 1 Target 2 Target 3 Target 4 Target 5 Target 6 Target 7 Target 8 Target 9 Target 10 Probe 1 FAM + + - - + - - + + - VIC - - + - - + - - - + HEX - - - + - - + - - - Probe 2 FAM - - - - + - - - - - VIC - - - - - + - + - - HEX - - - - - - + - + +

As shown in Table 5, when PCR was performed using two probes having fluorescent substances of FAM, VIC or HEX and capable of detecting different target nucleic acids, it was confirmed that 10 single nucleotide polymorphisms were correctly detected Respectively.

In particular, when two probes with the same fluorescent material were used, one amplification curve was observed, and it was found that the amplified quantitation line was increased about twice as compared with the case of using one probe.

On the other hand, when two probes with different fluorescent materials were used, two types of amplification curves were observed.

Example 2: Detection of single nucleotide polymorphic site using a nucleic acid probe according to the present invention

In the presence of 10 pmol of the probe prepared in Preparation Example 2-1 and 5 units of heat-resistant RNase H, bovine casein cDNA, the primer set prepared in Preparation Example 2-2, 50 μl Taq polymerase buffer, 0.2 mM dNTP And 2.5 units of Taq polymerase were subjected to polymerase chain reaction (PCR), and fluorescence intensity was monitored.

As a result, the amplification curve appeared only in the fluorescence channel of HEX, and it was found that the mutation occurred at the third genetic mutation site.

Example  3: Genotype analysis of single nucleotide polymorphism site

In order to analyze the genotypes of the genetic mutation sites present on the bovine casein cDNA identified in Example 2 above, three fluorescent substances (FAM, VIC, or HEX) Quot ;, " CTGT ", and " G "in the nucleotide sequence of the " CTGT ..." Then, bovine casein cDNA, the primer set prepared in Preparation Example 2-2, 50 Ta Taq Polymerase Buffer, 0.2 mM dNTP and 2.5 unit of the thermostable RNase H in the presence of 10 pmol of the prepared probe and 5 units of heat resistant RNase H The polymerase chain reaction (PCR) was performed using Taq polymerase, and fluorescence intensity was monitored.

Probe 1 Prof 2 Probe 3 Probe 4 Fluorescent material - FAM VIC HEX The base sequence of the probe that binds to the genetic mutation site R ₁ -rGrArCrA-R ₂ R ₁ -rGrArArA-R ₂ R ₁ -rGrArTrA-R ₂ R ₁ -rGrArGrA-R ₂

As a result, the amplification curve appeared only in the fluorescence channel of FAM, and it was found that the wild type base "G" was changed to "T".

Claims (10)

a) obtaining a sample comprising a target nucleic acid;
b) a nucleic acid sequence having the structure of R ₁ -XR ₂ , wherein the same or at least two different detectable markers are attached to both ends or inside of the probe, and the complementary binding to the base sequence of the target nucleic acid obtained in step a) Wherein R ₁ and R ₂ of the probe are DNA or RNA consisting of 1 to 20 nucleotide sequences, X is a nucleotide sequence of 1 to 10 nucleotides, Characterized in that it is RNA or DNA which can be cleaved by a reagent;
c) mixing the sample obtained in step a), two or more nucleic acid probes prepared in step b), a primer set having a base sequence complementary to the sample obtained in step a), and a cleavage reagent Amplifying the target nucleic acid-probe complex through the reaction; And
and d) measuring the amount of the probe fragment isolated from the target nucleic acid-probe complex amplified in step c).
The method according to claim 1,
Wherein the target nucleic acid in step a) is RNA or DNA to be detected from a sample, or cDNA obtained by amplifying the RNA with a reverse transcription polymerase.
The method according to claim 1,
Wherein the detectable marker in step b) is a fluorescent substance or a fluorescent pair (fluorescent substance and small molecule substance).
The method according to claim 1,
When a fluorescent dye is used as a detectable marker in step b), a fluorescent dye and a quencher are attached to positions at a distance of 1 to 20 base pairs at both ends or inside of X of the nucleic acid probe. ≪ / RTI >
The method according to claim 1,
Wherein R ₁ and R ₂ of the nucleic acid probe having the structure of R ₁ -XR ₂ in step b) are DNA and X is RNA.
a) obtaining a sample to be tested for the presence or absence of a nucleic acid comprising a genetic variation;
b) determining the genetic mutation site and then identifying the nucleic acid sequence having a structure of R ₁ -XR ₂ , at least two different detectable markers attached to both ends or inside of the probe, Wherein R ₁ and R ₂ of the probe are DNA or RNA consisting of 1 to 20 nucleotide sequences, and X is a nucleotide sequence having 1 to 10 nucleotide sequences Characterized in that it is RNA or DNA which can be cleaved by a cleavage reagent;
c) mixing the sample obtained in step a), two or more nucleic acid probes prepared in step b), a primer set having a base sequence complementary to the sample obtained in step a), and a cleavage reagent Amplifying a nucleic acid-probe complex comprising a genetic mutation through a reaction; And
d) measuring the amount of the probe fragment isolated from the nucleic acid-probe complex comprising the genetic mutation amplified in step c).
The method according to claim 6,
Wherein the genetic variation is a phenomenon caused by an allele, a single nucleotide polymorphism (SNP), a mutation or a combination thereof.

The method according to claim 6,
Wherein the detectable marker in step b) is a fluorescent substance or a fluorescent pair (a fluorescent substance and a small molecule substance).
The method according to claim 6,
When a fluorescent dye is used as a detectable marker in step b), a fluorescent dye and a quencher are attached to positions at a distance of 1 to 20 base pairs at both ends or inside of X of the nucleic acid probe. Wherein the method comprises the steps of:
The method according to claim 6,
Wherein R ₁ and R ₂ in the nucleic acid probe having the structure of R ₁ -XR ₂ in step b) are DNA and X is RNA.