WO2016003197A1 - Method and apparatus for detecting sequence-specific nucleic acid using exonuclease, and kit used for same - Google Patents

Abstract

Disclosed is a method for detecting sequence-specific nucleic acid using exonuclease, the method for detecting sequence-specific nucleic acid using exonuclease comprising the steps of: mixing exonuclease with a double helical target nucleic acid sequence, wherein, among the double helices, the 5' end (a first end) of one strand is in a state of not being able to be degraded by the exonuclease, and the 5' end (a second end) of the other strand is in a state of being able to be degraded by the exonuclease; hybridizing the resulting product from the mixing step (mixture hereinafter) with a capture probe; and detecting the target nucleic acid sequence hybridized with the capture probe.

Description

 Specification

 NAME OF THE INVENTION: A method and apparatus for detecting sequence-specific nucleic acids using nucleic acid terminal enzymes and kits used therein

 [1] The present invention relates to a method and apparatus for detecting sequence-specific nucleic acids using nucleic acid protease with enhanced sensitivity and specificity, and a kit used therein.

 Background

 [2] Nucleic acid, an important characteristic of living organisms, has been studied in various fields of biology, and in practice, information on specific DNA sequences is very useful for biological research.

 [3] For example, information about specific DNA sequences can be used to identify genetic or infectious diseases.

 The technique of hybridizing labeled probes and nucleic acids is the only practical way to detect complementary nucleic acids in nucleic acid complexes.

 [4] Conventional Northern and Southern blot methods are very

 For the detection of specific and sensitive nucleic acids, the principle of hybridization is used.

[5] However, this method may wihin a diagnosis in the field to have a process that is time consuming and difficult to apply the ^first industrial complex. (Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis.J Mol Biol 98: 503-517, 1975, Kevil CG, Walsh L, Laroux FS, Kalogeris T, Grisham MB, Alexander JS.An Improved, Rapid Northern Protocol.Biochem Biophys Res Commun 238: 277-279, 1997)

 [6] On the other hand, the emergence of paper-based microfluidics has facilitated studies related to lateral flow testing techniques, as membrane-based equipment is promising for application in field diagnostics. A simple device for detecting the presence of target analytes. Recently, for widespread use of such strips, nucleic acid side-flow micro-chromatography has been developed as a new alternative screening test. (Mao X, Ma Y, Zhang A, Zhang L, Zeng L, Liu G. Disposable Nucleic Acid Biosensors Based on Gold Nanoparticle Probes and Lateral Flow Strip.Anal Chem 81: 1660-8, 2009, Arunrut N, Prombun P,

 Saksmerprome V, Flegel TW, Kiatpathomchai W. Rapid and sensitive detection of infectious hypodermal and hematopoietic necrosis virus by loop-mediated isothermal amplification combined with a lateral flow dipstick. J Virol Methods 171; 21-25, 2011)

FIG. 17 is a diagram for explaining a method of detecting a conventional target nucleic acid.

 [8] The method of A) of FIG. 17 is performed in a stationary phase at the end of two strands of two strands of nucleic acid sequence

 Combine the attachable material and the other end of the detection material

Combined with each other to measure and detect markers in nucleic acid sequences The method of FIG. 17B is at the end of two strands of the nucleic acid sequence.

 After the detection material is bound, the single-stranded nucleic acid sequence is hybridized to a capture probe having a complementary sequence bound to a stationary phase in the presence of a substance that dissociates the nucleic acid such as sodium hydroxide. Method of detecting and detecting labeling substance

[9] However, both methods described above have many substances that did not respond to the results.

 The background (backgound) value is so large that the nautical ball that remains fixed

 Detection is not available because it affects hybridization.

 Detailed description of the invention

 Technical task

 According to an embodiment of the present invention, a method for detecting sequence-specific nucleic acids using nucleic acid terminal enzymes having improved sensitivity and specificity may be provided.

[11] According to another embodiment of the present invention, sensitivity and specificity are improved.

 A sequence-specific nucleic acid detection device using nucleic acid protease can be provided.

 [12] According to another embodiment of the present invention, sensitivity and specificity are improved.

 Kits for sequence-specific nucleic acid detection using nucleic acid protease can be provided. Task solution

 [13] According to one embodiment of the present invention, a method for detecting sequence-specific nucleic acids using nucleic acid terminal enzymes,

 [14] The double-stranded target nucleic acid sequence—the 5 'end of either strand of a double-stranded helix

 Terminal) is in a state that cannot be degraded by nucleic acid terminalase, and the 5 'terminal (second terminal) of the other strand is in a state that can be degraded by nucleic acid terminalase-combining with nucleic acid terminalase;

 [15] Capture the result of the above mixing step (hereinafter referred to as a mixture) and the probe

 Hybridizing; and

 [16] A method for detecting sequence-specific nucleic acids using nucleic acid terminal enzymes, the method comprising detecting a target nucleic acid sequence localized in the capture probe.

 [17] According to another embodiment of the present invention, in a sequence-specific nucleic acid detection kit using nucleic acid terminal enzyme,

 [18] a pair of primers; and

 [19] comprising nucleic acid protease;

 [20] The 5 'end of one of the pairs of primers is combined with a phosphate group.

 And

 [21] The 5 ᅳ terminal of the remaining primers of the pair of primers has a blocker.

 Combined, and

[21] The nucleic acid protease is characterized by degrading phosphate-bonded strands in a double-stranded amplified product amplified by the pair of primers. Kits for sequence-specific nucleic acid detection using nucleic acid protease may be provided.

[23] According to another embodiment of the present invention, in a sequence-specific nucleic acid detection kit using nucleic acid terminal enzyme,

[24] a pair of primers; and

[25] comprising nucleic acid protease;

 [26] The pair of primers has a phosphate group attached to the 5 'end of one primer.

 [27] The 5 'end of the pair of primers has a hydroxyl group present, and

 [28] The nucleic acid protease is characterized by degrading phosphate-bonded strands in a double helix amplified product amplified by the pair of primers.

 Kits for sequence-specific nucleic acid detection using nucleic acid protease can be provided.

 [29] According to another embodiment of the present invention, there is provided a sequence-specific nucleic acid detection apparatus using nucleic acid terminal enzyme,

 [30] The double-stranded target nucleic acid sequence has a 5 'end (first end) of one strand of the double strand that cannot be decomposed by nucleic acid protease, and 5 의 of the other strand.

 The terminal (second end) is in a state that can be degraded by nucleic acid terminal enzyme-and

 A mixed portion in which nucleic acid protease is combined;

 A hybridization unit which hybridizes the mixed mixture mixed by the mixing unit with the probe; Y -1

 [32] A sequence-specific nucleic acid sequence detection device using a nucleic acid terminal enzyme can be provided, including a detection unit for detecting a target nucleic acid sequence from the hybridized hybridization by the hybridization unit.

 Effects of the Invention

 According to one or more embodiments of the present invention, the sensitivity and specificity are very high in detecting the target nucleic acid in the sample.

 Brief description of the drawings

FIG. 1 is a flowchart illustrating a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention.

2 is a diagram illustrating a sequence-specific nucleic acid detection apparatus using nucleic acid terminal enzyme according to an embodiment of the present invention.

3 is a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention, showing an embodiment using a primer in which nothing is bound (in the presence of a hydroxyl group). -.

4 shows an example of using a primer to which a detection material is bound as a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention. FIG. 5 shows an example of using a ligand-bound primer as a sequence-specific nucleic acid detection method using nucleic acid protease according to an embodiment of the present invention.

 FIG. 6 is a view showing the results of an experimental example to which the embodiment of FIG. 3 is applied.

FIG. 7 is a drawing showing the results of an experimental example to which the embodiment of FIG. 4 is applied.

FIG. 8 is a diagram showing the results of an experimental example to which the embodiment of FIG. 5 is applied. FIG.

FIG. 9 is a diagram showing the results of an experimental example in which the embodiment of FIG. 3 is applied but a detection probe is used. FIG.

 FIG. 10 is a diagram showing the results of another experimental example in which the embodiment of FIG. 4 is applied.

FIG. 11 is a diagram showing the results of an experimental example in which the embodiment of FIG. 3 is applied but a detection probe is used. FIG.

 12 is a diagram showing the results of another experimental example in which the embodiment of FIG. 5 is applied.

FIG. 13 is a diagram showing the results of an experimental example using the detection probe coupled with a ligand, applying the embodiment of FIG. 3.

 FIG. 14 is a diagram showing the results of another experimental example in which the embodiment of FIG. 3 is applied but using a detection probe coupled with a ligand. FIG.

FIG. 15 is a diagram showing the results of another experimental example in which the embodiment of FIG. 4 is applied. FIG.

FIG. 16 is a diagram showing an experimental example and a result using a microparticle probe while applying the embodiment of FIG. 3.

 FIG. 17 is a diagram for explaining a method for detecting a conventional target nucleic acid.

[51] [Description of the Signs]

 [52] 100: Sequence-Specific Nucleic Acid Detection Device Using Nucleic Acid Terminalase

[53] 101: amplification part

[54] 103: mixing part

[55] 105: hybridization

107: detection unit

 Best Mode for Carrying Out the Invention

[0057] The above and other objects, features, and advantages of the present invention will be readily understood through the following preferred embodiments, which are attached to the accompanying drawings. However, the present invention is not limited to the embodiments described herein. Other embodiments may be embodied. Rather, the embodiments described herein are provided so that the disclosure may be thorough and complete, and that the spirit of the present invention may be conveyed to those skilled in the art.

In various embodiments of the present specification, terms such as first and second have been used to describe various components, but these components should not be limited by such terms. It is only used to distinguish one component from another. Embodiments described and illustrated herein Its complementary embodiments are also included.

 [59] The terminology used herein is for the purpose of describing particular embodiments only.

 In this specification, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise.

 'Comprises' and / or 'comprising' components mentioned do not exclude the presence or addition of one or more other components.

 [60] Hereinafter, the present invention will be described in detail with reference to the drawings.

 In describing the embodiments, several specific details have been written to further illustrate and help explain the invention. However, readers with knowledge of this field to the fullest extent understand this invention. In some cases, it is commonly known in describing the invention that parts not significantly related to the invention are not described in detail to avoid confusion in the description of the invention. Put it.

 [61] Definition of terms

 [62] In the present specification, the term '5. terminal cannot be resolved' may mean any of the following.

 [63]-If a blocker is combined at the 5th end: where the blocker is

 Nucleic acid proteases cannot be degraded, for example, they can be substances such as detectors or ligands.

 [64]-when no substance is bound at the 5 'end; meaning that the hydroxyl groups remain at the 5' end.

 [65] In the present specification, '5' terminal decomposable state 'may mean any of the following.

 [66]-when a 5'-terminus is bound to a substance capable of nucleic acid protease:

 It may be the case that phosphate groups are combined.

 [67]

 1 is a flowchart illustrating a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention.

 Referring to Figure 1, using a nucleic acid terminal enzyme according to an embodiment of the present invention

 The sequence-specific nucleic acid detection method includes the step of amplifying a sample (S101), the output authentication product of step S101, that is, the 5 'end (first end) of one strand of a double-stranded target nucleic acid sequence-a double-stranded helix. The 5 'terminus (second terminus) of the other strand is incapable of digestion by nucleic acid terminase and can be resolved by nucleic acid terminase.

 State -Synthesis step with nucleic acid terminalase (S103), S103

Hybridizing the resultant (hereinafter referred to as a mixture) with the capture probe (S105), and detecting the target nucleic acid sequence hybridized with the capture probe from the hybrid of step S105 (S107). '^{; ■ ■} In step S101, amplification of a sample, which is a target to be checked whether a target (target nucleic acid sequence) is included. The amplification method is one of techniques for amplifying a known or future known nucleic acid. This can be used.

 [71] Examples of conventionally known amplification technology stones are polymerase chain reactions.

Reaction: PCR, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), or Recombinase Polymerase Amplification (RPA). These amplification techniques are exemplary. It will be readily understood by those skilled in the art to which the present invention pertains that the present invention is not limited to such.

 The specific conditions or substances necessary for PCR reactions, RT-PCR reactions, or RPA reactions are conventionally known, and thus detailed descriptions are omitted in this specification.

 [73] When the step S101 is performed, the primers used for amplification of nucleic acids may for example be constructed as follows.

 [74]

 [75] Primer according to the first embodiment

 [76] One of the pairs of primers used to amplify the sample may be in an unconnected state (i.e., in the presence of hydroxyl groups) at the 5 'end of either primer, and the pair of primers 5 'terminal is phosphate

 Can be combined.

 3 shows a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention using an primer (without hydroxyl group) to which nothing is bound.

 Referring to FIG. 3, no substance is bound to the 5 'end of one of the pairs of primers (a state in which a hydroxyl group exists), and a phosphate group is bonded to the 5' end of the other primer. The amplification when using it is shown first on the left.

 [79]

 [80] Primer according to the second embodiment

 [81] One pair of primers used to amplify a sample may have a blocker attached to the 5 'end of one primer, and the other may have a phosphate group attached to the 5' end of the primer. ,For example,

 Such as fluorescence or luminescence

 It may be a detecting material.

 4 shows an example using a primer bound to a detection material as a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention.

Referring to FIG. 4, one of the pair of primers used when amplifying a sample is used. Fluorescence at the 5 'end of the primer or

 The amplification is shown by way of example in which a detection material such as luminescence is bound and a phosphate group is used at the 5 'end of the other primer.

 [84]

 [85] Primer according to the third embodiment

 [86] One pair of primers used to amplify a sample may have a blocker attached to the 5 'end of one primer, and a phosphate group bound to the 5' end of the other primer. For example, it can be a ligand.

 FIG. 5 illustrates an example of using a ligand-bound primer as a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention.

 Referring to FIG. 5, one of a pair of primers used to amplify a sample

 An example of the amplification is shown when a ligand is bound to the 5 ends of the primer and a phosphate group is bound to the 5 'end of the other primer.

 [89]

 The sample amplified in step S101 may be inferred to contain, for example, DNA and / or RNA of a disease mediated organism. Here, the disease agent may be applicable not only to living things but also to DNA and / or RNA such as viruses.

 In step S103, the product authentication bomb of step S101, that is, the target nucleic acid sequence of the double helix, is mixed with the exonuclease.

 [92] The exonuclease plays a role in decomposing strands bound to phosphate groups. Therefore, in step S103, exonucleases bind phosphate groups in the amplification of step S101. Only the strands are selectively decomposed, so that only a single strand to which a blocker is bound or no substance is bound (i.e., a hydroxyl group is present).

 According to an embodiment, the exonuclease is a lambda.

 It may be a lambda exonuclease, but

 Nucleic acid protease is an example, and it is not limited to the present invention. Those skilled in the art belong to the present invention.

 [94] Nucleic acid protease (exonuclease) coupled with the amplification product performs the operation.

 Necessary Conditions Stones and materials are conventionally known and therefore detailed descriptions will be omitted here.

[95] Referring to Figs. 3 to 5, it is shown schematically that the strand bound to the phosphate group is decomposed by the nucleic acid terminal enzyme. In step S105, the mixture resulting from the performance of step S103 is hybridized with a capture probe.

 In the present embodiment, the capture probe is specific to the target to be found in the sample.

 For example, the capture probe may be fixedly coupled via UV cross-linking or drying, but this is illustrative, and the present invention is not limited to such fixedly coupled capture probes. Will be understood.

 3 to 5, a target coupled with a capture probe is illustratively shown.

 Appear.

 [99] In step S107, as a result of performing step S105, a target coupled to the capture probe is detected. For example, a technique that is widely known or known in the future is known as a technique for detecting a target coupled to the capture probe. Can be used.

The technique for detecting a target bound to the capture probe may be a technique for detecting a substance such as fluorescence or luminescence bound to the target, for example.

[101] For example, step S107 is captured by detecting the detection material bound to the target.

 The amount of target bound to the probe will be known (see Figure 4).

In another example, step S107 identifies a detection probe that is specifically bound to the target.

 The amount of target bound to the capture probe may be known (see Figure 3). For example, the detection probe may have a fluorescence or

 Detection substances such as luminescence may be bound, or ligands may be bound.

 [103] When a detection material such as fluorescence or luminescence is bound to the detection probe, the detection probe is bound to the detection probe.

 The amount of fluorescence or luminescence

 By detecting, the amount of target bound to the capture probe can be known.

 [104] On the other hand, when the ligand is coupled to the detection probe, the ligand is bound to the ligand.

 By detecting the amount of detection material such as fluorescence or luminescence bound to the receptor, the amount of target bound to the capture probe can be determined. In this case, the performance of step S105 and Additional steps can be performed to mix ligand receptors in which substances such as fluorescence or luminescence are bound together.

 [105] In another example, step S107 is performed when a target is bound to a ligand,

 Targets can be detected using ligand receptors combined with materials such as fluorescence or luminescence. In this case, the performance of step S105 and the fluorescence or

An additional step of mixing the detection probes in which materials such as luminescence are bound to each other is performed, which is then coupled to the detection probes. Fluorescence or luminescence is detected (see FIG. 5). _; f,

 FIG. 2 is a view for explaining a sequence-specific nucleic acid detection apparatus using nucleic acid terminal enzyme according to an embodiment of the present invention.

Referring to Figure 2, using a nucleic acid terminal enzyme according to an embodiment of the present invention

 The sequence-specific nucleic acid detection apparatus 100 may include an amplifier 101, a mixing section 103, a hybridization section 105, and a detection section 107.

The amplifier 101 amplifies the sample.

 The amplification unit 101 may perform an amplification operation using any one of conventionally known or future known nucleic acid amplification techniques. Examples of known amplification techniques include polymerase chain reaction ( polymerase chain reaction (PCR) technology, reverse transcription-polymerase chain reaction (RT-PCR) technology, or recombinase polymerase amplification (RPA) technology. It will be readily apparent to those skilled in the art that the technology is illustrative and not intended to be such.

 The specific conditions or substances necessary for PCR reactions, RT-PCR reactions, or RPA reactions are conventionally known, and thus detailed descriptions thereof will be omitted.

 When the sample is amplified by the amplifying unit 101, as described with reference to FIG.

 Primers (for example, a primer according to the first embodiment, a primer according to the second embodiment, or a blue primer according to the third embodiment) can be used.

 In the present specification, the amplification part 101 includes software for maintaining an area where a sample is located and amplified (called a “sample location area”); and a condition under which a sample existing in the sample location area can be amplified. And hardware unit; as a generic term, will be used.

 The mixing unit 103 mixes the amplified product (hybridized nucleic acid sequence) mass-nucleic acid protease that has been amplified by the amplifying unit 101.

[114] When amplification products (generated nucleic acid sequences) and nucleic acid terminal enzymes are combined, strands in which the phosphate groups are bound in the amplification product are selectively degraded by exonucleases.

 In the present specification, the mixing unit 103, the amplified by the amplification unit 101 and

 The region in which the mixture containing the nucleic acid protease is located (referred to as the 'mixture position region'), and in the mixture present in the mixture position region,

 The term "software and hardware" that maintains the reaction conditions for normal operation will be used as a generic term.

 The hybridization unit 105, the nucleic acid terminal enzyme is a phosphate group is bonded in the mixing unit 103

With all the strands remaining in the mixture, the capture probes can become active. Keep it.

 In this embodiment, the capture probe includes a sequence that is specifically bound to the target to be found in the sample. In other words, the capture probe may be coupled to a stationary phase, but this is merely illustrative. It will be readily apparent to those skilled in the art that this is not sufficient.

 In the specification, the hybridization unit 105 is mixed in the mixing unit 103.

 The mixture (the mixture in which the phosphate-linked strand is decomposed by the nucleic acid terminalase) and the region in which the capture probe is located ('hybrid position region'); the capture probe at the mass-, hybrid position region The term "software and hardware" that maintains the conditions for the hybridization reaction to be performed will be used as a generic term.

 The detection unit 107 detects a target coupled to the capture probe. For example,

 The detection unit 107 can directly or indirectly detect a target coupled to the capture probe in the hybridized by the shake unit 105.

 [120] The detection unit 107 is a fluorescence (fluorescence) bound to the target or

 It may be a technique for detecting a substance such as luminescence, but this is illustrative, and the present invention is not limited to such a technique.

 [121] As another example, the detection unit 107, when the ligand is coupled to the target,

 Targets can be detected using ligand receptors incorporating materials such as fluorescence or luminescence.

 In the present specification, the detection unit 107 is used as a generic term for both software and hardware units that directly or indirectly detect a target coupled to the capture probe.

 Although not described, the person skilled in the art further includes a means for the apparatus 100 to move a sample located in the sample location area to the mixture location area, and a means for moving a mixture located in the mixture location area to the hybrid location area. It can also be implemented.

 In the present embodiment, the device 100 includes an amplifier 101.

 As described above, except for the amplification part 101, the mixing part 103, the shaking part 105, and

 It may also be possible to implement to include a detector 107. In this case, the amplification operation is performed by an amplification device configured separately from the device 100,

 With respect to the result of the execution, the mixing section 103, the shaking section 105, and the detecting section 107 will perform the operation.

 In another example, the apparatus 100 has been described as including a detector 107, but without the detector 107, the amplifier 101, the mixer 103, and the hybridizer 105. It is also possible to implement to include. In this case the detection operation may be performed by a detection device configured separately from the device (100).

3 to 5 show time series flows when the method or apparatus for detecting sequence-specific nucleic acid using a nucleic acid terminal enzyme according to an embodiment of the present invention is applied. In the following, the present invention will be described in terms of time series with reference to these drawings.

 Referring to FIG. 3, a sequence-specific nucleic acid detection method or apparatus according to an embodiment of the present invention is applied to an amplified sample using the primer according to the first embodiment described with reference to FIG. 1. In this case, time series flow is represented.

 In other words, for amplified samples using the primer according to the first embodiment,

 The incorporation of nucleic acid proteases (e.g. lambda nucleic acid proteases) only degrades the strands to which the phosphate groups are bound (meaning the strands indicated by dashed lines in FIG. 3). When the capture probe maintains a condition that can be hybridized and mixed with the detection probe to which the detection material is bound, the target coupled to the capture probe is hybridized to the detection probe to which the detection material is bound as shown in the drawing on the right side of FIG. Thereafter, the detection material bound to the detection pro- bodied in the target can be detected.

 Referring to FIG. 4, a sample amplified using the primer according to the second embodiment described with reference to FIG. 1 according to an embodiment of the present invention.

 The time-series flow is shown when a sequence-specific nucleic acid detection method or apparatus using nuclear trimethylase is applied.

 In other words, for the amplified sample using the primer according to the second embodiment,

 Mixing nucleic acid proteases (e.g. lambda nucleic acid proteases) results in the degradation of only the strands to which the phosphate groups are bound (meaning the strands indicated by dashed lines in Figure 3). When the detection material is bound to and the conditions under which the capture probe is hybridized are maintained, the target of the capture probe is hybridized as shown in the first right side of FIG. 4. After that, the detection coupled to the hybridized target is detected. The substance can be detected.

 Referring to FIG. 5, a sample amplified using a primer according to the third embodiment described with reference to FIG. 1 according to an embodiment of the present invention.

 Time-series flow in the case of applying a sequence-specific nucleic acid detection method or apparatus using nucleic acid terminal enzyme is shown.

 That is, for an amplified sample using the primer according to the third embodiment,

 Incorporation of nucleic acid proteases (eg lambda nucleic acid proteases) results in the degradation of only the strands to which the phosphate groups are bound (meaning the strands indicated by dashed lines in FIG. 3). The ligand receptor is retained in the target ligand bound to the capture probe, as shown in the first right side of FIG. 5, when the ligand is bound to-and the capture probe is hybridized and mixed with the ligand receptor. Thereafter, the detection substance bound to the ligand receptor can be detected.

 [133]

Hereinafter, experimental examples according to the embodiments of the present invention described above will be described. Example 1 Detection of Hepatitis C Virus

[137]

A control base sequence prepared by cloning the pGMT vector was synthesized by TA cloning method. The forward primer used was 5'-CCTTGTTTGGCCCTTCCT-3 ', with a phosphate group bound to the 5' end. Is

 5'-TATTGCTACCGCGCATGA-3 ', which was tested for all three embodiments described with reference to FIG. 1. That is, when no other material was bound to the 5' end of the reverse primer (i.e., in the presence of hydroxyl groups). ), Both detection materials such as Cy5 and bound ligands such as biotin were tested. .

 [139] The length of the control base sequence synthesized by polymerase chain reaction (PCR) is 90 base pairs. The base sequence is as follows.

 [140] CCTTGTTTGGCCCTTCCTACCAAGGCTTGGAATGTGTTGCTGGTTCTTTAT CGTGGCATGTGTTCCATAGTTTCATGCGCGGTAGCAATA

 [141] The base sequence of the capture probe attached to the fixed phase to detect it is

 5'-GGCATGTGTTCCATAGTTGGCATGTGTTCCATAGTT-3'ID-.

 [142]

[143] 388 base sequences of Hepatitis C virus (HCV) (GenBank: GU451221.1)

 The containing HCV fragment base sequence was synthesized (Cosmogenetech, South Korea) and linked to the T7 promoter to create a target plasmid for HCV.

 [144] HCV fragment base sequence:

[145] 1 ggaattcccc tgatgggggc gacactccac catgaatcac tcccctgtga ggaactactg

[146] 61 tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggac

[147] 121 cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccg gaattgccag

[148] 181 gacgaccggg tcctttcttg gatcaacccg ctcaatgcct ggagatttgg gcgtgccccc

[149] 241 gcgagactgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg

[150] 301 gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg aatcctaaac

[151] 361 ctcaaagaaa aaccaaacgt aacaccaacc gccgtcgaca

[152]

[153] A forward primer having a phosphate group bound to the 5 'end by using a T7 RNA polymerase to generate a target RNA from the target plasmid.

 The three examples described above with 5'-GGGAGAGCCATAGTGGTCT-3 '(i.e., no substance is bound at the 5' end (with a hydroxyl group present), a detection substance such as Cy5 is bound, or the same as Biotin. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using reverse primers 5'-CGCAAGCACCCTATCAGG-3 '. The HCV target sequence made with RT-PCR was 179 base pairs.

1: 154] FIG. 6 shows a forward primer having a phosphate group bonded to the 5 ′ terminal. The electrophoresis results for the amplification of RT-PCR were obtained using a reverse primer of 5'-GGGAGAGCCATAGTGGTCT-3 'and no substance at the end of 5 (ie, in the presence of hydroxyl groups). On the other hand, for the sake of understanding of the present experiment, the time series flow of the present experiment is additionally shown in the upper part of FIG. Those skilled in the art will be able to easily understand the operation principle of the present experiment by referring to the time series flow shown in FIG.

On the other hand, the base sequence of the capture probe attached to the stationary phase to detect the HCV target sequence is

 5'-ATTTGGGCGTGCCCCCGCATTTGGGCGTGCCCCCGC-3 '. Detection probes were 5'-Alexa-GGGTCCTTTCTTGGA-3' and

 5'-Alexa-GGTACTGCCTGATAGG-3 was used.

 [156] The capture probe was dissolved in 14xSSC solution at a concentration of 100 pmol / ul, line-lined on nitrocellulose membrane using DCI-300 (ZETA Corporation, South Korea), and at 0.12 J with UV cross linker (Hoefer, USA). A membrane for lateral flow assay was prepared by cross linking for 10 minutes.

 [157] 10 U of lambda exonuclease from HCV RT-PCR

Addition to, for 10 min at 38 voyage warm to terminate the enzyme banung to then create a DNA single strand 80 ^'for 10 minutes at a thermostat banung. The lateral flow assay using a single-stranded DNA was made by nucleic acid lyase Proceeded.

 [158] Using a Cy5 conjugated primer for the lateral flow assay, 10 ul

 In addition to single-stranded DNA-90 ul of hybridization buffer was used. When using biotin-coupled primers, 10 ul of single-stranded DNA was mixed with 90 ul of hybridization buffer and 2 ul of streptavidin conjugated dark beads, and when Alexa detection probe was used. 90 ul of hybridization buffer and Alexa detection probe were injected into membrane cartridge in 10 ul of single stranded DNA. Hybridization reaction was performed at 40 ° C for 20 minutes to confirm the fluorescence value with a probe.

 [159] The preparation of Streptavidin conjugated dark beads induced conjugation reaction by adding 2 mg / ml of streptavidin (Sigma, USA) to 500 ul of conjugate solution, and streptavidin conjugated with streptavidin contained blocking buffer (lxPBS, 1% BSA, 0.2% Tween 20 ) Was performed by blocking the solution.

 7 shows a forward primer having a phosphate group bonded to the 5 'end.

 Reverse direction of Cy5 binding to 5'-GGGAGAGCCATAGTGGTCT-3 'and 5' end

 An example of the HCV detection experiment in the case of using the primer is shown. On the other hand, for the sake of easy understanding of the experiment, the time series flow of the experiment is further shown in the upper part of FIG.

 8 shows a forward primer having a phosphate group bonded to five ends thereof.

 Reverse direction of biotin at 5'-GGGAGAGCCATAGTGGTCT-3 'and 5' ends

The following shows an example of HCV detection experiment using a primer. For ease of understanding, the time series flow of the present experiment is further illustrated in the upper part of FIG.

 9 shows a forward primer having a phosphate group bonded to the 5 'end.

 Example of HCV detection when a reverse primer was used in which 5'-GGGAGAGCCATAGTGGTCT-3 'and 5' terminal were not combined (ie, in the presence of a hydroxyl group). Used. On the other hand, for the sake of easy understanding of the experiment, the time series flow of the experiment is further shown in the upper part of FIG.

 [163]

 [164] As can be seen from these detection results, the detection method according to the present invention is very

 Sensitive and specific targets can be detected.

 [165]

 Example 2 Detection of Hepatitis B Virus

[167]

 [168] pAAV / HBV1.2 plasmid containing DNA of hepatitis B virus (HBV) (Proc

Natl Acad Sci USA 107 (20): 9340-5, 2010) was used to confirm the base sequence, and ° plasmid was used to recombinase polymerase amplification (RPA).

 [169] The forward primer used

 5'-TGCTCGTGTTACAGGCGGGGTTTTTCTTGTTG-3 ', which has a phosphate group attached to the 5' end.

 5'-GAGGAATATGATAAAACGCCGCAGACACATCC-3 ", all three examples were described with reference to FIG. 1. That is, when no other material was bound to the 5 'end of the reverse primer (i.e., in the presence of hydroxyl groups). ), Both detection materials such as Cy5 and bound ligands such as Biotin were tested.

[170] pAAV / HBVl.2 plasmid and TwistAmp Basic kit from TwistAmp (UK)

 RPA reaction was conducted for 15 minutes during the 38 ° C term. To terminate the amplification reaction, the 80 ° C term added 10 U of lambda exonuclease, and the 38 ° C term reacted for 5 minutes. Single stranded DNA was synthesized.

 [171] The HBV target sequence made with RPA was 218 base pairs. The base sequence of the capture probe attached to the stationary phase to detect it was

 S'-TTTTTTTTTTTTTTTCCTCCAATTTGTCCTGGTTAT-S ', and the detection probes were 5'-AACCTCCAATCACTCACCAAC-Cy5-3' and

 5'-AACCTCCAATCACTCACCAAC-Biotin-3 'was used.

 Lateral flow assay was performed in the same manner as in Example 1.

[173] FIG. 10 shows an example of HBV detection in the case of using a reverse primer having Cy5 bonded to the 5 'end. On the other hand, for the sake of easy understanding of the present experiment, the upper part of FIG. The flow is additionally shown,

 11 shows a state in which no substance is bound to the 5 'end (there is a hydroxyl group present).

 The following shows an example of the HBV detection experiment when the reverse prime conditioner is used. For the sake of easy understanding of the experiment, the time series flow of the experiment is further illustrated in the upper part of FIG. U. Referring to this time series flow, the hybridization of the probe probe with the Cy5 coupled probe is performed. The principle of detecting a target can be easily understood.

 FIG. 12 shows an example of HBV detection in the case of using a reverse primer coupled with a ligand at the 5 'end. For the sake of easy understanding of the experiment, the upper part of FIG. 12 also shows the time series flow of the experiment. Further illustrated and referring to these time series flows, the principle of detecting targets hybridized to the capture probe can be easily understood by detecting the detection material bound to the ligand receptor.

 13 and 14 show that no material is bound to the 5 'end (i.e.,

 The results of the HBV detection using the reverse primer in the presence of a hydroxyl group and the ligand-coupled detection probe are shown. For the sake of easy understanding of the experiment, the time series flow of the experiment is also shown in the upper part of FIG. Further shown and referring to these time series flows, the principle of detecting targets localized in the capture probes can be easily understood by detecting the detection material bound to the ligand receptor.

 [178]

 [179] Third implementation 1: Detection of Mycoplasma pneumoniae

[180]

 [181] Synthesized Mycoplasma pneumoniae fragment base sequence containing 300 16S rRNA base sequences (GenBank: AF132740.1) and 240 SDC1 repetitive sequences (GenBank: M35024.1) from Mycoplasma pneumoniae strain (Cosmogenetech, Korea), The target polasmid for Mycop v! Ma pneumoniae was made by connecting to the T7 promoter.

 [182]

 [183] Mycoplasma pneumoniae fragment base:

 [184] 1 gaattccgca tgaatcaaag ttgaaaggac ctgcaagggt tcgttatttg atgagggtgc

[185] 61 gccatatcag ctagttggtg gggtaacggc ctaccaaggc aatgacgtgt agctatgctg

[186] 121 agaagtagaa tagccacaat gggactgaga cacggcccat actcctacgg gaggcagcag

[187] 181 tagggaattt ttcacaatga gcgaaagctt gatggagcaa tgccgcgtga acgatgaagg

[188] 241 tctttaagat tgtaaagttc ttttatttgg gaagaatgac tttagcaggt aatggctaga

[189] 301 gtttgattgg acagtgatgg ggggtacaag gccttggtgg aaaacacggc cgggctcaac

[190] 361 ggcccgatta atggcttgtt taccctgctc gacacctttg cgtatgtgac ccccgtgagt [191] 421 gggatgaaag gggggagtca gaataatgaa gaagtgcaaa cgacttaccc ggtcaagtcc

[192] 481 gaccaaaagg ccaccgccaa aattgcctcc ttaattaatg ccagcccact caacagttat

[193] 541 ggggatgtcg ac

[194]

 [195] A target primer is prepared by using a T7 RNA polymerase to form a target RNA, and a forward primer having a phosphate group bound to the 5 'end thereof.

 5'-GCTGAGAAGTAGAATAGCCACAATGGGACTGAG-3 ', a reverse primer in which no substance is bound to the 5' end (i.e.

 Reverse transcription-recombinase polymerase amplification (RT-RPA) was performed using 5'-TAGCCATTACCTGCTAAAGTCATTCTTCCCAAA-3 '.

 [196] A 15-boom RT-RPA reaction was performed at 38 ° C using a TwistAmp Basic kit from Mycoplasma pneumoniae target RNA, reverse transcriptase, RNase inhibitor and TwistAmp (UK). After 10 minutes and half of the reaction at 80 ° C, 10 U of lambda exonuclease was added and the reaction was performed at 38 ° C for 5 minutes to synthesize single-stranded DNA.

 [197] The target sequence of Mycoplasma pneumoniae made with RT-RPA was 182 base pairs. The base sequence of the capture probe attached to the stationary phase to detect this is

5'-TTTTTTTnTTTTTTACAATGAGCGAAAGCTTGA-3 'and 5'-GCGTGAACGATGAAGGTCTT-C6-NH ₂ -3' was used as a detection probe.

An amine group (NH ₂ ) group; the combined detection probe was used in conjugation with the red microparticles (microspheres).

 [198] This red microparticle probe was manufactured as follows.

 50 microliters of red microparticles (Bangs Lab. Cat. No. DC02R) with carboxylated turbidity for 30 seconds were placed in a microtube and centrifuged for 10 minutes at 14,500 rpm in a 4 ° C centrifuge. The supernatant is then carefully removed. A supernatant with a removed carboxyl group is 50 ul of pH 4.5

 After adding 0.1M MES buffer, perform ultrasonic turbidity.

[199] Ultra-suspended microparticles were added with 2 ul of probe having an amine group (NH ₂ ) bound to the 3 'end at 100 u concentration, followed by mixing with a mixed stirrer and amine at the 3' end. After adding 2.5 ul of EDC solution to the mixed microtube, stir with a stirrer and reacting for 30 minutes at dark room temperature.Add 2.5 ul of EDC solution and stir with stirrer. Repeat for 30 minutes in a dark room. After adding and stirring 0.02% Tween 20 500 ul to the reaction product, centrifugation for 10 minutes at 14,500 rpm at 4 ° C, and remove the supernatant.

After adding 500 ul of 0.1% SDS solution to the tube containing the fine particle probe, perform ultrasonic turbidity for 30 seconds, and centrifugation at 14,500 rpm at 4 degrees. Remove the supernatant, add 100 ul of TE solution of ρΗ 8.0, perform ultrasonic turbidity, and store it in a light-blocked 4 degree refrigerator. Lateral flow assay

 It proceeded in the same manner as in the first embodiment.

[200] Figure 15 is a "Mycoplasma pnetwio in the case of using the sum passed Cy5 at the ^terminal, the detection result 5 of the reverse primer, Figure 16 (state in which that is, a hydroxyl group is present), the state not bonded any substance to the 5 'end The results of the detection of Mycoplasma pneumoniae using the red microparticle detection probe when the reverse primer was used. 15 and 16, the time series flow of each experiment is additionally shown, and with reference to such time series flow, the principle of detection according to the present invention can be easily understood. will be.

 [201]

 [202] The experimental examples described above were described using the lateral flow assay as an example.

 By way of example, the invention may be applied to other assays.

 On the other hand, according to one embodiment of the present invention, a sequence-specific nucleic acid detection kit using a nucleic acid terminal enzyme can be provided, and such a sequence-specific nucleic acid detection kit may be provided as described above. Can be used in detection methods or devices.

 For example, the sequence-specific nucleic acid detection kit,

 [205] a pair of primers; and

 Wherein the 5 ′ end of the pair of primers is bound to a phosphate group and the 5 ′ end of the other primer of the pair of primers is a block. The blocker is bound, and the nucleic acid protease may be to decompose the phosphate bound strand in a double helix amplification amplified by the pair of primers.

 In this kit, the blocker may be a detection material or a ligand.

 [208] For example, the sequence-specific nucleic acid detection kit is

 [209] a pair of primers; and

 [210] a nucleic acid protease; wherein the pair of primers

 The 5 'end of one primer is bound to a phosphate group, the 5' end of the other primer of the pair of primers is free of other substances (i.e., in the presence of a hydroxyl group), and It may be the breakdown of phosphate-bonded strands in a double helix amplified by a pair of primers.

 The above-described kits may also be configured to further include a capture probe coupled to a stationary phase, a control probe coupled to a stationary phase, and / or a detection probe.

 On the other hand, the detection probe may be a combination of a detection material or a ligand.

 The above-mentioned kits may further include samples.

Thus, it is obvious to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention without departing from the spirit and scope of the invention. Examples or variations It should also belong to the claims of the invention.

Claims

Claim  [Claim 1] A method for detecting sequence-specific nucleic acid using nucleic acid terminal enzyme  In  Double-stranded target nucleic acid sequence-5 ᅳ terminal (first end) of one strand of the double helix cannot be resolved by nucleic acid terminal enzyme, and 5 단 terminal (second end) of the other strand is nucleic acid terminal decomposition Mixing the enzyme with a nucleic acid terminal enzyme, which is capable of being degraded by an enzyme;  Capturing and hybridizing the result of the mixing step (hereinafter referred to as a mixture) with the probe; and  Detecting a target nucleic acid sequence localized in the capture probe; and a method for detecting a sequence-specific nucleic acid using a nucleic acid protease.  [Claim 2] In paragraph 1,  A sequence-specific nucleic acid sequence detection method using a nucleic acid terminal enzyme, characterized in that the detection substance or ligand is bound to the first end.  [Claim 3] In paragraph 1,  Specific nucleic acid sequence detection method using a nucleic acid terminal enzyme, characterized in that the phosphate group is not bound to the first end, and the phosphate group is bound to the second end.  [Claim 4] In paragraph 1,  The nucleic acid terminal enzyme is a sequence-specific nucleic acid sequence detection method using a nucleic acid terminal enzyme, characterized in that lambda nucleic acid terminal enzyme.  [Claim 5] In paragraph 1, The capture probe is a sequence-specific nucleic acid sequence detection method using a nucleic acid protease characterized in that the stationary phase is linked.

In that U port,  Amplifying the sample; further comprising:  The double-stranded target nucleic acid sequence is the sample is amplified, and the step of amplifying the sample, using a pair of primers, one of the pair of primers, the phosphate group is coupled to the 5 'end of the primer, The other end of a pair of primers has a 5-terminus of a hydroxyl group, or sequence-specific nucleic acid sequence detection using nucleic acid terminal enzymes characterized by binding of blockers. Way. In paragraph 1, Mixing the resultant of the hybridizing step with the detection probe; further comprising: The detecting step, Detecting a detection substance bound to the detection probe, or A sequence-specific nucleic acid sequence detection method using nucleic acid terminalases, characterized in that the step of detecting the detection material bound to the ligand receptor bound to the ligand bound to the detection probe. Article ： In paragraph 2 ， The detecting step, Detecting a detection substance bound to the first end, or detecting a detection substance bound to the ligand receptor bound to the ligand, sequence-specific nucleic acid sequence detection using nucleic acid terminal enzymes Way. In the sequence-specific nucleic acid detection kit using nucleic acid terminal enzyme, A pair of primers; and Contains nucleic acid protease; The 5 'end of the pair of primers one primer is a phosphate group, The 5 'end of the remaining primers Blockers are combined, and The nucleic acid terminalase is a sequence-specific nucleic acid detection kit using a nucleic acid terminalase, characterized in that to decompose the strand bound to the phosphate group in the double helix amplified by the pair of primers. According to claim 9, The blocker is a sequence-specific nucleic acid detection kit using a nucleic acid terminal enzyme characterized in that the detection material or ligand. In paragraph 9, A kit for sequence-specific nucleic acid detection using nucleic acid protease, further comprising a capture probe coupled to a stationary phase. According to claim 10, A kit for sequence-specific nucleic acid detection using nucleic acid terminal enzymes further comprising a control probe bound to a stationary phase. In paragraph 10, Detecting probe; further comprising  Kit for sequence-specific nucleic acid detection using nucleic acid protease. [Claim 14] The method of claim 13,  The detection probe is a sequence-specific nucleic acid detection kit using a nucleic acid terminal enzyme, characterized in that the detection material or ligand is bound.  15. The method of any one of paragraphs 9 to 14,  Kit for sequence-specific nucleic acid detection using a nucleic acid terminal enzyme characterized in that it further comprises a sample.  [Claim 16] In a sequence-specific nucleic acid detection kit using nucleic acid terminal enzyme,  A pair of primers; and  Contains nucleic acid protease;  The 5 ′ end of one pair of primer pairs has a phosphate group,  The 5 'end of the remaining pair of prime primers is the presence of a hydroxyl group, and  The nucleic acid terminal degradation effect i, a sequence-specific nucleic acid detection kit using a nucleic acid terminal enzyme, characterized in that to decompose the strand bound to the phosphate group in the double helix amplified by the pair of primers.  [Claim 17] In paragraph 16,  A kit for sequence-specific nucleic acid detection using nucleic acid protease, further comprising a capture probe coupled to a stationary phase. [Claim 18] In paragraph 17,  A kit for sequence-specific nucleic acid detection using nucleic acid terminal enzymes further comprising a control probe bound to a stationary phase. [Claim 19] In paragraph 16,  Characterized by further comprising a detection probe;  Kit for sequence-specific nucleic acid detection using nucleic acid protease. [Claim 20] In paragraph 19,  The detection probe is a sequence-specific nucleic acid detection kit using a nucleic acid terminal enzyme, characterized in that the detection material or ligand is bound.  [Claim 21] In any of paragraphs 16 to 20,  Kit for sequence-specific nucleic acid detection using a nucleic acid terminal enzyme characterized in that it further comprises a sample. [Claim 22] In a Sequence-Specific Nucleic Acid Detection Device Using Nucleic Acid Teratase In  The double-stranded target nucleic acid sequence has a 5 'end (first end) of one strand of a double helix that cannot be resolved by nucleic acid protease, and the 5' end (second end) of the other strand  Can be degraded by nucleic acid protease-and  A mixing portion in which the nucleic acid protease is mixed;  A hybridization unit that hybridizes the mixture mixed by the mixing unit with the probe; and  And a detection unit for detecting a target nucleic acid sequence from the hybridized by the hybridization unit. A sequence-specific nucleic acid sequence detection device using a nucleic acid terminal enzyme. [Claim 23] In paragraph 22,  An amplifying part for amplifying a sample; wherein the amplifying spiral target nucleic acid sequence is a sample amplified by the amplifying part,  In the amplification, a pair of primers is used,  The 5 'end of any one of the pair and the primer is bound to a phosphate group,  5. A sequence-specific nucleic acid sequence detection apparatus using nucleic acid terminal enzymes characterized in that the 5 'end of the pair of primers is not bound to anything, the detection material is bound, or the ligand is bound.  [Claim 24] In paragraph 22,  An apparatus for detecting sequence-specific nucleic acids using nucleic acid terminal enzymes, characterized in that the detection material is bound to the first end, or the ligand is bound or nothing is bound.  [Claim 25] In paragraph 22,  A sequence-specific nucleic acid sequence detection device using a nucleic acid terminal enzyme, characterized in that the phosphate group is not bound to the first end, and that the phosphate group is bound to the second end.  [Claim 26] In paragraph 22,  The nucleic acid terminal enzyme is a sequence-specific nucleic acid sequence detection device using a nucleic acid terminal enzyme, characterized in that lambda nucleic acid terminal enzyme.  [Claim 27] In paragraph 22,  The capture probe is a sequence-specific nucleic acid sequence detection device using a nucleic acid terminal enzyme characterized in that the stationary phase is linked.