WO2022092013A1 - Nucleic acid probe set comprising multiple nucleic acid probes - Google Patents

Nucleic acid probe set comprising multiple nucleic acid probes Download PDF

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WO2022092013A1
WO2022092013A1 PCT/JP2021/039268 JP2021039268W WO2022092013A1 WO 2022092013 A1 WO2022092013 A1 WO 2022092013A1 JP 2021039268 W JP2021039268 W JP 2021039268W WO 2022092013 A1 WO2022092013 A1 WO 2022092013A1
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nucleic acid
acid probe
target nucleic
detection
probe
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PCT/JP2021/039268
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Japanese (ja)
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洋介 川嶋
真史 道渕
光生 前野
明生 杉山
広道 鈴木
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東洋紡株式会社
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention relates to a nucleic acid probe set for detecting a target to be inspected with high sensitivity and a method of using the same.
  • a nucleic acid probe is an artificial nucleic acid having a base sequence complementary to a part or all of the target nucleic acid in order to detect a target nucleic acid (DNA or RNA) consisting of a specific base sequence, and is usually one. It is a chain nucleic acid.
  • the nucleic acid probe is often labeled with some substance and the signal from that substance is detected.
  • a fluorescent substance is used as the substance.
  • Typical examples of the nucleic acid probe labeled with a fluorescent substance include TaqMan (registered trademark) Probe and Qprobe (registered trademark).
  • nucleic acid probe By adjusting the base sequence of the nucleic acid probe itself according to the base sequence of the target nucleic acid, highly specific nucleic acid detection is possible.
  • a nucleic acid probe labeled with a fluorescent substance When a nucleic acid probe labeled with a fluorescent substance is used, a plurality of target nucleic acids can be detected and identified at the same time by labeling a nucleic acid probe having a plurality of different base sequences with a fluorescent substance having a different detection wavelength. It will be possible to do.
  • the technique of amplifying a plurality of target nucleic acids by PCR is called multiplex PCR, and the technique of detecting and differentiating each target nucleic acid using a plurality of nucleic acid probes is called multiplex real-time PCR.
  • a plurality of nucleic acid probes in which each nucleic acid probe is labeled with a fluorescent dye having a different detection wavelength are used in combination, and this is sometimes referred to as multicolor multiple
  • nucleic acid probe labeled with one kind of fluorescent dye usually only one kind of base sequence of the nucleic acid probe is used. This is because there is no way to distinguish one type of fluorescent dye even if it is labeled on multiple nucleic acid probes.
  • Patent Document 1 a method of detecting and identifying different target nucleic acids by using a plurality of nucleic acid probes labeled with one kind of fluorescent dye is known (Patent Document 1). In this method, different target nucleic acids are detected by using a plurality of nucleic acid probes having different Tm values and combining complicated annealing temperature conditions and temperature change profile design.
  • the conventional method using a plurality of nucleic acid probes has been used for differentiating and detecting a plurality of target nucleic acids.
  • Nested PCR is a method in which a first PCR is performed on a target nucleic acid to obtain an amplification product, and a second PCR is performed using the amplification product as a sample.
  • the amplified product can be detected by repeating PCR, so that the amount is very small. It enables detection of target nucleic acid.
  • nested PCR is a method of performing PCR multiple times, nucleic acid amplification requires time. Therefore, it may not be suitable for situations that require rapid nucleic acid detection results. Further, even in such a nested PCR method, one type of nucleic acid probe usually labeled with one type of fluorescent dye, or a plurality of nucleic acid probes labeled with a fluorescent substance having a different detection wavelength for each target nucleic acid has been used. rice field.
  • An object of the present invention is to provide a further useful nucleic acid detection system capable of detecting a target to be tested with high sensitivity.
  • the present inventors have used a set of nucleic acid probes having a plurality of different base sequences labeled with fluorescent dyes that can be detected at the same wavelength, and detected them without distinction, thereby detecting a nucleic acid detection signal.
  • the method of the present invention does not require the design of complex annealing temperature conditions or temperature change profiles and can be utilized in general-purpose nucleic acid detection methods such as, for example, real-time PCR, real-time RT-PCR, or melting curve analysis. That is, the outline of the present invention is as follows.
  • nucleic acid probe set according to Item 1 wherein the nucleic acid probe set comprises the first target nucleic acid probe and the second target nucleic acid probe.
  • nucleic acid probe set according to Item 1 or 2 wherein the fluorescent dyes that can be detected at the same wavelength are the same fluorescent dyes.
  • the nucleic acid detection system of the present invention does not distinguish the detection signals generated by individual nucleic acid probes, it is possible to superimpose a plurality of detection signals derived from each nucleic acid probe, thereby obtaining a clearer detection signal than usual. Therefore, when the present invention is applied to, for example, detecting the presence of a specific microorganism, a plurality of target nucleic acid probes corresponding to a plurality of genes possessed by the microorganism are designed, and the detection of those target nucleic acid probes is attempted. Therefore, if any one of the target nucleic acids is detected, it can be determined that the microorganism is present. Since the present invention can be used in general-purpose real-time PCR, real-time RT-PCR, nucleic acid detection methods such as melting curve analysis, it is possible to easily construct a highly sensitive nucleic acid detection system.
  • FIG. 1 is a graph showing the results of melting curve analysis when reagent 10 (CT + NG) is used in Example 4.
  • FIG. 2 is a graph showing the results of melting curve analysis when reagent 11 (CT) is used in Example 4.
  • FIG. 3 is a graph showing the results of melting curve analysis when reagent 12 (NG) is used in Example 4.
  • the targeting primer may be referred to as a nucleic acid primer, an oligonucleotide primer or simply a primer
  • the target nucleic acid probe may be referred to as a nucleic acid probe, an oligonucleotide probe or simply a probe, and these are collectively referred to as an oligonucleotide. Also called.
  • nucleic acid probe set is a nucleic acid probe set comprising two or more nucleic acid probes.
  • the nucleic acid probe set includes at least a first target nucleic acid probe and a second target nucleic acid probe having the following characteristics (A) to (C):
  • A) The first target nucleic acid probe and the second target nucleic acid probe have different base sequences.
  • B) The first target nucleic acid probe and the second target nucleic acid probe are labeled with a fluorescent dye that can be detected at the same wavelength.
  • C The first target nucleic acid probe and the second target nucleic acid probe bind to different target nucleic acids, but are detected so as not to distinguish each target nucleic acid.
  • the nucleic acid probe set includes one or more additional target nucleic acid probes (eg, a third target nucleic acid probe, a fourth target nucleic acid probe, in addition to the first target nucleic acid probe and the second target nucleic acid probe. , Fifth target nucleic acid probe, etc.) may be included.
  • Such a further target nucleic acid probe may have all the characteristics of the above (A) to (C) like the first target nucleic acid probe and the second target nucleic acid probe, or may have the above-mentioned (A). )-(C) may not be provided (for example, those labeled with a fluorescent dye detected at different wavelengths).
  • the number of nucleic acid probes constituting the nucleic acid probe set of the present invention is not particularly limited as long as it is two or more. It is preferably two to five, and more preferably two or three. Among them, a nucleic acid probe set including the first target nucleic acid probe and the second target nucleic acid probe is preferable.
  • the nucleic acid probe set of the present invention may be used in combination with other nucleic acid probes.
  • the nucleic acid probes constituting the nucleic acid probe set have different base sequences.
  • “different” in the embodiment for example, when there are nucleic acid probe A and nucleic acid probe B, it means that there is even one difference in the base sequence of the oligonucleotides constituting each nucleic acid probe.
  • the identity of the base sequence of each nucleic acid probe is preferably 99% or less, and more preferably the identity of the base sequence of each nucleic acid probe is 95% or less, 90% or less, 85% or less. , 80% or less, 75% or less.
  • the identity of the base sequence of each nucleic acid probe may be 50% or less. In the present invention, by using a plurality of nucleic acid probes having low identity with each other as described above, more sensitive detection may be possible.
  • each nucleic acid probe is labeled with a fluorescent dye.
  • the labeling site in the base sequence of each nucleic acid probe is not particularly limited, and the labeling site of each nucleic acid probe may be the same or different.
  • one end of each nucleic acid probe is labeled with a fluorescent dye.
  • each nucleic acid probe can be used with only one end labeled with a fluorescent dye. A known labeled substance can be used as the fluorescent dye.
  • fluorescent dyes examples include rhodamine-based compounds; fluorosane-based compounds (eg, FAM (carboxyfluorescein), ALEXA FLUOR, 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-. Diaza-s-Indacene-3-propionic Acid (BODIPY-FL), Carboxyl Rhodamine 6G, TAMRA, Rhodamine 6G, Tetrabromosulfone Fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H- Examples thereof include, but are not limited to, 1-benzopyran-3-carboxylic acid (Pacific Blue).
  • fluorosane-based compounds eg, FAM (carboxyfluorescein), ALEXA FLUOR, 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-.
  • the terminal opposite to the fluorescently labeled end may be an unlabeled oligonucleotide or may be labeled with some substance. ..
  • labeling substances include quenching substances, and examples thereof include, but are not limited to, BHQ1 (BHQ: Black Hole Quencher (registered trademark)), BHQ2, and BHQ3.
  • the term "detectable at the same wavelength” means that a commercially available nucleic acid detection device (for example, a measuring device capable of real-time PCR or melting curve analysis) has a fluorescence wavelength similar to the extent that it can be detected on the same detection channel. ..
  • the device typically has multiple fluorescence detection channels. Taking Rotor-Gene Q (QIAGEN) as an example, Rotor-Gene Q has six types of fluorescence detection channels, each of which has a different detection wavelength. Then, a plurality of fluorescent dyes can be detected for each detection wavelength. Specifically, it is as shown in Table 1.
  • FAM and SYBR Green I are detected on the same fluorescent channel.
  • the maximum detection wavelength (maximum fluorescence wavelength) of FAM is 520 nm
  • the maximum detection wavelength of SYBR Green I is 521 nm
  • the maximum detection wavelength of Eva Green is 525 nm, but these fluorescent dyes are not distinguished when fluorescence is detected.
  • FAM, SYBR Green I, and Eva Green can be detected at the same wavelength.
  • a group of fluorescent dyes that can be detected in each channel as shown in Table 1 above can be used as fluorescent dyes that can be detected at the same wavelength, but the present invention is not limited to these, and an equivalent maximum detection wavelength can be used. It is possible to use any fluorescent dye having.
  • the difference in the numerical value of the maximum detection wavelength of each fluorescent dye is not particularly limited as long as the effect of the present invention is achieved, and as described above, when fluorescence is actually detected.
  • Fluorescent substances that are not normally distinguished in the art can be said to be fluorescent substances that can be detected at the same wavelength.
  • the difference in the maximum detection wavelengths of the plurality of different fluorescent dyes is preferably 40 nm or less, more preferably 30 nm or less, still more preferably 25 nm or less. Is good.
  • the difference in the maximum detection wavelength of each fluorescent dye may be, for example, within 20 nm, within 15 nm, within 10 nm, within 5 nm, or even if the maximum detection wavelengths of each are substantially the same and there is no difference. good.
  • the same fluorescent material is used as the fluorescent material that can be detected at the same wavelength.
  • by using a fluorescent substance that can be detected at the same wavelength as in the present invention it is possible to easily target an analyzer equipped with one or a small number of fluorescent channels, even if the analyzer is not equipped with many types of fluorescence detection channels. Can be detected.
  • each nucleic acid probe constituting the nucleic acid probe set binds to a different target nucleic acid. Therefore, each nucleic acid probe detects a different target nucleic acid by emitting or quenching by binding or dissociating to the target nucleic acid.
  • One of the features of the present invention is to design and use a plurality of nucleic acid probes so as to detect such different target nucleic acids without daring to discriminate them.
  • the target nucleic acid is a nucleic acid to be detected by a nucleic acid probe, and is usually an amplification product produced by a nucleic acid amplification method.
  • Nucleic acid amplification method is a technology that amplifies several copies of target nucleic acid to a level that can be visualized, that is, to hundreds of millions of copies or more, and is widely used not only in the field of life science research but also in the fields of clinical diagnosis, food hygiene inspection, environmental inspection, etc. It is used.
  • Examples of such nucleic acid amplification methods include PCR method, LAMP method, LCR method, TMA method, SDA method, RT-PCR method, RT-LAMP method, NASBA method, TRC method, TMA method and the like. These techniques have already been established in the relevant technical field, and the method can be selected according to the purpose.
  • the nucleic acid amplification method performed in the present invention is preferably, but not limited to, the PCR method (including the RT-PCR method).
  • a nucleic acid primer set is used to amplify the base sequence sandwiched between the primer sets. Therefore, amplification products produced using different nucleic acid primer sets have different base sequences and are therefore different target nucleic acids.
  • one or more primary target primers capable of amplifying the region containing the sequence of the first target nucleic acid to which the first target nucleic acid probe can bind, and the second target nucleic acid probe.
  • the amplification of the first target nucleic acid and the amplification of the second target nucleic acid may be performed simultaneously or separately. From the viewpoint that the nucleic acid amplification reaction can be carried out in a shorter time, it is preferable to simultaneously perform the amplification of the first target nucleic acid and the amplification of the second target nucleic acid.
  • the first target nucleic acid and the second target nucleic acid detected by the first target nucleic acid probe and the second target nucleic acid probe are not differentiated.
  • the detection signal generated by each target nucleic acid probe is detected as the same color.
  • the detection signals detected as the same color in this way are measured, for example, in melting curve analysis, the fluorescence peaks are superposed and the target nucleic acids are not discriminated by making the detection temperatures uniform. To detect.
  • the detection signal refers to a change in fluorescence caused by a nucleic acid probe detecting a target nucleic acid.
  • the mode of the detection signal differs depending on the fluorescent dye or light-dissipating substance labeled on the nucleic acid probe, the chemical reaction occurring between the nucleic acid probe and the target nucleic acid, the structure of the device used for detection, etc., but the present invention has a specific mode of the detection signal.
  • the detection signal may be any fluorescence change phenomenon indicating the detection of the target nucleic acid by the nucleic acid probe.
  • Examples of the aspect of the detection signal include luminescence over time in real-time PCR (including real-time RT-PCR).
  • real-time PCR the amplified product produced is detected by changes in fluorescence over time.
  • a method using a TaqMan probe can be mentioned.
  • the TaqMan probe is a nucleic acid probe in an artificially synthesized single-stranded DNA labeled with a fluorescent dye at one end and a quencher at the other end. In the TaqMan probe, the emission of the fluorescent dye is suppressed by the quenching substance.
  • the TaqMan probe bound to the target nucleic acid is degraded by the 5' ⁇ 3'exonuclease activity of the TaqDNA polymerase in the annealing step of the PCR cycle, which complementarily binds to the target nucleic acid, and in the subsequent extension step, it fluoresces.
  • the dye is released from the probe, the suppression by the quencher is released, and the fluorescence is emitted.
  • the amount of amplification product, which is the target nucleic acid increases, more TaqMan probes are degraded and luminescence is enhanced.
  • the number of PCR cycles when the fluorescence due to the decomposition of the TaqMan probe exceeds the range value is referred to as Ct (Threshold Cycle).
  • the detection signal in real-time PCR is not limited, but the emission of the probe as described above can be mentioned, and the strength of this detection signal can be evaluated by the output of Ct or the like.
  • melting curve analysis As a different aspect of the detection signal, detection of fluorescence change by melting curve analysis (also referred to as melting curve analysis) can be mentioned. Detection and analysis of target nucleic acid using Tm value is called melting curve analysis.
  • the Tm value refers to the temperature at which the proportion of an oligonucleotide forming a double strand with its complementary strand and the proportion of a single strand without forming a double strand are equal. Since the Tm value is a value unique to the base sequence, melting curve analysis is used, for example, as a method for analyzing the base sequence polymorphism of the target nucleic acid.
  • the base sequence polymorphism referred to here includes a single nucleotide polymorphism, a base substitution, a base defect, a base insertion, and the like.
  • the melting curve analysis is performed, for example, as follows. As the solution containing the double-stranded DNA is heated, the absorbance at 260 nm increases. This is because the hydrogen bonds between the two strands in the double-stranded DNA are unwound by heating and dissociated into the single-stranded DNA (melting of the DNA). Then, when all the double-stranded DNA is dissociated into a single-stranded DNA, the absorbance thereof shows about 1.5 times the absorbance at the start of heating (the absorbance of only the double-stranded DNA), thereby melting. Can be judged to be completed. In this case, the difference in absorbance before and after heating is regarded as a detection signal by melting curve analysis.
  • Another aspect of the melting curve analysis is a method using a nucleic acid probe labeled with a fluorescent dye, which may be suitable for target detection with a fluorescent signal in the present invention.
  • a nucleic acid probe having a characteristic that fluorescence is extinguished when a complex is formed with a target nucleic acid and fluorescence is emitted when released from the target nucleic acid is incorporated into the reaction system of the nucleic acid amplification method, and after the nucleic acid amplification method is completed.
  • the reaction system is cooled at a low temperature to bind the nucleic acid probe to the target nucleic acid.
  • the fluorescent dye of the nucleic acid probe is quenched.
  • the nucleic acid probe is released from the target nucleic acid at a temperature near the Tm value of the nucleic acid probe.
  • the fluorescence of the nucleic acid probe emits light, a significant change (increase) in fluorescence occurs at a temperature near the Tm value.
  • the change in fluorescence is regarded as a detection signal.
  • a labeled probe whose terminal base labeled with a fluorescent quenching dye is cytosine is exemplified.
  • a nucleic acid probe hybridizes to a target nucleic acid, it can be quenched by forming a base pair with a guanine base in the target nucleic acid sequence and interacting with the target nucleic acid sequence. Also known), etc., and it is preferable because the change in fluorescence intensity can be measured very easily.
  • the present invention is characterized in that these nucleic acid probes are designed so that the detection signals generated by the first target nucleic acid probe and the second target nucleic acid probe constituting the nucleic acid probe set overlap. This makes it possible to detect each target nucleic acid without differentiating even if the first target nucleic acid probe and the second target nucleic acid probe bind to different target nucleic acids.
  • Overlapping detection signals means, for example, in the case of real-time PCR, when multiple nucleic acid probes labeled with fluorescent dyes that can be detected at the same wavelength are used, the Ct is lower than when each nucleic acid probe is used alone.
  • the TaqMan probe decomposed after binding to the target nucleic acid emits fluorescence, and Ct is output when the amount of fluorescence exceeds the threshold value.
  • Multiple nucleic acid probes labeled with a fluorescent dye that can be detected at the same wavelength bind to different target nucleic acids and are degraded, thereby enhancing the fluorescence detected at the same wavelength in the entire reaction system and increasing Ct earlier. It will be easier to obtain.
  • a plurality of nucleic acid probes bind to each target nucleic acid and then release and emit light.
  • the detection temperature by each target nucleic acid probe is significantly different, and the detection signal may be differentiated. ..
  • the detection signal is detected at almost the same temperature when the melting curve analysis is performed. Is obtained, and each target nucleic acid can be detected so as not to be differentiated.
  • the difference in Tm value between the target nucleic acid probes for detecting the target nucleic acid so that the detection signal appears at substantially the same temperature and does not discriminate between the target nucleic acids is preferably within 5 ° C., more preferably. It is within 4 ° C, more preferably within 3 ° C.
  • the difference in Tm value between the target nucleic acid probes may be within 2.5 ° C, within 2 ° C, within 1.5 ° C, or may have substantially no difference in Tm value. ..
  • fluorescence that can be detected at the same wavelength at a predetermined detection temperature by keeping the difference between the Tm values of the first target nucleic acid probe and the second target nucleic acid probe within the above range. Detection signals due to changes can be obtained, and these detection signals can be superimposed.
  • the Tm value of the target nucleic acid probe can be obtained by a method known in the art, and those skilled in the art can easily design a target nucleic acid probe having a predetermined Tm value.
  • the Tm value can be determined by the total number of bases constituting the nucleic acid probe and the base ratio in the base sequence (what percentage of A, T, C, G are present).
  • As a method for calculating the Tm value for example, the closest base pair method, the Wallace method, the GC% method and the like are known.
  • the difference in Tm value between the first target nucleic acid probe and the second target nucleic acid probe when the same Tm value calculation method is used is used. Is preferably designed so as to be within the range specified above.
  • the difference in the base length between the base sequence of the first target nucleic acid probe and the base sequence of the second target nucleic acid probe is not particularly limited as long as the effect of the present invention is exhibited.
  • a base is preferably used between the first target nucleic acid probe and the second target nucleic acid probe. It is preferable that there is no extreme difference in length.
  • the difference in base length between the first target nucleic acid probe and the second target nucleic acid probe is preferably 8 mer or less, more preferably 5 mer or less, and further preferably 3 mer or less.
  • the lower limit of the difference in base length between the first target nucleic acid probe and the second target nucleic acid probe may be 0 mer with no difference in base length, 1 mer or more, or 2 mer or more. You may.
  • the first target nucleic acid probe and the second target nucleic acid probe are preferably composed of a base sequence that is completely complementary to each target nucleic acid.
  • a target nucleic acid probe composed of a completely complementary base sequence it may be possible to detect the target of interest more accurately.
  • completely complementary means that the base sequence constituting the target nucleic acid probe is complementary to the base sequence of the target nucleic acid over its entire length, and does not contain a mismatched base.
  • the method of the invention comprises performing a nucleic acid amplification reaction with a nucleic acid primer set to produce a nucleic acid amplification product.
  • Nucleic acid amplification method is a technology that amplifies several copies of target nucleic acid to a level that can be visualized, that is, to hundreds of millions of copies or more, and is widely used not only in the field of life science research but also in the fields of clinical diagnosis, food hygiene inspection, environmental inspection, etc. It is used.
  • nucleic acid amplification methods examples include PCR method, LAMP method, LCR method, TMA method, SDA method, RT-PCR method, RT-LAMP method, NASBA method, TRC method, TMA method and the like. These techniques have already been established in the relevant technical field, and the method can be selected according to the purpose.
  • the nucleic acid amplification method performed in the present invention is preferably, but not limited to, the PCR method (including the RT-PCR method).
  • the PCR reaction is a reaction mainly catalyzed by DNA polymerase, (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), and (2) primer to template single-stranded DNA.
  • the three steps of annealing and (3) extension of the primer using DNA polymerase are set as one cycle, and the target nucleic acid is amplified by repeating this cycle.
  • the DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT and their variants.
  • a DNA polymerase belonging to Family B it is preferable to use a DNA polymerase belonging to Family B from the viewpoint of rapidity, high sensitivity, and resistance to amplification inhibition by a sample. Further, when performing the melting curve analysis method, it is preferable to use a DNA polymerase belonging to Family B which does not have 5' ⁇ 3'exonuclease activity from the viewpoint of using a fluorescent quenching probe.
  • the conditions of the PCR reaction are not particularly limited as long as the effects of the present invention are exhibited.
  • the first thermal deformation step is at 80 to 100 ° C. for 0 seconds to 5 minutes
  • the repeated thermal deformation step is at 80 to 100 ° C. at 0.
  • the Annie Link is carried out at 35 to 80 ° C. for 1 to 300 seconds
  • the extension reaction step is carried out at 35 to 85 ° C. for 1 to 300 seconds, and this repetition is repeated 30 to 70 times.
  • the temperature and time of the cycle repeated here may be changed every one to several cycles.
  • the DNA polymerase used in the present invention is preferably, but is not limited to, a DNA polymerase belonging to Family B.
  • the DNA polymerase belonging to the family B is not particularly limited, but is preferably a DNA polymerase derived from archaea, and more preferably derived from a bacterium belonging to the genus Pyrococcus and the genus Thermococcus. Examples include DNA polymerase.
  • the present invention also includes variants thereof that have not lost their DNA polymerase activity from archaea belonging to Family B.
  • Variants of DNA polymerase include, but are not limited to, mutants for the purpose of enhancing polymerase activity, deficiency of exonuclease activity, adjusting substrate specificity, and the like.
  • Examples of the DNA polymerase derived from the genus Pyrococcus include Pyrococcus furiosus and Pyrococcus sp. DNA polymerases isolated from GB-D, Pyrococcus womeni, Pyrococcus abyssi, Pyrococcus horikoshii, and variants thereof that have not lost their DNA polymerase activity, but are not limited thereto.
  • Examples of the DNA polymerase derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermococcus gogonarius, Thermococcus litoralis, and Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), DNA polymerase isolated from Thermococcus siculi, and variants thereof that have not lost their DNA polymerase activity, but are not limited thereto.
  • a DNA polymerase derived from Thermococcus kodakaranesis and a variant thereof are excellent in extensibility and thermal stability.
  • PCR enzymes using these DNA polymerases are commercially available, Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), DeepBent (New) , Tgo (Roche), Pwo (Roche) and the like, all of which can be used in the present invention.
  • a KOD-derived DNA polymerase (also referred to as a KOD DNA polymerase) is a DNA polymerase derived from Thermococcus kodakaranesis and a variant thereof (for example, one or several amino acids are substituted or deleted in a naturally occurring amino acid sequence). , And / or KOD-derived DNA polymerase, etc., in which 3' ⁇ 5'exonuclease activity has been deleted by addition.
  • the present invention uses such a KOD-derived DNA polymerase to carry out a nucleic acid amplification reaction.
  • KOD DNA polymerase is superior to Taq DNA polymerase, which is a DNA polymerase belonging to Family A, in accuracy, amplification efficiency, extensibility, and resistance to amplification inhibition by a sample-derived inhibitor.
  • the present invention can be used in situations where nucleic acids are detected in a target from any sample.
  • the sample that can be used in the present invention is not particularly limited as long as it may contain a plurality of target nucleic acids, and examples thereof include not only biological samples, foods, and environmental samples, but also purified nucleic acids and the like.
  • the sample may be subjected to nucleic acid extraction and some pretreatment. Nucleic acid extraction and pretreatment of samples are commonly performed in the art. Examples of the pretreatment include, but are not limited to, filtration, centrifugation, dilution treatment, heat treatment, acid treatment, alkali treatment, organic solvent treatment, suspension treatment, crushing treatment, and grinding treatment.
  • biological samples include, but are not limited to, animal and plant tissues, body fluids, excrement, cells, bacteria, viruses and the like. Further, blood, blood culture medium, urine, pus, spinal fluid, pleural fluid, pharyngeal swab, nasal swab, sputum, tissue section, skin, vomitus, feces, isolated culture colony, catheter lavage fluid, cervical scraping , Urethral scrapes, male urethral scrapes, urine and the like.
  • Examples of foods include water, alcoholic beverages, soft drinks, processed foods, vegetables, livestock products, marine products, eggs, dairy products, raw meat, raw fish, prepared foods and the like.
  • a food is used as a measurement sample, not only a part or all of the food can be used, but also a food whose surface has been wiped off can be used. Further, a material for wiping off a cooking utensil or a doorknob or a cleaning liquid for cleaning them can also be used as a sample.
  • Examples of environmental samples include water, ice, soil, air and aerosols.
  • Examples of water here include tap water, seawater, and water collected from rivers, waterfalls, lakes, ponds, and the like.
  • a cleaning solution obtained by wiping the wall surface, floor surface, equipment / equipment, toilet bowl, etc. of the facility or cleaning them can also be used as a sample.
  • any sample as described above can be used in the present invention, but it is preferably a sample that can contain an exogenous target nucleic acid.
  • the present invention is particularly effective for a subject or a sample taken from a subject suspected of being infected with one or more infectious microorganisms.
  • the type of sample to be collected it is preferable to select an appropriate sample each time based on the symptoms of the infectious disease of the subject, the condition of the subject, and the like.
  • Biological samples such as saliva can be used.
  • the method for collecting a sample, the method for preparing a sample, and the like are not particularly limited, and a known method can be used depending on the type and purpose of the sample.
  • the present invention can be used in any situation where it is desired to detect a target to be inspected with high sensitivity.
  • it can be suitably used when it is desired to detect a nucleic acid derived from a microorganism that can be contained in a very small amount in a sample.
  • a plurality of target nucleic acid probes corresponding to each of a plurality of different regions in the genomic sequence of the infectious microorganism are designed and nucleic acid probes. Make a set.
  • a plurality of target nucleic acid probes corresponding to the target nucleic acids derived from each infectious microorganism should be designed and the nucleic acid probe. It can be a set.
  • the invention provides a reagent composition comprising said nucleic acid probe set.
  • the invention may be a kit comprising said nucleic acid probe set or reagent composition.
  • the reagent composition or kit of the present invention in addition to the nucleic acid probe set containing at least the first target nucleic acid probe and the second target nucleic acid probe, for example, a sequence of a first target nucleic acid to which the first target nucleic acid probe can bind.
  • One or more regions containing a first target primer (or primer set) capable of amplifying a region containing, and a region containing a sequence of a second target nucleic acid to which a second target nucleic acid probe can bind.
  • the present reagent composition or kit further preferably contains at least an inorganic salt such as DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and magnesium salt in order to efficiently perform nucleic acid amplification with high specificity.
  • concentration of each component can be adjusted as appropriate, but for example, the oligonucleotide probe (first target nucleic acid probe or second target nucleic acid probe) is preferably 0.1 to 1.5 ⁇ M, more preferably 0.2 to 0.5 ⁇ M.
  • the DNA polymerase is preferably 0.01 to 1 U / uL, more preferably 0.1 to 0.5 U / uL.
  • the oligonucleotide primer (primer for first target or primer for second target) is preferably 0.1 to 10 ⁇ M.
  • Deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM.
  • the inorganic salt such as magnesium salt is preferably 0.1 to 6 mM, more preferably 1 to 5 mM.
  • the reagent composition or kit of the present invention may further contain additives and the like known in the art for the purpose of suppressing non-specific amplification and promoting the reaction.
  • additives for the purpose of suppressing non-specific amplification include anti-DNA polymerase antibodies and phosphoric acid.
  • Additives aimed at promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur or acetic acid-containing compounds, dimethylsulfoxide (DMSO), glycerol, Ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectin, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMC), tetramethylammonium hydroxide (TMAH), tetramethylacetate Examples thereof include ammonium (TMA), polyethylene glycol, Triton, Tween 20 and Nonidet P40. In the present invention, one or more of these additives may be used in combination, but the present invention is not limited thereto.
  • BSA bovine serum albumin
  • SSB single strand binding protein
  • T4 gene 32 protein t
  • nucleic acid detection method or nucleic acid detection sensitivity improvement method provides a method for detecting nucleic acids using a nucleic acid probe set, reagent composition, and / or kit as described above.
  • nucleic acid probes having a plurality of different base sequences labeled with fluorescent dyes that can be detected at the same wavelength are used as a set, and by detecting them without distinction, the nucleic acid detection signals are duplicated and the test target is used.
  • Target can be detected with high sensitivity. Therefore, the nucleic acid detection method of the present invention can also be said to be a method for improving the nucleic acid detection sensitivity in the detection of a target to be inspected.
  • the nucleic acid detection method or the nucleic acid detection sensitivity improving method of the present invention can be carried out by any nucleic acid detection method known in the art. Preferably, it can be carried out in real-time PCR, real-time RT-PCR, or melting curve analysis, and in particular, it can be preferably carried out in a method of detecting fluorescence by melting curve analysis.
  • Example 1 Confirmation of effect of nucleic acid probe set
  • the experiment described in this example was carried out for the purpose of confirming whether or not the nucleic acid signal was enhanced more than the nucleic acid signal obtained when the same nucleic acid amplification and detection was performed.
  • MP Mycoplasma pneumoniae
  • GeneCube Test Basic (Toyobo) is used, and the following two nucleic acid probes and their corresponding nucleic acid primer sets and samples are included.
  • a solution (reagent 1) was prepared.
  • Reagent 2 a solution using the nucleic acid probe constituting the nucleic acid probe set and the corresponding nucleic acid primer set alone was prepared using GeneCube Test Basic.
  • the Tm value of the probe shown by SEQ ID NO: 5 used in this example is 54.2 ° C.
  • the Tm value of the probe shown by SEQ ID NO: 6 is 54.0 ° C., so that the detection temperatures in the melting curve analysis are close to each other. Designed for.
  • the Tm value was calculated by the closest base pairing method. In the following examples as well, those in which the Tm value was expressed were similarly calculated by the nearest base pairing method.
  • the probes of SEQ ID NOs: 5 and 6 both have a base sequence complementary to the DNA sequence of MP and are labeled with the same fluorescent dye.
  • Reagent 1 Gene Cube Test Basic 0.3 ⁇ M Primer represented by SEQ ID NO: 1 1.5 ⁇ M Primer represented by SEQ ID NO: 2 0.3 ⁇ M Primer represented by SEQ ID NO: 3 1.5 ⁇ M Primer shown in SEQ ID NO: 4 0.25 ⁇ M Probe shown in SEQ ID NO: 5 (labeled 3'end with BODIPY-FL) 0.25 ⁇ M Probe shown in SEQ ID NO: 6 (labeled 3'end with BODIPY-FL)
  • Reagent 3 Gene Cube Test Basic 0.3 ⁇ M Primer represented by SEQ ID NO: 3 1.5 ⁇ M Primer shown in SEQ ID NO: 4 0.25 ⁇ M Probe shown in SEQ ID NO: 6 (labeled 3'end with BODIPY-FL)
  • Nucleic acid amplification and melting curve analysis 97 ° C for 30 seconds (More than 1 cycle) 97 ° C for 1 second 58 ° C for 3 seconds 63 ° C for 5 seconds (More than 50 cycles) 94 ° C for 30 seconds 39 ° C for 30 seconds 40 ° C to 75 ° C (temperature rises at 0.09 ° C / sec)
  • Result Table 2 is a table summarizing the results of detection by nucleic acid amplification and melting curve analysis under the above conditions (the upper row shows the positive judgment result, and the lower row shows the fluorescence peak height (No peak confirms the peak). Indicates that it could not be done)).
  • Reagent 1 containing the nucleic acid probe set 80 copies / test MP DNA could be detected with 100% probability, and 40 copies / test DNA could be detected with 50% probability.
  • reagent 2 had 80 copies / test of 50%
  • reagent 3 had 80 copies / test DNA of 100%
  • reagent 2 and reagent 3 had a detection rate of 40 copies / test of 0%. From the above, it is clear that it is possible to improve the detection rate from a sample having a trace concentration by detecting two different types of target nucleic acids using the composition containing the nucleic acid probe set of the present invention. rice field.
  • Example 2 Confirmation of effect of nucleic acid probe set in real-time PCR
  • (1) Purpose In order to confirm that the composition containing the nucleic acid probe set of the present invention is also effective in real-time PCR, an experiment was conducted using a TaqMan probe.
  • (3) Nucleic acid amplification and nucleic acid detection Each of the above samples was added to the following reagents, and MP was detected under the following conditions.
  • a solution (reagent 4) containing the following two nucleic acid probes and their corresponding nucleic acid primer sets and samples was prepared. Further, as a comparison with the reagent 4, using THUNDERBIRD Probe qPCR Mix (Toyobo), a nucleic acid probe constituting the nucleic acid probe set and a solution (reagent 5 and reagent 6) using the corresponding nucleic acid primer set alone were prepared. ..
  • the probes of SEQ ID NO: 7 (Tm value 68.6 ° C) and 8 (Tm value 66.0 ° C) used in this example both have a base sequence complementary to the DNA sequence of MP and are the same. It is labeled with a fluorescent dye and a quenching substance.
  • Reagent 4 THUNDERBIRD Probe qPCR Mix 0.5 ⁇ M Primer represented by SEQ ID NO: 1 0.5 ⁇ M Primer represented by SEQ ID NO: 2 0.5 ⁇ M Primer represented by SEQ ID NO: 3 0.5 ⁇ M Primer shown in SEQ ID NO: 4.
  • 0.1 ⁇ M Probe represented by SEQ ID NO: 7 (labeled 5'end with FAM and 3'end with BHQ1)
  • 0.1 ⁇ M Probe represented by SEQ ID NO: 8 (labeled 5'end with FAM and 3'end with BHQ1)
  • Reagent 5 THUNDERBIRD Probe qPCR Mix 0.5 ⁇ M Primer represented by SEQ ID NO: 1 0.5 ⁇ M Primer represented by SEQ ID NO: 2 0.1 ⁇ M Probe represented by SEQ ID NO: 7 (labeled 5'end with FAM and 3'end with BHQ1)
  • Nucleic acid amplification and detection 95 ° C for 2 minutes (More than 1 cycle) 95 ° C for 10 seconds 60 ° C for 30 seconds (fluorescence detection in this step) (More than 50 cycles)
  • Result Table 3 is a table summarizing the Ct values obtained by performing nucleic acid amplification and fluorescence detection by real-time PCR under the above conditions. As shown in this result, smaller Ct was output by using a combination of multiple nucleic acid probes having the same detection wavelength. This indicates that the detection of a plurality of target nucleic acids using a plurality of nucleic acid probes enhances the fluorescence as compared with the case of using a single nucleic acid probe, and the detection signal is detected earlier.
  • Example 3 Confirmation of effect of improving detection system sensitivity by using nucleic acid probe set
  • (1) Purpose In order to confirm that the sensitivity for detecting nucleic acid is improved by using the nucleic acid probe set of the present invention, an experiment using a nucleic acid probe set targeting SARS-CoV-2 was conducted.
  • a solution (reagent 7) containing the following two nucleic acid probes and their corresponding nucleic acid primer sets and samples was prepared. Further, as a comparison with the reagent 7, using GeneCube Test Basic and RiverTraAce, a solution (reagent 8, reagent 9) using the nucleic acid probe constituting the nucleic acid probe set and the corresponding nucleic acid primer set alone was prepared. ..
  • the Tm value of the probe shown by SEQ ID NO: 11 used in this example is 52.8 ° C.
  • the Tm value of the probe shown by SEQ ID NO: 14 is 53.7 ° C.
  • the detection temperatures in the melting curve analysis are close to each other. Designed for.
  • the probes of SEQ ID NOs: 11 and 14 both have a base sequence complementary to the RNA sequence of SARS-CoV-2 and are labeled with the same fluorescent dye.
  • Reagent 7 Gene Cube Test Basic RiverTra Ace 0.2 ⁇ M Primer shown in SEQ ID NO: 9. 1.15 ⁇ M Primer shown in SEQ ID NO: 10. 0.2 ⁇ M Primer shown in SEQ ID NO: 12. 1.15 ⁇ M Primer represented by SEQ ID NO: 13. 0.1 ⁇ M Probe represented by SEQ ID NO: 11 (labeled 3'end with BODIPY-FL) 0.1 ⁇ M Probe shown in SEQ ID NO: 14 (labeled 3'end with BODIPY-FL)
  • Reagent 8 Gene Cube Test Basic RiverTra Ace 0.2 ⁇ M Primer shown in SEQ ID NO: 9. 1.15 ⁇ M Primer shown in SEQ ID NO: 10. 0.1 ⁇ M Probe represented by SEQ ID NO: 11 (labeled 3'end with BODIPY-FL)
  • Reagent 9 Gene Cube Test Basic RiverTra Ace 0.2 ⁇ M Primer shown in SEQ ID NO: 12. 1.15 ⁇ M Primer represented by SEQ ID NO: 13. 0.1 ⁇ M Probe shown in SEQ ID NO: 14 (labeled 3'end with BODIPY-FL)
  • Reverse transcription, nucleic acid amplification and melting curve analysis 42 ° C for 2 minutes 97 ° C for 15 seconds (More than 1 cycle) 97 ° C for 1 second 58 ° C for 3 seconds 63 ° C for 5 seconds (More than 60 cycles) 94 ° C for 30 seconds 39 ° C for 30 seconds 40 ° C to 75 ° C (temperature rises at 0.09 ° C / sec)
  • Result Table 4 is a table summarizing the results of detecting SARS-CoV-2 at a low concentration by performing fluorescence detection by nucleic acid amplification and melting curve analysis under the above conditions.
  • the reagent 7 using the nucleic acid probe set of the present invention has a higher positive detection rate than the reagents 8 and 9 using the nucleic acid probe alone. Therefore, by using a composition consisting of a plurality of nucleic acid probes having the same detection wavelength, which is an embodiment of the present invention, it is possible to improve the nucleic acid detection sensitivity even when the target has an extremely low concentration. It was found that highly sensitive detection was possible.
  • Example 4 Enhancement of detection signal by using nucleic acid probe set
  • CT Chlamydia trachomatis
  • nucleic acid probes for CT detection and their corresponding nucleic acid primer sets were prepared.
  • nucleic acid probes for NG detection and their corresponding nucleic acid primer sets were prepared.
  • Reagent 12 was prepared.
  • the probe of SEQ ID NO: 17 (Tm value 56.2 ° C.) or 20 (Tm value 58.5 ° C.) used in this example has a base sequence complementary to the CT DNA sequence or the NG DNA sequence, respectively. It is labeled with the same fluorescent dye.
  • Reagent 11 Gene Cube Test Basic 0.5 ⁇ M Primer shown in SEQ ID NO: 15. 2.5 ⁇ M Primer shown in SEQ ID NO: 16. 0.3 ⁇ M Probe shown in SEQ ID NO: 17 (labeled 3'end with BODIPY-FL)
  • Reagent 12 Gene Cube Test Basic 0.3 ⁇ M Primer shown in SEQ ID NO: 18. 2.0 ⁇ M Primer shown in SEQ ID NO: 19. 0.3 ⁇ M Probe shown in SEQ ID NO: 20 (labeled with BODIPY-FL at the 5'end and phosphorylated at the 3'end)
  • Nucleic acid amplification and melting curve analysis 97 ° C for 30 seconds (More than 1 cycle) 97 ° C for 1 second 58 ° C for 5 seconds 63 ° C for 5 seconds (More than 60 cycles) 94 ° C for 30 seconds 39 ° C for 30 seconds 40 ° C to 75 ° C (temperature rises at 0.09 ° C / sec)
  • FIG. 1 is a graph of reagent 10 (CT + NG)
  • FIG. 2 is a graph of reagent 11 (CT)
  • FIG. 3 is a graph of melting curve analysis results of reagent 12 (NG).
  • FIG. 1 shows that the detection peak is synergistically higher and is very clear (fluorescence peak (d / dt): about 73 for reagent 10, and for reagent 11).
  • About 40 about 25 in the case of reagent 12. This is to detect both CT and NG DNAs by using the nucleic acid probe set and the nucleic acid primer set group corresponding to the probe set in the reagent 10, and to superimpose and enhance the detection signals thereof. It is presumed that this was done.
  • the detection temperature (corresponding to the Tm value in the melting curve analysis) of each reagent was confirmed, it was 62.0 ° C. for the reagent 10, 60.6 ° C. for the reagent 11, and 61.8 ° C. for the reagent 12.
  • the difference in Tm value of the target nucleic acid probe used as the nucleic acid probe set was 1.2 ° C. from the difference between the reagent 11 and the reagent 12. From the above, it was shown that by fluorescently detecting a plurality of target nucleic acids using the nucleic acid probe set of the present invention, high-sensitivity detection is possible by overlapping and enhancing the detection signals.
  • the present invention it is possible to construct a nucleic acid detection system with higher sensitivity than before.
  • the present invention contributes to the construction of a nucleic acid detection system in which the fluorescence peak is clarified as a result of being able to enhance the detection signal by superimposing the detection signals, and the result determination is easier than before.

Abstract

The present invention addresses the problem of providing a nucleic acid detection method having high detection sensitivity. The present invention provides a nucleic acid probe set that includes at least a first target nucleic acid probe and a second target nucleic acid probe, and that has the following features (A)-(C): (A) the first target nucleic acid probe and the second target nucleic acid probe have mutually different base sequences; (B) the first target nucleic acid probe and the second target nucleic acid probe are labeled with fluorescent pigments that can be detected with the same wavelength; and (C) the first target nucleic acid probe and the second target nucleic acid probe bind to mutually different target nucleic acids, but detection is carried out in such a manner as not to distinguish between these target nucleic acids. In the present invention, by carrying out detection such that the target nucleic acids are not distinguishable, more intense fluorescent signals can be obtained and thus a highly-sensitive nucleic acid detection system can be obtained.

Description

複数の核酸プローブからなる核酸プローブセットNucleic acid probe set consisting of multiple nucleic acid probes

　本発明は、検査対象となる標的を高感度に検出するための核酸プローブセットおよびその使用方法等に関する。
The present invention relates to a nucleic acid probe set for detecting a target to be inspected with high sensitivity and a method of using the same.

　核酸プローブは特定の塩基配列からなる標的核酸（ＤＮＡまたはＲＮＡ）を検出するために、該標的核酸の一部または全部と相補的な塩基配列を有する、人工的な核酸であり、通常は一本鎖の核酸である。標的核酸の検出を容易にするため、核酸プローブはしばしば何らかの物質で標識され、該物質によるシグナルが検知される。前記物質としては例えば蛍光物質が用いられる。蛍光物質で標識された核酸プローブの代表的なものにＴａｑＭａｎ（登録商標）Ｐｒｏｂｅ、Ｑｐｒｏｂｅ（登録商標）などがある。
A nucleic acid probe is an artificial nucleic acid having a base sequence complementary to a part or all of the target nucleic acid in order to detect a target nucleic acid (DNA or RNA) consisting of a specific base sequence, and is usually one. It is a chain nucleic acid. To facilitate the detection of the target nucleic acid, the nucleic acid probe is often labeled with some substance and the signal from that substance is detected. As the substance, for example, a fluorescent substance is used. Typical examples of the nucleic acid probe labeled with a fluorescent substance include TaqMan (registered trademark) Probe and Qprobe (registered trademark).

　標的核酸の塩基配列に応じて核酸プローブ自身の塩基配列を調整することで、特異性の高い核酸検出が可能である。また、蛍光物質で標識された核酸プローブを使用する場合、複数の異なる塩基配列を有する核酸プローブにそれぞれ異なる検出波長の蛍光物質を標識させることで、同時に複数の標的核酸を検出しかつそれらを識別することが可能になる。ＰＣＲによって複数の標的核酸を増幅する技術はマルチプレックスＰＣＲと呼ばれ、これと複数の核酸プローブを用いて各標的核酸を検出し鑑別する技術はマルチプレックスリアルタイムＰＣＲと呼ばれる。通常は各核酸プローブに異なる検出波長の蛍光色素を標識させた複数の核酸プローブが併用され、これは多色マルチプレックスリアルタイムＰＣＲと呼称される場合もある。
By adjusting the base sequence of the nucleic acid probe itself according to the base sequence of the target nucleic acid, highly specific nucleic acid detection is possible. When a nucleic acid probe labeled with a fluorescent substance is used, a plurality of target nucleic acids can be detected and identified at the same time by labeling a nucleic acid probe having a plurality of different base sequences with a fluorescent substance having a different detection wavelength. It will be possible to do. The technique of amplifying a plurality of target nucleic acids by PCR is called multiplex PCR, and the technique of detecting and differentiating each target nucleic acid using a plurality of nucleic acid probes is called multiplex real-time PCR. Usually, a plurality of nucleic acid probes in which each nucleic acid probe is labeled with a fluorescent dye having a different detection wavelength are used in combination, and this is sometimes referred to as multicolor multiplex real-time PCR.

　一方、一種類の蛍光色素で標識された核酸プローブを使用する場合、核酸プローブの塩基配列は通常は一種類のみを使用する。これは、一種類の蛍光色素を複数の核酸プローブに標識しても、それを鑑別する方法がないことが理由である。例外として、一種類の蛍光色素で標識された核酸プローブを複数用いて、異なる標的核酸を検出し識別する方法が知られている（特許文献１）。この方法では、Ｔｍ値が異なる核酸プローブを複数用い、複雑なアニーリング温度条件と温度変化プロファイルの設計を組み合わせることで、異なる標的核酸の検出を行っている。
On the other hand, when a nucleic acid probe labeled with one kind of fluorescent dye is used, usually only one kind of base sequence of the nucleic acid probe is used. This is because there is no way to distinguish one type of fluorescent dye even if it is labeled on multiple nucleic acid probes. As an exception, a method of detecting and identifying different target nucleic acids by using a plurality of nucleic acid probes labeled with one kind of fluorescent dye is known (Patent Document 1). In this method, different target nucleic acids are detected by using a plurality of nucleic acid probes having different Tm values and combining complicated annealing temperature conditions and temperature change profile design.

　以上述べたように、複数の核酸プローブを使用する従来の方法は、複数の標的核酸を鑑別して検出するために用いられてきた。
As described above, the conventional method using a plurality of nucleic acid probes has been used for differentiating and detecting a plurality of target nucleic acids.

　他方、少数の標的核酸を効率的に増幅し検出したい場合、例えば試料中にごく微量に含まれうる微生物由来の核酸を検出したいような場合には、ＰＣＲを重ねて行うｎｅｓｔｅｄ　ＰＣＲといわれる方法が用いられることがある（非特許文献１）。ｎｅｓｔｅｄ　ＰＣＲは、標的核酸に対して第１のＰＣＲを行って増幅産物を得、該増幅産物を試料として第２のＰＣＲを行う方法である。この方法により、１回のＰＣＲのみでは検出に十分な量の増幅核酸が得られない状況であっても、ＰＣＲを重ねて実施することで、増幅産物を検出できるようになるため、ごく微量な標的核酸の検出が可能になる。
On the other hand, when it is desired to efficiently amplify and detect a small number of target nucleic acids, for example, when it is desired to detect nucleic acid derived from a microorganism that can be contained in a very small amount in a sample, a method called nested PCR in which PCR is repeated is performed. It may be used (Non-Patent Document 1). Nested PCR is a method in which a first PCR is performed on a target nucleic acid to obtain an amplification product, and a second PCR is performed using the amplification product as a sample. By this method, even in a situation where a sufficient amount of amplified nucleic acid cannot be obtained by only one PCR, the amplified product can be detected by repeating PCR, so that the amount is very small. It enables detection of target nucleic acid.

　しかしながら、ｎｅｓｔｅｄ　ＰＣＲはＰＣＲを複数回行う方法であるため、核酸増幅には時間を必要とする。そのため、迅速に核酸検出の結果を必要とする状況には適さない場合がある。そして、このようなｎｅｓｔｅｄ　ＰＣＲ法でも、通常は一種類の蛍光色素で標識された一種類の核酸プローブか、標的核酸毎にそれぞれ異なる検出波長の蛍光物質を標識した複数の核酸プローブが用いられてきた。
However, since nested PCR is a method of performing PCR multiple times, nucleic acid amplification requires time. Therefore, it may not be suitable for situations that require rapid nucleic acid detection results. Further, even in such a nested PCR method, one type of nucleic acid probe usually labeled with one type of fluorescent dye, or a plurality of nucleic acid probes labeled with a fluorescent substance having a different detection wavelength for each target nucleic acid has been used. rice field.

特許第６７２０１６１号公報Japanese Patent No. 6720161


　本発明は、検査対象となる標的を高感度に検出できる更なる有用な核酸検出系を提供することを目的とする。
An object of the present invention is to provide a further useful nucleic acid detection system capable of detecting a target to be tested with high sensitivity.

　本発明者等は鋭意研究の結果、同一の波長で検出できる蛍光色素で標識された複数の異なる塩基配列を有する核酸プローブをセットで用いて、これらを区別せずに検出することで核酸検出シグナルを重複させ、検査対象となる標的を高感度に検出できることを見出し、本発明を完成させるに至った。本発明の方法は、複雑なアニーリング温度条件や温度変化プロファイルの設計を必要とせず、例えば、リアルタイムＰＣＲ、リアルタイムＲＴ－ＰＣＲ、又は融解曲線解析等の汎用的な核酸検出法において利用され得る。即ち、本発明の概要は以下の通りである。
As a result of diligent research, the present inventors have used a set of nucleic acid probes having a plurality of different base sequences labeled with fluorescent dyes that can be detected at the same wavelength, and detected them without distinction, thereby detecting a nucleic acid detection signal. We have found that the target to be inspected can be detected with high sensitivity by duplicating the above, and have completed the present invention. The method of the present invention does not require the design of complex annealing temperature conditions or temperature change profiles and can be utilized in general-purpose nucleic acid detection methods such as, for example, real-time PCR, real-time RT-PCR, or melting curve analysis. That is, the outline of the present invention is as follows.

　［項１］　二つ以上の核酸プローブを含む核酸プローブセットであって、該核酸プローブセットは少なくとも第一標的核酸プローブ及び第二標的核酸プローブを含み、以下の（Ａ）～（Ｃ）の特徴を有する、核酸プローブセット：

（Ａ）第一標的核酸プローブ及び第二標的核酸プローブはそれぞれ異なる塩基配列を有する。

（Ｂ）第一標的核酸プローブ及び第二標的核酸プローブは同一波長で検出できる蛍光色素で標識されている。

（Ｃ）第一標的核酸プローブ及び第二標的核酸プローブはそれぞれ異なる標的核酸に結合するが、各標的核酸を鑑別しないように検出される。

　［項２］　前記核酸プローブセットが、前記第一標的核酸プローブ及び第二標的核酸プローブからなることを特徴とする、項１に記載の核酸プローブセット。

　［項３］　同一波長で検出できる蛍光色素が同一の蛍光色素である、項１または２に記載の核酸プローブセット。

　［項４］　前記特徴（Ｃ）における検出が、リアルタイムＰＣＲ、リアルタイムＲＴ－ＰＣＲ、又は融解曲線解析のいずれかの核酸検出法における検出である、項１～３のいずれかに記載の核酸プローブセット。

　［項５］　前記特徴（Ｃ）における検出が、融解曲線解析における検出である、項１～４のいずれかに記載の核酸プローブセット。

　［項６］　第一標的核酸プローブと第二標的核酸プローブとのＴｍ値の差が３℃以内である、項１～５のいずれかに記載の核酸プローブセット。

　［項７］　第一標的核酸プローブ及び第二標的核酸プローブが、それぞれの標的核酸に対して完全に相補的な塩基配列から構成される、項１～６のいずれかに記載の核酸プローブセット。

　［項８］　第一標的核酸プローブの塩基配列と第二標的核酸プローブの塩基配列との塩基長の差が８ｍｅｒ以内である、項１～７のいずれかに記載の核酸プローブセット。

　［項９］　項１～８のいずれかに記載の核酸プローブセットを含む試薬組成物。

　［項１０］　項１～８のいずれかに記載の核酸プローブセット又は項９に記載の試薬組成物を含むキット。

　［項１１］　項１～８のいずれかに記載の核酸プローブセット、項９に記載の試薬組成物、又は項１０に記載のキットを用いる、核酸の検出方法。

　［項１２］　項１～８のいずれかに記載の核酸プローブセット、項９に記載の試薬組成物、又は項１０に記載のキットを用いる、核酸の検出感度向上方法。
[Item 1] A nucleic acid probe set containing two or more nucleic acid probes, wherein the nucleic acid probe set includes at least a first target nucleic acid probe and a second target nucleic acid probe, and has the following features (A) to (C). Has a nucleic acid probe set:

(A) The first target nucleic acid probe and the second target nucleic acid probe have different base sequences.

(B) The first target nucleic acid probe and the second target nucleic acid probe are labeled with a fluorescent dye that can be detected at the same wavelength.

(C) The first target nucleic acid probe and the second target nucleic acid probe bind to different target nucleic acids, but are detected so as not to distinguish each target nucleic acid.

[Item 2] The nucleic acid probe set according to Item 1, wherein the nucleic acid probe set comprises the first target nucleic acid probe and the second target nucleic acid probe.

[Item 3] The nucleic acid probe set according to Item 1 or 2, wherein the fluorescent dyes that can be detected at the same wavelength are the same fluorescent dyes.

[Item 4] The nucleic acid probe set according to any one of Items 1 to 3, wherein the detection in the feature (C) is a detection in any of the nucleic acid detection methods of real-time PCR, real-time RT-PCR, or melting curve analysis. ..

[Item 5] The nucleic acid probe set according to any one of Items 1 to 4, wherein the detection in the feature (C) is the detection in the melting curve analysis.

[Item 6] The nucleic acid probe set according to any one of Items 1 to 5, wherein the difference in Tm value between the first target nucleic acid probe and the second target nucleic acid probe is within 3 ° C.

[Item 7] The nucleic acid probe set according to any one of Items 1 to 6, wherein the first target nucleic acid probe and the second target nucleic acid probe are composed of a base sequence completely complementary to each target nucleic acid.

[Item 8] The nucleic acid probe set according to any one of Items 1 to 7, wherein the difference in base length between the base sequence of the first target nucleic acid probe and the base sequence of the second target nucleic acid probe is within 8 mer.

[Item 9] A reagent composition containing the nucleic acid probe set according to any one of Items 1 to 8.

[Item 10] A kit containing the nucleic acid probe set according to any one of Items 1 to 8 or the reagent composition according to Item 9.

[Item 11] A method for detecting nucleic acid using the nucleic acid probe set according to any one of Items 1 to 8, the reagent composition according to Item 9, or the kit according to Item 10.

[Item 12] A method for improving nucleic acid detection sensitivity using the nucleic acid probe set according to any one of Items 1 to 8, the reagent composition according to Item 9, or the kit according to Item 10.

　本発明の核酸検出系では個々の核酸プローブによって生じる検出シグナルを区別しないため、各核酸プローブに由来する検出シグナルを複数重ねることができ、それによって通常よりも明瞭な検出シグナルを得ることができる。従って、本発明を例えば特定の微生物の存在を検知することに応用する場合、該微生物が有する複数の遺伝子にそれぞれ対応する複数の標的核酸プローブを設計して、それら標的核酸プローブの検出を試みることで、標的核酸のいずれか一つでも検出されれば該微生物が存在すると判定することができる。本発明は、汎用的なリアルタイムＰＣＲ、リアルタイムＲＴ－ＰＣＲ、融解曲線解析等の核酸検出法において利用され得るので、簡便に高感度な核酸検出系の構築が可能となる。
Since the nucleic acid detection system of the present invention does not distinguish the detection signals generated by individual nucleic acid probes, it is possible to superimpose a plurality of detection signals derived from each nucleic acid probe, thereby obtaining a clearer detection signal than usual. Therefore, when the present invention is applied to, for example, detecting the presence of a specific microorganism, a plurality of target nucleic acid probes corresponding to a plurality of genes possessed by the microorganism are designed, and the detection of those target nucleic acid probes is attempted. Therefore, if any one of the target nucleic acids is detected, it can be determined that the microorganism is present. Since the present invention can be used in general-purpose real-time PCR, real-time RT-PCR, nucleic acid detection methods such as melting curve analysis, it is possible to easily construct a highly sensitive nucleic acid detection system.

図１は、実施例４において試薬１０（ＣＴ＋ＮＧ）を用いた場合の融解曲線解析結果を示すグラフである。FIG. 1 is a graph showing the results of melting curve analysis when reagent 10 (CT + NG) is used in Example 4. 図２は、実施例４において試薬１１（ＣＴ）を用いた場合の融解曲線解析結果を示すグラフである。FIG. 2 is a graph showing the results of melting curve analysis when reagent 11 (CT) is used in Example 4. 図３は、実施例４において試薬１２（ＮＧ）を用いた場合の融解曲線解析結果を示すグラフである。FIG. 3 is a graph showing the results of melting curve analysis when reagent 12 (NG) is used in Example 4.

　以下、本発明の実施形態を示しつつ、本発明についてさらに詳説するが、本発明はこれらに限定されるものではない。なお、本明細書中に記載された非特許文献および特許文献の全てが、本明細書中において参考として援用される。また本明細書中の「～」は「以上、以下」を意味し、例えば明細書中で「Ｘ～Ｙ」と記載されていれば「Ｘ以上、Ｙ以下」を示す。また本明細書中の「及び／又は」は、いずれか一方または両方を意味する。
Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention, but the present invention is not limited thereto. All of the non-patent documents and patent documents described in the present specification are incorporated herein by reference. Further, "-" in the present specification means "greater than or equal to or less than", and for example, if "X to Y" is described in the present specification, it means "more than or equal to X and less than or equal to Y". In addition, "and / or" in the present specification means either one or both.

　また、本明細書では、標的用プライマーを、核酸プライマー、オリゴヌクレオチドプライマー又は単にプライマーといい、標的核酸プローブを、核酸プローブ、オリゴヌクレオチドプローブ又は単にプローブという場合があり、これらを総称してオリゴヌクレオチドともいう。
Further, in the present specification, the targeting primer may be referred to as a nucleic acid primer, an oligonucleotide primer or simply a primer, and the target nucleic acid probe may be referred to as a nucleic acid probe, an oligonucleotide probe or simply a probe, and these are collectively referred to as an oligonucleotide. Also called.

［核酸プローブセット］

　本発明の実施態様の一つは、二つ以上の核酸プローブを含む核酸プローブセットである。該核酸プローブセッは以下の（Ａ）～（Ｃ）の特徴を有する第一標的核酸プローブ及び第二標的核酸プローブを少なくとも含むものである：

（Ａ）第一標的核酸プローブ及び第二標的核酸プローブはそれぞれ異なる塩基配列を有する。

（Ｂ）第一標的核酸プローブ及び第二標的核酸プローブは同一波長で検出できる蛍光色素で標識されている。

（Ｃ）第一標的核酸プローブ及び第二標的核酸プローブはそれぞれ異なる標的核酸に結合するが、各標的核酸を鑑別しないように検出される。
[Nucleic acid probe set]

One embodiment of the present invention is a nucleic acid probe set comprising two or more nucleic acid probes. The nucleic acid probe set includes at least a first target nucleic acid probe and a second target nucleic acid probe having the following characteristics (A) to (C):

(A) The first target nucleic acid probe and the second target nucleic acid probe have different base sequences.

(B) The first target nucleic acid probe and the second target nucleic acid probe are labeled with a fluorescent dye that can be detected at the same wavelength.

(C) The first target nucleic acid probe and the second target nucleic acid probe bind to different target nucleic acids, but are detected so as not to distinguish each target nucleic acid.

　前記実施態様において、核酸プローブセットは、第一標的核酸プローブ及び第二標的核酸プローブ以外に、一つ又は二つ以上の更なる標的核酸プローブ（例えば、第三標的核酸プローブ、第四標的核酸プローブ、第五標的核酸プローブなど）を含んでいてもよい。このような更なる標的核酸プローブは、第一標的核酸プローブ及び第二標的核酸プローブと同様に前記（Ａ）～（Ｃ）の全ての特徴を備えたものであってもよいし、前記（Ａ）～（Ｃ）のいずれかの特徴を備えていないもの（例えば、異なる波長で検出される蛍光色素で標識されたもの等）であってもよい。本発明の核酸プローブセットを構成する核酸プローブの数は二つ以上であれば特に制限されない。好ましくは二つ～五つであり、より好ましくは二つ又は三つである。なかでも、前記第一標的核酸プローブ及び第二標的核酸プローブからなる核酸プローブセットであることが好ましい。本発明の核酸プローブセットは、更に他の核酸プローブと組み合わせて用いてもよい。
In the above embodiment, the nucleic acid probe set includes one or more additional target nucleic acid probes (eg, a third target nucleic acid probe, a fourth target nucleic acid probe, in addition to the first target nucleic acid probe and the second target nucleic acid probe. , Fifth target nucleic acid probe, etc.) may be included. Such a further target nucleic acid probe may have all the characteristics of the above (A) to (C) like the first target nucleic acid probe and the second target nucleic acid probe, or may have the above-mentioned (A). )-(C) may not be provided (for example, those labeled with a fluorescent dye detected at different wavelengths). The number of nucleic acid probes constituting the nucleic acid probe set of the present invention is not particularly limited as long as it is two or more. It is preferably two to five, and more preferably two or three. Among them, a nucleic acid probe set including the first target nucleic acid probe and the second target nucleic acid probe is preferable. The nucleic acid probe set of the present invention may be used in combination with other nucleic acid probes.

　前記実施態様において、核酸プローブセットを構成する核酸プローブは、それぞれ異なる塩基配列を有することが好ましい。該実施態様における「異なる」とは、例えば核酸プローブＡと核酸プローブＢがある場合、各核酸プローブを構成するオリゴヌクレオチドの塩基配列に一つでも相違があることをいう。特定の好ましい実施形態では、各核酸プローブの塩基配列の同一性が９９％以下であることが好ましく、より好ましくは各核酸プローブの塩基配列の同一性が９５％以下、９０％以下、８５％以下、８０％以下、７５％以下である。例えば、各核酸プローブの塩基配列の同一性は５０％以下であってもよい。本発明ではこのように互いの塩基配列の同一性が低い核酸プローブを複数用いることで、より高感度な検出が可能となり得る。
In the above embodiment, it is preferable that the nucleic acid probes constituting the nucleic acid probe set have different base sequences. By "different" in the embodiment, for example, when there are nucleic acid probe A and nucleic acid probe B, it means that there is even one difference in the base sequence of the oligonucleotides constituting each nucleic acid probe. In a specific preferred embodiment, the identity of the base sequence of each nucleic acid probe is preferably 99% or less, and more preferably the identity of the base sequence of each nucleic acid probe is 95% or less, 90% or less, 85% or less. , 80% or less, 75% or less. For example, the identity of the base sequence of each nucleic acid probe may be 50% or less. In the present invention, by using a plurality of nucleic acid probes having low identity with each other as described above, more sensitive detection may be possible.

　前記実施態様において、各核酸プローブは蛍光色素で標識されていることを一つの特徴とする。各核酸プローブの塩基配列における標識部位は特に限定されず、各核酸プローブの標識部位は同一であっても異なっていてもよい。好ましくは、各核酸プローブにおいていずれか一方の末端が蛍光色素で標識される。特定の実施形態では、各核酸プローブにおいていずれか一方の末端のみが蛍光色素で標識されたものを使用することができる。蛍光色素は公知の標識物を使用することができる。このような蛍光色素としては、例えば、ローダミン系化合物；フルオロセイン系化合物（例えば、ＦＡＭ（カルボキシフルオレセイン）、ＡＬＥＸＡ　ＦＬＵＯＲ、４，４－ジフルオロ－５，７－ジメチル－４－ボラ－３ａ，４ａ－ジアザ－ｓ－インダセン－３－プロピオン酸（ＢＯＤＩＰＹ－ＦＬ）、カルボキシローダミン６Ｇ，ＴＡＭＲＡ，ローダミン６Ｇ，テトラブロモスルホンフルオレセイン（ＴＢＳＦ）、及び２－オキソ－６，８－ジフルオロ－７－ジヒドロキシ－２Ｈ－１－ベンゾピラン－３－カルボン酸（Ｐａｃｉｆｉｃ　Ｂｌｕｅ）等が挙げられるが、これらに限定されない。
In the above embodiment, one feature is that each nucleic acid probe is labeled with a fluorescent dye. The labeling site in the base sequence of each nucleic acid probe is not particularly limited, and the labeling site of each nucleic acid probe may be the same or different. Preferably, one end of each nucleic acid probe is labeled with a fluorescent dye. In certain embodiments, each nucleic acid probe can be used with only one end labeled with a fluorescent dye. A known labeled substance can be used as the fluorescent dye. Examples of such fluorescent dyes include rhodamine-based compounds; fluorosane-based compounds (eg, FAM (carboxyfluorescein), ALEXA FLUOR, 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-. Diaza-s-Indacene-3-propionic Acid (BODIPY-FL), Carboxyl Rhodamine 6G, TAMRA, Rhodamine 6G, Tetrabromosulfone Fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H- Examples thereof include, but are not limited to, 1-benzopyran-3-carboxylic acid (Pacific Blue).

　いずれか一方の末端が蛍光色素で標識される場合において、蛍光標識された末端と反対側の末端については、標識されていないオリゴヌクレオチドであってもよいし、何らかの物質で標識されていてもよい。このような標識物質の例示としては消光物質があり、例えばＢＨＱ１（ＢＨＱ：Ｂｌａｃｋ　Ｈｏｌｅ　Ｑｕｅｎｃｈｅｒ（登録商標））、ＢＨＱ２、ＢＨＱ３等が挙げられるが、これらに限定されない。リアルタイムＰＣＲ又はリアルタイムＲＴ－ＰＣＲにおいて本発明を実施する場合には、このように蛍光色素と消光物質とで標識した標的核酸プローブを用いることが好ましい。
When either end is labeled with a fluorescent dye, the terminal opposite to the fluorescently labeled end may be an unlabeled oligonucleotide or may be labeled with some substance. .. Examples of such labeling substances include quenching substances, and examples thereof include, but are not limited to, BHQ1 (BHQ: Black Hole Quencher (registered trademark)), BHQ2, and BHQ3. When carrying out the present invention in real-time PCR or real-time RT-PCR, it is preferable to use a target nucleic acid probe thus labeled with a fluorescent dye and a quencher.

［同一波長で検出できる蛍光色素］

　本明細書において、同一波長で検出できるとは、市販の核酸検出機器（例えば、リアルタイムＰＣＲや融解曲線解析を行える測定機器）において同じ検出チャンネルで検出できる程度に類似した蛍光波長を有することを指す。例えばリアルタイムＰＣＲ装置を使用する場合、該装置は複数の蛍光検出チャンネルを有することが一般的である。Ｒｏｔｏｒ－Ｇｅｎｅ　Ｑ（ＱＩＡＧＥＮ社）を例にとると、Ｒｏｔｏｒ－Ｇｅｎｅ　Ｑは６種類の蛍光検出用チャンネルを有しており、それぞれ異なる検出波長を備えている。そして、検出波長ごとに複数の蛍光色素が検出可能である。具体的には表１の通りである。
[Fluorescent dye that can be detected at the same wavelength]

As used herein, the term "detectable at the same wavelength" means that a commercially available nucleic acid detection device (for example, a measuring device capable of real-time PCR or melting curve analysis) has a fluorescence wavelength similar to the extent that it can be detected on the same detection channel. .. For example, when using a real-time PCR device, the device typically has multiple fluorescence detection channels. Taking Rotor-Gene Q (QIAGEN) as an example, Rotor-Gene Q has six types of fluorescence detection channels, each of which has a different detection wavelength. Then, a plurality of fluorescent dyes can be detected for each detection wavelength. Specifically, it is as shown in Table 1.

Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001

　上記表１に示される通り、例えばＦＡＭとＳＹＢＲ　Ｇｒｅｅｎ　Ｉは同じ蛍光チャンネルで検出される。ＦＡＭの最大検出波長（最大蛍光波長）は５２０ｎｍ、ＳＹＢＲ　Ｇｒｅｅｎ　Iの最大検出波長は５２１ｎｍ、Ｅｖａ　Ｇｒｅｅｎの最大検出波長は５２５ｎｍであるが、蛍光が検出される時にこれらの蛍光色素は区別されない。この場合にＦＡＭ、ＳＹＢＲ　Ｇｒｅｅｎ　Ｉ、Ｅｖａ　Ｇｒｅｅｎは同一波長で検出できることになる。本発明では、上記表１に示されるような各チャンネルで検出可能な蛍光色素の群を同一波長で検出できる蛍光色素として使用することができるが、これらに限定されず、同等の最大検出波長を有する任意の蛍光色素を用いることが可能である。
As shown in Table 1 above, for example, FAM and SYBR Green I are detected on the same fluorescent channel. The maximum detection wavelength (maximum fluorescence wavelength) of FAM is 520 nm, the maximum detection wavelength of SYBR Green I is 521 nm, and the maximum detection wavelength of Eva Green is 525 nm, but these fluorescent dyes are not distinguished when fluorescence is detected. In this case, FAM, SYBR Green I, and Eva Green can be detected at the same wavelength. In the present invention, a group of fluorescent dyes that can be detected in each channel as shown in Table 1 above can be used as fluorescent dyes that can be detected at the same wavelength, but the present invention is not limited to these, and an equivalent maximum detection wavelength can be used. It is possible to use any fluorescent dye having.

　複数の異なる蛍光色素が同一波長で検出できるという場合、各蛍光色素の最大検出波長の数値の差は、本発明の効果を奏する限り特に制限されず、前述の通り実際に蛍光が検出される際に当該分野で通常区別されない蛍光物質は、同一波長で検出できる蛍光物質ということができる。より確実に本発明の効果が得られ易いという観点から、好ましくは複数の異なる蛍光色素は互いの最大検出波長の差が４０ｎｍ以内であり、より好ましくは３０ｎｍ以内、更に好ましくは２５ｎｍ以内であるのがよい。各蛍光色素の最大検出波長の差は、例えば、２０ｎｍ以内、１５ｎｍ以内、１０ｎｍ以内、５ｎｍ以内であってもよいし、互いの最大検出波長が実質的に同一で差が無いものであってもよい。特定の好ましい実施形態では、同一波長で検出できる蛍光物質として同一の蛍光物質を用いる。同一の蛍光物質を用いることで、より低コストに効率よく本発明を実施することが可能となる。さらに本発明のように同一波長で検出できる蛍光物質を使用することで、多種類の蛍光検出チャンネルを備えた分析装置でなくても、一又は少数の蛍光チャンネルを備えた分析装置でも簡便に標的を検出することが可能となり得る。
When a plurality of different fluorescent dyes can be detected at the same wavelength, the difference in the numerical value of the maximum detection wavelength of each fluorescent dye is not particularly limited as long as the effect of the present invention is achieved, and as described above, when fluorescence is actually detected. Fluorescent substances that are not normally distinguished in the art can be said to be fluorescent substances that can be detected at the same wavelength. From the viewpoint that the effect of the present invention can be more reliably obtained, the difference in the maximum detection wavelengths of the plurality of different fluorescent dyes is preferably 40 nm or less, more preferably 30 nm or less, still more preferably 25 nm or less. Is good. The difference in the maximum detection wavelength of each fluorescent dye may be, for example, within 20 nm, within 15 nm, within 10 nm, within 5 nm, or even if the maximum detection wavelengths of each are substantially the same and there is no difference. good. In certain preferred embodiments, the same fluorescent material is used as the fluorescent material that can be detected at the same wavelength. By using the same fluorescent substance, it is possible to efficiently carry out the present invention at a lower cost. Furthermore, by using a fluorescent substance that can be detected at the same wavelength as in the present invention, it is possible to easily target an analyzer equipped with one or a small number of fluorescent channels, even if the analyzer is not equipped with many types of fluorescence detection channels. Can be detected.

　前記実施態様において、前記核酸プローブセットを構成する各核酸プローブはそれぞれ異なる標的核酸に結合する。従って、各核酸プローブは標的核酸への結合又は解離で発光又は消光することによって、異なる標的核酸を検出することになる。本発明では、このような異なる標的核酸の検出を、敢えて鑑別せずに検出するように複数の核酸プローブを設計して用いることを一つの特徴とする。標的核酸とは核酸プローブによって検出される対象となる核酸であり、通常は核酸増幅法によって生成される増幅産物である。核酸増幅法は数コピーの標的核酸を可視化可能なレベル、すなわち数億コピー以上に増幅する技術であり、生命科学研究分野のみならず、臨床診断、食品衛生検査、環境検査等の分野においても広く用いられている。そのような核酸増幅法としては、ＰＣＲ法、ＬＡＭＰ法、ＬＣＲ法、ＴＭＡ法、ＳＤＡ法、ＲＴ－ＰＣＲ法、ＲＴ－ＬＡＭＰ法、ＮＡＳＢＡ法、ＴＲＣ法、ＴＭＡ法等が挙げられる。これらの技術は既に当該技術分野において確立されており、目的に合わせて方法を選択することができる。本発明で行う核酸増幅法はＰＣＲ法（ＲＴ－ＰＣＲ法を含む）が好ましいが、これに限定されない。
In the above embodiment, each nucleic acid probe constituting the nucleic acid probe set binds to a different target nucleic acid. Therefore, each nucleic acid probe detects a different target nucleic acid by emitting or quenching by binding or dissociating to the target nucleic acid. One of the features of the present invention is to design and use a plurality of nucleic acid probes so as to detect such different target nucleic acids without daring to discriminate them. The target nucleic acid is a nucleic acid to be detected by a nucleic acid probe, and is usually an amplification product produced by a nucleic acid amplification method. Nucleic acid amplification method is a technology that amplifies several copies of target nucleic acid to a level that can be visualized, that is, to hundreds of millions of copies or more, and is widely used not only in the field of life science research but also in the fields of clinical diagnosis, food hygiene inspection, environmental inspection, etc. It is used. Examples of such nucleic acid amplification methods include PCR method, LAMP method, LCR method, TMA method, SDA method, RT-PCR method, RT-LAMP method, NASBA method, TRC method, TMA method and the like. These techniques have already been established in the relevant technical field, and the method can be selected according to the purpose. The nucleic acid amplification method performed in the present invention is preferably, but not limited to, the PCR method (including the RT-PCR method).

　核酸増幅法を用いて増幅産物を生成する場合には、例えば、核酸プライマーセットを用いて、該プライマーセットに挟まれた塩基配列を増幅する。従って、異なる核酸プライマーセットを用いて生成された増幅産物は各々異なる塩基配列を有するため、それぞれ異なる標的核酸である。特定の実施形態では、第一標的核酸プローブが結合可能な第一標的核酸の配列を含む領域を増幅可能な１つ以上の第一標的用プライマー（又はプライマーセット）、及び、第二標的核酸プローブが結合可能な第二標的核酸の配列を含む領域を増幅可能な１つ以上の第二標的用プライマー（又はプライマーセット）を用いて、第一標的核酸及び第二標的核酸をそれぞれ増幅することが好ましい。第一標的核酸の増幅と第二標的核酸の増幅は同時に実施されても別々に実施されてもよい。より短時間で核酸増幅反応を行うことができるという観点からは、第一標的核酸の増幅と第二標的核酸の増幅を同時に実施することが好ましい。
When an amplification product is produced by using a nucleic acid amplification method, for example, a nucleic acid primer set is used to amplify the base sequence sandwiched between the primer sets. Therefore, amplification products produced using different nucleic acid primer sets have different base sequences and are therefore different target nucleic acids. In certain embodiments, one or more primary target primers (or primer sets) capable of amplifying the region containing the sequence of the first target nucleic acid to which the first target nucleic acid probe can bind, and the second target nucleic acid probe. Amplify the first and second target nucleic acids, respectively, with one or more second target primers (or primer sets) capable of amplifying the region containing the sequence of the second target nucleic acid to which the nucleic acid can bind. preferable. The amplification of the first target nucleic acid and the amplification of the second target nucleic acid may be performed simultaneously or separately. From the viewpoint that the nucleic acid amplification reaction can be carried out in a shorter time, it is preferable to simultaneously perform the amplification of the first target nucleic acid and the amplification of the second target nucleic acid.

　本発明において、第一標的核酸プローブ及び第二標的核酸プローブにより検出された第一標的核酸及び第二標的核酸は鑑別されない。前記の通り、第一標的核酸プローブ及び第二標的核酸プローブにはいずれも同一の波長で検出できる蛍光色素が標識されるため、各標的核酸プローブによって生じる検出シグナルは同じ色として検知される。本発明では、このように同じ色として検知された検出シグナルを、例えば融解曲線解析において測定する場合に、それらの検出温度を揃えるようにすることで蛍光ピークを重ね合わせて各標的核酸を鑑別しないように検出する。
In the present invention, the first target nucleic acid and the second target nucleic acid detected by the first target nucleic acid probe and the second target nucleic acid probe are not differentiated. As described above, since the first target nucleic acid probe and the second target nucleic acid probe are both labeled with a fluorescent dye that can be detected at the same wavelength, the detection signal generated by each target nucleic acid probe is detected as the same color. In the present invention, when the detection signals detected as the same color in this way are measured, for example, in melting curve analysis, the fluorescence peaks are superposed and the target nucleic acids are not discriminated by making the detection temperatures uniform. To detect.

［検出シグナル］

　本明細書において検出シグナルとは、核酸プローブが標的核酸を検出することで生じる蛍光の変化のことをいう。検出シグナルの様式は核酸プローブに標識される蛍光色素や消光物質、核酸プローブと標的核酸との間に生じる化学反応、検出に用いる機器の構造等によって異なるが、本発明は特定の検出シグナルの様式に限定されず、核酸プローブによる標的核酸の検出を示す任意の蛍光変化現象による検出シグナルであり得る。
[Detection signal]

As used herein, the detection signal refers to a change in fluorescence caused by a nucleic acid probe detecting a target nucleic acid. The mode of the detection signal differs depending on the fluorescent dye or light-dissipating substance labeled on the nucleic acid probe, the chemical reaction occurring between the nucleic acid probe and the target nucleic acid, the structure of the device used for detection, etc., but the present invention has a specific mode of the detection signal. The detection signal may be any fluorescence change phenomenon indicating the detection of the target nucleic acid by the nucleic acid probe.

　前記検出シグナルの態様として、例えばリアルタイムＰＣＲ（リアルタイムＲＴ－ＰＣＲを含む）での経時的な発光が挙げられる。リアルタイムＰＣＲでは生成される増幅産物を経時的に蛍光変化により検出する。その方法としては、例えばＴａｑＭａｎプローブを用いた方法が挙げられる。ＴａｑＭａｎプローブは人工的に合成された一本鎖ＤＮＡにおいて、いずれかの末端が蛍光色素で、もう片方の末端が消光物質で標識された核酸プローブである。ＴａｑＭａｎプローブは消光物質によって蛍光色素の発光が抑制されている。しかしながら、ＰＣＲサイクルのアニーリングステップで標的核酸に相補的に結合し、その後の伸長ステップでＴａｑＤＮＡ　ポリメラーゼのもつ５’→３’エキソヌクレアーゼ活性により、標的核酸に結合したＴａｑＭａｎプローブが分解されると、蛍光色素がプローブから遊離し、消光物質による抑制が解除されて蛍光が発光する。標的核酸である増幅産物が増えるほど多くのＴａｑＭａｎプローブが分解され、発光が強化される。ＴａｑＭａｎプローブの分解による蛍光が域値を超えたときのＰＣＲサイクル数をＣｔ（Ｔｈｒｅｓｈｏｌｄ　Ｃｙｃｌｅ）と称する。Ｃｔが小さいほど、少ないＰＣＲで蛍光が生じていることを示しており、より高感度に標的を検出できていることを意味し得る。リアルタイムＰＣＲにおける検出シグナルとしては限定はされないが、前記の様なプローブの発光を挙げることができ、この検出シグナルの強さはＣｔの出力等により評価することが可能である。
Examples of the aspect of the detection signal include luminescence over time in real-time PCR (including real-time RT-PCR). In real-time PCR, the amplified product produced is detected by changes in fluorescence over time. As the method, for example, a method using a TaqMan probe can be mentioned. The TaqMan probe is a nucleic acid probe in an artificially synthesized single-stranded DNA labeled with a fluorescent dye at one end and a quencher at the other end. In the TaqMan probe, the emission of the fluorescent dye is suppressed by the quenching substance. However, when the TaqMan probe bound to the target nucleic acid is degraded by the 5'→ 3'exonuclease activity of the TaqDNA polymerase in the annealing step of the PCR cycle, which complementarily binds to the target nucleic acid, and in the subsequent extension step, it fluoresces. The dye is released from the probe, the suppression by the quencher is released, and the fluorescence is emitted. As the amount of amplification product, which is the target nucleic acid, increases, more TaqMan probes are degraded and luminescence is enhanced. The number of PCR cycles when the fluorescence due to the decomposition of the TaqMan probe exceeds the range value is referred to as Ct (Threshold Cycle). The smaller the Ct, the less PCR is required to generate fluorescence, which may mean that the target can be detected with higher sensitivity. The detection signal in real-time PCR is not limited, but the emission of the probe as described above can be mentioned, and the strength of this detection signal can be evaluated by the output of Ct or the like.

　前記検出シグナルの異なる態様として、融解曲線解析（融解曲線分析ともいう）による蛍光変化の検出が挙げられる。Ｔｍ値を用いた標的核酸の検出、分析等を融解曲線解析という。一般的に、Ｔｍ値は、オリゴヌクレオチドがその相補鎖と二本鎖を形成している割合と二本鎖を形成せず一本鎖である割合が等しいときの温度をいう。Ｔｍ値は、塩基配列に固有の値であるため、融解曲線解析は例えば、標的核酸の塩基配列多型を分析する手法として使用されている。ここでいう塩基配列多型とは、一塩基多型、塩基置換、塩基欠損、塩基挿入等を含む。融解曲線解析は、例えば、以下のように行う。

　二本鎖ＤＮＡを含む溶液を加熱していくと、２６０ｎｍにおける吸光度が上昇する。これは、二本鎖ＤＮＡにおける両鎖間の水素結合が加熱によってほどけ、一本鎖ＤＮＡに解離（ＤＮＡの融解）することが原因である。そして、全ての二本鎖ＤＮＡが解離して一本鎖ＤＮＡになると、その吸光度は、加熱開始時の吸光度（二本鎖ＤＮＡのみの吸光度）の約１．５倍程度を示し、これによって融解が完了したと判断できる。この場合は、加熱前と加熱後の吸光度の差が融解曲線解析による検出シグナルと捉えられる。
As a different aspect of the detection signal, detection of fluorescence change by melting curve analysis (also referred to as melting curve analysis) can be mentioned. Detection and analysis of target nucleic acid using Tm value is called melting curve analysis. In general, the Tm value refers to the temperature at which the proportion of an oligonucleotide forming a double strand with its complementary strand and the proportion of a single strand without forming a double strand are equal. Since the Tm value is a value unique to the base sequence, melting curve analysis is used, for example, as a method for analyzing the base sequence polymorphism of the target nucleic acid. The base sequence polymorphism referred to here includes a single nucleotide polymorphism, a base substitution, a base defect, a base insertion, and the like. The melting curve analysis is performed, for example, as follows.

As the solution containing the double-stranded DNA is heated, the absorbance at 260 nm increases. This is because the hydrogen bonds between the two strands in the double-stranded DNA are unwound by heating and dissociated into the single-stranded DNA (melting of the DNA). Then, when all the double-stranded DNA is dissociated into a single-stranded DNA, the absorbance thereof shows about 1.5 times the absorbance at the start of heating (the absorbance of only the double-stranded DNA), thereby melting. Can be judged to be completed. In this case, the difference in absorbance before and after heating is regarded as a detection signal by melting curve analysis.

　融解曲線解析の別の態様として、蛍光色素で標識された核酸プローブを用いる方法が挙げられ、本発明における蛍光シグナルでの標的検出に好適であり得る。本方法では例えば、標的核酸と複合体を形成すると蛍光が消光し、標的核酸から遊離すると蛍光が発光するという特色を有する核酸プローブを核酸増幅法の反応系に組み込み、核酸増幅法が終了した後で反応系を低温で冷却して、前記核酸プローブを標的核酸に結合させる。この時点では核酸プローブの蛍光色素は消光している。その後、徐々に温度を上昇させると、核酸プローブのＴｍ値近辺の温度で核酸プローブが標的核酸から遊離する。このとき核酸プローブの蛍光が発光するため、Ｔｍ値近辺の温度では蛍光の著しい変化（増加）が生じる。蛍光標識された核酸プローブを用いる融解曲線解析においては、前記蛍光の変化が検出シグナルと捉えられる。

　前記の様に標的核酸と複合体を形成すると消光し、標的核酸から解離して単独で存在すると発光する核酸プローブとして、蛍光消光色素で標識されている末端塩基がシトシンである標識プローブが例示される。このような核酸プローブは、標的核酸にハイブリダイズした際に、標的核酸配列中のグアニン塩基と塩基対を形成して相互作用することで消光できるため、グアニン消光プローブ（Ｑ　Ｐｒｏｂｅ（登録商標）としても公知）等とも呼ばれ、非常に簡便に蛍光強度の変化を測定することができるので好ましい。
Another aspect of the melting curve analysis is a method using a nucleic acid probe labeled with a fluorescent dye, which may be suitable for target detection with a fluorescent signal in the present invention. In this method, for example, a nucleic acid probe having a characteristic that fluorescence is extinguished when a complex is formed with a target nucleic acid and fluorescence is emitted when released from the target nucleic acid is incorporated into the reaction system of the nucleic acid amplification method, and after the nucleic acid amplification method is completed. The reaction system is cooled at a low temperature to bind the nucleic acid probe to the target nucleic acid. At this point, the fluorescent dye of the nucleic acid probe is quenched. After that, when the temperature is gradually increased, the nucleic acid probe is released from the target nucleic acid at a temperature near the Tm value of the nucleic acid probe. At this time, since the fluorescence of the nucleic acid probe emits light, a significant change (increase) in fluorescence occurs at a temperature near the Tm value. In the melting curve analysis using a fluorescently labeled nucleic acid probe, the change in fluorescence is regarded as a detection signal.

As a nucleic acid probe that quenches when it forms a complex with the target nucleic acid as described above and emits light when it dissociates from the target nucleic acid and exists alone, a labeled probe whose terminal base labeled with a fluorescent quenching dye is cytosine is exemplified. To. When such a nucleic acid probe hybridizes to a target nucleic acid, it can be quenched by forming a base pair with a guanine base in the target nucleic acid sequence and interacting with the target nucleic acid sequence. Also known), etc., and it is preferable because the change in fluorescence intensity can be measured very easily.

　一つの実施態様において、本発明では、前記核酸プローブセットを構成する第一標的核酸プローブ及び第二標的核酸プローブによって生じる検出シグナルが重なるようにこれらの核酸プローブを設計することを特徴とする。これにより、第一標的核酸プローブ及び第二標的核酸プローブがそれぞれ異なる標的核酸に結合しても、各標的核酸を鑑別しないように検出することが可能となり得る。検出シグナルが重なるとは、例えばリアルタイムＰＣＲの場合は、同一波長で検出できる蛍光色素で標識された複数の核酸プローブを用いた場合に、それぞれの核酸プローブを単独で用いる場合よりもＣｔが低下すれば、蛍光変化の増大が起きたと判断でき、検出シグナルが重なったと解される。Ｃｔの低下が起きる理由は以下の通りである。前記の通り、標的核酸と結合したのち分解されたＴａｑＭａｎプローブが蛍光を発し、蛍光量が閾値を超えた時点でＣｔが出力される。同一波長で検出できる蛍光色素で標識された複数の核酸プローブが別々の標的核酸と結合し分解されることで、反応系全体での同一波長で検出される蛍光が増強され、より早期にＣｔが得られやすくなる。
In one embodiment, the present invention is characterized in that these nucleic acid probes are designed so that the detection signals generated by the first target nucleic acid probe and the second target nucleic acid probe constituting the nucleic acid probe set overlap. This makes it possible to detect each target nucleic acid without differentiating even if the first target nucleic acid probe and the second target nucleic acid probe bind to different target nucleic acids. Overlapping detection signals means, for example, in the case of real-time PCR, when multiple nucleic acid probes labeled with fluorescent dyes that can be detected at the same wavelength are used, the Ct is lower than when each nucleic acid probe is used alone. For example, it can be determined that an increase in fluorescence change has occurred, and it is understood that the detection signals overlap. The reason why the decrease in Ct occurs is as follows. As described above, the TaqMan probe decomposed after binding to the target nucleic acid emits fluorescence, and Ct is output when the amount of fluorescence exceeds the threshold value. Multiple nucleic acid probes labeled with a fluorescent dye that can be detected at the same wavelength bind to different target nucleic acids and are degraded, thereby enhancing the fluorescence detected at the same wavelength in the entire reaction system and increasing Ct earlier. It will be easier to obtain.

　融解曲線解析の場合は、複数の核酸プローブがそれぞれの標的核酸と結合したのち遊離し発光する。このとき、第一標的核酸プローブと第二標的核酸プローブとの間のＴｍ値に大きな相違があれば、個々の標的核酸プローブによる検出温度が大きく相違し、検出シグナルは鑑別される可能性がある。一方、核酸プローブセットを構成する第一標的核酸プローブと第二標的核酸プローブとの間でＴｍ値の差が無い又は小さい場合は、融解曲線解析を行った場合に、ほぼ同一の温度で検出シグナルが得られ、各標的核酸を鑑別しないように検出することができる。このように、ほぼ同一の温度で検出シグナルが現れるようにし、各標的核酸を鑑別しないように検出するための標的核酸プローブ間のＴｍ値の差は、好ましくは５℃以内であり、より好ましくは４℃以内であり、更に好ましくは３℃以内である。例えば、標的核酸プローブ間のＴｍ値の差は、２．５℃以内、２℃以内、１．５℃以内であってもよいし、実質的にＴｍ値に差がないものであってもよい。融解曲線解析において本発明を実施する場合、第一標的核酸プローブと第二標的核酸プローブのＴｍ値の差を上記のような範囲内にすることで、所定の検出温度で同一波長で検出できる蛍光変化による検出シグナルが得られることとなり、これらの検出シグナルを重ね合わせることができる。
In the case of melting curve analysis, a plurality of nucleic acid probes bind to each target nucleic acid and then release and emit light. At this time, if there is a large difference in the Tm value between the first target nucleic acid probe and the second target nucleic acid probe, the detection temperature by each target nucleic acid probe is significantly different, and the detection signal may be differentiated. .. On the other hand, when there is no or small difference in Tm value between the first target nucleic acid probe and the second target nucleic acid probe constituting the nucleic acid probe set, the detection signal is detected at almost the same temperature when the melting curve analysis is performed. Is obtained, and each target nucleic acid can be detected so as not to be differentiated. In this way, the difference in Tm value between the target nucleic acid probes for detecting the target nucleic acid so that the detection signal appears at substantially the same temperature and does not discriminate between the target nucleic acids is preferably within 5 ° C., more preferably. It is within 4 ° C, more preferably within 3 ° C. For example, the difference in Tm value between the target nucleic acid probes may be within 2.5 ° C, within 2 ° C, within 1.5 ° C, or may have substantially no difference in Tm value. .. When the present invention is carried out in the melting curve analysis, fluorescence that can be detected at the same wavelength at a predetermined detection temperature by keeping the difference between the Tm values of the first target nucleic acid probe and the second target nucleic acid probe within the above range. Detection signals due to changes can be obtained, and these detection signals can be superimposed.

　標的核酸プローブのＴｍ値は、当該分野で公知の方法により求めることができ、当業者は所定のＴｍ値を有する標的核酸プローブを容易に設計することができる。具体的には、Ｔｍ値は、核酸プローブを構成する全長塩基数と塩基配列内の塩基比率（Ａ、Ｔ、Ｃ、Ｇが何パーセントあるか）によって決定することができる。Ｔｍ値の計算方法としては、例えば最近接塩基対法、Ｗａｌｌａｃｅ法、ＧＣ％法等が公知である。本発明において、特に融解曲線解析での標的の検出に用いられる場合には、同じＴｍ値計算法を用いたときに、第一標的核酸プローブと第二標的核酸プローブとの間のＴｍ値の差が上記で規定した範囲内となるように設計することが好ましい。
The Tm value of the target nucleic acid probe can be obtained by a method known in the art, and those skilled in the art can easily design a target nucleic acid probe having a predetermined Tm value. Specifically, the Tm value can be determined by the total number of bases constituting the nucleic acid probe and the base ratio in the base sequence (what percentage of A, T, C, G are present). As a method for calculating the Tm value, for example, the closest base pair method, the Wallace method, the GC% method and the like are known. In the present invention, especially when used for target detection in melting curve analysis, the difference in Tm value between the first target nucleic acid probe and the second target nucleic acid probe when the same Tm value calculation method is used. Is preferably designed so as to be within the range specified above.

　本発明において、第一標的核酸プローブの塩基配列と第二標的核酸プローブの塩基配列との塩基長の差は、本発明の効果を奏する限り特に限定されない。各標的核酸を区別せずに検出することで核酸検出シグナルを重複させ、対象の標的を高感度に検出できるという観点から、好ましくは第一標的核酸プローブと第二標的核酸プローブとの間で塩基長に極端な差が無いことが好ましい。このような観点から、第一標的核酸プローブと第二標的核酸プローブとの塩基長の差は８ｍｅｒ以下であることが好ましく、５ｍｅｒ以下であることがより好ましく、３ｍｅｒ以下であることが更に好ましい。第一標的核酸プローブと第二標的核酸プローブとの塩基長の差の下限値としては、塩基長に差がない０ｍｅｒであってもよいし、１ｍｅｒ以上であってもよいし、２ｍｅｒ以上であってもよい。
In the present invention, the difference in the base length between the base sequence of the first target nucleic acid probe and the base sequence of the second target nucleic acid probe is not particularly limited as long as the effect of the present invention is exhibited. From the viewpoint that nucleic acid detection signals can be duplicated by detecting each target nucleic acid without distinction, and the target of interest can be detected with high sensitivity, a base is preferably used between the first target nucleic acid probe and the second target nucleic acid probe. It is preferable that there is no extreme difference in length. From such a viewpoint, the difference in base length between the first target nucleic acid probe and the second target nucleic acid probe is preferably 8 mer or less, more preferably 5 mer or less, and further preferably 3 mer or less. The lower limit of the difference in base length between the first target nucleic acid probe and the second target nucleic acid probe may be 0 mer with no difference in base length, 1 mer or more, or 2 mer or more. You may.

　一つの実施形態において、第一標的核酸プローブと第二標的核酸プローブは、それぞれの標的核酸に対して完全に相補的な塩基配列から構成されることが好ましい。完全に相補的な塩基配列から構成される標的核酸プローブとすることで、対象の標的をより確度よく検出することが可能になり得る。本明細書において、完全に相補的であるとは標的核酸プローブを構成する塩基配列がその全長にわたって標的核酸の塩基配列と相補的であることをいい、ミスマッチ塩基を含まないことをいう。
In one embodiment, the first target nucleic acid probe and the second target nucleic acid probe are preferably composed of a base sequence that is completely complementary to each target nucleic acid. By using a target nucleic acid probe composed of a completely complementary base sequence, it may be possible to detect the target of interest more accurately. As used herein, "completely complementary" means that the base sequence constituting the target nucleic acid probe is complementary to the base sequence of the target nucleic acid over its entire length, and does not contain a mismatched base.

［核酸増幅反応］

　一つの実施態様において、本発明の方法は、核酸プライマーセットを用いて核酸増幅反応を行い、核酸増幅産物を生成させることを含む。核酸増幅法は数コピーの標的核酸を可視化可能なレベル、すなわち数億コピー以上に増幅する技術であり、生命科学研究分野のみならず、臨床診断、食品衛生検査、環境検査等の分野においても広く用いられている。そのような核酸増幅法としては、ＰＣＲ法、ＬＡＭＰ法、ＬＣＲ法、ＴＭＡ法、ＳＤＡ法、ＲＴ－ＰＣＲ法、ＲＴ－ＬＡＭＰ法、ＮＡＳＢＡ法、ＴＲＣ法、ＴＭＡ法等が挙げられる。これらの技術は既に当該技術分野において確立されており、目的に合わせて方法を選択することができる。本発明で行う核酸増幅法はＰＣＲ法（ＲＴ－ＰＣＲ法を含む）が好ましいが、これに限定されない。
[Nucleic acid amplification reaction]

In one embodiment, the method of the invention comprises performing a nucleic acid amplification reaction with a nucleic acid primer set to produce a nucleic acid amplification product. Nucleic acid amplification method is a technology that amplifies several copies of target nucleic acid to a level that can be visualized, that is, to hundreds of millions of copies or more, and is widely used not only in the field of life science research but also in the fields of clinical diagnosis, food hygiene inspection, environmental inspection, etc. It is used. Examples of such nucleic acid amplification methods include PCR method, LAMP method, LCR method, TMA method, SDA method, RT-PCR method, RT-LAMP method, NASBA method, TRC method, TMA method and the like. These techniques have already been established in the relevant technical field, and the method can be selected according to the purpose. The nucleic acid amplification method performed in the present invention is preferably, but not limited to, the PCR method (including the RT-PCR method).

［ＰＣＲ反応］

　ＰＣＲ反応は、主にＤＮＡポリメラーゼによって触媒される反応であり、（１）熱処理によるＤＮＡ変性（２本鎖ＤＮＡから１本鎖ＤＮＡへの乖離）、（２）鋳型１本鎖ＤＮＡへのプライマーのアニーリング、（３）ＤＮＡポリメラーゼを用いた前記プライマーの伸長、という３ステップを１サイクルとし、このサイクルを繰り返すことによって標的核酸を増幅する。ＤＮＡポリメラーゼとしては、Ｔａｑ、Ｔｔｈ、Ｂｓｔ、ＫＯＤ、Ｐｆｕ、Ｐｗｏ、Ｔｂｒ、Ｔｆｉ、Ｔｆｌ、Ｔｍａ、Ｔｎｅ、Ｖｅｎｔ、ＤＥＥＰＶＥＮＴやその変異体が挙げられる。本発明では、迅速、高感度、かつ検体による増幅阻害耐性を有するという観点から、ファミリーＢに属するＤＮＡポリメラーゼを用いることが好ましい。また、融解曲線解析法を行う場合には、蛍光消光プローブを用いる観点からも、５’→３’エキソヌクレアーゼ活性を有しないファミリーＢに属するＤＮＡポリメラーゼを用いることが好ましい。
[PCR reaction]

The PCR reaction is a reaction mainly catalyzed by DNA polymerase, (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), and (2) primer to template single-stranded DNA. The three steps of annealing and (3) extension of the primer using DNA polymerase are set as one cycle, and the target nucleic acid is amplified by repeating this cycle. Examples of the DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT and their variants. In the present invention, it is preferable to use a DNA polymerase belonging to Family B from the viewpoint of rapidity, high sensitivity, and resistance to amplification inhibition by a sample. Further, when performing the melting curve analysis method, it is preferable to use a DNA polymerase belonging to Family B which does not have 5'→ 3'exonuclease activity from the viewpoint of using a fluorescent quenching probe.

　ＰＣＲ反応の条件は、本発明の効果を奏する限り特に限定されないが、例えば、最初の熱変形工程が８０～１００℃で０秒～５分、繰り返しの熱変形工程が８０～１００℃で０．５～３００秒、アニーリンクが３５～８０℃で１～３００秒、伸長反応工程が３５～８５℃で１～３００秒程度行い、この繰り返しを３０～７０回繰り返すことが好ましい。ここで繰り返し行うサイクルの温度及び時間は、１～数サイクル毎に変化させてもよい。
The conditions of the PCR reaction are not particularly limited as long as the effects of the present invention are exhibited. For example, the first thermal deformation step is at 80 to 100 ° C. for 0 seconds to 5 minutes, and the repeated thermal deformation step is at 80 to 100 ° C. at 0. It is preferable that the Annie Link is carried out at 35 to 80 ° C. for 1 to 300 seconds and the extension reaction step is carried out at 35 to 85 ° C. for 1 to 300 seconds, and this repetition is repeated 30 to 70 times. The temperature and time of the cycle repeated here may be changed every one to several cycles.

［ファミリーＢに属するＤＮＡポリメラーゼ］

　本発明で用いるＤＮＡポリメラーゼは、ファミリーＢに属するＤＮＡポリメラーゼが好ましいが、これに限定されない。前記ファミリーＢに属するＤＮＡポリメラーゼは、特に制限されないが、好ましくは古細菌（Ａｒｃｈｅａ）由来のＤＮＡポリメラーゼであり、より好ましくは、パイロコッカス（Ｐｙｒｏｃｏｃｃｕｓ）属およびサーモコッカス（Ｔｈｅｒｍｏｃｏｃｃｕｓ）属の細菌に由来するＤＮＡポリメラーゼが挙げられる。また、本発明には、ファミリーＢに属する古細菌由来のＤＮＡポリメラーゼ活性を失っていないその変異体も含まれる。ＤＮＡポリメラーゼの変異体には、ポリメラーゼ活性の増強、エキソヌクレアーゼ活性の欠損、基質特異性の調整等を目的とした変異体が挙げられるが、これらに限定されない。

　パイロコッカス属由来のＤＮＡポリメラーゼとしては、Ｐｙｒｏｃｏｃｃｕｓ　ｆｕｒｉｏｓｕｓ、Ｐｙｒｏｃｏｃｃｕｓ　ｓｐ．ＧＢ－Ｄ、Ｐｙｒｏｃｏｃｃｕｓ　ｗｏｅｓｅｉ、Ｐｙｒｏｃｏｃｃｕｓ　ａｂｙｓｓｉ、Ｐｙｒｏｃｏｃｃｕｓ　ｈｏｒｉｋｏｓｈｉｉから単離されたＤＮＡポリメラーゼ、及びこれらに由来するＤＮＡポリメラーゼ活性を失っていないその変異体を含むが、これらに限定されない。

　サーモコッカス属に由来するＤＮＡポリメラーゼとしては、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｋｏｄａｋａｒａｅｎｓｉｓ、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｇｏｒｇｏｎａｒｉｕｓ、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｌｉｔｏｒａｌｉｓ、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．ＪＤＦ－３、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．９ｄｅｇｒｅｅｓ　Ｎｏｒｔｈ－７（Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．９°Ｎ－７）、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｉｃｕｌｉから単離されたＤＮＡポリメラーゼ、及びこれらに由来するＤＮＡポリメラーゼ活性を失っていないその変異体を含むが、これらに限定されない。好ましくは、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｋｏｄａｋａｒａｅｎｓｉｓ由来のＤＮＡポリメラーゼ及びその変異体（例えば、３’→５’エキソヌクレアーゼ活性を欠失させたＫＯＤ由来ＤＮＡポリメラーゼ等）が、伸長性や熱安定性に優れているという観点から、本発明においてとりわけ好適に用いることができる。

　これらのＤＮＡポリメラーゼを用いたＰＣＲ酵素は市販されており、Ｐｆｕ（Ｓｔａｒａｇｅｎｅ社）、ＫＯＤ（Ｔｏｙｏｂｏ社）、Ｐｆｘ（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）、Ｖｅｎｔ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ社）、Ｄｅｅｐ　Ｖｅｎｔ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ社）、Ｔｇｏ（Ｒｏｃｈｅ社）、Ｐｗｏ（Ｒｏｃｈｅ社）などが挙げられ、そのいずれもが本発明に用いられ得る。
[DNA polymerase belonging to Family B]

The DNA polymerase used in the present invention is preferably, but is not limited to, a DNA polymerase belonging to Family B. The DNA polymerase belonging to the family B is not particularly limited, but is preferably a DNA polymerase derived from archaea, and more preferably derived from a bacterium belonging to the genus Pyrococcus and the genus Thermococcus. Examples include DNA polymerase. The present invention also includes variants thereof that have not lost their DNA polymerase activity from archaea belonging to Family B. Variants of DNA polymerase include, but are not limited to, mutants for the purpose of enhancing polymerase activity, deficiency of exonuclease activity, adjusting substrate specificity, and the like.

Examples of the DNA polymerase derived from the genus Pyrococcus include Pyrococcus furiosus and Pyrococcus sp. DNA polymerases isolated from GB-D, Pyrococcus womeni, Pyrococcus abyssi, Pyrococcus horikoshii, and variants thereof that have not lost their DNA polymerase activity, but are not limited thereto.

Examples of the DNA polymerase derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermococcus gogonarius, Thermococcus litoralis, and Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), DNA polymerase isolated from Thermococcus siculi, and variants thereof that have not lost their DNA polymerase activity, but are not limited thereto. Preferably, a DNA polymerase derived from Thermococcus kodakaranesis and a variant thereof (for example, a KOD-derived DNA polymerase lacking 3'→ 5'exonuclease activity) are excellent in extensibility and thermal stability. , Can be particularly preferably used in the present invention.

PCR enzymes using these DNA polymerases are commercially available, Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), DeepBent (New) , Tgo (Roche), Pwo (Roche) and the like, all of which can be used in the present invention.

［ＫＯＤ由来のＤＮＡポリメラーゼ］

　本明細書において、ＫＯＤ由来のＤＮＡポリメラーゼ（ＫＯＤ　ＤＮＡポリメラーゼともいう）とは、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｋｏｄａｋａｒａｅｎｓｉｓ由来のＤＮＡポリメラーゼ及びその変異体（例えば、天然由来のアミノ酸配列において１又は数個のアミノ酸を置換、欠失、及び／又は付加することにより３’→５’エキソヌクレアーゼ活性を欠失させたＫＯＤ由来ＤＮＡポリメラーゼ等）をいう。一つの好ましい実施形態において、本発明は、このようなＫＯＤ由来のＤＮＡポリメラーゼを使用して核酸増幅反応を行う。ＫＯＤ　ＤＮＡポリメラーゼは、ファミリーＡに属するＤＮＡポリメラーゼであるＴａｑ　ＤＮＡポリメラーゼに比べて、正確性、増幅効率、伸長性、検体由来の阻害物質による増幅阻害耐性に優れている。
[DNA polymerase derived from KOD]

As used herein, a KOD-derived DNA polymerase (also referred to as a KOD DNA polymerase) is a DNA polymerase derived from Thermococcus kodakaranesis and a variant thereof (for example, one or several amino acids are substituted or deleted in a naturally occurring amino acid sequence). , And / or KOD-derived DNA polymerase, etc., in which 3'→ 5'exonuclease activity has been deleted by addition. In one preferred embodiment, the present invention uses such a KOD-derived DNA polymerase to carry out a nucleic acid amplification reaction. KOD DNA polymerase is superior to Taq DNA polymerase, which is a DNA polymerase belonging to Family A, in accuracy, amplification efficiency, extensibility, and resistance to amplification inhibition by a sample-derived inhibitor.

［試料］

　本発明は、任意の試料から標的を核酸検出する場面において利用され得る。本発明において使用できる試料は複数の標的核酸を含む可能性のあるものであれば特に限定されず、例えば、生体試料や食品、環境試料だけでなく、精製核酸等が挙げられる。試料は核酸抽出やいくつかの前処理を行ってもよい。試料の核酸抽出や前処理は、当該技術分野で一般的に行われている。前処理としては、ろ過、遠心分離、希釈処理、加熱処理、酸処理、アルカリ処理、有機溶媒処理、懸濁処理、破砕処理、磨砕処理等が挙げられるが、本発明ではこれらに限定されない。
[sample]

The present invention can be used in situations where nucleic acids are detected in a target from any sample. The sample that can be used in the present invention is not particularly limited as long as it may contain a plurality of target nucleic acids, and examples thereof include not only biological samples, foods, and environmental samples, but also purified nucleic acids and the like. The sample may be subjected to nucleic acid extraction and some pretreatment. Nucleic acid extraction and pretreatment of samples are commonly performed in the art. Examples of the pretreatment include, but are not limited to, filtration, centrifugation, dilution treatment, heat treatment, acid treatment, alkali treatment, organic solvent treatment, suspension treatment, crushing treatment, and grinding treatment.

　生体試料の例として、特に制限されないが、動植物組織、体液、排泄物、細胞、細菌、ウイルス等が挙げられる。さらに挙げると、血液、血液培養液、尿、膿、髄液、胸水、咽頭拭い液、鼻腔拭い液、喀痰、組織切片、皮膚、吐瀉物、糞便、分離培養コロニー、カテーテル洗浄液、子宮頸管擦過物、尿道擦過物、男性尿道擦過物尿等が挙げられる。
Examples of biological samples include, but are not limited to, animal and plant tissues, body fluids, excrement, cells, bacteria, viruses and the like. Further, blood, blood culture medium, urine, pus, spinal fluid, pleural fluid, pharyngeal swab, nasal swab, sputum, tissue section, skin, vomitus, feces, isolated culture colony, catheter lavage fluid, cervical scraping , Urethral scrapes, male urethral scrapes, urine and the like.

　食品の例として、水、アルコール飲料、清涼飲料水、加工食品、野菜、畜産物、海産物、卵、乳製品、生肉、生魚、惣菜等が挙げられる。また、食品を測定試料とする場合、その食品の一部あるいは全部を使用できるだけでなく、食品表面を拭き取ったものも使用できる。さらに、調理器具やドアノブを拭き取った材料あるいはそれらを洗浄した洗浄液も試料として用いることができる。
Examples of foods include water, alcoholic beverages, soft drinks, processed foods, vegetables, livestock products, marine products, eggs, dairy products, raw meat, raw fish, prepared foods and the like. When a food is used as a measurement sample, not only a part or all of the food can be used, but also a food whose surface has been wiped off can be used. Further, a material for wiping off a cooking utensil or a doorknob or a cleaning liquid for cleaning them can also be used as a sample.

　環境試料の例として、水、氷、土壌、空気やエアゾール等が挙げられる。ここでいう水とは、例として、水道水、海水あるいは川や滝、湖、池等から採取した水等が挙げられる。また、施設の壁面、床面、設備や備品、便器等を拭き取ったものあるいはそれらを洗浄した洗浄液も試料として用いることができる。
Examples of environmental samples include water, ice, soil, air and aerosols. Examples of water here include tap water, seawater, and water collected from rivers, waterfalls, lakes, ponds, and the like. Further, a cleaning solution obtained by wiping the wall surface, floor surface, equipment / equipment, toilet bowl, etc. of the facility or cleaning them can also be used as a sample.

　本発明には上記のようないずれの試料も用いることができるが、外来性の標的核酸を含み得る試料であることが好ましい。例えば、一つ又は複数の感染性微生物に感染したことが疑われる被験者又は被験物から採取した試料に対して特に本発明は有効である。採取する試料の種類としては、被験者の感染症の症状又は被験物の状態等から、適切な試料をその都度選択することが好ましい。例えば、呼吸器感染症の感染性微生物（例えば、Ｍｙｃｏｐｌａｓｍａ　ｐｎｅｕｍｏｎｉａｅ、ＳＡＲＳ－ＣｏＶ－２、インフルエンザウイルス等）に由来する複数の標的核酸を検出する場合は、鼻咽頭拭い液、鼻腔拭い液、喀痰、唾液等の生体試料を用いることができる。試料の採取方法、調製方法等は、特に制限されず、試料の種類、目的に応じて公知の方法を用いることができる。
Any sample as described above can be used in the present invention, but it is preferably a sample that can contain an exogenous target nucleic acid. For example, the present invention is particularly effective for a subject or a sample taken from a subject suspected of being infected with one or more infectious microorganisms. As the type of sample to be collected, it is preferable to select an appropriate sample each time based on the symptoms of the infectious disease of the subject, the condition of the subject, and the like. For example, when detecting multiple target nucleic acids derived from infectious microorganisms of respiratory infections (eg, Mycoplasma pneumoniae, SARS-CoV-2, influenza virus, etc.), nasopharyngeal swab, nasal swab, sputum, etc. Biological samples such as saliva can be used. The method for collecting a sample, the method for preparing a sample, and the like are not particularly limited, and a known method can be used depending on the type and purpose of the sample.

　本発明は、検査対象となる標的を高感度に検出することが望まれる任意の場面で利用され得る。例えば、試料中に極微量に含まれうる微生物由来の核酸を検出したいような場合において好適に使用され得る。例えば、一つの感染性微生物の感染の有無を検査する場合には、該感染性微生物のゲノム配列における複数の異なる領域を標的配列として、それぞれに対応する複数の標的核酸プローブを設計して核酸プローブセットとする。また、もし複数の感染性微生物を特に鑑別する必要がなく感染症の有無を検査する場合には、それぞれの感染性微生物に由来する標的核酸に対応する複数の標的核酸プローブを設計して核酸プローブセットとすることができる。
The present invention can be used in any situation where it is desired to detect a target to be inspected with high sensitivity. For example, it can be suitably used when it is desired to detect a nucleic acid derived from a microorganism that can be contained in a very small amount in a sample. For example, when inspecting the presence or absence of infection by one infectious microorganism, a plurality of target nucleic acid probes corresponding to each of a plurality of different regions in the genomic sequence of the infectious microorganism are designed and nucleic acid probes. Make a set. In addition, if it is not necessary to specifically distinguish multiple infectious microorganisms and the presence or absence of an infectious disease is to be inspected, a plurality of target nucleic acid probes corresponding to the target nucleic acids derived from each infectious microorganism should be designed and the nucleic acid probe. It can be a set.

［試薬組成物又はキット]

　一つの実施形態において、本発明は、前記核酸プローブセットを含む試薬組成物を提供する。更に別の態様では、本発明は、前記核酸プローブセット又は試薬組成物を含むキットであり得る。本発明の試薬組成物又はキットは、前記の第一標的核酸プローブ及び第二標的核酸プローブを少なくとも含む核酸プローブセットの他に、例えば、第一標的核酸プローブが結合可能な第一標的核酸の配列を含む領域を増幅可能な１つ以上の第一標的用プライマー（又はプライマーセット）、及び、第二標的核酸プローブが結合可能な第二標的核酸の配列を含む領域を増幅可能な１つ以上の第二標的用プライマー（又はプライマーセット）を更に含むことが好ましい。本試薬組成物又はキットは更に、効率よく特異性の高い核酸増幅を行うために、例えばＤＮＡポリメラーゼや、デオキシリボヌクレオシド三リン酸（ｄＮＴＰｓ）、マグネシウム塩等の無機塩を少なくとも含むことが好ましい。各成分の濃度は適宜調整できるが、例えば、オリゴヌクレオチドプローブ（第一標的核酸プローブ又は第二標的核酸プローブ）は０．１～１．５μＭが好ましく、０．２～０．５μＭがより好ましい。ＤＮＡポリメラーゼは０．０１～１Ｕ／ｕＬが好ましく、０．１～０．５Ｕ／ｕＬがより好ましい。オリゴヌクレオチドプライマー（第一標的用プライマー又は第二標的用プライマー）は、０．１～１０μＭが好ましい。デオキシリボヌクレオシド三リン酸（ｄＮＴＰｓ）は０．０２～１ｍＭが好ましく、０．１～０．５ｍＭがより好ましい。マグネシウム塩等の無機塩は０．１～６ｍＭが好ましく、１～５ｍＭがより好ましい。
[Reagent composition or kit]

In one embodiment, the invention provides a reagent composition comprising said nucleic acid probe set. In yet another aspect, the invention may be a kit comprising said nucleic acid probe set or reagent composition. In the reagent composition or kit of the present invention, in addition to the nucleic acid probe set containing at least the first target nucleic acid probe and the second target nucleic acid probe, for example, a sequence of a first target nucleic acid to which the first target nucleic acid probe can bind. One or more regions containing a first target primer (or primer set) capable of amplifying a region containing, and a region containing a sequence of a second target nucleic acid to which a second target nucleic acid probe can bind. It is preferable to further include a second target primer (or primer set). The present reagent composition or kit further preferably contains at least an inorganic salt such as DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and magnesium salt in order to efficiently perform nucleic acid amplification with high specificity. The concentration of each component can be adjusted as appropriate, but for example, the oligonucleotide probe (first target nucleic acid probe or second target nucleic acid probe) is preferably 0.1 to 1.5 μM, more preferably 0.2 to 0.5 μM. The DNA polymerase is preferably 0.01 to 1 U / uL, more preferably 0.1 to 0.5 U / uL. The oligonucleotide primer (primer for first target or primer for second target) is preferably 0.1 to 10 μM. Deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. The inorganic salt such as magnesium salt is preferably 0.1 to 6 mM, more preferably 1 to 5 mM.

　本発明の試薬組成物又はキットはさらに、非特異増幅の抑制や反応促進を目的として、当該技術分野で知られる添加物等を加えてもよい。非特異増幅の抑制を目的とする添加物として、抗ＤＮＡポリメラーゼ抗体やリン酸等が挙げられる。反応促進を目的とする添加物として、ウシ血清アルブミン（ＢＳＡ）、プロテアーゼインヒビター、シングルストランド結合タンパク質（ＳＳＢ）、Ｔ４遺伝子３２タンパク質、ｔＲＮＡ、硫黄または酢酸含有化合物類、ジメチルスルホキシド（ＤＭＳＯ）、グリセロール、エチレングリコール、プロピレングリコール、トリメチレングリコール、ホルムアミド、アセトアミド、ベタイン、エクトイン、トレハロース、デキストラン、ポリビニルピロリドン（ＰＶＰ）、ゼラチン、塩化テトラメチルアンモニウム（ＴＭＡＣ）、水酸化テトラメチルアンモニウム（ＴＭＡＨ）、酢酸テトラメチルアンモニウム（ＴＭＡＡ）、ポリエチレングリコール、トリトン（Ｔｒｉｔｏｎ）、ツイーン（Ｔｗｅｅｎ２０）、ノニデットＰ４０などが挙げられる。本発明では、これらの添加物を１種類以上組み合わせて使用してもよいが、これらに限定されない。
The reagent composition or kit of the present invention may further contain additives and the like known in the art for the purpose of suppressing non-specific amplification and promoting the reaction. Examples of additives for the purpose of suppressing non-specific amplification include anti-DNA polymerase antibodies and phosphoric acid. Additives aimed at promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur or acetic acid-containing compounds, dimethylsulfoxide (DMSO), glycerol, Ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectin, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMC), tetramethylammonium hydroxide (TMAH), tetramethylacetate Examples thereof include ammonium (TMA), polyethylene glycol, Triton, Tween 20 and Nonidet P40. In the present invention, one or more of these additives may be used in combination, but the present invention is not limited thereto.

［核酸検出方法又は核酸の検出感度向上方法］

　一つの実施形態において、本発明は、前記のような核酸プローブセット、試薬組成物、及び／又はキットを用いる核酸の検出方法を提供する。本発明では、同一波長で検出できる蛍光色素で標識された複数の異なる塩基配列を有する核酸プローブをセットで用いて、これらを区別せずに検出することで核酸検出シグナルを重複させ、検査対象となる標的を高感度に検出できる。従って、本発明の核酸検出方法は、検査対象となる標的の検出において、核酸の検出感度を向上させる方法ともいうことができる。本発明の核酸の検出方法又は核酸の検出感度向上方法は、当該分野で公知の任意の核酸検出法において実施することができる。好ましくは、リアルタイムＰＣＲ、リアルタイムＲＴ－ＰＣＲ、又は融解曲線解析において実施することができ、なかでも融解曲線解析で蛍光検出する方法において好適に実施され得る。
[Nucleic acid detection method or nucleic acid detection sensitivity improvement method]

In one embodiment, the invention provides a method for detecting nucleic acids using a nucleic acid probe set, reagent composition, and / or kit as described above. In the present invention, nucleic acid probes having a plurality of different base sequences labeled with fluorescent dyes that can be detected at the same wavelength are used as a set, and by detecting them without distinction, the nucleic acid detection signals are duplicated and the test target is used. Target can be detected with high sensitivity. Therefore, the nucleic acid detection method of the present invention can also be said to be a method for improving the nucleic acid detection sensitivity in the detection of a target to be inspected. The nucleic acid detection method or the nucleic acid detection sensitivity improving method of the present invention can be carried out by any nucleic acid detection method known in the art. Preferably, it can be carried out in real-time PCR, real-time RT-PCR, or melting curve analysis, and in particular, it can be preferably carried out in a method of detecting fluorescence by melting curve analysis.

　以下、本発明の実施例に基づき具体的に説明する。本発明は下記実施例に限定されるものではない。
Hereinafter, a specific description will be given based on examples of the present invention. The present invention is not limited to the following examples.

〔実施例１：核酸プローブセットの効果確認〕

（１）目的

　同一波長で検出できる蛍光色素で標識した２本の核酸プローブから成る核酸プローブセットを含む組成物を用いて核酸増幅（ＰＣＲ）及び検出を行った場合に得られる検出シグナルが、該核酸プローブを個々に用いて同様の核酸増幅及び検出を行った場合に得られる核酸シグナルよりも増強されるかを確認することを目的として、本実施例に記載の実験を行った。

（２）試料の調製

　Ｍｙｃｏｐｌａｓｍａ　ｐｎｅｕｍｏｎｉａｅ （以下、ＭＰ）から抽出されたＤＮＡを８０又は４０コピー／テストとなるように調製し、試料とした。

（３）核酸増幅、融解曲線解析、及び判定

　上記試料をそれぞれ下記試薬に添加して、下記条件によりＭＰの検出を行った。核酸増幅および融解曲線解析には東洋紡製ＧＥＮＥＣＵＢＥ（登録商標）を使用した。判定は、融解曲線解析にてピークが得られ、ピークの高さを示す蛍光変化量が５以上となった場合に陽性と判定した。測定は試料ごとにＮ＝２で行った。
[Example 1: Confirmation of effect of nucleic acid probe set]

(1) Purpose

The detection signal obtained when nucleic acid amplification (PCR) and detection are performed using a composition containing a nucleic acid probe set consisting of two nucleic acid probes labeled with a fluorescent dye that can be detected at the same wavelength individually indicates the nucleic acid probe. The experiment described in this example was carried out for the purpose of confirming whether or not the nucleic acid signal was enhanced more than the nucleic acid signal obtained when the same nucleic acid amplification and detection was performed.

(2) Preparation of sample

DNA extracted from Mycoplasma pneumoniae (hereinafter referred to as MP) was prepared to be 80 or 40 copies / test and used as a sample.

(3) Nucleic acid amplification, melting curve analysis, and determination

Each of the above samples was added to the following reagents, and MP was detected under the following conditions. GENECUBE® manufactured by Toyobo Co., Ltd. was used for nucleic acid amplification and melting curve analysis. The judgment was positive when a peak was obtained by melting curve analysis and the amount of change in fluorescence indicating the height of the peak was 5 or more. The measurement was performed with N = 2 for each sample.

　具体的には、核酸プローブセットを用いた場合の検出シグナルの増強を確認するため、ジーンキューブ　テストベーシック（東洋紡）を用い、以下の２本の核酸プローブ及びそれに対応する核酸プライマーセット及び試料を含む溶液（試薬１）を調製した。また、試薬１との比較として、ジーンキューブ　テストベーシックを用い、核酸プローブセットを構成する核酸プローブ、およびそれに対応する核酸プライマーセットを単独で用いる溶液（試薬２、試薬３）を調製した。本実施例で使用する配列番号５で示されるプローブのＴｍ値は５４．２℃、配列番号６で示されるプローブのＴｍ値は５４．０℃であり、融解曲線解析における検出温度が近接するように設計した。なお、Ｔｍ値は最近接塩基対法で計算した。以降の実施例でもＴｍ値を表記したものは同様に最近接塩基対法で計算した。配列番号５及び６のプローブはいずれもＭＰのＤＮＡ配列に対して相補的な塩基配列を有し、同一の蛍光色素で標識したものである。
Specifically, in order to confirm the enhancement of the detection signal when using the nucleic acid probe set, GeneCube Test Basic (Toyobo) is used, and the following two nucleic acid probes and their corresponding nucleic acid primer sets and samples are included. A solution (reagent 1) was prepared. In addition, as a comparison with Reagent 1, a solution (Reagent 2, Reagent 3) using the nucleic acid probe constituting the nucleic acid probe set and the corresponding nucleic acid primer set alone was prepared using GeneCube Test Basic. The Tm value of the probe shown by SEQ ID NO: 5 used in this example is 54.2 ° C., the Tm value of the probe shown by SEQ ID NO: 6 is 54.0 ° C., so that the detection temperatures in the melting curve analysis are close to each other. Designed for. The Tm value was calculated by the closest base pairing method. In the following examples as well, those in which the Tm value was expressed were similarly calculated by the nearest base pairing method. The probes of SEQ ID NOs: 5 and 6 both have a base sequence complementary to the DNA sequence of MP and are labeled with the same fluorescent dye.

試薬１

ジーンキューブ　テストベーシック

０．３μＭ　配列番号１で示されるプライマー

１．５μＭ　配列番号２で示されるプライマー

０．３μＭ　配列番号３で示されるプライマー

１．５μＭ　配列番号４で示されるプライマー

０．２５μＭ　配列番号５で示されるプローブ（３’末端をＢＯＤＩＰＹ－ＦＬで標識）

０．２５μＭ　配列番号６で示されるプローブ（３’末端をＢＯＤＩＰＹ－ＦＬで標識）
Reagent 1

Gene Cube Test Basic

0.3 μM Primer represented by SEQ ID NO: 1

1.5 μM Primer represented by SEQ ID NO: 2

0.3 μM Primer represented by SEQ ID NO: 3

1.5 μM Primer shown in SEQ ID NO: 4

0.25 μM Probe shown in SEQ ID NO: 5 (labeled 3'end with BODIPY-FL)

0.25 μM Probe shown in SEQ ID NO: 6 (labeled 3'end with BODIPY-FL)

試薬２

ジーンキューブ　テストベーシック

０．３μＭ　配列番号１で示されるプライマー

１．５μＭ　配列番号２で示されるプライマー

０．２５μＭ　配列番号５で示されるプローブ（３’末端をＢＯＤＩＰＹ－ＦＬで標識）
Reagent 2

Gene Cube Test Basic

0.3 μM Primer represented by SEQ ID NO: 1

1.5 μM Primer represented by SEQ ID NO: 2

0.25 μM Probe shown in SEQ ID NO: 5 (labeled 3'end with BODIPY-FL)

試薬３

ジーンキューブ　テストベーシック

０．３μＭ　配列番号３で示されるプライマー

１．５μＭ　配列番号４で示されるプライマー

０．２５μＭ　配列番号６で示されるプローブ（３’末端をＢＯＤＩＰＹ－ＦＬで標識）
Reagent 3

Gene Cube Test Basic

0.3 μM Primer represented by SEQ ID NO: 3

1.5 μM Primer shown in SEQ ID NO: 4

0.25 μM Probe shown in SEQ ID NO: 6 (labeled 3'end with BODIPY-FL)

核酸増幅および融解曲線解析

９７℃・３０秒

（以上１サイクル）

９７℃・１秒

５８℃・３秒

６３℃・５秒

（以上５０サイクル）

９４℃・３０秒

３９℃・３０秒

４０℃～７５℃（０．０９℃／秒で温度上昇）
Nucleic acid amplification and melting curve analysis

97 ° C for 30 seconds

(More than 1 cycle)

97 ° C for 1 second

58 ° C for 3 seconds

63 ° C for 5 seconds

(More than 50 cycles)

94 ° C for 30 seconds

39 ° C for 30 seconds

40 ° C to 75 ° C (temperature rises at 0.09 ° C / sec)

（４）結果

　下記の表２は、上記の条件で核酸増幅及び融解曲線解析による検出を行った結果をまとめた表である（上段に陽性判定結果、下段に蛍光ピーク高さを示す（No peakはピークが確認できなかったことを示す））。核酸プローブセットを含む試薬１では８０コピー／テストのＭＰ　ＤＮＡが１００％、４０コピー／テストのＤＮＡが５０％の確率で検出できた。一方、試薬２では８０コピー／テストが５０％、試薬３では８０コピー／テストのＤＮＡが１００％で、試薬２及び試薬３とも、４０コピー／テストの検出率は０％であった。以上から、本発明の核酸プローブセットを含む組成物を用いて異なる２種類の標的核酸を検出することで、微量な濃度の試料からの検出率を向上させることが可能であることが明らかとなった。
(4) Result

Table 2 below is a table summarizing the results of detection by nucleic acid amplification and melting curve analysis under the above conditions (the upper row shows the positive judgment result, and the lower row shows the fluorescence peak height (No peak confirms the peak). Indicates that it could not be done)). In Reagent 1 containing the nucleic acid probe set, 80 copies / test MP DNA could be detected with 100% probability, and 40 copies / test DNA could be detected with 50% probability. On the other hand, reagent 2 had 80 copies / test of 50%, reagent 3 had 80 copies / test DNA of 100%, and reagent 2 and reagent 3 had a detection rate of 40 copies / test of 0%. From the above, it is clear that it is possible to improve the detection rate from a sample having a trace concentration by detecting two different types of target nucleic acids using the composition containing the nucleic acid probe set of the present invention. rice field.

Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002

〔実施例２：リアルタイムＰＣＲにおける核酸プローブセットの効果確認〕

（１）目的

　リアルタイムＰＣＲにおいても本発明の核酸プローブセットを含む組成物が有効であることを確認するため、ＴａｑＭａｎプローブを用いて実験を行った。

（２）試料の調製

　ＭＰから抽出されたＤＮＡを５０コピー／テストとなるように調製し、試料とした。

（３）核酸増幅及び核酸検出

　上記試料をそれぞれ下記試薬に添加して、下記条件によりＭＰの検出を行った。核酸増幅および検出にはリアルタイムＰＣＲ装置であるＲｏｔｏｒ－Ｇｅｎｅ　Ｑ（ＱＩＡＧＥＮ社）を使用した。
[Example 2: Confirmation of effect of nucleic acid probe set in real-time PCR]

(1) Purpose

In order to confirm that the composition containing the nucleic acid probe set of the present invention is also effective in real-time PCR, an experiment was conducted using a TaqMan probe.

(2) Preparation of sample

DNA extracted from MP was prepared to be 50 copies / test and used as a sample.

(3) Nucleic acid amplification and nucleic acid detection

Each of the above samples was added to the following reagents, and MP was detected under the following conditions. A real-time PCR device, Rotor-Gene Q (QIAGEN), was used for nucleic acid amplification and detection.

　具体的には、ＴＨＵＮＤＥＲＢＩＲＤ Ｐｒｏｂｅ ｑＰＣＲ Ｍｉｘ（東洋紡）を用い、以下の２本の核酸プローブ及びそれに対応する核酸プライマーセット及び試料を含む溶液（試薬４）を調製した。また、試薬４との比較として、ＴＨＵＮＤＥＲＢＩＲＤ Ｐｒｏｂｅ ｑＰＣＲ Ｍｉｘ（東洋紡）を用い、核酸プローブセットを構成する核酸プローブ、およびそれに対応する核酸プライマーセットを単独で用いる溶液（試薬５、試薬６）を調製した。本実施例で使用する配列番号７（Ｔｍ値６８．６℃）及び８（Ｔｍ値６６．０℃）のプローブはいずれもＭＰのＤＮＡ配列に対して相補的な塩基配列を有し、同一の蛍光色素及び消光物質で標識したものである。
Specifically, using THUNDERBIRD Probe qPCR Mix (Toyobo), a solution (reagent 4) containing the following two nucleic acid probes and their corresponding nucleic acid primer sets and samples was prepared. Further, as a comparison with the reagent 4, using THUNDERBIRD Probe qPCR Mix (Toyobo), a nucleic acid probe constituting the nucleic acid probe set and a solution (reagent 5 and reagent 6) using the corresponding nucleic acid primer set alone were prepared. .. The probes of SEQ ID NO: 7 (Tm value 68.6 ° C) and 8 (Tm value 66.0 ° C) used in this example both have a base sequence complementary to the DNA sequence of MP and are the same. It is labeled with a fluorescent dye and a quenching substance.

試薬４

ＴＨＵＮＤＥＲＢＩＲＤ Ｐｒｏｂｅ ｑＰＣＲ Ｍｉｘ

０．５μＭ　配列番号１で示されるプライマー

０．５μＭ　配列番号２で示されるプライマー

０．５μＭ　配列番号３で示されるプライマー

０．５μＭ　配列番号４で示されるプライマー

０．１μＭ　配列番号７で示されるプローブ（５’末端をＦＡＭで、３’末端をＢＨＱ１で標識）

０．１μＭ　配列番号８で示されるプローブ（５’末端をＦＡＭで、３’末端をＢＨＱ１で標識）
Reagent 4

THUNDERBIRD Probe qPCR Mix

0.5 μM Primer represented by SEQ ID NO: 1

0.5 μM Primer represented by SEQ ID NO: 2

0.5 μM Primer represented by SEQ ID NO: 3

0.5 μM Primer shown in SEQ ID NO: 4.

0.1 μM Probe represented by SEQ ID NO: 7 (labeled 5'end with FAM and 3'end with BHQ1)

0.1 μM Probe represented by SEQ ID NO: 8 (labeled 5'end with FAM and 3'end with BHQ1)

試薬５

ＴＨＵＮＤＥＲＢＩＲＤ Ｐｒｏｂｅ ｑＰＣＲ Ｍｉｘ

０．５μＭ　配列番号１で示されるプライマー

０．５μＭ　配列番号２で示されるプライマー

０．１μＭ　配列番号７で示されるプローブ（５’末端をＦＡＭで、３’末端をＢＨＱ１で標識）
Reagent 5

THUNDERBIRD Probe qPCR Mix

0.5 μM Primer represented by SEQ ID NO: 1

0.5 μM Primer represented by SEQ ID NO: 2

0.1 μM Probe represented by SEQ ID NO: 7 (labeled 5'end with FAM and 3'end with BHQ1)

試薬６

ＴＨＵＮＤＥＲＢＩＲＤ Ｐｒｏｂｅ ｑＰＣＲ Ｍｉｘ

０．５μＭ　配列番号３で示されるプライマー

０．５μＭ　配列番号４で示されるプライマー

０．１μＭ　配列番号８で示されるプローブ（５’末端をＦＡＭで、３’末端をＢＨＱ１で標識）
Reagent 6

THUNDERBIRD Probe qPCR Mix

0.5 μM Primer represented by SEQ ID NO: 3

0.5 μM Primer shown in SEQ ID NO: 4.

0.1 μM Probe represented by SEQ ID NO: 8 (labeled 5'end with FAM and 3'end with BHQ1)

核酸増幅および検出

９５℃・２分

（以上１サイクル）

９５℃・１０秒

６０℃　３０秒（このステップで蛍光検出）

（以上５０サイクル）
Nucleic acid amplification and detection

95 ° C for 2 minutes

(More than 1 cycle)

95 ° C for 10 seconds

60 ° C for 30 seconds (fluorescence detection in this step)

(More than 50 cycles)

（４）結果

　下記の表３は、上記の条件で核酸増幅及びリアルタイムＰＣＲでの蛍光検出を行い、Ｃｔ値をまとめた表である。この結果に示されるように、同一の検出波長を有する核酸プローブを複数組み合わせて使用することによってより小さなＣｔが出力された。これは、複数の核酸プローブを用いて複数の標的核酸を検出することで、単独の核酸プローブを用いる場合よりも蛍光が増強され、より早期に検出シグナルが検知されたことを示す。
(4) Result

Table 3 below is a table summarizing the Ct values obtained by performing nucleic acid amplification and fluorescence detection by real-time PCR under the above conditions. As shown in this result, smaller Ct was output by using a combination of multiple nucleic acid probes having the same detection wavelength. This indicates that the detection of a plurality of target nucleic acids using a plurality of nucleic acid probes enhances the fluorescence as compared with the case of using a single nucleic acid probe, and the detection signal is detected earlier.

Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003

〔実施例３：核酸プローブセットの使用による検出系感度向上効果の確認〕

（１）目的

　本発明の核酸プローブセットを用いることで、核酸を検出する感度が向上することを確認するため、ＳＡＲＳ－ＣｏＶ－２を検出対象とする核酸プローブセットを用いた実験を行った。

（２）試料の調製

　ＳＡＲＳ－ＣｏＶ－２（ｓｅｖｅｒｅ　ａｃｕｔｅ　ｒｅｓｐｉｒａｔｏｒｙ　ｓｙｎｄｒｏｍｅ　ｃｏｒｏｎａｖｉｒｕｓ　２）の陽性コントロールＲＮＡ（ＥＤＸ　ＳＡＲＳ－ＣｏＶ－２　Ｓｔａｎｄａｒｄ（バイオ・ラッド　ラボラトリーズ株式会社））を２．５コピー／テストとなるように調製し、試料とした。

（３）逆転写、核酸増幅、融解曲線解析、及び判定

　上記試料をそれぞれ下記試薬に添加して、下記条件によりＳＡＲＳ－ＣｏＶ－２の検出を行った。逆転写、核酸増幅および融解曲線解析には東洋紡製ＧＥＮＥＣＵＢＥ（登録商標）を使用した。判定は、融解曲線解析にてピークが得られた場合に陽性と判定した。ｎ＝８で測定を行い、陽性数を８で除算して陽性検出率を算出した。
[Example 3: Confirmation of effect of improving detection system sensitivity by using nucleic acid probe set]

(1) Purpose

In order to confirm that the sensitivity for detecting nucleic acid is improved by using the nucleic acid probe set of the present invention, an experiment using a nucleic acid probe set targeting SARS-CoV-2 was conducted.

(2) Preparation of sample

A positive control RNA (EDX SARS-CoV-2 Standards Co., Ltd.) of SARS-CoV-2 (severe acute respiratory syndrome 2) was prepared for 2.5 copies / test and used as a sample. bottom.

(3) Reverse transcription, nucleic acid amplification, melting curve analysis, and determination

Each of the above samples was added to the following reagents, and SARS-CoV-2 was detected under the following conditions. Toyobo GENECUBE® was used for reverse transcription, nucleic acid amplification and melting curve analysis. The judgment was positive when a peak was obtained by the melting curve analysis. The measurement was performed at n = 8, and the positive number was divided by 8 to calculate the positive detection rate.

　具体的には、ジーンキューブ　テストベーシック及びＲｅｖｅｒＴｒａ Ａｃｅ（東洋紡）を用い、以下の２本の核酸プローブ及びそれに対応する核酸プライマーセット及び試料を含む溶液（試薬７）を調製した。また、試薬７との比較として、ジーンキューブ　テストベーシック及びＲｅｖｅｒＴｒａ Ａｃｅを用い、核酸プローブセットを構成する核酸プローブ、およびそれに対応する核酸プライマーセットを単独で用いる溶液（試薬８、試薬９）を調製した。本実施例で使用する配列番号１１で示されるプローブのＴｍ値は５２．８℃、配列番号１４で示されるプローブのＴｍ値は５３．７℃であり、融解曲線解析における検出温度が近接するように設計した。配列番号１１及び１４のプローブはいずれもＳＡＲＳ－ＣｏＶ－２のＲＮＡ配列に対して相補的な塩基配列を有し、同一の蛍光色素で標識したものである。
Specifically, using GeneCube Test Basic and RiverTra Ace (Toyobo), a solution (reagent 7) containing the following two nucleic acid probes and their corresponding nucleic acid primer sets and samples was prepared. Further, as a comparison with the reagent 7, using GeneCube Test Basic and RiverTraAce, a solution (reagent 8, reagent 9) using the nucleic acid probe constituting the nucleic acid probe set and the corresponding nucleic acid primer set alone was prepared. .. The Tm value of the probe shown by SEQ ID NO: 11 used in this example is 52.8 ° C., the Tm value of the probe shown by SEQ ID NO: 14 is 53.7 ° C., and the detection temperatures in the melting curve analysis are close to each other. Designed for. The probes of SEQ ID NOs: 11 and 14 both have a base sequence complementary to the RNA sequence of SARS-CoV-2 and are labeled with the same fluorescent dye.

試薬７

ジーンキューブ　テストベーシック

ＲｅｖｅｒＴｒａ Ａｃｅ

０．２μＭ　配列番号９で示されるプライマー

１．１５μＭ　配列番号１０で示されるプライマー

０．２μＭ　配列番号１２で示されるプライマー

１．１５μＭ　配列番号１３で示されるプライマー

０．１μＭ　配列番号１１で示されるプローブ（３’末端をＢＯＤＩＰＹ－ＦＬで標識）

０．１μＭ　配列番号１４で示されるプローブ（３’末端をＢＯＤＩＰＹ－ＦＬで標識）
Reagent 7

Gene Cube Test Basic

RiverTra Ace

0.2 μM Primer shown in SEQ ID NO: 9.

1.15 μM Primer shown in SEQ ID NO: 10.

0.2 μM Primer shown in SEQ ID NO: 12.

1.15 μM Primer represented by SEQ ID NO: 13.

0.1 μM Probe represented by SEQ ID NO: 11 (labeled 3'end with BODIPY-FL)

0.1 μM Probe shown in SEQ ID NO: 14 (labeled 3'end with BODIPY-FL)

試薬８

ジーンキューブ　テストベーシック

ＲｅｖｅｒＴｒａ Ａｃｅ

０．２μＭ　配列番号９で示されるプライマー

１．１５μＭ　配列番号１０で示されるプライマー

０．１μＭ　配列番号１１で示されるプローブ（３’末端をＢＯＤＩＰＹ－ＦＬで標識）
Reagent 8

Gene Cube Test Basic

RiverTra Ace

0.2 μM Primer shown in SEQ ID NO: 9.

1.15 μM Primer shown in SEQ ID NO: 10.

0.1 μM Probe represented by SEQ ID NO: 11 (labeled 3'end with BODIPY-FL)

試薬９

ジーンキューブ　テストベーシック

ＲｅｖｅｒＴｒａ Ａｃｅ

０．２μＭ　配列番号１２で示されるプライマー

１．１５μＭ　配列番号１３で示されるプライマー

０．１μＭ　配列番号１４で示されるプローブ（３’末端をＢＯＤＩＰＹ－ＦＬで標識）
Reagent 9

Gene Cube Test Basic

RiverTra Ace

0.2 μM Primer shown in SEQ ID NO: 12.

1.15 μM Primer represented by SEQ ID NO: 13.

0.1 μM Probe shown in SEQ ID NO: 14 (labeled 3'end with BODIPY-FL)

逆転写、核酸増幅および融解曲線解析

４２℃・２分

９７℃・１５秒

（以上１サイクル）

９７℃・１秒

５８℃・３秒

６３℃・５秒

（以上６０サイクル）

９４℃・３０秒

３９℃・３０秒

４０℃～７５℃（０．０９℃／秒で温度上昇）
Reverse transcription, nucleic acid amplification and melting curve analysis

42 ° C for 2 minutes

97 ° C for 15 seconds

(More than 1 cycle)

97 ° C for 1 second

58 ° C for 3 seconds

63 ° C for 5 seconds

(More than 60 cycles)

94 ° C for 30 seconds

39 ° C for 30 seconds

40 ° C to 75 ° C (temperature rises at 0.09 ° C / sec)

（４）結果

　下記の表４は、上記の条件で核酸増幅及び融解曲線解析による蛍光検出を行い、ＳＡＲＳ－ＣｏＶ－２を低濃度で検出した結果をまとめた表である。ｎ＝８測定において検出された陽性検出率を示している。表４の結果より、本発明の核酸プローブセットを用いた試薬７は、核酸プローブを単独で用いた試薬８、試薬９よりも陽性検出率が高いことが明らかとなった。従って、本発明の実施態様である同一の検出波長を有する複数の核酸プローブからなる組成物を使用することで、標的が極めて低濃度である場合であっても、核酸検出感度を向上させることができ高感度な検出が可能であることが分かった。
(4) Result

Table 4 below is a table summarizing the results of detecting SARS-CoV-2 at a low concentration by performing fluorescence detection by nucleic acid amplification and melting curve analysis under the above conditions. The positive detection rate detected in the n = 8 measurement is shown. From the results in Table 4, it was clarified that the reagent 7 using the nucleic acid probe set of the present invention has a higher positive detection rate than the reagents 8 and 9 using the nucleic acid probe alone. Therefore, by using a composition consisting of a plurality of nucleic acid probes having the same detection wavelength, which is an embodiment of the present invention, it is possible to improve the nucleic acid detection sensitivity even when the target has an extremely low concentration. It was found that highly sensitive detection was possible.

Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004

〔実施例４：核酸プローブセットの使用による検出シグナル増強〕

（１）目的

　本発明の核酸プローブセットを用いて融解曲線解析を行うことで、核酸プローブを単独で使用するよりも検出シグナルが増強されることを確認するため、Ｃｈｌａｍｙｄｉａ ｔｒａｃｈｏｍａｔｉｓ（以下、ＣＴ）を検出するための核酸プローブ及び核酸プライマーセットと、Ｎｅｉｓｓｅｒｉａ ｇｏｎｏｒｒｈｏｅａｅ（以下、ＮＧ）を検出するための核酸プローブ及び核酸プライマーセットとを用いて実験を行った。

（２）試料の調製

　ＣＴから抽出されたＤＮＡを１００コピー／テスト、ＮＧから抽出されたＤＮＡを１００コピー／テストとなるように調製し、試料とした。

（３）核酸増幅、融解曲線解析、及び判定

　上記試料をそれぞれ下記試薬に添加して、下記条件により標的核酸の検出を行った。核酸増幅および融解曲線解析には東洋紡製ＧＥＮＥＣＵＢＥ（登録商標）を使用した。判定は、融解曲線解析にてピークが得られた場合に陽性と判定した。
[Example 4: Enhancement of detection signal by using nucleic acid probe set]

(1) Purpose

In order to confirm that the detection signal is enhanced by performing the melting curve analysis using the nucleic acid probe set of the present invention as compared with the use of the nucleic acid probe alone, in order to detect Chlamydia trachomatis (hereinafter referred to as CT). An experiment was carried out using a nucleic acid probe and a nucleic acid primer set, and a nucleic acid probe and a nucleic acid primer set for detecting Neisseria gonorrohoeae (hereinafter referred to as NG).

(2) Preparation of sample

DNA extracted from CT was prepared to be 100 copies / test, and DNA extracted from NG was prepared to be 100 copies / test, and used as a sample.

(3) Nucleic acid amplification, melting curve analysis, and determination

Each of the above samples was added to the following reagents, and the target nucleic acid was detected under the following conditions. GENECUBE® manufactured by Toyobo Co., Ltd. was used for nucleic acid amplification and melting curve analysis. The judgment was positive when a peak was obtained by the melting curve analysis.

　具体的には、ジーンキューブ　テストベーシックを用い、以下のＣＴ検出用の核酸プローブ及びそれに対応する核酸プライマーセット、ＮＧ検出用の核酸プローブ及びそれに対応する核酸プライマーセット、そしてＣＴのＤＮＡ及びＮＧのＤＮＡを含む溶液（試薬１０）を調製した。また、試薬１０との比較として、ジーンキューブ　テストベーシックを用い、ＣＴ検出用の核酸プローブ及びそれに対応する核酸プライマーセット、そしてＣＴのＤＮＡを含む溶液（試薬１１）、同様にＮＧに対応した溶液（試薬１２）を調製した。本実施例で使用する配列番号１７（Ｔｍ値５６．２℃）又は２０（Ｔｍ値５８．５℃）のプローブはそれぞれＣＴ　ＤＮＡ配列又はＮＧ　ＤＮＡ配列に対して相補的な塩基配列を有し、同一の蛍光色素で標識したものである。
Specifically, using the GeneCube Test Basic, the following nucleic acid probes for CT detection and their corresponding nucleic acid primer sets, nucleic acid probes for NG detection and their corresponding nucleic acid primer sets, and CT DNA and NG DNA. A solution (nucleic acid 10) containing the above was prepared. In addition, as a comparison with Reagent 10, using GeneCube Test Basic, a nucleic acid probe for CT detection, a corresponding nucleic acid primer set, a solution containing CT DNA (reagent 11), and a solution corresponding to NG (reagent 11). Reagent 12) was prepared. The probe of SEQ ID NO: 17 (Tm value 56.2 ° C.) or 20 (Tm value 58.5 ° C.) used in this example has a base sequence complementary to the CT DNA sequence or the NG DNA sequence, respectively. It is labeled with the same fluorescent dye.

試薬１０

ジーンキューブ　テストベーシック

０．５μＭ　配列番号１５で示されるプライマー

２．５μＭ　配列番号１６で示されるプライマー

０．３μＭ　配列番号１７で示されるプローブ（３’末端をＢＯＤＩＰＹ－ＦＬで標識）

０．３μＭ　配列番号１８で示されるプライマー

２．０μＭ　配列番号１９で示されるプライマー

０．３μＭ　配列番号２０で示されるプローブ（５’末端をＢＯＤＩＰＹ－ＦＬで標識、３’末端をリン酸化）
Reagent 10

Gene Cube Test Basic

0.5 μM Primer shown in SEQ ID NO: 15.

2.5 μM Primer shown in SEQ ID NO: 16.

0.3 μM Probe shown in SEQ ID NO: 17 (labeled 3'end with BODIPY-FL)

0.3 μM Primer shown in SEQ ID NO: 18.

2.0 μM Primer shown in SEQ ID NO: 19.

0.3 μM Probe shown in SEQ ID NO: 20 (labeled with BODIPY-FL at the 5'end and phosphorylated at the 3'end)

試薬１１

ジーンキューブ　テストベーシック

０．５μＭ　配列番号１５で示されるプライマー

２．５μＭ　配列番号１６で示されるプライマー

０．３μＭ　配列番号１７で示されるプローブ（３’末端をＢＯＤＩＰＹ－ＦＬで標識）
Reagent 11

Gene Cube Test Basic

0.5 μM Primer shown in SEQ ID NO: 15.

2.5 μM Primer shown in SEQ ID NO: 16.

0.3 μM Probe shown in SEQ ID NO: 17 (labeled 3'end with BODIPY-FL)

試薬１２

ジーンキューブ　テストベーシック

０．３μＭ　配列番号１８で示されるプライマー

２．０μＭ　配列番号１９で示されるプライマー

０．３μＭ　配列番号２０で示されるプローブ（５’末端をＢＯＤＩＰＹ－ＦＬで標識、３’末端をリン酸化）
Reagent 12

Gene Cube Test Basic

0.3 μM Primer shown in SEQ ID NO: 18.

2.0 μM Primer shown in SEQ ID NO: 19.

0.3 μM Probe shown in SEQ ID NO: 20 (labeled with BODIPY-FL at the 5'end and phosphorylated at the 3'end)

核酸増幅および融解曲線解析

９７℃・３０秒

（以上１サイクル）

９７℃・１秒

５８℃・５秒

６３℃・５秒

（以上６０サイクル）

９４℃・３０秒

３９℃・３０秒

４０℃～７５℃（０．０９℃／秒で温度上昇）
Nucleic acid amplification and melting curve analysis

97 ° C for 30 seconds

(More than 1 cycle)

97 ° C for 1 second

58 ° C for 5 seconds

63 ° C for 5 seconds

(More than 60 cycles)

94 ° C for 30 seconds

39 ° C for 30 seconds

40 ° C to 75 ° C (temperature rises at 0.09 ° C / sec)

（３）結果

　図１は試薬１０（ＣＴ＋ＮＧ）、図２は試薬１１（ＣＴ）、そして図３は試薬１２（ＮＧ）の融解曲線解析結果のグラフである。図２、図３と比較して図１は検出ピークが相乗効果的に高くなっており非常に明瞭である（蛍光ピーク（ｄ／ｄｔ）：試薬１０の場合は約７３、試薬１１の場合は約４０、試薬１２の場合は約２５）。これは、試薬１０において、核酸プローブセット及び該プローブセットに対応する核酸プライマーセット群を使用することで、ＣＴとＮＧの両方のＤＮＡが検出され、かつそれらの検出シグナルを重ね合わせて増強することができたためと推測される。なお、各試薬での検出温度（融解曲線解析におけるＴｍ値に対応する）を確認したところ、試薬１０では６２．０℃、試薬１１では６０．６℃、そして試薬１２では６１．８℃であり、核酸プローブセットとして用いる標的核酸プローブのＴｍ値の差は、試薬１１と試薬１２の差から１．２℃であった。

　以上から、本発明の核酸プローブセットを用いて複数の標的核酸を蛍光検出することで、検出シグナルが重なり増強されることで高感度な検出が可能になることが示された。
(3) Result

FIG. 1 is a graph of reagent 10 (CT + NG), FIG. 2 is a graph of reagent 11 (CT), and FIG. 3 is a graph of melting curve analysis results of reagent 12 (NG). Compared with FIGS. 2 and 3, FIG. 1 shows that the detection peak is synergistically higher and is very clear (fluorescence peak (d / dt): about 73 for reagent 10, and for reagent 11). About 40, about 25 in the case of reagent 12. This is to detect both CT and NG DNAs by using the nucleic acid probe set and the nucleic acid primer set group corresponding to the probe set in the reagent 10, and to superimpose and enhance the detection signals thereof. It is presumed that this was done. When the detection temperature (corresponding to the Tm value in the melting curve analysis) of each reagent was confirmed, it was 62.0 ° C. for the reagent 10, 60.6 ° C. for the reagent 11, and 61.8 ° C. for the reagent 12. The difference in Tm value of the target nucleic acid probe used as the nucleic acid probe set was 1.2 ° C. from the difference between the reagent 11 and the reagent 12.

From the above, it was shown that by fluorescently detecting a plurality of target nucleic acids using the nucleic acid probe set of the present invention, high-sensitivity detection is possible by overlapping and enhancing the detection signals.

　本発明により、従来よりも高感度な核酸検出系の構築が可能となる。本発明は、検出シグナルを重ね合わせることにより検出シグナルを増強できる結果、蛍光ピークが明瞭化され、従来よりも結果判定が容易な核酸検出系の構築にも貢献する。
INDUSTRIAL APPLICABILITY According to the present invention, it is possible to construct a nucleic acid detection system with higher sensitivity than before. The present invention contributes to the construction of a nucleic acid detection system in which the fluorescence peak is clarified as a result of being able to enhance the detection signal by superimposing the detection signals, and the result determination is easier than before.

Claims (12)


  1. 　二つ以上の核酸プローブを含む核酸プローブセットであって、該核酸プローブセットは少なくとも第一標的核酸プローブ及び第二標的核酸プローブを含み、以下の（Ａ）～（Ｃ）の特徴を有する、核酸プローブセット：

    （Ａ）第一標的核酸プローブ及び第二標的核酸プローブはそれぞれ異なる塩基配列を有する。

    （Ｂ）第一標的核酸プローブ及び第二標的核酸プローブは同一波長で検出できる蛍光色素で標識されている。

    （Ｃ）第一標的核酸プローブ及び第二標的核酸プローブはそれぞれ異なる標的核酸に結合するが、各標的核酸を鑑別しないように検出される。
    A nucleic acid probe set containing two or more nucleic acid probes, the nucleic acid probe set containing at least a first target nucleic acid probe and a second target nucleic acid probe, and having the following characteristics (A) to (C). Probe set:

    (A) The first target nucleic acid probe and the second target nucleic acid probe have different base sequences.

    (B) The first target nucleic acid probe and the second target nucleic acid probe are labeled with a fluorescent dye that can be detected at the same wavelength.

    (C) The first target nucleic acid probe and the second target nucleic acid probe bind to different target nucleic acids, but are detected so as not to distinguish each target nucleic acid.

  2. 　前記核酸プローブセットが、前記第一標的核酸プローブ及び第二標的核酸プローブからなることを特徴とする、請求項１に記載の核酸プローブセット。
    The nucleic acid probe set according to claim 1, wherein the nucleic acid probe set comprises the first target nucleic acid probe and the second target nucleic acid probe.

  3. 　同一波長で検出できる蛍光色素が同一の蛍光色素である、請求項１または２に記載の核酸プローブセット。
    The nucleic acid probe set according to claim 1 or 2, wherein the fluorescent dyes that can be detected at the same wavelength are the same fluorescent dyes.

  4. 　前記特徴（Ｃ）における検出が、リアルタイムＰＣＲ、リアルタイムＲＴ－ＰＣＲ、又は融解曲線解析のいずれかの核酸検出法における検出である、請求項１～３のいずれかに記載の核酸プローブセット。
    The nucleic acid probe set according to any one of claims 1 to 3, wherein the detection in the feature (C) is a detection in any of the nucleic acid detection methods of real-time PCR, real-time RT-PCR, or melting curve analysis.

  5. 　前記特徴（Ｃ）における検出が、融解曲線解析における検出である、請求項１～４のいずれかに記載の核酸プローブセット。
    The nucleic acid probe set according to any one of claims 1 to 4, wherein the detection in the feature (C) is the detection in the melting curve analysis.

  6. 　第一標的核酸プローブと第二標的核酸プローブとのＴｍ値の差が３℃以内である、請求項１～５のいずれかに記載の核酸プローブセット。
    The nucleic acid probe set according to any one of claims 1 to 5, wherein the difference in Tm value between the first target nucleic acid probe and the second target nucleic acid probe is within 3 ° C.

  7. 　第一標的核酸プローブ及び第二標的核酸プローブが、それぞれの標的核酸に対して完全に相補的な塩基配列から構成される、請求項１～６のいずれかに記載の核酸プローブセット。
    The nucleic acid probe set according to any one of claims 1 to 6, wherein the first target nucleic acid probe and the second target nucleic acid probe are composed of a base sequence completely complementary to each target nucleic acid.

  8. 　第一標的核酸プローブの塩基配列と第二標的核酸プローブの塩基配列との塩基長の差が８ｍｅｒ以内である、請求項１～７のいずれかに記載の核酸プローブセット。
    The nucleic acid probe set according to any one of claims 1 to 7, wherein the difference in base length between the base sequence of the first target nucleic acid probe and the base sequence of the second target nucleic acid probe is within 8 mer.

  9. 　請求項１～８のいずれかに記載の核酸プローブセットを含む試薬組成物。
    A reagent composition comprising the nucleic acid probe set according to any one of claims 1 to 8.

  10. 　請求項１～８のいずれかに記載の核酸プローブセット又は請求項９に記載の試薬組成物を含むキット。
    A kit comprising the nucleic acid probe set according to any one of claims 1 to 8 or the reagent composition according to claim 9.

  11. 　請求項１～８のいずれかに記載の核酸プローブセット、請求項９に記載の試薬組成物、又は請求項１０に記載のキットを用いる、核酸の検出方法。
    A method for detecting nucleic acid using the nucleic acid probe set according to any one of claims 1 to 8, the reagent composition according to claim 9, or the kit according to claim 10.

  12. 　請求項１～８のいずれかに記載の核酸プローブセット、請求項９に記載の試薬組成物、又は請求項１０に記載のキットを用いる、核酸の検出感度向上方法。
    A method for improving nucleic acid detection sensitivity using the nucleic acid probe set according to any one of claims 1 to 8, the reagent composition according to claim 9, or the kit according to claim 10.
PCT/JP2021/039268 2020-10-30 2021-10-25 Nucleic acid probe set comprising multiple nucleic acid probes WO2022092013A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004041191A (en) * 2002-05-21 2004-02-12 Adgene Co Ltd Method for identifying nucleic acid
JP2009039105A (en) * 2007-07-13 2009-02-26 Toyobo Co Ltd Oligonucleotide for detection of acid-fast bacterium and use thereof
JP2009039106A (en) * 2007-07-13 2009-02-26 Toyobo Co Ltd Oligonucleotide for species identification of acid-fast bacterium and use thereof
JP2016531550A (en) * 2013-09-23 2016-10-13 クエスト ダイアグノスティクス インベストメンツ インコーポレイテッド Detection of methicillin-resistant Staphylococcus aureus in biological samples

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004041191A (en) * 2002-05-21 2004-02-12 Adgene Co Ltd Method for identifying nucleic acid
JP2009039105A (en) * 2007-07-13 2009-02-26 Toyobo Co Ltd Oligonucleotide for detection of acid-fast bacterium and use thereof
JP2009039106A (en) * 2007-07-13 2009-02-26 Toyobo Co Ltd Oligonucleotide for species identification of acid-fast bacterium and use thereof
JP2016531550A (en) * 2013-09-23 2016-10-13 クエスト ダイアグノスティクス インベストメンツ インコーポレイテッド Detection of methicillin-resistant Staphylococcus aureus in biological samples

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