WO2022126721A1 - Procédé de détection à haute spécificité d'une séquence d'acide nucléique cible - Google Patents

Procédé de détection à haute spécificité d'une séquence d'acide nucléique cible Download PDF

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WO2022126721A1
WO2022126721A1 PCT/CN2020/140053 CN2020140053W WO2022126721A1 WO 2022126721 A1 WO2022126721 A1 WO 2022126721A1 CN 2020140053 W CN2020140053 W CN 2020140053W WO 2022126721 A1 WO2022126721 A1 WO 2022126721A1
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sequence
mediator
probe
nucleic acid
hairpin
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PCT/CN2020/140053
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Chinese (zh)
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李庆阁
杜琛
廖逸群
许晔
周淑娟
宋甲宝
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厦门大学
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    • 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
    • C12Q1/6832—Enhancement of hybridisation reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the present application relates to multiplex detection of nucleic acid molecules.
  • the present application provides a method for detecting a target nucleic acid sequence, which is capable of simultaneously detecting the presence of multiple target nucleic acid sequences in a sample with high specificity.
  • the present application also provides a probe set, and a kit comprising one or more of the probe sets, which can be used to implement the methods of the present invention.
  • Real-time fluorescent PCR is a common method for nucleic acid detection. It is easy to operate and widely used. At the same time, as a closed-tube detection mode, the chance of contamination of amplification products is low. Multiplex real-time PCR can simultaneously detect multiple target sequences in a single reaction tube. , not only the detection efficiency is improved, but the cost is further reduced.
  • the detection principle of real-time fluorescent PCR is based on the specific binding of fluorescently labeled oligonucleotide probes to target sequences between primers, which can avoid interference signals generated by non-specific amplification such as primer dimers, and ensure the specificity of detection results.
  • different fluorescent groups are used to label probes that specifically bind to target sequences, and the corresponding target sequences can be identified by detecting different fluorescent signals of the probes.
  • detection mode selection the real-time detection mode has no other steps except amplification, which is simple and direct, but the maximum number of target sequences that can be detected in this mode is limited by the number of fluorescence detection channels of the fluorescence real-time PCR instrument, generally no more than 6.
  • Another detection mode is the melting curve analysis after amplification.
  • a temperature change process is added after amplification.
  • the maximum number of target sequences that can be detected is equal to the increase of the number of probe and target sequences on the basis of the number of fluorescence detection channels. Compared with the real-time detection mode, the dimension of melting point is greatly improved.
  • CN108823287A discloses a multiplex real-time PCR detection system for detecting target nucleic acid sequences, which is characterized in that the number of detection probes used is less than the target sequence.
  • the above-mentioned multiplex real-time PCR detection system adopts a unique detection system, that is, for each target sequence, a non-fluorescently labeled target sequence-specific mediator probe is designed, and mediator sequences on multiple mediator probes are designed. It can be combined and extended with the same fluorescent detection probe, and the extension products have different melting points. Since each mediator probe corresponds to one target sequence, these target sequences are actually detected simultaneously by one fluorescent probe.
  • non-specific signals ie, non-specific melting peaks
  • the inventors of the present application have noticed that in some cases, non-specific signals (ie, non-specific melting peaks) may appear in the detection results obtained by this method, which may affect the interpretation of the results to a certain extent. Therefore, there is a need to improve the multiplex real-time PCR detection system and detection method to further reduce or even eliminate the generation of non-specific signals (ie, non-specific melting peaks) without affecting the detection sensitivity.
  • non-specific signals ie, non-specific melting peaks
  • the detection probe binds non-specifically to non-target sequences, so that the detection probe emits signal (eg, a self-quenching fluorescent probe nonspecifically binds to a non-target nucleic acid molecule such that the fluorophore it carries is separated from the quencher group and thus fluoresces).
  • the detection probe can usually be modified, such as designing a hairpin structure on the detection probe to inhibit the non-specific binding of the detection probe to non-target nucleic acid molecules.
  • the present application provides a method of detecting the presence of n target nucleic acid sequences in a sample, wherein n is an integer > 1 (eg, n is 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or larger integers), and the method includes the following step:
  • an upstream oligonucleotide sequence and a mediator probe are provided; wherein, the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence sequence;
  • the mediator probe comprises a mediator sequence and a target-specific sequence from the 5' to 3' direction, the mediator subsequence comprises a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is located upstream of the target-specific sequence; and, all mediator probes comprise The mediator subsequences of are different from each other;
  • At least one mediator probe itself can form a hairpin structure
  • the at least one mediator probe has characteristics selected from the group consisting of:
  • the mediator probe further comprises a first hairpin-forming sequence downstream or at the 3' end of its target-specific sequence, the first hairpin-forming sequence and the mediator sequence of the mediator probe or It is partially complementary, whereby the mediator probe is capable of forming a hairpin structure through the mediator sequence and the first hairpin-forming sequence;
  • the mediator probe further comprises a second hairpin-forming sequence upstream or 5' of its mediator sequence, the second hairpin-forming sequence and the target-specific sequence of the mediator probe or It is partially complementary, whereby the mediator probe is capable of forming a hairpin structure through the second hairpin-forming sequence and the target-specific sequence;
  • the mediator probe further comprises a third hairpin-forming sequence upstream or 5' end of its mediator sequence, and further comprises a fourth hairpin-forming sequence downstream or 3' end of its target-specific sequence , and the third hairpin-forming sequence or part thereof is complementary to the fourth hairpin-forming sequence or part thereof, whereby the mediator probe can pass through the third hairpin-forming sequence and the third hairpin-forming sequence Four hairpin forming sequences form a hairpin structure;
  • step (2) (2) contacting the product of step (1) with an enzyme having 5' nuclease activity under conditions that allow cleavage of the mediator probe;
  • step (3) providing m kinds of detection probes, and contacting the product of step (2) with the m kinds of detection probes under conditions that allow nucleic acid hybridization, wherein m is an integer greater than 0,
  • each detection probe independently comprises, from the 3' to 5' direction, one or more capture sequences complementary to one or more mediator subsequences or portions thereof, and a templating sequence;
  • the m detection probes comprise at least n capture sequences, which are respectively complementary to the mediator sequences of the n mediator probes provided in step (1) or parts thereof;
  • Each detection probe is independently labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the reporter group emitting and, each detection probe emits a different signal when hybridized to its complementary sequence than when not hybridized to its complementary sequence; and,
  • step (3) (4) contacting the product of step (3) with the nucleic acid polymerase under conditions that allow the nucleic acid polymerase to carry out the extension reaction;
  • step (4) Perform melting curve analysis on the product of step (4); and determine whether the n target nucleic acid sequences exist in the sample according to the result of the melting curve analysis.
  • m is an integer less than n and greater than zero. In such embodiments, the number of detection probes used is less than the number of mediator probes. Thus, at least one detection probe comprises two or more capture sequences. In certain embodiments, m equals n. In such embodiments, the number of detection probes used is equal to the number of mediator probes. Therefore, the detection probes can be in a one-to-one correspondence with the mediator probes.
  • m is an integer >1, >2, >3, >4, >5, >6, >8, >10 (eg, m is 1, 2, 3, 4, 5, or 6 ); preferably, when m ⁇ 2, each of the m detection probes is labeled with a different reporter group.
  • step (1) provides at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 mediator probes; and, step (3) provides at least 1, at least 2, at least 3, at least 4 , at least 5, at least 6, at least 8, or at least 10 detection probes.
  • the mediator probes are each independently capable of forming a hairpin structure, for example each independently having features (i), (ii) or (iii) as defined in claim 1 .
  • each mediator probe is independently capable of forming a hairpin structure, eg, each independently has features (i), (ii) or (iii) as defined in claim 1 .
  • the first, second, third or fourth hairpin forming sequences are each independently 5-140nt in length, eg, 5-10nt, 10-20nt, 20-30nt, 30-40nt , 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the m detection probes comprise the same reporter group; and, in step (5), melting curve analysis is performed on the product of step (4), and according to the obtained melting curve the melting peak (melting point) to determine the presence of a certain target nucleic acid sequence.
  • the reporter groups contained in the m detection probes are different from each other; and, in step (5), when the melting curve analysis is performed on the product of step (4), each real-time monitoring A signal of a reporter group, thereby obtaining a plurality of melting curves corresponding to the signal of a reporter group; the presence of a target nucleic acid sequence.
  • n is an integer > 1 (eg, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 or larger integers).
  • the method includes the steps of:
  • an upstream oligonucleotide sequence and a mediator probe are provided; wherein, the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence sequence; and, the mediator probe comprises a mediator sequence and a target-specific sequence from the 5' to 3' direction, the mediator sequence comprises a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is located upstream of the target-specific sequence; and, all mediator probes The included mediator subsequences differ from each other;
  • the at least one mediator probe itself is capable of forming a hairpin structure; for example, the at least one mediator probe has features (i), (ii) or (iii) as defined in claim 1;
  • step (2) (2) contacting the product of step (1) with an enzyme having 5' nuclease activity under conditions that allow cleavage of the mediator probe;
  • step (3) contacting the product of step (2) with a detection probe comprising from the 3' to 5' direction, under conditions allowing nucleic acid hybridization, with each of the mediator subsequences or portions thereof a complementary capture sequence, and a templating sequence; and the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of signaling, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and, the detection probe emits a signal when hybridized to its complementary sequence that is different from the signal emitted when it is not hybridized to its complementary sequence;
  • step (3) (4) contacting the product of step (3) with the nucleic acid polymerase under conditions that allow the nucleic acid polymerase to carry out the extension reaction;
  • step (4) Perform melting curve analysis on the product of step (4); and determine whether the n target nucleic acid sequences exist in the sample according to the result of the melting curve analysis.
  • the sample can be any sample to be detected.
  • the sample comprises or is a mixture of DNA, or RNA, or nucleic acids.
  • the target nucleic acid sequence is DNA or RNA; and/or, the target nucleic acid sequence is single-stranded or double-stranded.
  • the sample or target nucleic acid sequence is obtained from a source selected from prokaryotes, eukaryotes, viruses, or viroids.
  • the mediator probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the mediator probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the mediator probes comprise modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine pyrimidine.
  • the mediator probe comprises non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitro pyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitro pyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • the mediator probe is 15-150nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80 -90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
  • the target-specific sequence in the mediator probe is 10-140nt in length, eg, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt , 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the length of the mediator sequence in the mediator probe can be 5-140nt, such as 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt , 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the inventors of the present application believe that although the detection probe has been designed to have a stable hairpin structure, non-specific binding of the mediator probe to the detection probe may still occur, which can make the hairpin of the detection probe possible.
  • the clip structure is opened, resulting in the separation of the fluorophore and the quencher group it carries, which in turn leads to the generation of a non-specific signal.
  • the mediator probe ie, adding a first hairpin-forming sequence downstream or 3' of its target-specific sequence, or adding a second hairpin upstream or 5' of its mediator sequence forming sequence, or adding a third hairpin forming sequence upstream or 5' end of its mediator sequence, and adding a fourth hairpin forming sequence downstream or 3' end of its target-specific sequence
  • making the mediator probe Capable of forming a hairpin structure by itself (by means of complementary base pairing between an intermediary subsequence and a first hairpin forming sequence, or base pairing between a second hairpin forming sequence and a target-specific sequence, or a third
  • the complementary base pairing between the hairpin-forming sequence and the fourth hairpin-forming sequence can further reduce or even eliminate the non-specific binding between the mediator probe and the detection probe, thereby further reducing or even eliminating the generation of non-specific signals.
  • the first, second, third or fourth hairpin-forming sequence can be of any length without limitation as long as it enables the mediator probe to form a hairpin structure.
  • the stability of the hairpin structure formed by the mediator probe will decrease, which may affect (eg increase the ) non-specific binding of the mediator probe to the detection probe.
  • the stability of the hairpin structure formed by the mediator probe will increase, which may affect (eg reduce) the mediator. Specific binding of probes to target nucleic acid sequences.
  • the first, second, third or fourth hairpin forming sequences are each independently 5-140nt in length, eg, 5-10nt, 10-20nt, 20-30nt, 30-40nt , 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • mediator probes with hairpin structures may bring additional potential benefits: the ability to enhance the intensity of positive signals and improve the sensitivity of detection.
  • the inventors of the present application believe that the use of the mediator probe with a hairpin structure reduces the non-specific binding between the mediator probe and the detection probe, which allows more detection probes in the reaction system. The needle can bind to the cleaved mediator fragment, resulting in a stronger positive signal for the same positive sample.
  • a linker is also included between the target-specific sequence of the mediator probe and the first hairpin-forming sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is further included between the second hairpin-forming sequence of the mediator probe and the mediator sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is also included between the third hairpin-forming sequence of the mediator probe and the mediator sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is further included between the target-specific sequence of the mediator probe and the fourth hairpin-forming sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • the first hairpin-forming sequence is fully or partially complementary to the mediator sequence.
  • the mediator probe is capable of forming a hairpin structure through the mediator sequence and the first hairpin-forming sequence, wherein the arms of the hairpin structure have blunt ends (ie, do not with an overhang), or the arm has a 5' overhang (eg, it has at least 1, at least 2, or more free bases at its 5' end), or the arm has a 3' overhang (eg, , its 3' end has at least 1, at least 2, or more free bases).
  • the arm consists of a fully complementary first hairpin-forming sequence and a mediator subsequence.
  • the arms are symmetrical or have blunt ends.
  • the arm consists of a partially complementary first hairpin-forming sequence and a mediator subsequence.
  • the arms are asymmetric or have 5' or 3' overhangs.
  • the first hairpin-forming sequence is the same length as the mediator subsequence.
  • the arms may have blunt ends.
  • the first hairpin-forming sequence is different in length from the mediator subsequence.
  • the length of the first hairpin forming sequence and the length of the mediator subsequence may differ by 1-10 nt, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt.
  • the arms may have 5' or 3' overhangs.
  • the length of the first hairpin-forming sequence is 1-10 nt longer than the length of the mediator subsequence, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt .
  • the length of the mediator subsequence is 1-10 nt longer than the length of the first hairpin forming sequence, eg, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10nt.
  • the second hairpin-forming sequence is fully or partially complementary to the target-specific sequence.
  • the mediator probe is capable of forming a hairpin structure via the second hairpin-forming sequence and the target-specific sequence, wherein the arms of the hairpin structure have blunt ends (ie, no overhang), or the arm has a 5' overhang (eg, it has at least 1, at least 2, or more free bases at its 5' end), or the arm has a 3' overhang ( For example, it has at least 1, at least 2, or more free bases at its 3' end).
  • the arm consists of a fully complementary second hairpin-forming sequence and a target-specific sequence.
  • the arms are symmetrical or have blunt ends.
  • the arm consists of a partially complementary second hairpin-forming sequence and a target-specific sequence.
  • the arms are asymmetric or have 5' or 3' overhangs.
  • the second hairpin-forming sequence is the same length as the target-specific sequence.
  • the arms may have blunt ends.
  • the second hairpin-forming sequence is not the same length as the target-specific sequence.
  • the length of the second hairpin forming sequence may differ from the length of the target-specific sequence by 1-10 nt, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt.
  • the arms may have 5' or 3' overhangs.
  • the length of the second hairpin-forming sequence is 1-10 nt longer than the length of the target-specific sequence, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt.
  • the length of the target-specific sequence is 1-10 nt longer than the length of the second hairpin forming sequence, eg, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt.
  • the third hairpin-forming sequence is fully or partially complementary to the fourth hairpin-forming sequence.
  • the mediator probe is capable of forming a hairpin structure via the third hairpin-forming sequence and the fourth hairpin-forming sequence, wherein the arms of the hairpin structure have blunt ends ( That is, no overhang), or the arm has a 5' overhang (eg, it has at least 1, at least 2, or more free bases at its 5' end), or the arm has a 3' overhang Overhang (eg, having at least 1, at least 2, or more free bases at its 3' end).
  • the arms are comprised of fully complementary third and fourth hairpin-forming sequences.
  • the arms are symmetrical or have blunt ends.
  • the arms are comprised of partially complementary third and fourth hairpin-forming sequences.
  • the arms are asymmetric or have 5' or 3' overhangs.
  • the third hairpin-forming sequence is the same length as the fourth hairpin-forming sequence.
  • the arms may have blunt ends.
  • the third hairpin-forming sequence is different in length from the fourth hairpin-forming sequence.
  • the length of the third hairpin forming sequence may differ from the length of the fourth hairpin forming sequence by 1-10 nt, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt.
  • the arms may have 5' or 3' overhangs.
  • the length of the third hairpin-forming sequence is 1-10 nt longer than the length of the fourth hairpin-forming sequence, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt.
  • the length of the fourth hairpin-forming sequence is 1-10 nt longer than the length of the third hairpin-forming sequence, eg, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt.
  • the mediator probe has a 3'-OH terminus, or its 3'-terminus is blocked. In certain preferred embodiments, the 3'-terminus of the mediator probe is blocked to inhibit its extension.
  • the 3'-terminus of nucleic acids can be blocked by various methods. For example, the 3'-terminus of the mediator probe can be blocked by modifying the 3'-OH of the last nucleotide of the mediator probe. In certain embodiments, the 3'-terminus of the mediator probe can be blocked by adding a chemical moiety (eg, biotin or an alkyl group) to the 3'-OH of the last nucleotide of the mediator probe . In certain embodiments, the mediator probe can be blocked by removing the 3'-OH of the last nucleotide of the mediator probe, or by replacing the last nucleotide with a dideoxynucleotide 3'-end.
  • a chemical moiety eg, biotin or an alkyl group
  • the upstream oligonucleotide sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the upstream oligonucleotide sequence comprises or consists of natural nucleotides such as deoxyribonucleotides or ribonucleotides.
  • the upstream oligonucleotide sequence comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethyl base cytosine.
  • the upstream oligonucleotide sequence comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3 - Nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3 - Nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • the upstream oligonucleotide sequence is not limited by its length, as long as it can specifically hybridize to the target nucleic acid sequence.
  • the upstream oligonucleotide sequence is 15-150nt in length, eg, 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt , 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
  • the upstream oligonucleotide sequence after hybridization to the target nucleic acid sequence, is located at the upstream distal end of the mediator probe, or is located upstream adjacent to the mediator probe, or is adjacent to the mediator probe.
  • Target-specific sequences have partially overlapping sequences.
  • the upstream oligonucleotide sequence is a primer specific for a target nucleic acid sequence or a probe specific for a target nucleic acid sequence.
  • step (2) the enzyme with 5' nuclease activity cleaves the mediator probe hybridized to the target nucleic acid sequence, and releases the entire mediator sequence or a portion of the mediator sequence (5'-end portion) of the mediator fragment.
  • the enzyme having 5' nuclease activity is a nucleic acid polymerase (eg, a DNA polymerase, particularly a thermostable) having 5' nuclease activity (eg, 5' exonuclease activity) DNA polymerase).
  • a nucleic acid polymerase eg, a DNA polymerase, particularly a thermostable
  • 5' nuclease activity eg, 5' exonuclease activity
  • the DNA polymerase is obtained from a bacterium selected from the group consisting of: Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, Thermus flavus,Thermus igniterrae,Thermus lacteus,Thermus oshimai,Thermus ruber,Thermus rubens,Thermus scotoductus,Thermus silvanus,Thermus thermophllus,Thermotoga maritima,Thermotoga neapolitana,Thermosipho africanus,Thermococcus litoralis,Thermococcus barossi,Thermococcus gorgonarius,Thermotoga maritima,Thermotoga bacterium
  • step (2) the nucleic acid polymerase having 5' nuclease activity catalyzes the extension of the upstream oligonucleotide sequence and induces cleavage of the mediator probe.
  • a nucleic acid polymerase is used to catalyze the extension of the upstream oligonucleotide sequence using the target nucleic acid sequence as a template, and then the enzyme having 5' nuclease activity is bound to the upstream An extension product of an oligonucleotide sequence and catalyzes cleavage of the mediator probe.
  • the sample in steps (1) and/or (2), is also contacted with a downstream oligonucleotide sequence (or downstream primer) specific for the target nucleic acid sequence.
  • a downstream oligonucleotide sequence or downstream primer
  • the use of nucleic acid polymerases and downstream oligonucleotide sequences (or downstream primers) is particularly advantageous.
  • the nucleic acid polymerase can use the target nucleic acid sequence as a template and the upstream oligonucleotide sequence and the downstream oligonucleotide sequence as primers to generate additional target nucleic acid sequences, thereby improving the sensitivity of the method of the present invention.
  • step (1) in addition to the upstream oligonucleotide sequences and mediator probes as defined above, for each target nucleic acid sequence to be detected , also provides a downstream oligonucleotide sequence; wherein, the downstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized with the target nucleic acid sequence, the downstream oligonucleotide sequence The nucleotide sequence is located downstream of the target-specific sequence; the sample is then contacted with the provided upstream oligonucleotide sequence, mediator probe, and downstream oligonucleotide sequence under conditions that allow nucleic acid hybridization .
  • step (2) the product of step (1) is contacted with a nucleic acid polymerase having 5' nuclease activity under conditions that allow nucleic acid amplification.
  • the downstream oligonucleotide sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the downstream oligonucleotide sequence comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the downstream oligonucleotide sequence comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethyl base cytosine.
  • the downstream oligonucleotide sequence comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3 - Nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3 - Nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • the downstream oligonucleotide sequence is not limited by its length, as long as it can specifically hybridize to the target nucleic acid sequence.
  • the downstream oligonucleotide sequence is 15-150nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt , 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
  • all upstream and downstream oligonucleotide sequences provided in step (1) have an identical stretch of oligonucleotide sequence at the 5' end.
  • step (1) in addition to the upstream oligonucleotide sequence, the mediator probe and the downstream oligonucleotide sequence, a universal primer is provided, the universal primer has a sequence complementary to the same oligonucleotide sequence; the sample is then combined with the provided upstream oligonucleotide sequence, mediator probe, downstream oligonucleotide sequence under conditions that allow nucleic acid hybridization Contact with universal primers.
  • the identical oligonucleotide sequences are 8-50 nt in length, eg, 8-15 nt, 15-20 nt, 20-30 nt, 30-40 nt, or 40-50 nt.
  • the length of the universal primer may be 8-50nt, eg, 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.
  • universal primers may comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the universal primers comprise or consist of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the universal primers comprise modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine.
  • the universal primers comprise non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole , 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole , 5-nitroindole or locked nucleic acid (LNA).
  • the universal primer is not limited by its length, as long as it can specifically hybridize to the same oligonucleotide sequence contained in the upstream and downstream oligonucleotide sequences.
  • a universal primer can be 8-50nt in length, such as 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.
  • the detection probe comprises a plurality of capture sequences; and, the plurality of capture sequences are arranged adjacently, separated by linker sequences, or overlapping.
  • the detection probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the detection probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the detection probes comprise modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine .
  • the detection probe comprises non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitro Pyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitro Pyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • the detection probe is 15-1000nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80- 90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt, 900-1000nt.
  • the capture sequence in the detection probe can be of any length as long as it can specifically hybridize to the mediator fragment.
  • the capture sequence in the detection probe is 10-500nt in length, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-nt 80nt, 80-90nt, 90-100nt, 100-150nt, 150-200nt, 200-250nt, 250-300nt, 300-350nt, 350-400nt, 400-450nt, 450-500nt.
  • the template sequence in the detection probe can be of any length as long as it can be used as a template for extending the mediator subfragment.
  • the length of the template sequence in the detection probe is 1-900nt, such as 1-5nt, 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-nt 60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt.
  • the detection probe has a 3'-OH terminus, or its 3'-terminus is blocked to inhibit its extension.
  • the 3'-terminus of nucleic acids can be blocked by various methods.
  • the 3'-terminus of the detection probe can be blocked by modifying the 3'-OH of the last nucleotide of the detection probe.
  • the 3'-terminus of the detection probe can be blocked by adding a chemical moiety (eg, biotin or an alkyl group) to the 3'-OH of the last nucleotide of the detection probe.
  • the 3'-OH of the detection probe can be blocked by removing the 3'-OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a dideoxynucleotide. '-end.
  • the detection probe is a self-quenching probe; eg, the detection probe is labeled with a reporter group at its 5' end or upstream and labeled with a quencher at its 3' end or downstream group, or a reporter group is labeled at its 3' end or downstream and a quencher group is labeled at its 5' end or upstream.
  • the reporter group and the quencher group are separated by a distance of 10-80 nt or more.
  • the reporter group in the detection probe is a fluorophore (eg ALEX-350, FAM, VIC, TET, CAL Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and a quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg, DABCYL, BHQ (eg, BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA).
  • a quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg, DABCYL, BHQ (eg, BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA).
  • the detection probes are modified or not.
  • the detection probe is resistant to nuclease activity (eg, 5' nuclease activity, eg, 5' to 3' exonuclease activity);
  • the backbone of the detection probe comprises nuclease resistant Reactive modifications such as phosphorothioate bonds, alkyl phosphotriester bonds, aryl phosphotriester bonds, alkyl phosphonate bonds, aryl phosphonate bonds, hydrophosphate bonds, alkyl phosphoramidate bonds , aryl phosphoramidate linkages, 2'-O-aminopropyl modifications, 2'-O-alkyl modifications, 2'-O-allyl modifications, 2'-O-butyl modifications, and 1-( 4'-thio-PD-ribofuranosyl) modification.
  • nuclease activity eg, 5' nuclease activity, eg, 5' to 3' exonuclease activity
  • the detection probe is linear, or has a hairpin structure. In certain preferred embodiments, the detection probe is linear. In certain preferred embodiments, the detection probe has a hairpin structure. Hairpin structures can be natural or artificially introduced.
  • the nucleic acid polymerase uses the detection probe as a template to extend the mediator fragment hybridized to the detection probe , and thus form a duplex.
  • the enzyme with 5' nuclease activity used is a nucleic acid polymerase with 5' nuclease activity, and is the same as the nucleic acid polymerase used in step (4) .
  • steps (1)-(5) are carried out by a scheme comprising the following steps (I)-(VII):
  • step (II) of the method the sample is mixed with the upstream oligonucleotide sequences, the mediator probe and downstream oligonucleotide sequences, and a nucleic acid polymerase, And carry out the PCR reaction, then, after the PCR reaction is completed, add the detection probe to the product of step (VI), and carry out melting curve analysis; or, in step (II), the sample and the upstream
  • the oligonucleotide sequence, the mediator probe, the downstream oligonucleotide sequence, the detection probe, and the nucleic acid polymerase are mixed and subjected to a PCR reaction, and then, after the PCR reaction is completed, a melting curve analysis is performed.
  • step (II) of the method the sample is combined with the upstream oligonucleotide sequences, the mediator probe and downstream oligonucleotide sequences, a nucleic acid polymerase, and a universal
  • the primers are mixed, and a PCR reaction is performed, and then, after the PCR reaction is completed, the detection probe is added to the product of step (VI), and a melting curve analysis is performed; or, in step (II), the sample is mixed with.
  • the upstream oligonucleotide sequence, the mediator probe, the downstream oligonucleotide sequence and the detection probe, nucleic acid polymerase, and universal primer are mixed, and a PCR reaction is performed, and then, after the PCR reaction is completed, a PCR reaction is performed. Melting curve analysis.
  • step (III) the product of step (II) is incubated at a temperature of 80-105°C, thereby denaturing the nucleic acid.
  • step (III) the product of step (II) is incubated for 10-20s, 20-40s, 40-60s, 1-2 min, or 2-5 min.
  • step (IV) at 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, or 65-70°C
  • the product of step (III) is incubated at a temperature to allow nucleic acid annealing or hybridization.
  • step (IV) the product of step (III) is incubated for 10-20s, 20-40s, 40-60s, 1-2 min, or 2-5 min.
  • step (V) at 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, 65-70°C, The product of step (IV) is incubated at temperatures of 70-75°C, 75-80°C, 80-85°C, thereby allowing nucleic acid extension.
  • step (V) the product of step (IV) is incubated for 10-20s, 20-40s, 40-60s, 1-2min, 2-5min, 5-10min, 10-20min or 20-30min.
  • steps (IV) and (V) are performed at the same or different temperatures.
  • steps (III)-(V) are repeated at least once, eg, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times. In certain embodiments, when steps (III)-(V) are repeated one or more times, the conditions used in each cycle of steps (III)-(V) are each independently the same or different.
  • step (VII) the product of step (VI) is gradually heated or cooled and the signal emitted by the reporter group on each detection probe is monitored in real time, thereby obtaining each A plot of the signal intensity of the reporter group as a function of temperature.
  • the obtained curve is derived to obtain a melting curve for the product of step (VI).
  • the presence of a mediator subfragment corresponding to the melting peak (melting point) is determined based on the melting peak (melting point) in the melting curve; then, the mediator subsequence in the mediator subfragment and the target nucleic acid sequence are determined The corresponding relationship is determined to determine the existence of the target nucleic acid sequence corresponding to the mediator fragment.
  • the application provides a probe set comprising a detection probe, and one or more (eg, at least two) mediator probes, wherein,
  • the mediator probes each independently comprise, in the 5' to 3' direction, a mediator sequence comprising a sequence complementary to a target nucleic acid sequence and a target-specific sequence comprising a non-target nucleic acid sequence. a sequence complementary to the target nucleic acid sequence, and the mediator sequences contained in all mediator probes are different from each other;
  • the at least one mediator probe itself is capable of forming a hairpin structure; preferably, the at least one mediator probe has features (i), (ii) or (iii) as defined in claim 1; and
  • the detection probe comprises, from 3' to 5', a capture sequence complementary to each mediator subsequence or a portion thereof, and a templating sequence; and the detection probe is labeled with a reporter group and a quenching group, wherein the reporter group can emit a signal, and the quenching group can absorb or quench the signal emitted by the reporter group; and the detection probe is hybridized to its complementary sequence The signal emitted is different from the signal emitted in the absence of hybridization to its complementary sequence.
  • the probe set comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, or at least 20 mediator probes.
  • each mediator probe is independently capable of forming a hairpin structure, eg, each independently has features (i), (ii) or (iii) as defined in claim 1 .
  • the mediator probe is 15-150nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80 -90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
  • the target-specific sequence in the mediator probe is 10-140nt in length, eg, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt , 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the length of the mediator sequence in the mediator probe can be 5-140nt, such as 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt , 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the first, second, third or fourth hairpin forming sequences are each independently 5-140nt in length, eg, 5-10nt, 10-20nt, 20-30nt, 30-40nt , 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the mediator probe has a 3'-OH terminus, or its 3'-terminus is blocked. In certain preferred embodiments, the 3'-terminus of the mediator probe is blocked to inhibit its extension.
  • the 3'-terminus of nucleic acids can be blocked by various methods. For example, the 3'-terminus of the mediator probe can be blocked by modifying the 3'-OH of the last nucleotide of the mediator probe. In certain embodiments, the 3'-terminus of the mediator probe can be blocked by adding a chemical moiety (eg, biotin or an alkyl group) to the 3'-OH of the last nucleotide of the mediator probe . In certain embodiments, the mediator probe can be blocked by removing the 3'-OH of the last nucleotide of the mediator probe, or by replacing the last nucleotide with a dideoxynucleotide 3'-end.
  • a chemical moiety eg, biotin or an alkyl group
  • a linker is also included between the target-specific sequence of the mediator probe and the first hairpin forming sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is further included between the second hairpin-forming sequence of the mediator probe and the mediator sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is also included between the third hairpin-forming sequence of the mediator probe and the mediator sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is further included between the target-specific sequence of the mediator probe and the fourth hairpin-forming sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • all mediator probes comprise target-specific sequences that differ from each other.
  • all mediator probes each target a different target nucleic acid sequence.
  • the probe set comprises a mediator probe as defined above.
  • the probe set comprises detection probes as defined above.
  • the application provides a kit comprising one or more probe sets as previously defined.
  • the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 probe sets.
  • all mediator sequences in the kit each target a different target nucleic acid sequence.
  • all the mediator probes in the kit contain mediator sequences that are different from each other.
  • all mediator probes in the kit comprise target-specific sequences that differ from each other.
  • all detection probes in the kit are each independently labeled with the same or different reporter groups.
  • kits can be used to practice the methods of the invention described in detail above. Therefore, the various technical features described in detail above for the mediator probe and the detection probe can also be applied to the mediator probe and the detection probe in the kit. Furthermore, such kits may also contain other reagents required for carrying out the methods of the present invention.
  • the kit may further comprise upstream oligonucleotide sequences, downstream oligonucleotide sequences, universal primers, enzymes with 5' nuclease activity, nucleic acids as defined above A polymerase, or any combination thereof.
  • the kit may further comprise, reagents for nucleic acid hybridization, reagents for mediator probe cleavage, reagents for nucleic acid extension, nucleic acid amplification reagents, or any combination thereof.
  • Such reagents can be routinely determined by one of skill in the art, and include, but are not limited to, working buffers for enzymes (eg, nucleic acid polymerases), dNTPs, water, solutions containing ions (eg, Mg 2+ ), single-stranded DNA binding proteins (Single Strand DNA-Binding Protein, SSB), or any combination thereof.
  • enzymes eg, nucleic acid polymerases
  • dNTPs eg, dNTPs
  • water solutions containing ions
  • solutions ions eg, Mg 2+
  • Single-stranded DNA binding proteins Single Strand DNA-Binding Protein
  • the term “mediator probe” refers to a single-stranded chain containing a mediator sequence and a targeting sequence (ie, a target-specific sequence) from the 5' to 3' direction A nucleic acid molecule, which optionally may also comprise a first hairpin-forming sequence downstream or 3' of the targeting sequence, or a second hairpin-forming sequence upstream or 5' of the mediator sequence, or at the mediator A third hairpin-forming sequence is included upstream or 5' of the sequence, and a fourth hairpin-forming sequence is included downstream or 3' of the target-specific sequence.
  • the mediator probe When the mediator probe contains a first, second, third or fourth hairpin-forming sequence, the mediator probe can pass through the mediator sequence and the first hairpin-forming sequence, or the second hairpin-forming sequence
  • the clip-forming sequence and the target-specific sequence, or the third hairpin-forming sequence and the fourth hairpin-forming sequence form a hairpin structure.
  • the mediator subsequence does not contain a sequence complementary to the target nucleic acid sequence
  • the target specific sequence comprises a sequence complementary to the target nucleic acid sequence.
  • the mediator probe hybridizes or anneals (ie, forms a double-stranded structure) to the target nucleic acid sequence through the target-specific sequence under conditions that allow nucleic acid hybridization, annealing, or amplification, and the mediator sequence in the mediator probe Does not hybridize to the target nucleic acid sequence.
  • targeting sequence and “target-specific sequence” refer to those capable of selectively/specifically hybridizing or annealing to a target nucleic acid sequence under conditions that permit hybridization, annealing, or amplification of the nucleic acid.
  • a sequence comprising a sequence complementary to a target nucleic acid sequence.
  • targeting sequence and “target-specific sequence” have the same meaning and are used interchangeably. It is readily understood that a targeting sequence or target-specific sequence is specific for a target nucleic acid sequence.
  • a targeting sequence or target-specific sequence only hybridizes or anneals to a specific target nucleic acid sequence, and not to other nucleic acid sequences, under conditions that allow nucleic acid hybridization, annealing, or amplification.
  • the term "mediator sequence” refers to a stretch of oligonucleotide sequence in the mediator probe that is not complementary to the target nucleic acid sequence and is located upstream (5' end) of the target-specific sequence.
  • a unique mediator probe is designed or provided, which has a unique mediator sequence (in other words, the mediator sequences in all mediator probes used are different from each other)
  • each target nucleic acid sequence corresponds to a unique mediator probe (unique mediator sequence).
  • upstream oligonucleotide sequence refers to an oligonucleotide sequence comprising a sequence complementary to a target nucleic acid sequence under conditions that allow nucleic acid hybridization (or annealing) or amplification , is capable of hybridizing (or annealing) to the target nucleic acid sequence and, when hybridized to the target nucleic acid sequence, is located upstream of the mediator probe.
  • the term “complementary” means that two nucleic acid sequences are capable of forming hydrogen bonds between each other according to the principles of base pairing (Waston-Crick principle), and thereby forming duplexes.
  • the term “complementary” includes “substantially complementary” and “completely complementary”.
  • the term “completely complementary” means that every base in one nucleic acid sequence is capable of pairing with bases in another nucleic acid strand without mismatches or gaps.
  • the term "substantially complementary” means that a majority of bases in one nucleic acid sequence are capable of pairing with bases in the other nucleic acid strand, which allows for mismatches or gaps (eg, one or mismatches or gaps of several nucleotides).
  • two nucleic acid sequences that are "complementary” eg, substantially complementary or fully complementary
  • non-complementary means that two nucleic acid sequences cannot hybridize or anneal under conditions that permit hybridization, annealing, or amplification of the nucleic acids to form a duplex.
  • not perfectly complementary means that bases in one nucleic acid sequence cannot perfectly pair with bases in another nucleic acid strand, at least one mismatch or gap exists.
  • hybridization and “annealing” mean the process by which complementary single-stranded nucleic acid molecules form a double-stranded nucleic acid.
  • hybridization and “annealing” have the same meaning and are used interchangeably.
  • two nucleic acid sequences that are completely complementary or substantially complementary can hybridize or anneal.
  • the complementarity required for hybridization or annealing of two nucleic acid sequences depends on the hybridization conditions used, in particular the temperature.
  • PCR reaction has the meaning commonly understood by those skilled in the art, which refers to a reaction (polymerase chain reaction) that amplifies a target nucleic acid using a nucleic acid polymerase and primers.
  • multiplex amplification refers to the amplification of multiple target nucleic acids in the same reaction system.
  • asymmetric amplification means that in the amplification product obtained by amplifying a target nucleic acid, the amount of two complementary nucleic acid strands is different, and the amount of one nucleic acid strand is greater than that of the other. nucleic acid strands.
  • detection probe is labeled with a reporter group and a quencher group.
  • detection probes are capable of forming duplexes with their complementary sequences through base pairing.
  • the reporter group such as a fluorophore
  • the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (such as a fluorescent signal) emitted by the reporter group. to the strongest signal (e.g. fluorescence signal).
  • the two strands of the duplex begin to dissociate (ie, the detection probe gradually dissociates from its complementary sequence), and the dissociated detection probe is in a single-stranded free coil state.
  • the reporter group (eg, fluorophore) and the quencher group on the dissociated detection probe are in close proximity to each other, whereby the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) absorbed by the quenching group. Therefore, as the temperature increases, the detected signal (eg, the fluorescent signal) gradually becomes weaker.
  • the two strands of the duplex are completely dissociated, all detection probes are in a single-stranded free coil state. In this case, all the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) on the detection probe is absorbed by the quencher group.
  • the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) is substantially undetectable.
  • melting curve analysis has the meaning commonly understood by those skilled in the art and refers to the analysis of the presence or identity of a double-stranded nucleic acid molecule by determining the melting curve of the double-stranded nucleic acid molecule. method, which is commonly used to assess the dissociation characteristics of double-stranded nucleic acid molecules during heating. Methods for performing melting curve analysis are well known to those skilled in the art (see, eg, The Journal of Molecular Diagnostics 2009, 11(2):93-101). In this application, the terms “melting curve analysis” and “melting analysis” have the same meaning and are used interchangeably.
  • melting curve analysis can be performed by using detection probes labeled with reporter and quencher groups.
  • detection probes are capable of forming duplexes with their complementary sequences through base pairing.
  • the reporter group such as a fluorophore
  • the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (such as a fluorescent signal) emitted by the reporter group. to the strongest signal (e.g. fluorescence signal).
  • the two strands of the duplex begin to dissociate (ie, the detection probe gradually dissociates from its complementary sequence), and the dissociated detection probe assumes a single-stranded free coil state.
  • the reporter group (eg, fluorophore) and the quencher group on the dissociated detection probe are in close proximity to each other, whereby the signal (eg, fluorescence signal) emitted by the reporter group (eg, fluorophore) absorbed by the quenching group. Therefore, as the temperature increases, the detected signal (eg, the fluorescent signal) gradually becomes weaker.
  • the two strands of the duplex are completely dissociated, all detection probes are in a single-stranded free coil state. In this case, all the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) on the detection probe is absorbed by the quencher group.
  • the signal eg, fluorescent signal
  • the reporter group eg, fluorophore
  • the hybridization and dissociation process of the detection probe and its complementary sequence can be observed, and the signal intensity changes with temperature. changing curve.
  • a curve with the change rate of signal intensity as the ordinate and the temperature as the abscissa ie, the melting curve of the duplex
  • the peak in the melting curve is the melting peak
  • the corresponding temperature is the melting point (T m value) of the duplex.
  • the more closely the detection probe matches the complementary sequence eg, fewer bases are mismatched and more bases are paired
  • the terms "melting peak”, “melting point” and “ Tm value” have the same meaning and are used interchangeably.
  • the method of the present application can effectively reduce (or even eliminate) the non-specific signal of the multiplex real-time PCR detection method using the mediator probe and the detection probe, and significantly improve the specificity of the detection method.
  • the method of the present application can also increase the intensity of the positive signal to a certain extent, thereby improving the detection sensitivity. Therefore, the methods of the present application are particularly advantageous, particularly suitable for multiplex detection of target nucleic acid sequences.
  • Figure 1 shows the structures of three hairpin mediator probes of the present invention.
  • 1A shows that when the mediator probe contains the mediator sequence, the target-specific sequence and the first hairpin forming sequence, the mediator subsequence is in a hairpin structure before being cleaved.
  • Figure IB shows that when the mediator probe contains a mediator sequence, a target-specific sequence and a second hairpin forming sequence, the mediator sequence is in a hairpin structure before the mediator sequence is cleaved.
  • Figure 1C shows that the mediator probe assumes a hairpin structure before the mediator sequence is cleaved when the mediator probe contains a mediator sequence, a target-specific sequence, a third hairpin-forming sequence, and a fourth hairpin-forming sequence.
  • Figure 2 schematically shows the principle of the system of the present invention.
  • Figure 2A shows that when the mediator probe contains a mediator sequence and a target-specific sequence, before the mediator sequence is cleaved, it has a linear structure, which is easy to non-specifically bind to the detection probe.
  • Figure 2B shows that when the mediator probe contains a mediator sequence, a target-specific sequence and a first hairpin-forming sequence, the mediator sequence is in a hairpin structure before the mediator sequence is cleaved, significantly reducing non-specificity with the detection probe combine.
  • Figure 3 shows the detection results of the reaction system of Example 1, wherein Figure 3A is the detection result produced by the Gp8wzx detection system using the linear mediator probe; Figure 3B is the Gp8wzx detection system using the hairpin mediator probe The detection results generated; Figure 3C shows the detection results generated by the O52wzm detection system using the linear mediator probes; Figure 3D shows the detection results generated by the O52wzm detection system using the hairpin mediator probes.
  • the solid line in the figure represents the detection result using the plasmid (4 samples) as the template; the dotted line represents the detection result (negative control) using the TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template.
  • Figure 4 shows the detection results of the Gp8wzx detection system, wherein Figure 4A is the detection result using the linear mediator probe; Figure 4B is the detection result using the hairpin mediator probe with 6 complementary bases; Figure 4C is the detection result. The detection result using the hairpin mediator probe with 8 complementary bases; Fig. 4D is the detection result using the hairpin mediator probe with 10 complementary bases.
  • the solid line in the figure represents the detection result using Escherichia coli Gp8wzx plasmid as the template; the dotted line represents the detection result (negative control) using TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template.
  • Figure 5 shows the detection results of the reaction system of Example 3, wherein Figure 5A is the detection system using the linear mediator probe; Figure 5B is the detection system using the hairpin mediator probe.
  • the solid line in the figure represents the detection result using the plasmid containing the target gene 1-6 as the template; the dotted line represents the detection result using the TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template (negative control) .
  • Figure 6 shows the detection results of the reaction system of Example 4, wherein Figure 6A is the detection system using the linear mediator probe; Figure 6B is the detection system using the hairpin mediator probe.
  • the solid line in the figure represents the detection result using the plasmid containing the target gene 7-12 as the template; the dashed line represents the detection result using the TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template (negative control) .
  • the hairpin mediator probe can be used for dual-probe real-time PCR detection, in this example, a linear mediator probe and a hairpin mediator probe are used for comparison, and the detection probe is the same.
  • the experiment used Escherichia coli O antigen-specific genes Gp8wzx and O52wzm as detection objects, and each 25 ⁇ L PCR reaction system included 1 ⁇ PCR buffer (Zhishan Biotechnology Co., Ltd., Xiamen), 7.0 mM MgCl 2 , 0.2 mM dNTPs, 3 U Taq01 enzyme ( Zhishan Biotechnology Co., Ltd., Xiamen), primers and probes (see Table 1 for dosage), and 5 ⁇ L of templates (the templates of positive control are E.
  • coli Gp8wzx plasmid and O52wzm plasmid (10000 copies/ul), respectively, will be as SEQ ID NO:
  • the nucleotide sequences shown in 56 and SEQ ID NO: 57 were inserted into the multi-cloning site of the puc57 vector to obtain the Escherichia coli Gp8wzx plasmid and the O52wzm plasmid respectively; the template of the negative control was TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5).
  • the PCR reaction program is: incubation at 50 °C for 2 min, denaturation at 95 °C for 5 min, then 40 cycles of 20 s at 95 °C and 1 min at 60 °C, fluorescence collection at 60 °C, and the procedure for melting analysis after PCR is as follows: Hybridization at 35 °C Extend for 20min, then denature at 95°C for 2min, keep at 45°C for 2min, then heat up from 45°C to 95°C at a heating rate of 0.04°C/s, and collect the fluorescence signal of the CY5 channel.
  • the experimental instrument is a SLAN 96S real-time PCR instrument (Hongshi Medical Technology Co., Ltd., Shanghai), primers and probes were synthesized by Shanghai Bioengineering Co., Ltd.
  • Reaction A is a Gp8wzx singleplex detection system, using a linear mediator probe
  • Reaction B is a Gp8wzx singleplex detection system, using a hairpin mediator probe.
  • the results showed that the negative control (dotted line) of reaction A (Fig. 3A) produced an obvious non-specific melting peak at 52°C, while the negative control (dotted line) of reaction B (Fig. 3B) had no obvious non-specific melting peak.
  • the experimental results show that the hairpin mediator probe can significantly reduce the non-specific melting signal compared with the linear mediator probe.
  • Reaction C is an O52wzm singleplex detection system, using a linear mediator probe
  • Reaction D is an O52wzm singleplex detection system, using a hairpin mediator probe.
  • the universal primer Tag is used in both the Gp8 wzx singleplex detection system and the O52 wzm singleplex detection system.
  • Example 2 the Gp8 wzx singleplex detection system described in Example 1 was used to investigate the use of the linear mediator probe and the mediator probe with hairpin structure complementary bases of 6, 8, and 10 bases, respectively, for melting curves. Feasibility of analysis.
  • the composition of the reaction system and the PCR reaction conditions are the same as those in Example 1.
  • the linear mediator probes used in this example are also the same as those in Example 1.
  • Table 2 shows the hairpin mediator probes.
  • a six-fold PCR melting curve analysis system was used to detect and distinguish 6 different target sequences.
  • Five target genes (target genes 1-5) and one positive quality control gene (target gene 6) were used as detection objects.
  • the nucleotide sequences shown in SEQ ID NO: 58 to SEQ ID NO: 63 were inserted into the multiple cloning sites of the puc57 vector, respectively, to obtain plasmids containing target genes 1-6.
  • reaction system A was a control system, using 6 linear mediator probes and 2 fluorescent probes.
  • Reaction system B is an experimental system, using 4 linear mediator probes, 2 hairpin mediator probes and 2 fluorescent probes, wherein the 4 linear mediator probes correspond to target genes 1, 2, and 3 respectively And 5, 2 hairpin mediator probes correspond to positive quality control gene and target gene 4.
  • the composition of the reaction system and the PCR reaction conditions were the same as those in Example 1, and the detected templates were 6 positive plasmids (concentration: 1000 copies/ul) carrying target genes or quality control genes.
  • the primers and probes used in this example are specifically shown in Table 3.
  • reaction system A Fig. 5A
  • reaction system B Fig. 5B
  • the melting peak signal of the sub-probe is generally higher than that of the reaction system A (all of which are linear mediator sub-probes).
  • the non-specific melting signal of the reaction system B at 45-50 °C was significantly reduced.
  • a six-fold PCR melting curve analysis system is used to detect and distinguish the target genes of 6 different microorganisms.
  • the target gene 7-12 was used as the detection object.
  • the nucleotide sequences shown in SEQ ID NO: 64 to SEQ ID NO: 69 were inserted into the multiple cloning sites of the puc57 vector, respectively, to obtain plasmids containing target genes 7-12.
  • reaction system A was a control system, using 6 linear mediator probes and 2 fluorescent probes
  • reaction system B was an experimental system, using 5 linear mediator probes, 1 hairpin mediator probe and 2 fluorescent probes.
  • the composition of the reaction system and the PCR reaction conditions were the same as those in Example 1, and the melting curve analysis system used the ROX fluorescence channel.
  • the detected templates were 6 positive plasmids (concentration of 1000 copies/ul) carrying the target gene.
  • the primers and probes used in this example are specifically shown in Table 4.
  • reaction system A (Fig. 6A) and reaction system B (Fig. 6B) detected melting peaks with the same Tm value for each target gene, however, reaction system B (with a hairpin medium)
  • the melting peak signal of the sub-probe is generally higher than that of the reaction system A (all of which are linear mediator sub-probes).
  • the non-specific melting signal of the reaction system B at 45-50°C was completely eliminated.

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Abstract

La présente invention concerne un procédé de détection d'une séquence d'acide nucléique cible. Le procédé permet de détecter simultanément la présence de plusieurs séquences d'acide nucléique cibles dans un échantillon avec une spécificité élevée. L'invention concerne en outre un ensemble de sondes et un kit comprenant un ou plusieurs des ensembles de sondes. L'ensemble sonde et le kit peuvent être utilisés pour mettre en œuvre le procédé de la présente invention.
PCT/CN2020/140053 2020-12-14 2020-12-28 Procédé de détection à haute spécificité d'une séquence d'acide nucléique cible WO2022126721A1 (fr)

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