WO2022126760A1 - Procédé pour effectuer une détection multiplex sur des acides nucléiques - Google Patents

Procédé pour effectuer une détection multiplex sur des acides nucléiques Download PDF

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WO2022126760A1
WO2022126760A1 PCT/CN2020/141201 CN2020141201W WO2022126760A1 WO 2022126760 A1 WO2022126760 A1 WO 2022126760A1 CN 2020141201 W CN2020141201 W CN 2020141201W WO 2022126760 A1 WO2022126760 A1 WO 2022126760A1
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probe
nucleic acid
sequence
mediator
group
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PCT/CN2020/141201
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Chinese (zh)
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李庆阁
刘巧巧
宋甲宝
许晔
廖逸群
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present application relates to multiplex detection of nucleic acid molecules.
  • the present application provides a method for detecting target nucleic acid sequences, which can simultaneously qualitatively and quantitatively detect multiple target nucleic acid sequences in a single reaction system.
  • Real-time quantitative PCR developed at the end of the 20th century has the advantages of high sensitivity, good specificity and easy operation.
  • Real-time PCR is a common tool for nucleic acid detection. As a closed-tube detection mode, it is easy to operate, has low chance of contamination by amplification products, and is widely used.
  • the multiplex real-time fluorescent quantitative PCR developed on this basis which integrates the detection of various pathogens into one tube for reaction, not only saves the cost, but also has the accuracy and sensitivity of fluorescent quantitative PCR.
  • the maximum number of target sequences that can be detected in this mode is limited by the number of fluorescence detection channels of the real-time PCR instrument. Therefore, although many studies or patents reduce costs by using multiple quantitative detection systems, the number of detection objects in one reaction tube is still relatively large. few.
  • Another detection mode of PCR nucleic acid detection is the melting curve analysis after amplification. This mode is to add a temperature change process after amplification, and the maximum number of target sequences that can be detected is equal to the number of fluorescence detection channels. Compared with the real-time detection mode, the dimension of melting point between the needle and the target sequence is greatly improved, and the number of detection objects has also increased. Faltin et al.
  • a detection probe corresponds to more mediators, which greatly improves the detection throughput. Because the non-fluorescent labeled mediator probes are low in cost, and fluorescent probes can be used as universal probes for different target sequences. Therefore, it is different from detection. Compared with the single-plex real-time PCR system of the target sequence, a common fluorescent probe can be shared, thereby reducing the cost. This method has been applied to various detections, for example, but the disadvantage of this method is that it cannot quantitatively analyze the detection object.
  • nucleic acid detection In many aspects of nucleic acid detection, only qualitative analysis or only quantitative analysis cannot meet the needs. For example, in pathogen detection, there are pathogenic bacteria and colonizing bacteria. In this case, colonization and infection need to be distinguished. In terms of transgenic detection, for different transgenic product detection marker genes, some only need qualitative detection, and some require quantitative detection. For the establishment of multiple nucleic acid detection reactions, the detection of nucleic acids to be quantified cannot be satisfied by simply relying on media probes, and the cost of all reactions with linear probes is higher.
  • CN102559868B discloses a method for qualitative and quantitative detection of multiple target nucleic acid sequences in a single tube. The regions with large differences are selected to design specific probes that can recognize various target nucleic acid sequences. Each group of probes is equipped with the same fluorophore as a detection channel, and the target sequences are distinguished by melting point curve analysis. This method needs to adjust the melting point of each probe, which is difficult in probe design, and each set of probes needs to be added with a fluorophore, which is more expensive than medium probes. Therefore, there remains a need for accurate methods for high-throughput multiplex detection of target nucleic acids.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • target nucleic acid sequence refers to a target nucleic acid sequence to be detected.
  • probe refers to a polynucleotide sequence capable of hybridizing or annealing to a target nucleic acid of interest and allowing specific detection of the target nucleic acid.
  • the probes described herein refer to oligonucleotide probes.
  • the term "mediator probe” refers to a sequence that contains a mediator sequence and a targeting sequence from the 5' to 3' direction; ie, a target-specific sequence ) of single-stranded nucleic acid molecules.
  • the mediator sequence contains no sequence complementary to the target nucleic acid sequence
  • the target specific sequence contains the 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, but is in a free state (ie, maintains a single-stranded structure).
  • the mediator probe does not contain a reporter molecule, and a general probe containing a reporter molecule needs to be used for fluorescence signal generation.
  • the terms "mediator probe” and “mediator probe” have the same meaning and are used interchangeably.
  • 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).
  • the terms “mediator system” and “mediator sub-system” refer to nucleic acids that detect a target nucleic acid using a mediator probe that does not contain a reporter molecule and produce a detectable signal using a universal probe that contains a reporter molecule Detection method.
  • upstream primer refers to an oligonucleotide sequence comprising a sequence complementary to a target nucleic acid sequence, which is capable of interacting with the target under conditions that permit hybridization (or annealing) or amplification of the nucleic acid.
  • the nucleic acid sequence hybridizes (or anneals) and, when hybridized to the target nucleic acid sequence, is located upstream of the mediator probe.
  • the upstream primer serves as the starting point for nucleic acid synthesis.
  • 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
  • the target-specific sequences in the upstream primer and the mediator probe each comprise a sequence that is complementary (eg, substantially complementary or fully complementary) to the target nucleic acid sequence.
  • target-specific sequences in the upstream primer and mediator probe will selectively/specifically hybridize or anneal to target nucleic acid sequences under conditions that allow nucleic acid hybridization, annealing, or amplification.
  • 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.
  • an intermediary subsequence comprises a sequence that is not complementary to a target nucleic acid sequence.
  • the mediator sequence does not hybridize or anneal to the target nucleic acid sequence, cannot form a duplex, but is in a free state (ie, maintains a single-stranded structure).
  • 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.
  • condition that allow nucleic acid hybridization have the meaning commonly understood by those skilled in the art, and can be determined by conventional methods.
  • two nucleic acid molecules having complementary sequences can hybridize under suitable hybridization conditions.
  • Such hybridization conditions may involve factors such as temperature, pH, composition and ionic strength of the hybridization buffer, etc., and may be determined based on the length and GC content of the two nucleic acid molecules that are complementary.
  • low stringency hybridization conditions can be used when the lengths of the two complementary nucleic acid molecules are relatively short and/or the GC content is relatively low.
  • High stringency hybridization conditions can be used when the lengths of the two complementary nucleic acid molecules are relatively long and/or the GC content is relatively high.
  • hybridization conditions are well known to those skilled in the art and can be found in, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999).
  • “hybridization” and “annealing” have the same meaning and are used interchangeably. Accordingly, the expressions “conditions allowing nucleic acid hybridization” and “conditions allowing nucleic acid annealing” also have the same meaning and are used interchangeably.
  • condition that allow nucleic acid amplification has the meaning commonly understood by those skilled in the art, which means that a nucleic acid polymerase (eg, DNA polymerase) is allowed to synthesize another nucleic acid using one nucleic acid strand as a template chain and the conditions for duplex formation. Such conditions are well known to those skilled in the art and may relate to factors such as temperature, pH, composition, concentration and ionic strength of the hybridization buffer, among others. Suitable nucleic acid amplification conditions can be determined by routine methods (see, e.g., Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)). In the method of the present invention, "conditions allowing nucleic acid amplification” are preferably working conditions of a nucleic acid polymerase (eg, DNA polymerase).
  • a nucleic acid polymerase eg, DNA polymerase
  • condition that allow a nucleic acid polymerase to perform an extension reaction has the meaning commonly understood by those skilled in the art, which means that a nucleic acid polymerase (eg, a DNA polymerase) is allowed to extend a nucleic acid strand as a template Another nucleic acid strand (eg, a primer or probe), and the conditions under which it forms a duplex.
  • a nucleic acid polymerase eg, a DNA polymerase
  • Another nucleic acid strand eg, a primer or probe
  • Such conditions are well known to those skilled in the art and may relate to factors such as temperature, pH, composition, concentration and ionic strength of the hybridization buffer, among others.
  • Suitable nucleic acid amplification conditions can be determined by routine methods (see, e.g., Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)).
  • "conditions that allow the nucleic acid polymerase to carry out the extension reaction” are preferably working conditions of the nucleic acid polymerase (eg, DNA polymerase).
  • the expressions “conditions that allow nucleic acid polymerase to carry out the extension reaction” and “conditions that allow nucleic acid extension” have the same meaning and are used interchangeably.
  • the working conditions of various enzymes can be determined by those skilled in the art by routine methods, and can generally involve the following factors: temperature, pH of buffer, composition, concentration, ionic strength, and the like. Alternatively, conditions recommended by the manufacturer of the enzyme can be used.
  • nucleic acid denaturation has the meaning commonly understood by those skilled in the art and refers to the process by which double-stranded nucleic acid molecules are dissociated into single strands.
  • condition that allow denaturation of nucleic acid refers to conditions under which a double-stranded nucleic acid molecule is dissociated into single strands. Such conditions can be routinely determined by those skilled in the art (see, e.g., Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)).
  • nucleic acids can be denatured by conventional techniques such as heat, alkali treatment, urea treatment, enzymatic methods (eg, methods using helicase).
  • the nucleic acid is preferably denatured under heating.
  • nucleic acids can be denatured by heating to 80-105°C.
  • upstream is used to describe the relative positional relationship of two nucleic acid sequences (or two nucleic acid molecules) and has the meaning commonly understood by those skilled in the art.
  • expression “a nucleic acid sequence is located upstream of another nucleic acid sequence” means that when arranged in a 5' to 3' direction, the former is located at a more forward position (ie, closer to the 5' end) than the latter. Location).
  • downstream has the opposite meaning to "upstream.”
  • PCR cycle refers to a single round of (1) unwinding of DNA strands called “denaturation” followed by (2) oligonucleotide primers to the resulting single Strand DNA hybridization, a process called “annealing", and (3) polymerization of a new DNA strand starting from the 3' end of the oligonucleotide primer and moving in the 5' to 3' direction, called “amplification” or The process of "extending".
  • polymerization uses a DNA polymerase such as Taq polymerase to catalyze the formation of phosphodiester bonds between adjacent deoxynucleotide triphosphates ("dNTPs"), which are joined by hydrogen bonds along the The exposed single-stranded template DNA is placed. Denaturation, annealing, and amplification are performed at certain temperatures based in part on the GC content of the DNA template and oligonucleotide primers and the length of the DNA strand to be replicated.
  • the term "quantitative PCR” or “qPCR” or “Q-PCR” is a type of PCR capable of monitoring amplicon formation during the PCR cycling process. Q-PCR can be used to quantify the amount of specific template DNA in a sample.
  • Q-PCR also introduces at least one oligonucleotide detection probe into the reaction mixture.
  • the detection probe is a single-stranded oligonucleotide that hybridizes to the sense or antisense strand of the target template DNA somewhere between the forward primer binding site and the reverse primer binding site. During the annealing step, the oligonucleotide detection probe anneals to the single-stranded template.
  • 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, e.g., 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 a universal probe labeled with a reporter group and a quencher group.
  • a universal probe labeled with a reporter group and a quencher group are capable of forming duplexes with their complementary sequences through base pairing.
  • the reporter group such as a fluorophore
  • the quencher group on the universal probe are separated from each other, and the quencher group cannot absorb the signal (such as a fluorescent signal) emitted by the reporter group. At this time, it is possible to detect 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
  • the quencher group on the dissociated universal 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.
  • all universal probes are in a single-stranded free coil state.
  • the signals (eg, fluorescent signals) emitted by reporter groups (eg, fluorophores) on the universal probe are absorbed by the quencher groups.
  • the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) is substantially undetectable. Therefore, by detecting the signal (such as a fluorescent signal) emitted by the duplex containing the universal probe during the heating or cooling process, the hybridization and dissociation process of the universal probe and its complementary sequence can be observed, and the signal intensity changes with temperature. changing curve.
  • a curve 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.
  • T m value the melting point of the duplex.
  • the term "self-quenching probe” refers to an oligonucleotide labeled with a reporter group and a quencher group.
  • the quencher group is located in a position capable of absorbing or quenching the signal of the reporter group (eg, the quencher group is located adjacent to the reporter group), thereby absorbing or quenching the reporter group signal from the group. In this case, the probe does not emit a signal.
  • the quencher group when the probe is hybridized to its complementary sequence, the quencher group is located in a position that cannot absorb or quench the signal of the reporter group (eg, the quencher group is located away from the reporter group), so that it cannot absorb or quench the signal of the reporter group. Quench the signal from the reporter group. In this case, the probe emits a signal.
  • the present invention provides a method for detecting a target nucleic acid in a sample, comprising the steps of:
  • the upstream primer comprises a sequence complementary to the target nucleic acid sequence
  • the detection probe comprises a sequence complementary to the target nucleic acid sequence and is labeled with a quantitative reporter group and a quencher group; and, when hybridized to the target nucleic acid sequence, the upstream primer is positioned upstream of the detection probe;
  • the quantitative reporter groups labeled with all detection probes are different from each other;
  • the upstream primer comprises a sequence complementary to the target nucleotide sequence
  • the 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 comprises a sequence that is complementary to the target nucleic acid sequence. a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the upstream primer is positioned upstream of the target-specific sequence; and all the mediator probes comprise mediator sequences that are different from each other;
  • each universal probe independently comprises from 3' to 5' direction, with one or more universal probes one or more capture sequences complementary to a seed mediator subsequence or a portion thereof, and a template sequence; and, the one or more universal probes comprise capture sequences capable of being respectively compatible with each mediator described in (ii)
  • the mediator subsequences or portions thereof of the subprobes are complementary (i.e., the one or more universal probes contain a one-to-one correspondence between the species of capture sequences and the species of mediator subprobes); and, each universal probe each independently labeled with a qualitative reporter group and a quencher group; and each universal probe emits a different signal when hybridized to its complementary sequence than when not hybridized to its complementary sequence;
  • a melting curve analysis is performed on the amplification product, the melting curve analysis comprising measuring the signal from the qualitative reporter group described in (a)(iii), based on the determined qualitative reporter The signal of the group determines the presence of the corresponding target nucleic acid sequence.
  • the number of detection probes can be at least 1, at least 2, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or greater.
  • the number of universal probes may be 1, 2, 3, 4, 5, or 6.
  • the number of mediator probes may be at least 1, at least 2, eg, 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.
  • the sample can be any sample to be detected.
  • the sample comprises or is DNA (eg, genomic DNA or cDNA).
  • the sample comprises or is RNA (eg, mRNA).
  • the sample comprises or is a mixture of nucleic acids (eg, a mixture of DNA, a mixture of RNA, or a mixture of DNA and RNA).
  • the target nucleic acid sequence to be detected is not limited by its sequence composition or length.
  • the target nucleic acid sequence can be a DNA (eg, genomic DNA or cDNA) or an RNA molecule (eg, mRNA).
  • the target nucleic acid sequence to be detected may be single-stranded or double-stranded.
  • a reverse transcription reaction is performed to obtain cDNA complementary to the mRNA.
  • a detailed description of reverse transcription reactions can be found, for example, in Joseph Sam-brook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
  • the sample or target nucleic acid sequence to be detected can be obtained from any source, including but not limited to prokaryotes (eg, bacteria), eukaryotes (eg, protozoa, parasites, fungi, yeast, plants, animals including mammals and humans) or Viruses (eg, Herpes virus, HIV, influenza virus, Epstein-Barr virus, hepatitis virus, polio virus, etc.) or viroids.
  • the sample or target nucleic acid sequence to be detected can also be a nucleic acid sequence in any form, such as a genomic sequence, an artificially isolated or fragmented sequence, a synthetic sequence, and the like.
  • the detection probe described in (i) can be any oligonucleotide probe used for quantitative PCR.
  • the detection probe is labeled with a quantitative reporter group and a quencher group, wherein the quantitative reporter group can emit a signal, and the quencher group can absorb or quench the The signal from the quantitative reporter group is described.
  • the quencher group is located in a position capable of absorbing or quenching the signal of the quantitative reporter group, thereby absorbing or quenching the signal emitted by the quantitative reporter group, when the detection probe is not hybridized to other sequences . In this case, the detection probe does not emit a signal.
  • the detection probe when the detection probe is hybridized with its complementary sequence and undergoes an extension reaction, the detection probe is cleaved by enzymes, so that the quantitative reporter group and the quenching group are separated, so that the quantitative reporter group cannot be absorbed or quenched. or, when the detection probe hybridizes with its complementary sequence, the quantitative reporter group and the quencher group are separated by a sufficient distance from each other, so that the signal emitted by the quantitative reporter group cannot be quenched by the quencher group. absorb. In this case, the detection probe emits a signal.
  • a quantitative reporter group may be labeled at the 5' end of the detection probe and a quencher group at the 3' end, or a quantitative reporter group may be labeled at the 3' end of the detection probe and a quantitative reporter group at the 5' end End-labeled quencher groups. It should be understood, however, that the quantitative reporter and quencher groups do not have to be labeled at the ends of the detection probe. Quantitative reporter groups and/or quencher groups can also be labeled on the interior of the detection probe.
  • the quantitative reporter group can be labeled upstream (or downstream) of the detection probe, while the quencher group can be labeled downstream (or upstream) of the detection probe.
  • the quantitative reporter group and the quencher group are separated by a distance of 10-80nt or more, eg, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt , 70-80nt.
  • the quantitative reporter group and the quencher group are no more than 80 nt, no more than 70 nt, no more than 60 nt, no more than 50 nt, no more than 40 nt, no more than 30 nt, or no more than 20 nt apart.
  • the quantitative reporter group and the quencher group are separated by at least 5 nt, at least 10 nt, at least 15 nt, or at least 20 nt. In certain embodiments, at least one of a quantitative reporter group and a quencher group is located at the terminus (eg, the 5' or 3' terminus) of the detection probe. In certain embodiments, one of the quantitative reporter group and the quencher group is located at or 1-10 nt from the 5' end of the detection probe, and the quantitative reporter group and the quencher group are separated The appropriate distance is such that the quenching group absorbs or quenches the signal of the quantitative reporter group prior to hybridization of the detection probe to its complementary sequence.
  • one of the quantitative reporter group and the quencher group is located at the 3' end of the detection probe or at a position 1-10 nt from the 3' end, and the quantitative reporter group and the quencher group are separated The appropriate distance is such that the quenching group absorbs or quenches the signal of the quantitative reporter group prior to hybridization of the detection probe to its complementary sequence.
  • one of the quantitative reporter group and the quencher group is located at the 5' end of the detection probe and the other is located at the 3' end.
  • the quantitative reporter group and quenching group can be any suitable group or molecule known in the art, specific examples of which include but are not limited to Cy2 TM (506), YO-PRO TM -l(509), YOYO TM -l(509), Calcein(517), FITC(518), FluorX TM (519), Alexa TM (520), Rhodamine 110(520), Oregon Green TM 500(522), Oregon Green TM 488 (524), RiboGreen TM (525), Rhodamine Green TM (527), Rhodamine 123 (529), Magnesium Green TM (531), Calcium Green TM (533), TO-PRO TM -1 (533) ,TOTOl(533),JOE(548),BODIPY530/550(550),Dil(565),BODIPYTMR(568),BODIPY558/568(568),BODIPY564/570(570), Cy3TM (570),
  • the quantitative reporter group is a fluorophore.
  • the signal emitted by the quantitative reporter group is fluorescence
  • the quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg, another fluorophore capable of absorbing the fluorescence molecule, or a quencher capable of quenching the fluorescence).
  • the fluorophore includes, but is not limited to, various fluorescent molecules, such as 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, etc.
  • the quenching group includes, but is not limited to, various quenchers, such as DABCYL, BHQ (eg, BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA, among others.
  • the detection probe described in (i) can be degraded by an enzyme (eg, DNA polymerase).
  • an enzyme eg, DNA polymerase
  • the detection probe described in (i) may be linear, or may have a hairpin structure.
  • the detection probe is linear.
  • the detection probe has a hairpin structure.
  • Hairpin structures can be natural or artificially introduced.
  • detection probes with hairpin structures can be constructed using routine methods in the art.
  • the detection probe can form a hairpin structure by adding two complementary oligonucleotide sequences to the two ends (5' and 3' ends) of the detection probe.
  • the complementary 2 stretches of oligonucleotide sequences constitute the arms (stems) of the hairpin structure.
  • the arms of the hairpin structure can be of any desired length, for example the length of the arms can be 2-15nt, eg 3-7nt, 4-9nt, 5-10nt, 6-12nt.
  • the detection probes described in (i) may comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-naturally occurring nucleotides nucleotides (eg, peptide nucleic acid (PNA) or locked nucleic acid), or any combination thereof.
  • the detection probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the detection probes comprise modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 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).
  • the detection probe described in (i) is not limited by its length.
  • the length of the detection probe can be 10-100 nt, such as 15-50 nt, 20-30 nt.
  • the detection probe described in (i) has a 3'-OH terminus.
  • the 3'-end of the detection probe 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. In certain embodiments, the detection probes are selected from Taqman probes, TaqMan MGB probes, or molecular beacons.
  • the mediator probe refers to a probe used in the mediator system to detect target nucleic acid and unlabeled reporter molecule.
  • the mediator probe can comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), 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, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine.
  • the mediator probes comprise non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole , 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole , 5-nitroindole or locked nucleic acid (LNA).
  • the mediator probe is not limited by its length.
  • the mediator probe can be 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 can be of any length as long as it can specifically hybridize to the target nucleic acid sequence.
  • the target-specific sequence in the mediator probe can be 10-140nt in length, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80nt -90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the mediator sequence in the mediator probe can be of any length as long as it can specifically hybridize and extend the universal probe.
  • the mediator sequence in the mediator probe can be 5-140nt in length, such as 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-nt 80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the target-specific sequence in the mediator probe is 10-100 nt in length (eg, 10-90 nt, 10-80 nt, 10-50 nt, 10-40 nt, 10-30 nt, 10-20 nt)
  • the length of the mediator subsequence is 5-100 nt (eg, 10-90 nt, 10-80 nt, 10-50 nt, 10-40 nt, 10-30 nt, 10-20 nt).
  • the mediator probe has a 3'-OH terminus.
  • 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.
  • the 3'-terminus of the mediator probe can be blocked by modifying the 3'-OH of the last nucleotide of the mediator probe.
  • 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 .
  • 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 universal probe refers to a probe labeled with a reporter molecule for generating a detectable signal in a mediator subsystem.
  • a universal probe may comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (eg, peptides) nucleic acid (PNA) or locked nucleic acid), or any combination thereof.
  • the universal probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • universal probes comprise modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine.
  • the universal probe comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitropyrrole, 5-Nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitropyrrole, 5-Nitroindole or locked nucleic acid (LNA).
  • the universal probe is not limited by its length.
  • a universal probe can be 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 universal probe can be of any length as long as it can specifically hybridize to the mediator fragment.
  • a capture sequence in a universal probe can be 10-500nt in length, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 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 universal probe can be of any length as long as it can be used as a template for extending the mediator subfragment.
  • a template sequence in a universal probe can be 1-900nt in length, such as 1-5nt, 5-10nt, 10-20nt, 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.
  • the capture sequence in the universal probe is 10-200nt in length (eg, 10-190nt, 10-180nt, 10-150nt, 10-140nt, 10-130nt, 10-120nt, 10-100nt , 10-90nt, 10-80nt, 10-50nt, 10-40nt, 10-30nt, 10-20nt), and, the length of the template sequence is 5-200nt (eg, 10-190nt, 10-180nt, 10-150nt , 10-140nt, 10-130nt, 10-120nt, 10-100nt, 10-90nt, 10-80nt, 10-50nt, 10-40nt, 10-30nt, 10-20nt).
  • the universal probe has a 3'-OH terminus.
  • the 3'-end of the universal probe is blocked to inhibit its extension.
  • the 3'-terminus of nucleic acids can be blocked by various methods.
  • the 3'-terminus of the universal probe can be blocked by modifying the 3'-OH of the last nucleotide of the universal probe.
  • the 3'-terminus of the universal 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 universal probe.
  • the 3'-OH of the universal probe can be blocked by removing the 3'-OH of the last nucleotide of the universal probe, or by replacing the last nucleotide with a dideoxynucleotide. '-end.
  • the mediator fragment hybridizes to the universal probe, and thereby initiates an extension reaction by a nucleic acid polymerase.
  • uncleaved mediator probes are also capable of hybridizing to the universal probe via the mediator sequence
  • mediator probes also contain target-specific sequences that are downstream of the mediator sequence and do not hybridize to the universal probe (ie, at the free state), so that the nucleic acid polymerase cannot extend the mediator probe hybridized to the universal probe.
  • the universal probe is labeled with a qualitative reporter group and a quencher group, wherein the qualitative reporter group is capable of signaling and the quencher group is capable of absorbing or quenching the qualitative reporter
  • the universal probe is a self-quenching probe.
  • the quenching group when the universal probe is not hybridized to other sequences, the quenching group is located in a position capable of absorbing or quenching the signal of the qualitative reporter group (eg, in the vicinity of the qualitative reporter group), thereby absorbing or to quench the signal from a qualitative reporter group. In this case, the universal probe emits no signal. Further, when the universal probe hybridizes to its complementary sequence, the quencher group is located at a position that cannot absorb or quench the signal of the qualitative reporter group (eg, is located away from the qualitative reporter group), and thus cannot absorb or quench the signal of the qualitative reporter group. Deactivate the signal from the qualitative reporter group. In this case, the universal probe emits a signal.
  • a qualitative reporter group may be labeled at the 5' end of the universal probe and a quencher group at the 3' end, or a qualitative reporter group may be labeled at the 3' end of the universal probe and the 5' end End-labeled quencher groups.
  • the qualitative reporter group and the quencher group are close to each other and interact with each other, so that the signal emitted by the qualitative reporter group is absorbed by the quencher group , so that the universal probe does not emit a signal; and when the universal probe hybridizes with its complementary sequence, the qualitative reporter group and the quencher group are separated from each other, so that the qualitative reporter group emits The signal cannot be absorbed by the quencher group, thereby allowing the universal probe to signal.
  • the qualitative reporter and quencher groups do not have to be labeled at the ends of the universal probe.
  • Qualitative reporter and/or quencher groups can also be labeled internal to a universal probe, as long as the universal probe emits a different signal when hybridized to its complementary sequence than it does when it is not hybridized to its complementary sequence signal of.
  • the qualitative reporter group can be labeled upstream (or downstream) of the universal probe, and the quencher group can be labeled downstream (or upstream) of the universal probe, and the two are sufficiently separated (eg, 10- 20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, or longer distances).
  • the qualitative reporter group and the quencher group are mutually exclusive due to the free coiling of the probe molecule or the formation of a secondary structure (eg, a hairpin structure) of the probe. approach and interact so that the signal from the qualitative reporter group is absorbed by the quencher group, so that the universal probe does not emit a signal; and, when the universal probe hybridizes to its complement, the The qualitative reporter group and the quencher group are separated from each other by a sufficient distance so that the signal emitted by the qualitative reporter group cannot be absorbed by the quencher group, so that the universal probe emits a signal.
  • a secondary structure eg, a hairpin structure
  • the qualitative reporter group and the quencher group are separated by a distance of 10-80nt or more, eg, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt , 70-80nt.
  • the qualitative reporter group and the quencher group are no more than 80 nt, no more than 70 nt, no more than 60 nt, no more than 50 nt, no more than 40 nt, no more than 30 nt, or no more than 20 nt apart.
  • the qualitative reporter group and the quencher group are separated by at least 5 nt, at least 10 nt, at least 15 nt, or at least 20 nt.
  • the qualitative reporter and quencher groups can be labeled at any suitable location on the universal probe, so long as the universal probe emits a different signal when hybridized to its complementary sequence than when it is not hybridized to its complementary sequence signal below.
  • at least one of the qualitative reporter group and the quencher group is located at the terminus (eg, the 5' or 3' terminus) of the universal probe.
  • one of the qualitative reporter group and the quencher group is located at the 5' end of the universal probe or at a position 1-10 nt from the 5' end, and the qualitative reporter group and the quencher group are separated The appropriate distance is such that the quencher group can absorb or quench the signal of the qualitative reporter group before the universal probe hybridizes to its complementary sequence.
  • one of the qualitative reporter group and the quencher group is located at the 3' end of the universal probe or at a position 1-10 nt from the 3' end, and the qualitative reporter group and the quencher group are separated The appropriate distance is such that the quencher group can absorb or quench the signal of the qualitative reporter group before the universal probe hybridizes to its complementary sequence.
  • the qualitative reporter group and the quencher group may be separated by a distance as defined above (eg, a distance of 10-80 nt or more).
  • one of the qualitative reporter group and the quencher group is located at the 5' end of the universal probe and the other is located at the 3' end.
  • the qualitative reporter group and quencher group can be any suitable group or molecule known in the art, specific examples of which include but are not limited to Cy2 TM (506), YO-PRO TM -l(509), YOYO TM -l(509), Calcein(517), FITC(518), FluorX TM (519), Alexa TM (520), Rhodamine 110(520), Oregon Green TM 500(522), Oregon Green TM 488 (524), RiboGreen TM (525), Rhodamine Green TM (527), Rhodamine 123 (529), Magnesium Green TM (531), Calcium Green TM (533), TO-PRO TM -1 (533) ,TOTOl(533),JOE(548),BODIPY530/550(550),Dil(565),BODIPYTMR(568),BODIPY558/568(568),BODIPY564/570(570), Cy3TM (570),
  • the qualitative reporter group is a fluorescent group, that is, the universal probe is selected from fluorescent probes.
  • the signal emitted by the qualitative reporter group is fluorescence
  • the quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg, another fluorophore capable of absorbing the fluorescence molecule, or a quencher capable of quenching the fluorescence).
  • the fluorophore includes, but is not limited to, various fluorescent molecules, such as ALEX-350, FAM, VIC, TET, CAL Gold 540, JOE, HEX, CAL Fluor Orange560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705, etc.
  • the quenching group includes, but is not limited to, various quenchers, such as DABCYL, BHQ (eg, BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA, among others.
  • general probes (such as fluorescent probes) can also be modified, such as phosphorothioate linkages, alkyl phosphotriester linkages, aryl phosphotriester linkages, alkylphosphonate linkages, Aryl Phosphonate Bond, Hydrogen Phosphate Bond, Alkyl Phosphoramidate Bond, Aryl Phosphoramidate Bond, 2'-O-Aminopropyl Modification, 2'-O-Alkyl Modification, 2'-O- Allyl modification, 2'-O-butyl modification, or 1-(4'-thio-PD-ribofuranosyl) modification.
  • universal probes may be linear, or may have a hairpin structure.
  • the universal probe is linear.
  • the universal probe has a hairpin structure.
  • Hairpin structures can be natural or artificially introduced.
  • detection probes with hairpin structures can be constructed using routine methods in the art.
  • the universal probe can form a hairpin structure by adding complementary 2-segment oligonucleotide sequences to the two ends (5' and 3' ends) of the universal probe.
  • the complementary 2 stretches of oligonucleotide sequences constitute the arms (stems) of the hairpin structure.
  • the arms of the hairpin structure can be of any desired length, for example the length of the arms can be 2-15nt, eg 3-7nt, 4-9nt, 5-10nt, 6-12nt.
  • the qualitative reporter group in the universal probe described in (iii) and the quantitative reporter group in the detection probe described in (i) may be the same or different.
  • the quencher group in the universal probe described in (iii) and the quencher group in the detection probe described in (i) may be the same or different.
  • 1 universal probe is provided for the mediator probe described in (ii), the universal probe ranging from 3' to 5' The 'direction comprises, a capture sequence complementary to each mediator sequence or a portion thereof, and a template sequence; and the universal probe is labeled with a qualitative reporter group and a quencher group.
  • the upstream primer may comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any of these Combination composition.
  • the upstream primer comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the upstream primer comprises modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine.
  • the upstream primer comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitropyrrole, 5 - Nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitropyrrole, 5 - Nitroindole or locked nucleic acid (LNA).
  • the upstream primer is not limited by its length as long as it can specifically hybridize to the target nucleic acid sequence.
  • the upstream primer can be 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.
  • a mediator system it is desirable to induce cleavage of a mediator probe that hybridizes to the target nucleic acid sequence.
  • an enzyme with 5' nuclease activity can be used to induce cleavage of a mediator probe that hybridizes to a target nucleic acid sequence using an upstream primer or an extension product thereof that hybridizes to the target nucleic acid sequence.
  • the upstream primer described in (ii) is positioned upstream of the mediator probe after hybridization to the target nucleic acid sequence.
  • the upstream primer directly induces enzymatic cleavage of the mediator probe with 5' nuclease activity without the need for an extension reaction.
  • two adjacent nucleic acid sequences are no more than 30 nt apart, eg, no more than 20 nt, eg, no more than 15 nt, eg, no more than 10 nt, eg, no more than 5 nt, eg 4nt, 3nt, 2nt, 1nt.
  • the upstream primer described in (ii) has a partially overlapping sequence with the target-specific sequence of the mediator probe after hybridization to the target nucleic acid sequence.
  • the upstream primer directly induces enzymatic cleavage of the mediator probe with 5' nuclease activity without the need for an extension reaction.
  • the partially overlapping sequences are 1-10 nt in length, eg, 1-5 nt, or 1-3 nt in length.
  • the upstream primer described in (ii) is located at the upstream distal end of the mediator probe after hybridization to the target nucleic acid sequence.
  • the upstream primer is extended by a nucleic acid polymerase, and the resulting extension product induces enzymatic cleavage of the mediator probe with 5' nuclease activity.
  • distal is intended to mean that two nucleic acid sequences are distant from each other, eg, at least 30 nt, at least 50 nt, at least 80 nt, at least 100 nt or more apart from each other.
  • step (a) (i) in addition to the upstream primer and the detection probe, a downstream primer is also provided for each target nucleic acid sequence that needs to be quantitatively detected; wherein , the downstream primer comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized with the target nucleic acid sequence, the downstream primer is located downstream of the detection probe.
  • the nucleic acid polymerase will use the upstream and downstream primers as primers to amplify the target nucleic acid sequence.
  • the nucleic acid polymerase induces cleavage of the detection probe hybridized to the target nucleic acid sequence through its own 5' nuclease activity, thereby releasing the signal of the quantitative reporter group.
  • a downstream primer is also provided for each target nucleic acid sequence that needs to be qualitatively detected;
  • the downstream primer comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the downstream primer is located downstream of the target-specific sequence.
  • the nucleic acid polymerase will use the upstream and downstream primers as primers to amplify the target nucleic acid sequence.
  • the nucleic acid polymerase induces cleavage of the mediator probe hybridized to the target nucleic acid sequence through its own 5' nuclease activity, thereby releasing the mediator comprising the mediator sequence or a portion thereof subfragment.
  • the downstream primers described in (i) or (ii) may comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides , non-natural nucleotides, or any combination thereof.
  • the downstream primer comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the downstream primer comprises modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine.
  • the downstream primer comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitropyrrole, 5 - Nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitropyrrole, 5 - Nitroindole or locked nucleic acid (LNA).
  • the downstream primer is not limited by its length as long as it can specifically hybridize to the target nucleic acid sequence.
  • the length of the downstream primer can be 15-150nt, 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.
  • step (a) all upstream primers and downstream primers described in (i) and (ii) have an identical stretch of oligonucleotide sequence at the 5' end; and, Step (a) also includes providing the following components: (iv) for all upstream primers and downstream primers described in (i) and (ii), providing a universal primer having the same oligo A sequence that is complementary to a nucleotide sequence.
  • the universal primer comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the universal primer is 8-50nt in length, eg, 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.
  • PCR reaction conditions may include conditions that allow nucleic acid hybridization, conditions that allow nucleic acid amplification, conditions that allow nucleic acid polymerase to perform extension reactions, and conditions that allow nucleic acid denaturation.
  • the PCR reaction conditions described in step (b) include the use of a nucleic acid polymerase having 5' nuclease activity, and the nucleic acid polymerase can both use the target nucleic acid sequence as a template to catalyze the extension of the upstream primer and/or capable of inducing cleavage of the probe.
  • the nucleic acid polymerase has, eg, 5' exonuclease activity.
  • the nucleic acid polymerase is a DNA polymerase.
  • the nucleic acid polymerase is a thermostable DNA polymerase available from various bacterial species, eg, Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus 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 litoraripolitana,Thermosphos Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosi
  • the PCR reaction is performed in a three-step method. In such embodiments, each round of nucleic acid amplification requires three steps: nucleic acid denaturation at a first temperature, nucleic acid annealing at a second temperature, and nucleic acid extension at a third temperature. In certain embodiments, the PCR reaction is performed in a two-step process. In such embodiments, each round of nucleic acid amplification requires two steps: nucleic acid denaturation at a first temperature, and nucleic acid annealing and extension at a second temperature.
  • Temperatures suitable for nucleic acid denaturation, nucleic acid annealing, and nucleic acid extension can be readily determined by those skilled in the art by routine methods (see, e.g., Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)).
  • step (b) symmetric PCR amplification is performed.
  • the upstream primer (ie, the primer for the DNA strand in the same direction as the probe) and the downstream primer (ie, the primer for the DNA strand that is complementary to the probe) described in (i) are substantially equivalent.
  • the upstream primer (ie, the primer for the DNA strand in the same direction as the probe) and the downstream primer (ie, the primer for the DNA strand that is complementary to the probe) described in (ii) are substantially equivalent.
  • step (b) asymmetric PCR amplification is performed.
  • the upstream primer described in (i) is in excess (eg, the primer on the DNA strand complementary to the probe) relative to the downstream primer (ie, the primer on the DNA strand complementary to the probe). at least 1-fold, at least 2-fold, at least 5-fold, at least 8-fold, at least 10-fold, eg, 1-10-fold excess).
  • the upstream primer described in (i) is 2-10 times the size of the downstream primer (ie, the primer on the DNA strand complementary to the probe) ( For example 2-8 times or 2-6 times, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times or 8 times).
  • the downstream primer described in (i) is in excess (eg, at least 1-fold, at least 2-fold, at least 5-fold, at least 8-fold, at least 10-fold excess, eg, excess) relative to the upstream primer 1-10 times).
  • the upstream primer described in (ii) is in excess (eg, the primer on the DNA strand complementary to the probe) relative to the downstream primer (ie, the primer on the DNA strand complementary to the probe). at least 1-fold, at least 2-fold, at least 5-fold, at least 8-fold, at least 10-fold, eg, 1-10-fold excess).
  • the upstream primer described in (ii) is 2-10 times the size of the downstream primer (ie, the primer on the DNA strand complementary to the probe) ( For example 2-8 times or 2-6 times, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times or 8 times).
  • the downstream primer described in (ii) is in excess (eg, at least 1-fold, at least 2-fold, at least 5-fold, at least 8-fold, at least 10-fold excess, eg, excess) relative to the upstream primer 1-10 times).
  • signal acquisition in a PCR cycle is performed at a temperature above the annealing and extension temperatures, eg, at least 10°C, at least 15°C, or at least 20°C above the annealing and extension temperatures.
  • the specific temperature described in step (c) is a denaturation temperature. In certain embodiments, the denaturation temperature is 94-98°C.
  • pre-amplification is performed before the measurement of the signal of the quantitative reporter group, which may be beneficial to eliminate early lighting instability without affecting the final lighting effect.
  • step (c) the signal emitted by the quantitative reporter group on each of the detection probes described in (i) is monitored in real time to obtain a signal for each quantitative reporter group A curve of intensity as a function of cycle number (ie, amplification curve).
  • step (3) allows the enzyme with 5' nuclease activity to cleave the mediator probe hybridized to the target nucleic acid sequence to be qualitatively detected, and release the nucleic acid fragment containing the mediator sequence or a part thereof .
  • the qualitative detection of the target nucleic acid sequence is realized by melting curve analysis based on the mediator subsystem.
  • the one or more universal probes may comprise the same qualitative reporter group.
  • the melting curve analysis includes: determining the presence of a certain target nucleic acid sequence according to a melting peak (melting point) in the obtained melting curve.
  • the one or more universal probes comprise qualitative reporter groups that are different from each other.
  • the melting curve analysis includes: monitoring the signal of each qualitative reporter group in real time respectively, thereby obtaining a plurality of melting points corresponding to the signal of each qualitative reporter group curve; then, the presence of a certain target nucleic acid sequence is determined based on the signal species of the qualitative reporter group and the melting peak (melting point) in the melting curve.
  • the melting curve analysis comprises: gradually heating or cooling the PCR product and monitoring in real time the qualitative reporting on each of the universal probes described in (iii) The signal intensity of each qualitative reporter group as a function of temperature was obtained.
  • the obtained curve is derived to obtain a melting curve.
  • 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 invention makes full use of the respective advantages of the medium probe system and the linear quantitative system, and provides a method for qualitatively and quantitatively detecting multiple targets simultaneously in a single reaction tube, which has the advantages of high throughput, low cost, flexible probe selection, high sensitivity and high sensitivity. high specificity.
  • Figure 1 shows that the medium system in Example 1 produces an amplification curve in addition to melting peaks when the medium system is annealed at the annealing temperature, and the amplification curve can be eliminated by lighting at the denaturation temperature.
  • FIG. 2 shows the amplification curves of the linear fluorescent probe or the hairpin fluorescent probe in Example 2 in the presence of the universal probe of the medium system with annealing temperature lighting and denaturing temperature lighting.
  • Figure 3 shows that in Example 3, by adjusting the amount of upstream and downstream primers, the melting peak generated by the quantitative probe can be eliminated.
  • FIG. 4 shows the results of qualitative and quantitative detection in a single reaction using the combination of linear fluorescent probes or hairpin fluorescent probes and mediator systems in Example 4.
  • FIG. 5 shows the results of the sensitivity investigation of the qualitative analysis of viruses and atypical pathogens in Example 5.
  • FIG. 6 shows the results of the sensitivity examination of the quantitative analysis of bacteria in Example 5.
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Refined Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995, was performed as described; restriction enzymes were used according to the conditions recommended by the product manufacturer.
  • restriction enzymes were used according to the conditions recommended by the product manufacturer.
  • the primers, media probes and universal probes used in PCR amplification were synthesized by Xiamen Boshang Biotechnology Co., Ltd. or Shanghai Sangon Biotechnology Co., Ltd.; dNTPs were purchased from Shanghai Linglan Biotechnology Co., Ltd.; Taq 01 enzyme was purchased from Xiamen Zhishan Biotechnology Co., Ltd., column-type viral nucleic acid extraction kit was purchased from Shanghai Sangon Bioengineering Co., Ltd.; other conventional chemical reagents were domestic analytical reagents.
  • Primer design of this system adopts Primer Premier 5.0.
  • the Tm values of primers were predicted online using IDT Biophysics.
  • the HAND system is introduced, that is, after the specific primers are designed, a tag sequence is added to the 5' end of the primers.
  • the internationally recognized Filmarray pneumonia detection kit adopts the Taqman single-plex real-time detection system as a control method at the beginning of the establishment of the system.
  • the Taqman single-plex real-time detection system can give qualitative and quantitative detection results of pathogens, which is suitable as a control for this system Therefore, we refer to the literature to establish a Taqman single-plex control system for 19 pathogens on the basis of the existing system in the laboratory. For the specific sequence, please refer to Table 3.
  • the established single-plex real-time control system was 25 ⁇ L.
  • the reaction system includes 1 ⁇ SSP buffer, 5.0mM MgCl 2 , 0.2mM dNTPs, 40 ⁇ M dUTP, 0.4 ⁇ M upstream and downstream primers, 0.2 ⁇ M TaqMan fluorescent probe, 1.0U TaqHS DNA polymerase (TaKaRa, Beijing), 5 ⁇ L template,
  • the reaction system was made up to 25 ⁇ L with sterile water.
  • the primer/probe information of each detection object involved in the following examples is shown in the table below.
  • Table 1 Simultaneous qualitative and quantitative detection of multiple target nucleic acid sequence system primers and probes
  • P-U1-ROX respiratory syncytial virus
  • P-U2-ROX influenza A, rhinovirus
  • P-U-CY5 influenza B
  • P-U-HEX internal control RPP30
  • P-U1-FAM Adenovirus
  • P-U2-FAM Mycoplasma pneumoniae.
  • Detection object F R MP/P Haemophilus influenzae 80 80 800 Staphylococcus aureus 60 60 60 Streptococcus pneumoniae 20 20 100 Pseudomonas aeruginosa 80 80 800 External control SUC2 40 40 400 Mycoplasma pneumoniae 40 400 A stream 40 40 400 B flow 80 80 800 respiratory syncytial virus type A 40 40 400 Respiratory syncytial virus type B 40 40 400 Rhinovirus 40 400 Adenovirus 60 60 600 Internal control gene RPP30 40 40 400 400
  • a 25- ⁇ L PCR reaction system was used, including: 7.0 mM MgCl 2 , 0.2 mM dNTPs, 40 ⁇ M dUTP, 3.0 U Taq01 (Zhishan Biotechnology Co., Ltd., Xiamen), 0.01 U UNG enzyme, 1 universal primer 0.8 ⁇ M, 1 Universal probe 0.04 ⁇ M, the sequences and concentrations of the upstream primer (F), downstream primer (R) and fluorescent probe (P) for human adenovirus are shown in Table 1 and Table 2.
  • the reaction program was 50 °C for 2 min, pre-denaturation at 95 °C for 10 min, 95 °C ⁇ 15 s, 60 °C ⁇ 20 s, and 72 °C ⁇ 20 s for 50 cycles.
  • Example 2 Investigation on the lighting temperature of the media probe system and the combined detection system of the fluorescent quantitative probe
  • Example 1 The inventors found in Example 1 that using light at denaturation temperature can eliminate the amplification curve generated by qualitative probes. All use the annealing temperature for lighting, so the lighting at the denaturing temperature may interfere with the quantitative detection. In order to ensure the accuracy of simultaneous qualitative and quantitative detection in the same system, the inventors investigated the light harvesting of the probe from the annealing temperature to the denaturation temperature, and added a general probe of the same channel to investigate the quantitative detection under this condition. Needle test results.
  • a 25- ⁇ L PCR reaction system including: 7.0 mM MgCl 2 , 0.2 mM dNTPs, 40 ⁇ M dUTP, 3.0 U Taq01 (Zhishan Biotechnology Co., Ltd., Xiamen), 0.01 U UNG enzyme, 1 universal primer 0.8 ⁇ M, 1 universal probe 0.04 ⁇ M, the sequences and concentrations of upstream primer (F), downstream primer (R) and fluorescent probe (P) against Haemophilus influenzae are shown in Table 1 and shown in Table 2.
  • the reaction program was 50 °C for 2 min, pre-denaturation at 95 °C for 10 min, 95 °C ⁇ 15 s, 60 °C ⁇ 20 s, and 72 °C ⁇ 20 s for 50 cycles.
  • Embodiment 3 The influence of the amount of upstream and downstream primers on the melting peak produced by the quantitative detection object
  • Example 2 In the investigation of Example 2, it was found that in addition to generating an amplification curve, the quantitative detection object also generated a melting peak. In order to further expand the detection throughput, we tried to eliminate the melting peaks generated by quantitative probes by adjusting the amount of upstream and downstream primers of quantitative detection objects. Taking the detection of Haemophilus influenzae (HIB) as an example to investigate, three amplification methods are used: symmetric amplification, forward asymmetric amplification and reverse asymmetric amplification.
  • HAI Haemophilus influenzae
  • primers complementary to the probe DNA strands The ratio of primers to the DNA strand in the same direction as the probe is 4 to 1, and for reverse asymmetry: the ratio of the primers of the DNA strand in the same direction as the probe to the primer of the complementary DNA strand of the probe is 4 to 1.
  • a 25- ⁇ L PCR reaction system was used, including: 7.0 mM MgCl 2 , 0.2 mM dNTPs, 40 ⁇ M dUTP, 3.0 U Taq01 (Zhishan Biotechnology Co., Ltd., Xiamen), 0.01 U UNG enzyme, 1 universal primer 0.8 ⁇ M, 1 Universal probe 0.04 ⁇ M, the sequences of upstream primer (F), downstream primer (R) and fluorescent probe (P) against Haemophilus influenzae are shown in Table 1 and Table 2.
  • the reaction program was 50 °C for 2 min, pre-denaturation at 95 °C for 10 min, 95 °C ⁇ 15 s, 60 °C ⁇ 20 s, and 72 °C ⁇ 20 s for 50 cycles.
  • Example 4 Qualitative and quantitative detection by combined use of media probe and fluorescent quantitative probe
  • the denaturation temperature as the lighting temperature, in order to further investigate whether the combined use of multiple fluorescent quantitative probes (such as linear fluorescent probes or hairpin fluorescent probes) and the media system can achieve Achieving qualitative and quantitative detection in one reaction, select common respiratory viruses and bacteria to investigate.
  • the medium probe system is used to qualitatively detect the virus, and only the melting curve is generated when the virus is positive, but the amplification curve is not generated; the fluorescent quantitative probe is used to quantitatively detect the bacteria, and the amplification curve can be generated. There is no melting peak, and the method in Example 3 can be used to solve the melting peak.
  • the 25- ⁇ L PCR reaction system includes 7.0 mM MgCl 2 , 0.2 mM dNTPs, 40 ⁇ M dUTP, 3.0 U Taq01 (Zhishan Biotechnology Co., Ltd., Xiamen), 0.01 U UNG enzyme, 1 universal primer 0.8 ⁇ M, and 5 universal probes 0.04 ⁇ M each, and the sequences and concentrations of other primer probes are shown in Table 1 and Table 2.
  • the PCR reaction program was: denaturation at 95 °C for 5 min, followed by 50 cycles of 95 °C for 20 s, 60 °C for 1 min, and fluorescence collection at 95 °C.
  • the procedure of melting analysis after PCR is as follows: hybridization and extension at 35°C for 20 min, then denaturation at 95°C for 2 min, incubation at 40°C for 2 min, and then the temperature is increased from 45°C to 95°C at a heating rate of 0.5°C/step, and the fluorescence signal is collected during the melting process.
  • the experimental instrument was a SLAN real-time fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.), and the primers and probes were synthesized by Shanghai Bioengineering Co., Ltd.
  • the experimental results are shown in Figure 4, adding bacterial plasmid standards or externally controlled plasmid standards to generate an amplification curve, with or without a melting peak. Positive plasmid standards for virus are added to produce only melting curves, not amplification curves.
  • the experimental results show that the combination of the media system and the linear fluorescent probe or the hairpin fluorescent probe can realize quantitative detection and qualitative detection in one reaction.
  • Example 5 Sensitivity investigation of simultaneous qualitative and quantitative detection by the combined use of media probes and fluorescent quantitative probes
  • the sensitivity of a multiplex detection system consisting of a medium probe and a fluorescent quantitative probe (such as a linear fluorescent probe or a hairpin fluorescent probe) refers to the lowest copy number that the system can stably detect.
  • the plasmid standards were diluted with TE to 1000 copies/ ⁇ L, 100 copies/ ⁇ L, and 10 copies/ ⁇ L. To exclude experimental errors, the experiments were repeated three times with 3 parallel wells of 10 copies/ ⁇ L per experiment. Two parallel wells were repeated at 1000copies/ ⁇ L and 100copies/ ⁇ L. 5 ⁇ L of plasmid standard was added to each reaction well as a template, and TE was added to 5 wells as a negative control to indicate experimental contamination.
  • the primer probe sequences and concentrations of the detection objects involved are shown in Table 1 and Table 2, and the remaining reaction conditions are as described in Example 2.
  • Example 6 Stability investigation of simultaneous qualitative and quantitative detection by the combined use of media probes and fluorescent quantitative probes
  • the repeatability of the multiplex detection system consisting of media probes and fluorescent quantitative probes (such as linear fluorescent probes or hairpin fluorescent probes) is mainly examined by the Tm value of the qualitative detection object and the Ct value of the real-time quantitative detection object.
  • the primer probe sequences and concentrations of the detection objects involved are shown in Table 1 and Table 2, and the remaining reaction conditions are as described in Example 2.
  • the results of the stability investigation of the qualitative detection of viruses, atypical bacteria and internal control genes are shown in Table 4.
  • the SD maximum value of the Tm value is 0.31, and the CV value is less than 0.22%.
  • the results show that the multiple detection system of the present invention has good stability for the detection of viruses, atypical bacteria and internal control genes.
  • each experiment was repeated three times in parallel wells at 1000 copies/ ⁇ L, 100 copies/ ⁇ L and 10 copies/ ⁇ L, and the experiments were repeated three times on three instruments.
  • the reaction system and experimental procedure were the same as those in Example 1.
  • the experimental results are shown in Table 5, and the overall CV value is less than 1.3%, indicating that the multiple detection system of the present invention can better quantify bacteria.
  • Example 7 Investigation of the detection ability of mixed nucleic acid samples for simultaneous qualitative and quantitative detection by the combined use of media probes and fluorescent quantitative probes
  • the lower respiratory tract bacteria As the research object, the lower respiratory tract was considered to be in a sterile environment in the past, but in recent years, the literature reported that, like the upper respiratory tract, the lower respiratory tract also has an asymptomatic state of bacteria. Therefore, the nucleic acid detection method has a high probability of detecting more than one type of bacteria in the lower respiratory tract specimens, and there is also a mixed infection of bacteria in the clinic. Therefore, the system is particularly important for the detection ability of bacterial mixed infection.
  • the bacteria covered by the system were mixed at equal concentrations to obtain 1000copies/ ⁇ L, 100copies/ ⁇ L, and 10copies/ ⁇ L mixed standards. Three parallel wells were set up for each standard, and the average of the Ct values was subtracted.
  • the primer probe sequences and concentrations of the detection objects involved are shown in Table 1 and Table 2, and the remaining reaction conditions are as described in Example 2.
  • the experimental results are shown in Table 6.
  • the difference between the Ct value of each object in the mixed infection and the single infection is less than 1. It shows that the system can also quantify the mixed infection of bacteria (mixed nucleic acid) more accurately.
  • ⁇ Ct is equal to the Ct value of mixed infection minus the Ct value of one bacterial infection.

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Abstract

L'invention concerne un procédé pour effectuer une détection sur des séquences d'acides nucléiques cibles. Le procédé peut détecter simultanément, qualitativement et quantitativement, diverses séquences d'acides nucléiques cibles dans un seul système de réaction, et présente les caractéristiques suivantes : haut débit, faible coût, sélection de sonde flexible, haute sensibilité et haute spécificité.
PCT/CN2020/141201 2020-12-17 2020-12-30 Procédé pour effectuer une détection multiplex sur des acides nucléiques WO2022126760A1 (fr)

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