WO2022126760A1

WO2022126760A1 - Method for performing multiplex detection on nucleic acids

Info

Publication number: WO2022126760A1
Application number: PCT/CN2020/141201
Authority: WO
Inventors: 李庆阁; 刘巧巧; 宋甲宝; 许晔; 廖逸群
Original assignee: 厦门大学
Priority date: 2020-12-17
Filing date: 2020-12-30
Publication date: 2022-06-23
Also published as: CN112592964A

Abstract

Provided is a method for performing detection on target nucleic acid sequences. The method can qualitatively and quantitatively detect various target nucleic acid sequences in a single reaction system at the same time, and has the characteristics of a high throughput, a low cost, flexible probe selection, high sensitivity and a high specificity.

Description

Method for performing multiplex detection of nucleic acids

technical field

The present application relates to multiplex detection of nucleic acid molecules. In particular, 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.

Background technique

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. In recent years, 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. However, 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. Clinical Chemistry 2012, 58(11): 1546-1556 describe a real-time PCR detection mode of "mediator probe PCR" - this mode we call a single-probe detection system, which uses a A dual-probe detection system—that is, a target sequence requires two probes—includes a non-fluorescently labeled target sequence-specific mediator probe and another fluorescently labeled probe that does not bind to the target sequence. However, the method of Faltin et al. also suffers from significant drawbacks. In particular, when the method of Faltin et al. is used to perform multiplex real-time PCR that needs to distinguish each target sequence, it is necessary to design a mediator probe carrying a target-specific sequence and a corresponding Fluorescent probes with unique fluorescent signals. In this case, the method of Faltin et al. requires the use of double the number of probes compared to traditional multiplex real-time PCR using a single probe for each target sequence. Correspondingly, the entire reaction system becomes more complicated, and the detection cost becomes higher. However, Li Qingge et al. have used this principle to invent a method to simultaneously detect the presence of multiple target nucleic acid sequences in a sample. In the CN108823287B patent, a probe set was provided, and "detection probes" with different fluorescent labels were designed. 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.

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.

It is necessary to develop a method for simultaneous qualitative and quantitative detection of multiple targets in a single reaction tube. Regarding simultaneous qualitative and quantitative detection, 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.

Therefore, there is still a need to develop new methods for multiplex detection of target nucleic acids, so as to achieve high-throughput, high-sensitivity and high-specificity target nucleic acid detection with simpler reaction systems and lower detection costs.

SUMMARY OF THE INVENTION

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the nucleic acid chemistry laboratory operation steps used herein are all routine steps widely used in the corresponding fields. Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the terms "target nucleic acid sequence," "target nucleic acid," and "target sequence" refer to a target nucleic acid sequence to be detected. In this application, the terms "target nucleic acid sequence", "target nucleic acid" and "target sequence" have the same meaning and are used interchangeably.

As used herein, the term "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. In some embodiments, the probes described herein refer to oligonucleotide probes.

As used herein, 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. In the present application, the mediator sequence contains no sequence complementary to the target nucleic acid sequence, and the target specific sequence contains the sequence complementary to the target nucleic acid sequence. Thus, 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. In this application, the terms "mediator probe" and "mediator probe" have the same meaning and are used interchangeably.

As used herein, the terms "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. In this application, the terms "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. In other words, 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.

As used herein, 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. In this application, for each target nucleic acid 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) Thus, each target nucleic acid sequence corresponds to a unique mediator probe (unique mediator sequence). Thus, by detecting the unique mediator sequence, the corresponding target nucleic acid sequence can be detected.

As used herein, 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.

As used herein, the term "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. In the case of amplification, the upstream primer serves as the starting point for nucleic acid synthesis.

As used herein, 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. In this application, the term "complementary" includes "substantially complementary" and "completely complementary". As used herein, 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. As used herein, 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). Typically, two nucleic acid sequences that are "complementary" (eg, substantially complementary or fully complementary) will selectively/specifically hybridize or anneal under conditions that allow nucleic acid hybridization, annealing, or amplification, and form a duplex . For example, in the present application, 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. Thus, 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. Accordingly, the term "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. For example, in the present application, an intermediary subsequence comprises a sequence that is not complementary to a target nucleic acid sequence. Thus, under conditions that allow nucleic acid hybridization, annealing, or amplification, 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).

As used herein, the terms "hybridization" and "annealing" mean the process by which complementary single-stranded nucleic acid molecules form a double-stranded nucleic acid. In this application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Typically, 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.

As used herein, "conditions that allow nucleic acid hybridization" have the meaning commonly understood by those skilled in the art, and can be determined by conventional methods. For example, 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. For example, 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. Such 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). In this application, "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.

As used herein, the expression "conditions 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).

As used herein, the expression "conditions 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. 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 that allow the nucleic acid polymerase to carry out the extension reaction" are preferably working conditions of the nucleic acid polymerase (eg, DNA polymerase). In this application, 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.

As used herein, the term "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. The expression "conditions 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)). For example, nucleic acids can be denatured by conventional techniques such as heat, alkali treatment, urea treatment, enzymatic methods (eg, methods using helicase). In the present application, the nucleic acid is preferably denatured under heating. For example, nucleic acids can be denatured by heating to 80-105°C.

As used herein, the term "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. For example, the 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). As used herein, the term "downstream" has the opposite meaning to "upstream."

As used herein, the term "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". Typically, 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.

As used herein, the term "quantitative PCR" or "qPCR" or "Q-PCR" (also known as "real-time 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. In addition to the forward and reverse oligonucleotide primers, 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.

As used herein, the term "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.

In certain embodiments of the present application, melting curve analysis can be performed by using a universal probe labeled with a reporter group and a quencher group. Briefly, at ambient temperature, universal probes are capable of forming duplexes with their complementary sequences through base pairing. In this case, the reporter group (such as a fluorophore) and 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). As the temperature increases, 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. In this case, the reporter group (eg, fluorophore) and 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. When the two strands of the duplex are completely dissociated, all universal probes are in a single-stranded free coil state. In this case, all the signals (eg, fluorescent signals) emitted by reporter groups (eg, fluorophores) on the universal probe are absorbed by the quencher groups. Thus, 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. Further, by performing derivation analysis on the obtained curve, a curve (ie, the melting curve of the duplex) with the change rate of the signal intensity as the ordinate and the temperature as the abscissa can be obtained. The peak in the melting curve is the melting peak, and the corresponding temperature is the melting point (T _m value) of the duplex. In general, the more closely the universal probe matches the complementary sequence (eg, fewer bases are mismatched and more bases are paired), the higher the _Tm value of the duplex will be. Thus, by detecting the _Tm value of the duplex, the presence and identity of the sequence complementary to the universal probe in the duplex can be determined. Herein, the terms "melting peak", "melting point" and " _Tm value" have the same meaning and are used interchangeably.

As used herein, the term "self-quenching probe" refers to an oligonucleotide labeled with a reporter group and a quencher group. When the probe is not hybridized to other sequences, 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. Further, 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.

Detection method

Therefore, in one aspect, the present invention provides a method for detecting a target nucleic acid in a sample, comprising the steps of:

(a) The following reaction components are provided:

(i) for each of the target nucleic acid sequences to be quantitatively determined, provide an upstream primer and a detection probe; wherein the upstream primer comprises a sequence complementary to the target nucleic acid sequence; the detection probe The needle 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; Moreover, the quantitative reporter groups labeled with all detection probes are different from each other;

(ii) for each of the target nucleic acid sequences to be qualitatively determined, provide an upstream primer and a mediator probe; wherein, 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;

(iii) providing one or more universal probes for the mediator probes described in (ii); wherein 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;

(b) contacting a sample containing the target nucleic acid sequence to be detected with the components described in (a) in a single reaction vessel, and implementing PCR reaction conditions;

(c) measuring the signal from the quantitative reporter group described in (a)(i) at a specific temperature in the PCR cycle, the specific temperature being above the annealing temperature and extension temperature; based on the quantitative reporter determined a signal of a group that determines the presence or amount of the corresponding target nucleic acid sequence (eg, by the signal from multiple PCR cycles to detect the presence or amount of the corresponding target nucleic acid sequence);

(d) After PCR is completed, 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.

In certain embodiments, 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.

In certain embodiments, there are fewer universal probes than mediator probes. In certain embodiments, the number of universal probes may be 1, 2, 3, 4, 5, or 6. In certain embodiments, 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.

In the methods of the present invention, the sample can be any sample to be detected. For example, in certain embodiments, the sample comprises or is DNA (eg, genomic DNA or cDNA). In certain embodiments, the sample comprises or is RNA (eg, mRNA). In certain embodiments, 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).

In the method of the present invention, the target nucleic acid sequence to be detected is not limited by its sequence composition or length. For example, the target nucleic acid sequence can be a DNA (eg, genomic DNA or cDNA) or an RNA molecule (eg, mRNA). Furthermore, the target nucleic acid sequence to be detected may be single-stranded or double-stranded.

When the sample or target nucleic acid sequence to be detected is mRNA, preferably, before performing the method of the present invention, 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.

step (a)

detection probe

In the method of the present invention, quantitative detection of various target nucleic acid sequences is achieved by quantitative PCR. Thus, the detection probe described in (i) can be any oligonucleotide probe used for quantitative PCR.

In certain embodiments, 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. In certain embodiments, 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. Further, 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.

Designing such detection probes is well within the purview of those skilled in the art. For example, 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. For example, 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. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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.

In the method of the present invention, 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),Alexa ^TM 546(570), TRITC(572), Magnesium Orange ^TM (575), Phycoerythrin R&B(575), Rhodamine Phalloidin(575), Calcium Orange ^TM (576), PyroninY(580), Rhodamine B(580), TAMRA( 582), Rhodamine Red ^TM (590), Cy3.5 ^TM (596), ROX (608), Calcium Crimson ^TM (615), Alexa ^TM 594 (615), Texas Red (615), Nile Red (628), YO -PRO ^TM -3(631), YOYO ^TM -3(631), R-phycocyanin(642), C-Phycocyanin(648), TO-PRO ^TM -3(660), T0T03(660), DiD DilC(5 )(665), Cy5 ^TM (670), Thiadicarbocyanine(671), Cy5.5(694), HEX(556), TET(536), Biosearch Blue(447), CAL Fluor Gold 540(544), CAL Fluor Orange 560(559), CAL Fluor Red 590(591), CAL Fluor Red 610(610), CAL Fluor Red 635(637), FAM(520), Fluorescein(520), Fluorescein-C3(520), Pulsar 650(566 ), Quasar 570(667), Quasar 670(705), and Quasar 705(610). The numbers in parentheses indicate the maximum emission wavelength in nm.

Furthermore, various suitable pairings of quantitative reporter and quencher groups are known in the art, see eg Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Spectroscopy Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color AND Constitution of Oiganic Molecules (Academic Press, New York, 1971); York, 1976); Bishop, editor, Indicators (Peigamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949) ); Haugland, R.P., Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996); U.S. Patents 3,996,345 and 4,351,760.

In certain embodiments, the quantitative reporter group is a fluorophore. In such embodiments, the signal emitted by the quantitative reporter group is fluorescence, and 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). In certain embodiments, 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. In certain embodiments, 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.

In certain embodiments, the detection probe described in (i) can be degraded by an enzyme (eg, DNA polymerase).

In the method of the present invention, the detection probe described in (i) may be linear, or may have a hairpin structure. In certain embodiments, the detection probe is linear. In certain embodiments, the detection probe has a hairpin structure. Hairpin structures can be natural or artificially introduced. In addition, detection probes with hairpin structures can be constructed using routine methods in the art. For example, 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. In such embodiments, 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.

In certain embodiments, 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. In certain embodiments, the detection probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides). In certain embodiments, the detection probes comprise modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine . In certain preferred embodiments, the detection probe comprises non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitro Pyrrole, 5-nitroindole or locked nucleic acid (LNA).

In the method of the present invention, the detection probe described in (i) is not limited by its length. For example, the length of the detection probe can be 10-100 nt, such as 15-50 nt, 20-30 nt.

In certain embodiments, the detection probe described in (i) has a 3'-OH terminus. In certain embodiments, the 3'-end of the detection probe is blocked to inhibit its extension. The 3'-terminus of nucleic acids (eg, detection probes) can be blocked by various methods. For example, the 3'-terminus of the detection probe can be blocked by modifying the 3'-OH of the last nucleotide of the detection probe. In certain embodiments, 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. In certain embodiments, 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.

In certain embodiments, 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.

Mediator Probes and Universal Probes

In the methods of the present invention, qualitative detection of various target nucleic acid sequences is achieved by melting curve analysis based on a mediator sub-system that uses a mediator probe to detect the target nucleic acid and a universal probe to generate a detectable signal . Detailed teachings about the mediation subsystem can be found in, for example, Chinese patent application CN201710299957.4.

In the method of the present invention, the mediator probe refers to a probe used in the mediator system to detect target nucleic acid and unlabeled reporter molecule.

In certain embodiments, the mediator probe can comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain embodiments, the mediator probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides). In certain embodiments, the mediator probes comprise modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine. In certain embodiments, 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).

In the method of the present invention, the mediator probe is not limited by its length. For example, 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. For example, 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. For example, 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. In certain embodiments, 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) , and 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).

In certain embodiments, the mediator probe has a 3'-OH terminus. In certain embodiments, the 3'-terminus of the mediator probe is blocked to inhibit its extension. The 3'-terminus of nucleic acids (eg, mediator probes) 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.

In the method of the present invention, a universal probe refers to a probe labeled with a reporter molecule for generating a detectable signal in a mediator subsystem.

In certain embodiments, 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. In certain embodiments, the universal probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides). In certain embodiments, universal probes comprise modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine. In certain embodiments, the universal probe comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-Nitroindole or locked nucleic acid (LNA).

In the method of the present invention, the universal probe is not limited by its length. For example, 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. For example, 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. For example, 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. In certain embodiments, 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).

In certain embodiments, the universal probe has a 3'-OH terminus. In certain embodiments, the 3'-end of the universal probe is blocked to inhibit its extension. The 3'-terminus of nucleic acids (eg, universal probes) can be blocked by various methods. For example, the 3'-terminus of the universal probe can be blocked by modifying the 3'-OH of the last nucleotide of the universal probe. In certain embodiments, 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. In certain embodiments, 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.

In the methods of the present invention, the mediator fragment hybridizes to the universal probe, and thereby initiates an extension reaction by a nucleic acid polymerase. While 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.

As described above, 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 signal emitted by the group; and the signal emitted by the universal probe when hybridized to its complementary sequence is different from the signal emitted when not hybridized to its complementary sequence.

In certain embodiments, the universal probe is a self-quenching probe. In such embodiments, 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.

The design of such self-quenching universal probes is within the purview of those skilled in the art. For example, 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. Thus, when the universal probe exists alone, 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.

It should be understood, however, that 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. For example, 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). Thus, when the universal probe exists alone, 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. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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.

Thus, 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. However, in certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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). In certain embodiments, 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.

In the method of the present invention, 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),Alexa ^TM 546(570), TRITC(572), Magnesium Orange ^TM (575), Phycoerythrin R&B(575), Rhodamine Phalloidin(575), Calcium Orange ^TM (576), PyroninY(580), Rhodamine B(580), TAMRA( 582), Rhodamine Red ^TM (590), Cy3.5 ^TM (596), ROX (608), Calcium Crimson ^TM (615), Alexa ^TM 594 (615), Texas Red (615), Nile Red (628), YO -PRO ^TM -3(631), YOYO ^TM -3(631), R-phycocyanin(642), C-Phycocyanin(648), TO-PRO ^TM -3(660), T0T03(660), DiD DilC(5 )(665), Cy5 ^TM (670), Thiadicarbocyanine(671), Cy5.5(694), HEX(556), TET(536), Biosearch Blue(447), CAL Fluor Gold 540(544), CAL Fluor Orange 560(559), CAL Fluor Red 590(591), CAL Fluor Red 610(610), CAL Fluor Red 635(637), FAM(520), Fluorescein(520), Fluorescein-C3(520), Pulsar 650(566 ), Quasar 570(667), Quasar 670(705), and Quasar 705(610). The numbers in parentheses indicate the maximum emission wavelength in nm.

In addition, various suitable pairings of qualitative reporter and quencher groups are known in the art, see eg Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Spectroscopy Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color AND Constitution of Oiganic Molecules (Academic Press, New York, 1971); York, 1976); Bishop, editor, Indicators (Peigamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949) ); Haugland, R.P., Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996); U.S. Patents 3,996,345 and 4,351,760.

In certain embodiments, the qualitative reporter group is a fluorescent group, that is, the universal probe is selected from fluorescent probes. In such embodiments, the signal emitted by the qualitative reporter group is fluorescence, and 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). In certain embodiments, 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. In certain embodiments, 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.

In the method of the present invention, 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.

In the methods of the present invention, universal probes (eg, fluorescent probes) may be linear, or may have a hairpin structure. In certain embodiments, the universal probe is linear. In certain embodiments, the universal probe has a hairpin structure. Hairpin structures can be natural or artificially introduced. In addition, detection probes with hairpin structures can be constructed using routine methods in the art. For example, 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. In such embodiments, 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.

In the method of the present invention, 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. In certain embodiments, 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.

In certain embodiments of the invention, in (iii) of step (a), 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.

upstream primer

In the methods of the invention, 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. In certain embodiments, the upstream primer comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides). In certain embodiments, the upstream primer comprises modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine. In certain embodiments, the upstream primer comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5 - Nitroindole or locked nucleic acid (LNA).

In the method of the present invention, the upstream primer is not limited by its length as long as it can specifically hybridize to the target nucleic acid sequence. For example, 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.

In a mediator system, it is desirable to induce cleavage of a mediator probe that hybridizes to the target nucleic acid sequence. In general, 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. Thus, in certain embodiments, the upstream primer described in (ii) is positioned upstream of the mediator probe after hybridization to the target nucleic acid sequence. In such embodiments, the upstream primer directly induces enzymatic cleavage of the mediator probe with 5' nuclease activity without the need for an extension reaction. As used herein, the term "adjacent" is intended to mean that two nucleic acid sequences are adjacent to each other, forming a gap. In certain embodiments, two adjacent nucleic acid sequences (eg, an upstream primer and a mediator probe) 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. In certain embodiments, 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. In such embodiments, the upstream primer directly induces enzymatic cleavage of the mediator probe with 5' nuclease activity without the need for an extension reaction. In certain embodiments, the partially overlapping sequences are 1-10 nt in length, eg, 1-5 nt, or 1-3 nt in length. In certain embodiments, 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. In such embodiments, 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. As used herein, the term "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.

Various methods of utilizing upstream oligonucleotides to induce cleavage of downstream oligonucleotides are known to those skilled in the art and can be used in the present invention. Detailed descriptions of such methods can be found in, eg, US Patents 5,210,015, 5,487,972, 5,691,142, 5,994,069 and 7,381,532, and US Application US 2008/0241838.

downstream primer

In the method of the present invention, in 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. In such embodiments, the nucleic acid polymerase will use the upstream and downstream primers as primers to amplify the target nucleic acid sequence. Also, during the amplification of the target nucleic acid, 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.

In the method of the present invention, in step (a) (ii), in addition to the upstream primer and the mediator probe, a downstream primer is also provided for each target nucleic acid sequence that needs to be qualitatively detected; Wherein, 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. In such embodiments, the nucleic acid polymerase will use the upstream and downstream primers as primers to amplify the target nucleic acid sequence. Also, during the amplification of the target nucleic acid, 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.

In certain embodiments, 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. In certain embodiments, the downstream primer comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides). In certain embodiments, the downstream primer comprises modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine. In certain embodiments, the downstream primer comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5 - Nitroindole or locked nucleic acid (LNA).

In the method of the present invention, the downstream primer is not limited by its length as long as it can specifically hybridize to the target nucleic acid sequence. For example, 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.

Universal primer

In certain embodiments of the invention, in 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.

In certain embodiments, the universal primer comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof. In certain embodiments, the universal primer is 8-50nt in length, eg, 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.

step (b)

In the method of the present invention, 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.

In certain embodiments, 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. In certain embodiments, the nucleic acid polymerase has, eg, 5' exonuclease activity. In certain embodiments, the nucleic acid polymerase is a DNA polymerase. In certain embodiments, 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, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifex aeoolieus. Particularly preferably, the DNA polymerase with 5' nuclease activity is Taq polymerase.

In certain embodiments, 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)).

In certain embodiments, in step (b), symmetric PCR amplification is performed. In certain embodiments, 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. In certain embodiments, 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.

In certain embodiments, in step (b), asymmetric PCR amplification is performed.

In certain embodiments, the upstream primer described in (i) (ie, the primer on the DNA strand in the same direction as the probe) 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). In certain exemplary embodiments, the upstream primer described in (i) (ie, the primer on the DNA strand in the same direction as the probe) 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). In certain embodiments, 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).

In certain embodiments, the upstream primer described in (ii) (ie, the primer on the DNA strand in the same direction as the probe) 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). In certain exemplary embodiments, the upstream primer described in (ii) (ie, the primer on the DNA strand in the same direction as the probe) 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). In certain embodiments, 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).

step (c)

In the method of the present invention, quantitative detection of the target nucleic acid sequence is achieved by monitoring the signal released by the detection probe described in (i) in the PCR cycle.

In certain embodiments, 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.

In certain embodiments, the specific temperature described in step (c) is a denaturation temperature. In certain embodiments, the denaturation temperature is 94-98°C.

In certain embodiments, in step (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.

In certain embodiments, in 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).

In the method of the present invention, 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 .

step (d)

In the method of the present invention, the qualitative detection of the target nucleic acid sequence is realized by melting curve analysis based on the mediator subsystem.

In certain embodiments, the one or more universal probes may comprise the same qualitative reporter group. In this case, in step (d), 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.

In certain embodiments, the one or more universal probes comprise qualitative reporter groups that are different from each other. In this case, in step (d), 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.

In certain embodiments, in step (d), 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. In certain embodiments, the obtained curve is derived to obtain a melting curve. In certain embodiments, 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.

Beneficial Effects of Invention

Although methods for multiplex nucleic acid detection have been reported in the prior art, in many aspects of nucleic acid detection, only qualitative analysis or only quantitative analysis cannot meet the needs. 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.

The embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention, rather than limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Description of drawings

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.

FIG. 5 shows the results of the sensitivity investigation of the qualitative analysis of viruses and atypical pathogens in Example 5. FIG.

FIG. 6 shows the results of the sensitivity examination of the quantitative analysis of bacteria in Example 5. FIG.

Detailed ways

The present invention will now be described with reference to the following examples, which are intended to illustrate, but not limit, the invention.

Unless otherwise specified, 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. Those skilled in the art appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

The experimental methods of nucleic acid detection involved in the following examples are as follows:

1. Instruments and reagents

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.

2. Design and synthesis of primer probes

Primer design of this system adopts Primer Premier 5.0. The Tm values of primers were predicted online using IDT Biophysics. In order to reduce the non-specificity of the system, 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. Probe design: for qualitative detection: universal probes and media probes for media systems; for quantitative detection: fluorescent probes (such as linear fluorescent probes or hairpin-type fluorescent probes) for real-time detection of objects.

3. Establishment of single-plex control system

At present, 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 μL. The reaction system includes 1×SSP buffer, 5.0mM MgCl ₂ , 0.2mM dNTPs, 40μM dUTP, 0.4μM upstream and downstream primers, 0.2μM TaqMan fluorescent probe, 1.0U TaqHS DNA polymerase (TaKaRa, Beijing), 5μL template, The reaction system was made up to 25 μL with sterile water. 5 μL of 10000copies/μL, 1000copies/μL, 100copies/μL, 10copies/μL, and 1copies/μL plasmid standards were added to each experiment to make a standard curve for quantification of samples, and at the same time, it was used as a positive quality control to indicate whether the PCR reaction solution was qualified. Three no-template controls were set in each experiment, and 5 μL of TE was added to the no-template controls to indicate whether there was contamination during the experiment. 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.

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

Note: The correspondence between universal probes and media probes is: 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.

Table 2: Final Concentrations of Primers/Probes

检测对象Detection object	FF	RR	MP/PMP/P
流感嗜血杆菌Haemophilus influenzae	8080	8080	800800
金黄色葡萄球菌 Staphylococcus aureus	6060	6060	6060
肺炎链球菌 Streptococcus pneumoniae	2020	2020	100100
铜绿假单胞菌 Pseudomonas aeruginosa	8080	8080	800800
外控SUC2 External control SUC2	4040	4040	400400
肺炎支原体 Mycoplasma pneumoniae	4040	4040	400400
甲流A stream	4040	4040	400400
乙流 B flow	8080	8080	800800
甲型呼吸道合胞病毒respiratory syncytial virus type A	4040	4040	400400
乙型呼吸道合胞病毒Respiratory syncytial virus type B	4040	4040	400400
鼻病毒 Rhinovirus	4040	4040	400400
腺病毒 Adenovirus	6060	6060	600600
内控基因RPP30Internal control gene RPP30	4040	4040	400400

Table 3: Control system primers and probes

Example 1: Investigation on the Lighting Temperature of the Media Probe System

Generally, real-time quantitative PCR requires light harvesting at the annealing temperature to achieve real-time monitoring of fluorescent signals. However, when using media probes and linear fluorescent probes or hairpin fluorescent probes in the same system, the high-temperature media probes will generate amplification curves at the annealing temperature, which will affect the accuracy of quantitative detection, as shown in Figure 1. As shown, taking human adenovirus (hAdV) as an example, when using the vehicle system and lighting at the annealing temperature, not only the peak of interest but also the amplification curve is generated. In order to solve the above problems, the present inventors investigated the light harvesting of the probe from the annealing temperature to the denaturing temperature. A 25-μL PCR reaction system was used, including: 7.0 mM MgCl ₂ , 0.2 mM dNTPs, 40 μM dUTP, 3.0 U Taq01 (Zhishan Biotechnology Co., Ltd., Xiamen), 0.01 U UNG enzyme, 1 universal primer 0.8 μM, 1 Universal probe 0.04 μM, 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.

The experimental results are shown in Figure 1. The plasmid standard of the virus is added, and light is collected at the annealing temperature in the amplification stage, and the qualitative probe will generate an amplification curve. At denaturing temperatures, the qualitative probe does not generate an amplification curve.

Example 2: Investigation on the lighting temperature of the media probe system and the combined detection system of the fluorescent quantitative probe

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. Taking the detection of Haemophilus influenzae as an example, a 25-μL PCR reaction system was used, including: 7.0 mM MgCl ₂ , 0.2 mM dNTPs, 40 μM dUTP, 3.0 U Taq01 (Zhishan Biotechnology Co., Ltd., Xiamen), 0.01 U UNG enzyme, 1 universal primer 0.8 μM, 1 universal probe 0.04 μM, 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.

The experimental results are shown in Figure 2. The bacterial plasmid standard is added, and the sample wells will generate amplification curves at the annealing temperature and the denaturing temperature in the amplification stage, and the existence of the universal probe will not affect the linear fluorescent probe. Fluorescence acquisition with needle or hairpin-type fluorescent probes.

Embodiment 3: The influence of the amount of upstream and downstream primers on the melting peak produced by the quantitative detection object

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. Forward asymmetry: 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-μL PCR reaction system was used, including: 7.0 mM MgCl ₂ , 0.2 mM dNTPs, 40 μM dUTP, 3.0 U Taq01 (Zhishan Biotechnology Co., Ltd., Xiamen), 0.01 U UNG enzyme, 1 universal primer 0.8 μM, 1 Universal probe 0.04 μM, 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.

The experimental results are shown in Figure 3. The three amplification methods of symmetric amplification, forward asymmetric amplification and reverse asymmetric amplification were investigated, and bacterial plasmid standards were added. The results showed that symmetric amplification and reverse asymmetric amplification were used. Both the methods of increasing can realize quantitative detection of the object, only generate an amplification curve without generating a melting peak.

Example 4: Qualitative and quantitative detection by combined use of media probe and fluorescent quantitative probe

Through the investigation in Examples 1-3, we selected 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. Among them, 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-μL PCR reaction system includes 7.0 mM MgCl ₂ , 0.2 mM dNTPs, 40 μM dUTP, 3.0 U Taq01 (Zhishan Biotechnology Co., Ltd., Xiamen), 0.01 U UNG enzyme, 1 universal primer 0.8 μM, and 5 universal probes 0.04 μM 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. To examine the sensitivity of the system, the plasmid standards were diluted with TE to 1000 copies/μL, 100 copies/μL, and 10 copies/μL. To exclude experimental errors, the experiments were repeated three times with 3 parallel wells of 10 copies/μL per experiment. Two parallel wells were repeated at 1000copies/μL and 100copies/μL. 5 μL 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.

The experimental results are shown in Figure 5 and Figure 6. Whether it is semi-quantitatively detected bacteria or qualitatively detected viruses and atypical pathogens, 10 copies/μL of template can be stably raised, so the sensitivity of the system can reach 50 copies/reaction.

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.

In order to examine the stability of the Tm value of each subject, three positive wells were made for each subject in each experiment, and the concentrations were 1000 copies/μL, 100 copies/μL, and 10 copies/μL, respectively. The experiments were repeated three times on different instruments. After the experiment, the 9 Tm values of each subject were counted, and the mean (Mean), standard deviation (SD) and coefficient of variation (CV) were calculated. The smaller the standard deviation and the coefficient of variation, the more stable the Tm value of the subject.

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.

Table 4: Stability investigation of viruses and atypical pathogens

In order to investigate the stability of the Ct value of each object, each experiment was repeated three times in parallel wells at 1000 copies/μL, 100 copies/μL and 10 copies/μL, 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.

Table 5: Investigation on the stability of Ct value of bacteria and external control genes

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

Taking the 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.

In order to examine the ability of the system to detect bacterial mixed nucleic acid samples, the bacteria covered by the system were mixed at equal concentrations to obtain 1000copies/μL, 100copies/μL, and 10copies/μL 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.

Table 6: Investigation of bacterial mixed nucleic acid samples

Note: ΔCt is equal to the Ct value of mixed infection minus the Ct value of one bacterial infection.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings that have been published, and that these changes are all within the scope of the present invention . The full division of the invention is given by the appended claims and any equivalents thereof.

Claims

A method for detecting a target nucleic acid in a sample, comprising the steps of:

(a) The following reaction components are provided:

(i) for each of the target nucleic acid sequences to be quantitatively determined, provide an upstream primer and a detection probe; wherein the upstream primer comprises a sequence complementary to the target nucleic acid sequence; the detection probe The needle 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 first target nucleic acid sequence, the upstream primer is positioned on the detection probe. upstream; and, the quantitative reporter groups labeled with all detection probes are different from each other;

(ii) for each of the target nucleic acid sequences to be qualitatively determined, provide an upstream primer and a mediator probe; wherein, 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;

(iii) providing one or more universal probes for the mediator probe described in (ii), wherein each universal probe independently comprises from 3' to 5' direction: with one or more one or more capture sequences complementary to the seed mediator subsequence or a portion thereof, and a template sequence; and the one or more universal probes comprise capture sequences capable of being The mediator sequences of the mediator probes or parts thereof are complementary; and each universal probe is independently labeled with a qualitative reporter group and a quencher group;

(b) contacting a sample containing the target nucleic acid sequence to be detected with the components described in (a) in a single reaction vessel, and implementing PCR reaction conditions;

(c) measuring the signal from the quantitative reporter group described in (a)(i) at a specific temperature in the PCR cycle, the specific temperature being above the annealing temperature and extension temperature; based on the quantitative reporter determined The signal of the group determines the presence or amount of the corresponding target nucleic acid sequence;

(d) After PCR is completed, 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 method of claim 1, wherein the specified temperature in step (c) is at least 10°C, at least 15°C, or at least 20°C above the annealing temperature and the extension temperature;

Preferably, the specific temperature described in step (c) is a denaturation temperature;

Preferably, the denaturation temperature is 94-98°C.
The method of claim 1 or 2, wherein the PCR reaction conditions described in step (b) comprise using a nucleic acid polymerase with 5' nuclease activity;

Preferably, the nucleic acid polymerase has 5' exonuclease activity;

Preferably, the nucleic acid polymerase is a DNA polymerase;

Preferably, the nucleic acid polymerase is a thermostable DNA polymerase.
The method described in any one of claims 1-3, wherein, in step (a), the quantitative reporter group in the detection probe described in (i) is a fluorescent group (for example ALEX-350, FAM, VIC, TET,
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), the quenching Groups are molecules or groups capable of absorbing/quenching the fluorescence (eg DABCYL, BHQ (eg BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA);

Preferably, the detection probe described in (i) is a self-quenching probe;

Preferably, the detection probe described in (i) is labeled with a quantitative reporter group at its 5' end or upstream and labeled with a quencher group at its 3' end or downstream, or labeled at its 3' end or downstream a quantitative reporter group and a quencher group labeled at the 5' end or upstream; preferably, the quantitative reporter group and the quencher group are separated by a distance of 10-80 nt or more;

Preferably, the detection probe described in (i) is linear, or has a hairpin structure;

Preferably, the detection probes described in (i) can be degraded by enzymes such as DNA polymerases.
The method described in any one of claims 1-4, wherein, in step (a), the qualitative reporter group in the universal probe described in (iii) is a fluorescent group (for example ALEX-350, FAM, VIC, TET,
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, the 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);

Preferably, the universal probe described in (iii) is a self-quenching probe;

Preferably, the universal probe described in (iii) is labeled with a qualitative reporter group at its 5' end or upstream and a quencher group at its 3' end or downstream, or labeled at its 3' end or downstream A qualitative reporter group and a quencher group labeled at the 5' end or upstream; preferably, the qualitative reporter group and the quencher group are separated by a distance of 10-80 nt or more;

Preferably, the universal probes described in (iii) are linear or have a hairpin structure.
The method of any one of claims 1-5, wherein the one or more universal probes comprise the same qualitative reporter group; and, in step (d), the melting curve analysis comprises: according to The melting peak (melting point) in the obtained melting curve is used to determine the presence of a certain target nucleic acid sequence.
The method of any one of claims 1-5, wherein the qualitative reporter groups contained in the one or more universal probes are different from each other; and, in step (d), the melting curve analysis includes : Monitor the signal of each qualitative reporter group in real time, thereby obtaining multiple melting curves corresponding to the signal of one qualitative reporter group; then, according to the signal type of the qualitative reporter group and the melting curve in the melting curve Peak (melting point) to determine the presence of a certain target nucleic acid sequence.
7. The method of any one of claims 1-7, wherein the number of universal probes is less than that of mediator probes.
The method of any one of claims 1-7, wherein, in (iii) of step (a), 1 universal probe is provided for the mediator probe described in (ii), the universal probe Comprising from 3' to 5' direction, 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 method according to any one of claims 1-9, wherein, in step (c), the signal emitted by the quantitative reporter group on each detection probe described in (i) is monitored in real time, thereby obtaining each signal. A curve of the signal intensity of a quantitative reporter group as a function of cycle number (ie, amplification curve).
The method according to any one of claims 1-10, wherein, in step (d), the melting curve analysis comprises: gradually increasing or decreasing the temperature of the PCR product and monitoring each of the PCR products in real time The signal emitted by the qualitative reporter group on the universal probe can be obtained, so as to obtain the curve of the signal intensity of each qualitative reporter group as a function of temperature;

Preferably, the obtained curve is derived to obtain a melting curve;

Preferably, according to the melting peak (melting point) in the melting curve, the existence of the mediator sub-fragment corresponding to the melting peak (melting point) is determined; then, through the correspondence between the mediator subsequence in the mediator sub-fragment and the target nucleic acid sequence, The presence of a target nucleic acid sequence corresponding to the mediator fragment is determined.
The method of any one of claims 1-11, wherein the detection probe described in (i) has one or more characteristics selected from the group consisting of:

(1) the detection probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof;

(2) the length of the detection probe is 10-100nt, such as 15-50nt, 20-30nt; and

(3) The detection probe has a 3'-OH end, or its 3'-end is blocked.
The method of any one of claims 1-12, wherein the mediator probe described in (ii) has one or more characteristics selected from the group consisting of:

(1) the mediator probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof;

(2) The length of the mediator probe is 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;

(3) The length of the target-specific sequence in the mediator probe is 10-140nt, such as 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;

(4) 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; and

(5) The mediator probe has a 3'-OH end, or its 3'-end is blocked.
The method of any one of claims 1-13, wherein the universal probe described in (iii) has one or more characteristics selected from the group consisting of:

(1) the universal probe comprises a plurality of capture sequences; and, the plurality of capture sequences are arranged in an adjacent manner, in a manner of being spaced by a linker sequence, or in an overlapping manner;

(2) the universal probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof;

(3) The length of the universal probe is 15-1000nt, 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;

(4) The length of the capture sequence in the universal probe is 10-500nt, 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;

(5) The length of the template sequence in the universal probe is 1-900nt, such as 1-5nt, 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-nt 70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt;

(6) The universal probe has a 3'-OH terminal, or its 3'-terminal is blocked.
The method of any one of claims 1-14, wherein the upstream primer described in (i) or (ii) has one or more features selected from the group consisting of:

(1) the upstream primer comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof;

(2) The length of the upstream primer is 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.
The method of any one of claims 1-15, wherein,

In (i) of step (a), in addition to the upstream primer and 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 and 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 detection probe; and/or,

In (ii) of step (a), in addition to the upstream primer and the mediator probe, a downstream primer is also provided for each target nucleic acid sequence that needs to be qualitatively detected; wherein, 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 positioned downstream of the target-specific sequence.
The method of claim 16, wherein the downstream primer has one or more characteristics selected from the group consisting of:

(1) the downstream primer comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; and

(2) The length of the downstream primer is 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.
The method of any one of claims 1-17, wherein, in step (b), symmetric or asymmetric PCR amplification is performed;

Preferably, the upstream and downstream primers described in (i) are in equal amounts, or the upstream primers described in (i) are in excess relative to the downstream primers.
The method of any one of claims 16-18, wherein, in step (a), all upstream primers and downstream primers described in (i) and (ii) have an identical stretch of oligonucleotides at the 5' end acid sequence; 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, the universal primer has the same a sequence complementary to the same oligonucleotide sequence;

Preferably, the universal primer has one or more characteristics selected from the group consisting of:

(1) the universal primer comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; and

(2) The length of the universal primer is 8-50nt, such as 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.
The method of any one of claims 1-19, wherein, in step (c), pre-amplification is performed prior to measuring the signal of the quantitative reporter group.
20. The method of any one of claims 1-20, wherein the sample comprises or is a mixture of DNA, or RNA, or nucleic acids.
The method of any one of claims 1-21, wherein the target nucleic acid sequence is DNA or RNA; and/or the target nucleic acid sequence is single-stranded or double-stranded.