Method for detecting existence of X target nucleic acid sequences in sample, probe tag group and application thereof, kit and application thereof

Abstract

The invention relates to the technical field of nucleic acid molecule detection, in particular to a method for detecting existence of X target nucleic acid sequences in a sample, a probe tag group and application thereof, and a kit and application thereof. The method comprises the following preparation steps: aiming at each target nucleic acid sequence to be detected, providing an upstream oligonucleotide sequence, a downstream oligonucleotide sequence, an I nDex tag sequence, a tag primer oligonucleotide sequence and a probe which are specifically combined with the target nucleic acid sequence; carrying out PCR (Polymerase Chain Reaction) amplification on a sample through the provided oligonucleotide sequence to obtain a PCR amplification product; providing Y detection probes, and contacting the PCR amplification product with one of the detection probes to obtain a hybrid; wherein Y is an integer greater than 0; carrying out melting curve analysis on the hybrids; and determining whether the X target nucleic acid sequences exist in the sample or not according to a melting curve analysis result. The invention provides a simple, efficient and low-cost multiple detection method which can simply, efficiently and simultaneously detect the existence of multiple target nucleic acid sequences in a sample.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid molecule detection, and in particular to a method for detecting the presence of X target nucleic acid sequences in a sample, a probe label set and its use, a kit and its use. Background Art

[0002] Real-time PCR is a common method for detecting nucleic acids. It is easy to operate and widely used. In addition, by using multiplex real-time PCR, multiple target sequences can be detected simultaneously in a single reaction tube, which not only improves detection efficiency but also reduces costs.

[0003] In the real-time PCR method, the target sequence can be detected by using a fluorescently labeled oligonucleotide probe. Generally speaking, the fluorescently labeled probe specifically binds to the target sequence between the two primers used for PCR amplification to avoid interference signals caused by the non-specific amplification of primer dimers, thereby improving the specificity of the detection result. For multiple real-time PCR, when using oligonucleotide probes specific to a variety of target sequences, different fluorescent groups can be used to label each oligonucleotide probe, thus, by detecting the unique fluorescent signal carried by each probe, the target sequence specifically identified by each probe can be detected. In the real-time PCR method, the target sequence can be detected by two modes, that is, the real-time detection mode and the melting curve analysis (also referred to as the post-PCR MCA mode) after amplification. In the real-time detection mode, the detection of the target sequence and the PCR amplification are carried out simultaneously, without the need to carry out additional steps. Therefore, the real-time detection mode is simple and direct. However, the number of the maximum target sequences that this mode can detect in a single round of detection is limited to the number of fluorescence detection channels of the real-time PCR instrument, which is generally no more than 6. In the MCA mode, an additional step is required after PCR amplification, that is, analyzing the melting point of the duplex consisting of the probe and the target sequence. The MCA mode can identify or distinguish the target sequence by fluorescent color and / or melting point.

[0004] Therefore, although the MCA mode is relatively cumbersome (i.e., an extra step is added), the maximum number of target sequences that can be detected in a single round of detection is increased. However, there are also some problems with multiple real-time PCR based on fluorescent probe detection: first, the preparation of fluorescent probes involves complex chemical modification and purification processes, and its cost is much higher than that of unlabeled probes. Therefore, the use of multiple fluorescent probes will lead to an increase in detection costs. Secondly, in the multiple real-time PCR method, the coexistence of multiple fluorescent probes will increase the background fluorescence of the reaction system, which in turn can lead to a decrease in detection sensitivity. Therefore, it is necessary to improve the multiple real-time PCR method in order to detect as many target sequences as possible with as few fluorescent probes as possible.

[0005] The patent application with publication number US 20130109588 A1 discloses a real-time PCR assay that can be used for melting curve analysis, which is similar to the method of Faltin et al., and detects the target sequence by two probes (PTO probe, which corresponds to the label primer; and CTO probe, which corresponds to the fluorescent probe). Accordingly, the method of US 2013 / 0109588 A1 has similar advantages and disadvantages to the method of Faltin et al. In particular, when the method described in the patent application is used to implement multiple real-time PCR that needs to distinguish each target sequence, a PTO probe and a CTO probe need to be designed for each target sequence; that is, a double number of probes are used. For example, the patent application describes a dual real-time PCR for the simultaneous detection of Neisseria gonorrhoeae and Staphylococcus aureus, in which two PTO probes and two CTO probes are used. In this case, compared with the traditional multiple real-time PCR using a single probe for each target sequence, the method of US 2013 / 0109588 A1 is more complicated and costly. The patent application with publication number US 20140057264 A1 discloses another real-time PCR method using two probes. In this method, the fluorescent signal is generated by the cutting of the labeled probe, so the method can only be used in the real-time detection mode, but not in the MCA mode. In addition, similar to the method of Faltin et al., when the method described in US2014 / 0057264 A1 is used to implement multiple real-time PCR that needs to distinguish each target sequence, two probes need to be designed for each target sequence, which makes the reaction system more complicated and expensive. The patent application with publication number US20150072887 A1 discloses a real-time PCR assay that can be used for melting curve analysis, which detects the target sequence by 3 probes. However, when the method described in the patent application is used to implement multiple real-time PCR that needs to distinguish each target sequence, 3 probes need to be designed for each target sequence, which makes the reaction system more complicated and expensive. Similar real-time PCR assays using three probes are also disclosed in US 20150167060 A1 and US 20160060690 A1. However, these methods, like the method disclosed in US 20150072887 A1, are more complicated and costly than conventional real-time PCR methods when used to perform multiplex real-time PCR that requires distinguishing each target sequence.

[0006] In summary, compared with the traditional real-time PCR method, the improved real-time PCR method using two or three probes (such as the method of Faltin et al.) has significant advantages when performing single real-time PCR or multiple real-time PCR without identifying or distinguishing each target sequence: that is, multiple label primers carrying the same label sequence but different target-specific sequences and a universal fluorescent probe can be used, thereby significantly reducing the detection cost. However, when performing multiple real-time PCR that requires distinguishing each target sequence, these improved real-time PCR methods require the use of double or even triple the number of probes, which is more complicated and more expensive than the traditional real-time PCR method.

[0007] Therefore, it is necessary to develop a new real-time PCR assay method so that multiplex real-time PCR that needs to distinguish each target sequence can be performed with a simpler reaction system and lower detection cost. Summary of the invention

[0008] In order to solve the deficiencies of the prior art mentioned in the above background technology, the present invention provides a method for detecting a target nucleic acid sequence (i.e., a method for detecting the presence of X target nucleic acid sequences in a sample), which can simultaneously detect the presence of multiple target nucleic acid sequences in a sample. In addition, the present application also provides a probe label set, and a kit comprising one or more of the probe label sets, which can be used to implement the method of the present invention.

[0009] The method for detecting the presence of X target nucleic acid sequences in a sample (where X≥1, for example, X is a natural number such as 2, 3, 4, 5, 6, 7, 8, ...), the technical scheme of which is as follows:

[0010] The method comprises the following preparation steps:

[0011] For each target nucleic acid sequence to be detected, an upstream oligonucleotide sequence, a downstream oligonucleotide sequence (Reverse primer, R), an InDex tag sequence (InDex, ID), a tag primer oligonucleotide sequence (InDex Forward primer, IDF) and a probe (Probe) that specifically bind to the target nucleic acid sequence are provided;

[0012] Wherein, the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; the tag primer comprises a probe tag sequence and a target-specific sequence from the 5' to 3' direction, the probe tag sequence comprises a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence comprises a sequence complementary to the target nucleic acid sequence; when the tag primer oligonucleotide sequence hybridizes with the target nucleic acid sequence, the tag sequence is located upstream of the target-specific sequence; and the sequences contained in all InDex tag sequences are different from each other;

[0013] The sample is subjected to PCR amplification through the provided oligonucleotide sequence to obtain a PCR amplification product; wherein the oligonucleotide sequence refers to an upstream oligonucleotide sequence, a downstream oligonucleotide sequence, an InDex tag sequence, a tag primer oligonucleotide sequence and an oligonucleotide sequence of a probe;

[0014] Providing Y detection probes, contacting the PCR amplification product with one of the detection probes to obtain a hybrid; wherein Y is an integer greater than 0;

[0015] Wherein, each PCR amplification product independently comprises a label primer oligonucleotide sequence and a template sequence from the 5' to 3' direction; the detection probe comprises at least N InDex label sequences, and N is greater than or equal to Y, and the detection probes are respectively consistent with the sequence of the provided probe or a part of its sequence; each detection probe is independently labeled with a reporter group and a blocking group, wherein the reporter group can emit a signal, and the blocking group can hinder PCR amplification; when the detection probe hybridizes with the complementary sequence of each label primer oligonucleotide sequence, the signals emitted are different.

[0016] Performing melting curve analysis on the hybrid: determining whether the X target nucleic acid sequences exist in the sample based on the result of the melting curve analysis.

[0017] In one embodiment, when X≥2, each of the Y detection probes is labeled with the same or different reporter groups.

[0018] In one embodiment, at least N InDex tag sequences are provided, and at least Y detection probes are provided.

[0019] In one embodiment, the Y detection probes contain the same or different reporter groups; when performing melting curve analysis on the hybrid, the presence of a certain target nucleic acid sequence is determined based on the melting peak (melting point) in the obtained melting curve;

[0020] In one embodiment, the reporter groups contained in the Y detection probes are different from each other; when performing melting curve analysis on the hybrid, the signal of each reporter group is monitored in real time, and multiple melting curves corresponding to the complementary chain of the label sequence in the corresponding amplification product and the signal of a reporter group are obtained, and then the presence of a certain target nucleic acid sequence is determined based on the signal type of the reporter group and the melting peak (melting point) in the melting curve.

[0021] In one embodiment, the sample is one of DNA and RNA or a mixture of both;

[0022] In one embodiment, the target nucleic acid sequence is DNA or RNA;

[0023] In one embodiment, the target nucleic acid sequence is single-stranded or double-stranded.

[0024] In one embodiment, the sample or target nucleic acid sequence is obtained from a source selected from the group consisting of prokaryotes, eukaryotes, viruses or viroids, and the like.

[0025] In one embodiment, the probe or detection probe or tag primer oligonucleotide sequence has at least one or more of the following characteristics:

[0026] (1) The probe comprises one or more of naturally occurring nucleotides, modified nucleotides, and non-natural nucleotides;

[0027] (2) The length of the probe is 15-150 nt, for example, 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, 140-150 nt;

[0028] (3) The length of the target-specific sequence in the tag primer oligonucleotide sequence is 15-150 nt; for example, 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, 140-150 nt;

[0029] (4) The length of the probe tag sequence in the tag primer oligonucleotide sequence may be 15-150 nt; for example, 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, 140-150 nt;

[0030] (5) Each detection probe of the probe is independently labeled with a reporter group and a blocking group;

[0031] (6) The reporter group in the detection probe is a fluorescent group; (e.g., ALEX-350, FAM, VIC, TET, CALGold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CALFluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar705, etc.) and the blocking group is a molecule or group that enables the probe to be used as an amplification primer; (e.g., 3' phosphorylation modification PO4 or 3' amino modification NH2C7, etc.);

[0032] (7) The detection probe has resistance to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity); for example, the backbone of the detection probe contains a modification that is resistant to nuclease activity, such as a thiophosphate bond, an alkyl phosphotriester bond, an aryl phosphotriester bond, an alkyl phosphoester bond, an aryl phosphoester bond, a hydrogenated phosphate bond, an alkyl phosphoramidate bond, an aryl phosphoramidate bond, a 2'-O-aminopropyl modification, a 2'-O-alkyl modification, a 2'-O-allyl modification, a 2'-O-butyl modification, and a 1-(4'-thio-PD-ribofuranosyl) modification;

[0033] (8) The detection probe is linear or has a hairpin structure.

[0034] In one embodiment, under conditions that allow polymerase chain reaction, the sample is subjected to PCR amplification using the provided oligonucleotide sequence to obtain a PCR amplification product; under conditions that allow nucleic acid hybridization, the PCR amplification product is contacted with one of the detection probes.

[0035] In one embodiment, under conditions that allow the nucleic acid polymerase to perform an extension reaction, the nucleic acid polymerase uses the target nucleic acid sequence in the sample as a template to extend the tag primer hybridized to the template, thereby forming a duplex to obtain a PCR amplification product. Preferably, in the PCR amplification step, the nucleic acid polymerase used is a nucleic acid polymerase with 5' nuclease activity.

[0036] In one embodiment, the step of obtaining the PCR amplification product is performed by a scheme comprising the following steps (a)-(f):

[0037] (a) providing Y detection probes and N tags, and for each target nucleic acid sequence to be detected, providing an upstream oligonucleotide sequence, a probe, a tag primer oligonucleotide sequence and a downstream oligonucleotide sequence;

[0038] (b) mixing the sample to be detected with the detection probe, upstream oligonucleotide sequence, tag primer oligonucleotide sequence, probe, downstream oligonucleotide sequence and template-dependent nucleic acid polymerase having 5' nuclease activity (e.g., DNA polymerase, particularly thermostable DNA polymerase) provided in step (a);

[0039] (c) incubating the mixture of (b) under conditions that allow nucleic acid denaturation;

[0040] (d) incubating the product of step (c) under conditions that allow annealing or hybridization of the nucleic acids;

[0041] (e) incubating the product of step (d) under conditions that allow for extension of the nucleic acid;

[0042] (f) Repeating steps (c)-(e) one or more times.

[0043] In one embodiment, the operation process of the melting curve analysis step is:

[0044] The hybrid is gradually heated or cooled, and the signal emitted by the reporter group on each detection probe is monitored in real time, so as to obtain a curve showing the change of the signal intensity of each reporter group with the change of temperature;

[0045] The obtained curve is differentiated to obtain the melting curve of the hybrid;

[0046] According to the melting peak (melting point) in the melting curve, the presence of the InDex tag corresponding to the melting peak (melting point) is determined; then, the presence of the target nucleic acid sequence corresponding to the tag is determined through the correspondence between the tag sequence in the tag primer and the target nucleic acid sequence.

[0047] The present invention also provides a probe label set (InDexset) for detecting the presence of X target nucleic acid sequences in a sample, where X is ≥1 (for example, X is a natural number such as 2, 3, 4, 5, 6, 7, 8, ...), and the scheme is:

[0048] The probe tag group includes a detection probe; the detection probe corresponds to at least one probe tag; wherein the tag primers of the probe tags each independently include a probe tag sequence and a target-specific sequence from 5' to 3', wherein the target-specific sequence includes a sequence complementary to a target nucleic acid sequence, and the probe tag sequence includes a sequence not complementary to the target nucleic acid sequence; the detection probe includes from 5' to 3': a capture sequence and a template sequence complementary to each tag group subsequence or part thereof; and the detection probe is labeled with a reporter group and a blocking group, wherein the reporter group can emit a signal, and the blocking group or modification can prevent the probe from being extended; wherein the tag group subsequences included in the probe are different from each other. wherein the probe tag group includes at least 2, at least 3, at least 4, at least 5, at least 6, etc. tags.

[0049] In one embodiment, the tag sequences contained in all the tag primers are different from each other; and the target-specific sequences contained in all the tag primers are different from each other. And / or, the primers contained in all the tag primers each target a different target nucleic acid sequence.

[0050] In one embodiment, the tag primer has at least one or more of the following characteristics:

[0051] (1) The length of the target-specific sequence in the tag primer oligonucleotide sequence is 15-150 nt, for example, 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, 140-150 nt;

[0052] (2) The length of the probe tag sequence in the tag primer oligonucleotide sequence can be 15-150 nt, for example, 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, 140-150 nt;

[0053] The detection probe has at least one or more of the following characteristics:

[0054] (1) The reporter group in the detection probe is a fluorescent group (e.g., ALEX-350, FAM, VIC, TET, CALGold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CALFluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar705, etc.); and the blocking group is a molecule or group that enables the probe to be used as an amplification primer (e.g., 3' phosphorylation modification PO4 or 3' amino modification NH2C7, etc.);

[0055] (2) the detection probe has resistance to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity); for example, the backbone of the detection probe comprises a modification that is resistant to nuclease activity, such as a thiophosphate bond, an alkyl phosphotriester bond, an aryl phosphotriester bond, an alkyl phosphoester bond, an aryl phosphoester bond, a hydrogenated phosphate bond, an alkyl phosphoramidate bond, an aryl phosphoramidate bond, a 2'-O-aminopropyl modification, a 2'-O-alkyl modification, a 2'-O-allyl modification, a 2'-O-butyl modification, and a 1-(4'-thio-PD-ribofuranosyl) modification;

[0056] (3) The detection probe is linear or has a hairpin structure.

[0057] The present invention also provides a kit, and its technical solution is as follows:

[0058] The kit comprises one or more probe label sets; wherein the probe label set adopts the probe label set described above.

[0059] In one embodiment, the kit comprises at least one (for example, at least one, at least two, at least three, at least four, at least five, at least six, etc.) probe label set; wherein all label primers of the probe label set each target a different target nucleic acid sequence, the label sequences contained in all label primers are different from each other, the target-specific sequences contained in all label primers are different from each other, and all detection probes are independently labeled with the same or different reporter groups.

[0060] In one embodiment, the kit further comprises an upstream oligonucleotide sequence (e.g., an upstream oligonucleotide sequence as defined in claim 1 or 11), a downstream oligonucleotide sequence (e.g., a downstream oligonucleotide sequence as defined in claim 15 or 17), a universal primer (e.g., a universal primer as defined in claim 18), an enzyme having 5' nuclease activity, and a nucleic acid polymerase, any combination of one or more thereof.

[0061] In one embodiment, the kit further comprises any combination of one or more reagents for nucleic acid hybridization, reagents for nucleic acid extension, and reagents for nucleic acid amplification.

[0062] The present invention also provides the use of the probe tag set or the kit as described above:

[0063] The probe tag set or kit is used to implement the method for detecting the presence of X target nucleic acid sequences in a sample as described above.

[0064] Based on the above, compared with the prior art, the present invention has the following beneficial effects:

[0065] (1) The method of the present invention can achieve simultaneous detection (multiplex detection) of multiple target nucleic acid sequences using only one labeled probe (ie, detection probe) and a corresponding tag set.

[0066] (2) The method of the present invention can realize the simultaneous detection of multiple target nucleic acid sequences (multiple detection), and the maximum number of target nucleic acid sequences that can be detected simultaneously far exceeds the number of labeled probes (i.e., detection probes) used and the corresponding number of labels and targets.

[0067] Therefore, the present invention provides a simple, efficient, low-cost multiplex detection method. The maximum number of target nucleic acid sequences that the method of the present invention can detect is not limited by the number of label probes (i.e., detection probes) used, that is, the method of the present invention can realize simultaneous detection (multiple detection) of significantly more target nucleic acid sequences based on a relatively limited number of label probes (i.e., detection probes). BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 A schematic diagram of the basic principle of an exemplary embodiment of the present invention;

[0069] Figure 2 In an exemplary embodiment of the present invention, a two-step method ( Figure 2 A and 2B) or three-step method ( Figure 2 C and 2D) Melting curve plots of real-time PCR reactions for amplification;

[0070] Figure 3 The first on the left is a melting curve diagram of a real-time PCR reaction using two blocking fluorescent probes Probe1 and Probe2 for 4-plex detection using a three-step method and asymmetric PCR in an exemplary embodiment of the present invention;

[0071] Figure 3The first on the right is a melting curve diagram of a real-time PCR reaction using two blocking fluorescent probes Probe1 and Probe2 for 8-plex detection using a three-step method and asymmetric PCR in an exemplary embodiment of the present invention;

[0072] Figure 4 This is a melting curve diagram of a real-time PCR reaction using two blocking fluorescent probes, Probe1 and Probe2, in an exemplary embodiment of the present invention, using a three-step method and asymmetric PCR for 12-plex detection. DETAILED DESCRIPTION

[0073] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; the technical features designed in different implementation modes of the present invention described below can be combined with each other as long as they do not conflict with each other; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0074] Provide definitions and explanations of relevant terms in the present invention:

[0075] 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;

[0076] "InDex sequence", "InDex", "tag" and "ID" refer to the specific tag sequence of each primer corresponding to the probe Probe; "tag primer sequence" and "tag primer" refer to the combined sequence of the primer (target sequence) of the target nucleic acid sequence to be detected and the probe tag, that is, a single-stranded nucleic acid molecule containing a tag sequence (ID) and a targeting sequence (i.e., a target-specific sequence) from the 5' to 3' direction. In this application, the terms "target nucleic acid sequence", "target nucleic acid" and "target sequence" have the same meaning and can be used interchangeably.

[0077] In the present application, the tag sequence does not contain a sequence complementary to the target nucleic acid sequence, and the target-specific sequence contains a sequence complementary to the target nucleic acid sequence. Therefore, under conditions that allow nucleic acid hybridization, annealing or amplification, the probe hybridizes with the ID complementary chain (ie, forms a double-stranded structure).

[0078] In the present application, for each target nucleic acid sequence, a unique ID is designed or provided, which has a unique tag sequence (in other words, all the tag sequences corresponding to the probes used are different from each other); thus, each target nucleic acid sequence corresponds to a unique ID (unique tag sequence). Therefore, by detecting the unique tag sequence, the target nucleic acid sequence corresponding thereto can be detected.

[0079] As used herein, the term "complementary" means that two nucleic acid sequences can form hydrogen bonds between each other according to the base pairing principle (Waston-Crick principle), and thus form a duplex. In the present application, the term "complementary" includes "substantially complementary" and "completely complementary". As used herein, the term "completely complementary" means that each base in a nucleic acid sequence can be paired with a base in another nucleic acid chain without mismatches or gaps. As used herein, the term "substantially complementary" means that most of the bases in a nucleic acid sequence can be paired with bases in another nucleic acid chain, which allows the presence of mismatches or gaps (for example, mismatches or gaps of one or several nucleotides). Generally, under conditions that allow nucleic acid hybridization, annealing or amplification, two nucleic acid sequences of "complementary" (for example, substantially complementary or completely complementary) will selectively / specifically hybridize or anneal, and form a duplex. For example, in the present application, the target-specific sequence in the upstream oligonucleotide sequence and the tag primer comprises a sequence complementary to the target nucleic acid sequence (for example, substantially complementary or completely complementary). Therefore, under conditions that allow nucleic acid hybridization, annealing or amplification, the upstream oligonucleotide sequence and the target-specific sequence in the tag primer will selectively / specifically hybridize or anneal with the target nucleic acid sequence. Accordingly, the term "non-complementary" means that the two nucleic acid sequences cannot hybridize or anneal under conditions that allow nucleic acid hybridization, annealing or amplification, and cannot form a duplex. For example, in the present application, the tag sequence contains a sequence that is not complementary to the target nucleic acid sequence. Therefore, under conditions that allow nucleic acid hybridization, annealing or amplification, the tag sequence does not hybridize or anneal with the target nucleic acid sequence, cannot form a duplex, but is in a free state (i.e., maintains a single-stranded structure).

[0080] As used herein, the terms "hybridization" and "annealing" refer to the process by which complementary single-stranded nucleic acid molecules form double-stranded nucleic acids. In this application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Typically, two nucleic acid sequences that are fully 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, particularly the temperature.

[0081] As used herein, "conditions that allow nucleic acid hybridization (annealing)" have the meaning commonly understood by those skilled in the art and can be determined by conventional methods. For example, two nucleic acid molecules with complementary sequences can hybridize under suitable hybridization conditions. Such hybridization conditions may involve the following factors: temperature, pH value, composition and ionic strength of the hybridization buffer, etc., and can be determined based on the length and GC content of the two complementary nucleic acid molecules. For example, when the length of the two complementary nucleic acid molecules is relatively short and / or the GC content is relatively low, low stringency hybridization conditions can be used. When the length of the two complementary nucleic acid molecules is relatively long and / or the GC content is relatively high, high stringency hybridization conditions can be used. Such hybridization conditions are well known to those skilled in the art and can be found, for example, in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999). In the present application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Accordingly, the expressions "conditions that allow nucleic acid hybridization" and "conditions that allow nucleic acid annealing" also have the same meaning and are used interchangeably.

[0082] As used herein, the expression "conditions that allow nucleic acid amplification" has a meaning generally understood by those skilled in the art, which refers to conditions that allow a nucleic acid polymerase (e.g., a DNA polymerase) to synthesize another nucleic acid chain using one nucleic acid chain as a template and form a duplex. Such conditions are well known to those skilled in the art and may involve the following factors: temperature, pH value, composition, concentration and ionic strength of the hybridization buffer, etc. In the method of the present invention, "conditions that allow nucleic acid amplification" are preferably working conditions for a nucleic acid polymerase (e.g., a DNA polymerase).

[0083] As used herein, the expression "conditions that allow nucleic acid polymerase to perform an extension reaction" has the meaning generally understood by those skilled in the art, which refers to conditions that allow nucleic acid polymerase (e.g., DNA polymerase) to extend another nucleic acid chain (e.g., primer or probe) using one nucleic acid chain as a template and form a duplex. Such conditions are well known to those skilled in the art and may involve the following factors: temperature, pH value, composition, concentration and ionic strength of the hybridization buffer, etc. In the method of the present invention, "conditions that allow nucleic acid polymerase to perform an extension reaction" are preferably working conditions of nucleic acid polymerase (e.g., DNA polymerase). In the present application, the expressions "conditions that allow nucleic acid polymerase to perform an extension reaction" and "conditions that allow nucleic acid extension" have the same meaning and can be used interchangeably.

[0084] The working conditions of the enzyme can be determined by a person skilled in the art by routine methods, and generally may involve the following factors: temperature, pH value, composition, concentration, ionic strength of the buffer, etc. Alternatively, the conditions recommended by the manufacturer of the enzyme may be used.

[0085] As used herein, the term "nucleic acid denaturation" has a meaning generally understood by those skilled in the art, and refers to the process of dissociating a double-stranded nucleic acid molecule into a single strand. The expression "conditions allowing nucleic acid denaturation" refers to conditions that allow a double-stranded nucleic acid molecule to dissociate into a single strand. For example, nucleic acids can be denatured by conventional techniques such as heating, alkali treatment, urea treatment, enzymatic methods (such as methods using helicases). In the present application, preferably, nucleic acids are denatured under heating conditions. For example, nucleic acids can be denatured by heating to 90-105°C.

[0086] 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 a meaning generally 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 in a more forward position (i.e., a position closer to the 5' end) than the latter. As used herein, the term "downstream" has the opposite meaning to "upstream".

[0087] As used herein, the term "PCR reaction" has a meaning generally understood by those skilled in the art, and refers to a reaction (polymerase chain reaction) using a nucleic acid polymerase and a primer to amplify a target nucleic acid. As used herein, the term "multiplex amplification" refers to amplifying multiple target nucleic acids in the same reaction system. As used herein, the term "asymmetric amplification" refers to amplification products obtained by amplifying a target nucleic acid in which the amounts of two complementary nucleic acid chains are different, and the amount of one nucleic acid chain is greater than the amount of the other nucleic acid chain.

[0088] As used herein, the term "fluorescent probe" refers to an oligonucleotide carrying a fluorescent group and capable of generating a fluorescent signal. In the present application, the fluorescent probe is used as a detection probe.

[0089] As used herein, the term "melting curve analysis" has a meaning generally understood by those skilled in the art, which refers to a method for analyzing the presence or identity of a double-stranded nucleic acid molecule by measuring the melting curve of the double-stranded nucleic acid molecule, which is generally used to assess the dissociation characteristics of the double-stranded nucleic acid molecule during heating. The method for performing melting curve analysis is well known to those skilled in the art (see, for example, 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.

[0090] In certain preferred embodiments of the present application, melting curve analysis can be performed by using a detection probe labeled with a reporter group and a blocking group. In short, at ambient temperature, the detection probe can form a duplex with its complementary sequence through base pairing. As the temperature rises, the two chains of the duplex begin to dissociate (i.e., the detection probe gradually dissociates from its complementary sequence). In this case, the detection probe under dissociation is in a single-stranded free curling state, and the signal (e.g., fluorescent signal) emitted by the reporter group (e.g., fluorescent group) on the dissociated detection probe changes. Therefore, by detecting the signal (e.g., fluorescent signal) emitted by the duplex containing the detection probe during the heating or cooling process, the hybridization and dissociation process of the detection probe and its complementary sequence can be observed, forming a curve in which the signal intensity changes with temperature. Further derivative analysis of the obtained curve can be obtained, and a curve with the signal intensity change rate as the ordinate and the temperature as the abscissa (i.e., the melting curve of the duplex) can be obtained. The peak in the melting curve is the melting peak, and the temperature corresponding to it is the melting point (Tm value) of the duplex. Generally speaking, the higher the degree of match between the detection probe and the complementary sequence (e.g., the fewer mismatched bases, the more paired bases), the higher the Tm value of the duplex. Therefore, by detecting the Tm value of the duplex, the presence and identity of the sequence complementary to the detection probe in the duplex can be determined. In this article, the terms "melting peak", "melting point" and "Tm value" have the same meaning and can be used interchangeably.

[0091] In combination with a multiple asymmetric amplification system and probe melting curve analysis technology, the present invention provides a method for detecting the presence of at least two target nucleic acid sequences in a sample, and a kit for implementing the method for achieving simple and rapid amplification and detection of multiple genes.

[0092] The present invention provides an example of a method for detecting the presence of X target nucleic acid sequences in a sample, comprising the following steps:

[0093] For each target nucleic acid sequence to be detected, an upstream oligonucleotide sequence, a downstream oligonucleotide sequence, an InDex tag sequence, a tag primer oligonucleotide sequence and a detection probe that specifically bind to the target nucleic acid sequence are provided;

[0094] (1) contacting the sample with a first upstream oligonucleotide sequence, a first tag primer, a second upstream oligonucleotide sequence, and a second tag primer under conditions that allow nucleic acid hybridization;

[0095] in:

[0096] (i) the first upstream oligonucleotide sequence comprises a sequence complementary to the first target nucleic acid sequence; and the first tag primer comprises a first tag sequence and a first target-specific sequence from the 5' to the 3' direction, wherein the first tag sequence comprises a sequence that is not complementary to the first target nucleic acid sequence, and the first target-specific sequence comprises a sequence complementary to the first target nucleic acid sequence; and when the tag primer oligonucleotide sequence hybridizes with the first target nucleic acid sequence, the first tag sequence is located upstream of the first target-specific sequence;

[0097] (ii) the second upstream oligonucleotide sequence comprises a sequence complementary to the second target nucleic acid sequence; and the second tag primer comprises a second tag sequence and a second target-specific sequence from the 5' to the 3' direction, wherein the second tag sequence comprises a sequence that is not complementary to the second target nucleic acid sequence, and the second target-specific sequence comprises a sequence complementary to the second target nucleic acid sequence; and when hybridizing with the second target nucleic acid sequence, the second tag sequence is located upstream of the second target-specific sequence;

[0098] (iii) the first tag sequence is different from the second tag sequence;

[0099] (2) contacting the product of step (1) with a nucleic acid polymerase under conditions that allow the nucleic acid polymerase to perform an extension reaction, performing PCR amplification, and obtaining a PCR amplification product, that is, performing PCR amplification on the sample through the provided oligonucleotide sequence to obtain a PCR amplification product;

[0100] (3) providing a detection probe, and contacting the product of step (2) with the detection probe under conditions that allow nucleic acid hybridization to obtain a hybrid;

[0101] Wherein, the detection probe comprises, from 5' to 3' direction: a first capture sequence complementary to the first tag sequence or a part thereof, a second capture sequence complementary to the second tag sequence or a part thereof, and a template sequence; and the detection probe is labeled with a reporter group and a blocking group, wherein the reporter group can emit a signal, and the blocking group can prevent the probe from being extended; and the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence;

[0102] (4) performing a melting curve analysis on the product of step (3); and determining whether the first target nucleic acid sequence and the second target nucleic acid sequence are present in the sample based on the results of the melting curve analysis.

[0103] In step (1) of the method of the present invention, since the first upstream oligonucleotide sequence comprises a sequence complementary to the first target nucleic acid sequence, and the first target-specific sequence comprises a sequence complementary to the first target nucleic acid sequence, when the first target nucleic acid sequence is present, the first upstream oligonucleotide sequence and the first tag primer both hybridize with the first target nucleic acid sequence. Similarly, since the second upstream oligonucleotide sequence comprises a sequence complementary to the second target nucleic acid sequence, and the second target-specific sequence comprises a sequence complementary to the second target nucleic acid sequence, when the second target nucleic acid sequence is present, the second upstream oligonucleotide sequence and the second tag primer both hybridize with the second target nucleic acid sequence.

[0104] In steps (1) and (2) of the method of the present invention, when a first target nucleic acid sequence is present, the first tag primer hybridizes with the first target nucleic acid sequence. Further, since the first tag primer sequence contains a sequence that is not complementary to the first target nucleic acid sequence. Similarly, when a second target nucleic acid sequence is present, the second tag primer sequence hybridizes with the second target nucleic acid sequence, and the second tag sequence in the second tag primer is in a free state and does not hybridize with the second target nucleic acid sequence. In step (3) of the method of the present invention, when a first tag primer fragment is present, since the first tag primer fragment contains the first tag sequence or a portion thereof, and the detection probe contains a sequence consistent with the first tag sequence or a portion thereof, the complementary chain of the first tag primer fragment hybridizes with the detection probe. Similarly, when a second tag primer fragment is present, since the second tag primer fragment contains the second tag sequence or a portion thereof, and the detection probe contains a sequence consistent with the second tag sequence or a portion thereof, the complementary chain of the second tag fragment hybridizes with the detection probe.

[0105] In step (4) of the method of the present invention, when the complementary strand of the first tag primer fragment is present, the complementary strand of the first tag primer fragment hybridizes with the detection probe to form a first duplex. Similarly, when the second tag fragment is present, the complementary strand of the second tag fragment hybridizes with the detection probe to form a second duplex.

[0106] In step (5) of the method of the present invention, when the first duplex is present, a melting peak corresponding to the first duplex can be detected. Therefore, the presence or absence of the first target nucleic acid sequence in the sample can be determined by the presence or absence of the melting peak corresponding to the first duplex. For example, when the melting peak corresponding to the first duplex is detected or not detected, the presence or absence of the first target nucleic acid sequence in the sample is determined. Similarly, the presence or absence of the second target nucleic acid sequence in the sample can be determined by the presence or absence of the melting peak corresponding to the second duplex. For example, when the melting peak corresponding to the second duplex is detected or not detected, the presence or absence of the second target nucleic acid sequence in the sample is determined.

[0107] In particular, in the method of the present invention, since the first tag sequence used is different from the second tag sequence, the first tag primer fragment and the second tag fragment formed have different sequences, and the first tag primer fragment and the second tag fragment have different abilities to hybridize to the detection probe with their respective complementary chains. Thus, the first duplex comprising the extension product of the first tag primer fragment and the detection probe is also different in structure (sequence) from the second duplex comprising the extension product of the second tag fragment and the detection probe. Accordingly, the first duplex will have a different melting point (Tm value) from the second duplex. Therefore, in the melting curve analysis, the first duplex shows a different melting peak from the second duplex. Thus, by detecting the melting peak of the first duplex or the second duplex, the presence of the first target nucleic acid sequence or the second target nucleic acid sequence in the sample can be determined.

[0108] In addition, since the first tag sequence, the second tag sequence, and the sequence of the detection probe are all known or predetermined, the melting points (Tm values) of the first duplex and the second duplex can be calculated in advance. Thus, by detecting the melting peak having the melting point (Tm value) of the first duplex or the second duplex in the melting curve analysis, the presence of the first target nucleic acid sequence or the second target nucleic acid sequence in the sample can be determined.

[0109] Based on the same principle as above, by designing more tag primers, the method of the present invention can be used to detect more target nucleic acid sequences simultaneously. Therefore, in certain preferred embodiments, in step (1), in addition to the first upstream oligonucleotide sequence, the first tag primer, the second upstream oligonucleotide sequence and the second tag primer, the sample is contacted with a third upstream oligonucleotide sequence and a third tag primer under conditions that allow nucleic acid hybridization, wherein:

[0110] Similarly, the third upstream oligonucleotide sequence comprises a sequence complementary to the third target nucleic acid sequence; and the third tag primer comprises a third tag sequence and a third target-specific sequence from the 5' to the 3' direction, wherein the third tag sequence comprises a sequence that is not complementary to the third target nucleic acid sequence, and the third target-specific sequence comprises a sequence complementary to the third target nucleic acid sequence;

[0111] Furthermore, when hybridizing with the third target nucleic acid sequence, the third tag sequence fragment is freed, and the third target-specific sequence fragment hybridizes with the third target nucleic acid sequence; and, the third tag sequence is different from the first and second tag sequences;

[0112] Furthermore, in step (3), the detection probe used further comprises a third capture sequence that is complementary to the third tag sequence or a portion thereof.

[0113] In such embodiments, in steps (1) and (2), when a third target nucleic acid sequence is present, the third upstream oligonucleotide sequence and the third tag primer hybridize with the third target nucleic acid sequence. Further, in step (2), when a third target nucleic acid sequence is present, the third tag primer sequence is extended.

[0114] Further, in steps (3) and (4), when the third tag fragment is present, the complementary strand of the third tag fragment hybridizes with the detection probe to form a third duplex. Further, in step (5), when a melting peak corresponding to the third duplex is detected or not detected, it is determined that the third target nucleic acid sequence is present or not in the sample.

[0115] Similarly, in the method of the present invention, since the first, second and third tag sequences used are different, the first tag primer fragment, the second tag fragment and the third tag fragment formed have different sequences and hybridize to different positions of the detection probe. Thus, the first duplex comprising the extension product of the first tag primer fragment and the detection probe, the second duplex comprising the extension product of the second tag fragment and the detection probe, and the third duplex comprising the extension product of the third tag fragment and the detection probe are different from each other in structure (sequence). Accordingly, the first, second and third duplexes have different melting points (Tm values) from each other. Therefore, in the melting curve analysis, the first, second and third duplexes show three melting peaks that can be distinguished from each other. Thus, by detecting the melting peaks of the first, second and third duplexes, the presence of the first, second and third target nucleic acid sequences in the sample can be determined.

[0116] In addition, since the first, second, and third tag sequences and the sequences of the detection probe are all known or predetermined, the melting points (Tm values) of the first, second, and third duplexes can be calculated in advance. Thus, by detecting the melting peak having the melting point (Tm value) of the first, second, or third duplex in the melting curve analysis, the presence of the first, second, or third target nucleic acid sequence in the sample can be determined.

[0117] Similarly, in the method of the present invention, since the first, second and third tag sequences used are different, the first tag primer fragment, the second tag fragment and the third tag fragment formed have different sequences and hybridize to the detection probe. Thus, the first duplex containing the extension product of the first tag primer fragment and the detection probe, the second duplex containing the extension product of the second tag fragment and the detection probe, and the third duplex containing the extension product of the third tag fragment and the detection probe are different from each other in structure (sequence). Accordingly, the first, second and third duplexes have different melting points (Tm values) from each other. Therefore, in the melting curve analysis, the first, second and third duplexes show three melting peaks that can be distinguished from each other. Thus, by detecting the melting peaks of the first, second and third duplexes, the presence of the first, second and third target nucleic acid sequences in the sample can be determined.

[0118] In addition, since the first, second and third tag sequences and the sequences of the detection probe are all known or predetermined, the melting points (Tm values) of the first, second and third duplexes can be calculated in advance. Thus, by detecting the melting peaks having the melting points (Tm values) of the first, second and third duplexes in the melting curve analysis, the presence of the first, second and third target nucleic acid sequences in the sample can be determined.

[0119] Similarly, more upstream oligonucleotide sequences and more tag primers can be used to implement the method of the present invention. For example, in certain embodiments, at least 5 upstream oligonucleotide sequences, at least 5 tag primers and a detection probe can be used to implement the method of the present invention, wherein:

[0120] Each upstream oligonucleotide sequence contains a sequence complementary to a target nucleic acid sequence; and each label probe contains a label sequence and a target-specific sequence from the 5' to 3' direction, wherein the label sequence contains a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence contains a sequence complementary to a target nucleic acid sequence; thus, when a certain target nucleic acid sequence is present, the upstream oligonucleotide sequence and the label probe corresponding to the target nucleic acid sequence are both able to hybridize with the target nucleic acid sequence; and the label sequences contained in all label probes are different from each other. In such embodiments, the method of the present invention can be used to simultaneously detect at least 6 target nucleic acid sequences.

[0121] In certain embodiments, the method of the present invention may use at least 6 upstream oligonucleotide sequences, at least 6 tags and one detection probe; preferably, at least 7 upstream oligonucleotide sequences, at least 7 tags and one detection probe; preferably, at least 8 upstream oligonucleotide sequences, at least 8 tags and one / two detection probes; preferably, at least 9 upstream oligonucleotide sequences, at least 9 tags and one / two detection probes; preferably, at least 10 upstream oligonucleotide sequences, at least 10 tag probes and one / two detection probes; preferably, at least 12 upstream oligonucleotide sequences, at least 12 tag probes and one / two detection probes; preferably, at least 15 upstream oligonucleotide sequences, at least 15 tag probes and one / two detection probes; preferably, at least 20 upstream oligonucleotide sequences, at least 20 tag probes and two / three detection probes; wherein the upstream oligonucleotide sequences, tags and detection probes are as defined above. In such embodiments, the methods of the invention can be used to simultaneously detect at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, or at least 20 target nucleic acid sequences.

[0122] Thus, in certain embodiments, the present invention provides a method for detecting the presence of X target nucleic acid sequences in a sample, wherein X is an integer ≥ 1 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or a greater integer).

[0123] The basic principle of the method of the present invention is briefly summarized above. Now, the method of the present invention is explained and illustrated in detail with reference to each step of the method of the present invention.

[0124] Regarding steps (1) and (2)

[0125] In the method of the present invention, the target nucleic acid sequence in the sample (e.g., the first or second target nucleic acid sequence; if present) is first hybridized with the corresponding upstream oligonucleotide sequence (e.g., the first or second upstream oligonucleotide sequence) and the corresponding tag primer (e.g., the first or second tag primer).

[0126] In the method of the present invention, the sample can be any sample to be detected. For example, in certain preferred embodiments, the sample comprises or is DNA (e.g., genomic DNA or cDNA). In certain preferred embodiments, the sample comprises or is RNA (e.g., mRNA). In certain preferred embodiments, the sample comprises or is a mixture of nucleic acids (e.g., a mixture of DNA, a mixture of RNA, or a mixture of DNA and RNA).

[0127] In the method of the present invention, the target nucleic acid sequence to be detected is not limited to its sequence composition or length. For example, the target nucleic acid sequence can be a DNA (e.g., genomic DNA or cDNA) or an RNA molecule (e.g., mRNA). In addition, the target nucleic acid sequence to be detected can be single-stranded or double-stranded.

[0128] When the sample or target nucleic acid sequence to be detected is mRNA, preferably, before carrying out the method of the present invention, a reverse transcription reaction is performed to obtain cDNA complementary to the mRNA. For a detailed description of the reverse transcription reaction, see, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

[0129] The sample or target nucleic acid sequence to be detected can be obtained from any source, including but not limited to prokaryotes, eukaryotes (e.g., protozoa, parasites, fungi, yeast, plants; animals include mammals and humans) or viruses (e.g., 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 any form of nucleic acid sequence, such as genomic sequence, artificially separated or fragmented sequence, synthetic sequence, etc.

[0130] In certain embodiments of the invention, tags and probes may comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.

[0131] In certain preferred embodiments, the tags and probes comprise or consist of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the tags and probes comprise modified nucleotides, e.g., modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the tags and probes comprise non-natural nucleotides, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0132] In the method of the present invention, the probe is not limited by its length. For example, the length of the probe 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. The target-specific sequence in the tag primer can be of any length as long as it can specifically hybridize with the target nucleic acid sequence. For example, the length of the target-specific sequence in the index primer can be 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. The index sequence in the index primer can be of any length as long as it can specifically hybridize with the detection probe and be extended. For example, the length of the tag sequence in the tag primer 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. In certain preferred embodiments, the length of the target-specific sequence in the tag primer is 10-100nt (e.g., 10-90nt, 10-80nt, 10-50nt, 10-40nt, 10-30nt, 10-20nt), and the length of the tag sequence is 5-100nt (e.g., 10-90nt, 10-80nt, 10-50nt, 10-40nt, 10-30nt, 10-20nt).

[0133] In certain preferred embodiments, the probe has a phosphate group terminal. In certain preferred embodiments, the 3'-end of the probe is blocked to inhibit its extension. The 3'-end of a nucleic acid (e.g., a probe) can be blocked by various methods. For example, the 3'-PO4 of the last nucleotide of the probe can be modified to block the 3'-end of the probe. In certain embodiments, the 3'-end of the probe can be blocked by adding a chemical moiety (e.g., an amine group, biotin, or an alkyl group) to the 3'-PO4 of the last nucleotide of the tag primer. In certain embodiments, the 3'-end of the probe can be blocked by replacing the last nucleotide of the probe with a dideoxynucleotide.

[0134] In certain embodiments of the present invention, the upstream oligonucleotide sequence may comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the upstream oligonucleotide sequence comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the upstream oligonucleotide sequence comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the upstream oligonucleotide sequence comprises non-natural nucleotides, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0135] In the method for the present invention, upstream oligonucleotide sequence and downstream oligonucleotide sequence are not limited by their length, as long as they can be specifically hybridized with the target nucleic acid sequence. For example, the length of upstream oligonucleotide sequence and downstream oligonucleotide sequence 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.

[0136] In the method of the present invention, the conditions for allowing nucleic acid hybridization can be routinely determined by those skilled in the art. For example, suitable hybridization conditions can be determined based on the target nucleic acid sequence to be detected, the upstream oligonucleotide sequence used, the target-specific sequence in the label primer, the probe, and the target-specific sequence complementary chain. In certain embodiments of the present invention, the conditions for allowing nucleic acid hybridization are such stringent conditions: it allows the upstream oligonucleotide sequence and the target-specific sequence in the label primer to hybridize with the corresponding target nucleic acid sequence through base complementary pairing, and the label sequence in the label primer does not hybridize with the target nucleic acid sequence. In certain preferred embodiments, under high stringency conditions, the sample is contacted with various upstream oligonucleotide sequences and various label primers.

[0137] In the method of the present invention, after contacting the sample with various upstream oligonucleotide sequences and various tag primers, an enzyme having polymerization activity can generally be used to perform extension amplification using the upstream oligonucleotide sequences hybridized to the target nucleic acid sequence and the tag primers.

[0138] In particular, in step (1),

[0139] When the upstream oligonucleotide and the tag primer contact the target nucleic acid sequence, the target-specific sequence contained therein hybridizes with the target nucleic acid sequence and forms a double-stranded structure, while the tag sequence does not hybridize with the target nucleic acid sequence and maintains a single-stranded structure. Therefore, an enzyme with polymerization can be used for extension amplification.

[0140] In certain preferred embodiments, in step (1) and / or (2), the sample is also contacted with a downstream oligonucleotide sequence (or downstream primer) specific to the target nucleic acid sequence. In certain embodiments, the use of a nucleic acid polymerase and a downstream oligonucleotide sequence (or downstream primer) is particularly advantageous. In particular, the nucleic acid polymerase can use the target nucleic acid sequence as a template, the upstream oligonucleotide sequence, the tag primer, and the downstream oligonucleotide sequence as primers to generate additional target nucleic acid sequences, thereby improving the sensitivity of the method of the present invention.

[0141] Therefore, in certain preferred embodiments, in step (1), in addition to the upstream oligonucleotide sequence and label primer defined above, a downstream oligonucleotide sequence is also provided for each target nucleic acid sequence to be detected; wherein the downstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; and when hybridizing with the target nucleic acid sequence, the downstream oligonucleotide sequence is located downstream of the target-specific sequence; then, under conditions allowing nucleic acid hybridization, the sample is contacted with the provided upstream oligonucleotide sequence, label primer and downstream oligonucleotide sequence.

[0142] For example, when the method of the present invention is used to detect the first and second target nucleic acid sequences, first and second downstream oligonucleotide sequences can be provided, which respectively include sequences complementary to the first and second target nucleic acid sequences. Similarly, a third downstream oligonucleotide sequence can be provided for the third target nucleic acid sequence, which includes a sequence complementary to the third target nucleic acid sequence.

[0143] In certain preferred embodiments, the target nucleic acid sequence is amplified in a symmetrical amplification mode. In such embodiments, for a certain target nucleic acid sequence, an equal amount of upstream and downstream oligonucleotide sequences are used to amplify. In certain preferred embodiments, the target nucleic acid sequence is amplified in an asymmetrical amplification mode. In such embodiments, for a certain target nucleic acid sequence, an unequal amount of upstream and downstream oligonucleotide sequences are used to amplify. In certain embodiments, the upstream oligonucleotide sequence is excessive (e.g., excessive at least 1 times, at least 2 times, at least 5 times, at least 8 times, at least 10 times, for example, excessive 1-10 times, etc.) relative to the downstream oligonucleotide sequence. In certain embodiments, the downstream oligonucleotide sequence is excessive (e.g., excessive at least 1 times, at least 2 times, at least 5 times, at least 8 times, at least 10 times, for example, excessive 1-10 times, etc.) relative to the upstream oligonucleotide sequence.

[0144] In certain preferred embodiments, the target nucleic acid sequence is amplified 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 preferred embodiments, the target nucleic acid sequence is amplified in a two-step method. 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. The temperature suitable for nucleic acid denaturation, nucleic acid annealing, and nucleic acid extension can be easily determined by a person skilled in the art by conventional methods (see, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)).

[0145] In the method of the present invention, the label primers used are usually one-to-one corresponding to the target nucleic acid sequences. In other words, a unique label is provided for each target nucleic acid sequence to be detected. In the method of the present invention, for different target nucleic acid sequences, the same or different upstream oligonucleotide sequences can be used; and / or, the same or different downstream oligonucleotide sequences can be used.

[0146] About step (3)

[0147] In the present application, the detection probe includes, from 5' to 3', a tag sequence with site-directed mutations of each tag sequence or part thereof. Thus, in steps (2) and (3), under the action of a nucleic acid polymerase, the upstream oligonucleotide sequence extension product is used as a template, and the tag primer is used for extension; and after the extension reaction is completed, the extension product of the tag primer hybridizes with the detection probe to form a nucleic acid duplex.

[0148] In the method of the present invention, the detection probe comprises a plurality of capture sequences with a plurality of tag sequences or partial base mutations thereof (e.g., a first capture sequence with a first tag sequence or partial base mutations thereof, a second capture sequence with a second tag sequence or partial base mutations thereof, and a third capture sequence with a third tag sequence or partial base mutations thereof). It is easy to understand that the individual capture sequences can be arranged in any order.

[0149] In certain preferred embodiments, the reporter group is a fluorescent group. In such embodiments, the signal emitted by the reporter group is fluorescence. In certain preferred embodiments, the fluorescent group includes, but is not limited to, various fluorescent molecules, such as ALEX-350, FAM, VIC, TET, CALGold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar670, CY3, CY5, CY5.5, and Quasar705. In certain preferred embodiments, the blocking group includes but is not limited to various chemical modifications, such as phosphorylation modification, amino modification, phosphorothioate bond, alkyl phosphotriester bond, aryl phosphotriester bond, alkyl phosphoester bond, aryl phosphoester bond, hydrogenated phosphate bond, alkyl phosphoramidate bond, aryl phosphoramidate bond, 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification and 1-(4'-thio-PD-ribofuranosyl) modification, etc.

[0150] In the method of the present invention, the detection probe can be linear or can have a hairpin structure. In certain preferred embodiments, the detection probe is linear. In certain preferred embodiments, the detection probe has a hairpin structure. The hairpin structure can be natural or artificially introduced. In addition, conventional methods in the art can be used to construct a detection probe with a hairpin structure. For example, two complementary oligonucleotide sequences can be added to the two ends (5' end and 3' end) of the detection probe so that the detection probe can form a hairpin structure. In such embodiments, two complementary oligonucleotide sequences constitute the arms (stems) of the hairpin structure. The arms of the hairpin structure can have any desired length, for example, the length of the arm can be 2-15nt, for example 3-7nt, 4-9nt, 5-10nt, 6-12nt.

[0151] Furthermore, in the method of the present invention, "hybridization", "extension", "PCR reaction", "amplification", "nucleic acid hybridization" and "conditions allowing nucleic acid hybridization" in step (3) may be as defined above.

[0152] As described in detail above, each probe contains a unique tag sequence, and each tag primer is extended by a nucleic acid polymerase, and its product forms a duplex with the detection probe. Thus, for each tag, when its corresponding target sequence is present, a unique duplex will be generated in step (4), which contains the detection probe (as one chain) and the extension product corresponding to the tag primer (as the other chain). Therefore, each duplex generated in step (4) has a different structure (sequence) from each other, and thus has a different Tm value from each other, and shows a different melting peak in the melting curve analysis.

[0153] In certain preferred embodiments, the nucleic acid polymerase used in step (4) is a template-dependent nucleic acid polymerase (eg, a DNA polymerase, in particular a thermostable DNA polymerase). In certain preferred embodiments, the nucleic acid polymerase is a thermostable DNA polymerase, which can be obtained from various bacterial species, for example, Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermus flavus, Thermus literalis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus ru bens, Thermus scotoductus, Thermus silvanus, Thermus thermophllus, Thermotoga maritima, Thermotoganeapolitana, Thermosipho africanus, Thermococcus litoralis, Thermococcus ba rossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga Preferably, the template-dependent nucleic acid polymerase is Taq polymerase.

[0154] About step (4)

[0155] In step (4) of the method according to the present invention, a melting curve analysis is performed on the product of step (3); and based on the result of the melting curve analysis, it is determined whether the n target nucleic acid sequences exist in the sample.

[0156] As discussed above, melting curve analysis can be performed by using detection probes labeled with reporter and blocker groups.

[0157] In certain embodiments, the product of step (3) can be gradually heated and the signal emitted by the reporter group on the detection probe can be monitored in real time to obtain a curve showing how the signal intensity of the product of step (3) changes with temperature.

[0158] For example, the product of step (3) can be gradually heated from 25°C or lower (e.g., no more than 25°C, no more than 30°C, no more than 35°C, no more than 40°C, no more than 45°C) to 75°C or higher (e.g., at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C), and the signal emitted by the reporter group on the detection probe can be monitored in real time to obtain a curve showing that the signal intensity of the reporter group changes with temperature. The rate of heating can be routinely determined by those skilled in the art. For example, the rate of temperature increase can be: 0.01-1°C (e.g., 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, such as 0.01°C, 0.02°C, 0.03°C, 0.04°C, 0.05°C, 0.06°C, 0.07°C, 0.08°C, 0.09°C, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C or 1.0°C) per step, and each step is maintained for 0.5-15s (e.g., 0.5-1s , 1-2s, 2-3s, 3-4s, 4-5s, 5-10s, 10-15s); or the temperature rises by 0.01-1°C per second (for example, 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, for example, 0.01°C, 0.02°C, 0.03°C, 0.04°C, 0.05°C, 0.06°C, 0.07°C, 0.08°C, 0.09°C, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C or 1.0°C).

[0159] In certain embodiments, the product of step (3) can be gradually cooled and the signal emitted by the reporter group on the detection probe can be monitored in real time to obtain a curve showing how the signal intensity of the product of step (3) changes with temperature.

[0160] For example, the product of step (3) can be gradually cooled from 75°C or higher (e.g., at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C) to 45°C or lower (e.g., no more than 45°C, no more than 40°C, no more than 35°C, no more than 30°C, no more than 25°C), and the signal emitted by the reporter group on the detection probe is monitored in real time, thereby obtaining a curve showing that the signal intensity of the reporter group changes with temperature. The rate of cooling can be routinely determined by those skilled in the art. For example, the cooling rate can be: cooling by 0.01-1°C per step (e.g., 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, such as 0.01°C, 0.02°C, 0.03°C, 0.04°C, 0.05°C, 0.06°C, 0.07°C, 0.08°C, 0.09°C, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C or 1.0°C), and each step is maintained for 0.5-15s (e.g., 0.5-1s , 1-2s, 2-3s, 3-4s, 4-5s, 5-10s, 10-15s); or cooling by 0.01-1°C per second (for example, 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, for example, 0.01°C, 0.02°C, 0.03°C, 0.04°C, 0.05°C, 0.06°C, 0.07°C, 0.08°C, 0.09°C, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C or 1.0°C).

[0161] Subsequently, the obtained curve can be derived to obtain the melting curve of the product of step (3). According to the melting peak (melting point) in the melting curve, the presence of the tag corresponding to the melting peak (melting point) can be determined. Subsequently, the presence of the target nucleic acid sequence corresponding to the tag can be determined through the correspondence between the tag and the target nucleic acid sequence.

[0162] For example, when the result of the melting curve analysis shows the presence or absence of a melting peak corresponding to the first duplex comprising the detection probe and the first tag primer extension product, it can be determined that the first target nucleic acid sequence is present or absent in the sample. Similarly, when the result of the melting curve analysis shows the presence or absence of a melting peak corresponding to the second duplex comprising the detection probe and the second tag primer extension product, it can be determined that the second target nucleic acid sequence is present or absent in the sample. When the result of the melting curve analysis shows the presence or absence of a melting peak corresponding to the third duplex comprising the detection probe and the third tag primer extension product, it can be determined that the third target nucleic acid sequence is present or absent in the sample. Thus, the method of the present invention can achieve simultaneous detection (multiple detection) of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) target nucleic acid sequences by using one detection probe and at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) labels.

[0163] Without being limited by theory, the resolution or accuracy of the melting curve analysis can reach 0.5°C or higher. In other words, the melting curve analysis can distinguish two melting peaks with melting points that differ by only 0.5°C or lower (e.g., 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C). Therefore, in certain embodiments of the method of the present invention, the melting point difference between any two duplexes (e.g., the first duplex and the second duplex) can be at least 0.5°C (e.g., by designing the sequence of the first molecular tag sequence, the second molecular tag sequence, and the detection probe), so that the any two duplexes (e.g., the first duplex and the second duplex) can be distinguished and discriminated by melting curve analysis. However, for the purpose of facilitating distinction and discrimination, a greater melting point difference between two duplexes (e.g., the first duplex and the second duplex) is preferred in some cases. Therefore, in certain embodiments of the methods of the present invention, the difference in melting point between two duplexes (e.g., a first duplex and a second duplex) can be any desired value (e.g., at least 0.5°C, at least 1°C, at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 8°C, at least 10°C, at least 15°C, or at least 20°C), as long as the difference in melting point can be distinguished and discerned by melting curve analysis.

[0164] Simultaneous use of one or more detection probes

[0165] In the method described above, a detection probe is used to achieve multiple detection of multiple target nucleic acid sequences. However, it is easy to understand that the method of the present invention is not limited to the number of detection probes used and corresponding probe label groups. The method of the present invention can use one or more detection probes (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or more detection probes).

[0166] Furthermore, based on the same principles as above, at least two or more (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more detection probes) labels can be designed for each detection probe. Thus, the method of the present invention can be used to simultaneously detect the presence of multiple target nucleic acid sequences, and the maximum number of target nucleic acid sequences that can be simultaneously detected by the method of the present invention far exceeds the number of detection probes used, and is equal to the sum of the number of labels designed for each detection probe (i.e., the number of all labels used).

[0167] Therefore, in certain embodiments, the present invention provides a method for detecting the presence of n target nucleic acid sequences in a sample, wherein n is an integer ≥ 1 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or a greater integer), and the method comprises the following steps:

[0168] For each target nucleic acid sequence to be detected, an upstream oligonucleotide sequence, a downstream oligonucleotide sequence, an InDex tag sequence, a tag primer oligonucleotide sequence and a detection probe that specifically bind to the target nucleic acid sequence are provided;

[0169] (1) providing an upstream oligonucleotide sequence and a label primer for each target nucleic acid sequence to be detected; contacting the sample with the provided upstream oligonucleotide sequence and label primer under conditions that allow nucleic acid hybridization;

[0170] Wherein, the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; and the tag primer comprises a tag sequence and a target-specific sequence from the 5' to the 3' direction, the tag sequence comprises a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence comprises a sequence complementary to the target nucleic acid sequence; and the tag sequences comprised by all tag primers are different from each other;

[0171] (2) extending the hybridization product of step (1) and performing PCR amplification under conditions that allow nucleic acid polymerase to perform an extension reaction; that is, performing PCR amplification on the sample through the provided oligonucleotide sequence to obtain a PCR amplification product;

[0172] (3) providing m detection probes, and contacting the product of step (2) with the m detection probes under conditions that allow nucleic acid hybridization to obtain a hybrid;

[0173] Wherein, m is an integer less than n and greater than 0, and each detection probe independently comprises, from 5' to 3' direction, one or more tag sequences; and the m detection probes comprise at least n capture sequences, which are respectively complementary to the tag sequences or parts thereof of the n tag primers provided in step (2); and each detection probe is independently labeled with a reporter group and a blocking group, wherein the reporter group is capable of emitting a signal; and the signal emitted by each detection probe when hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence;

[0174] (4) performing a melting curve analysis on the product of step (3); and determining whether the n target nucleic acid sequences are present in the sample based on the results of the melting curve analysis.

[0175] In step (1) of such an embodiment, when a certain target nucleic acid sequence is present, the upstream oligonucleotide sequence corresponding to the target nucleic acid sequence (i.e., the upstream oligonucleotide sequence comprising a sequence complementary to the target nucleic acid sequence) and the tag primer corresponding to the target nucleic acid sequence (i.e., the tag primer whose target-specific sequence comprises a sequence complementary to the target nucleic acid sequence) both hybridize with the target nucleic acid sequence.

[0176] Further, in steps (2) and (3) of such embodiments, when there is a label primer sequence corresponding to a certain target nucleic acid sequence, the complementary chain of the label sequence hybridizes with the detection probe (i.e., a detection probe containing a capture sequence complementary to the label sequence or a portion thereof in the label primer), and the nucleic acid polymerase will use the complementary chain of the label primer as a template to extend the label primer to form a double helix corresponding to the target nucleic acid sequence.

[0177] Furthermore, in step (4) of such embodiment, when a melting peak of a duplex corresponding to a certain target nucleic acid sequence is detected or not detected, it is determined that the target nucleic acid sequence exists in the sample or not.

[0178] In step (3) of such an embodiment, the m detection probes contain at least n capture sequences, which are respectively complementary to the tag sequences or parts of the n tag primers provided in step (1); thus, any capture sequence generated in step (2) can hybridize with at least one detection probe (which contains a sequence complementary to the tag sequence or part of the tag primer) and form a duplex for subsequent extension and detection. In certain preferred embodiments,

[0179] The m detection probes contain n capture sequences, which are respectively complementary to the tag sequences or parts of the n tag primers.

[0180] In certain preferred embodiments, the m detection probes do not contain the same capture sequence. In this case, for each tag primer, there is only one detection probe (which contains a capture sequence complementary to the tag sequence in the tag primer) that hybridizes with the tag sequence derived from the tag primer, and after the extension reaction, only one duplex is generated. Subsequently, by detecting the presence of the duplex in step (4), the presence of the target nucleic acid sequence corresponding to the tag can be determined.

[0181] In certain preferred embodiments, the m detection probes comprise different tag sequences from each other (ie, a tag set for each detection probe).

[0182] In this case, for each probe, there are one or more tag sequences that hybridize with the extension product fragments derived from the tag primer; and after the extension reaction, one or more duplexes are generated. Subsequently, by detecting the presence of the one or more duplexes in step (4), the presence of the target nucleic acid sequence corresponding to the tag can be determined.

[0183] In step (4) of such embodiments, the duplexes can be distinguished and identified by the melting point of the duplexes and / or the reporter group in the detection probe. In certain preferred embodiments, the m detection probes contain different reporter groups and label groups. In this case, the product of step (4) can be subjected to melting curve analysis, and the presence of a certain duplex can be determined based on the melting peak (melting point) in the melting curve, thereby determining the presence of the target nucleic acid sequence corresponding to the duplex. In certain preferred embodiments, the reporter groups contained in the m detection probes are different from each other. In this case, when the product of step (3) is subjected to melting curve analysis, the signal of each reporter group can be monitored in real time, thereby obtaining multiple melting curves (derived from the melting points of different labels in the label group under the same probe) each corresponding to the signal of a reporter group. Subsequently, the presence of a certain duplex can be determined based on the signal type of the reporter group and the melting peak (melting point) in the melting curve, thereby determining the presence of the target nucleic acid sequence corresponding to the duplex.

[0184] In certain exemplary embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10 detection probes can be used (i.e., m is an integer of ≥1, ≥2, ≥3, ≥4, ≥5, ≥6, ≥8, ≥10). In certain exemplary embodiments, 1-6 detection probes can be used (i.e., m is an integer of 1-6; for example, m is 1, 2, 3, 4, 5 or 6). Further preferably, each of the detection probes used is labeled with a different reporter group.

[0185] For example, in certain exemplary embodiments, the method of the present invention may use a first and a second detection probe, which are labeled with a first reporter group and a second reporter group, respectively. Thus, in step (4), the signal changes of the first reporter group and the second reporter group with temperature are respectively monitored in real time to obtain a first melting curve and a second melting curve. Subsequently, based on the melting peak in the first (or second) melting curve, the presence of a duplex containing the first (or second) detection probe can be determined, and then the presence of a target nucleic acid sequence corresponding to the label primer hybridized with the first (or second) detection probe can be determined.

[0186] In certain exemplary embodiments, the methods of the present invention use at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 9, or at least 10 detection probes, etc.; and at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 45 tag groups, etc. Thus, the methods of the present invention can achieve simultaneous detection of multiple target nucleic acid sequences (multiple detection), wherein the maximum number of detectable target nucleic acid sequences is equal to the number of tags used. For example,

[0187] In Example 4 of the present application, the method of the present invention uses 2 detection probes and 12 molecular tags to achieve simultaneous detection of 12 target nucleic acid sequences (twelve-plex detection).

[0188] It is also easy to understand that the various technical features described in detail for the method using one detection probe can also be applied to the method using two or more detection probes. For example, the various detailed descriptions made above for the sample to be detected, the target nucleic acid sequence, the label, the upstream oligonucleotide sequence, the downstream oligonucleotide sequence, the detection probe, the conditions allowing nucleic acid hybridization, the conditions allowing nucleic acid polymerase to perform an extension reaction, the nucleic acid polymerase, the melting curve analysis, the repetition of the steps, etc. can all be applied to the method using two or more detection probes. Therefore, in certain preferred embodiments, the method using two or more detection probes of the present invention may involve any one or more technical features as described in detail above, or any combination of the technical features.

[0189] This probe labeling method also provides probe labeling sets and kits

[0190] In another aspect, the present invention provides a probe tag set (InDex set), which comprises a detection probe and a plurality of corresponding probe tags, wherein:

[0191] The tag primers each independently comprise a tag sequence and a target-specific sequence from the 5' to 3' direction, the target-specific sequence comprises a sequence complementary to a target nucleic acid sequence, and the probe tag sequence comprises a sequence not complementary to the target nucleic acid sequence;

[0192] The detection probe comprises, from 5' to 3' direction, a capture sequence complementary to each tag subsequence or a part thereof; and the detection probe is labeled with a reporter group and a blocking group, wherein the reporter group is capable of emitting a signal, and the blocking group is capable of preventing the probe from extending; and the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence.

[0193] In certain preferred embodiments, the probe tag set comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, or at least 20 molecular tags.

[0194] In certain preferred embodiments, all the tag primers each target a different target nucleic acid sequence. In certain preferred embodiments, the tag sequences contained in all the tag primers are different from each other; and the target-specific sequences contained in all the tag primers are different from each other.

[0195] It is easy to understand that such probe label sets can be used to implement the method of the present invention described in detail above. Therefore, the various technical features described in detail above for label primers and detection probes can also be applied to the labels and detection probes in the label set. Therefore, in certain preferred embodiments, the probe label set comprises a label as defined above. In certain preferred embodiments, the probe test kit contains a detection probe as defined above.

[0196] In another aspect, the present invention provides a kit comprising one or more probes and a label set as defined above.

[0197] In certain preferred embodiments, the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 probe tag sets as described above.

[0198] In certain preferred embodiments, all tag sequences in the kit target different target nucleic acid sequences. In certain preferred embodiments, the tag sequences contained in all tag primers in the kit are different from each other. In certain preferred embodiments, the target-specific sequences contained in all tag primers in the kit are different from each other.

[0199] In certain preferred embodiments, all detection probes in the kit contain the same reporter group and correspond to different labels. In certain preferred embodiments, different detection probes in the kit contain different reporter groups and correspond to different labels.

[0200] In certain preferred embodiments, all detection probes in the kit are independently labeled with the same or different reporter groups. In certain preferred embodiments, the reporter groups contained in all detection probes in the kit are different from each other.

[0201] In certain preferred embodiments, the kit comprises 1-6 probe sets. Preferably, the reporter groups contained in all detection probes in the kit are different from each other. Further preferably, the tag sequences contained in all tag primers in the kit are different from each other, and the target-specific sequences contained in all tag primers in the kit are different from each other.

[0202] It is easy to understand that such a kit can be used to implement the method of the present invention described in detail above. Therefore, the various technical features described in detail above for the label primer and the detection probe can also be applied to the label primer and the detection probe in the kit. In addition, such a kit may also contain other reagents required for implementing the method of the present invention.

[0203] For example, in certain preferred embodiments, the kit may further comprise an upstream oligonucleotide sequence, a downstream oligonucleotide sequence, an enzyme having 5' nuclease activity, a nucleic acid polymerase, or any combination thereof as defined above. In certain preferred embodiments, the kit may further comprise reagents for nucleic acid extension and hybridization, reagents for nucleic acid extension, reagents for nucleic acid amplification, or any combination thereof. Such reagents may be routinely determined by those skilled in the art, and include, but are not limited to, working buffers for enzymes (e.g., nucleic acid polymerases), dNTPs, water, ions (e.g., Mg), and the like. 2+ ) or any combination thereof.

[0204] Those skilled in the art may modify, replace or combine the various technical features of the technical solution of the present invention based on the principles described in detail in this application without departing from the spirit and scope of the present invention. All such technical solutions and their variations are included in the scope of the claims of this application or their equivalents.

[0205] In order to experimentally verify the feasibility and effect of the present application, the present application provides the following specific exemplary implementation scheme:

[0206] 1. Exemplary Embodiments of the Methods of the Invention

[0207] (1) Diagram:

[0208] Figure 1 Exemplary embodiments of the method of the present invention are schematically described in order to illustrate the basic principles of the method of the present invention. Figure 1 A(The above picture is Figure 1 A, the following figure is Figure 1 B) schematically depicts an exemplary embodiment of detecting one target nucleic acid molecule using one detection probe and one tag primer.

[0209] It should be noted that:

[0210] Figure 1 The annotations for each symbol in A are as follows:

[0211] 1) IDx: Derived from Probe, mismatched bases are introduced to make its complementary chain C-IDx specifically recognized by Probe, and the melting curve Tx is distinguished by its specific C-IDx.

[0212] 2) F / R: Target sequence-specific upstream primer, which varies depending on the specificity of different target sequences.

[0213] 3) FAX / HEX etc.: indicates different fluorescent group modifications, including but not limited to FAX, HEX, etc.

[0214] 4) PO4 / C7 etc.: represents different groups or other modifications, including but not limited to PO4, C7, etc.

[0215] Figure 1 The annotations for each symbol in B are as follows:

[0216] The differences between TXx generated by C-IDx and the fluorescence signal channels (FAX and HEX, etc.) are integrated to form a three-dimensional matrix-specific TX, -d(Ft) / dT, and RX discrimination.

[0217] ① Melting curve analysis of C-IDx combined with probe Probe, distinguishing according to its TX value

[0218] ②Depending on the different signal channels of the probe modification group, further interpretation can be achieved by combining -d(Ft) / dT and RX.

[0219] (2) Implementation process principles and results analysis

[0220] like Figure 1 As shown, in this embodiment:

[0221] Step 1:

[0222] A blocking fluorescent probe (carrying a fluorescent group and a blocking group phosphorylation group / C7NH2) is provided, and for each target nucleic acid molecule (T1-T6-X), an upstream primer (upstream primer 1-6-X), a downstream primer (downstream primer 1-6-X), a class of molecular label groups (ID1-ID6-IDX) are respectively designed and provided, and the label primers and the blocking fluorescent probe are formed together with the primers;

[0223] Each tag primer contains a unique molecular tag sequence (molecular tag sequence 1-6-X), which can hybridize with the detection probe. The position where each molecular tag sequence hybridizes with the detection probe is unique, but there may be overlapping regions between them. For example, Figure 1 As shown in A, the specific primer binds to the target, and the complementary chain of the label (ID1-ID6-IDX) in the corresponding label primer binds to the probe, but each label in the label group has different binding abilities to the probe. The hybridization position of the molecular label sequence 1 on the detection probe overlaps with the molecular label sequence 2, and the hybridization position of the molecular label sequence 3 on the detection probe overlaps with the molecular label sequence 4. Similarly, the label IDX in the label group overlaps with any other label in the label group.

[0224] Step 2: PCR amplification:

[0225] During the detection process, 6 upstream primers and 6 downstream primers hybridize (anneal) with their corresponding target nucleic acid molecules respectively; then, under the action of nucleic acid polymerase, all upstream primers and downstream primers are extended to form extension templates of molecular tag primers, and the sub-tag primers are extended by nucleic acid polymerase to produce 6 extension products (i.e., PCR amplification products).

[0226] Step 3: contacting the PCR amplification product with the detection probe under conditions that allow nucleic acid hybridization:

[0227] For example, Figure 1 As shown in B, the molecular tag probe can hybridize (anneal) with the six molecular tag product nucleic acid molecule sequences respectively, and together with the detection probe form six duplexes with different Tm values.

[0228] Step 4: Perform melting curve analysis on the product of step 3:

[0229] By melting curve analysis, the presence of a duplex with a specific Tm value can be determined, and the presence of a target nucleic acid molecule corresponding to the duplex can be determined. Therefore, in the method of the present invention, one detection probe and six molecular tags can be used to detect six target nucleic acid molecules.

[0230] Exemplary embodiments are as follows:

[0231] Example 1. Selection of PCR amplification conditions

[0232] In this example, the effect of PCR amplification conditions on PCR MCA assay based on different primer ratios and amplification methods was evaluated.

[0233] (1) Symmetric and asymmetric amplification

[0234] The experimental process includes the following steps:

[0235] Step 1, prepare a reaction system of corresponding concentration according to the corresponding raw materials provided in Table 1, and distinguish between preparing a symmetric reaction system (upstream primer concentration: downstream primer concentration = 1:1) and an asymmetric reaction system (upstream primer concentration: downstream primer concentration = 1:10);

[0236] Step 2: Two-step and three-step PCR were used to compare symmetric amplification and asymmetric amplification.

[0237] The amplification conditions used for the two-step PCR were: 95°C, 5 min; followed by 40 cycles (95°C, 15 s and 60°C, 40 s).

[0238] The amplification conditions used for the three-step PCR were: 95°C, 5 min; then 40 cycles (95°C, 15 s; 60°C, 30 s; and 72°C, 10 s);

[0239] Step 3. After the PCR is completed, perform denaturation and melting curve analysis according to the following procedure: 95°C, 2mins30s; cool to 28°C at a rate of 4.0°C / s and maintain for 10mins, then increase the temperature of the reaction system from 28°C to 76°C at a heating rate of 0.04°C / s, and collect fluorescent signals such as FAM and HEX during this process.

[0240] Experimental conditions:

[0241] Briefly, a 25-μL PCR reaction system was used to perform real-time PCR and subsequent MCA assay, wherein the detection probes used were Probe1 and Probe2, the polymerase used was Taq, and the template used was a specified amount (1 pg) of plasmid DNA.

[0242] In symmetric PCR, the primers used are an upstream primer and a downstream primer each having a concentration of 100 nM to 1000 nM, and the concentration of the upstream or downstream primer to which the molecular tag is linked is 20 nM to 100 nM.

[0243] In asymmetric PCR, the primers used are 20nM to 100nM of upstream primer (the primer is opposite to the probe, that is, its extension product binds to the probe) and 100nM to 1000nM of downstream primer. The concentration of the upstream or downstream primer connected with the molecular tag is 20nM to 100nM. The experimental results are as follows Figure 2 As shown:

[0244] Shown is a symmetrical amplification ( Figure 2 ①③) or asymmetric amplification ( Figure 2 ②④) melting curve of real-time PCR reaction; wherein, the real-time PCR reaction uses detection probes Probe1 and Probe2, polymerase Taq and a specified amount (1 pg) of plasmid DNA; and, the ★ line in the figure represents the experimental result using plasmid DNA as a template; the ┃ line represents the experimental result using water as a template (negative control).

[0245] result: Figure 2 The results show:

[0246] Under the conditions of symmetric amplification or asymmetric amplification, the post-PCR MCA assay based on the detection probes used being Probe1 and Probe2 can detect specific target peaks; under the two conditions, there are certain differences in the specific peaks produced by the PCR MCA assay. Therefore, in the post-PCR MCA assay, PCR amplification can be performed in an asymmetric manner.

[0247] (2) Two-step PCR and three-step PCR

[0248] Experimental conditions:

[0249] Briefly, a 25-μL PCR reaction system was used to perform real-time PCR and subsequent MCA assay, wherein the detection probes used were Probe1 and Probe2, the polymerase used was Taq, and the template used was a specified amount (1 pg) of plasmid DNA. The specific experimental conditions are shown in Table 1:

[0250] Table 1 Double detection primer probe and added concentration

[0251]

[0252]

[0253] Except for the PCR reaction conditions, the PCR reaction system and MCA assay conditions used in the two-step PCR and three-step PCR were the same as those in Example 1 (1):

[0254] The amplification conditions used for the two-step PCR were: 95°C, 5 min; followed by 40 cycles (95°C, 15 s and 60°C, 40 s).

[0255] The amplification conditions used for the three-step PCR were: 95°C, 5 min; then 40 cycles of (95°C, 15 s; 60°C, 30 s; and 72°C, 10 s);

[0256] After the PCR was completed, the following procedures were used for melting curve analysis: 95°C for 2 mins30 s; cooling to 28°C at a rate of 4.0°C / s and maintaining for 10 mins, then heating the reaction system from 28°C to 75°C at a rate of 0.04°C / s, and collecting FAM and HEX fluorescence signals during this process. Figure 2 shown.

[0257] Experimental results:

[0258] Figure 2 The two-step method ( Figure 2 A and 2B) or three-step method ( Figure 2C and 2D) for amplification; wherein, the real-time PCR reaction uses molecular label probes Probe1 and Probe2, polymerase Taq and specified amounts (1 pg) of Sau plasmid and 16S plasmid; and, the ★ line in the figure represents the experimental results using Sau plasmid as a template, and the ┃ line represents the experimental results using 16S plasmid as a template; ①③ represent the experimental results using water as a template (negative control).

[0259] Figure 2 The results show that under the conditions of two-step PCR or three-step PCR, the post-PCR MCA assay based on the detection of fluorescent probes can detect specific target peaks. In addition, under the conditions of two-step PCR, the post-PCR MCA assay showed mixed peaks (non-specific peaks); under the conditions of three-step PCR, the post-PCR MCA assay did not show non-specific peaks.

[0260] These results indicate that, in the post-PCR MCA assay, PCR amplification can be performed in an asymmetric amplification manner; and that the post-PCR MCA assay is more specific when a three-step method is used to perform PCR amplification.

[0261] Example 2. Quadruple Detection

[0262] Experimental conditions:

[0263] In this embodiment, four molecular tag primers and two detection fluorescent probes are used to achieve simultaneous detection and differentiation of four target sequences in a single post-PCR MCA assay (i.e., quadruple detection). In this experiment, three detection objects (Sau plasmid, Cpn plasmid and Hin plasmid) and one sample assumed internal reference (16S plasmid) are used as exemplary target sequences to be detected, and a specified amount of 1pg plasmid DNA is used as the sample to be detected.

[0264] Briefly, real-time PCR was performed using a 25-μL PCR reaction system, which included 5× buffer A, 5.0 U of polymerase TaqHS (TaKaRa, Dalian), primers (8 primers and 4 molecular tag primers) and probes (Probe1 and Probe2) as shown in Table 2, and a specified amount of 1 pg of plasmid DNA; 3 replicates were set.

[0265] Table 2 Quadruple detection primers, probes and added concentrations

[0266]

[0267]

[0268] Except for the PCR reaction conditions, the PCR reaction system and MCA assay conditions used in this example are the same as those in Example 1(1); the PCR reaction conditions used are as follows: the amplification conditions used for the three-step PCR are: 95°C, 5 min; then 40 cycles of (95°C, 15 s; 60°C, 30 s; and 72°C, 10 s).

[0269] After PCR was completed, the following procedures were used for melting curve analysis: 95°C for 2 mins30 s; cooling to 28°C at a rate of 4.0°C / s and maintaining for 10 mins, then heating the reaction system from 28°C to 76°C at a rate of 0.04°C / s, and collecting FAM and HEX fluorescence signals during this process. The experimental instrument used was Hongshi 96P. The results are shown in Figure 2. Figure 3 Shown on the left.

[0270] Experimental results:

[0271] Figure 3 The first left shows the melting curve of a real-time PCR reaction using two blocking fluorescent probes Probe1 and Probe2 (which carry fluorescent groups and blocking group phosphorylation groups / C7NH2) for 4-plex detection using a three-step method and asymmetric PCR; wherein the real-time PCR reaction uses 8 primers (SauF, SauR, 16SF, 16SR, CpnF, CpnR, HinF and HinR), 4 molecular tag primers (SauTF, 16STF, CpnTF and HinTF), polymerase Taq and a specified amount (1 pg) of Sau plasmid, 16S plasmid, Cpn plasmid and Hin plasmid; and the ★ line in the figure represents the experimental results using Sau plasmid and Cpn plasmid DNA as templates; the ┃ line in the figure represents the experimental results using 16S plasmid and HinTF plasmid DNA as templates.

[0272] Figure 3 The result on the left shows that four specific peaks that can be distinguished from each other appear in the melting curve (two peaks in the FAM signal channel are Sau and Cpn respectively; two peaks in the HEX signal channel are 16S and Hin respectively, a total of four peaks), and their melting points correspond to the duplex formed by the extension product of the fluorescent probe and the molecular tag sequence in the tag primer. This result shows that using four molecular tag sequences and two fluorescent probes, four target sequences can be detected and distinguished simultaneously in a single post-PCR MCA assay (i.e., quadruple detection).

[0273] Example 3. Eight-fold detection

[0274] Experimental conditions:

[0275] In this embodiment, 8 molecular tag primers and 2 detection fluorescent probes are used to achieve simultaneous detection and differentiation of 8 target sequences in a single post-PCR MCA assay (i.e., eightfold detection). In this experiment, 7 detection objects (Sau plasmid, Mca plasmid, Mtb plasmid, Cpn plasmid, Kpn plasmid, Spn plasmid and Hin plasmid) and 1 sample assumed internal reference (16S plasmid) are used as exemplary target sequences to be tested, and 1pg of plasmid DNA in a specified amount is used as the sample to be tested. In brief, a 25-μL PCR reaction system is used for real-time PCR, and the PCR reaction system includes 5×buffer A, 5.0U of polymerase TaqHS (TaKaRa, Dalian), primers (16 primers and 8 molecular tag primers) and probes (Probe1 and Probe2) as shown in Table 3, and a specified amount of 1pg plasmid DNA; 3 replicates are set.

[0276] Table 3 Eightfold detection primers, probes and added concentrations

[0277]

[0278]

[0279] Except for the PCR reaction conditions, the PCR reaction system and MCA assay conditions used in this example are the same as those in Example 1(1); the PCR reaction conditions used are as follows: the amplification conditions used for the three-step PCR are: 95°C, 5 min; then 40 cycles of (95°C, 15 s; 60°C, 30 s; and 72°C, 10 s).

[0280] After PCR was completed, the following procedures were used for melting curve analysis: 95°C for 2 mins30 s; cooling to 28°C at a rate of 4.0°C / s and maintaining for 10 mins, then heating the reaction system from 28°C to 76°C at a rate of 0.04°C / s, and collecting FAM and HEX fluorescence signals during this process. The experimental instrument used was Hongshi 96P. The results are shown in Figure 2. Figure 3 Shown on the right.

[0281] Experimental results:

[0282] Figure 3The first figure on the right shows the melting curve of the real-time PCR reaction using two blocking fluorescent probes Probe1 and Probe2 (which carry fluorescent groups and blocking group phosphorylation groups / C7NH2) for 8-plex detection using the three-step method and asymmetric PCR; wherein the real-time PCR reaction uses 16 primers (SauF, SauR, 16SF, 16SR, McaF, McaR, HinF, HinR, MtbF, MtbR, CpnF, CpnR, KpnF, KpnR, SpnF, SpnR), 8 molecular tag primers (SauTF , 16STF, McaTF, HinTF, MtbTF, CpnTF, KpnTF, SpnTF), polymerase Taq and specified amount (1pg) of Sau plasmid, 16S plasmid, Mca plasmid, Hin plasmid, Mtb plasmid, Cpn plasmid, Kpn plasmid and Spn plasmid; and, the ★ line in the figure represents the experimental results using Sau plasmid, Mca plasmid, Sau plasmid and Cpn plasmid DNA as templates; the ┃ line in the figure represents the experimental results using 16S plasmid, HinTF plasmid, Kpn plasmid and Spn plasmid DNA as templates.

[0283] Figure 3 The result on the right shows that four specific peaks that can be distinguished from each other appear in the melting curve (the four peaks of the FAM signal channel are Sau, Mtb, Mca and Cpn, respectively; the four peaks of the HEX signal channel are Kpn, 16S, Hin and Spn, respectively, for a total of 8 peaks), and their melting points correspond to the duplexes formed by the extension products of the fluorescent probe and the molecular tag sequence in the tag primer. This result shows that using 8 molecular tag sequences and 2 fluorescent probes, it is possible to simultaneously detect and distinguish 8 target sequences in a single post-PCR MCA assay (i.e., octal detection).

[0284] Example 5. Twelve-fold detection

[0285] Experimental conditions:

[0286] In this embodiment, 12 molecular tag primers and 2 detection fluorescent probes are used to achieve simultaneous detection and differentiation of 12 target sequences in a single post-PCR MCA assay (i.e., twelve-fold detection). In this experiment, 11 detection objects (Aba plasmid, Sau plasmid, Mtb plasmid, Mca plasmid, Cpn plasmid, Sma plasmid, Kpn plasmid, Mpn plasmid, Pae plasmid, Hin plasmid and Spn) and 1 sample assumed internal reference (16S plasmid) are exemplary target sequences to be detected, and a specified amount of 1pg of plasmid DNA is used as the sample to be detected.

[0287] Briefly, real-time PCR was performed using a 25-μL PCR reaction system, which included 5× buffer A, 5.0 U of polymerase TaqHS (TaKaRa, Dalian), primers (24 primers and 12 molecular tag primers) and probes (Probe1 and Probe2) as shown in Table 4, and a specified amount of 1 pg of plasmid DNA; 3 replicates were set.

[0288] Table 4 Twelve-plex detection primers, probes and added concentrations

[0289]

[0290]

[0291] Except for the PCR reaction conditions, the PCR reaction system and MCA determination conditions used in this example are the same as those in Example 1 (1); the PCR reaction conditions used are as follows: the amplification conditions used in the three-step PCR are: 95°C, 5min; then 40 cycles of (95°C, 15s; 60°C, 30s; and 72°C, 10s). After the PCR is completed, the denaturation and melting curve analysis are performed according to the following procedures: 95°C, 2mins30s; cool to 28°C at a rate of 4.0°C / s and maintain for 10mins, then increase the temperature of the reaction system from 28°C to 76°C at a heating rate of 0.04°C / s, and collect fluorescent signals such as FAM and HEX during this process. The experimental instrument used is Hongshi 96P. The results are as follows: Figure 4 shown.

[0292] Experimental results:

[0293] Figure 4The melting curve of the real-time PCR reaction using 12-plex detection using the three-step method and asymmetric PCR and using two blocking fluorescent probes Probe1 and Probe2 (which carry a fluorescent group and a blocking group phosphorylation group / C7NH2) is shown; wherein the real-time PCR reaction uses 24 primers (AbaF, AbaR, SauF, SauR, MtbF, MtbR, McaF, McaR, CpnF, CpnR, KpnF, KpnR, 16SF, 16SR, MpnF, MpnR, PaeF, PaeR, HinF, HinR, SpnF, SpnR), 12 molecular tag primers (AbaTF, SauTF, MtbTF, McaTF, C pnTF, SmaTF, KpnTF, 16STF, MpnTF, PaeTF, HinTF, SpnTF), polymerase Taq and specified amount (1pg) of Aba plasmid, Sau plasmid, Mtb plasmid, Mca plasmid, Cpn plasmid, Sma plasmid, Kpn plasmid, 16S plasmid, Mpn plasmid, Pae plasmid, Hin plasmid and Spn plasmid; and, the ★ line in the figure represents the experimental results using Aba plasmid, Sau plasmid, Mtb plasmid, Mca plasmid and Cpn plasmid DNA as templates; the ┃ line in the figure represents the experimental results using Sma plasmid, Kpn plasmid, 16S plasmid, Mpn plasmid, Pae plasmid, Hin plasmid and Spn plasmid DNA as templates.

[0294] Figure 4 The results showed that five specific peaks that can be distinguished from each other appeared in the melting curve (the 5 peaks of the FAM signal channel are Aba, Sau, Mtb, Mca and Cpn, respectively; the 7 peaks of the HEX signal channel are Sma, Kpn, 16S, Mpn, Pae, Hin and Spn, a total of 12 peaks), whose melting points correspond to the duplexes composed of the extension products of the fluorescent probe and the molecular tag sequence in the tag primer. This result shows that using 12 molecular tag sequences and 2 fluorescent probes, 12 target sequences can be detected and distinguished simultaneously in a single post-PCR MCA assay (i.e., twelve-fold detection).

[0295] In summary, the present invention has the following beneficial effects:

[0296] The present invention provides a simple, efficient, low-cost multiplex detection method, which can simultaneously detect the presence of multiple target nucleic acid sequences in a sample, and the maximum number of target nucleic acid sequences that can be detected is not limited by the number of label probes (i.e., detection probes) used, that is, the method of the present invention can achieve simultaneous detection (multiple detection) of significantly more target nucleic acid sequences based on a relatively limited number of label probes (i.e., detection probes). The present invention also provides a probe label set and a kit, which can be used to implement the above-mentioned method for detecting the presence of X types of target nucleic acid sequences in a sample, solving the problems of high detection instrument price, complex operation, easy contamination, high detection cost, etc. existing in traditional detection technology and finished detection kits.

Claims

1. A method for detecting the presence of X target nucleic acid sequences in a sample, X ≥ 1, characterized in that: The method comprises the following preparation steps: For each target nucleic acid sequence to be detected, an upstream oligonucleotide sequence, a downstream oligonucleotide sequence, an InDex tag sequence, a tag primer oligonucleotide sequence and a probe that specifically bind to the target nucleic acid sequence are provided; Performing PCR amplification on the sample using the provided oligonucleotide sequence to obtain a PCR amplification product; Providing Y detection probes, contacting the PCR amplification product with one of the detection probes to obtain a hybrid; wherein Y is an integer greater than 0; Performing melting curve analysis on the hybrid: determining whether the X target nucleic acid sequences exist in the sample based on the result of the melting curve analysis.

2. The method according to claim 1, characterized in that: The upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; the tag primer comprises a probe tag sequence and a target-specific sequence from the 5' to the 3' direction, the probe tag sequence comprises a sequence not complementary to the target nucleic acid sequence, and the target-specific sequence comprises a sequence complementary to the target nucleic acid sequence; when the tag primer oligonucleotide sequence hybridizes with the target nucleic acid sequence, the tag sequence is located upstream of the target-specific sequence; and the sequences contained in all InDex tag sequences are different from each other; Wherein, each PCR amplification product independently comprises a label primer oligonucleotide sequence and a template sequence from the 5' to 3' direction; the detection probe comprises at least N InDex label sequences, and N is greater than or equal to Y, and the detection probes are respectively consistent with the sequence of the provided probe or a part of its sequence; each detection probe is independently labeled with a reporter group and a blocking group, wherein the reporter group can emit a signal, and the blocking group can hinder PCR amplification; when the detection probe hybridizes with the complementary sequence of each label primer oligonucleotide sequence, the signals emitted are different.

3. The method according to claim 1, characterized in that: When X≥2, the Y detection probes are each labeled with the same or different reporter groups.

4. The method according to claim 1, characterized in that: The Y detection probes contain the same or different reporter groups; when performing melting curve analysis on the hybrid, the presence of a certain target nucleic acid sequence is determined based on the melting peak in the obtained melting curve; Alternatively, the reporter groups contained in the Y detection probes are different from each other; when performing melting curve analysis on the hybrid, the signal of each reporter group is monitored in real time, and multiple melting curves corresponding to the complementary chain of the label sequence in the corresponding amplification product and the signal of a reporter group are obtained, and then the presence of a certain target nucleic acid sequence is determined based on the signal type of the reporter group and the melting peak in the melting curve.

5. The method according to claim 2, characterized in that: The probe or detection probe or tag primer oligonucleotide sequence has at least one or more of the following characteristics: (1) The probe comprises one or more of naturally occurring nucleotides, modified nucleotides, and non-natural nucleotides; (2) The probe is 15-150 nt in length; (3) the length of the target-specific sequence in the tag primer oligonucleotide sequence is 15-150 nt; (4) The length of the probe tag sequence in the tag primer oligonucleotide sequence may be 15-150 nt; (5) Each detection probe of the probe is independently labeled with a reporter group and a blocking group; (6) The reporter group in the detection probe is a fluorescent group; and the blocking group is a molecule or group that enables the probe to be used as an amplification primer; (7) The detection probe has resistance to nuclease activity; (8) The detection probe is linear or has a hairpin structure.

6. The method according to claim 1, characterized in that: Under conditions that allow polymerase chain reaction, subjecting the sample to PCR amplification via the provided oligonucleotide sequence to obtain a PCR amplification product; The PCR amplification product is contacted with one of the detection probes under conditions that allow nucleic acid hybridization.

7. The method according to claim 1, characterized in that: Under conditions that allow the nucleic acid polymerase to perform an extension reaction, the nucleic acid polymerase uses the target nucleic acid sequence in the sample as a template to extend the label primer hybridized to the template, thereby forming a duplex to obtain a PCR amplification product.

8. The method according to claim 1, characterized in that: The step of obtaining the PCR amplification product is carried out by a scheme comprising the following steps (a)-(f): (a) providing Y detection probes and N tags, and for each target nucleic acid sequence to be detected, providing an upstream oligonucleotide sequence, a probe, a tag primer oligonucleotide sequence and a downstream oligonucleotide sequence; (b) mixing the sample to be detected with the detection probe, the upstream oligonucleotide sequence, the tag primer oligonucleotide sequence, the probe, the downstream oligonucleotide sequence and a template-dependent nucleic acid polymerase having 5' nuclease activity provided in step (a); (c) incubating the mixture of step (b) under conditions that allow nucleic acid denaturation; (d) incubating the product of step (c) under conditions that allow annealing or hybridization of the nucleic acids; (e) incubating the product of step (d) under conditions that allow nucleic acid extension (f) Repeating steps (c)-(e) one or more times.

9. The method according to claim 2, characterized in that: The operation process of the melting curve analysis step is: The hybrid is gradually heated or cooled, and the signal emitted by the reporter group on each detection probe is monitored in real time, so as to obtain a curve showing the change of the signal intensity of each reporter group with the change of temperature; The obtained curve is differentiated to obtain the melting curve of the hybrid; According to the melting peak in the melting curve, the presence of the InDex tag corresponding to the melting peak is determined; then, the presence of the target nucleic acid sequence corresponding to the tag is determined through the correspondence between the tag sequence in the tag primer and the target nucleic acid sequence.

10. A probe tag set for detecting the presence of X target nucleic acid sequences in a sample, X≥1, characterized in that: It comprises a detection probe; the detection probe corresponds to at least one probe label; Wherein, the tag primers of the probe tag each independently comprise a probe tag sequence and a target-specific sequence from the 5' to 3' direction, the target-specific sequence comprises a sequence complementary to a target nucleic acid sequence, and the probe tag sequence comprises a sequence not complementary to the target nucleic acid sequence; The detection probe comprises from 5' to 3' direction: a capture sequence complementary to each tag group subsequence or part thereof, and a template sequence; and the detection probe is labeled with a reporter group and a blocking group, wherein the reporter group can emit a signal, and the blocking group or modification can prevent the probe from being extended.

11. The probe tag set according to claim 10, characterized in that: The tag sequences contained in all the tag primers are different from each other; and the target-specific sequences contained in all the tag primers are different from each other; And / or, the primers contained in all the tag primers each target a different target nucleic acid sequence.

12. The probe tag set according to any one of claims 10 to 11, characterized in that: The tag primer has at least one or more of the following characteristics: (1) The length of the target-specific sequence in the tag primer oligonucleotide sequence is 15-150 nt; (2) The length of the probe tag sequence in the tag primer oligonucleotide sequence may be 15-150 nt; The detection probe has at least one or more of the following characteristics: (1) The reporter group in the detection probe is a fluorescent group; and the blocking group is a molecule or group that enables the probe to be used as an amplification primer; (2) The detection probe has resistance to nuclease activity; (3) The detection probe is linear or has a hairpin structure.

13. A kit, characterized in that: comprising one or more probe tag sets; Wherein, the probe tag group adopts the probe tag group as described in any one of claims 10-12.

14. The kit according to claim 13, characterized in that: Contains at least one probe tag set; Among them, all label primers of the probe label group target different target nucleic acid sequences, the label sequences contained in all label primers are different from each other, the target specific sequences contained in all label primers are different from each other, and all detection probes are independently labeled with the same or different reporter groups.

15. The kit according to claim 13, characterized in that: It also comprises any combination of one or more of an upstream oligonucleotide sequence, a downstream oligonucleotide sequence, a universal primer, an enzyme having 5' nuclease activity, and a nucleic acid polymerase.

16. The kit according to any one of claims 13 to 15, characterized in that: It also includes any combination of one or more reagents for nucleic acid hybridization, reagents for nucleic acid extension, and reagents for nucleic acid amplification.

17. A use of a probe tag set or a kit, characterized in that: The probe tag set or the kit is used to implement the method according to claims 1-9; Wherein, the probe tag group adopts the probe tag group as described in any one of claims 10-12; and the kit adopts the kit as described in any one of claims 13-16.

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