A probe set, a kit, and a method for detecting two or more target nucleic acids

Abstract

This application belongs to the field of multiplex detection of nucleic acid molecules, and relates to a probe set, a kit, and a method for detecting two or more target nucleic acids. The probe set includes at least one linear fluorescent probe and at least two mediator probes. Each mediator probe independently contains a mediator sequence and a target probe sequence from the 5' to 3' direction. The linear fluorescent probe contains a label sequence and a positioning sequence from the 5' to 3' direction. When multiple mediator sequences are simultaneously paired on the linear fluorescent probe, there is at least 2 nt of overlapping nucleotides between each pair of mediator sequences; or, if the downstream mediator sequence and the upstream mediator sequence have less than 2 nt of non-overlapping nucleotides, the 5' end of the upstream mediator sequence is modified with a nuclease-resistant modification. The technical solution provided by this application can prevent the mediator sequence of the mediator probe from being cleaved by enzymes with 5' nuclease activity, resulting in a relatively flat baseline for the melting analysis curve, which is beneficial for the identification of the melting peak and the reading of the Tm value.

Description

Technical Field

[0001] This application relates to the field of nucleic acid molecular multiplex detection technology, and more specifically, to a probe set, a kit, and a method for detecting two or more target nucleic acids. Background Technology

[0002] PCR technology is an effective means of detecting specific nucleic acid sequences. The introduction of TaqMan probes allows for the simultaneous detection of amplified fluorescent signals of five different target nucleic acid sequences in a single-tube reaction system. In the TaqMan probe method disclosed in US Patent 5538848A, the fluorescent probe hybridizes with the target nucleic acid and its amplicons. When the extension reaction initiated by the upstream primer, carrying DNA polymerase, touches the 5' end of the fluorescent probe, it progressively cleaves the fluorescent probe from 5' to 3', causing the fluorescent group to separate from the quenching group and thus releasing a fluorescent signal. When the 5' local sequence of the fluorescent probe does not pair with the target nucleic acid, that 5' fragment is also cleaved.

[0003] Furthermore, Faltin et al. (Clinical Chemistry 2012, 58(11):1546-1556) first used DNA fragments cleaved by DNA polymerase 5' nuclease activity to trigger the fluorescence signal of fluorescently modified probes, introducing "mediator probes" into the fluorescent PCR system.

[0004] US Patent 2013 / 0109588 A1 discloses a fluorescent PCR method for melting curve analysis. Similar to the method of Faltin et al., the "PTO" probe in this patent is equivalent to a mediator probe, and there is also a "CTO" probe used as a fluorescent probe. The difference is that after the mediator reacts with the fluorescent probe, it does not cause the fluorescent probe to be cleaved, but rather they work together to form a double strand. This causes the fluorescent probe to unwind from its original single-stranded coiled state and release a fluorescent signal. In particular, the DNA double-stranded fragment generated by the reaction of the mediator and the fluorescent probe can dissociate back into single strands after being raised to a specific temperature. The fluorescent probe returns to its coiled state, causing fluorescence regression quenching, which manifests as a characteristic melting peak in melting analysis. In actual detection, each target nucleic acid sequence corresponds to one mediator probe and one fluorescent probe, which increases the number of probes compared to the TaqMan probe method. However, nucleic acid diagnostic products developed using this technology can utilize 5 fluorescence channels to detect 15 target nucleic acid sequences in a single tube.

[0005] Chinese patent CN 108823287 A discloses a melting curve method for detecting multiple target nucleic acids using a mediator probe. It also utilizes melting analysis to distinguish double strands formed by different mediators with fluorescent probes, thus enabling single-tube detection of multiple target nucleic acids. The innovation lies in allowing one fluorescent probe to bind with multiple mediators to form their own specific double strands. In the implementation method, when detecting n target nucleic acids, n mediator primers and m fluorescent probes can be used, where m < n. Reducing the number of fluorescent probe types in a single reaction system means a reduction in the total amount of fluorescent probes used. Furthermore, the patent uses a hairpin-structured fluorescent probe. In the fluorescence quenching state, the distance between the fluorescent group modified in the "stem" region and the quenching group is smaller than that of a naturally curled fluorescent probe, thus achieving better quenching effects. Further, the patent introduces a homology tag-assisted primer-free dimer system, effectively solving the problem of inequivalence between primer dimers and multiplex amplification reactions. A related patent, CN 112094930 A, which also uses this technology, describes a method for detecting 92 serotypes of Streptococcus pneumoniae using a two-tube PCR reaction.

[0006] However, current real-time fluorescence amplification curve methods based on medium probes require the addition of one corresponding medium probe and one fluorescent probe for each target nucleic acid, which does not reduce the use of fluorescent probes. In fact, the number of probes used is doubled compared to traditional real-time fluorescence amplification curve methods, and the number of target nucleic acid types detected is still limited by the number of fluorescence channels in the instrument. The introduction of melting curve analysis removes the limitation on the number of target types that can be detected by this method. While existing technologies have achieved the detection of 15 or more target nucleic acids in a single reaction, some shortcomings still exist in practical testing. Initially, when melting curve methods were used, each target nucleic acid still corresponded to one fluorescent probe. However, increasing the types and amounts of fluorescent probes would lead to excessively high background fluorescence of the reagents or increase the design complexity, which limits the development of this technology. While using multi-position fluorescent probes has somewhat mitigated the problem of excessive probe types when detecting multiple target nucleic acids, this technique presents a challenge. If the mediator probe is continuously cleaved and the mediator extends within the same reaction system, the extension of the mediator upstream of the multi-position fluorescent probe by DNA polymerase containing 5' nuclease activity will inevitably cleave the mediator downstream. This will unavoidably affect the downstream mediator and the resulting double strand. The double strand affected by the upstream mediator sequence typically exhibits a "collapse" of its melting peak towards lower Tm values ​​on the melting peak curve, resulting in a significant reduction in the normally specific Tm value, or even making it difficult to read. Furthermore, some patents favor hairpin structures as fluorescent probes. While this structure offers better quenching effects than linear probes, when using such probes for melting analysis, the baseline of most melting curves, especially for blank samples, is not flat or horizontal. A possible cause of this phenomenon is the structural instability of the hairpin structure during melting and heating. Summary of the Invention

[0007] The technical problems to be solved by the embodiments of this application are: the mediator subsequence of the medium probe is cleaved by an enzyme with 5' nuclease activity during the amplification process, making it difficult to read the melting temperature value; and the use of a fluorescent probe with a hairpin structure that has a better quenching effect causes unevenness in the melting curve baseline.

[0008] To address the aforementioned technical problems, this application provides a probe set, employing the technical solution described below:

[0009] It includes at least one linear fluorescent probe and at least two mediator probes; wherein each of the mediator probes independently comprises a mediator sub-sequence and a target probe sequence from the 5' to 3' direction, the target probe sequence comprising a sequence that is identical or complementary to the target nucleic acid to be tested, and the mediator sub-sequence comprising a sequence complementary to the linear fluorescent probe;

[0010] The linear fluorescent probe contains a labeling sequence and a mooring sequence from 5' to 3', the mooring sequence from 3' to 5' includes a capture sequence complementary to each of the mediator subsequences or a portion of the mediator subsequence, and the labeling sequence is labeled with a fluorescent group and a quenching group;

[0011] When multiple mediator sequences are simultaneously paired on the linear fluorescent probe, there is at least 2 nt of overlapping nucleotides between each pair of mediator sequences. Alternatively, if the mediator sequence downstream of the linear fluorescent probe and the mediator sequence upstream of the linear fluorescent probe have less than 2 nt of non-overlapping nucleotides, then the 5' end of the mediator sequence upstream of the linear fluorescent probe is modified with a nuclease-resistant modification.

[0012] To address the aforementioned technical problems, this application also provides a reagent kit, which employs the following technical solution:

[0013] The kit includes the probe set as described above, an enzyme with 5' nuclease activity, and a nucleic acid polymerase.

[0014] To address the aforementioned technical problems, this application also provides a method for detecting two or more target nucleic acids, employing the technical solution described below:

[0015] Step S10: Hybridize the target nucleic acid to be tested with the upstream primer and the medium probe, wherein the target probe sequence of the upstream primer and the medium probe is paired with the target nucleic acid to be tested, and the remaining medium subsequence in the medium probe is in a free single-stranded state.

[0016] In step S20, after the upstream primer contacts an enzyme with 5' nuclease activity, it extends from the 3' end. During the extension reaction, the enzyme with 5' nuclease activity contacts the 5' end of the target probe sequence and begins cleaving from the 3' end of the first nucleotide of the target probe sequence, releasing the complete mediator sequence; or, when the enzyme with 5' nuclease activity contacts the upstream primer, it also contacts the 5' end of the target probe sequence, cleaving the mediator probe and releasing the complete mediator sequence.

[0017] In step S30, the free mediator sequence targets a specific site on the linear fluorescent probe and, under the action of nucleic acid polymerase, extends from the 3' end of the mediator sequence to form a specific double strand.

[0018] Step S40: Perform melting curve analysis on the double strand and determine whether the target nucleic acid to be tested exists based on the results of the melting curve analysis.

[0019] Compared with the prior art, this application has the following main advantages:

[0020] The probe set provided in this application has higher detection throughput potential. The same detection system can detect mixed samples of multiple target nucleic acids as well as samples of a single target nucleic acid. The linear fluorescent probes used have narrower peaks, which facilitates multiplex detection resolution and solves the problem of uneven melting curve baseline. The melting analysis curve of the probe set in this application has a relatively flat baseline, which avoids the cleavage of the mediator subsequence of the mediator probe by enzymes with 5' nuclease activity, which is beneficial for the identification of melting peaks and Tm value reading, and has higher sensitivity. The amount of fluorescent probes used in this application is reduced, the detection throughput per reaction is increased, the cost of reagents and consumables and labor costs are reduced, and thus the detection cost is reduced. Attached Figure Description

[0021] To more clearly illustrate the solutions in this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 AG is a schematic diagram of the melting curve obtained by performing a PCR reaction using the probe set according to the first embodiment provided in this application;

[0023] Figure 2 AG is a schematic diagram of the melting curve obtained by performing a PCR reaction using the probe set according to the second embodiment provided in this application;

[0024] Figure 3 AG is a schematic diagram of the melting curve obtained by performing a PCR reaction using the probe set according to the third embodiment provided in this application;

[0025] Figure 4 AG is a schematic diagram of the melting curve obtained by PCR reaction based on the first comparative probe set provided in this application;

[0026] Figure 5 This is a schematic diagram of the corresponding binding sites of the mediator subsequence and the fluorescent probe according to the first comparative example provided in this application;

[0027] Figure 6 AG is a schematic diagram of the melting curve obtained by PCR reaction based on the probe set of the second comparative example provided in this application. Detailed Implementation

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0029] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0030] The nucleic acid chemistry experimental procedures described in this application are all standard procedures widely used in the relevant fields. To better understand this application, definitions and explanations of some key terms are provided below.

[0031] As used herein, the term "target nucleic acid" refers to the target nucleic acid sequence to be detected, typically the sequence amplified by PCR primers. Nucleic acid molecules (amplifiers) synthesized with the same sequence during the PCR process can also be considered "target nucleic acids." Target nucleic acids can be double-stranded or single-stranded, and can exist alone or as part of a larger nucleic acid molecule, such as a genome, transcriptome, plasmid, or artificially synthesized nucleic acid molecule. In this application, the detection of target nucleic acids refers only to the sequence amplified in the detection reaction; the detection of target nucleic acids does not necessarily indicate the presence of nucleic acid sequences other than the target nucleic acid in the sample.

[0032] As used in this article, the term "targeting" refers to the process or function by which a nucleic acid molecule specifically binds to a target nucleic acid or a sequence complementary to the target nucleic acid under conditions such as hybridization, annealing, or extension. Specific binding means that a nucleic acid molecule, under conditions such as hybridization, annealing, or extension, binds only to its corresponding target nucleic acid or a sequence complementary to the target nucleic acid, and not to other nucleic acid sequences.

[0033] As used herein, the term "mediator probe" refers to a single-stranded nucleic acid molecule containing a mediator sequence and a target probe sequence in the 5' to 3' direction. The mediator sequence and target probe sequence on the same mediator probe are corresponding. In the embodiments of this application, the 3' of the mediator sequence and the 5' of the target probe sequence share a single nucleotide, i.e., there is a 1 nt overlap. The mediator sequence targets only the designated site on the fluorescent probe. The target probe sequence targets the target nucleic acid or its amplicons. Therefore, under conditions allowing nucleic acid hybridization, annealing, or extension, the target probe sequence of the mediator probe hybridizes or anneals with the target nucleic acid sequence to form a double-stranded structure, while the remaining mediator sequence does not hybridize with the target nucleic acid sequence and remains a single-stranded structure.

[0034] As used herein, the term "fluorescent probe" refers to an oligonucleotide sequence modified with a fluorescent group. The preferred linear fluorescent probe used in this application contains a labeling sequence and a mooring sequence from the 5' to 3' direction. The labeling sequence may label the fluorescent group and quencher group respectively in the middle or on both sides of the sequence, maintaining an appropriate distance between the fluorescent group and the quencher group. The labeling sequence may also modify only the fluorescent group and use base quenching according to the method of patent CN 107365850B. The mooring sequence may be complementary to one or more mediator sequences. The pairing sequence of each mediator in the mooring sequence is called a mooring; probes with multiple moorings are called multi-mooring fluorescent probes.

[0035] As used in this article, the term "complementary" refers to the ability of two nucleic acid sequences to form hydrogen bonds and thus a double helix. "Complementary" in this article can refer to two nucleic acid sequences that fully conform to the base-pairing principle, or two nucleic acid sequences that only partially conform to the base-pairing principle but can specifically form a stable double helix under certain conditions. The terms "complementary" and "pairing" in this article can be interpreted in the same way.

[0036] As used herein, the term "hybridization" refers to the process by which complementary single-stranded nucleic acid molecules form double-stranded nucleic acids. Typically, two completely complementary or substantially complementary nucleic acid sequences can hybridize. The complementarity required for hybridization of two nucleic acid sequences depends on the hybridization conditions used, particularly temperature.

[0037] As used herein, the term “upstream” describes the relative position of two nucleic acid sequences (or two nucleic acid molecules) and has the meaning commonly understood by those skilled in the art. For example, stating that “one nucleic acid sequence is upstream of another nucleic acid sequence” means that, when aligned in a 5’ to 3’ direction, the former is located further forward (i.e., closer to the 5’ end) than the latter. As used herein, the term “downstream” has the opposite meaning to “upstream”.

[0038] As used herein, the term “melting curve analysis” has the meaning commonly understood by those skilled in the art as referring to the method of analyzing the presence or identity of double-stranded nucleic acid molecules by measuring their melting curves, which is typically used to assess the dissociation characteristics of double-stranded nucleic acid molecules during heating.

[0039] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0040] This application provides a probe set, including at least one linear fluorescent probe and at least two mediator probes; wherein each mediator probe independently includes a mediator sub-sequence and a target probe sequence from the 5' to 3' direction, the target probe sequence includes a sequence that is the same as or complementary to the target nucleic acid to be tested, and the mediator sub-sequence includes a sequence that is complementary to the linear fluorescent probe;

[0041] The linear fluorescent probe contains a labeling sequence and a mooring sequence from 5' to 3'. The mooring sequence from 3' to 5' contains a capture sequence that is complementary to each mediator subsequence or part of the mediator subsequence. The labeling sequence is labeled with a fluorescent group and a quenching group.

[0042] When multiple mediator sequences are paired simultaneously on a linear fluorescent probe, there must be at least 2 nt of overlapping nucleotides between each pair of mediator sequences. Alternatively, if the mediator sequence downstream of the linear fluorescent probe and the mediator sequence upstream of the linear fluorescent probe have less than 2 nt of non-overlapping nucleotides, then the 5' end of the mediator sequence upstream of the linear fluorescent probe is modified with a nuclease-resistant modification.

[0043] The probe set provided in this application can detect all target nucleic acids in a single system, or it can be divided into multiple systems for collaborative detection of all target nucleic acids. Each target nucleic acid is typically complementary to one targeting probe sequence, but multiple targeting probe sequences are also allowed to be complementary to the same target nucleic acid, or one targeting probe sequence is complementary to multiple target nucleic acids. Each targeting probe sequence typically corresponds to one mediator subsequence, but one targeting probe sequence is also allowed to correspond to multiple mediator subsequences, or one mediator subsequence is allowed to correspond to multiple targeting probe sequences. Each mediator subsequence targets a specific mooring site on a linear fluorescent probe. Each mooring site is typically present on only one fluorescent probe, but it is also allowed to be present on multiple fluorescent probes.

[0044] When a linear fluorescent probe can pair with multiple mediator sequences simultaneously, it is called a multi-position fluorescent probe. When multiple mediator sequences need to be paired on the same multi-position fluorescent probe, each pair of these mediator sequences must overlap by at least 2 nucleotides. Each downstream mediator sequence must be upstream of another mediator sequence that does not overlap by more than 2 nucleotides. Since the mediator and the linear fluorescent probe are reverse complementary, this upstream mediator sequence must be resistant to 5' nuclease modification. This prevents 5' nuclease activity guided by the upstream mediator sequence from cleaving the downstream mediator sequence.

[0045] In some embodiments, when multiple mediator sequences need to be paired on the same multi-position fluorescent probe, there are at least 2 nt of overlapping nucleotides between each pair of mediator sequences. This approach can also avoid the 5' nuclease activity guided by the upstream mediator sequence from cleaving the downstream mediator sequence.

[0046] In some embodiments, modifications that are resistant to nucleases include, but are not limited to, at least one of the following: thiophosphate bond, locked nucleic acid, alkyl phosphate triester bond, aryl phosphate triester bond, alkyl phosphonate bond, aryl phosphonate bond, hydrogenated phosphate bond, alkyl amino phosphate bond, aryl amino phosphate bond, 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-I-yl modification, and 1-4'-thio-PD-furan ribosyl modification.

[0047] The labeled sequence can be labeled with a fluorescent group and a quencher group respectively in the middle or on both sides of the sequence, while maintaining an appropriate distance between the fluorescent group and the quencher group. In some embodiments, the fluorescent group and the quencher group are separated by a nucleotide interval of 10-30 nt.

[0048] In some embodiments, the labeling sequence may also be modified only with fluorescent groups and a base quenching method may be used.

[0049] In some embodiments, the fluorescent group is selected from at least one of ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, Yellow, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, AP593, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, and Quasar 705.

[0050] In some embodiments, the quenching group is selected from at least one of DABCYL, BHQ1, BHQ2, BHQ3, ECLIPSE, and TAMRA.

[0051] In some embodiments, the target nucleic acid to be tested is selected from at least one of modified and / or unmodified DNA, RNA, and LNA. Specifically, the target nucleic acid to be tested can be RNA, DNA, or LNA; it can be chemically synthesized or biosynthesized (e.g., derived from animals, plants, or microorganisms); DNA, RNA, and LNA can be unmodified or modified (e.g., methylated or otherwise chemically modified); or a combination thereof.

[0052] It should be noted that when the target nucleic acid to be tested is an RNA sequence, it is necessary to first synthesize paired cDNA under the action of reverse transcriptase or DNA polymerase with reverse transcriptase activity before proceeding with subsequent reactions.

[0053] In some embodiments, the mediator probe and the linear fluorescent probe each have a 3'-OH end, or the 3'-end is closed.

[0054] This application introduces a homologous tag-assisted primer-free dimer system to address the inequivalence between primer dimers and multiplex amplification reactions. Specifically, the probe set also includes homologous tag-assisted primers, which consist of a homologous tag sequence and a specific sequence, the specific sequence containing a sequence complementary to the target nucleic acid.

[0055] Homologous tag sequences are typically a dozen to several dozen nucleotides long and are added to both ends of the target DNA sequence. This tag sequence is identical in different target sequences and can therefore be recognized by the same set of primers.

[0056] In some embodiments, the probe set further includes an upstream primer and a downstream primer; wherein:

[0057] The upstream primer contains a sequence complementary to the target nucleic acid. When hybridizing with the target nucleic acid, the upstream primer is located upstream of the target probe sequence of the medium probe.

[0058] The downstream primer contains a sequence complementary to the target nucleic acid. When hybridizing with the target nucleic acid, the downstream primer is located downstream of the target probe sequence of the medium probe.

[0059] It should be noted that the primers and probes used in this application can be DNA, RNA, or LNA (locked nucleic acid), or a combination thereof.

[0060] In some embodiments, the probe set is used to detect human papillomavirus (HPV), and the target nucleic acid to be tested is the target nucleic acid sequence of HPV. Since HPV has different subtypes, including 18, 68, 16, 31, 66, and 59, corresponding primers and probes need to be designed for the subtype to be detected.

[0061] In this embodiment, the medium probe is selected from the nucleotide sequences shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:12, and / or from the nucleotide sequences shown in SEQ ID NO:17 and SEQ ID NO:20;

[0062] The linear fluorescent probe is selected from the nucleotide sequences shown in SEQ ID NO:13 and / or SEQ ID NO:21;

[0063] The upstream primer is selected from the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:10, and / or from the nucleotide sequences shown in SEQ ID NO:15 and SEQ ID NO:18;

[0064] The downstream primer is selected from the nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:11, and / or from the nucleotide sequences shown in SEQ ID NO:16 and SEQ ID NO:19;

[0065] The homologous tag auxiliary primers are selected from the nucleotide sequence shown in SEQ ID NO:14.

[0066] Based on the probe set described above, this application provides a kit comprising the probe set as described above, an enzyme with 5' nuclease activity, and a nucleic acid polymerase.

[0067] Among them, the enzyme with 5' nuclease activity is a nucleic acid polymerase with 5' nuclease activity (e.g., 5' exonuclease activity), such as DNA polymerase, preferably Taq polymerase; the nucleic acid polymerase is a common nucleic acid polymerase, such as T4 DNA polymerase, reverse transcriptase, etc.

[0068] Based on the above-mentioned kit, this application provides a method for detecting two or more target nucleic acids, comprising the following steps:

[0069] Step S10: Hybridize the target nucleic acid to be tested with the upstream primer and the medium probe, wherein the target probe sequence of the upstream primer and the medium probe is paired with the target nucleic acid to be tested, and the remaining medium subsequence in the medium probe is in a free single-stranded state.

[0070] In step S20, after the upstream primer comes into contact with an enzyme with 5' nuclease activity, it begins to extend from the 3' end. During the extension reaction, the enzyme with 5' nuclease activity comes into contact with the 5' end of the target probe sequence and begins to cleave from the 3' end of the first nucleotide of the target probe sequence, releasing the complete mediator sequence; or, when the enzyme with 5' nuclease activity comes into contact with the upstream primer, it also comes into contact with the 5' end of the target probe sequence, cleaving the mediator probe and releasing the complete mediator sequence.

[0071] In step S30, the free mediator sequence targets a specific site on the linear fluorescent probe and, under the action of nucleic acid polymerase, extends from the 3' end of the mediator sequence to form a specific double strand.

[0072] Step S40: Perform melting curve analysis on the double strand and determine whether the target nucleic acid to be tested exists based on the results of the melting curve analysis.

[0073] In step S20, a DNA polymerase with 5' nuclease activity is used to cleave the medium probe. Alternatively, other enzymes with 5' nuclease activity can be used to cleave the medium probe. In step S30, a specific double-stranded structure is formed, at which point the linear fluorescent probe changes from its single-stranded, naturally coiled state (fluorescence quenching) to its double-stranded state (fluorescence release).

[0074] The resulting double strands have specific Tm values, so in melting curve analysis, each double strand will produce a specific melting peak, thereby determining the detection results of the target nucleic acid.

[0075] In some embodiments, the number of upstream primers and vector probes is greater than or equal to the number of target nucleic acids to be tested; and / or,

[0076] The number of free mediator sequences is greater than or equal to the number of target nucleic acids to be tested.

[0077] Steps S10 and S20 mainly occur during the PCR process and can target one or more nucleic acids. The number of primer and probe types should be no less than the number of target nucleic acids to be tested. The number of free probe sequences generated in step S20 should be no less than the number of target nucleic acids to be tested. Step S30 can occur during the PCR process or as an extension reaction at a constant temperature after the PCR reaction. Step S40 is performed under melting curve analysis conditions, which can be either a heating or cooling reaction. The data after melting analysis can be analyzed by the built-in software of the fluorescence PCR instrument to generate melting peaks and corresponding Tm values ​​for each fluorescence channel, or it can be analyzed using other software or calculated manually.

[0078] In step S40, specifically, the presence of a certain target nucleic acid is determined based on the melting peak of the obtained melting curve.

[0079] In this application, the interpretation of test results requires information from two dimensions. The first dimension is the fluorescence channels of the fluorescence PCR instrument. In step S40, linear fluorescent probes with different sequences can modify fluorescent groups with different excitation wavelengths, and the melting peaks of the test results can be found in one or more fluorescence channels of the fluorescence PCR instrument. The second dimension is the melting temperature. Each melting peak has a corresponding Tm value, which allows for a certain fluctuation (e.g., ±1℃). The Tm values ​​are then used to distinguish each melting peak in the same fluorescence channel. Since melting peaks have a width, maintaining an appropriate Tm difference between each melting peak in the same fluorescence channel is beneficial for separating and distinguishing each specific melting peak. Therefore, the Tm of each melting peak in each fluorescence channel usually represents the detection result of one target nucleic acid, but it can also represent the detection result of multiple target nucleic acids depending on the actual design requirements.

[0080] The following section provides a further explanation of the method for detecting two or more target nucleic acids through specific implementation details.

[0081] It should be noted that the reaction system of this application embodiment can be optimized using the reaction conditions recommended by the DNA polymerase manufacturer. The concentration of each component in the system can be adjusted, and components can be added, removed, or replaced. The reaction procedure of this application embodiment includes a PCR reaction procedure and a melting reaction procedure. A reverse transcription reaction procedure or a UDG enzyme reaction procedure can be added before the PCR reaction procedure. An isothermal extension procedure can be added between the PCR reaction procedure and the melting reaction procedure to promote mediator extension. The melting reaction procedure can be a heating reaction or a cooling reaction. The heating or cooling rates of each reaction step can be appropriately varied, and the temperature of each reaction step can be appropriately adjusted.

[0082] Example 1

[0083] In this embodiment, a 5' locked nucleic acid modified and blocked medium probe is used. The 5' of a portion of the medium probe is modified with locked nucleic acid to resist nuclease cleavage.

[0084] In this embodiment, the genomes of human papillomavirus (HPV) types 18, 68, 16, and 31 were used as test samples, and the primer and probe sequences used are shown in Table 1. This embodiment, through the implementation of the technology of this application, achieves genotyping detection of HPV types 18, 68, 16, and 31, aiming to illustrate the necessity of using 5' nuclease-resistant probes.

[0085] Table 1 Primers and probes used in Example 1

[0086]

[0087]

[0088] Note: In the table, in the sequence column, lowercase letters are homologous tag sequences, bold letters are mediator subsequences, underlined letters are target probe sequences, and letters with a "+" to the left are locked nucleic acids.

[0089] The reaction was performed using a fluorescence PCR instrument with melting analysis capability. The reaction program was as follows: 95℃, 5 min; then 45 cycles (95℃, 10 s; 60℃, 30 s); 35℃, 10 min; 95℃, 1 min; 45℃, 2 min; followed by a heating rate of 0.04℃ / s from 50℃ to 90℃, during which fluorescence signals were collected. The raw melting curves obtained from the collected fluorescence were automatically analyzed by the fluorescence PCR instrument's software to generate melting peak curves.

[0090] In this embodiment, seven sample combinations were tested. The concentration of each type in the tested samples was 1000 copies / mL. The melting curve analysis results are as follows: Figure 1 As shown, the solid line represents the sample test result curve, and the dashed line represents the blank sample test reference. Figure 1 A represents the melting analysis curve of the human papillomavirus type 18 genome; Figure 1 B represents the melting analysis results curve for testing the genome of human papillomavirus type 68; Figure 1 C represents the melting analysis result curve of the human papillomavirus type 16 genome; Figure 1 D represents the melting analysis curve of the human papillomavirus type 31 genome; Figure 1 E represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 68 and 31; Figure 1 F represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 18 and 16; Figure 1 G represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 18, 68, 16, and 31.

[0091] Correspondingly, the peak value of each melting curve analysis corresponds to the temperature Tm, as shown in Table 2.

[0092] Table 2. Tm values ​​of melting curves

[0093]

[0094] like Figure 1 A, Figure 1 B Figure 1 C and Figure 1In D, when testing a single template of the genomes of human papillomavirus types 18, 68, 16, and 31, the melting peak morphology in the results was good, and the peak values ​​corresponding to Tm were easily identifiable. Figure 1 E shows that when testing a mixed whole genome sample of human papillomavirus types 68 and 31, the peak shapes of types 68 and 31 were good, and no abnormal broadening was observed. The Tm values ​​were less than 1°C different from those of types 68 or 31 when testing the genomes separately. Figure 1 As shown in F, when testing a mixed whole genome sample of human papillomavirus types 18 and 16, the peaks of types 18 and 16 maintained good morphology, and the Tm value was less than 1°C compared with the time variation of the type 18 or type 16 genome tested alone. Figure 1 G shows the results of testing a mixed whole genome sample of human papillomavirus types 18, 68, 16, and 31. The peak shapes for types 18, 68, 16, and 31 are good, and the Tm values ​​relative to the individual type genome test results are all less than 1°C. Because the four type tests share a single linear fluorescent probe, the upper limit of the overall peak height is limited. Figure 1 The peak height in G will be significantly lower than the results of testing a single genome type, which is normal.

[0095] As shown in Table 2, by modifying the mediator probes HPV+C1M1-18L1-P and HPV+C1M6-68L1-P corresponding to types 18 and 68 with locked nucleic acids, it was ensured that the 5' end of the mediator sequence was not cleaved by enzymes when targeting the linear fluorescent probe. The constructed system showed no significant changes in specific Tm in the detection results of different samples when detecting single or multiple mixed target nucleic acids, and the peak shapes maintained good morphology.

[0096] This embodiment demonstrates that locking nucleic acid modification of the 5' mediator sequence effectively resists the 5' nuclease activity of DNA polymerase, and that this nuclease-resistant modification effect is necessary for the use of multiple mediator probes on "multi-position" fluorescent probes.

[0097] Example 2

[0098] This embodiment uses a 5' thio-modified blocking medium probe. Specifically, the 5' end of a portion of the medium probe is thio-modified to resist nuclease cleavage.

[0099] In this embodiment, the genomes of human papillomavirus types 18, 68, 16, and 31 were used as test samples, and the primer and probe sequences and reaction conditions used were the same as in Example 1.

[0100] A 25 μL reaction system was used for sequential PCR reactions and melting curve analysis. The 25 μL reaction system was an aqueous solution containing the following solutes: 70 mM Tris·Cl (pH 8.5), 50 mM KCl, 3 mM MgCl2, 14 mM (NH4)2SO4, 0.05% (m / w) Tween 20, 0.2 mM dNTPs, 2 U Taq DNA polymerase, 80 nM upstream and downstream primers, 160 nM or 320 nM mediator probes, 160 nM linear fluorescent probes, 3.2 μM homology tag auxiliary primers, and 5 μL of the sample to be tested. The primer and probe sequences used are shown in Table 3. Unlike Example 1, the 5' end of the 18 and 68 mediator probes was thiolated.

[0101] Table 3 Primers and probes used in Example 2

[0102]

[0103]

[0104]

[0105] Note: In the table, in the sequence column, lowercase letters are homologous tag sequences, bold letters are mediator subsequences, underlined letters are target probe sequences, and "*" indicates thiomodification.

[0106] The reaction was performed using a fluorescence PCR instrument with melting analysis capability. The reaction program was as follows: 95℃, 5 min; then 45 cycles (95℃, 10 s; 60℃, 30 s); 35℃, 10 min; 95℃, 1 min; 45℃, 2 min; followed by a heating rate of 0.04℃ / s from 50℃ to 90℃, during which fluorescence signals were collected. The raw melting curves obtained from the collected fluorescence were automatically analyzed by the fluorescence PCR instrument's software to generate melting peak curves.

[0107] In this embodiment, seven sample combinations were tested. The concentration of each type of the tested sample was 1000 copies / mL. The melting curve analysis results are as follows: Figure 2 As shown, the solid line represents the sample test result curve, and the dashed line represents the blank sample test reference. Figure 2 A represents the melting analysis curve of the human papillomavirus type 18 genome; Figure 2 B represents the melting analysis results curve for testing the genome of human papillomavirus type 68; Figure 2 C represents the melting analysis result curve of the human papillomavirus type 16 genome; Figure 2 D represents the melting analysis curve of the human papillomavirus type 31 genome; Figure 2E represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 68 and 31; Figure 2 F represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 18 and 16; Figure 2 G represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 18, 68, 16, and 31.

[0108] Correspondingly, the peak value of each melting curve analysis corresponds to the temperature Tm, which is the same as... Figure 2 The corresponding Tm values ​​are listed in Table 4.

[0109] Table 4. Statistics of Tm values ​​for melting curves

[0110]

[0111] like Figure 2 A, Figure 2 B Figure 2 C and Figure 2 In D, when testing a single template of the genomes of human papillomavirus types 18, 68, 16, and 31, the melting peak morphology in the results was good, and the peak values ​​corresponding to Tm were easily identifiable. Figure 2 E shows that when testing a mixed whole genome sample of human papillomavirus types 68 and 31, the peak shapes of types 68 and 31 were good, and no abnormal broadening was observed. The Tm values ​​were less than 1°C different from those of types 68 or 31 when testing the genomes separately. Figure 2 As shown in F, when testing a mixed whole genome sample of human papillomavirus types 18 and 16, the peaks of types 18 and 16 maintained good morphology, and the Tm value was less than 1°C compared to testing the type 18 or type 16 genome separately. Figure 2 G shows the results of testing a mixed whole genome sample of human papillomavirus types 18, 68, 16, and 31. The peak shapes for types 18, 68, 16, and 31 are good, and the Tm values ​​relative to the individual type genome test results are all less than 1°C. Because the four type tests share a single linear fluorescent probe, the upper limit of the overall peak height is limited. Figure 4 The peak height in G will be significantly lower than the results of testing a single genome type, which is normal.

[0112] As shown in Table 3, by thiolation modification of the mediator probes HPV*C1M1-18L1-P and HPV*C1M6-68L1-P corresponding to types 18 and 68, it is ensured that the 5' end of the mediator sequence is not cleaved by enzymes when targeting the fluorescent probe. In the constructed system, when detecting single or multiple mixed target nucleic acids, the specific Tm in the detection results of different samples showed no significant changes, and the peak shape remained good.

[0113] This example demonstrates that the thiolation of the 5' mediator sequence effectively resists the 5' nuclease activity of DNA polymerase, and that this nuclease-resistant modification is necessary for the use of multiple mediator probes on "multi-position" fluorescent probes.

[0114] Example 3

[0115] This embodiment uses two linear fluorescent probes.

[0116] In this embodiment, the genomes of human papillomavirus types 18, 68, 16, 31, 66, and 59 are used as test samples to achieve genotyping detection.

[0117] A 25 μL reaction system was used for sequential PCR reactions and melting curve analysis. The 25 μL reaction system was an aqueous solution containing the following solutes: 80 mM Tris·Cl (pH 8.5), 60 mM KCl, 5 mM MgCl2, 14 mM (NH4)2SO4, 0.2% (m / w) Tween 20, 2% (m / w) formamide, 0.2 mM dNTPs, 0.4 mM dUTP, 2 U Taq DNA polymerase, 0.02 U UDG enzyme, and the amounts of primers and probes are shown in Table 5, along with 5 μL of the sample to be tested. Table 5 shows that the mediators generated after cleavage of mediator probes HPV*C1M1-18L1-P, HPV*C1M6-68L1-P, HPV C1M12-16L1-P, and HPV C1M20-31L1-P are all targeted by the linear fluorescent probe Cat-L1, and the mediators generated after cleavage of mediator probes HPV*C23M40-66L1-BP28 and HPV C23M51-59L1-BP18 are all targeted by the linear fluorescent probe Cat-L23. HPV*C1M1-18L1-P, HPV*C1M6-68L1-P, and HPV*C23M40-66L1-BP28 in the mediator probes underwent thiolation modification at the 5' end.

[0118] Table 5 Primers and probes used in Example 3

[0119]

[0120]

[0121]

[0122] Note: In the table, in the sequence column, lowercase letters are homologous tag sequences, bold letters are mediator subsequences, underlined letters are target probe sequences, and "*" indicates thiomodification.

[0123] The reaction was performed using a fluorescence PCR instrument with melting analysis capability. The reaction program was as follows: 50℃, 2 min; 95℃, 5 min; then 45 cycles (95℃, 10 s; 60℃, 30 s); 35℃, 10 min; 95℃, 1 min; 45℃, 2 min; subsequently, the temperature was increased from 50℃ to 90℃ at a rate of 0.04℃ / s, and fluorescence signals were collected during this heating process. The raw melting curves obtained from the collected fluorescence were automatically analyzed by the fluorescence PCR instrument's software to generate melting peak curves.

[0124] In this embodiment, seven sample combinations were tested. The concentration of each type of the tested sample was 1000 copies / mL. The melting curve analysis results are as follows: Figure 3 As shown, the solid line represents the sample test result curve, and the dashed line represents the blank sample test reference. Figure 3 A represents the melting analysis curve of the human papillomavirus type 18 genome; Figure 3 B represents the melting analysis results curve for testing the genome of human papillomavirus type 68; Figure 3 C represents the melting analysis result curve of the human papillomavirus type 16 genome; Figure 3 D represents the melting analysis curve of the human papillomavirus type 31 genome; Figure 3 E represents the melting analysis result curve of the human papillomavirus type 66 genome; Figure 3 F represents the melting analysis curve of the human papillomavirus type 59 genome; Figure 3 G represents the melting analysis results curve of a mixed sample of human papillomavirus (HPV) genomes of types 18, 68, 16, 31, 66, and 59.

[0125] Correspondingly, the peak value of each melting curve analysis corresponds to the temperature Tm, which is the same as... Figure 3 The corresponding Tm values ​​are listed in Table 6.

[0126] Table 6. Statistics of Tm values ​​for melting curves

[0127]

[0128]

[0129] like Figure 3 A to Figure 3In G, when testing single template samples of human papillomavirus (HPV) types 18, 68, 16, 31, 66, and 59, the melting peak morphology was good, and the corresponding Tm values ​​were easily identifiable. When testing samples containing mixtures of HPV types 18, 68, 16, 31, 66, and 59, each type had its corresponding detection peak and Tm. Compared to the Tm values ​​of single-genome samples, the Tm variation for each type in these mixed genome samples was less than 1°C.

[0130] This embodiment uses two fluorescent probes in a single channel: the fluorescent probe Cat-L1 and its associated four mediator probes HPV*C1M1-18L1-P, HPV*C1M6-68L1-P, HPV C1M12-16L1-P, and HPV C1M20-31L1-P, which is the same as in Example 2. The newly added fluorescent probe Cat-L23 is associated with the mediator probes HPV*C23M40-66L1-BP28 and HPV C23M51-59L1-BP18, and the mediator probe HPV*C23M40-66L1-BP28 requires 5' nuclease resistance modification. In addition, UDG enzyme and dUTP are added to the system to prevent nucleic acid contamination.

[0131] Comparative Example 1

[0132] The probe used in this comparative example was not modified at the 5' end to resist 5' nucleases.

[0133] In this comparative example, the genomes of human papillomavirus types 18, 68, 16, and 31 were used as test samples. Primers and probes were designed based on the gene sequence encoding the late protein L1 according to existing technical solutions.

[0134] A 25 μL reaction system was used for sequential PCR reactions and melting curve analysis. The 25 μL reaction system was an aqueous solution containing the following solutes: 70 mM Tris·Cl (pH 8.5), 50 mM KCl, 3 mM MgCl2, 14 mM (NH4)2SO4, 0.05% (m / w) Tween 20, 0.2 mM dNTPs, 2 U Taq DNA polymerase, 80 nM upstream and downstream primers, 160 nM or 320 nM mediator probes, 160 nM linear fluorescent probes, 3.2 μM homology tag auxiliary primers, and 5 μL of the sample to be tested. The primer and probe sequences used are shown in Table 7.

[0135] Table 7 shows the primers and probes used in Comparative Example 1.

[0136]

[0137]

[0138] The reaction was performed using a fluorescence PCR instrument with melting analysis capability. The reaction program was as follows: 95℃, 5 min; then 45 cycles (95℃, 10 s; 60℃, 30 s); 35℃, 10 min; 95℃, 1 min; 45℃, 2 min; followed by a heating rate of 0.04℃ / s from 50℃ to 90℃, during which fluorescence signals were collected. The raw melting curves obtained from the collected fluorescence were automatically analyzed by the fluorescence PCR instrument's software to generate melting peak curves.

[0139] This comparative test included seven sample combinations. The concentration of each type in the tested samples was 1000 copies / mL. The melting curve analysis results are as follows: Figure 4 As shown, the solid line represents the sample test result curve, and the dashed line represents the blank sample test reference. Among them, Figure 4 A represents the melting analysis curve of the human papillomavirus type 18 genome; Figure 4 B represents the melting analysis results curve for testing the genome of human papillomavirus type 68; Figure 4 C represents the melting analysis result curve of the human papillomavirus type 16 genome; Figure 4 D represents the melting analysis curve of the human papillomavirus type 31 genome; Figure 4 E represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 68 and 31; Figure 4 F represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 18 and 16; Figure 4 G represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 18, 68, 16, and 31.

[0140] Correspondingly, the peak value of each melting curve analysis corresponds to the temperature Tm, which is the same as... Figure 4 The corresponding Tm values ​​are listed in Table 8.

[0141] Table 8. Statistics of Tm values ​​for melting curves

[0142]

[0143] like Figure 4 A, Figure 4 B Figure 4 C Figure 4 As shown in D, when testing a single template of the genomes of human papillomavirus types 18, 68, 16, and 31, the melting peak morphology in the results was good, and the peak values ​​corresponding to Tm were easily identifiable. Figure 4E shows that when testing a mixed whole genome sample of human papillomavirus types 68 and 31, the peak of type 31 maintained a good shape, and the Tm value was less than 1°C compared to testing the type 31 genome alone; however, the peak shape of type 68 was relatively... Figure 4 The peak Tm was significantly broadened in B, and the peak Tm was reduced by more than 2°C compared to the test of the 68-type genome alone. Figure 4 F shows that when testing a mixed whole genome sample of human papillomavirus types 18 and 16, the peak of type 16 maintained a good shape, and the Tm value was less than 1°C compared with the test of type 16 genome alone; although the peak shape of type 18 did not widen significantly, the peak Tm was reduced by more than 2°C compared with the test of type 18 genome alone. Figure 4 G shows the results of testing a mixture of whole genome samples of human papillomavirus types 18, 68, 16, and 31. Types 16 and 31 showed good peak shapes and Tm values ​​less than 1°C compared to the genome test results of individual types 16 or 31. However, the peak shape of type 68 was significantly broadened and the Tm was more than 2°C lower than that of type 68 alone. The peak of type 18 was difficult to interpret, with a broad and low peak in the target region and a Tm more than 2°C lower than that of type 18 alone.

[0144] After the probe sequence of the mediator probe is cleaved, a free mediator sequence is released. Similar to primers binding to target nucleic acids, the mediator sequence targets a mooring site on the fluorescent probe and extends under the action of DNA polymerase. When testing a single target nucleic acid, a corresponding mediator and a Tm-specific double strand are formed. However, when testing multiple target nucleic acids simultaneously, the resulting reaction becomes more complex. For example, in this comparative example, when testing a sample containing a mixture of human papillomavirus type 68 and type 31 genomes, two mediators corresponding to type 68 and type 31 are generated. These two mediators can each target the fluorescent probe Cat-L1, forming their own specific double strands. However, if they both target the same Cat-L1, problems may arise.

[0145] according to Figure 5Based on the binding sites of the mediators and fluorescent probes listed, the following analysis can be made: 1) When both type 68 and type 31 mediators bind to the same fluorescent probe molecule for extension, the type 68 mediator will be cleaved by the 5' nuclease activity of the DNA polymerase carried by the upstream type 31 mediator; 2) When the type 68 mediator and fluorescent probe extend to form a double strand, the binding site of the type 31 mediator upstream of the fluorescent probe is still exposed. Upon encountering a free type 31 mediator, it will bind and extend to cleave the downstream type 68 double strand, still starting the cleavage from the 5' of the type 68 mediator; 3) When the type 31 mediator and fluorescent probe extend to form a double strand, the binding site of the type 68 mediator downstream of the fluorescent probe is blocked by the double strand and can no longer bind to the type 68 mediator. These reactions occur simultaneously, but in any case, the double strand corresponding to type 31 will not be interfered with, while the mediator or double strand corresponding to type 68 is easily cleaved by the DNA polymerase carried by the upstream type 31 mediator. Therefore... Figure 4 In E, the melting peak shape of type 31 remains unchanged, but the melting peak shape and peak value Tm of type 68 change significantly. Because some double strands of type 68 are cut to varying degrees, the melting peak widens or shifts to the left of the horizontal axis, and the peak value Tm also decreases accordingly. Figure 5 In the study, the binding sites of type 18 and type 16 mediators on the fluorescent probes did not overlap. When testing mixed samples of type 18 and type 16 genomes, the binding site of the type 16 mediator was upstream, and its melting curve peak shape remained unchanged. The binding site of the type 18 mediator sequence was relatively downstream, and its melting peak shifted to the left and Tm decreased. Furthermore, when testing samples of mixed samples of type 18, type 68, type 16, and type 31 genomes, according to... Figure 5 The binding sites of the mediator sequences indicated in the diagram show that the melting peaks and Tm values ​​of types 18 and 68 change relative to the test genome template. Although the binding site of the type 16 mediator is downstream of type 31, its 5' sequence overlaps with the 3' sequence of type 31 mediator by 4 nucleotides, so the melting peaks and Tm values ​​of types 16 and 31 do not change significantly relative to the test genome template.

[0146] Therefore, to prevent downstream mediators from being cleaved by DNA polymerase carried by upstream mediators on the same fluorescent probe, a certain degree of sequence overlap between the upstream and downstream mediator sequences is allowed through design. When only two mediator binding sites are designed on the same fluorescent probe, partial overlap of the two mediator sequences is feasible. However, when three or more mediator binding sites need to be designed on the same fluorescent probe, the upstream mediator sequence needs to overlap with the downstream mediator sequence. To ensure that Tm remains unchanged, the 5' sequence of each mediator needs to remain unchanged to ensure that the final double strands remain unchanged. Therefore, the upstream mediator needs to be lengthened from 3' to partially overlap with the downstream mediator sequence. However, while this lengthened upstream mediator targets the fluorescent probe, it also covers almost all the intermediate sites, inhibiting mediators targeting these intermediate sites.

[0147] According to the scheme of this application, the 5' of the mediator probe can be modified without extending the mediator sequence to resist the 5' nuclease activity of DNA polymerase.

[0148] Comparative Example 2

[0149] This comparative example modifies the linear fluorescent probe in Example 2 into a hairpin structure, retaining the original "berth" sequence and adding a paired arm to each end of the "berth" sequence, as detailed in Table 9 for the hairpin fluorescent probe Cat-U1.

[0150] In this comparative example, the genomes of human papillomavirus types 18, 68, 16, and 31 were used as test samples. The primer and probe sequences and reaction conditions used were the same as in Example 2. Serial PCR reactions and melting curve analysis were performed using a 25 μL reaction system.

[0151] The 25 μL reaction system was an aqueous solution containing the following solutes: 70 mM Tris·Cl (pH 8.5), 50 mM KCl, 3 mM MgCl2, 14 mM (NH4)2SO4, 0.05% (m / w) Tween 20, 0.2 mM dNTPs, 2 U Taq DNA polymerase, 80 nM upstream and downstream primers, 160 nM or 320 nM mediator probes, 160 nM hairpin fluorescent probes, 3.2 μM homology tag auxiliary primers, and 5 μL of the sample to be tested. The primer and probe sequences used are shown in Table 9.

[0152] Table 9 shows the primers and probes used in Comparative Example 2.

[0153]

[0154]

[0155]

[0156] The reaction was performed using a fluorescence PCR instrument with melting analysis capability. The reaction program was as follows: 95℃, 5 min; then 45 cycles (95℃, 10 s; 60℃, 30 s); 35℃, 10 min; 95℃, 1 min; 45℃, 2 min; followed by a heating rate of 0.04℃ / s from 50℃ to 90℃, during which fluorescence signals were collected. The raw melting curves obtained from the collected fluorescence were automatically analyzed by the fluorescence PCR instrument's software to generate melting peak curves.

[0157] In this comparative example, seven sample combinations were tested. The concentration of each type of the tested sample was 1000 copies / mL. The melting curve analysis results are as follows: Figure 6 As shown, the solid line represents the sample test result curve, and the dashed line represents the blank sample test reference. Among them, Figure 6 A represents the melting analysis curve of the human papillomavirus type 18 genome; Figure 6 B represents the melting analysis results curve for testing the genome of human papillomavirus type 68; Figure 6 C represents the melting analysis result curve of the human papillomavirus type 16 genome; Figure 6 D represents the melting analysis curve of the human papillomavirus type 31 genome; Figure 6 E represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 68 and 31; Figure 6 F represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 18 and 16; Figure 6 G represents the melting analysis results curve for testing the mixed genomes of human papillomavirus types 18, 68, 16, and 31.

[0158] Correspondingly, the peak value of each melting curve analysis corresponds to the temperature Tm, which is the same as... Figure 6 The corresponding Tm values ​​are listed in Table 10.

[0159] Table 10. Statistics of Tm values ​​for melting curves

[0160]

[0161]

[0162] Figure 6 In the test results, the blank sample curve showed a downward-sloping shape, forming a large negative peak, while the curves of other samples all showed varying degrees of downward slope. Compared with Example 2, the only possible cause of this result is the use of a hairpin fluorescent probe.

[0163] In the Tm results summarized in Table 10, when testing mixed templates of human papillomavirus types 18, 68, 16, and 31, the Tm value of type 68 was greater than 1°C compared to the test of a single genome, while the Tm differences for other test results of the same type were all less than 1°C. This test still used the same thiomodified probe as in Example 2. Although the peak shape was affected by the hairpin probe, the Tm value variation remained relatively stable.

[0164] In summary, the method of this application can avoid the mediator sequence being cleaved by DNA polymerase 5' nuclease activity. At the same time, the use of a modified linear fluorescent probe can solve the problem of uneven melting curve baseline.

[0165] Obviously, the embodiments described above are only some embodiments of this application, not all embodiments. The accompanying drawings show preferred embodiments of this application, but do not limit the patent scope of this application. This application can be implemented in many different forms; rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this application's specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this application.

Claims

1. A probe assembly, characterized in that, It includes at least one linear fluorescent probe and at least two mediator probes; wherein each of the mediator probes independently comprises a mediator sub-sequence and a target probe sequence from the 5' to 3' direction, the target probe sequence comprising a sequence that is identical or complementary to the target nucleic acid to be tested, and the mediator sub-sequence comprising a sequence complementary to the linear fluorescent probe; The linear fluorescent probe contains a labeling sequence and a mooring sequence from 5' to 3', the mooring sequence from 3' to 5' includes a capture sequence complementary to each of the mediator subsequences or a portion of the mediator subsequence, and the labeling sequence is labeled with a fluorescent group and a quenching group; When multiple mediator sequences are simultaneously paired on the linear fluorescent probe, there is at least 2 nt of overlapping nucleotides between each pair of mediator sequences. Alternatively, if the mediator sequence downstream of the linear fluorescent probe and the mediator sequence upstream of the linear fluorescent probe have less than 2 nt of non-overlapping nucleotides, then the 5' end of the mediator sequence upstream of the linear fluorescent probe is modified with a nuclease-resistant modification.

2. The probe assembly according to claim 1, characterized in that, The modifications that provide resistance to nucleases include at least one of the following: thiophosphate bond, locked nucleic acid, alkyl phosphate triester bond, aryl phosphate triester bond, alkyl phosphonate bond, aryl phosphonate bond, hydrogenated phosphate bond, alkyl amino phosphate bond, aryl amino phosphate bond, 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-I-yl modification, and 1-4'-thio-PD-furan ribosyl modification.

3. The probe assembly according to claim 1, characterized in that, A nucleotide spaced 10-30 nt between the fluorescent group and the quencher group; and / or, The fluorescent group is selected from at least one of ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, Yellow, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, AP593, CAL Fluor Red 635, Quasar670, CY3, CY5, CY5.5, and Quasar705; and / or, The quenching group is selected from at least one of DABCYL, BHQ1, BHQ2, BHQ3, ECLIPSE, and TAMRA.

4. The probe assembly according to claim 1, characterized in that, The target nucleic acid to be tested is selected from at least one of modified and / or unmodified DNA, RNA, and LNA; and / or, The medium probe and the linear fluorescent probe each have a 3'-OH end, or the 3'-end is closed.

5. The probe assembly according to claim 1, characterized in that, The probe set further includes homology tag auxiliary primers, which include a homology tag sequence and a specific sequence, wherein the specific sequence contains a sequence complementary to the target nucleic acid to be tested; and / or, The probe set also includes upstream and downstream primers; wherein, The upstream primer contains a sequence complementary to the target nucleic acid to be tested, and when hybridizing with the target nucleic acid to be tested, the upstream primer is located upstream of the target probe sequence of the medium probe; The downstream primer contains a sequence complementary to the target nucleic acid and is located downstream of the target probe sequence of the medium probe when hybridizing with the target nucleic acid.

6. The probe assembly according to claim 5, characterized in that, The target nucleic acid to be tested is the target nucleic acid sequence of human papillomavirus; The medium probe is selected from the nucleotide sequences shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:12, and / or from the nucleotide sequences shown in SEQ ID NO:17 and SEQ ID NO:20; The linear fluorescent probe is selected from nucleotide sequences as shown in SEQ ID NO:13 and / or SEQ ID NO:21; The upstream primer is selected from the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:10, and / or from the nucleotide sequences shown in SEQ ID NO:15 and SEQ ID NO:18; The downstream primer is selected from the nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:11, and / or from the nucleotide sequences shown in SEQ ID NO:16 and SEQ ID NO:19; The homologous tag auxiliary primer is selected from the nucleotide sequence shown in SEQ ID NO:

14.

7. A reagent kit, characterized in that, The kit comprises a probe set as described in any one of claims 1 to 6, an enzyme having 5' nuclease activity, and a nucleic acid polymerase.

8. A method for detecting two or more target nucleic acids, characterized in that, Includes the following steps: Step S10: Hybridize the target nucleic acid to be tested with the upstream primer and the medium probe, wherein the target probe sequence of the upstream primer and the medium probe is paired with the target nucleic acid to be tested, and the remaining medium subsequence in the medium probe is in a free single-stranded state. In step S20, after the upstream primer contacts an enzyme with 5' nuclease activity, it extends from the 3' end. During the extension reaction, the enzyme with 5' nuclease activity contacts the 5' end of the target probe sequence and begins cleaving from the 3' end of the first nucleotide of the target probe sequence, releasing the complete mediator sequence; or, when the enzyme with 5' nuclease activity contacts the upstream primer, it also contacts the 5' end of the target probe sequence, cleaving the mediator probe and releasing the complete mediator sequence. In step S30, the free mediator sequence targets a specific site on the linear fluorescent probe and, under the action of nucleic acid polymerase, extends from the 3' end of the mediator sequence to form a specific double strand. Step S40: Perform melting curve analysis on the double strand and determine whether the target nucleic acid to be tested exists based on the results of the melting curve analysis.

9. The method according to claim 8, characterized in that, The number of types of the upstream primer and the medium probe is greater than or equal to the number of types of the target nucleic acid to be tested; and / or, The number of types of the free mediator subsequences is greater than or equal to the number of types of the target nucleic acid to be tested.

10. The method according to claim 8, characterized in that, In step S40, the presence of a certain target nucleic acid is determined based on the melting peak of the obtained melting curve.

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