A genotyping method

Abstract

The present application relates to genotyping. In particular, the present application provides a method of genotyping that is capable of simultaneously detecting the presence or absence of one or more of a plurality of genotypes in a sample. In addition, the present application provides a set of probes, and a kit comprising one or more of the set of probes, which can be used to practice the method of the present application.

Description

Technical Field

[0001] This application relates to the field of genotyping. Specifically, this application provides a method for genotyping capable of simultaneously detecting the presence of one or more of multiple genotypes in a sample. Furthermore, this application provides a probe set and a kit comprising one or more of the probe set, which can be used to implement the method of the present invention. Background Technology

[0002] Human papillomavirus (HPV) is a small, non-enveloped, circular double-stranded DNA virus with a genome length of approximately 80,000 base pairs (bp). 99.7% of cervical cancers are caused by persistent infection with high-risk HPV types. Currently, HPV DNA detection methods mainly include fluorescence PCR (qPCR), gene chips, and PCR melting curve analysis. These three techniques are described below:

[0003] qPCR is a technique limited by the absorption wavelength of the fluorescent labeling probe, and the types of genotypes that can be detected are limited by the fluorescence channel.

[0004] Gene chip technology is the most commonly used method for detecting PCR products. Solid-phase chip technology utilizes the hybridization of biotin-labeled PCR products with probes immobilized on nitrocellulose or nylon membranes, generating a detection signal through chemical color development. However, gene chip technology involves multiple steps, requires opening the container during the reaction, carries a high risk of PCR product contamination, and is prone to human error in interpreting the color development results.

[0005] PCR melting curve technology is a post-PCR endpoint fluorescence signal detection technique. It generates a melting curve by gradually increasing the temperature after the PCR reaction and simultaneously monitoring the fluorescence signal at each step. Genotypes are identified by the hybridization melting temperature of the fluorescent probe and PCR product. This technique requires no opening of the container, eliminates the risk of contamination, and allows for instrument automation. Currently, it mainly includes asymmetric PCR melting curve technology and symmetric PCR melting curve technology.

[0006] Asymmetric PCR melting curve technology uses upstream and downstream primers with significant differences in concentration for PCR amplification. After PCR, the enriched single-stranded PCR product binds to a fluorescent probe to form double-stranded DNA. The corresponding characteristic peaks can be obtained by analyzing the melting curve. Different lengths and fluorescently labeled fluorescent probes can be designed for different genotypes to obtain different characteristic peaks, thus achieving genotyping. The disadvantage of this technology is its susceptibility to gene mutations, leading to inaccurate HPV genotyping. Specifically, when the amplified sequence contains gene mutations, the bases of the PCR product also change. Since the fluorescent probe directly binds to the PCR product, the melting temperature of the double-stranded DNA also changes, resulting in altered characteristic peaks.

[0007] Symmetric PCR melting curve techniques mainly include two approaches: multiplex probe amplification (MPA) and mediator-guided PCR melting curve techniques. The drawback of MPA is that result interpretation requires comparison with a negative control, thus the negative result must be controllable. The disadvantage of mediator-guided PCR melting curve techniques is that the background is affected by the sample matrix, resulting in uneven baselines when analyzing multi-infected samples, making result interpretation difficult.

[0008] Therefore, considering the problems existing in the current technology, it is necessary to develop a method that can detect multiple genotypes in one tube with accurate typing, thereby achieving highly sensitive and specific genotyping. Summary of the Invention

[0009] Terminology Definition

[0010] As used herein, the term "genotyping" refers to the process of identifying and classifying an organism by recognizing its genotype through the identification of its nucleic acid sequences (e.g., specific genetic markers in its genome). Genotyping has a wide range of applications, including but not limited to genetic disease screening (e.g., patent US20220187372A1), cancer screening (e.g., patent EP3565903B1), pathogen detection (e.g., patent WO2022101302A1), transgenic crop detection (e.g., patent EP3947609A1), individual identification, and environmental biodiversity assessment. Therefore, the range of organisms to which genotyping is applicable includes, but is not limited to, eukaryotes such as mammals (e.g., humans, mice, giant pandas) and plants (e.g., corn); prokaryotes such as Staphylococcus aureus, Escherichia coli, and deep-sea hydrothermal vent archaea; and viruses such as HIV, HPV, RSV, influenza virus, and SARS-CoV-2. The nucleic acid sequences of organisms can be identified using molecular biology detection techniques such as PCR and nucleic acid sequencing. It should be understood that the nucleic acid sequence selected for identifying organisms varies depending on the application scenario. For example, when performing genotyping for different HPV subtypes, the nucleic acid sequence may be a sequence derived from the variable region of the HPV genome. Similarly, when performing individual identification for different subjects, the nucleic acid sequence may be a sequence derived from the individual subject's genome containing SNP sites.

[0011] As used herein, the terms “target nucleic acid sequence,” “target nucleic acid,” and “target sequence” refer to the target nucleic acid sequence to be detected. In this application, the terms “target nucleic acid sequence,” “target nucleic acid,” and “target sequence” have the same meaning and are used interchangeably.

[0012] As used herein, the term "selective probe" refers to a single-stranded nucleic acid molecule consisting of a tag sequence and a target-specific sequence (target sequence) in the 5' to 3' direction. In this application, the tag sequence does not contain a sequence complementary to the target nucleic acid sequence, the target-specific sequence contains a sequence complementary to the target nucleic acid sequence, and one or more consecutive nucleotides at the 5' end of the target-specific sequence are completely complementary to the target nucleic acid sequence. Therefore, under conditions allowing nucleic acid hybridization, annealing, or amplification, the selective probe hybridizes or anneals to the target nucleic acid sequence via the target-specific sequence (i.e., forming a double-stranded structure), and the tag sequence in the selective probe does not hybridize with the target nucleic acid sequence and remains in a free state (i.e., maintaining a single-stranded structure), while the 5' end of the target-specific sequence is in a hybridized state (i.e., not in a free state) because one or more consecutive nucleotides thereon are completely complementary to the target nucleic acid sequence.

[0013] As used herein, the terms "target sequence" and "target-specific sequence" refer to a sequence capable of selectively / specifically hybridizing or annealing with a target nucleic acid sequence under conditions allowing nucleic acid hybridization, annealing, or amplification, and which includes a sequence complementary to the target nucleic acid sequence. In this application, the terms "target sequence" and "target-specific sequence" have the same meaning and are used interchangeably. It is readily understood that a target sequence or target-specific sequence is specific to the target nucleic acid sequence. In other words, under conditions allowing nucleic acid hybridization, annealing, or amplification, the target sequence or target-specific sequence hybridizes or anneals only with the specific target nucleic acid sequence and not with other nucleic acid sequences.

[0014] As used herein, the term "tag sequence" refers to an oligonucleotide sequence in the selectable probe that is not complementary to the target nucleic acid sequence and is located upstream (5' end) of the target-specific sequence. In this application, a unique selectable probe with a unique tag sequence is designed or provided for each target nucleic acid sequence (i.e., different due to the sequence of the tag sequence itself, or different due to the fluorescent group labeled on the tag sequence); thus, each target nucleic acid sequence corresponds to a unique selectable probe (unique tag sequence). Therefore, by detecting the unique tag sequence, the corresponding target nucleic acid sequence can be detected, thereby detecting the corresponding genotype.

[0015] As used herein, the term "upstream primer" refers to an oligonucleotide sequence containing a sequence complementary to the target nucleic acid sequence, which is capable of hybridizing (or annealing) with the target nucleic acid sequence under conditions that allow nucleic acid hybridization (or annealing) or amplification, and which, when hybridizing with the target nucleic acid sequence, is located upstream of the selectable probe.

[0016] As used herein, the term "complementary" means that two nucleic acid sequences are capable of forming hydrogen bonds with each other according to the Watson-Crick principle, thereby forming a double helix. In this application, the term "complementary" includes both "substantially complementary" and "completely complementary." As used herein, the term "completely complementary" means that every base in the region referred to in the nucleic acid sequence is capable of pairing with a base in the other nucleic acid strand, with no mismatches or gaps. As used herein, the term "substantially complementary" means that a majority of the bases in one nucleic acid sequence are capable of pairing with bases in the other nucleic acid strand, allowing for mismatches or gaps (e.g., mismatches or gaps of one or more nucleotides). Typically, under conditions that allow nucleic acid hybridization, annealing, or amplification, two "complementary" (e.g., substantially complementary or completely complementary) nucleic acid sequences will selectively / specifically hybridize or anneal and form a double helix. For example, in this application, the target-specific sequences in the upstream primer and the selective probe each contain a sequence complementary to (e.g., substantially complementary or completely complementary) the target nucleic acid sequence. Therefore, under conditions that allow nucleic acid hybridization, annealing, or amplification, the target-specific sequences in the upstream primers and selective probes will selectively / specifically hybridize or anneal with the target nucleic acid sequence. Correspondingly, the term "non-complementary" means that two nucleic acid sequences cannot hybridize or anneal under conditions that allow nucleic acid hybridization, annealing, or amplification, and cannot form a double strand. For example, in this 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 double strand, and remains in a free state (i.e., maintaining a single-stranded structure).

[0017] 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. Generally, two completely complementary or substantially complementary nucleic acid sequences can hybridize or anneal. The complementarity required for two nucleic acid sequences to hybridize or anneal depends on the hybridization conditions used, particularly the temperature.

[0018] As used herein, “conditions that allow nucleic acid hybridization” has 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 factors such as temperature, pH, composition, and ionic strength of the hybridization buffer, and can be determined based on the lengths and GC contents of the two complementary nucleic acid molecules. For example, those skilled in the art can select low-tightness or high-tightness hybridization conditions based on the relative lengths and / or GC contents of the two complementary nucleic acid molecules. Such hybridization conditions are well known to those skilled in the art and can be found, for example, 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 this application, “hybridization” and “annealing” have the same meaning and are used interchangeably. Correspondingly, the expressions "conditions that allow nucleic acid hybridization" and "conditions that allow nucleic acid annealing" have the same meaning and can be used interchangeably.

[0019] As used herein, the expression "conditions that allow nucleic acid polymerase extension" has the meaning commonly understood by those skilled in the art; it refers to conditions that allow a nucleic acid polymerase (e.g., DNA polymerase) to extend one nucleic acid strand (e.g., primer or probe) using one nucleic acid strand as a template and form a double helix. Such conditions are well known to those skilled in the art and may involve factors such as temperature, pH, composition, concentration, and ionic strength of the hybridization buffer, etc. Suitable nucleic acid amplification conditions can be determined using conventional methods (see, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)). In the method of this invention, "conditions that allow nucleic acid polymerase to perform the extension reaction" preferably refers to the operating conditions of the nucleic acid polymerase (e.g., DNA polymerase). In this application, the expressions "conditions that allow nucleic acid polymerase to perform the extension reaction" and "conditions that allow nucleic acid extension" have the same meaning and are used interchangeably.

[0020] As used herein, the phrase "conditions allowing cleavage of the selected probe" refers to conditions that allow an enzyme with 5' nuclease activity to cleave the selected probe hybridized to the target nucleic acid sequence and release a cleavage product containing the tag sequence or a portion thereof. In the method of the present invention, the conditions allowing cleavage of the selected probe are preferably the operating conditions of a nucleic acid polymerase with 5' nuclease activity. For example, when the enzyme with 5' nuclease activity used is a nucleic acid polymerase with 5' nuclease activity, the conditions allowing cleavage of the selected probe can be the operating conditions of the nucleic acid polymerase.

[0021] The operating conditions for various enzymes can be determined by those skilled in the art using conventional methods, and typically involve the following factors: temperature, pH of the buffer solution, composition, concentration, ionic strength, etc. Alternatively, the conditions recommended by the enzyme manufacturer can be used.

[0022] As used herein, the term "nucleic acid denaturation" has the meaning commonly understood by those skilled in the art as referring to the process by which a double-stranded nucleic acid molecule dissociates into a single strand. The expression "conditions that allow nucleic acid denaturation" refers to conditions that cause a double-stranded nucleic acid molecule to dissociate into a single strand. Such conditions can be routinely determined by those skilled in the art (see, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)). For example, nucleic acids can be denatured using conventional techniques such as heating, alkali treatment, urea treatment, and enzymatic methods (e.g., methods using helicases). In this application, nucleic acid denaturation is preferably performed under heating conditions. For example, nucleic acid denaturation can be achieved by heating to 80-105°C.

[0023] 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”.

[0024] As used herein, the term "variation site" refers to a change in the nucleic acid sequence at a specific reference coordinate location in the genome, including but not limited to single nucleotide polymorphisms (SNPs), single nucleotide variations (SNVs), nucleotide insertions, and nucleotide deletions.

[0025] As used herein, the term "melting curve analysis" has the meaning commonly understood by those skilled in the art, referring to the method of analyzing the presence or identity of double-stranded nucleic acid molecules by measuring their melting curves, typically used to assess the dissociation characteristics of double-stranded nucleic acid molecules during heating. Methods for performing melting curve analysis are well known to those skilled in the art (see, 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.

[0026] In certain preferred embodiments of this application, melting curve analysis can be performed using a select probe labeled with a fluorescent group on a tag sequence and a detection probe labeled with a quencher group on a capture sequence. In short, at ambient temperature, the detection probe and the select probe can form a double strand through their complementary sequences (i.e., the capture sequence and the tag sequence) via base pairing. In this case, the quencher group on the detection probe and the fluorescent group on the select probe are close to each other, and all or most of the fluorescence signal emitted by the fluorescent group on the tag sequence is absorbed by the quencher group. Therefore, the fluorescence signal emitted by the fluorescent group is essentially undetectable. As the temperature increases, the two strands of the double strand begin to dissociate (i.e., the capture sequence and the tag sequence gradually dissociate), and the two dissociated strands are in a single-stranded free state. Therefore, the dissociation causes the quencher group on the capture sequence and the fluorescent group on the tag sequence to move away from each other, thereby the fluorescent group will gradually emit a fluorescence signal. Therefore, as the temperature increases, the detected fluorescence signal gradually intensifies. When both strands of the double strand have completely dissociated, the strongest fluorescence signal can be detected. Therefore, by detecting the fluorescence signal emitted by the duplex during heating or cooling, the hybridization and dissociation processes of the capture and tag sequences can be observed, forming a curve showing the signal intensity changing with temperature. Furthermore, by performing negative derivative analysis on the obtained curve, a curve with the rate of change of signal intensity on the ordinate and temperature on the abscissa (i.e., the melting curve of the duplex) can be obtained. The peak in this melting curve is the melting peak, and the corresponding temperature is the dissolution temperature of the duplex, also known as the melting point or T0. m Value. Generally speaking, the higher the match between the detection probe and the complementary sequence (e.g., fewer mismatched bases and more paired bases), the higher the T value of the double strand. m The higher the value, the better. Therefore, by detecting the T value of the double strand... m The value can determine the presence and identity of the sequence complementary to the detection probe in the duplex.

[0027] As used herein, the terms "negative peak" or "negative melting peak" refer to the melting peak obtained by taking the negative derivative of the melting curve. When the melting peak is negative, it is below the baseline of the melting curve. However, regardless of whether the melting peak is negative or positive, the melting point (T0) of the double-chain polymer remains constant. m (value) and melting peak height (R) m The absolute values ​​of the values ​​are all the same. Furthermore, based on the formation principle of the melting curve described above, if the melting peak shows a negative peak, it can be determined that during the formation of the double strand, the quenching group and the fluorescent group gradually approach each other, causing the detected fluorescence signal to gradually weaken. This indicates that the quenching group and the fluorescent group are on different strands within the double strand (the double strand formed by the capture sequence and the tag sequence). That is, one group is labeled on the capture sequence, and the other group is labeled on the tag sequence.

[0028] Genotyping methods

[0029] Therefore, in one aspect, the present invention provides a genotyping method, characterized in that, by detecting target nucleic acid sequences of n genotypes, it is determined whether one or more of the n genotypes are present in a sample, the method comprising the following steps:

[0030] (1) For each of the target nucleic acid sequences, an upstream primer and a selection probe are provided;

[0031] The upstream primer contains a sequence complementary to the target nucleic acid sequence; the selective probe consists of a tag sequence and a target-specific sequence from the 5' to 3' direction, wherein the tag sequence contains a sequence not complementary to the target nucleic acid sequence, the target-specific sequence contains a sequence complementary to the target nucleic acid sequence, and one or more consecutive nucleotides at the 5' end of the target-specific sequence are completely complementary to the target nucleic acid sequence; the tag sequence is labeled with a fluorescent group; and when the upstream primer and the selective probe hybridize with the target nucleic acid sequence, the upstream primer is located upstream of the target-specific sequence.

[0032] Furthermore, under conditions that allow nucleic acid hybridization, the sample is contacted with the upstream primer and the selective probe;

[0033] (2) Under conditions that allow cleavage of the selected probe, the product of step (1) is contacted with an enzyme having 5' nuclease activity, wherein the enzyme having 5' nuclease activity is able to cleave the selected probe that hybridizes with the target nucleic acid sequence and release the tag sequence labeled with the fluorescent group.

[0034] (3) Provide m types of detection probes, among which,

[0035] Each of the detection probes comprises one or more capture sequences complementary to one or more of the tag sequences or portions thereof; and the m detection probes comprise at least n capture sequences, each complementary to the tag sequences or portions thereof of the n selected probes in step (1); and each capture sequence is independently labeled with a quenching group such that the fluorescence signal emitted when the capture sequence of each detection probe and its corresponding tag sequence hybridize is different from the fluorescence signal emitted when no hybridization product is generated with the tag sequence.

[0036] Furthermore, under conditions that allow nucleic acid hybridization, the product of step (2) is contacted with the m detection probes;

[0037] (4) Under conditions that allow the nucleic acid polymerase to extend, the product of step (3) is brought into contact with the nucleic acid polymerase, so that the hybridization product present in step (3) is extended to generate a double strand.

[0038] (5) Perform melting curve analysis on the product of step (4); and determine whether one or more of the n genotypes exist in the sample based on the results of the melting curve analysis.

[0039] Wherein, n is an integer greater than or equal to 2, and m is an integer greater than or equal to 1; optionally, the design of the upstream primer and the target-specific sequence is based on the differences in the target nucleic acid sequences of the n genotypes.

[0040] In step (1) of the method of the present invention, since the first upstream primer contains a sequence complementary to the first target nucleic acid sequence, and the first target-specific sequence contains a sequence complementary to the first target nucleic acid sequence, both the first upstream primer and the first selective probe hybridize with the first target nucleic acid sequence when the first target nucleic acid sequence is present. Specifically, since one or more consecutive nucleotides at the 5' end of the first target-specific sequence are completely complementary to the first target nucleic acid sequence, the one or more consecutive nucleotides at the 5' end of the first target-specific sequence will form a double strand with the first target nucleic acid and will not be in a free state. Similarly, since the second upstream primer contains a sequence complementary to the second target nucleic acid sequence, and the second target-specific sequence contains a sequence complementary to the second target nucleic acid sequence, both the second upstream primer and the second selective probe hybridize with the second target nucleic acid sequence when the second target nucleic acid sequence is present. Specifically, since one or more consecutive nucleotides at the 5' end of the second target-specific sequence are completely complementary to the first target nucleic acid sequence, the one or more consecutive nucleotides at the 5' end of the second target-specific sequence will form a double strand with the second target nucleic acid and will not be in a free state.

[0041] In step (2) of the method of the present invention, when a first target nucleic acid sequence is present, both the first upstream primer and the first selective probe hybridize with the first target nucleic acid sequence. Further, since the first tag sequence contains a sequence that is not complementary to the first target nucleic acid sequence, the first tag sequence in the first selective probe is in a free state and does not hybridize with the first target nucleic acid sequence. In this case, under the action of an enzyme with 5' nuclease activity, the first tag sequence or a portion thereof will be cleaved from the first selective probe hybridized with the first target nucleic acid sequence due to the presence of the first upstream primer or its extension product, forming a first cleavage product. Similarly, when a second target nucleic acid sequence is present, both the second upstream primer and the second selective probe hybridize with the second target nucleic acid sequence, and the second tag sequence in the second selective probe is in a free state and does not hybridize with the second target nucleic acid sequence. In this case, under the action of an enzyme with 5' nuclease activity, the second tag sequence or a portion thereof will be cleaved from the second selective probe hybridized with the second target nucleic acid sequence due to the presence of the second upstream primer or its extension product, forming a second cleavage product.

[0042] In step (3) of the method of the present invention, when a first cleavage product is present, since the first cleavage product contains a first tag sequence or a portion thereof, and the detection probe contains a first capture sequence complementary to the first tag sequence or a portion thereof, the first tag sequence hybridizes with the detection probe. Similarly, when a second cleavage product is present, since the second cleavage product contains a second tag sequence or a portion thereof, and the detection probe contains a second capture sequence complementary to the second tag sequence or a portion thereof, the second tag sequence hybridizes with the detection probe.

[0043] In step (4) of the method of the present invention, when a first cleavage product is present, since the first tag sequence hybridizes with the detection probe and the detection probe contains an additional sequence (e.g., a template sequence), the nuclease will use the detection probe as a template to extend the first tag sequence to form a first double strand. Similarly, when a second cleavage product is present, since the second tag sequence hybridizes with the detection probe and the detection probe contains an additional sequence (e.g., a template sequence), the nuclease will use the detection probe as a template to extend the second tag sequence to form a second double strand.

[0044] In step (5) of the method of the present invention, when the first double-stranded protein is present, a melting peak corresponding to the first double-stranded protein can be detected. Therefore, the presence or absence of the melting peak corresponding to the first double-stranded protein can be used to determine whether the first target nucleic acid sequence is present or not in the sample, thereby determining whether the genotype of the first target nucleic acid sequence is present in the sample. Similarly, the presence or absence of the melting peak corresponding to the second double-stranded protein can be used to determine whether the second target nucleic acid sequence is present or not in the sample, thereby determining whether the genotype of the second target nucleic acid sequence is present in the sample.

[0045] Based on the same principles described above, the method of the present invention can be implemented using more upstream primers and more selective probes to simultaneously detect more genotypes. Therefore, in some preferred embodiments, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or greater, and m is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater.

[0046] In some implementations, m is less than or equal to n.

[0047] Sensitivity and specificity

[0048] This application has experimentally demonstrated that, compared with methods where the selection probe's tag sequence is labeled with a quenching group and the detection probe's capture sequence is labeled with a fluorescent group, and compared with methods where the detection probe is simultaneously labeled with both a quenching group and a fluorescent group, the method and probe set of this application have better detection performance.

[0049] Therefore, in some embodiments, the genotyping method has higher sensitivity compared to a method where the tag sequence of the selection probe is labeled with a quencher group and the capture sequence of the detection probe is labeled with a fluorescent group. In some embodiments, the genotyping method has higher sensitivity compared to a method where the detection probe is simultaneously labeled with a quencher group and a fluorescent group.

[0050] In some embodiments, the sensitivity of the genotyping method is 1 × 10⁻⁶. 4 copies / mL, 1×10 3 copies / mL, 1×10 2 Copies / mL or 1×10 copies / mL. In some embodiments, the sensitivity of the genotyping method is 1×10 2 copies / mL.

[0051] Therefore, in some embodiments, the genotyping method has higher specificity compared to a method where the tag sequence of the selection probe is labeled with a quencher group and the capture sequence of the detection probe is labeled with a fluorescent group. In some embodiments, the genotyping method has higher specificity compared to a method where the detection probe is simultaneously labeled with a quencher group and a fluorescent group.

[0052] In some embodiments, the higher specificity is at least 2 times the specificity. For example, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, the higher specificity is at least 5 times the specificity.

[0053] In some embodiments, at least two consecutive nucleotides at the 5' end of the target-specific sequence are not complementary to a variant site (e.g., a single nucleotide polymorphism (SNP) site, a single nucleotide variant (SNV) site) in the target nucleic acid sequence; or, at least two consecutive nucleotides at the 3' end of a fragment in the target nucleic acid sequence complementary to the target-specific sequence do not contain a variant site.

[0054] In some embodiments, the fluorescent group is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red610, TEXAS RED, CAL Fluor Red635, Quasar 670, CY3, CY5, CY5.5, Quasar 705, or any combination thereof; and the quenching group is a molecule or group capable of quenching the fluorescence signal emitted by the fluorescent group (e.g., DABCYL, BHQ, ECLIPSE, and TAMRA).

[0055] In some embodiments, the melting curve analysis of the product of step (4) includes determining the presence of a certain genotype based on the melting peaks in the obtained melting curve and / or the melting temperature of the duplex.

[0056] In some implementations, the melting peak is obtained by taking the negative derivative of the melting curve, and the melting peak is negative (e.g., the melting peak is below the baseline of the melting curve) to indicate the presence of the genotype.

[0057] In some embodiments, the 5' end or 5' portion of each of the said tag sequences is labeled with the same or different fluorescent groups.

[0058] In some embodiments, each of the selected probes contains a different tag sequence from one another; and each of the selected probes targets a different target nucleic acid sequence of the genotype.

[0059] Detection probe

[0060] In this document, the detection probe is not limited to a specific structure. In some embodiments, the detection probe comprises, from 5' to 3', a template sequence and one or more capture sequences complementary to one or more of the tag sequences or portions thereof. In some embodiments, the 3' end or 3' portion of each capture sequence is independently labeled with the same or different quenching groups.

[0061] The detection probes in the detection system of this application can be designed to include one capture sequence or multiple capture sequences. The detection system of this application can also include both types of detection probes simultaneously. Those skilled in the art are capable of arbitrarily combining the exemplary embodiments provided below to form different specific embodiments. Therefore, it should be understood that the specific embodiments described below are for illustration and explanation only and are not intended to limit this application.

[0062] This application provides three exemplary implementations when some or all of the detection probes in the detection system are designed to include a capture sequence complementary to one or more of the tag sequence.

[0063] In a first exemplary embodiment, the selected probes have different tag sequences, but the tag sequences carry the same fluorescent group. Therefore, different tag sequences hybridize to different detection probes. Consequently, the extended products containing the tag sequences and the duplexes of the detection probes are structurally (sequence-wise) different from each other. Accordingly, the duplexes have their own different melting points (T0). m Therefore, under the same fluorescence signal, the presence of a melting point (T0) with a double chain can be detected in melting curve analysis. m The melting peak of the sample (value) is used to determine the presence of the corresponding target nucleic acid sequence in the sample, thereby determining whether the corresponding genotype exists in the sample.

[0064] In such embodiments, each of the tag sequences labels the same fluorescent group, and each of the capture sequences labels the same or different quenching groups; and the melting temperature of the duplex formed after hybridization of each tag sequence and its corresponding capture sequence differs (e.g., the melting temperature of the duplex formed after hybridization of one tag sequence and its corresponding capture sequence is T). m1The melting temperature of the double strand formed by the hybridization of the other tag sequence and its corresponding capture sequence is T. m2 And T m1 Greater than T m2 );

[0065] Furthermore, the melting curve analysis of the product of step (4) includes determining the presence of a certain genotype based on the melting temperature of the obtained double strand.

[0066] In such implementations, the tag sequences contained in each of the selected probes are different from each other.

[0067] In some implementations, each of the captured sequences is labeled with the same quenching group.

[0068] In some implementations, the number of selection probes is the same as the number of detection probes.

[0069] In this preferred embodiment, since both the tag sequence and the detection probe sequence are known or predetermined, the melting point (T0) of each bichain can be calculated in advance. m (Value). Therefore, by detecting the melting point (T) of the double-chain structure in melting curve analysis... m The melting peak of the target nucleic acid sequence can be used to determine the presence of the target nucleic acid sequence in the sample, thereby determining whether the genotype exists in the sample.

[0070] In such preferred embodiments, an exemplary schematic diagram is shown below. Figure 1 As shown.

[0071] In a second exemplary embodiment, the selected probes have the same tag sequence but different fluorescent groups. Therefore, the presence of a double-stranded structure can be detected by melting curve analysis under different fluorescent signals corresponding to the fluorescent groups. m The melting peak of the sample (value) is used to determine the presence of the corresponding target nucleic acid sequence in the sample, thereby determining whether the corresponding genotype exists in the sample.

[0072] In such embodiments, at least two of the tag sequences are labeled with different fluorescent groups; and the melting curve analysis of the product of step (4) includes: monitoring the fluorescence signal of each fluorescent group in real time, thereby obtaining the melting curve corresponding to the fluorescence signal of the fluorescent group and the melting temperature of the duplex; and then determining the presence of a certain genotype based on the type of fluorescence signal of the different fluorescent groups and the melting temperature of the duplex.

[0073] Wherein, the melting temperature of the double strand formed after hybridization of each of the tag sequences and the corresponding capture sequence is the same or different; each of the capture sequences is labeled with the same or different quenching groups.

[0074] In such preferred embodiments, the detection system contains an excess of detection probes.

[0075] In such preferred embodiments, an exemplary schematic diagram is shown below. Figure 2 As shown.

[0076] In a third exemplary embodiment, the selected probes have different tag sequences, each carrying a different fluorescent group. Therefore, the presence of a double-stranded structure can be detected by melting curve analysis under the fluorescence signals corresponding to different fluorescent groups. m The melting peak (value) is used to determine the presence of the target nucleic acid sequence in the sample, thereby determining whether the genotype exists in the sample.

[0077] In such embodiments, the tag sequence is labeled with different fluorescent groups; and the melting temperature of the double strand formed after hybridization of each tag sequence and its corresponding capture sequence is different.

[0078] Furthermore, the melting curve analysis of the product of step (4) includes: monitoring the fluorescence signal of each fluorescent group in real time to obtain the melting curve corresponding to the fluorescence signal of the fluorescent group and the melting temperature of the double strand; then, determining the presence of a certain genotype based on the fluorescence signal type of the different fluorescent groups and the melting temperature of the double strand.

[0079] In this context, all the selected probes contain different tag sequences; each of the capture sequences is labeled with the same or different quenching groups.

[0080] This application provides an exemplary implementation when at least one of the detection probes in the detection system contains at least two of the capture sequences that are complementary to at least two of the tag sequences or portions thereof.

[0081] In such implementations, different tag sequences are bound to the same detection probe. Therefore, the resulting cleavage products have different sequences and hybridize to different positions on the detection probe. Consequently, the extended product containing the tag sequence and the bistrand containing the detection probe are structurally (sequence-wise) different. Accordingly, the bistrands have their own different melting points (T0). m Therefore, in melting curve analysis, different duplexes exhibit different melting peaks. Thus, the presence of a duplex can be detected by the type of fluorescence signal and / or by detecting the melting point (T0) in melting curve analysis. mThe melting peak of the sample (value) can be used to determine the presence of the corresponding target nucleic acid sequence in the sample, and thus determine whether the corresponding genotype exists in the sample.

[0082] In such embodiments, the 3' end or 3' portion of each of the captured sequences is independently labeled with the same or different quenching groups.

[0083] Furthermore, the melting temperatures of the duplexes formed after hybridization of different tag sequences with different capture sequences in the same detection probe differ (for example, the melting temperature of the duplex formed after hybridization of one tag sequence and its corresponding capture sequence is T). m1 The melting temperature of the double strand formed after hybridization of the other tag sequence and its corresponding capture sequence is T. m2 And T m1 Greater than T m2 Furthermore, different tag sequences hybridized to the same detection probe are labeled with the same or different fluorescent groups;

[0084] Furthermore, the melting curve analysis of the product of step (4) includes determining the presence of a certain genotype based on the melting temperature of the obtained double strand and / or the type of fluorescence signal of the fluorescent group.

[0085] In such implementations, the tag sequences hybridized to the same detection probe are different from each other.

[0086] In such implementations, the number of detection probes is less than the number of selection probes.

[0087] In such preferred embodiments, the quenching group is the same for each of the captured sequence markers.

[0088] In such preferred embodiments, the tag sequences hybridized to the same detection probe are labeled with the same fluorescent group, and the tag sequences hybridized to different detection probes are labeled with different fluorescent groups;

[0089] Furthermore, the melting curve analysis of the product of step (4) includes: monitoring the fluorescence signal of each fluorescent group in real time, thereby obtaining multiple melting curves corresponding to the fluorescence signals of the different fluorescent groups and the melting temperature of the double strand; subsequently, determining the presence of a certain genotype based on the type of fluorescence signal of the fluorescent group and the melting temperature of the double strand.

[0090] In such preferred embodiments, an exemplary schematic diagram is shown below. Figure 3 As shown.

[0091] Enzymes with 5' nuclease activity

[0092] In this application, various enzymes or combinations thereof with 5' nuclease activity can be used to implement the methods of the present invention.

[0093] In some embodiments, the enzyme having 5' nuclease activity is a DNA polymerase.

[0094] In some embodiments, the DNA polymerase is obtained from the following eubacteria: *Thermus aquaticus* (Taq), *Thermus thermophiles* (Tth), *Thermus filiformis*, *Thermus flavus*, *Thermococcus literalis*, *Thermus antranildanii*, *Thermus caldophllus*, *Thermus chliarophilus*, *Thermus flavus*, *Thermus igniterrae*, *Thermus lacteus*, *Thermusoshimai*, *Thermus ruber*, *Thermus rubens*, *Thermus scotoductus*, *Thermus silvanus*, *Thermus thermophllus*, *Thermotoga maritima*, *Thermotoga neapolitana*, *Thermosiphoafricanus*, *Thermococcus litoralis*, *Thermococcus barossi*, *Thermococcus gorgonarius*, *Thermotoga maritima*, *Thermotoga... neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictiumoccultum, Aquifexpyrophilus and Aquifex aeolieus.

[0095] In some embodiments, the DNA polymerase is a Taq polymerase.

[0096] Samples and target nucleic acids

[0097] In the method of this invention, the sample can be any sample to be tested. For example, in some preferred embodiments, the sample contains DNA (e.g., genomic DNA or cDNA). In some preferred embodiments, the sample contains RNA (e.g., mRNA). In some preferred embodiments, the sample contains a mixture of nucleic acids (e.g., a mixture of DNA, a mixture of RNA, or a mixture of DNA and RNA).

[0098] When the sample to be tested contains mRNA, preferably, a reverse transcription reaction is performed before performing the method of the present invention to obtain cDNA complementary to the mRNA. A detailed description of the reverse transcription reaction can be found, for example, Joseph Sam-brook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

[0099] In some embodiments, the sample is selected from blood, saliva, urine, feces, cerebrospinal fluid, pleural fluid, breast milk, lymph, sputum, semen, cells, or any combination thereof.

[0100] In the method of this invention, the target nucleic acid sequence to be detected is not limited by its sequence composition or length. Furthermore, the target nucleic acid sequence to be detected can be single-stranded or double-stranded. The target nucleic acid sequence to be detected can be obtained from any source, including but not limited to prokaryotes (e.g., bacteria), eukaryotes (e.g., fungi, yeast, plants, animals including mammals and humans), or viruses or viroids.

[0101] In some implementations, the target nucleic acid sequence is a nucleic acid sequence obtained from a virus.

[0102] In some embodiments, the virus is selected from human papillomavirus (HPV), hepatitis B virus (HBV), rotavirus, norovirus, Coxsackievirus, measles virus, varicella-zoster virus, influenza virus, or any combination thereof.

[0103] In some implementations, the target nucleic acid sequence is a nucleic acid sequence obtained from the variable region of the viral genome.

[0104] In some implementations, the virus is HPV.

[0105] In some implementations, the n genotypes are selected from any of the following HPV subtypes: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, and 83.

[0106] In some implementations, the n genotypes are selected from any of the following HPV subtypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

[0107] In some implementations, each of the target nucleic acid sequences is a nucleic acid sequence obtained from the variable region of the genome of the corresponding HPV subtype.

[0108] In some implementations, the sample is a sample obtained from the subject's cervix. For example, cervical exfoliated cells.

[0109] Selecting probes and detection probes

[0110] In some embodiments, the select probe and the detect probe each independently comprise naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-naturally occurring nucleotides (e.g., peptide nucleic acids (PNAs) or locked nucleic acids), or any combination thereof.

[0111] In this document, neither the selection probe nor the detection probe is limited in length. In some embodiments, the lengths of the selection probe and the detection probe are each independently 11-20 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, 100-200 nt, 200-300 nt, 300-400 nt, 400-500 nt, 500-600 nt, 600-700 nt, 700-800 nt, 800-900 nt, or 900-1000 nt.

[0112] The target-specific sequence in the selective probe can be of any length, as long as it can specifically hybridize with the target nucleic acid sequence. In some embodiments, the length of the target-specific sequence in the selective probe is 10-140 nt, for example, 10-20 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, or 130-140 nt.

[0113] The tag sequence in the selector probe can be of any length, as long as it can specifically hybridize with and extend the capture probe. In some embodiments, the tag sequence in the selector probe is 5-140 nt in length, for example, 5-10 nt, 10-20 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.

[0114] In some embodiments, the upstream primer comprises naturally occurring nucleotides, modified nucleotides, non-naturally occurring nucleotides, or any combination thereof.

[0115] In the method of this invention, the upstream primer is not limited in length, as long as it can specifically hybridize with the target nucleic acid sequence. In some embodiments, the length of each upstream primer is independently 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, or 140-150 nt.

[0116] In some embodiments, after hybridization with the target nucleic acid sequence, the upstream primer is located distal upstream of the select probe, or adjacent upstream of the select probe, or has a partially overlapping sequence with the select probe.

[0117] In some implementations, the upstream primer is a primer specific to the target nucleic acid sequence.

[0118] In some implementations, steps (1) and (2) are performed simultaneously; for example, the upstream primer, the selective probe, and the enzyme with 5' nuclease activity are provided and contacted with the target nucleic acid sequence, provided that nucleic acid extension and cleavage of the selective probe are permitted.

[0119] In some embodiments, the enzyme with 5' nuclease activity used in step (2) is a nucleic acid polymerase with 5' nuclease activity, and is the same as the nucleic acid polymerase used in step (4).

[0120] In some embodiments of the present invention, the upstream primer and the downstream primer serve as target-specific primer pairs for amplifying the target nucleic acid. Depending on the method for amplifying the target nucleic acid, one or more pairs of target-specific primers may be used for each target nucleic acid. Therefore, in some embodiments, in step (1), in addition to the upstream primer and the selection probe, a downstream primer is provided for each target nucleic acid sequence; wherein the downstream primer contains a sequence complementary to the target nucleic acid sequence; and, when the downstream primer hybridizes with the target nucleic acid sequence, the downstream primer is located downstream of the target-specific sequence.

[0121] In some embodiments, the nucleic acid polymerase having 5' nuclease activity catalyzes the upstream and downstream primers to amplify the target nucleic acid sequence and induces cleavage of the selected probe.

[0122] In some embodiments, the downstream primer comprises naturally occurring nucleotides, modified nucleotides, non-naturally occurring nucleotides, or any combination thereof.

[0123] In the method of this invention, the downstream primers are not limited in length, as long as they can specifically hybridize with the target nucleic acid sequence. In some embodiments, the lengths of the downstream primers are each independently 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, or 140-150 nt.

[0124] In some implementations, the downstream primer is a primer specific to the target nucleic acid sequence.

[0125] In some embodiments, in step (4), under conditions that allow for extension of the nucleic acid polymerase, the nucleic acid polymerase extends the hybridization product present in step (3) using the detection probe as a template, thereby forming the double strand.

[0126] In some embodiments, in step (5), the product of step (4) is gradually heated or cooled and the fluorescence signal emitted by the fluorescent group hybridized to each of the detection probes is monitored in real time, thereby obtaining a melting curve of the fluorescence signal intensity of the fluorescent group as a function of temperature.

[0127] In some implementations, the presence of a tag sequence corresponding to the melting peak and / or melting temperature is determined based on the melting peak and / or melting temperature in the melting curve; subsequently, the presence of the genotype corresponding to the tag sequence is determined by the correspondence between the tag sequence and the target nucleic acid sequence and the correspondence between the target nucleic acid sequence and the genotype.

[0128] probe group

[0129] In a second aspect, the present invention provides a probe set comprising at least one detection probe and at least two select probes, the probe set being used to detect at least two target nucleic acid sequences, wherein...

[0130] The selection probe consists of a tag sequence and a target-specific sequence from the 5' to 3' direction. The tag sequence contains a sequence that is not complementary to the corresponding target nucleic acid sequence, and the target-specific sequence contains a sequence that is complementary to the corresponding target nucleic acid sequence. Furthermore, one or more consecutive nucleotides at the 5' end of the target-specific sequence are completely complementary to the corresponding target nucleic acid sequence. In addition, the tag sequence is labeled with a fluorescent group.

[0131] The detection probe includes one or more capture sequences that are complementary to one or more of the tag sequences or portions thereof; and each capture sequence is independently labeled with a quenching group such that the fluorescence signal emitted when the capture sequence of the detection probe and its corresponding tag sequence hybridize is different from the fluorescence signal emitted when it does not hybridize with the tag sequence.

[0132] It is readily understood that the probe set can be used to implement the genotyping methods described in detail above. Therefore, the various technical features described in detail above regarding the genotyping methods can also be applied to the probe set.

[0133] In some embodiments, the probe set includes m types of detection probes and n types of selection probes, the probe set being used to detect the target nucleic acid sequences of n genotypes. Wherein, n is an integer greater than or equal to 2, and m is an integer greater than or equal to 1.

[0134] In some implementations, the probe set determines the presence of one or more of the n genotypes in the sample by detecting the target nucleic acid sequences of the n genotypes.

[0135] In some implementations, the m detection probes comprise n capture sequences, each of which is complementary to a tag sequence or a portion thereof of the n select probes.

[0136] In some implementations, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or greater, and m is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater.

[0137] In some implementations, m is less than or equal to n.

[0138] In some embodiments, the tag sequences contained in each of the selected probes are different from each other. Furthermore, each of the selected probes targets a different target nucleic acid sequence for a different genotype.

[0139] In some embodiments, at least two consecutive nucleotides at the 5' end of the target-specific sequence are not complementary to any variant site (e.g., single nucleotide polymorphism (SNP) site, single nucleotide variant (SNV) site) in the target nucleic acid sequence. Alternatively, at least two consecutive nucleotides at the 3' end of the segment in the target nucleic acid sequence complementary to the target-specific sequence do not contain any variant site.

[0140] In some embodiments, the fluorophore is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red610, TEXAS RED, CAL Fluor Red635, Quasar 670, CY3, CY5, CY5.5, Quasar 705, or any combination thereof. Furthermore, the quenching group is a molecule or group capable of quenching the fluorescence signal emitted by the fluorophore (e.g., DABCYL, BHQ, ECLIPSE, and TAMRA).

[0141] In some embodiments, the detection probe comprises, from 5' to 3', a template sequence and one or more capture sequences complementary to one or more of the tag sequences or portions thereof; wherein the 3' end or 3' portion of each capture sequence is independently labeled with the same or different quenching groups.

[0142] In some embodiments, at least one of the detection probes comprises at least two capture sequences complementary to at least two of the tag sequences or portions thereof, and the 3' end or 3' portion of each of the capture sequences is independently labeled with the same or different quenching groups;

[0143] Furthermore, the melting temperatures of the duplexes formed after hybridization of different tag sequences with different capture sequences in the same detection probe differ (for example, the melting temperature of the duplex formed after hybridization of one tag sequence and its corresponding capture sequence is T). m1 The melting temperature of the double strand formed after hybridization of the other tag sequence and its corresponding capture sequence is T. m2 And T m1 Greater than T m2 Furthermore, different tag sequences hybridized to the same detection probe are labeled with the same or different fluorescent groups.

[0144] In some implementations, the tag sequences hybridized to the same detection probe are different from each other.

[0145] In some embodiments, the quenching group is the same for each of the captured sequence markers.

[0146] In some embodiments, the tag sequences hybridized to the same detection probe are labeled with the same fluorescent group, and the tag sequences hybridized to different detection probes are labeled with different fluorescent groups.

[0147] In some embodiments, at least one of the detection probes comprises a plurality of capture sequences complementary to a plurality of the tag sequences or portions thereof, and the 3' end or 3' portion of each of the capture sequences is independently labeled with the same or different quenching groups;

[0148] Furthermore, the melting temperatures of the duplexes formed after hybridization of different tag sequences with different capture sequences in the same detection probe differ (for example, the melting temperature of the duplex formed after hybridization of a tag sequence with a corresponding capture sequence in a detection probe is T). m1 The melting temperature of the double strand formed after the other tag sequence hybridizes with the corresponding capture sequence in the detection probe is T. m2 And T m1 Greater than T m2 Furthermore, different tag sequences hybridized to the same detection probe are labeled with the same or different fluorescent groups.

[0149] In some embodiments, the tag sequences hybridized to the same detection probe are labeled with the same fluorescent group, and the tag sequences hybridized to different detection probes are labeled with different fluorescent groups.

[0150] Reagent test kit

[0151] In a third aspect, the present invention provides a kit comprising one or more probe sets as defined in any of the second aspects.

[0152] In some embodiments, the kit contains one, two, three, four, five, six, seven, eight, nine, ten or more of the probe sets.

[0153] In some embodiments, the kit further comprises an upstream primer containing a sequence complementary to the target nucleic acid sequence, and the upstream primer is located upstream of the target-specific sequence when the upstream primer and the selectable probe hybridize with the target nucleic acid sequence.

[0154] In some embodiments, the kit further comprises a downstream primer containing a sequence complementary to the target nucleic acid sequence. Furthermore, when the downstream primer hybridizes with the target nucleic acid sequence, the downstream primer is located downstream of the target-specific sequence.

[0155] In some embodiments, the kit further comprises an enzyme having 5' nuclease activity. In some embodiments, the enzyme having 5' nuclease activity is a DNA polymerase.

[0156] It is readily understood that the kit can be used to implement the genotyping methods described in detail above. Therefore, the various technical features of the enzymes, upstream primers, and downstream primers with 5' nuclease activity described in detail above for the genotyping methods can also be applied to the kit. Attached Figure Description

[0157] Figure 1 A first exemplary embodiment of the method of the present invention is illustrated to explain the basic principles of the method of the present invention.

[0158] Figure 2 A second exemplary embodiment of the method of the present invention is illustrated to explain the basic principles of the method of the present invention.

[0159] Figure 3 A third exemplary embodiment of the method of the present invention is illustrated to explain the basic principles of the method of the present invention.

[0160] Figure 4 The results are for test group 1 in Example 1.

[0161] Figure 5 The results are for experimental group 2 in Example 1.

[0162] Figure 6 The results are the detection results of HPV52 genomic DNA in experimental group 1 of Example 2.

[0163] Figure 7 The results of HPV52 genomic DNA detection in experimental group 2 of Example 2 are shown.

[0164] Figure 8 The results are for the negative samples in experimental group 1 of Example 2.

[0165] Figure 9 The results are for the negative samples in experimental group 2 of Example 2.

[0166] Figure 10 The results are for HPV31 detection in Example 3.

[0167] Figure 11 The results are for HPV66 detection in Example 3.

[0168] Figure 12 The results are for HPV52 detection in Example 3.

[0169] Figure 13 The results are for HPV31 / 52 / 66 in Example 3.

[0170] Figure 14 The results are for the detection of HPV31 / 52 / 66 in the FAM fluorescence channel in Example 4.

[0171] Figure 15 The results are the detection results of HPV33 / 35 / 45 / 59 in the VIC fluorescence channel in Example 4.

[0172] Figure 16 The results are the detection results of HPV56 / 18 / 58 / 51 in the ROX fluorescence channel in Example 4.

[0173] Figure 17 The results are for the detection of HPV16 / 39 / 68 in the CY5 fluorescence channel in Example 4.

[0174] Beneficial effects

[0175] The method of this invention enables simultaneous detection of multiple genotypes (multiplex detection), and compared with existing technologies (e.g., methods where the selector probe's tag sequence is labeled with a quencher group and the detection probe's capture sequence is labeled with a fluorescent group, and methods where the detection probe is simultaneously labeled with a quencher group and a fluorescent group), the method and probe set of this application have better detection performance. Specifically, the method and probe set of this application have higher sensitivity (e.g., sensitivity can reach 1×10⁻⁶). 2 (copies / mL), and higher specificity (e.g., R) m (The value is at least 5 times higher). Therefore, this invention provides a simple, efficient, and low-cost multiple genotyping method. Detailed Implementation

[0176] The specific embodiments of this application are described in detail below. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this application.

[0177] Those skilled in the art will understand that the embodiments are described by way of example and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed under conventional conditions or conditions recommended by the manufacturer, such as at room temperature and pressure. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0178] Example 1

[0179] This application evaluates the sensitivity and specificity of its detection protocol compared to a control protocol.

[0180] Specifically, in the detection scheme of this application (Experimental Group 2 below), the selector probe's tag sequence is labeled with a fluorescent group, and the detection probe's capture sequence is labeled with a quenching group. In the control detection scheme (Experimental Group 1 below), the rest of the probe design remains unchanged, the only difference being that the selector probe's tag sequence is labeled with a quenching group, and the detection probe's capture sequence is labeled with a fluorescent group.

[0181] Experimental Materials: Using HPV18 as the detection target, the 30 μL PCR-melting curve analysis reaction system included: 1×buffer (100 mM Tris-HCl, 50 mM KCl, and 0.33 mg / mL BSA), 3.0 mM MgCl2, 0.3 mM dNTPs, 3.6 U of Taq polymerase (Baorui Biotechnology Co., Ltd., Zhuhai), 300 nM each of upstream and downstream primers, 100 nM of select probe, 100 nM of detection probe, and 5 μL of HPV18 genomic DNA at different concentrations. The primer and probe sequences are shown in Table 1.

[0182] Table 1 Primer and probe sequences

[0183]

[0184]

[0185] Note: -PHO is used to block the 3'-OH of the selective probe (the same applies below), -BHQ2 is a quenching group, and -ROX is a fluorescent group.

[0186] Experimental process

[0187] Experimental group 1 used selector probe 1 and detection probe 1 in the reaction solution, and experimental group 2 used selector probe 2 and detection probe 2 in the reaction solution. 5 μL of each solution was taken at a concentration of 1×10⁻⁶. 6 1×10 5 1×10 4 1×10 3 and 1×10 2 HPV18 genomic DNA was detected at copies / mL, with three replicates per sample. The PCR-melting curve reaction was performed in Shanghai Hongshi's fully automated medical PCR analysis system according to the reaction conditions in Table 2.

[0188] Table 2 Reaction Procedure

[0189]

[0190] Experimental results

[0191] After the reaction was completed, the Tm value and Rm value of the melting peak were statistically analyzed, and the results are shown in Table 3. Figure 4 (Experimental Group 1) and Figure 5 As shown in (Experimental Group 2).

[0192] Table 3. Sensitivity detection results for different modification methods of the selected and detection probes.

[0193]

[0194]

[0195] Experimental conclusion: The results show that the sensitivity of the reagent with selected probe modified with quencher group and detection probe modified with fluorescent group is only 1×10⁻⁶. 4 copies / mL. However, by selecting probes modified with fluorescent groups and detecting probes modified with quenching groups, the reagent sensitivity can reach 1×10⁻⁶. 2 Copies / mL. Furthermore, when the sample concentrations of the two modification schemes were the same, the melting peak height Rm value produced by the modification scheme of selecting the probe to modify fluorescence and the detection probe to modify quenching was at least 5 times that of the opposite modification scheme, demonstrating a significant improvement in specificity.

[0196] Example 2

[0197] The specificity evaluation of the test protocol and the control test protocol in this application.

[0198] Specifically, in the detection scheme of this application (Experimental Group 1 below), the selector probe is labeled with a fluorescent group, and the capture sequence of the detection probe is labeled with a quenching group. In the control detection scheme (Experimental Group 2 below), the rest of the probe design remains unchanged, the only difference being that the selector probe is not labeled with any group, while the detection probe is labeled with both a fluorescent group and a quenching group.

[0199] Experimental Materials: Using HPV52 as the detection target, the 30 μL PCR-melting curve analysis reaction system included: 1×buffer (100 mM Tris-HCl, 50 mM KCl, and 0.33 mg / mL BSA), 3.0 mM MgCl2, 0.3 mM dNTPs, 3.6 U of polymerase Taq (Baorui Biotechnology Co., Ltd., Zhuhai), 300 nM each of upstream and downstream primers, 100 nM of select probe, 100 nM of detection probe, and 5 μL of HPV52 genomic DNA and clinical HPV52 negative samples. Primers and probes are shown in Table 4.

[0200] Table 4 Primer and probe sequences

[0201]

[0202]

[0203] Note: / int- / indicates intermediate modification, the same below; -FAM is a fluorescent group, -BHQ1 is a quenching group, the same below; int-6-FAM dT indicates that the T base at this position modifies the FAM fluorescent group.

[0204] Experimental process

[0205] In experimental group 1, the reaction solution used selective probe 3 and detection probe 3; in experimental group 2, the reaction solution used selective probe 4 and detection probe 4. 5 μL of each solution was taken at a concentration of 1×10⁻⁶. 4 HPV52 genomic DNA and clinical HPV52 negative samples were tested at copies / mL. Each sample was tested in triplicate, and PCR-melting curve reactions were performed using Shanghai Hongshi's fully automated medical PCR analysis system according to the reaction conditions in Table 2.

[0206] Experimental results

[0207] After the reaction was completed, the Tm value and Rm value of the melting peak were statistically analyzed. The results are shown in Table 5. Figure 6 (Experimental group 1, HPV52 genomic DNA) Figure 7 (Experimental Group 2, HPV52 genomic DNA) Figure 8 (Experimental group 1, negative sample) and Figure 9 (Experimental group 2, negative sample) is shown.

[0208] Table 5

[0209]

[0210] Experimental Conclusion: The experimental results show that modifying the fluorescent group with the selected probe and modifying the quencher group with the detection probe results in a higher melting peak height (Rm value) for detecting the HPV52 target compared to selecting an unmodified probe and modifying the detection probe with a fluorescent quencher group. Furthermore, the baseline of negative samples is flatter. This indicates that the modification method of the present invention has higher specificity.

[0211] Example 3

[0212] An exploration of genotyping using a single detection probe combined with multiple selective probes (Schematic C).

[0213] Experimental materials: The 30 μL PCR-melting curve analysis reaction system included: 1×buffer (100 mM Tris-HCl, 50 mM KCl, and 0.33 mg / mL BSA), 3.0 mM MgCl2, 0.3 mM dNTPs, 3.6 U of Taq polymerase (Baorui Biotechnology Co., Ltd., Zhuhai), 300 nM upstream and downstream primers, 100 nM select probes, 300 nM detection probes, and 5 μL of HPV18, 56, and 58 genomic DNA. Primer and probe sequences are shown in Table 6.

[0214] Table 6 Primer and probe sequences

[0215]

[0216] Experimental process

[0217] Experimental group 1 used 5 μL of a concentration of 1×10 4 HPV31 genomic DNA copies / mL, and 5 μL of 1×10⁻⁶ HPV31 genomic DNA in experimental group 2. 4 HPV66 genomic DNA copies / mL, and 5 μL of 1×10⁻⁶ HPV66 genomic DNA in experimental group 3. 4 HPV52 genomic DNA copies / mL, and 5 μL of 1×10⁻⁶ HPV52 genomic DNA was taken from experimental group 4. 4 HPV31 / 66 / 52 mixed genomic DNA (copies / mL) was subjected to PCR-melting curve reaction in Shanghai Hongshi's fully automated medical PCR analysis system according to the reaction conditions in Table 2.

[0218] Experimental results

[0219] After the reaction was completed, the Tm value and Rm value of the melting peak were statistically analyzed, and the results are shown in Table 7. Figure 10 (HPV31) Figure 11 (HPV66) Figure 12 (HPV52) and Figure 13 As shown in (HPV31 / 52 / 66).

[0220] Table 7

[0221]

[0222] Experimental conclusion: The results of melting analysis show that when the detection probe is modified with a quenching group, it can bind to multiple detection probes to achieve the purpose of genotyping.

[0223] Example 4

[0224] This invention achieves the goal of detecting 14 types of HPV and identifying specific types in a single tube.

[0225] Experimental materials: The 30 μL PCR-melting curve analysis reaction system included: 1×buffer (100 mM Tris-HCl, 50 mM KCl, and 0.33 mg / mL BSA), 3.0 mM MgCl2, 0.3 mM dNTPs, 3.6 U of Taq polymerase (Baorui Biotechnology Co., Ltd., Zhuhai), 300 nM upstream and downstream primers, 100 nM selection probes, 100 nM detection probes, and 5 μL of genomic DNA. The primers and probes are shown in Table 8 below.

[0226] Table 8

[0227]

[0228]

[0229] Note: -ROX, -FAM, -VIC, and CY5 are different fluorescent groups; -BHQ1 and -BHQ2 are different quenching groups.

[0230] Experimental process

[0231] Take 5 μL of each concentration of 1×10 4 HPV genomic DNA of 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 copies / mL were subjected to PCR-melting curve reaction in the fully automated medical PCR analysis system of Shanghai Hongshi according to the reaction conditions in Table 2.

[0232] Experimental results

[0233] After the reaction was completed, the Tm value and Rm value of the melting peak were statistically analyzed, and the results are shown in Table 9. Figure 14 (FAM fluorescence channels, HPV31 / 52 / 66) Figure 15 (VIC fluorescence channel, HPV33 / 35 / 45 / 59) Figure 16 (ROX fluorescence channels, HPV56 / 18 / 58 / 51) and Figure 17(CY5 fluorescence channel, HPV16 / 39 / 68) shown.

[0234] Table 9

[0235]

[0236] Experimental conclusion: Using the method of the present invention, it is possible to detect 14 types of HPV and achieve genotyping in one tube.

[0237] Example 5

[0238] The kit obtained in this invention was used to test 359 clinical samples. At the same time, a parallel comparative experiment was conducted using the human papillomavirus nucleic acid typing detection kit produced by Sansure Biotech Co., Ltd. (this kit is currently the mainstream detection kit used in hospitals, using the PCR-fluorescent probe method) to evaluate the effectiveness of the kit.

[0239] The detection principle of Sansure reagent is qPCR. In this example, 14 subtypes are detected through a 4-tube reaction system, while the present invention detects 14 subtypes through a 1-tube system.

[0240] Sansure Reagents Testing Process

[0241] 1. Preparation of amplification reagents

[0242] Remove the reagents from the -20℃ freezer, melt and mix each reagent, and then briefly centrifuge. Prepare reaction solutions 1, 2, 3, and 4 (38 μL PCR reaction solution + 2 μL enzyme mixture) separately, mix thoroughly to form PCR mixtures, centrifuge at 2000 rpm for 10 seconds, and transfer the aliquoted PCR mixtures into the transfer window for later use.

[0243] 2. Nucleic acid extraction

[0244] Mix the samples thoroughly, and take 200 μL of each sample for nucleic acid extraction. After nucleic acid extraction, take 10 μL of the extracted product from the same case and add it to reaction solutions 1, 2, 3, and 4 respectively. Tightly cap the tubes, vortex and briefly centrifuge, and then transfer them to the transfer window.

[0245] 3. PCR amplification (between stages)

[0246] (1) Place the PCR reaction tube into the amplification instrument and set the sample name.

[0247] (2) Perform PCR amplification analysis according to the parameters in the table below.

[0248] The amplification procedure for Sansure HPV reagent is shown in Table 10.

[0249] Table 10

[0250]

[0251] The reagent detection process of this invention is as follows: Prepare the reaction solution according to the experimental material requirements, take 5 μL of the nucleic acid extraction product of each sample, and perform PCR-melting curve reaction in the fully automated medical PCR analysis system of Shanghai Hongshi according to the reaction conditions in Table 2.

[0252] Based on the positive and negative results of different subtypes detected by the two methods, the positive concordance rate and the total negative concordance rate were calculated respectively. Taking subtype HPV16 as an example, the positive concordance rate was: the number of positive subtype HPV16 detected by the reagent of this invention / 37 (the number of positive subtype 16 detected by the Sansure reagent); the total negative concordance rate was: the number of cases where both the reagent of this invention and the Sansure reagent were negative / the number of cases where the Sansure reagent was negative.

[0253] Specifically, the detection and calculation results of the two methods are shown in Tables 11 and 12.

[0254] Table 11

[0255]

[0256] Table 12 Detection Results of Negative Samples

[0257]

[0258] After completion, the genotype concordance rate was 427 / 430*100% = 99.30%, and the overall negative concordance rate was 105 / 106*100% = 99.1%. The experimental results indicate that the method and probe set of this application have good detection performance and equivalent detection accuracy compared to mainstream detection methods currently used in hospitals.

Claims

1. A genotyping method, characterized in that, The method for determining the presence of one or more of the n genotypes in a sample by detecting the target nucleic acid sequences of n genotypes includes the following steps: (1) For each of the target nucleic acid sequences, an upstream primer and a selection probe are provided; The upstream primer contains a sequence complementary to the target nucleic acid sequence; the selective probe consists of a tag sequence and a target-specific sequence from the 5' to 3' direction, wherein the tag sequence contains a sequence not complementary to the target nucleic acid sequence, the target-specific sequence contains a sequence complementary to the target nucleic acid sequence, and one or more consecutive nucleotides at the 5' end of the target-specific sequence are completely complementary to the target nucleic acid sequence; the tag sequence is labeled with a fluorescent group; and when the upstream primer and the selective probe hybridize with the target nucleic acid sequence, the upstream primer is located upstream of the target-specific sequence. Furthermore, under conditions that allow nucleic acid hybridization, the sample is contacted with the upstream primer and the selective probe; (2) Under conditions that allow cleavage of the selected probe, the product of step (1) is contacted with an enzyme having 5' nuclease activity, wherein the enzyme having 5' nuclease activity is able to cleave the selected probe that hybridizes with the target nucleic acid sequence and release the tag sequence labeled with the fluorescent group. (3) Provide m types of detection probes, among which, Each of the detection probes comprises one or more capture sequences complementary to one or more of the tag sequences or portions thereof; and the m detection probes comprise at least n capture sequences, each complementary to the tag sequences or portions thereof of the n selected probes in step (1); and each capture sequence is independently labeled with a quenching group such that the fluorescence signal emitted when the capture sequence of each detection probe and its corresponding tag sequence hybridize is different from the fluorescence signal emitted when no hybridization product is generated with the tag sequence. Furthermore, under conditions that allow nucleic acid hybridization, the product of step (2) is contacted with the m detection probes; (4) Under conditions that allow the nucleic acid polymerase to extend, the product of step (3) is brought into contact with the nucleic acid polymerase, so that the hybridization product present in step (3) is extended to generate a double strand. (5) Perform melting curve analysis on the product of step (4); and determine whether one or more of the n genotypes exist in the sample based on the results of the melting curve analysis. Wherein, n is an integer greater than or equal to 2, and m is an integer greater than or equal to 1; optionally, the design of the upstream primer and the target-specific sequence is based on the differences in the target nucleic acid sequences of the n genotypes.

2. The method of claim 1, wherein, Compared with methods where the selection probe's tag sequence is labeled with a quenching group and the detection probe's capture sequence is labeled with a fluorescent group, the genotyping method has higher sensitivity. Preferably, the sensitivity of the genotyping method is 1×10⁻⁶. 4 copies / mL, 1×10 3 copies / mL, 1×10 2 copies / mL or 1×10 copies / mL; preferably, the sensitivity of the genotyping method is 1×10 2 copies / mL.

3. The method of claim 1 or 2, wherein, Compared with methods where the selection probe's tag sequence is labeled with a quenching group and the detection probe's capture sequence is labeled with a fluorescent group, the genotyping method has higher specificity; Preferably, the higher specificity is at least 2 times the specificity; for example, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or at least 10 times; preferably, the higher specificity is at least 5 times the specificity.

4. The method according to any one of claims 1-3, wherein, At least two consecutive nucleotides at the 5' end of the target-specific sequence are not complementary to any variant site (e.g., single nucleotide polymorphism (SNP) site, single nucleotide variant (SNV) site) in the target nucleic acid sequence; or, at least two consecutive nucleotides at the 3' end of the segment in the target nucleic acid sequence that is complementary to the target-specific sequence do not contain any variant site. Preferably, the fluorescent group is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705, or any combination thereof; and the quenching group is a molecule or group capable of quenching the fluorescence signal emitted by the fluorescent group (e.g., DABCYL, BHQ, ECLIPSE, and TAMRA).

5. The method according to any one of claims 1-4, wherein, The melting curve analysis of the product of step (4) includes: determining the presence of a certain genotype based on the melting peak in the obtained melting curve and / or the melting temperature of the double strand; Preferably, the melting peak is obtained by taking the negative derivative of the melting curve, and the melting peak is a negative peak (e.g., the melting peak is below the baseline of the melting curve) to indicate the presence of the genotype.

6. The method according to any one of claims 1-5, wherein, The method has one or more features selected from the following: (1) The detection probe comprises, from 5' to 3', a template sequence and one or more capture sequences that are complementary to one or more of the tag sequences or a portion thereof; (2) The 5' end or 5' portion of each of the aforementioned tag sequences is labeled with the same or different fluorescent groups; (3) The 3' end or 3' portion of each of the captured sequences is independently labeled with the same or different quenching groups.

7. The method according to any one of claims 1-6, wherein, Each of the detection probes includes a capture sequence that is complementary to one of the tag sequences or a portion thereof.

8. The method of claim 7, wherein, Each of the stated tag sequences labels the same fluorescent group, and each of the stated capture sequences labels the same or different quenching groups; furthermore, the melting temperature of the duplex formed after hybridization of each stated tag sequence and its corresponding capture sequence differs (e.g., the melting temperature of the duplex formed after hybridization of one stated tag sequence and its corresponding capture sequence is T). m1 The melting temperature of the double strand formed by the hybridization of the other tag sequence and its corresponding capture sequence is T. m2 And T m1 Greater than T m2 ); Furthermore, the melting curve analysis of the product of step (4) includes: determining the presence of a certain genotype based on the melting temperature of the obtained double strand; Preferably, the tag sequences contained in each of the selected probes are different from each other; Preferably, each of the captured sequences is labeled with the same quenching group; Preferably, the number of selection probes is the same as the number of detection probes.

9. The method of claim 7, wherein, At least two of the tag sequences are labeled with different fluorescent groups; and the melting curve analysis of the product of step (4) includes: monitoring the fluorescence signal of each fluorescent group in real time, thereby obtaining the melting curve corresponding to the fluorescence signal of the fluorescent group and the melting temperature of the duplex; and then determining the presence of a certain genotype based on the type of fluorescence signal of the different fluorescent groups and the melting temperature of the duplex. Wherein, the melting temperature of the double strand formed after hybridization of each of the tag sequences and the corresponding capture sequence is the same or different; the tag sequences contained in all the selected probes are the same or different; and each of the capture sequences is labeled with the same or different quenching groups. Preferably, the number of selection probes is the same as the number of detection probes.

10. The method according to any one of claims 1-6, wherein, At least one of the detection probes comprises at least two capture sequences that are complementary to at least two of the tag sequences or portions thereof, and the 3' end or 3' portion of each of the capture sequences is independently labeled with the same or different quenching groups; Furthermore, the melting temperatures of the duplexes formed after hybridization of different tag sequences with different capture sequences in the same detection probe differ (for example, the melting temperature of the duplex formed after hybridization of one tag sequence and its corresponding capture sequence is T). m1 The melting temperature of the double strand formed after hybridization of the other tag sequence and its corresponding capture sequence is T. m2 And T m1 Greater than T m2 Furthermore, different tag sequences hybridized to the same detection probe are labeled with the same or different fluorescent groups; Furthermore, the melting curve analysis of the product of step (4) includes: determining the presence of a certain genotype based on the melting temperature of the obtained double strand and / or the type of fluorescence signal of the fluorescent group; Preferably, the tag sequences hybridized onto the same detection probe are different from each other; Preferably, the number of detection probes is less than the number of selection probes; Preferably, the quenching group is the same for each of the captured sequence markers.

11. The method of claim 10, wherein, The tag sequences hybridized to the same detection probe are labeled with the same fluorescent group, and the tag sequences hybridized to different detection probes are labeled with different fluorescent groups. Furthermore, the melting curve analysis of the product of step (4) includes: monitoring the fluorescence signal of each fluorescent group in real time, thereby obtaining multiple melting curves corresponding to the fluorescence signals of the different fluorescent groups and the melting temperature of the double strand; subsequently, determining the presence of a certain genotype based on the type of fluorescence signal of the fluorescent group and the melting temperature of the double strand.

12. The method according to any one of claims 1-10, wherein, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or greater, and m is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater; Preferably, m is less than or equal to n; Preferably, the tag sequences contained in each of the selected probes are different from each other; and each of the selected probes targets a different target nucleic acid sequence of the genotype.

13. The method according to any one of claims 1-12, wherein, The enzyme with 5' nuclease activity is DNA polymerase; Preferably, the DNA polymerase is obtained from the following eubacteria: Thermus aquaticus (Taq), Thermusthermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, Thermusflavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai,Thermus ruber,Thermus rubens,Thermus scotoductus,Thermus silvanus,Thermus thermophllus,Thermotoga maritima,Thermotoga neapolitana,Thermosipho africanus,Thermococcuslitoralis,Thermococcus barossi,Thermococcus gorgonarius,Thermotoga maritima,Thermotoga neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcushorikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifexaeolieus; Particularly preferred is that the DNA polymerase is Taq polymerase.

14. The method according to any one of claims 1-13, wherein, The method has one or more features selected from the following: (1) The sample contains a mixture of DNA, or RNA, or nucleic acids; (2) The target nucleic acid sequence is DNA or RNA; (3) The target nucleic acid sequence is obtained from prokaryotes, eukaryotes, viruses or viroids; (4) The sample is selected from blood, saliva, urine, feces, cerebrospinal fluid, pleural fluid, milk, lymph, sputum, semen, cells, or any combination thereof.

15. The method according to any one of claims 1-14, wherein, The target nucleic acid sequence is a nucleic acid sequence obtained from a virus; Preferably, the virus is selected from human papillomavirus (HPV), hepatitis B virus (HBV), rotavirus, norovirus, Coxsackievirus, measles virus, varicella-zoster virus, influenza virus, or any combination thereof; Preferably, the target nucleic acid sequence is a nucleic acid sequence obtained from the variable region of the viral genome.

16. The method of claim 15, wherein, The virus in question is HPV; Preferably, the n genotypes are selected from any of the following HPV subtypes: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, and 83; Preferably, the n genotypes are selected from any of the following HPV subtypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68; Preferably, each of the target nucleic acid sequences is a nucleic acid sequence obtained from the variable region of the genome of the corresponding HPV subtype; Preferably, the sample is a sample obtained from the cervix of the subject; for example, cervical exfoliated cells.

17. The method according to any one of claims 1-16, wherein, The selection probe and the detection probe each independently have one or more features selected from the following: (1) The select probe and the detection probe each independently comprise naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-naturally occurring nucleotides (e.g., peptide nucleic acids (PNAs) or locked nucleic acids), or any combination thereof; (2) The lengths of the selection probe and the detection probe are each independently 11-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt or 900-1000nt; (3) The length of the target-specific sequence in the selected probe is 10-140 nt, for example, 10-20 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, or 130-140 nt; and (4) The length of the tag sequence in the selected probe is 5-140nt, for example, 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.

18. The method according to any one of claims 1-17, wherein, The upstream primer has one or more characteristics selected from the following: (1) The upstream primer contains naturally occurring nucleotides, modified nucleotides, non-naturally occurring nucleotides, or any combination thereof; (2) The length of each upstream primer is independently 15-150nt, for example, 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, or 140-150nt; (3) After the upstream primer hybridizes with the target nucleic acid sequence, the upstream primer is located at the distal upstream end of the select probe, or at the adjacent upstream end of the select probe, or has a partially overlapping sequence with the select probe. (4) The upstream primer is a primer specific to the target nucleic acid sequence.

19. The method according to any one of claims 1-18, wherein, Steps (1) and (2) are performed simultaneously; for example, under conditions that allow nucleic acid extension and allow cleavage of the selective probe, the upstream primer, the selective probe, and the enzyme with 5' nuclease activity are provided and contacted with the target nucleic acid sequence. Preferably, in step (2), the enzyme with 5' nuclease activity used is a nucleic acid polymerase with 5' nuclease activity, and is the same as the nucleic acid polymerase used in step (4); Preferably, in step (1), in addition to the upstream primer and the selective probe, a downstream primer is provided for each target nucleic acid sequence; wherein the downstream primer contains a sequence complementary to the target nucleic acid sequence; and, when the downstream primer hybridizes with the target nucleic acid sequence, the downstream primer is located downstream of the target-specific sequence; Preferably, the nucleic acid polymerase with 5' nuclease activity catalyzes the upstream and downstream primers to amplify the target nucleic acid sequence and induces the cleavage of the selective probe; Preferably, the downstream primer has one or more characteristics selected from the following: (1) The downstream primer contains naturally occurring nucleotides, modified nucleotides, non-naturally occurring nucleotides, or any combination thereof; (2) The length of each of the downstream primers is independently 15-150nt, for example, 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, or 140-150nt; (3) The downstream primer is a primer specific to the target nucleic acid sequence.

20. The method of any one of claims 1-19, wherein, In step (4), under conditions that allow the nucleic acid polymerase to extend, the nucleic acid polymerase uses the detection probe as a template to extend the hybridization product present in step (3), thereby forming the double strand; Preferably, in step (5), the product of step (4) is gradually heated or cooled and the fluorescence signal emitted by the fluorescent group hybridized to each of the detection probes is monitored in real time, so as to obtain the melting curve of the fluorescence signal intensity of the fluorescent group as a function of temperature. Preferably, the presence of a tag sequence corresponding to the melting peak and / or melting temperature is determined based on the melting peak and / or melting temperature in the melting curve; subsequently, the presence of the genotype corresponding to the tag sequence is determined by the correspondence between the tag sequence and the target nucleic acid sequence and the correspondence between the target nucleic acid sequence and the genotype.

21. A probe set comprising at least one detection probe and at least two selectable probes, said probe set being used to detect at least two target nucleic acid sequences, wherein, The selection probe consists of a tag sequence and a target-specific sequence from the 5' to 3' direction. The tag sequence contains a sequence that is not complementary to the corresponding target nucleic acid sequence, and the target-specific sequence contains a sequence that is complementary to the corresponding target nucleic acid sequence. Furthermore, one or more consecutive nucleotides at the 5' end of the target-specific sequence are completely complementary to the corresponding target nucleic acid sequence. In addition, the tag sequence is labeled with a fluorescent group. The detection probe includes one or more capture sequences that are complementary to one or more of the tag sequences or portions thereof; and each capture sequence is independently labeled with a quenching group such that the fluorescence signal emitted when the capture sequence of the detection probe and its corresponding tag sequence hybridize is different from the fluorescence signal emitted when it does not hybridize with the tag sequence.

22. The probe assembly of claim 21, wherein, The probe set includes m types of detection probes and n types of selection probes, and the probe set is used to detect the target nucleic acid sequence of n genotypes; where n is an integer greater than or equal to 2, and m is an integer greater than or equal to 1; Preferably, the probe set determines whether one or more of the n genotypes are present in the sample by detecting the target nucleic acid sequences of the n genotypes; Preferably, the m detection probes include n capture sequences, and the n capture sequences are complementary to the tag sequences or portions thereof of the n selection probes; Preferably, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or larger, and m is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or larger; Preferably, m is less than or equal to n; Preferably, the tag sequences contained in each of the selected probes are different from each other; and each of the selected probes targets a different target nucleic acid sequence of the genotype.

23. The probe assembly according to any one of claims 21-22, wherein, At least two consecutive nucleotides at the 5' end of the target-specific sequence are not complementary to any variant site (e.g., single nucleotide polymorphism (SNP) site, single nucleotide variant (SNV) site) in the target nucleic acid sequence; or, at least two consecutive nucleotides at the 3' end of the segment in the target nucleic acid sequence that is complementary to the target-specific sequence do not contain any variant site. Preferably, the fluorescent group is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705, or any combination thereof; and the quenching group is a molecule or group capable of quenching the fluorescence signal emitted by the fluorescent group (e.g., DABCYL, BHQ, ECLIPSE, and TAMRA).

24. The probe assembly according to any one of claims 21-23, wherein, The detection probe comprises, from 5' to 3', a template sequence and one or more capture sequences complementary to one or more of the tag sequences or portions thereof; wherein the 3' end or 3' portion of each capture sequence is independently labeled with the same or different quenching groups.

25. The probe assembly according to any one of claims 21-24, wherein, At least one of the detection probes comprises at least two capture sequences that are complementary to at least two of the tag sequences or portions thereof, and the 3' end or 3' portion of each of the capture sequences is independently labeled with the same or different quenching groups; Furthermore, the melting temperatures of the duplexes formed after hybridization of different tag sequences with different capture sequences in the same detection probe differ (for example, the melting temperature of the duplex formed after hybridization of one tag sequence and its corresponding capture sequence is T). m1 The melting temperature of the double strand formed after hybridization of the other tag sequence and its corresponding capture sequence is T. m2 And T m1 Greater than T m2 Furthermore, different tag sequences hybridized to the same detection probe are labeled with the same or different fluorescent groups; Preferably, the tag sequences hybridized onto the same detection probe are different from each other; Preferably, the quenching group is the same for each of the captured sequence markers; Preferably, the tag sequences hybridized to the same detection probe are labeled with the same fluorescent group, and the tag sequences hybridized to different detection probes are labeled with different fluorescent groups.

26. The probe assembly according to any one of claims 21-25, wherein, At least one of the detection probes comprises a plurality of said capture sequences complementary to a plurality of said tag sequences or portions thereof, and the 3' end or 3' portion of each said capture sequence is independently labeled with the same or different said quenching groups; Furthermore, the melting temperatures of the duplexes formed after hybridization of different tag sequences with different capture sequences in the same detection probe differ (for example, the melting temperature of the duplex formed after hybridization of a tag sequence with a corresponding capture sequence in a detection probe is T). m1 The melting temperature of the double strand formed after the other tag sequence hybridizes with the corresponding capture sequence in the detection probe is T. m2 And T m1 Greater than T m2 Furthermore, different tag sequences hybridized to the same detection probe are labeled with the same or different fluorescent groups; Preferably, the tag sequences hybridized to the same detection probe are labeled with the same fluorescent group, and the tag sequences hybridized to different detection probes are labeled with different fluorescent groups.

27. A kit comprising one or more probe sets as defined in any one of claims 21-26; preferably, the kit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the probe sets; Preferably, the kit further comprises an upstream primer, wherein, The upstream primer contains a sequence complementary to the target nucleic acid sequence, and when the upstream primer and the selective probe hybridize with the target nucleic acid sequence, the upstream primer is located upstream of the target-specific sequence; Preferably, the kit further comprises a downstream primer, wherein the downstream primer contains a sequence complementary to the target nucleic acid sequence; and, when the downstream primer hybridizes with the target nucleic acid sequence, the downstream primer is located downstream of the target-specific sequence. Preferably, the kit further comprises an enzyme with 5' nuclease activity; preferably, the enzyme with 5' nuclease activity is a DNA polymerase.

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