Multiplexed nucleic acid detection system and uses thereof

Abstract

This invention relates to a multiplex nucleic acid detection system and its applications. The invention develops a multiplex nucleic acid detection system by designing specific structures for restriction primers, excess primers, hairpin probes, and signal probes. This system can simultaneously achieve efficient parallel detection of multiple target nucleic acids without multiple batch operations. While ensuring detection specificity and sensitivity, it significantly shortens detection time and reduces detection costs, making it suitable for rapid screening of large-scale samples and better meeting the practical needs of simultaneous detection of multiple target nucleic acids in different scenarios.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a multiplex nucleic acid detection system and its application. Background Technology

[0002] Real-time quantitative PCR (qPCR) technology, since its introduction in the 1990s, has been widely used in many fields such as clinical medical diagnosis, pathogen detection, gene expression analysis, food safety monitoring, forensic identification, and basic scientific research due to its outstanding advantages such as high sensitivity, high specificity, and accurate quantification.

[0003] The core technical feature of qPCR lies in its "real-time monitoring" capability. During the PCR amplification of the target gene in the sample, a dedicated instrument continuously monitors the changes in the intensity of the fluorescence signal generated within the reaction system through an optical system. As the number of PCR cycles increases, the target sequence is amplified exponentially, and the fluorescence signal intensifies accordingly. The instrument can record the fluorescence value of each cycle in real time, generating an amplification curve. By analyzing the amplification curve, the cycle threshold (Ct value) can be determined, which is the number of cycles required for the fluorescence signal to reach a set threshold. The Ct value has a linear relationship with the logarithm of the initial template amount, thus enabling accurate quantification of the initial template. qPCR is simple to operate and widely used. During the PCR amplification of the target gene in the sample, the instrument monitors the fluorescence signal generated in the reaction system in real time, allowing for real-time detection of the PCR progress without the need for additional steps after amplification.

[0004] The main technical approaches to fluorescence signal detection fall into two categories: probe methods and dye methods. Each has its own characteristics and is suitable for different detection scenarios. Dye methods achieve detection by adding fluorescent dyes that specifically bind to double-stranded DNA to the reaction system. For example, SYBR Green I fluorescent dye can specifically embed into the minor groove of the DNA double helix structure. The fluorescence signal of the dye in its free state is extremely weak, but when the dye binds to double-stranded DNA, the fluorescence intensity increases significantly, reaching more than 1000 times. The fluorescence signal intensity is directly proportional to the amount of double-stranded DNA in the system. However, its specificity is relatively low; non-specific amplification can also produce signals, making multiplexing impossible. All double-stranded DNA produces the same fluorescence, and it is easily interfered with by primer dimers. Probe methods achieve signal detection by adding specific oligonucleotide probes labeled with fluorescent groups to the reaction system. The probes are typically designed to bind complementary to regions within the target sequence, with a reporter fluorescent group labeled at the 5' end and a quencher group labeled at the 3' end. In the intact probe state, the reporter group and the quencher group are close to each other, and fluorescence resonance energy transfer occurs, quenching the fluorescence of the reporter group. During PCR extension, the 5'→3' exonuclease activity of Taq DNA polymerase hydrolyzes the probe, separating the reporter group and the quencher group, allowing the fluorescence signal to be released and detected by the instrument.

[0005] On the other hand, most probe methods employ probe hydrolysis to generate signal changes, which often results in high background fluorescence signals. This amplifies interference between signals in multiplex systems. The main problem lies in the fact that the probe undergoes a certain degree of spontaneous hydrolysis in solution, and the quenching efficiency of fluorophores and quencher groups is not 100%. Furthermore, non-specific fluorescent substances may be present in the reaction system. These factors lead to high background fluorescence signals, reduced signal-to-noise ratio, and increased interference between channels in multiplex detection systems. This also decreases the detection sensitivity of low-abundance targets and affects quantitative accuracy.

[0006] In qPCR detection, PCR amplification and target sequence detection occur simultaneously, requiring no additional steps, making real-time detection simple and direct. However, this simple and direct detection mode also has significant technical bottlenecks. The maximum number of target sequences that can be detected simultaneously in a single tube is mainly limited by the number of fluorescence detection channels in the qPCR instrument. Different fluorescent dyes require different combinations of excitation and emission wavelengths, and the instrument's optical system needs to be equipped with corresponding filters and detectors. Overlapping fluorescence spectra can lead to signal crosstalk, limiting the number of channels. Currently, mainstream commercial qPCR instruments are generally equipped with 4 to 6 fluorescence detection channels, meaning that a single tube can only detect a maximum of 4 to 6 target sequences simultaneously. For applications requiring the simultaneous detection of dozens or even hundreds of targets, such as pathogen lineage identification and gene expression profiling analysis, this limitation is particularly pronounced.

[0007] In conclusion, developing a convenient and efficient multiplex nucleic acid detection system is of great significance to the field of nucleic acid testing. Summary of the Invention

[0008] In response to the shortcomings of existing technologies and practical needs, this invention provides a multiplex nucleic acid detection system and its application, aiming to develop a convenient, efficient, and highly specific and sensitive multiplex nucleic acid detection solution.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a multiplex nucleic acid detection system, the multiplex nucleic acid detection system comprising an amplification primer set and a detection probe set targeting a target nucleic acid sequence;

[0011] The amplification primer set includes restriction primers and excess primers. The restriction primers, from the 5' end to the 3' end, sequentially include: a 5' end region, a fulcrum anchoring sequence region, an upstream target sequence binding region, and a 3' end region. The excess primers, from the 5' end to the 3' end, sequentially include: a 5' end region, a downstream target sequence binding region, and a 3' end region. The upstream target sequence binding region binds upstream of the target nucleic acid sequence, and the downstream target sequence binding region binds downstream of the target nucleic acid sequence.

[0012] The detection probe group includes a hairpin probe and a signal probe. The hairpin probe includes, from the 5' end to the 3' end, the following in sequence: 5' end region, mediating primer sequence region, 5' end hairpin sequence region, fulcrum sequence region, 3' end hairpin sequence region, target sequence binding region and 3' end region.

[0013] The signal probe comprises, from 5' to 3', a 5' end region, an artificial sequence region, and a 3' end region, wherein the 5' end of the 5' end region of the signal probe is modified with a fluorescent quenching group, and the 3' end of the 3' end region is modified with a fluorescent reporter group.

[0014] The 5-end hairpin sequence region can hybridize with the 3-end hairpin sequence region, the artificial sequence region can hybridize with the mediator primer sequence region, and the fulcrum anchoring sequence region can hybridize with the fulcrum sequence region.

[0015] This invention develops a multiplex nucleic acid detection system, designing restriction primers, excess primers, hairpin probes, and signal probes with specific structures. During detection, when the target sequence is absent, the 5' hairpin sequence region and the 3' hairpin sequence region of the hairpin probe undergo reverse complementary pairing to form a hairpin structure. When the target sequence is present, the "3' hairpin sequence region and target sequence binding region" of the hairpin probe preferentially hybridize with the target sequence template during annealing. Amplification then occurs under the action of restriction primers and excess primers. The hairpin probe is hydrolyzed by Taq enzyme, leaving only the "mediating primer sequence". The region consists of the "5' hairpin sequence region and the fulcrum sequence region". At the same time, a large number of single-stranded amplification products have a fulcrum anchoring sequence at their 3' end. Subsequently, the fulcrum sequence anchors and extends with the fulcrum anchoring sequence, adding the mediator primer sequence to the end of the 3' end of the single-stranded amplification product. Finally, the mediator primer sequence hybridizes with the artificial sequence region in the signal probe, extends, and produces changes in fluorescence signal at a specific temperature. Different targets correspond to different "artificial sequence regions", and the Tm values ​​of the double-stranded stability formed by the signal probe are different, so multiple detection can be achieved in a single fluorescence channel by the Tm value.

[0016] It is understood that the lengths and specific base sequences of the primers and probes in this invention can be designed according to actual needs, as long as they meet the above-mentioned structural functions. For example, the length of the fulcrum anchoring sequence region in the restriction primer can be 10-50 nt, the length of the upstream target sequence binding region can be 10-50 nt, the length of the downstream target sequence binding region in the excess primer can be 10-50 nt, the length of the intermediate primer sequence region in the hairpin probe can be 10-50 nt, the length of the 5-end hairpin sequence region can be 5-30 nt, the length of the fulcrum sequence region can be 8-25 nt, the length of the 3-end hairpin sequence region can be 5-30 nt, the length of the target sequence binding region can be 10-50 nt, and the length of the artificial sequence region in the signal probe can be 40-60 nt.

[0017] In one specific embodiment of the present invention, a detection system can be designed for multiple target nucleic acids such as HPV 16, HPV 18, Flu A, and Flu B. The specific sequences of primers and probes are shown in SEQ ID NO.1 to SEQ ID NO.40.

[0018] Optionally, the 3' end of the 3' end region of the hairpin probe is modified with C3.

[0019] Optionally, the fluorescence quenching group includes any one of Dabcyl, QYS-7, BHQ1, BHQ2, or BHQ3.

[0020] Optionally, the fluorescent reporter group includes any one of TET, Bodipy R6G-X, JOE, FAM, HEX, TAMRA, ROX, CY5, CY5.5, or QUASAR 705.

[0021] Secondly, the present invention provides the application of the multiplex nucleic acid detection system described in the first aspect in multiplex nucleic acid detection.

[0022] Thirdly, the present invention provides a multiplex nucleic acid detection kit, the multiplex nucleic acid detection kit comprising the multiplex nucleic acid detection system described in the first aspect.

[0023] Optionally, the kit may also include PCR amplification reagents.

[0024] Optionally, the PCR amplification reagents include DNA polymerase, dNTPs, and Mg. 2+ and buffer solution.

[0025] Fourthly, the present invention provides a multiplex nucleic acid detection method, the multiplex nucleic acid detection method comprising:

[0026] The multiplex nucleic acid detection system described in the first aspect is used to mix the nucleic acid sample to be tested and PCR amplification reagents to perform a PCR reaction. The melting curve of the PCR reaction product is analyzed, and the type of target nucleic acid sequence present in the reaction system is determined based on the melting curve analysis results.

[0027] Optionally, the ratio of the restriction primer: excess primer: hairpin probe: signal probe input is 1:(3-50):(3-50):(3-50).

[0028] The multiplex nucleic acid detection method developed based on the designed multiplex nucleic acid detection system in this invention has broad application prospects and can be applied to various nucleic acid detection fields, such as multiplex nucleic acid detection of pathogens, as well as detection for non-disease diagnostic purposes, such as food microbiology detection, animal and plant gene detection, and nucleic acid typing detection of environmental samples.

[0029] Fifthly, the present invention provides a multiplex nucleic acid detection device, which includes a nucleic acid extraction module, an amplification module, and an analysis module;

[0030] The nucleic acid extraction module is used to perform the following: extracting nucleic acids from the sample;

[0031] The amplification module is used to perform the following: using the obtained nucleic acid as a template, performing a PCR reaction using the multiplex nucleic acid detection system described in the first aspect;

[0032] The analysis module is used to perform the following: performing melting curve analysis on the PCR reaction products obtained by the amplification module, and determining the type of target nucleic acid sequence present in the reaction system based on the melting curve analysis results.

[0033] Compared with the prior art, the present invention has at least the following beneficial effects:

[0034] This invention develops a multiplex nucleic acid detection system, which designs specific structures of restriction primers, excess primers, hairpin probes, and signal probes. This system can simultaneously achieve efficient parallel detection of multiple target nucleic acids without the need for multiple batch operations. While ensuring detection specificity and sensitivity, it significantly shortens the detection time and reduces detection costs, making it suitable for rapid screening of large-scale samples and better meeting the practical needs of simultaneous detection of multiple target nucleic acids in different scenarios. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the primer and probe structures in a multiplex nucleic acid detection system.

[0036] Figure 2 This is a schematic diagram illustrating the principle of multiplex nucleic acid detection.

[0037] Figure 3 This is the melting curve diagram from Example 1.

[0038] Figure 4 This is the amplification curve from Example 1.

[0039] Figure 5 This is a melting curve diagram of the six mixed target samples in Example 2.

[0040] Figure 6 This is an amplification curve of the six mixed target samples in Example 2.

[0041] Figure 7 This is a melting curve of the template-free sample in Example 2.

[0042] Figure 8 This is the amplification curve of the template-free sample in Example 2.

[0043] Figure 9 This is a melting curve diagram of the three mixed target samples in Example 3.

[0044] Figure 10 This is an amplification curve of the three mixed target samples in Example 3.

[0045] Figure 11 This is a melting curve of the template-free sample in Example 3.

[0046] Figure 12 This is the amplification curve of the template-free sample in Example 3. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0048] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0049] Unless otherwise defined, scientific and technical terms and their abbreviations used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Some of the terms and abbreviations used in this invention are listed below.

[0050] PCR (Polymerase Chain Reaction): Polymerase chain reaction.

[0051] Real-time quantitative PCR (qPCR): a method that uses fluorescent chemicals to measure the total amount of product after each PCR cycle.

[0052] Primers: In PCR, macromolecules with specific sequences used to stimulate synthesis.

[0053] Probe: A probe binds to a target sequence through molecular hybridization, thereby enabling the detection of the target sequence.

[0054] 5' end region: In nucleic acid molecules (DNA or RNA), the fifth carbon atom (5' carbon) of the terminal nucleotide is not attached, and is in a free state or carries an additional chemical group.

[0055] 3' end region: The 3' end region refers to the end of a nucleic acid molecule (DNA or RNA) where the third carbon atom (3' carbon) of the terminal nucleotide is not attached, is in a free state, or has a hydroxyl group (-OH).

[0056] Multiplex nucleic acid testing refers to a molecular detection technology that simultaneously detects multiple (two or more) different target nucleic acid sequences (such as DNA / RNA of specific pathogens, multiple gene loci, different mutation types, etc.) in a single reaction.

[0057] Melting curve analysis: a technique that obtains target nucleic acid sequence information by monitoring the melting process of double-stranded nucleic acids (DNA or RNA) during heating, based on the physical property that double-stranded nucleic acids (DNA or RNA) undergo melting (denaturation).

[0058] C3 (C3 Spacer): refers to three CH2 atoms that can be modified at the 5' end, 3' end, and middle of the primer. When modified at the 3' end of the primer, it can block DNA polymerase extension.

[0059] Fluorescence quenching group: A chemical molecule that can absorb (or consume) the fluorescence energy emitted by a fluorescent reporter group and release it in a non-radiative form (such as heat energy), thereby inhibiting fluorescence emission.

[0060] Fluorescent reporter group: A chemical molecule that emits fluorescence of a specific wavelength when excited by light of a specific wavelength.

[0061] Target sequence: refers to the target nucleic acid sequence to be amplified and detected.

[0062] HPV 16 (Human papillomavirus 16): Human papillomavirus type 16.

[0063] HPV 18 (Human papillomavirus 18): Human papillomavirus type 18.

[0064] HPV 31 (Human papillomavirus 31): Human papillomavirus type 31.

[0065] CT (Chlamydia trachomatis): Chlamydia trachomatis.

[0066] UU (Ureaplasma Urealyticum): Ureaplasma urealyticum.

[0067] NG (Neisseria gonorrhoeae): Neisseria gonorrhoeae.

[0068] C677T: The C677T site of the methylenetetrahydrofolate reductase gene.

[0069] Flu A (Influenza A): Influenza A virus.

[0070] Flu B (Influenza B): Influenza B virus.

[0071] SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2): Novel coronavirus.

[0072] To achieve more convenient and efficient multiplex nucleic acid detection, this invention provides a multiplex nucleic acid detection system, including an amplification primer set and a detection probe set targeting the target nucleic acid sequence, as shown in the schematic diagram below. Figure 1 As shown, the amplification primer set includes restriction primers and excess primers. The restriction primers are composed of the following from 5' to 3': 5' end region, anchoring sequence region, upstream target sequence binding region, and 3' end region. The excess primers are composed of the following from 5' to 3': 5' end region, downstream target sequence binding region, and 3' end region. The upstream target sequence binding region binds upstream of the target nucleic acid sequence, and the downstream target sequence binding region binds downstream of the target nucleic acid sequence.

[0073] The detection probe set includes a hairpin probe and a signal probe (also named a Beacon probe). The hairpin probe, from its 5' end to its 3' end, consists of: a 5' region, a mediating primer sequence region, a 5' hairpin sequence region, a fulcrum sequence region, a 3' hairpin sequence region, a target sequence binding region, and a 3' region. The 3' end of the 3' region is modified with C3. The signal probe, from its 5' end to its 3' end, consists of: a 5' region, an artificial sequence region, and a 3' region. The 5' end of the 5' region is modified with a fluorescence quencher, and the 3' end of the 3' region is modified with a fluorescent reporter group. The 5' hairpin sequence region of the hairpin probe can hybridize with the 3' hairpin sequence region, and the artificial sequence region can hybridize with the mediating primer sequence region. Different artificial sequence regions can be designed for different target sequences. The three hybridization modes of the hairpin probe correspond to three hybridization sequences: the target sequence binding region has the highest annealing temperature (Tm value), followed by the hairpin sequence region, and the lowest is the fulcrum sequence region. The three Tm values ​​can be set to a pairwise interval of 8-10℃ (e.g., 75℃, 65℃, 55℃).

[0074] This invention further develops a multiplex nucleic acid detection method based on the aforementioned multiplex nucleic acid detection system, comprising: acquiring nucleic acid from a biological sample to be tested; mixing the biological sample nucleic acid, DNA polymerase, and the aforementioned multiplex nucleic acid detection system to prepare a PCR reaction system, performing a PCR reaction to obtain PCR reaction products; and performing melting curve analysis on the aforementioned PCR products to determine the types of target nucleic acid sequences present in the reaction system, thereby achieving multiplex nucleic acid detection.

[0075] Specifically, during the detection process, a schematic diagram of the cascade reaction and signal transduction process of the multiplex nucleic acid detection system is shown below. Figure 2As shown, when the target sequence is absent, the 5-end hairpin sequence region of the hairpin probe and the 3-end hairpin sequence region are paired in reverse complementary manner to form a hairpin structure. At this point, the fulcrum sequence is confined to a limited space and cannot hybridize with other sequences. When the target sequence is present, the "3' hairpin sequence region and target sequence binding region" of the hairpin probe preferentially hybridize with the target sequence template during annealing. Next, amplification is performed under the action of restriction primers and excess primers. The hairpin probe is hydrolyzed by Taq enzyme, leaving only the "mediator primer sequence region - 5' hairpin sequence region - fulcrum sequence region". At the same time, a large number of single-stranded amplification products have a fulcrum anchoring sequence at the 3' end. Subsequently, the fulcrum sequence anchors with the fulcrum anchoring sequence and extends again, adding the mediator primer sequence to the end of the 3' end of the single-stranded amplification product. Finally, the mediator primer sequence hybridizes with the artificial sequence region in the signal probe, extends, and produces a change in fluorescence signal at a specific temperature. Since the "artificial sequence region" corresponding to different targets is different, the Tm value of the double-stranded stability formed by the signal probe is different, thus enabling multiple detection through Tm value in a single fluorescence channel.

[0076] In one specific embodiment of the present invention, a multiplex nucleic acid detection kit is provided, the kit comprising the multiplex nucleic acid detection system and PCR amplification reagents.

[0077] In specific embodiments of the present invention, the detection of the C677T site of the methylenetetrahydrofolate reductase (MTHFR) gene in folic acid metabolism and common reproductive tract pathogens are used as examples to verify the multiplex nucleic acid detection system and method of the present invention.

[0078] Example 1

[0079] This embodiment detects the C677T site of the methylenetetrahydrofolate reductase gene in human folic acid metabolism.

[0080] Test sample: Nucleic acid is extracted from human whole blood and diluted to a set of 10-10. 8 Template concentrations were gradients of copies / mL. The 25 μL PCR reaction system used consisted of: 75 mmol / L Tris-HCl (pH 8.5), 20 mmol / L (NH4)2SO4, 0.1% Tween20, 2 mmol / L MgCl2, 200 μmol / L dNTPs, 200 μmol / L dUTP, 1 U Uracil DNA Glycosylase, 2 U Taq polymerase, 0.04 μmol / L restriction primers, 0.4 μmol / L excess primers, 0.4 μmol / L hairpin probe, and 0.2 μmol / L signal probe.

[0081] After the amplification cycle was completed, melting curve analysis was performed, and the fluorescence signal of the FAM channel was acquired. The specific reaction procedure is shown in Table 1.

[0082] Table 1

[0083]

[0084] The melting curve and amplification curve results are as follows: Figure 3 and Figure 4 As shown, this detection method is effective for samples with a molecular weight of 10⁻¹⁰. 8 It can effectively detect targets within the range of copies / mL and generates no signal when there is no template, thus exhibiting high sensitivity.

[0085] Example 2

[0086] This embodiment performs multiple target detection of common reproductive tract pathogens.

[0087] Test samples: 6 types of 10 4 Pathogen copies / mL: Full-length plasmids of HPV16, HPV18, HPV31, CT, UU, and NG were mixed in equal proportions and used as templates. The 25 μL PCR reaction system used was as follows: 75 mmol / L Tris-HCl (pH 8.5), 20 mmol / L (NH4)2SO4, 0.1% Tween 20, 2 mmol / L MgCl2, 200 μmol / L dNTPs, 200 μmol / L dUTP, 1 U Uracil DNA Glycosylase, 2 U Taq polymerase, 0.04 μmol / L restriction primers, 0.4 μmol / L excess primers, 0.4 μmol / L hairpin probe, and 0.2 μmol / L signal probe. A reaction system without template was used as a control.

[0088] After the amplification cycle was completed, melting curve analysis was performed, and fluorescence signals of the FAM and HEX channels were collected. The specific reaction procedure is shown in Table 2.

[0089] Table 2

[0090]

[0091] The results are as follows Figures 5-8 As shown, all six DNA mixed targets were detected normally, with melting peaks appearing within their respective designed temperature ranges. There was no overlap, and no signal was detected without a template, demonstrating good specificity.

[0092] Example 3

[0093] This embodiment performs multiple target detection of common reproductive tract pathogens.

[0094] The test samples were: 3 types of 10 4 Copies / mL: Flu A, Flu B, and SARS-CoV-2 pseudoviruses were mixed in equal proportions and used as templates. The 25 μL PCR reaction system used contained: 75 mmol / L Tris-HCl (pH 8.5), 20 mmol / L (NH4)2SO4, 0.1% Tween 20, 2 mmol / L MgCl2, 200 μmol / L dNTPs, 200 μmol / L dUTP, 1 U Reverse Transcriptase, 1 U Uracil DNA Glycosylase, 2 U Taq polymerases, 0.04 μmol / L restriction primers, 0.4 μmol / L excess primers, 0.4 μmol / L hairpin probe, and 0.2 μmol / L signal probe. A reaction system without template was used as a control.

[0095] After the amplification cycle was completed, melting curve analysis was performed, and the fluorescence signal of the FAM channel was acquired. The specific reaction procedure is shown in Table 3.

[0096] Table 3

[0097]

[0098] The results are as follows Figures 9-12 As shown, all three RNA mixed targets can be detected normally, the melting peaks appear within their respective designed temperature ranges, there is no superposition, and there is no signal when there is no template. This method has good specificity while also taking into account PCR reactions involving reverse transcription.

[0099] The multiplex nucleic acid detection systems used in Examples 1-3 are shown below.

[0100] The C677T site detection system is shown in Table 4.

[0101] Table 4

[0102]

[0103] The HPV 16 detection system is shown in Table 5.

[0104] Table 5

[0105]

[0106] The HPV 18 detection system is shown in Table 6.

[0107] Table 6

[0108]

[0109] The HPV 31 detection system is shown in Table 7.

[0110] Table 7

[0111]

[0112] The CT (Chlamydia trachomatis) detection system is shown in Table 8.

[0113] Table 8

[0114]

[0115] The UU (Ureaplasma urealyticum) detection system is shown in Table 9.

[0116] Table 9

[0117]

[0118] The NG (gonococcal) detection system is shown in Table 10.

[0119] Table 10

[0120]

[0121] The Flu A detection system is shown in Table 11.

[0122] Table 11

[0123]

[0124] The Flu B detection system is shown in Table 12.

[0125] Table 12

[0126]

[0127] The SARS-CoV-2 detection system is shown in Table 13.

[0128] Table 13

[0129]

[0130] In summary, this invention develops a multiplex nucleic acid detection system by designing specific structures for restriction primers, excess primers, hairpin probes, and signal probes. Through asymmetric amplification using restriction and excess primers, a large number of single-stranded DNA products with "fulcrum anchoring sequence regions" are generated. The hairpin sequence region at the 3' end of the hairpin probe and the target sequence binding region can hybridize with the target sequence and are hydrolyzed during the PCR extension stage. The fulcrum sequence region is no longer spatially restricted and can hybridize with the fulcrum anchoring sequence region, causing the product to extend again. The signal probe, acting as a "signal reporter," can hybridize with the final extension product, generating a fluorescent signal with a specific Tm value. The detection specificity is improved based on a dual mechanism: firstly, it is determined by the specificity of the primer design itself; secondly, only when the hairpin probe correctly hybridizes with the target sequence and is hydrolyzed by Taq enzyme can the hairpin structure be prevented from being generated, allowing the fulcrum sequence to hybridize. Further development of multiplex nucleic acid detection methods combines PCR amplification with melting curve analysis. After PCR amplification, a melting curve is generated by slowly heating and monitoring fluorescence changes in real time. The presence of target nucleic acids is determined by analyzing the melting peaks in the melting curve. Since different targets correspond to different "artificial sequences," the Tm values ​​of the double-stranded stability formed by the signal probes are different. Thus, multiplex detection can be achieved in a single fluorescence channel by using the Tm value. Compared with hydrolyzed fluorescent probes, the system using this fluorescent probe has lower background noise, resulting in more sensitive monitoring of signal changes and higher detection sensitivity.

[0131] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A multiplex nucleic acid detection system, characterized in that, The multiplex nucleic acid detection system includes an amplification primer set and a detection probe set targeting the target nucleic acid sequence; The amplification primer set includes restriction primers and excess primers. The restriction primers, from the 5' end to the 3' end, sequentially include: a 5' end region, a fulcrum anchoring sequence region, an upstream target sequence binding region, and a 3' end region. The excess primers, from the 5' end to the 3' end, sequentially include: a 5' end region, a downstream target sequence binding region, and a 3' end region. The upstream target sequence binding region binds upstream of the target nucleic acid sequence, and the downstream target sequence binding region binds downstream of the target nucleic acid sequence. The detection probe group includes a hairpin probe and a signal probe. The hairpin probe includes, from the 5' end to the 3' end, the following in sequence: 5' end region, mediating primer sequence region, 5' end hairpin sequence region, fulcrum sequence region, 3' end hairpin sequence region, target sequence binding region and 3' end region. The signal probe comprises, from 5' to 3', a 5' end region, an artificial sequence region, and a 3' end region, wherein the 5' end of the 5' end region of the signal probe is modified with a fluorescent quenching group, and the 3' end of the 3' end region is modified with a fluorescent reporter group. The 5-end hairpin sequence region can hybridize with the 3-end hairpin sequence region, the artificial sequence region can hybridize with the mediator primer sequence region, and the fulcrum anchoring sequence region can hybridize with the fulcrum sequence region.

2. The multiplex nucleic acid detection system according to claim 1, characterized in that, The 3' end of the 3' end region of the hairpin probe is modified with C3.

3. The multiplex nucleic acid detection system according to claim 1, characterized in that, The fluorescence quenching group includes any one of Dabcyl, QYS-7, BHQ1, BHQ2, or BHQ3.

4. The multiplex nucleic acid detection system according to claim 1, characterized in that, The fluorescent reporter group includes any one of TET, Bodipy R6G-X, JOE, FAM, HEX, TAMRA, ROX, CY5, CY5.5, or QUASAR 705.

5. The application of the multiplex nucleic acid detection system according to any one of claims 1-4 in multiplex nucleic acid detection.

6. A multiplex nucleic acid detection kit, characterized in that, The multiplex nucleic acid detection kit includes the multiplex nucleic acid detection system according to any one of claims 1-4.

7. The reagent kit according to claim 6, characterized in that, The kit also includes PCR amplification reagents; The PCR amplification reagents include DNA polymerase, dNTPs, and Mg. 2+ and buffer solution.

8. A multiplex nucleic acid detection method, characterized in that, The multiplex nucleic acid detection method includes: The multiplex nucleic acid detection system according to any one of claims 1-4 is used to mix the nucleic acid sample to be tested and PCR amplification reagents to perform a PCR reaction. The melting curve of the PCR reaction product is analyzed, and the type of target nucleic acid sequence present in the reaction system is determined based on the melting curve analysis results.

9. The multiplex nucleic acid detection method according to claim 8, characterized in that, The molar ratio of the restriction primer: excess primer: hairpin probe: signal probe is 1:(3-50):(3-50):(3-50).

10. A multiplex nucleic acid detection device, characterized in that, The multiplex nucleic acid detection device includes a nucleic acid extraction module, an amplification module, and an analysis module; The nucleic acid extraction module is used to perform the following: extracting nucleic acids from the sample; The amplification module is used to perform the following: using the obtained nucleic acid as a template, performing a PCR reaction using the multiplex nucleic acid detection system according to any one of claims 1-4; The analysis module is used to perform the following: performing melting curve analysis on the PCR reaction products obtained by the amplification module, and determining the type of target nucleic acid sequence present in the reaction system based on the melting curve analysis results.

Images