Method and kit for isothermal cascade amplification melting curve analysis to detect RNA.

Abstract

This invention discloses a method and kit for isothermal cascade amplification melting curve analysis to detect RNA. The method includes designing a first primer, a second primer, a circular amplification probe, and a detection probe based on the sequence of the circular amplification probe. Isothermal amplification is performed in an amplification system containing the first primer, the second primer, the circular amplification probe, the detection probe, the sample, a nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. This invention ingeniously combines an isothermal detection system, RNA polymerase-mediated transcription amplification technology, and a melting curve analysis system, avoiding the heating delay caused by the large heat capacity of the metal module in traditional thermal cyclers, thus significantly reducing the required time and enabling rapid detection.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and more specifically, to a method and kit for detecting RNA under test by isothermal cascade amplification melting curve analysis. Background Technology

[0002] Nucleic acid testing, as a core technology in molecular diagnostics, plays an irreplaceable role in infectious disease control, early cancer screening, genetic disease diagnosis, and forensic identification. Current mainstream nucleic acid testing methods include real-time fluorescence PCR, isothermal amplification (such as LAMP), digital PCR, nucleic acid aptamer detection, and multicolor melting curve analysis, each exhibiting differentiated advantages in sensitivity, throughput, and specificity.

[0003] Real-time fluorescence PCR is currently the most widely used nucleic acid detection technology. Its core principle is to introduce fluorescently labeled probes (such as TaqMan probes or molecular beacons) into the PCR system, and to quantify the target by monitoring the dynamic changes in fluorescence signals during amplification. This method has a high degree of standardization and quantifiable results, and has been widely used for the rapid detection of pathogens such as COVID-19 and HPV. However, it is essentially still a single-dimensional fluorescence signal reading—a single fluorescence channel can only correspond to one target. Multiplex detection requires multi-channel instruments, which not only limits the number of targets detected per tube (usually ≤6), but also restricts its widespread adoption in primary healthcare settings due to the high cost of the instruments.

[0004] Digital PCR uses microfluidic chips to divide the reaction system into tens of thousands of single-molecule droplets, enabling absolute quantitative detection. Compared to real-time fluorescence PCR, its sensitivity is increased by 10-100 times, and it can detect mutation frequencies as low as 0.01%, making it widely used in the monitoring of minimal residual disease (MRD) in cancer. However, microfluidic chips are expensive to manufacture, have limited throughput (a single chip can typically detect only tens to hundreds of targets), and require complex sample pretreatment, making it difficult to meet the needs of high-throughput screening.

[0005] Nucleic acid aptamer detection technology is based on the specific binding of artificially designed single-stranded oligonucleotides (aptamers) to targets, outputting detection results through fluorescence resonance energy transfer (FRET) or electrochemical signals. This method requires no amplification, has a short detection time (<30 minutes), and aptamers can be screened in vitro to optimize affinity, making it suitable for rapid detection of highly specific biomarkers (such as circulating tumor DNA). However, the in vivo stability of aptamers and their cross-reactivity with complex samples (such as blood and tissue homogenates) still need further resolution.

[0006] Traditional isothermal amplification (AAMP) technology uses multiple pairs of specific primers to drive exponential DNA amplification at a constant temperature, avoiding the reliance on thermal cycling equipment found in traditional PCR. Its advantages include ease of operation, no need for precise temperature control, and suitability for on-site testing in resource-scarce areas. However, this technology suffers from low detection sensitivity, difficulty in achieving simultaneous amplification of multiple targets in the same reaction system, and limited detection throughput.

[0007] Patent CN101333565B reports a real-time fluorescent nucleic acid isothermal amplification detection technology. This method focuses on the detection of RNA targets. It uses M-MLV reverse transcriptase to generate a double-stranded DNA copy of the target nucleic acid (RNA), and then uses T7 RNA polymerase to generate multiple (100-1000) RNA copies from this DNA copy. Each RNA copy then enters the next amplification cycle starting from reverse transcription. Simultaneously, a fluorescently labeled probe specifically binds to these RNA copies, generating fluorescence. This fluorescence signal can be captured in real time by a fluorescence detection instrument, reflecting the amplification cycle in real time. This method has advantages such as high amplification efficiency, short reaction time, simple reaction conditions, easy operation, and no need for temperature cycling. However, this method has low detection throughput; each detection channel can only detect one target, and its detection throughput is heavily dependent on the instrument. Secondly, its amplification product is RNA, which is easily degraded, requiring high levels of degradation prevention and inhibitors.

[0008] The publicly reported multicolor melting curve analysis technique combines real-time fluorescence PCR with melting curve analysis, analyzing two dimensions: fluorescence change and T. m The value is used for detection and analysis to achieve multiplex detection in a single channel. The principle is based on the T value generated by the hybridization of a probe with a large number of single-stranded DNA molecules. m Melting curve analysis was performed. Therefore, asymmetric PCR was used. By adjusting the concentration ratio of upstream and downstream primers, a large number of single-stranded products complementary to the fluorescent probe were obtained. This asymmetric amplification was then combined with the DNA melting temperature to form the characteristic peak of the melting curve. This method avoids the limitations of the PCR instrument's fluorescence channel in real-time fluorescence PCR multiplexing, and has advantages such as high throughput, simple operation, low cost, and reliable accuracy. However, multicolor melting curve analysis technology still faces many challenges. Because asymmetric amplification is linear, non-exponential amplification is prone to low amplification yield and low sensitivity. Furthermore, optimizing the upstream and downstream primer ratio is difficult and design is challenging. The molecular beacon used in asymmetric melting curves undergoes a process of hairpin structure, free single strand, hybrid double strand, and free single strand during the reaction. The fluorescence signal has two parts: weak to strong and strong to weak, which can easily lead to uneven baselines and inverted peaks. Therefore, it is necessary to improve the asymmetric melting curve method to improve detection sensitivity using a symmetric melting curve approach.

[0009] Patents CN119685459B and CN119685458B disclose a method for detecting target nucleic acids using symmetric melting curves. This method generates reporter primers by introducing nickase-specific recognition sequences / restriction endonuclease-specific recognition sequences. The reporter primers then complementaryly pair with the detection probe to form a double-stranded product, and the melting curve of this double-stranded product is obtained. This melting curve corresponds to the melting curve of the target nucleic acid. While the methods disclosed in these patents effectively solve the problem of low yield caused by linear amplification using asymmetric melting curves, they also have significant unresolved drawbacks and pain points.

[0010] First, the aforementioned method is based on real-time fluorescence PCR, which inherently suffers from the limitation of PCR amplification relying on a heating and cooling system, resulting in a bottleneck in thermal cycling efficiency and a long reaction time. If the target is RNA, an additional reverse transcription step is required, further complicating the diagnostic testing process. Second, the core limitation of this method lies in its single-pass PCR amplification—the reporter primer directly binds to the detection probe—essentially relying on the amplification of low-abundance signals from the initial template. However, the sensitivity requirements for clinical diagnosis are continuously increasing with the development of detection technology (e.g., early infection markers, trace residual lesions). The signal gain of a single amplification is limited, making it difficult to break through the current detection limit and thus failing to meet the growing demand for accurate diagnosis. Finally, the patented method makes it difficult to achieve secondary amplification and amplification of the reporter primer. The main obstacles are: 1) the inherent contradiction between the thermal cycling mechanism and isothermal conditions. PCR relies on periodic heating and cooling (denaturation 95℃ → annealing 55℃ → extension 72℃), while isothermal amplification requires amplification at a constant temperature. If forced fusion is attempted, the isothermal amplification enzyme will be inactivated during the high-temperature denaturation stage of PCR (>90℃), while the thermostable Taq DNA polymerase used in PCR cannot achieve template denaturation during the isothermal stage; 2) Thermodynamic parameters are incompatible; the rapid heating and cooling rates of PCR are incompatible with the steady-state conditions of isothermal amplification; 3) There are compatibility barriers in the reaction system. PCR relies on thermostable DNA polymerases (such as Taq DNA polymerase), while isothermal amplification technology uses different enzyme systems (such as Bst enzyme in LAMP, recombinase-polymerase complex in RPA). When used in combination, the buffer systems of different enzymes may inhibit each other. PCR requires staged temperature control (such as pre-denaturation, cyclic amplification, and final extension), while isothermal amplification requires continuously stable reaction conditions. Fusion procedures require frequent switching of temperature modes, increasing the risk of operational errors.

[0011] This reveals a significant technological gap in existing methods. While traditional isothermal amplification methods can achieve isothermal amplification, their single-stage amplification efficiency limit, lacking a cascaded signal amplification system, fails to meet the sensitivity requirements of clinical diagnosis and struggles to increase throughput. Although melting curve techniques based on traditional PCR amplification can improve throughput, they remain limited by temperature dependence, leading to equipment complexity and long reaction times. More critically, existing methods face a dual bottleneck in direct RNA detection—the introduction of the reverse transcription step not only prolongs detection time but also increases the risk of aerosol contamination, severely restricting clinical diagnostic efficacy.

[0012] Therefore, there is a need to develop technical methods that simultaneously meet the requirements of high amplification efficiency, short reaction time, amplification process that does not depend on periodic temperature rise and fall, can achieve cascaded multiple amplifications, and can realize single-channel multi-target detection, and are suitable for RNA targets. Summary of the Invention

[0013] To address the aforementioned technical problems, this invention provides a method for isothermal cascade amplification melting curve analysis to detect RNA, the method comprising the following steps: Step 1: Designing a first primer, a second primer, and a circular amplification probe for the target nucleic acid sequence, wherein the target nucleic acid sequence is RNA, wherein: a. The first primer consists of an RNA polymerase promoter sequence and a target nucleic acid-specific binding sequence, sequentially from the 5' end to the 3' end; b. The second primer consists of a tag sequence, a nicking enzyme-specific recognition sequence, and a target nucleic acid antisense strand recognition sequence, sequentially from the 5' end to the 3' end; the tag sequence cannot bind to the target nucleic acid, the nicking enzyme-specific recognition sequence refers to a single-stranded oligonucleotide sequence in a double-stranded oligonucleotide sequence that is not cleaved by the nicking enzyme, and the target nucleic acid antisense strand recognition sequence refers to a sequence that specifically binds to the antisense strand of the target nucleic acid; c. A circular amplification probe is a circular DNA segment whose sequence consists of two parts. The first part of the sequence completely contains a continuous sequence composed of the tag sequence and the nicking enzyme-specific recognition sequence in the second primer. The second part of the sequence is called the mediator sequence, which does not bind to the first primer, the second primer, or the target nucleic acid. Step 2: Design a detection probe based on the sequence of the circular amplification probe described in Step 1. The detection probe sequence can specifically bind to all sequences of the circular amplification probe or only to a portion of the circular amplification probe sequence to form a stable double strand. The detection probe is modified with a reporter group to facilitate the formation of a melting curve peak during the detection stage. Step 3: Isothermal amplification is performed in an amplification system containing the first primer, the second primer, a circular amplification probe and a detection probe, the sample to be tested, nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. If the sample to be tested contains the target nucleic acid, the first primer specifically binds to the target nucleic acid and extends thereto to generate a new DNA single strand, which is the target nucleic acid antisense strand. The 5' end of the target nucleic acid antisense strand carries the RNA polymerase promoter sequence. Under the action of H, the target nucleic acid is degraded, leaving only the antisense strand. The second primer specifically binds to the antisense strand and extends thereto to generate a double-stranded amplification product A, whose 5' end contains the tag sequence and the nicking enzyme-specific recognition sequence. The double-stranded amplification product A also carries an RNA polymerase promoter sequence. Using this as a template, RNA polymerase transcribes more RNA products complementary to the second primer. The second primer then uses this as a template to extend and generate a complementary cDNA strand, whose 5' end contains the tag sequence and the nicking enzyme-specific recognition sequence. Subsequently, the first primer uses this cDNA as a template for a new round of primer extension to form the double-stranded amplification product A. Then, using the newly generated double-stranded DNA as a template, a new round of transcription reaction is carried out to generate more negative-strand RNA, and this cycle is repeated. The nicking enzyme specifically recognizes the nicking enzyme-specific recognition sequence in the double-stranded amplification product A and digests its complementary sequence to generate a nick. The cleaved complementary sequence fragment is released under the action of the enzyme, and this released fragment serves as a reporter primer. Step 4: The reporter primers generated in Step 3 are complementary to the circular amplification probe and undergo rolling circle amplification in the strand displacement polymerase system. The reporter primers continue to extend along the circular amplification probe and replace the original strand, forming a linear single-stranded DNA composed of multiple tandem repeat sequences complementary to the circular amplification probe sequence. Step 5: The linear single-stranded DNA generated in Step 4 is complementary to the detection probe to form a double-stranded product. The melting curve of the double-stranded product is obtained. The melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be tested, thereby realizing the detection of the target nucleic acid to be tested.

[0014] In one embodiment, the present invention provides a method for isothermal cascade amplification melting curve analysis to detect multiple RNAs, the method comprising: Step 1: When multiple target nucleic acid sequences A1, A2...An are tested simultaneously as a group of target nucleic acids, the target nucleic acid sequences are RNA; for each target nucleic acid sequence A1, A2...An, corresponding first primers B1, B2...Bn and second primers C1, C2...Cn are designed, along with a shared circular amplification probe D and a shared detection probe J, where n is an integer not less than 2; a. The first primers B1, B2...Bn, from the 5' end to the 3' end, are the RNA polymerase promoter sequence and their respective target nucleic acid A1, A2...An specific binding sequences; b. The second primer C1 consists of a tag sequence, a nick enzyme-specific recognition sequence, and a target nucleic acid recognition sequence, from the 5' end to the 3' end. The tag sequence cannot bind to the target nucleic acid to be tested. The nick enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence that is not cleaved by the nick enzyme. The target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be tested. The second primers C1, C2...Cn have the same structure and contain the same nick enzyme-specific recognition sequence. The second primers C1, C2...Cn have corresponding target nucleic acid recognition sequences that bind to the target nucleic acid sequences A1, A2...An, respectively. The tag sequences in the second primers C1, C2...Cn are different. Compared with the tag sequence of the second primer C1, the tag sequence of the second primer C2...Cn has at least one SNP site to facilitate the formation of melting curve peaks with different Tm values. c. Circular amplification probe D, wherein the sequence of the circular amplification probe D consists of two parts. The first part of the sequence completely contains a continuous sequence consisting of the tag sequence and the nicking enzyme-specific recognition sequence in the second primer B1. The second part of the sequence is a mediator sequence, which does not bind to the first primer B1, B2...Bn, the second primer C1, C2...Cn, or the target nucleic acid. d. Detection probe J, wherein the sequence of detection probe J specifically binds to the first part of the sequence of the cyclic amplification probe, and the detection probe is modified with a reporter group to facilitate the formation of a melting curve peak during the detection stage; Step 2: Isothermal amplification is performed in an amplification system containing the first primers B1, B2…Bn, the second primers C1, C2…Cn, a shared circular amplification probe D, a shared detection probe J, the sample to be tested, nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. If the sample to be tested contains multiple target nucleic acid sequences A1, A2…An, the target nucleic acid recognition sequences of B1, B2…Bn in the first primers specifically bind to the corresponding target nucleic acids A1, A2…An, and extend to the corresponding target nucleic acids to generate new corresponding single strands E1, E2…En, which are the antisense strands of target nucleic acids A1, A2…An. The 5' end of the target nucleic acid antisense strands E1, E2…En carries an RNA polymerase promoter sequence. Under the action of H, the target nucleic acids A1, A2...An are degraded, leaving only the antisense strands E1, E2...En. The second primers C1, C2...Cn specifically bind to the single strands E1, E2...En generated above, and extend them to generate double-stranded amplification products F1, F2...Fn, whose 5' ends contain the tag sequence and the nicking enzyme-specific recognition sequence, respectively. Subsequently, using the double-stranded amplification products F1, F2...Fn as templates, RNA polymerase transcribes more RNA products G1, G2..., which are complementary to the second primers C1, C2...Cn, respectively. …Gn, the second primers C1, C2…Cn use G1, G2…Gn as templates to extend complementary cDNA strands H1, H2…Hn. The 5' ends of H1, H2…Hn are tag sequences and nicking enzyme-specific recognition sequences. Subsequently, the first primers B1, B2…Bn use cDNA strands H1, H2…Hn as templates for a new round of primer extension to form double-stranded amplification products F1, F2…Fn, and this cycle is repeated. The nicking enzyme specifically recognizes the nicking enzyme-specific recognition sequence in the double-stranded amplification products F1, F2…Fn and digests its complementary sequence to produce a nick. Under the action of the enzyme, the cleaved oligonucleotide chain fragments are released, and the released fragments are reporter primers I1, I2…In. Step 3: The generated reporter primers I1, I2...In are complementary to the circular amplification probe D, respectively, and undergo rolling circle amplification in the chain displacement polymerase system to form linear single strands K1, K2...Kn composed of different tandem repeat sequences complementary to the probe sequence of the circular amplification probe D; Step 4: The generated linear single-stranded K1, K2...Kn are complementary to the detection probe J to form different double-stranded products L1, L2...Ln, and melting curves of different double-stranded products L1, L2...Ln are obtained. The melting curves of different double-stranded products L1, L2...Ln are the melting curves of the corresponding target nucleic acids A1, A2...An, thereby realizing the simultaneous detection of multiple target nucleic acid sequences A1, A2...An.

[0015] In one embodiment, the present invention provides a method for isothermal cascade amplification melting curve analysis to detect multiple target RNAs, the method comprising: Step 1: When multiple target nucleic acid sequences A1, A2...An are detected simultaneously as a group of target nucleic acids, the target nucleic acid sequences are RNA; for each target nucleic acid sequence A1, A2...An, corresponding first primers B1, B2...Bn and second primers C1, C2...Cn, circular amplification probes D1, D2...Dn, and detection probes J1, J2...Jn are designed, where n is an integer not less than 2; a. The first primers B1, B2...Bn, from the 5' end to the 3' end, are the RNA polymerase promoter sequence and their respective target nucleic acid A1, A2...An specific binding sequences; b. The second primer C1 consists of a tag sequence, a nick enzyme-specific recognition sequence, and a target nucleic acid recognition sequence, from the 5' end to the 3' end. The tag sequence cannot bind to the target nucleic acid to be tested. The nick enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence recognized by the nick enzyme that is not cleaved by the nick enzyme. The target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be tested. The second primers C1, C2...Cn have the same structure and contain the same nick enzyme-specific recognition sequence. The second primers C1, C2...Cn each have a corresponding target nucleic acid recognition sequence that binds to the target nucleic acid sequence A1, A2...An. The tag sequences in the second primers C1, C2...Cn are different to form melting curve peaks with different Tm values, and there is no specific binding between the tag sequences. c. The sequence of the circular amplification probes D1, D2...Dn consists of two parts. The first part of the sequence completely contains the tag sequence and the nicking enzyme-specific recognition sequence of the first primers B1, B2...Bn, respectively. The second part of the sequence is the medium sequence. The medium sequences in the circular amplification probes D1, D2...Dn are all different, and the medium sequence does not specifically bind to the first primers B1, B2...Bn, the second primers C1, C2...Cn, and the target nucleic acid. d. The sequences of the detection probes J1, J2...Jn can specifically bind to all sequences of the cyclic amplification probes D1, D2...Dn, or they can specifically bind to only some sequences of the cyclic amplification probes D1, D2...Dn, forming a stable double-stranded structure; the detection probes are modified with reporter groups to facilitate the formation of melting curve peaks during the detection stage; Step 2: Isothermal amplification is performed in an amplification system containing the first primers B1, B2…Bn, the second primers C1, C2…Cn, circular amplification probes D1, D2…Dn, detection probes J1, J2…Jn, the sample to be tested, nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. If the sample to be tested contains multiple target nucleic acid sequences A1, A2…An, the target nucleic acid recognition sequences of B1, B2…Bn in the first primers specifically bind to the corresponding target nucleic acids A1, A2…An, and extend to the corresponding target nucleic acids to generate new single strands E1, E2…En, which are the antisense strands of the target nucleic acids A1, A2…An. The 5' end of the target nucleic acid antisense strands E1, E2…En carries the RNA polymerase promoter sequence. Under the action of RNase H, the target nucleic acids A1, A2…An are degraded, leaving only the target nucleic acid antisense strand E1. E2……En, the second primers C1, C2……Cn specifically bind to the single-stranded E1, E2……En generated above, and extend thereon to generate double-stranded amplified product F1 with the tag sequence and the nicking enzyme-specific recognition sequence at the 5' end. F2…Fn; subsequently, using the double-stranded amplification products F1, F2…Fn as templates, RNA polymerase transcribes more RNA products G1, G2…Gn, which are complementary to the second primers C1, C2…Cn, respectively. The second primers C1, C2…Cn then use G1, G2…Gn as templates, respectively, and extend via the second primers C1, C2…Cn to generate complementary cDNA strands H1, H2…Hn. The 5' ends of H1, H2…Hn are tag sequences and nicking enzyme-specific recognition sequences. Subsequently, the first primers B1, B2…Bn use cDNA strands H1, H2…Hn as templates for a new round of primer extension to form double-stranded amplification products F1, F2…Fn, and this cycle is repeated. The nicking enzyme specifically recognizes the double-stranded amplification product F1…Fn. The nicking enzyme in F2……Fn specifically recognizes the sequence and performs enzyme digestion on its complementary sequence to generate a nick. Under the action of the enzyme, the cleaved oligonucleotide chain fragment is released, and the released fragment is the reporter primer I1, I2……In; Step 3: The generated reporter primers I1, I2...In are complementary to the circular amplification probes D1, D2...Dn, respectively, and undergo rolling circle amplification in the chain substitution polymerase system to form linear single strands K1, K2...Kn composed of different tandem repeat sequences complementary to the probe sequences of the circular amplification probes D1, D2...Dn. Step 4: The generated linear single-stranded K1, K2...Kn are complementary to the detection probes J1, J2...Jn to form different double-stranded products L1, L2...Ln, and the melting curves of different double-stranded products L1, L2...Ln are obtained. The melting curves of different double-stranded products L1, L2...Ln are the melting curves of the corresponding target nucleic acids A1, A2...An, thereby realizing the simultaneous detection of multiple target nucleic acid sequences A1, A2...An.

[0016] In one implementation, in step 1, a corresponding Taqman probe is designed for each target nucleic acid sequence. This Taqman probe is used to generate a real-time amplification curve during the isothermal amplification phase, facilitating the detection of the amplification reaction.

[0017] In one embodiment, the circular amplification probe and detection probe sequences comprise or consist of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

[0018] In one embodiment, the tag sequence portion of the first primer is 4-30 nt in length, and the nicking enzyme-specific recognition sequence is 1-40 nt in length.

[0019] In one embodiment, the sequence length of the ring-shaped amplification probe is 10 nt to 10000 nt.

[0020] In one embodiment, the RNA polymerase promoter sequence is a T7 promoter sequence, a T3 promoter sequence, an M13 promoter sequence, or an SP6 promoter sequence.

[0021] In one embodiment, the reporter group modified on the detection probe is a pair of paired quencher groups and fluorescent groups. The fluorescent groups include, but are not limited to, various commonly used fluorescent markers such as Pacific Blue, Oregon Green, Bodipy FL-X, FAM, VIC, TET, Bodipy R6G-X, JOE, HEX, Cy3, Cy3B, Rhodamine Red, TAMRA, Texas Red-X, ROX, Cy3.5, Cy5, etc.; the quencher groups include, but are not limited to, various commonly used quenchers such as Dabcyl, Eclipse, BHQ-1, BHQ-2, QYS-7, etc.

[0022] In one embodiment, the nicking enzyme includes at least one of the following nicking enzymes: Nb.BbvCI, Nb.Bpu10I, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI, Nb.Mva1269I, Nt.AlwI, Nt.BbvCI, Nt.Bpu10I, Nt.BsmAI, Nt.BspQI, Nt.Bst9I, Nt.BstNBI, Nt.CviPII, etc.

[0023] In one embodiment, the RNA polymerase is a T7 RNA polymerase, a T3 RNA polymerase, an M13 RNA polymerase, or an SP6 RNA polymerase.

[0024] In one embodiment, the RNA-dependent DNA polymerase includes at least one of the following RNA-dependent DNA polymerases: MMLV reverse transcriptase, AMV reverse transcriptase, etc.

[0025] In one embodiment, the strand substitution polymerase includes at least one of the following strand substitution polymerases: φ29 DNA polymerase, Bst DNA polymerase, Bsu DNA polymerase, Klenow fragment, etc.

[0026] In one embodiment, the present invention provides a kit for isothermal cascade amplification melting curve analysis to detect RNA, the kit comprising: a first primer, a second primer, and a circular amplification probe designed for a target nucleic acid sequence, wherein the target nucleic acid sequence is RNA, a detection probe, and various enzymes for amplification and detection, wherein: a. The first primer, wherein the first primer consists of an RNA polymerase promoter sequence and a target nucleic acid-specific binding sequence, from the 5' end to the 3' end; b. A second primer, wherein the second primer comprises, from the 5' end to the 3' end, a tag sequence, a nicking enzyme-specific recognition sequence, and a target nucleic acid antisense strand recognition sequence, respectively; the tag sequence cannot bind to the target nucleic acid to be tested; the nicking enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence that is not cleaved by the nicking enzyme; and the target nucleic acid antisense strand recognition sequence refers to the sequence that specifically binds to the antisense strand of the target nucleic acid to be tested. c. A circular amplifying probe, wherein the circular amplifying probe is a circular DNA segment whose sequence consists of two parts. The first part of the sequence completely contains a continuous sequence consisting of the tag sequence and the nicking enzyme-specific recognition sequence in the second primer. The second part of the sequence is called the mediator sequence, which does not bind to the first primer, the second primer, or the target nucleic acid. d. Detection probe, wherein the detection probe sequence can specifically bind to all sequences of the cyclic amplification probe or only to a portion of the cyclic amplification probe sequence to form a stable double strand, and the detection probe is modified with a reporter group to facilitate the formation of melting curve peaks during the detection stage; e. Various enzymes used for amplification detection include nicking enzymes, RNA-dependent DNA polymerases, ribonuclease H, RNA polymerases, and strand displacement polymerases.

[0027] In one embodiment, the present invention provides a kit for isothermal cascade amplification melting curve analysis to detect multiple target RNAs. The kit includes: when multiple target nucleic acid sequences A1, A2…An are detected simultaneously as a group of target nucleic acids, the target nucleic acid sequences are RNA; for each target nucleic acid sequence A1, A2…An, corresponding first primers B1, B2…Bn and second primers C1, C2…Cn are designed, along with a shared circular amplification probe D and a shared detection probe J, where n is an integer not less than 2; and various enzymes for amplification and detection. a. The first primers B1, B2...Bn, from the 5' end to the 3' end, are the RNA polymerase promoter sequence and their respective target nucleic acid A1, A2...An specific binding sequences; b. The second primer C1 consists of a tag sequence, a nick enzyme-specific recognition sequence, and a target nucleic acid recognition sequence, from the 5' end to the 3' end. The tag sequence cannot bind to the target nucleic acid to be tested. The nick enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence that is not cleaved by the nick enzyme. The target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be tested. The second primers C1, C2...Cn have the same structure and contain the same nick enzyme-specific recognition sequence. The second primers C1, C2...Cn have corresponding target nucleic acid recognition sequences that bind to the target nucleic acid sequences A1, A2...An, respectively. The tag sequences in the second primers C1, C2...Cn are different. Compared with the tag sequence of the second primer C1, the tag sequence of the second primer C2...Cn has at least one SNP site to facilitate the formation of melting curve peaks with different Tm values. c. Circular amplification probe D, wherein the sequence of the circular amplification probe D consists of two parts. The first part of the sequence completely contains a continuous sequence consisting of the tag sequence and the nicking enzyme-specific recognition sequence in the second primer B1. The second part of the sequence is a mediator sequence, which does not bind to the first primer B1, B2...Bn, the second primer C1, C2...Cn, or the target nucleic acid. d. Detection probe J, wherein the sequence of detection probe J specifically binds to the first part of the sequence of the cyclic amplification probe, and the detection probe is modified with a reporter group to facilitate the formation of a melting curve peak during the detection stage; e. Various enzymes used for amplification detection include nicking enzymes, RNA-dependent DNA polymerases, ribonuclease H, RNA polymerases, and strand displacement polymerases.

[0028] In one embodiment, the present invention provides a kit for isothermal cascade amplification melting curve analysis to detect multiple target RNAs. The kit includes: when multiple target nucleic acid sequences A1, A2…An are detected simultaneously as a group of target nucleic acids, the target nucleic acid sequences are RNA; for each target nucleic acid sequence A1, A2…An, corresponding first primers B1, B2…Bn and second primers C1, C2…Cn, circular amplification probes D1, D2…Dn, and detection probes J1, J2…Jn are designed, where n is an integer not less than 2; and various enzymes for amplification and detection. a. The first primers B1, B2...Bn, from the 5' end to the 3' end, are the RNA polymerase promoter sequence and their respective target nucleic acid A1, A2...An specific binding sequences; b. The second primer C1 consists of a tag sequence, a nick enzyme-specific recognition sequence, and a target nucleic acid recognition sequence, from the 5' end to the 3' end. The tag sequence cannot bind to the target nucleic acid to be tested. The nick enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence recognized by the nick enzyme that is not cleaved by the nick enzyme. The target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be tested. The second primers C1, C2...Cn have the same structure and contain the same nick enzyme-specific recognition sequence. The second primers C1, C2...Cn each have a corresponding target nucleic acid recognition sequence that binds to the target nucleic acid sequence A1, A2...An. The tag sequences in the second primers C1, C2...Cn are different to form melting curve peaks with different Tm values, and there is no specific binding between the tag sequences. c. The sequence of the circular amplification probes D1, D2...Dn consists of two parts. The first part of the sequence completely contains the tag sequence and the nicking enzyme-specific recognition sequence of the first primers B1, B2...Bn, respectively. The second part of the sequence is the medium sequence. The medium sequences in the circular amplification probes D1, D2...Dn are all different, and the medium sequence does not specifically bind to the first primers B1, B2...Bn, the second primers C1, C2...Cn, and the target nucleic acid. d. The sequences of the detection probes J1, J2...Jn can specifically bind to all sequences of the cyclic amplification probes D1, D2...Dn, or they can specifically bind to only some sequences of the cyclic amplification probes D1, D2...Dn, forming a stable double-stranded structure; the detection probes are modified with reporter groups to facilitate the formation of melting curve peaks during the detection stage; e. Various enzymes used for amplification detection include nicking enzymes, RNA-dependent DNA polymerases, ribonuclease H, RNA polymerases, and strand displacement polymerases.

[0029] This invention ingeniously combines an isothermal detection system, RNA polymerase-mediated transcription amplification technology, and a melting curve analysis system. Compared with existing technologies, this invention has the following advantages: a. This invention overcomes the bottleneck of traditional PCR technology, which relies on repeated heating and cooling cycles, by achieving a nucleic acid amplification process without temperature fluctuations throughout through an isothermal amplification mechanism. Compared to traditional PCR, which requires 25-40 heating and cooling cycles (each cycle taking approximately 1-2 minutes), this technology reduces the total amplification time to 30-60 minutes, increasing efficiency by over 80%. This scheme employs a strand displacement enzyme-assisted isothermal control system, enabling the entire process—RNA reverse transcription, reporter primer generation by nickase, and secondary amplification of the reporter primer—to be completed at a single temperature of 37°C. This avoids the heating delay (an average delay of 15-30 seconds per cycle) caused by the large heat capacity of the metal module in traditional thermal cyclers, thus significantly reducing the required time and enabling rapid detection. b. This invention solves the bottleneck of limited single-channel detection throughput in traditional isothermal amplification technology. Through the linkage system of isothermal amplification and RCA technology secondary amplification amplification system and multicolor melting curve analysis technology, multiple targets can be simultaneously amplified and detected in a single reaction channel, which greatly improves the detection throughput, significantly reduces the time for manual operation and waiting time, and effectively reduces reagent costs. c. This invention solves the sensitivity bottleneck problem caused by the asymmetric amplification mechanism of the traditional melting curve method. The traditional melting curve method relies on linear amplification, resulting in low amplification yield per reaction and consequently low sensitivity. Furthermore, it requires multiple thermal cycles to complete the amplification, taking up to 2-3 hours. In contrast, this technology employs rolling circle amplification and an exponential signal amplification system, achieving exponential amplification within 45 minutes without any temperature fluctuations throughout the process.

[0030] d. This invention solves the problem in the symmetrical melting curve method of temperature-controlled PCR, which only sets up a single PCR amplification in the amplification process, relying on the low abundance signal amplification of the initial template, making it difficult to break through the existing detection limit. In the technical solution used in this invention, a cascaded nucleic acid amplification system is constructed. Through the synergistic effect of rolling circle amplification and RNA polymerase-mediated transcriptional amplification, exponential signal amplification of the target nucleic acid is achieved. On the one hand, the RNA polymerase-mediated transcriptional amplification scheme enables the reporter primers to be generated exponentially. On the other hand, through the synergistic effect of circular DNA template design and strand displacement enzyme, the product of the single amplification—the reporter primer—can be amplified and amplified a second time, forming a linear single-stranded DNA composed of hundreds to thousands of tandem repeat sequences complementary to the circular amplification probe sequence. This breaks through the bottleneck of limited signal gain in a single amplification of the symmetrical melting curve, further improving detection sensitivity and providing a highly sensitive solution for the growing demand for precision diagnosis.

[0031] e. In the technical solution of this invention, the generated linear single-stranded DNA and the detection probe form a double-stranded product through base complementary pairing. The melting curve of this double-stranded product corresponds to the characteristic curve of the target nucleic acid to be tested, thereby realizing the detection of the target nucleic acid. Based on this, the method described in this invention belongs to the non-target-dependent melting curve technology—its core lies in the fact that the sequences of both the linear single-stranded DNA and the detection probe are artificially designed, known, and predetermined, so the melting point (Tm value) of each double-stranded combination can be calculated in advance. Specifically, in melting curve analysis, it is only necessary to detect the melting peak with a specific Tm value to determine whether the corresponding target nucleic acid sequence exists in the sample. This design fundamentally solves two major pain points of traditional melting curve technology: first, mutations in the target nucleic acid sequence can easily lead to melting peak shifts and misjudgments (because the Tm value of the double-stranded DNA is accurately calculated from the known sequence and is not affected by the target's own mutations); second, melting curve analysis is not applicable to RNA samples. By artificially designing linear single-stranded DNA and probes adapted to RNA reverse transcription products, effective detection of RNA targets can be achieved, thereby significantly improving detection accuracy. Meanwhile, since the sequences of the linear single strand and the detection probe are completely controllable and the Tm value can be pre-calibrated, this technology can simultaneously detect multiple targets in the same reaction system by designing multiple combinations of double strands with different Tm values, thus greatly improving the detection throughput.

[0032] f. The core of this invention lies in achieving precise detection through a target-dependent reporter primer triggering mechanism. Specifically, the generated reporter primer is synthesized only when the target nucleic acid is present, meaning there is a clear causal relationship between the reporter primer and the target. After binding to the circular amplification probe via complementary base pairing, rolling circle amplification (RCA) is initiated under the catalysis of a strand displacement polymerase. During this process, the reporter primer extends along the circular probe and replaces the original strand, forming a linear single-stranded DNA composed of hundreds to thousands of repeating units, whose sequence is completely complementary to the circular probe. Subsequently, this linear single-stranded DNA pairs complementaryly with the specific detection probe to form a double-stranded product, and a characteristic melting peak is obtained through melting curve analysis. This melting peak directly corresponds to the presence of the target nucleic acid—because the generation of the reporter primer strictly depends on the initial presence of the target nucleic acid, and the binding of the circular amplification probe and the detection probe is entirely mediated by the reporter primer, completely avoiding false positive interference caused by excessive mediator probes in traditional technologies. Compared to existing technologies that force the addition of artificially designed media probes, this technology achieves three major breakthroughs through a target-triggered reporter primer generation mechanism: 1) Zero false positive background: eliminating non-specific binding caused by excessive media probes; 2) Single tube multi-target detection: by designing reporter primer-circular amplification probe combinations with different Tm values, parallel detection of multiple targets within a single reaction system is achieved; 3) Direct RNA detection: compatible with RNA reverse transcription products as circular amplification probe templates, breaking through the limitation of traditional melting curve methods that are limited to DNA detection.

[0033] g. In the technical solution of this invention, a novel spatial distribution design of SNP sites in the tag sequence is adopted for the same detection channel. By pre-setting polymorphic site clusters in the tag region of each tag primer, that is, each tag sequence contains a different number of SNP sites, and the spacing between SNP sites is gradient-distributed, a single detection probe can efficiently distinguish multiple target nucleic acids. This design breaks through the limitations of traditional single-probe single-target detection, enabling a single detection probe to simultaneously identify multiple target nucleic acids. Compared with traditional multi-probe schemes, it has the advantages of reducing probe usage, reducing reagent costs, and reducing system complexity from O(n²) to O(n) (where n is the number of targets). It fundamentally solves the problem of cross-reaction interference in multi-target detection, providing a low-cost, high-precision solution for high-density multiplex nucleic acid detection. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1A schematic diagram of the first primer used in the method of this invention. Figure 2 This is a schematic diagram of the second primer used in the method of the present invention; Figure 3 This is a schematic diagram illustrating the basic principle of the method of the present invention; Figure 4 This is a schematic diagram of multiple second primers used in the method of the present invention to detect multiple target nucleic acids; Figure 5 This is a schematic diagram illustrating the basic principle of the method for detecting multiple target nucleic acids in this invention. Figure 1 ; Figure 6 This is a schematic diagram illustrating the basic principle of the method for detecting multiple target nucleic acids in this invention. Figure 2 ; Figure 7 This is a schematic diagram showing the detection results of EGFR mRNA by the method of the present invention; Figure 8 This is a schematic diagram of the detection results of GAPDH mRNA by the method of the present invention; Figure 9 This diagram illustrates the detection results of EGFR mRNA and GAPDH mRNA using the method of this invention. Figure 1 ; Figure 10 This diagram illustrates the detection results of EGFR mRNA and GAPDH mRNA using the method of this invention. Figure 2 . Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are merely some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application. Unless otherwise specified, the following embodiments are all conventional methods in the art.

[0037] Example 1: Basic Principles of the Invention like Figure 1 , Figure 2 and Figure 3 As shown, this invention provides a method for isothermal cascade amplification melting curve analysis to detect RNA, the method comprising the following steps: Step 1: Design a first primer, a second primer, and a circular amplification probe for the target nucleic acid sequence, wherein the target nucleic acid sequence is RNA, and: a. The first primer, wherein the first primer consists of an RNA polymerase promoter sequence and a target nucleic acid-specific binding sequence, from the 5' end to the 3' end; b. A second primer, wherein the second primer comprises, from the 5' end to the 3' end, a tag sequence, a nicking enzyme-specific recognition sequence, and a target nucleic acid antisense strand recognition sequence, respectively; the tag sequence cannot bind to the target nucleic acid to be tested; the nicking enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence that is not cleaved by the nicking enzyme; and the target nucleic acid antisense strand recognition sequence refers to the sequence that specifically binds to the antisense strand of the target nucleic acid to be tested. c. A circular amplifying probe, wherein the circular amplifying probe is a circular DNA segment whose sequence consists of two parts. The first part of the sequence completely contains a continuous sequence consisting of the tag sequence and the nicking enzyme-specific recognition sequence in the second primer. The second part of the sequence is called the mediator sequence, which does not bind to the first primer, the second primer, or the target nucleic acid. Step 2: Design a detection probe based on the sequence of the circular amplification probe described in Step 1. The detection probe sequence can specifically bind to all sequences of the circular amplification probe or only to a portion of the circular amplification probe sequence to form a stable double strand. The detection probe is modified with a reporter group to facilitate the formation of a melting curve peak during the detection stage. Step 3: Isothermal amplification is performed in an amplification system containing the first primer, the second primer, a circular amplification probe and a detection probe, the sample to be tested, nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. If the sample to be tested contains the target nucleic acid, the first primer specifically binds to the target nucleic acid and extends thereto to generate a new DNA single strand, which is the target nucleic acid antisense strand. The 5' end of the target nucleic acid antisense strand carries the RNA polymerase promoter sequence. Under the action of H, the target nucleic acid is degraded, leaving only the antisense strand. The second primer specifically binds to the antisense strand and extends thereto to generate a double-stranded amplification product A, whose 5' end contains the tag sequence and the nicking enzyme-specific recognition sequence. The double-stranded amplification product A also carries an RNA polymerase promoter sequence. Using this as a template, RNA polymerase transcribes more RNA products complementary to the second primer. The second primer then uses this as a template to extend and generate a complementary cDNA strand, whose 5' end contains the tag sequence and the nicking enzyme-specific recognition sequence. Subsequently, the first primer uses this cDNA as a template for a new round of primer extension to form the double-stranded amplification product A. Then, using the newly generated double-stranded DNA as a template, a new round of transcription reaction is carried out to generate more negative-strand RNA, and this cycle is repeated. The nicking enzyme specifically recognizes the nicking enzyme-specific recognition sequence in the double-stranded amplification product A and digests its complementary sequence to generate a nick. The cleaved complementary sequence fragment is released under the action of the enzyme, and this released fragment serves as a reporter primer. Step 4: The reporter primers generated in Step 3 are complementary to the circular amplification probe and undergo rolling circle amplification in the strand displacement polymerase system. The reporter primers continue to extend along the circular amplification probe and replace the original strand, forming a linear single-stranded DNA composed of multiple tandem repeat sequences complementary to the circular amplification probe sequence. Step 5: The linear single-stranded DNA generated in Step 4 is complementary to the detection probe to form a double-stranded product. The melting curve of the double-stranded product is obtained. The melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be tested, thereby realizing the detection of the target nucleic acid to be tested.

[0038] Similarly, such as Figure 4 and Figure 5 As shown, a method for isothermal cascade amplification melting curve analysis to detect multiple RNA molecules is described, the method comprising: Step 1: When multiple target nucleic acid sequences A1, A2...An are tested simultaneously as a group of target nucleic acids, the target nucleic acid sequences are RNA; for each target nucleic acid sequence A1, A2...An, corresponding first primers B1, B2...Bn and second primers C1, C2...Cn are designed, along with a shared circular amplification probe D and a shared detection probe J, where n is an integer not less than 2; a. The first primers B1, B2...Bn, from the 5' end to the 3' end, are the RNA polymerase promoter sequence and their respective target nucleic acid A1, A2...An specific binding sequences; b. The second primer C1 consists of a tag sequence, a nick enzyme-specific recognition sequence, and a target nucleic acid recognition sequence, from the 5' end to the 3' end. The tag sequence cannot bind to the target nucleic acid to be tested. The nick enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence that is not cleaved by the nick enzyme. The target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be tested. The second primers C1, C2...Cn have the same structure and contain the same nick enzyme-specific recognition sequence. The second primers C1, C2...Cn have corresponding target nucleic acid recognition sequences that bind to the target nucleic acid sequences A1, A2...An, respectively. The tag sequences in the second primers C1, C2...Cn are different. Compared with the tag sequence of the second primer C1, the tag sequence of the second primer C2...Cn has at least one SNP site to facilitate the formation of melting curve peaks with different Tm values. c. Circular amplification probe D, wherein the sequence of the circular amplification probe D consists of two parts. The first part of the sequence completely contains a continuous sequence consisting of the tag sequence and the nicking enzyme-specific recognition sequence in the second primer B1. The second part of the sequence is a mediator sequence, which does not bind to the first primer B1, B2...Bn, the second primer C1, C2...Cn, or the target nucleic acid. d. Detection probe J, wherein the sequence of detection probe J specifically binds to the first part of the sequence of the cyclic amplification probe, and the detection probe is modified with a reporter group to facilitate the formation of a melting curve peak during the detection stage; Step 2: Isothermal amplification is performed in an amplification system containing the first primers B1, B2…Bn, the second primers C1, C2…Cn, a shared circular amplification probe D, a shared detection probe J, the sample to be tested, nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. If the sample to be tested contains multiple target nucleic acid sequences A1, A2…An, the target nucleic acid recognition sequences of B1, B2…Bn in the first primers specifically bind to the corresponding target nucleic acids A1, A2…An, and extend to the corresponding target nucleic acids to generate new corresponding single strands E1, E2…En, which are the antisense strands of target nucleic acids A1, A2…An. The 5' end of the target nucleic acid antisense strands E1, E2…En carries an RNA polymerase promoter sequence. Under the action of H, the target nucleic acids A1, A2...An are degraded, leaving only the antisense strands E1, E2...En. The second primers C1, C2...Cn specifically bind to the single strands E1, E2...En generated above, and extend them to generate double-stranded amplification products F1, F2...Fn, whose 5' ends contain the tag sequence and the nicking enzyme-specific recognition sequence, respectively. Subsequently, using the double-stranded amplification products F1, F2...Fn as templates, RNA polymerase transcribes more RNA products G1, G2..., which are complementary to the second primers C1, C2...Cn, respectively. …Gn, the second primers C1, C2…Cn use G1, G2…Gn as templates to extend complementary cDNA strands H1, H2…Hn. The 5' ends of H1, H2…Hn are tag sequences and nicking enzyme-specific recognition sequences. Subsequently, the first primers B1, B2…Bn use cDNA strands H1, H2…Hn as templates for a new round of primer extension to form double-stranded amplification products F1, F2…Fn, and this cycle is repeated. The nicking enzyme specifically recognizes the nicking enzyme-specific recognition sequence in the double-stranded amplification products F1, F2…Fn and digests its complementary sequence to produce a nick. Under the action of the enzyme, the cleaved oligonucleotide chain fragments are released, and the released fragments are reporter primers I1, I2…In. Step 3: The generated reporter primers I1, I2...In are complementary to the circular amplification probe D, respectively, and undergo rolling circle amplification in the chain displacement polymerase system to form linear single strands K1, K2...Kn composed of different tandem repeat sequences complementary to the probe sequence of the circular amplification probe D; Step 4: The generated linear single-stranded K1, K2...Kn are complementary to the detection probe J to form different double-stranded products L1, L2...Ln, and melting curves of different double-stranded products L1, L2...Ln are obtained. The melting curves of different double-stranded products L1, L2...Ln are the melting curves of the corresponding target nucleic acids A1, A2...An, thereby realizing the simultaneous detection of multiple target nucleic acid sequences A1, A2...An.

[0039] Similarly, such as Figure 6 As shown, this invention provides a method for isothermal cascade amplification melting curve analysis to detect multiple RNA molecules, the method comprising: Step 1: When multiple target nucleic acid sequences A1, A2...An are detected simultaneously as a group of target nucleic acids, the target nucleic acid sequences are RNA; for each target nucleic acid sequence A1, A2...An, corresponding first primers B1, B2...Bn and second primers C1, C2...Cn, circular amplification probes D1, D2...Dn, and detection probes J1, J2...Jn are designed, where n is an integer not less than 2; a. The first primers B1, B2...Bn, from the 5' end to the 3' end, are the RNA polymerase promoter sequence and their respective target nucleic acid A1, A2...An specific binding sequences; b. The second primer C1 consists of a tag sequence, a nick enzyme-specific recognition sequence, and a target nucleic acid recognition sequence, from the 5' end to the 3' end. The tag sequence cannot bind to the target nucleic acid to be tested. The nick enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence recognized by the nick enzyme that is not cleaved by the nick enzyme. The target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be tested. The second primers C1, C2...Cn have the same structure and contain the same nick enzyme-specific recognition sequence. The second primers C1, C2...Cn each have a corresponding target nucleic acid recognition sequence that binds to the target nucleic acid sequence A1, A2...An. The tag sequences in the second primers C1, C2...Cn are different to form melting curve peaks with different Tm values, and there is no specific binding between the tag sequences. c. The sequence of the circular amplification probes D1, D2...Dn consists of two parts. The first part of the sequence completely contains the tag sequence and the nicking enzyme-specific recognition sequence of the first primers B1, B2...Bn, respectively. The second part of the sequence is the medium sequence. The medium sequences in the circular amplification probes D1, D2...Dn are all different, and the medium sequence does not specifically bind to the first primers B1, B2...Bn, the second primers C1, C2...Cn, and the target nucleic acid. d. The sequences of the detection probes J1, J2...Jn can specifically bind to all sequences of the cyclic amplification probes D1, D2...Dn, or they can specifically bind to only some sequences of the cyclic amplification probes D1, D2...Dn, forming a stable double-stranded structure; the detection probes are modified with reporter groups to facilitate the formation of melting curve peaks during the detection stage; Step 2: Isothermal amplification is performed in an amplification system containing the first primers B1, B2…Bn, the second primers C1, C2…Cn, circular amplification probes D1, D2…Dn, detection probes J1, J2…Jn, the sample to be tested, nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. If the sample to be tested contains multiple target nucleic acid sequences A1, A2…An, the target nucleic acid recognition sequences of B1, B2…Bn in the first primers specifically bind to the corresponding target nucleic acids A1, A2…An, and extend to the corresponding target nucleic acids to generate new single strands E1, E2…En, which are the antisense strands of the target nucleic acids A1, A2…An. The 5' end of the target nucleic acid antisense strands E1, E2…En carries the RNA polymerase promoter sequence. Under the action of RNase H, the target nucleic acids A1, A2…An are degraded, leaving only the target nucleic acid antisense strand E1. E2……En, the second primers C1, C2……Cn specifically bind to the single-stranded E1, E2……En generated above, and extend thereon to generate double-stranded amplified product F1 with the tag sequence and the nicking enzyme-specific recognition sequence at the 5' end. F2…Fn; subsequently, using the double-stranded amplification products F1, F2…Fn as templates, RNA polymerase transcribes more RNA products G1, G2…Gn, which are complementary to the second primers C1, C2…Cn, respectively. The second primers C1, C2…Cn then use G1, G2…Gn as templates, respectively, and extend via the second primers C1, C2…Cn to generate complementary cDNA strands H1, H2…Hn. The 5' ends of H1, H2…Hn are tag sequences and nicking enzyme-specific recognition sequences. Subsequently, the first primers B1, B2…Bn use cDNA strands H1, H2…Hn as templates for a new round of primer extension to form double-stranded amplification products F1, F2…Fn, and this cycle is repeated. The nicking enzyme specifically recognizes the double-stranded amplification product F1…Fn. The nicking enzyme in F2……Fn specifically recognizes the sequence and performs enzyme digestion on its complementary sequence to generate a nick. Under the action of the enzyme, the cleaved oligonucleotide chain fragment is released, and the released fragment is the reporter primer I1, I2……In; Step 3: The generated reporter primers I1, I2...In are complementary to the circular amplification probes D1, D2...Dn, respectively, and undergo rolling circle amplification in the chain substitution polymerase system to form linear single strands K1, K2...Kn composed of different tandem repeat sequences complementary to the probe sequences of the circular amplification probes D1, D2...Dn. Step 4: The generated linear single-stranded K1, K2...Kn are complementary to the detection probes J1, J2...Jn to form different double-stranded products L1, L2...Ln, and the melting curves of different double-stranded products L1, L2...Ln are obtained. The melting curves of different double-stranded products L1, L2...Ln are the melting curves of the corresponding target nucleic acids A1, A2...An, thereby realizing the simultaneous detection of multiple target nucleic acid sequences A1, A2...An.

[0040] In the above method, when multiple target nucleic acid sequences A1, A2...An are detected simultaneously as a group, they are detected in the same fluorescence channel. Of course, to further increase the detection throughput, a group of simultaneously detected target nucleic acids can be detected in multiple fluorescence detection channels. For example, multiple target nucleic acid sequences A1, A2...An can be detected in the FAM fluorescence channel, while multiple different target nucleic acid sequences A11, A12...A1n can be detected in the VIC fluorescence channel.

[0041] Example 2: EGFR mRNA Single Target Detection This embodiment takes the detection of EGFR mRNA as an example, and uses the method of the present invention to perform qualitative detection of EGFR mRNA. The specific method includes the following steps: I. Primer and probe sequence information The first primer, second primer, circular amplification probe, and detection probe were designed based on the EGFR mRNA sequence to be tested. The sequence information is shown in the table below.

[0042] Table 1: Primer and probe sequences involved in this embodiment .

[0043] Note: Lowercase bases are tag sequences, lowercase bolded bases are nicking enzyme (Nb.BtsI) specific recognition sequences, and uppercase bolded bases are RNA polymerase promoter sequences.

[0044] II. Reaction System Table 2: Amounts of Reagents Used in This Example Reagent Name concentration Amount added / reaction reverse transcriptase 200 U / μL 100 U RNase H 5 U / μL 10 U Nb.BtsI cleavage enzyme 10 U / μL 5 U phi29 DNA polymerase 10 U / μL 10 U T7 RNA polymerase 50 U / μL 125 U 10× buffer solution 10× 1× dNTPs 10 mM 100 μM EGFR-first primer 100 μM 100 nM EGFR-second primer 100 μM 100 nM EGFR-ring amplification probe 10 ng / μL 1 ng EGFR detection probe 100 μM 100 nM RNA template 5 ng / μL 5 ng Ultrapure water / Add 50 μL

[0045] III. Reaction Procedure After incubation at 37℃ for 45 minutes, melting curve analysis was performed. The melting curve program was 95℃ for 2 minutes, 40℃ for 5 minutes, and 40-90℃ for melting curve analysis, with fluorescence signals collected every 0.04℃. The instrument used in this example was a SLAN96S real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0046] IV. Test Results The EGFR detection results of the method of the present invention are as follows: Figure 7 As shown, the ROX channel at T m The peak value was 62.3±1℃. The baseline of this result was flat, the peak value was as expected, and there were no non-specific peaks.

[0047] Example 3: Detection of GAPDH mRNA Single Target This embodiment takes the detection of GAPDH mRNA as an example, and uses the method of the present invention to perform qualitative detection of GAPDH mRNA. The specific method includes the following steps: I. Primer and probe sequence information The first primer, second primer, circular amplification probe, and detection probe were designed based on the GAPDH mRNA sequence to be tested. The sequence information is shown in the table below.

[0048] Table 3: Primer and probe sequences involved in this embodiment .

[0049] II. Reaction System Table 4: Amounts of Reagents Used in This Example Reagent Name concentration Amount added / reaction reverse transcriptase 200 U / μL 100 U RNase H 5 U / μL 10 U Nb.BtsI cleavage enzyme 10 U / μL 5 U phi29 DNA polymerase 10 U / μL 10 U T7 RNA polymerase 50 U / μL 125 U 10× buffer solution 10× 1× dNTPs 10 mM 100 μM GAPDH - First Primer 100 μM 100 nM GAPDH-Second Primer 100 μM 100 nM GAPDH - Ring Amplifying Probe 10 ng / μL 1 ng GAPDH-Detection Probe 100 μM 100 nM RNA template 5 ng / μL 5 ng Ultrapure water / Add 50 μL

[0050] III. Reaction Procedure After incubation at 37℃ for 45 minutes, melting curve analysis was performed. The melting curve program was 95℃ for 2 minutes, 40℃ for 5 minutes, and 40-90℃ for melting curve analysis, with fluorescence signals collected every 0.04℃. The instrument used in this example was a SLAN96S real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0051] IV. Test Results The detection results of GAPDH by the method of the present invention are as follows: Figure 8 As shown, the ROX channel at T m The peak value was 62.1±1℃. The baseline of this result was flat, the peak value was as expected, and there were no non-specific peaks.

[0052] Example 4: Dual-target detection of EGFR mRNA and GAPDH mRNA This embodiment takes the detection of dual targets EGFR mRNA and GAPDH mRNA as an example, and uses the method of the present invention to perform qualitative detection of EGFR mRNA and GAPDH mRNA. The specific method includes the following steps: I. Primer and probe sequence information The first primer, second primer, circular amplification probe, and detection probe were designed based on the EGFR mRNA and GAPDH mRNA sequences to be tested. The sequence information is shown in the table below.

[0053] Table 5: Primer and probe sequences involved in this embodiment

[0054] Note: Lowercase bases are tag sequences, lowercase bolded bases are nicking enzyme (Nb.BtsI) specific recognition sequences, lowercase underlined parts are SNP sites, and uppercase bolded bases are RNA polymerase promoter sequences.

[0055] II. Reaction System Table 6: Amounts of Reagents Used in This Example Reagent Name concentration Amount added / reaction reverse transcriptase 200 U / μL 200 U RNase H 5 U / μL 10 U Nb.BtsI cleavage enzyme 10 U / μL 10 U phi29 DNA polymerase 10 U / μL 20 U T7 RNA polymerase 50 U / μL 200 U 10× buffer solution 10× 1× dNTPs 10 mM 100 μM EGFR-first primer 100 μM 100 nM EGFR-second primer 100 μM 100 nM GAPDH - First Primer 100 μM 100 nM GAPDH-Second Primer 100 μM 100 nM Circular magnifying probe 10 ng / μL 2 ng Detection probe 100 μM 150 nM RNA template 5 ng / μL 5 ng Ultrapure water / Add 50 μL

[0056] III. Reaction Procedure After incubation at 37℃ for 45 minutes, melting curve analysis was performed. The melting curve program was 95℃ for 2 minutes, 40℃ for 5 minutes, and 40-90℃ for melting curve analysis, with fluorescence signals collected every 0.04℃. The instrument used in this example was a SLAN96S real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0057] IV. Test Results The detection results of EGFR and GAPDH by the method of this invention are as follows: Figure 9 As shown, if the ROX channel has a melting peak T m If the peak value is found at 62.5±1℃, it indicates EGFR mRNA; if the melting peak T... m If the value peaks at 54.8±1℃, it indicates GAPDH mRNA.

[0058] Example 5: Dual-target detection of EGFR mRNA and GAPDH mRNA This embodiment takes the detection of dual targets EGFR mRNA and GAPDH mRNA as an example, and uses the method of the present invention to perform qualitative detection of EGFR mRNA and GAPDH mRNA. The specific method includes the following steps: I. Primer and probe sequence information The first primer, second primer, circular amplification probe, and detection probe were designed based on the EGFR mRNA and GAPDH mRNA sequences to be tested. The sequence information is shown in the table below.

[0059] Table 7: Primer and probe sequences involved in this embodiment .

[0060] Note: Lowercase bases are tag sequences, lowercase bolded bases are nicking enzyme (Nb.BtsI) specific recognition sequences, and uppercase bolded bases are RNA polymerase promoter sequences.

[0061] II. Reaction System Table 8: Amounts of Reagents Used in This Example Reagent Name concentration Amount added / reaction reverse transcriptase 200 U / μL 200 U RNase H 5 U / μL 10 U Nb.BtsI cleavage enzyme 10 U / μL 10 U phi29 DNA polymerase 10 U / μL 20 U T7 RNA polymerase 50 U / μL 200 U 10× buffer solution 10× 1× dNTPs 10 mM 100 μM EGFR-first primer 100 μM 100 nM EGFR-second primer 100 μM 100 nM EGFR-ring amplification probe 10 ng / μL 1 ng EGFR detection probe 100 μM 100 nM GAPDH - First Primer 100 μM 100 nM GAPDH-Second Primer 100 μM 100 nM GAPDH - Ring Amplifying Probe 10 ng / μL 1 ng GAPDH-Detection Probe 100 μM 100 nM RNA template 5 ng / μL 5 ng Ultrapure water / Add 50 μL

[0062] III. Reaction Procedure After incubation at 37℃ for 45 minutes, melting curve analysis was performed. The melting curve program was 95℃ for 2 minutes, 40℃ for 5 minutes, and 40-90℃ for melting curve analysis, with fluorescence signals collected every 0.04℃. The instrument used in this example was a SLAN96S real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0063] IV. Test Results The detection results of EGFR and GAPDH by the method of this invention are as follows: Figure 10 As shown, if the FAM channel has a melting peak T m If a peak appears at 58.9±1℃, it indicates EGFR mRNA. If the ROX channel shows a melting peak (T0), it indicates EGFR mRNA. m If the value peaks at 67.2±1℃, it indicates GAPDH mRNA.

[0064] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0065] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A method for detecting RNA under test using isothermal cascade amplification melting curve analysis, characterized in that, The method includes the following steps: Step 1: Design a first primer, a second primer, and a circular amplification probe for the target nucleic acid sequence, wherein the target nucleic acid sequence is RNA, and: a. The first primer, wherein the first primer consists of an RNA polymerase promoter sequence and a target nucleic acid-specific binding sequence, from the 5' end to the 3' end; b. A second primer, wherein the second primer comprises, from the 5' end to the 3' end, a tag sequence, a nicking enzyme-specific recognition sequence, and a target nucleic acid antisense strand recognition sequence, respectively; the tag sequence cannot bind to the target nucleic acid to be tested; the nicking enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence that is not cleaved by the nicking enzyme; and the target nucleic acid antisense strand recognition sequence refers to the sequence that specifically binds to the antisense strand of the target nucleic acid to be tested. c. A circular amplifying probe, wherein the circular amplifying probe is a circular DNA segment whose sequence consists of two parts. The first part of the sequence completely contains a continuous sequence consisting of the tag sequence and the nicking enzyme-specific recognition sequence in the second primer. The second part of the sequence is called the mediator sequence, which does not bind to the first primer, the second primer, or the target nucleic acid. Step 2: Design a detection probe based on the sequence of the circular amplification probe described in Step 1. The detection probe sequence can specifically bind to all sequences of the circular amplification probe or only to a portion of the circular amplification probe sequence to form a stable double strand. The detection probe is modified with a reporter group to facilitate the formation of a melting curve peak during the detection stage. Step 3: Isothermal amplification is performed in an amplification system containing the first primer, the second primer, a circular amplification probe and a detection probe, the sample to be tested, nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. If the sample to be tested contains the target nucleic acid, the first primer specifically binds to the target nucleic acid and extends thereto to generate a new DNA single strand, which is the target nucleic acid antisense strand. The 5' end of the target nucleic acid antisense strand carries the RNA polymerase promoter sequence. Under the action of H, the target nucleic acid is degraded, leaving only the antisense strand. The second primer specifically binds to the antisense strand and extends thereto to generate a double-stranded amplification product A, whose 5' end contains the tag sequence and the nicking enzyme-specific recognition sequence. The double-stranded amplification product A also carries an RNA polymerase promoter sequence. Using this as a template, RNA polymerase transcribes more RNA products complementary to the second primer. The second primer then uses this as a template to extend and generate a complementary cDNA strand, whose 5' end contains the tag sequence and the nicking enzyme-specific recognition sequence. Subsequently, the first primer uses this cDNA as a template for a new round of primer extension to form the double-stranded amplification product A. Then, using the newly generated double-stranded DNA as a template, a new round of transcription reaction is carried out to generate more negative-strand RNA, and this cycle is repeated. The nicking enzyme specifically recognizes the nicking enzyme-specific recognition sequence in the double-stranded amplification product A and digests its complementary sequence to generate a nick. The cleaved complementary sequence fragment is released under the action of the enzyme, and this released fragment serves as a reporter primer. Step 4: The reporter primers generated in Step 3 are complementary to the circular amplification probe and undergo rolling circle amplification in the strand displacement polymerase system. The reporter primers continue to extend along the circular amplification probe and replace the original strand, forming a linear single-stranded DNA composed of multiple tandem repeat sequences complementary to the circular amplification probe sequence. Step 5: The linear single-stranded DNA generated in Step 4 is complementary to the detection probe to form a double-stranded product. The melting curve of the double-stranded product is obtained. The melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be tested, thereby realizing the detection of the target nucleic acid to be tested.

2. A method for detecting multiple RNA molecules by isothermal cascade amplification melting curve analysis, characterized in that, The method includes: Step 1: When multiple target nucleic acid sequences A1, A2...An are tested simultaneously as a group of target nucleic acids, the target nucleic acid sequences are RNA; for each target nucleic acid sequence A1, A2...An, corresponding first primers B1, B2...Bn and second primers C1, C2...Cn are designed, along with a shared circular amplification probe D and a shared detection probe J, where n is an integer not less than 2; a. The first primers B1, B2...Bn, from the 5' end to the 3' end, are the RNA polymerase promoter sequence and their respective target nucleic acid A1, A2...An specific binding sequences; b. The second primer C1 consists of a tag sequence, a nick enzyme-specific recognition sequence, and a target nucleic acid recognition sequence, from the 5' end to the 3' end. The tag sequence cannot bind to the target nucleic acid to be tested. The nick enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence that is not cleaved by the nick enzyme. The target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be tested. The second primers C1, C2...Cn have the same structure and contain the same nick enzyme-specific recognition sequence. The second primers C1, C2...Cn have corresponding target nucleic acid recognition sequences that bind to the target nucleic acid sequences A1, A2...An, respectively. The tag sequences in the second primers C1, C2...Cn are different. Compared with the tag sequence of the second primer C1, the tag sequence of the second primer C2...Cn has at least one SNP site to facilitate the formation of melting curve peaks with different Tm values. c. Circular amplification probe D, wherein the sequence of the circular amplification probe D consists of two parts. The first part of the sequence completely contains a continuous sequence consisting of the tag sequence and the nicking enzyme-specific recognition sequence in the second primer B1. The second part of the sequence is a mediator sequence, which does not bind to the first primer B1, B2...Bn, the second primer C1, C2...Cn, or the target nucleic acid. d. Detection probe J, wherein the sequence of detection probe J specifically binds to the first part of the sequence of the cyclic amplification probe, and the detection probe is modified with a reporter group to facilitate the formation of a melting curve peak during the detection stage; Step 2: Isothermal amplification is performed in an amplification system containing the first primers B1, B2…Bn, the second primers C1, C2…Cn, a shared circular amplification probe D, a shared detection probe J, the sample to be tested, nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. If the sample to be tested contains multiple target nucleic acid sequences A1, A2…An, the target nucleic acid recognition sequences of B1, B2…Bn in the first primers specifically bind to the corresponding target nucleic acids A1, A2…An, and extend to the corresponding target nucleic acids to generate new corresponding single strands E1, E2…En, which are the antisense strands of target nucleic acids A1, A2…An. The 5' end of the target nucleic acid antisense strands E1, E2…En carries an RNA polymerase promoter sequence. Under the action of H, the target nucleic acids A1, A2...An are degraded, leaving only the antisense strands E1, E2...En. The second primers C1, C2...Cn specifically bind to the single strands E1, E2...En generated above, and extend them to generate double-stranded amplification products F1, F2...Fn, whose 5' ends contain the tag sequence and the nicking enzyme-specific recognition sequence, respectively. Subsequently, using the double-stranded amplification products F1, F2...Fn as templates, RNA polymerase transcribes more RNA products G1, G2..., which are complementary to the second primers C1, C2...Cn, respectively. …Gn, the second primers C1, C2…Cn use G1, G2…Gn as templates to extend complementary cDNA strands H1, H2…Hn. The 5' ends of H1, H2…Hn are tag sequences and nicking enzyme-specific recognition sequences. Subsequently, the first primers B1, B2…Bn use cDNA strands H1, H2…Hn as templates for a new round of primer extension to form double-stranded amplification products F1, F2…Fn, and this cycle is repeated. The nicking enzyme specifically recognizes the nicking enzyme-specific recognition sequence in the double-stranded amplification products F1, F2…Fn and digests its complementary sequence to produce a nick. Under the action of the enzyme, the cleaved oligonucleotide chain fragments are released, and the released fragments are reporter primers I1, I2…In. Step 3: The generated reporter primers I1, I2...In are complementary to the circular amplification probe D, respectively, and undergo rolling circle amplification in the chain displacement polymerase system to form linear single strands K1, K2...Kn composed of different tandem repeat sequences complementary to the probe sequence of the circular amplification probe D; Step 4: The generated linear single-stranded K1, K2...Kn are complementary to the detection probe J to form different double-stranded products L1, L2...Ln, and melting curves of different double-stranded products L1, L2...Ln are obtained. The melting curves of different double-stranded products L1, L2...Ln are the melting curves of the corresponding target nucleic acids A1, A2...An, thereby realizing the simultaneous detection of multiple target nucleic acid sequences A1, A2...An.

3. A method for detecting multiple RNA molecules by isothermal cascade amplification melting curve analysis, characterized in that, The method includes: Step 1: When multiple target nucleic acid sequences A1, A2...An are detected simultaneously as a group of target nucleic acids, the target nucleic acid sequences are RNA; for each target nucleic acid sequence A1, A2...An, corresponding first primers B1, B2...Bn and second primers C1, C2...Cn, circular amplification probes D1, D2...Dn, and detection probes J1, J2...Jn are designed, where n is an integer not less than 2; a. The first primers B1, B2...Bn, from the 5' end to the 3' end, are the RNA polymerase promoter sequence and their respective target nucleic acid A1, A2...An specific binding sequences; b. The second primer C1 consists of a tag sequence, a nick enzyme-specific recognition sequence, and a target nucleic acid recognition sequence, from the 5' end to the 3' end. The tag sequence cannot bind to the target nucleic acid to be tested. The nick enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence recognized by the nick enzyme that is not cleaved by the nick enzyme. The target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be tested. The second primers C1, C2...Cn have the same structure and contain the same nick enzyme-specific recognition sequence. The second primers C1, C2...Cn each have a corresponding target nucleic acid recognition sequence that binds to the target nucleic acid sequence A1, A2...An. The tag sequences in the second primers C1, C2...Cn are different to form melting curve peaks with different Tm values, and there is no specific binding between the tag sequences. c. The sequence of the circular amplification probes D1, D2...Dn consists of two parts. The first part of the sequence completely contains the tag sequence and the nicking enzyme-specific recognition sequence of the first primers B1, B2...Bn, respectively. The second part of the sequence is the medium sequence. The medium sequences in the circular amplification probes D1, D2...Dn are all different, and the medium sequence does not specifically bind to the first primers B1, B2...Bn, the second primers C1, C2...Cn, and the target nucleic acid. d. The sequences of the detection probes J1, J2...Jn can specifically bind to all sequences of the cyclic amplification probes D1, D2...Dn, or they can specifically bind to only some sequences of the cyclic amplification probes D1, D2...Dn, forming a stable double-stranded structure; the detection probes are modified with reporter groups to facilitate the formation of melting curve peaks during the detection stage; Step 2: Isothermal amplification is performed in an amplification system containing the first primers B1, B2…Bn, the second primers C1, C2…Cn, circular amplification probes D1, D2…Dn, detection probes J1, J2…Jn, the sample to be tested, nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. If the sample to be tested contains multiple target nucleic acid sequences A1, A2…An, the target nucleic acid recognition sequences of B1, B2…Bn in the first primers specifically bind to the corresponding target nucleic acids A1, A2…An, and extend to the corresponding target nucleic acids to generate new single strands E1, E2…En, which are the antisense strands of the target nucleic acids A1, A2…An. The 5' end of the target nucleic acid antisense strands E1, E2…En carries the RNA polymerase promoter sequence. Under the action of RNase H, the target nucleic acids A1, A2…An are degraded, leaving only the target nucleic acid antisense strand E1. E2……En, the second primers C1, C2……Cn specifically bind to the single-stranded E1, E2……En generated above, and extend thereon to generate double-stranded amplified product F1 with the tag sequence and the nicking enzyme-specific recognition sequence at the 5' end. F2…Fn; subsequently, using the double-stranded amplification products F1, F2…Fn as templates, RNA polymerase transcribes more RNA products G1, G2…Gn, which are complementary to the second primers C1, C2…Cn, respectively. The second primers C1, C2…Cn then use G1, G2…Gn as templates, respectively, and extend via the second primers C1, C2…Cn to generate complementary cDNA strands H1, H2…Hn. The 5' ends of H1, H2…Hn are tag sequences and nicking enzyme-specific recognition sequences. Subsequently, the first primers B1, B2…Bn use cDNA strands H1, H2…Hn as templates for a new round of primer extension to form double-stranded amplification products F1, F2…Fn, and this cycle is repeated. The nicking enzyme specifically recognizes the double-stranded amplification product F1…Fn. The nicking enzyme in F2……Fn specifically recognizes the sequence and performs enzyme digestion on its complementary sequence to generate a nick. Under the action of the enzyme, the cleaved oligonucleotide chain fragment is released, and the released fragment is the reporter primer I1, I2……In; Step 3: The generated reporter primers I1, I2...In are complementary to the circular amplification probes D1, D2...Dn, respectively, and undergo rolling circle amplification in the chain substitution polymerase system to form linear single strands K1, K2...Kn composed of different tandem repeat sequences complementary to the probe sequences of the circular amplification probes D1, D2...Dn. Step 4: The generated linear single-stranded K1, K2...Kn are complementary to the detection probes J1, J2...Jn to form different double-stranded products L1, L2...Ln, and the melting curves of different double-stranded products L1, L2...Ln are obtained. The melting curves of different double-stranded products L1, L2...Ln are the melting curves of the corresponding target nucleic acids A1, A2...An, thereby realizing the simultaneous detection of multiple target nucleic acid sequences A1, A2...An.

4. The method according to any one of claims 1-3, characterized in that, In step 1, a corresponding Taqman probe is designed for each target nucleic acid sequence to be tested.

5. The method according to any one of claims 1-3, characterized in that, The circular amplification probe and / or the detection probe sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

6. The method according to any one of claims 1-3, characterized in that, The tag sequence in the first primer is 4-30 nt in length, and / or the nickase-specific recognition sequence is 1-40 nt in length.

7. The method according to any one of claims 1-3, characterized in that, The sequence length of the ring-shaped amplification probe ranges from 10 nt to 10,000 nt.

8. The method according to any one of claims 1-3, characterized in that, The RNA polymerase promoter sequence is a T7 promoter sequence, a T3 promoter sequence, an M13 promoter sequence, or an SP6 promoter sequence.

9. The method according to any one of claims 1-3, characterized in that, The RNA polymerase is T7 RNA polymerase, T3 RNA polymerase, M13 RNA polymerase, or SP6 RNA polymerase.

10. A kit for isothermal cascade amplification melting curve analysis and detection of RNA, characterized in that, The kit includes: The test involves a first primer, a second primer, and a circular amplification probe designed for the target nucleic acid sequence, wherein the target nucleic acid sequence is RNA, a detection probe, and various enzymes for amplification and detection. a. The first primer, wherein the first primer consists of an RNA polymerase promoter sequence and a target nucleic acid-specific binding sequence, from the 5' end to the 3' end; b. A second primer, wherein the second primer comprises, from the 5' end to the 3' end, a tag sequence, a nicking enzyme-specific recognition sequence, and a target nucleic acid antisense strand recognition sequence, respectively; the tag sequence cannot bind to the target nucleic acid to be tested; the nicking enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence that is not cleaved by the nicking enzyme; and the target nucleic acid antisense strand recognition sequence refers to the sequence that specifically binds to the antisense strand of the target nucleic acid to be tested. c. A circular amplifying probe, wherein the circular amplifying probe is a circular DNA segment whose sequence consists of two parts. The first part of the sequence completely contains a continuous sequence consisting of the tag sequence and the nicking enzyme-specific recognition sequence in the second primer. The second part of the sequence is called the mediator sequence, which does not bind to the first primer, the second primer, or the target nucleic acid. d. Detection probe, wherein the detection probe sequence can specifically bind to all sequences of the cyclic amplification probe or only to a portion of the cyclic amplification probe sequence to form a stable double strand, and the detection probe is modified with a reporter group to facilitate the formation of melting curve peaks during the detection stage; e. Various enzymes used for amplification detection include nicking enzymes, RNA-dependent DNA polymerases, ribonuclease H, RNA polymerases, and strand displacement polymerases.

11. A kit for isothermal cascade amplification melting curve analysis and detection of multiple target RNAs, characterized in that, The kit includes: when multiple target nucleic acid sequences A1, A2...An are detected simultaneously as a group, the target nucleic acid sequences are RNA; for each target nucleic acid sequence A1, A2...An, corresponding first primers B1, B2...Bn and second primers C1, C2...Cn are designed, along with a shared circular amplification probe D and a shared detection probe J, where n is an integer not less than 2, and various enzymes for amplification and detection; a. The first primers B1, B2...Bn, from the 5' end to the 3' end, are the RNA polymerase promoter sequence and their respective target nucleic acid A1, A2...An specific binding sequences; b. The second primer C1 consists of a tag sequence, a nick enzyme-specific recognition sequence, and a target nucleic acid recognition sequence, from the 5' end to the 3' end. The tag sequence cannot bind to the target nucleic acid to be tested. The nick enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence that is not cleaved by the nick enzyme. The target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be tested. The second primers C1, C2...Cn have the same structure and contain the same nick enzyme-specific recognition sequence. The second primers C1, C2...Cn have corresponding target nucleic acid recognition sequences that bind to the target nucleic acid sequences A1, A2...An, respectively. The tag sequences in the second primers C1, C2...Cn are different. Compared with the tag sequence of the second primer C1, the tag sequence of the second primer C2...Cn has at least one SNP site to facilitate the formation of melting curve peaks with different Tm values. c. Circular amplification probe D, wherein the sequence of the circular amplification probe D consists of two parts. The first part of the sequence completely contains a continuous sequence consisting of the tag sequence and the nicking enzyme-specific recognition sequence in the second primer B1. The second part of the sequence is a mediator sequence, which does not bind to the first primer B1, B2...Bn, the second primer C1, C2...Cn, or the target nucleic acid. d. Detection probe J, wherein the sequence of detection probe J specifically binds to the first part of the sequence of the cyclic amplification probe, and the detection probe is modified with a reporter group to facilitate the formation of a melting curve peak during the detection stage; e. Various enzymes used for amplification detection include nicking enzymes, RNA-dependent DNA polymerases, ribonuclease H, RNA polymerases, and strand displacement polymerases.

12. A kit for isothermal cascade amplification melting curve analysis and detection of multiple target RNAs, characterized in that, The kit includes: when multiple target nucleic acid sequences A1, A2...An are detected simultaneously as a group, the target nucleic acid sequences are RNA; for each target nucleic acid sequence A1, A2...An, corresponding first primers B1, B2...Bn and second primers C1, C2...Cn, circular amplification probes D1, D2...Dn, and detection probes J1, J2...Jn are designed, where n is an integer not less than 2; and various enzymes for amplification and detection. a. The first primers B1, B2...Bn, from the 5' end to the 3' end, are the RNA polymerase promoter sequence and their respective target nucleic acid A1, A2...An specific binding sequences; b. The second primer C1 consists of a tag sequence, a nick enzyme-specific recognition sequence, and a target nucleic acid recognition sequence, from the 5' end to the 3' end. The tag sequence cannot bind to the target nucleic acid to be tested. The nick enzyme-specific recognition sequence refers to the single-stranded oligonucleotide sequence in the double-stranded oligonucleotide chain sequence recognized by the nick enzyme that is not cleaved by the nick enzyme. The target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be tested. The second primers C1, C2...Cn have the same structure and contain the same nick enzyme-specific recognition sequence. The second primers C1, C2...Cn each have a corresponding target nucleic acid recognition sequence that binds to the target nucleic acid sequence A1, A2...An. The tag sequences in the second primers C1, C2...Cn are different to form melting curve peaks with different Tm values, and there is no specific binding between the tag sequences. c. The sequence of the circular amplification probes D1, D2...Dn consists of two parts. The first part of the sequence completely contains the tag sequence and the nicking enzyme-specific recognition sequence of the first primers B1, B2...Bn, respectively. The second part of the sequence is the medium sequence. The medium sequences in the circular amplification probes D1, D2...Dn are all different, and the medium sequence does not specifically bind to the first primers B1, B2...Bn, the second primers C1, C2...Cn, and the target nucleic acid. d. The sequences of the detection probes J1, J2...Jn can specifically bind to all sequences of the cyclic amplification probes D1, D2...Dn, or they can specifically bind to only some sequences of the cyclic amplification probes D1, D2...Dn, forming a stable double-stranded structure; the detection probes are modified with reporter groups to facilitate the formation of melting curve peaks during the detection stage; e. Various enzymes used for amplification detection include nicking enzymes, RNA-dependent DNA polymerases, ribonuclease H, RNA polymerases, and strand displacement polymerases.

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