Cascaded Signal Amplification System Based on Transcription-Driven CRISPR / Cas13a and Its Applications

Abstract

This invention relates to the field of biodetection technology, specifically to a transcription-driven CRISPR / Cas13a cascade signal amplification system and its applications. Based on a T7 transcription-driven CRISPR / Cas13a system, this invention constructs a cascade signal amplification mechanism. Through T7 transcription, the target nucleic acid signal is converted into a large number of RNA intermediates, thereby activating the cleavage activity of the Cas13a protein, achieving highly efficient cascade amplification of the target nucleic acid. Compared to traditional methods, this invention achieves highly sensitive detection of trace amounts of miRNA in serum without additional pre-amplification steps, significantly reducing the detection limit. It completely avoids the necessary reverse transcription (RT) and PCR / isothermal pre-amplification steps in conventional nucleic acid detection, greatly shortening the detection time (approximately 15 minutes) and reducing reagent consumption, making it suitable for widespread application.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a cascaded signal amplification system based on transcription-driven CRISPR / Cas13a and its applications. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related morbidity and mortality worldwide. Studies have shown that the survival rate of lung cancer patients is closely related to the stage of diagnosis, and early detection and treatment can significantly improve patient survival. Currently, clinical lung cancer screening mainly relies on imaging techniques (such as low-dose spiral CT, LDCT) and serum tumor marker detection. However, LDCT has problems such as radiation exposure risks, high false positive rates, and difficulty in being widely implemented in areas with limited medical resources; traditional protein-based tumor markers (such as CEA, CYFRA21-1) often have unsatisfactory sensitivity and specificity in the early stages of lung cancer.

[0003] Molecular diagnostic technologies, particularly nucleic acid detection based on polymerase chain reaction (PCR) or next-generation sequencing (NGS), while highly sensitive, typically rely on expensive, large-scale precision instruments (such as thermal cyclers), specialized operators, and demanding laboratory environments. This results in lengthy processing times and high costs, hindering their widespread application in primary healthcare institutions or point-of-care testing (POCT) scenarios. MicroRNAs (miRNAs), a class of endogenous non-coding small RNA molecules approximately 19-25 nucleotides in length, exhibit specific changes in expression levels in the bloodstream during early-stage lung cancer and are considered highly promising biomarkers for liquid biopsies. However, due to the extremely low abundance and short sequences of miRNAs in serum, achieving high-sensitivity and high-specificity detection without complex pretreatment remains a significant challenge.

[0004] In recent years, the CRISPR / Cas system, consisting of clustered, regularly spaced short palindromic repeats and their associated proteins, has become a next-generation molecular diagnostic tool following PCR due to its high specificity and programmability. Based on the different effector proteins, the application mechanisms of the CRISPR system in nucleic acid detection can be mainly classified into the following categories:

[0005] 1. CRISPR / Cas9 system: Primarily recognizes double-stranded DNA. The Cas9 protein, guided by sgRNA, recognizes target DNA containing PAM sequences and performs site-specific cleavage. Although widely used in gene editing, Cas9's reliance on cis-cleavage activity and lack of trans-cleavage capability limits its direct application in ultrasensitive nucleic acid signal amplification detection.

[0006] 2. CRISPR / Cas12 and Cas14 Systems: Cas12 (e.g., Cas12a) and Cas14 (e.g., Cas12f) belong to type V systems and primarily target DNA. They are activated upon recognizing target DNA, exhibiting non-specific trans-cleavage activity against single-stranded DNA (ssDNA) (e.g., DETECTR technology). However, when used to detect RNA targets such as miRNAs, these systems typically require additional reverse transcription and amplification steps to convert RNA into DNA. This not only increases the complexity and cost of the procedure but also reduces detection efficiency.

[0007] 3. CRISPR / Cas13 System: Cas13, belonging to the type VI system (such as LwaCas13a), is a nuclease that targets single-stranded RNA (ssRNA). After recognizing the target RNA under the guidance of crRNA, Cas13a trans-cleaves the non-specific ssRNA reporter probe in the system, releasing a fluorescent signal. This characteristic has been applied in detection technologies such as SHERLOCK.

[0008] Although Cas13a-based SHERLOCK technology has shown great potential in RNA detection, most existing technologies still have the following significant limitations:

[0009] 1. Pre-amplification dependency: To achieve amolar (aM) level detection sensitivity, existing Cas13a detection systems typically require pre-amplification steps such as recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP). These steps are not only cumbersome and costly, but also highly susceptible to aerosol contamination.

[0010] 2. Difficulty in multiplex detection: Commonly used isothermal amplification systems are difficult to simultaneously amplify and detect multiple targets in a single reaction tube, which limits their application in the combined screening of multiple indicators for lung cancer.

[0011] 3. Equipment dependence: Existing high-sensitivity detection processes often still rely on electrically driven centrifuges and temperature control equipment, which makes it difficult to meet the needs of truly portable, low-resource-constraint POCT.

[0012] Therefore, developing a microfluidic detection system that does not require reverse transcription and PCR / isothermal pre-amplification, can directly amplify serum miRNA signals with high sensitivity, and has multiple detection capabilities and portable operation features is a key technical challenge that urgently needs to be solved in the field of in vitro diagnostics. Summary of the Invention

[0013] In view of this, the technical problem to be solved by the present invention is to provide a cascade signal amplification system based on transcription-driven CRISPR / Cas13a and its application. The present invention provides a detection system that uses specific amplicon in conjunction with LwaCas13a protein to detect target nucleic acids. This detection system is simple to operate, low in cost, and has high sensitivity and specificity.

[0014] The present invention provides an amplicon comprising a T7 promoter sequence, a transcription template sequence complementary to the target nucleic acid, and an RNA cleavage site.

[0015] In some embodiments, the amplicon is a stem-loop structure, with its stem containing the T7 promoter sequence and its loop containing the transcription template sequence and the RNA cleavage site;

[0016] The RNA cleavage site is an RNA uracil residue.

[0017] In some specific embodiments, the amplicon is a DNA-RNA chimeric nucleic acid strand that forms a stem-loop structure through partial complementary pairing of its own sequences; the T7 promoter sequence is located in the stem of the stem-loop structure, the RNA cleavage site is an RNA uracil residue (rU) embedded in the nucleic acid strand, and the RNA cleavage site and the transcription template sequence are located in the loop of the stem-loop structure.

[0018] This invention provides a transcription-driven CRISPR / Cas13a cascade signal amplification system, comprising: the aforementioned amplicon, Cas13a:crRNA complex, T7 RNA polymerase, and fluorescent reporter probe;

[0019] The Cas13a:crRNA complex comprises: Cas13 protein and crRNA targeting the nucleic acid;

[0020] The fluorescent reporter probe can be cleaved by activated Cas13 protein.

[0021] In some embodiments, the crRNA includes repeat sequences and spacer sequences;

[0022] The repeat sequence binds specifically to the Cas13 protein;

[0023] The spacer sequence specifically recognizes the target nucleic acid.

[0024] In some embodiments, the Cas13 protein includes at least one of LbaCas13a, LbuCas13a, LwaCas13a, and PsmCas13b.

[0025] In some specific embodiments, the Cas13 protein is LwaCas13a.

[0026] In some embodiments, the fluorescent reporter probe includes a fluorescent group and a quencher group;

[0027] The fluorescent group is selected from at least one of FAM, HEX, TET, ROX, Cy3, and Cy5;

[0028] The quenching group is selected from at least one of BHQ, Dabcyl, and Tamra.

[0029] In some specific embodiments, the fluorescent group is FAM, preferably 6-FAM;

[0030] The quenching group is BHQ, preferably BHQ-1.

[0031] In some embodiments, the target nucleic acid includes at least one of microRNA, mRNA, lncRNA, and circRNA.

[0032] In some specific embodiments, the target nucleic acid is microRNA.

[0033] In some embodiments, the target nucleic acid has a nucleotide sequence as shown in SEQ ID NO:2, the crRNA has a nucleotide sequence as shown in SEQ ID NO:3, the amplicon has a nucleotide sequence as shown in SEQ ID NO:4, and the fluorescent reporter probe has a nucleotide sequence as shown in SEQ ID NO:1;

[0034] and / or

[0035] The target nucleic acid has a nucleotide sequence as shown in SEQ ID NO:5, the crRNA has a nucleotide sequence as shown in SEQ ID NO:6, the amplicon has a nucleotide sequence as shown in SEQ ID NO:7, and the fluorescent reporter probe has a nucleotide sequence as shown in SEQ ID NO:1;

[0036] and / or

[0037] The target nucleic acid has a nucleotide sequence as shown in SEQ ID NO:8, the crRNA has a nucleotide sequence as shown in SEQ ID NO:9, the amplicon has a nucleotide sequence as shown in SEQ ID NO:10, and the fluorescent reporter probe has a nucleotide sequence as shown in SEQ ID NO:1.

[0038] This invention provides applications of the aforementioned amplicon and / or the aforementioned cascaded signal amplification system in the following ① and / or ②:

[0039] ① Nucleic acid testing for non-diagnostic purposes;

[0040] ② Prepare nucleic acid detection kits.

[0041] In some embodiments, the method for non-diagnostic nucleic acid detection includes: mixing the sample to be tested with the cascaded signal amplification system, incubating, and collecting fluorescence signals to detect the target nucleic acid in the sample.

[0042] The present invention provides a nucleic acid detection kit, comprising the aforementioned amplicon and / or the aforementioned cascaded signal amplification system.

[0043] In some embodiments, rNTPs and RNase inhibitors are also included.

[0044] In some embodiments, the kit includes 0.1–0.3 μM Cas13 protein, 1–2 U / μL T7 RNA polymerase, 2–6 U / μL RNase inhibitor, 1.25–3.75 mM rNTPs, 50–150 nM crRNA, 1–20 µM amplicon, and 800–1200 nM fluorescent reporter probe.

[0045] In some specific embodiments, the kit includes 0.2 μM Cas13 protein, 1.5 U / μL T7 RNA polymerase, 2 U / μL RNase inhibitor, 1.25 mM rNTPs, 100 nM crRNA, 10 µM amplicon, and 1000 nM fluorescent reporter probe.

[0046] In some embodiments, the kit also includes a reaction buffer and a lyophilization protectant.

[0047] This invention provides a method for nucleic acid detection for non-diagnostic purposes, comprising mixing the sample to be tested with the cascaded signal amplification system or the reagent kit, incubating, and collecting fluorescence signals to detect the target nucleic acid in the sample.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. This invention constructs a cascade signal amplification mechanism based on a T7 transcription-driven CRISPR / Cas13a system. T7 transcription converts the target nucleic acid signal into a large number of RNA intermediates, thereby activating the cleavage activity of the Cas13a protein, achieving highly efficient cascade amplification of the target nucleic acid. Compared with traditional methods, this invention achieves highly sensitive detection of trace amounts of miRNA in serum without additional pre-amplification steps, significantly reducing the detection limit. Experimental verification shows that the cascade signal amplification system provided by this invention can achieve attomolar-level ultrasensitive detection, and the dual specific recognition of T7 transcription and Cas13a cleavage ensures high accuracy and reproducibility of the detection results.

[0050] 2. This invention directly utilizes the LwaCas13a protein and specific amplicon to recognize target nucleic acids, completely avoiding the reverse transcription (RT) and PCR / isothermal pre-amplification steps required in conventional nucleic acid detection. This significantly shortens the detection time (approximately 15 minutes), reduces reagent consumption, and also reduces the risk of cross-contamination and bias introduced by amplification, making the detection system simpler and more stable.

[0051] 3. This invention can directly amplify miRNA signals in serum with high sensitivity, without the need for complex RNA extraction or purification of the sample. This feature overcomes the challenges of low miRNA abundance and numerous interfering substances in serum samples, providing an efficient technical tool for liquid biopsy and non-invasive diagnosis.

[0052] 4. The cascaded signal amplification system provided by this invention can realize the parallel detection of multiple target nucleic acids in the same reaction system. The detection system is integrated into a microfluidic chip and has the characteristics of small size, low reagent consumption, fast reaction and easy automation. It further improves the throughput and accuracy of multiplex detection and is suitable for application scenarios such as point-of-care testing (POCT), rapid screening in resource-limited environments and bedside diagnosis. It is suitable for widespread application. Attached Figure Description

[0053] Figure 1 A schematic diagram illustrating the principle of the nucleic acid detection method based on transcription-driven CRISPR / Cas13a cascade signal amplification (WASTON) provided in Example 1;

[0054] Figure 2The following is an example of the screening experiments and results of key components in the WASTON system in Example 3. Figure A shows a bar graph of fluorescence intensity at the reaction endpoint under different component combinations. The table below the figure lists the component composition corresponding to each group of experiments (including Cas13a, annealing treatment, matched target, mismatched target, and presence or absence of T7 RNA polymerase), used to examine the dependence of the cascade reaction on the above key components. Figure B shows the curves of fluorescence intensity changing with reaction time under different reaction conditions. Each curve corresponds to one of the five groups of experiments: "cascade reaction - matched", "non-cascade reaction", "cascade reaction - mismatched", "no annealing", and "annealing". Figure C shows the comparison of fluorescence intensity collected at 5, 10, 15, 20, and 30 mins after adding matched and mismatched targets to the cascade reaction system. Figure D shows the fluorescence intensity collected at 5, 10, 15, 20, and 30 mins after annealing and without annealing in the cascade reaction system. The comparison results of fluorescence intensity collected at min time;

[0055] Figure 3 This shows the concentration optimization results of the WASTON component provided in Example 4;

[0056] Figure 4 The following is a comparison of the sensitivity of the traditional single Cas13a detection method and the WASTON method in Example 4. Figure A shows the fluorescence intensity of the single Cas13a reaction system as a function of reaction time when the target nucleic acid concentrations are 5 nM, 10 nM, 100 nM, and 1 μM. Figure B shows the fluorescence intensity of the cascade reaction system as a function of reaction time when the target nucleic acid concentrations are 100 μM, 1 aM, 10 aM, 100 aM, 10 fM, 1 pM, 100 pM, 10 nM, and the negative control (Neg). Figure C shows the fluorescence intensity of the cascade reaction system and the traditional single Cas13a detection system as a function of reaction time when the target nucleic acid concentration is 10 nM. Figure D shows the fluorescence intensity of the cascade reaction system and the traditional single Cas13a detection system as a function of reaction time when the target nucleic acid concentration is 1 nM. Figure E shows the fluorescence intensity of the cascade reaction system and the traditional single Cas13a detection system as a function of reaction time when the target nucleic acid concentration is 1 nM. At pM, the fluorescence intensity of the cascade reaction system and the traditional single Cas13a detection system changes with reaction time. The fluorescence intensity of the cascade reaction system at the reaction endpoint is about 74.4 times that of the traditional single Cas13a detection system. Figure F is a scatter plot comparing the fluorescence intensity at the reaction endpoint of the cascade reaction system and the traditional single Cas13a detection system (SingleCas13a) under gradient target nucleic acid concentrations (the horizontal axis is the negative logarithm of the target nucleic acid concentration - Lg concentration (M), and a negative control Neg is set).

[0057] Figure 5 The results of verifying the target specificity of the WASTON method using artificially mismatched crRNA introduced in Example 4 are shown in Figure A. Figure A is a bar chart showing the fluorescence intensity at the reaction endpoint when a cascade reaction system was constructed using crRNA targeting miR-17 and its corresponding amplicons, followed by the addition of the target miR-17, and the sequence-similar miR-106a, miR-20a, miR-20b, and a blank control. Figure B is a bar chart showing the fluorescence intensity at the reaction endpoint when a cascade reaction system was constructed using crRNA targeting miR-17 and its corresponding amplicons, followed by the addition of a single sample of miR-17, a 1:1000 mixture of miR-17 and miR-155, a 1:1000 mixture of miR-17 and miR-19b, and a 1:1000 mixture of miR-17, miR-155, and miR-19b.

[0058] Figure 6 The diagram shows the structure of the hand-driven centrifugal microfluidic chip for nucleic acid detection provided in Example 5. In Figure A, the structure of the microfluidic chip is shown, and in Figure B, the position of the detection reagents pre-embedded in the microfluidic chip for different samples is shown.

[0059] Figure 7 The following is a graph showing the detection results of multiple miRNA markers (miR-17, miR-155, miR-19b) in serum samples from lung cancer patients and healthy controls provided in Example 6. In the graph, A shows the detection results of miR-17, B shows the detection results of miR-155, and C shows the detection results of miR-19b.

[0060] Figure 8 Example 6 illustrates the use of receiver operating characteristic (ROC) curves to evaluate the diagnostic accuracy of the detection system described in this invention. Detailed Implementation

[0061] This invention provides a cascaded signal amplification system based on transcription-driven CRISPR / Cas13a and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0062] This application provides a pre-amplification-free nucleic acid detection system and method based on CRISPR / Cas13a and T7 transcription cascade amplification technology (WASTON). The system includes:

[0063] The reaction system contains LwaCas13a protein, T7 RNA polymerase, ribonucleoside triphosphates (rNTPs), RNase inhibitors, guide RNA (crRNA) specific to the target microRNA, a universal fluorescent reporter probe, a transcription-driven signal amplification template, and a reaction buffer.

[0064] Hand-driven centrifugal microfluidic chip: The chip is pre-loaded with spatially encoded reaction units for different targets (such as miR-17, miR-155, miR-19b), and the reagents are pre-embedded in the form of lyophilized microspheres.

[0065] The detection method is as follows: the sample to be tested is added to a microfluidic chip, and centrifugal force generated manually drives fluid distribution and reconstitution. During the isothermal reaction, the target microRNA specifically activates the Cas13a protein, triggering its trans-cleavage activity. The activated Cas13a cleaves the signal amplification template, relieving its inhibition of the T7 promoter, causing the T7 polymerase to transcribe a large number of target RNA replicas, thus forming an exponential positive feedback signal amplification loop; on the other hand, it efficiently cleaves the fluorescent reporter probe, generating a detectable fluorescent signal.

[0066] Technical advantages: This system eliminates the need for nucleic acid pre-amplification steps such as PCR, enabling highly sensitive (aM-level) and highly specific detection of multiplex lung cancer-related microRNAs within approximately 15 minutes. This application features low cost, no need for electrically driven centrifugation, and ease of operation, making it particularly suitable for early lung cancer screening and point-of-care testing (POCT) in resource-scarce areas.

[0067] This invention provides a transcription-driven CRISPR / Cas13a cascade signal amplification system and its application. The method for detecting nucleic acids using the cascade amplification system can be called "WASTON" (Wide-ranging Amplification-free Specific Testing for Onco-lung Nucleic Acids).

[0068] In a first aspect, the present invention provides a pre-amplification-free multiplex nucleic acid detection system (named "WASTON") based on Cas13a and T7 transcriptional cascade amplification, which includes the following steps:

[0069] Provide a sample to be tested, wherein the sample contains a target nucleic acid sequence (such as a target miRNA).

[0070] Prepare a reaction system comprising LwaCas13a protein, a target-specific guide RNA (crRNA) designed for the target, T7 RNA polymerase, ribonucleoside triphosphates (rNTPs), a universal fluorescent reporter probe, a transcription-driven amplifier, and a reaction buffer.

[0071] The amplifier is a DNA / RNA chimeric oligonucleotide; the amplifier contains a T7 promoter sequence, a transcription template sequence, and an RNA cleavage sequence; in its initial state, the amplifier forms a stem-loop structure through intramolecular base pairing; the T7 promoter sequence is located in the stem of the stem-loop structure, and the RNA cleavage sequence is located in the loop of the stem-loop structure; the stem-loop structure spatially prevents the binding of T7 RNA polymerase to the T7 promoter sequence.

[0072] The reaction and detection process involves adding the sample to be tested into the reaction system and carrying out the reaction under isothermal conditions.

[0073] The target guide RNA guides the Cas13a protein to specifically recognize and bind to the target nucleic acid sequence, thereby activating the Cas13a protein's trans cleavage activity (Collateral Cleavage).

[0074] The activated Cas13a protein specifically cleaves the blocked RNA strand in the amplicon, removing its steric hindrance to the T7 promoter and exposing the functional T7 promoter sequence;

[0075] The T7 RNA polymerase recognizes the exposed promoter sequence and binds to the amplicon, initiating the transcription process and producing a large number of RNA replicas identical to the target sequence;

[0076] The released RNA replicas act as secondary activators, guiding the remaining Cas13a protein in the system to bind specifically and activate, forming a positive feedback loop system with exponential signal amplification.

[0077] During this process, the activated Cas13a protein simultaneously cleaves the universal fluorescent reporter probe, separating its fluorophore from its quenching group to generate a detectable fluorescent signal.

[0078] In embodiments of the present invention, the core components and reaction principle are detailed below:

[0079] 1. Regarding target guide RNA (crRNA): The crRNA comprises a direct repeat (DR) sequence and a spacer sequence. The direct repeat sequence has a specific secondary structure that can form a stable binary complex with the LwaCas13a protein; the spacer sequence contains a nucleotide sequence complementary to the target nucleic acid for specific target recognition.

[0080] 2. Regarding the transcription-driven amplifier: The amplifier is a core component in constructing the "WASTON" positive feedback loop. It is characterized by comprising a hybrid double-stranded structure consisting of one DNA strand and one RNA strand. The RNA strand contains a cleavage site (such as a Poly-U site) specifically recognized by an activated Cas13a protein. In its intact, uncut state, the RNA strand hybridizes with the T7 promoter region of the DNA strand or its adjacent region to form an inactive, closed structure, thereby shielding the transcriptional activity of T7 RNA polymerase.

[0081] 3. Principle of the Nucleic Acid Detection Method: The improved sensitivity of the method described in this invention relies on a three-step cascade mechanism of "Cas13a unlocking - T7 transcription - cascade feedback":

[0082] Step 1 (Initiation): The target nucleic acid (such as miRNA) binds to the Cas13a / crRNA complex, inducing a conformational change in the Cas13a protein and activating its non-specific RNase activity.

[0083] Step 2 (Unlocking): The activated Cas13a protein degrades the blocked RNA strand on the Amplifier. This process converts the Amplifier from a "silent state" to an "active state," releasing the DNA template that can be recognized by T7 RNA polymerase.

[0084] Step 3 (Cascade Amplification): T7 RNA polymerase uses exposed DNA as a template, consumes rNTPs in the system, and transcribes thousands of RNA replicas. These replicas are not only transcription products but also novel Cas13a activators. They rapidly activate the free Cas13a / crRNA complex in the system, prompting more amplifiers to be unlocked and more fluorescent reporter probes to be cleaved. This positive feedback loop causes the fluorescence signal to increase exponentially in a short time (e.g., within 15 minutes), thus achieving highly sensitive and specific detection of trace target nucleic acids without the need for PCR pre-amplification.

[0085] In this embodiment of the invention, regarding the target nucleic acid and sample source: the target nucleic acid sequence is selected from various ribonucleic acid fragments such as microRNA, messenger ribonucleic acid (mRNA), long non-coding ribonucleic acid (lncRNA), or circular RNA (circRNA). The sample to be tested can originate from the body fluids or tissues of mammals, including humans, or from biological environmental samples carrying specific pathogens.

[0086] Specifically, the samples to be tested include, but are not limited to, whole blood, serum, plasma, urine, saliva, bronchoalveolar lavage fluid, cell lysate, exosome extracts, or viral particle suspensions.

[0087] Preferably, the sample to be tested undergoes nucleic acid extraction and enrichment. For example, total nucleic acid (Total RNA) is extracted from the original sample using a magnetic bead or centrifuge column nucleic acid extraction kit to improve detection sensitivity.

[0088] In this embodiment of the invention, regarding the amplifier: the amplifier is designed as a DNA / RNA chimera or hybrid complex structure.

[0089] Sequence Design: The amplicon contains a DNA transcription template sequence that is complementary to the target nucleic acid sequence. This design ensures that, under the action of T7 RNA polymerase, the template can transcribe RNA replicas with a sequence highly consistent with the initial target, thereby achieving sustained activation of the Cas13a protein.

[0090] Structural features: The amplicon undergoes annealing before the reaction to pair and form a stable secondary structure (such as a stem-loop structure). This secondary structure spatially blocks the T7 promoter, preventing non-specific transcription in the absence of a target.

[0091] Nucleotide modification: The nucleotides in the amplicon can be independently selected from natural nucleotides (A, T, C, G, U).

[0092] In this embodiment of the invention, regarding the recognition characteristics of Cas13a: the recognition of the target nucleic acid sequence does not depend on strict pre-intermediate sequence adjacent motifs (PFS).

[0093] The LwaCas13a protein used in this invention exhibits broad adaptability to target RNA recognition. Upon binding to crRNA, it enables highly specific recognition and cleavage of RNA targets without requiring specific PAM or strict PFS sequence restrictions. This characteristic provides the method of this invention with great flexibility in target selection and crRNA design, making it particularly suitable for the detection of short-sequence miRNAs.

[0094] In this embodiment of the invention, regarding signal output: the detectable markers include, but are not limited to, fluorescent groups (Fluorophore), fluorescence quenchers (Quencher), biotin, digoxigenin, or colloidal gold particles.

[0095] Preferably, in the fluorescence detection system of the present invention, the marker is a fluorescence-quenching pair (such as FAM-BHQ1) marked on both ends of a universal reporter probe, used to generate a real-time fluorescence signal.

[0096] In this embodiment of the invention, regarding the structure and modification of the fluorescent detection probe (Reporter): the fluorescent detection probe is a single-stranded nucleic acid sequence, preferably a single-stranded RNA (ssRNA) sequence (such as a Poly-U or Poly-A sequence) that can be non-specifically cleaved by the Cas13a protein. The probe is simultaneously modified with a fluorescent group (Fluorophore) and a fluorescence quencher group (Quencher).

[0097] The fluorescent group and quencher group are modified at the 5' and 3' ends of the fluorescent detection probe, respectively. For example, the 5' end is modified with a fluorescent group and the 3' end with a quencher group; or, the 5' end is modified with a quencher group and the 3' end with a fluorescent group. This end modification method is simple to synthesize and can form an effective fluorescence resonance energy transfer (FRET) effect when the probe is not cleaved.

[0098] In this embodiment of the invention, regarding the selection of detectable markers: the fluorescent group and the quenching group should be selected according to the matching principle of fluorescence resonance energy transfer (FRET) to ensure high quenching efficiency and low background signal.

[0099] Fluorescent group: can be selected from one or more of carboxyfluorescein (FAM, such as 5-FAM or 6-FAM), hexachloro-6-methylfluorescein (HEX), tetrachloro-6-carboxyfluorescein (TET), rhodamine (ROX), Cy3, Cy5, etc.

[0100] Quenching group: can be selected from one or more of black hole quenchers (BHQ, such as BHQ-1, BHQ-2, BHQ-3), Dabcyl, Tamra, etc.

[0101] Preferred combination: For example, when the fluorescent group is FAM, the quenching group is preferably BHQ-1;

[0102] In this embodiment of the invention, regarding the mechanism of action of the fluorescent detection probe: the signal generation mechanism of the fluorescent detection probe is based on the collateral cleavage activity of the Cas13a protein.

[0103] Suppressed state (background signal): In the initial stage of the reaction or in the absence of a target, the fluorescent detection probe maintains an intact single-chain structure. Due to the spatial proximity of the fluorescent group and the quenching group (within the FRET distance), the energy emitted by the fluorescent group is absorbed by the quenching group or undergoes static quenching, resulting in the fluorescence signal being suppressed to an extremely low background level.

[0104] Activated state (signal release): When the target protein or a target copy generated by transcription activates the Cas13a protein, the activated Cas13a protein serves as a fluorescent detection probe in a nuclease-specific degradation system. The breakage of the probe backbone leads to irreversible spatial separation of the fluorophore and quencher groups, releasing the quenching effect and thus releasing a significant fluorescent signal.

[0105] In this embodiment of the invention, regarding the acquisition of Cas13a effector proteins: the Cas13a protein (such as LwaCas13a) can be obtained through genetic engineering recombinant expression and protein purification.

[0106] Commercial availability: Optionally, the Cas13a protein can also be obtained directly through commercial channels as a highly active formulation.

[0107] In this embodiment of the invention, the universal fluorescent reporter probe is characterized as follows: The fluorescent detection probe is a single-stranded ribonucleic acid (ssRNA) with fluorescent groups and quenching groups modified at both ends, and its sequence is designed as a nucleotide sequence rich in uracil (U) to respond to the non-specific trans-cleavage activity of the Cas13a protein.

[0108] Specific sequence: In this embodiment, the sequence of the fluorescent detection probe is 5'- / 6-FAM / rUrUrUrUrU / BHQ1 / -3' (SEQ ID NO: 1). Here, "rU" represents ribouracil nucleotide, and the continuous "rU" sequence constitutes a highly efficient cleavage site for Cas13a.

[0109] In this embodiment of the invention, the design principle for the amplifier is as follows: the amplifier contains a specific DNA transcription template sequence. The base composition of this DNA template sequence needs to be designed with reverse complementation based on the specific target microRNA sequence.

[0110] Design objective: To ensure that the RNA product (i.e., the target copy) transcribed from this template by T7 RNA polymerase is sequence-identical or highly homologous to the initial target microRNA. This design forms the material basis for the in-situ positive feedback amplification loop of "target activation of Cas13a—Cas13a unlocking of the amplifier—generation of more target copies—reactivation of Cas13a."

[0111] Regarding the detection performance and technical advantages in this invention: The nucleic acid detection method of this invention, through a cascaded signal amplification strategy (WASTON), has the following significant advantages:

[0112] 1. Ultra-high sensitivity: No pre-amplification steps such as PCR, LAMP, or RPA are required; its limit of detection (LOD) can be as low as 1 aM (i.e., 10⁻⁶). -18 At the M level, nucleic acid detection at the single-molecule level has been achieved.

[0113] 2. Single-base resolution specificity: Combined with engineered crRNA, the method of this invention can accurately distinguish miRNA homologues with highly similar sequences, achieving specific recognition with single-base resolution (e.g., effectively distinguishing lung cancer-related miR-17 from its family member miR-106a).

[0114] 3. Low cost and convenience: This method has a short reaction time (about 15 minutes) and when used with a hand-driven microfluidic chip, it eliminates the dependence on expensive precision instruments and electricity, greatly reducing the cost of a single test.

[0115] In this embodiment of the invention, regarding the application field: the nucleic acid detection method and system have broad clinical and epidemiological application value.

[0116] Tumor screening: Particularly suitable for early screening, auxiliary diagnosis, and prognostic monitoring of lung cancer. Specific detection targets include tumor-associated miRNAs in serum or exosomes, such as miR-17, miR-155, miR-19b, or combinations thereof.

[0117] Extended applications: Based on the programmability of Cas13a, by changing the sequences of crRNA and Amplifier, the method can be flexibly extended to high-sensitivity field detection of other disease biomarkers, epidemic pathogens (such as influenza virus), or emerging RNA viruses.

[0118] In this embodiment of the invention, a nucleic acid detection method and reagent combination targeting the lung cancer-related biomarker miR-17 are provided:

[0119] Target sequence: The target nucleic acid is miR-17 (e.g., hsa-miR-17-5p), and its nucleotide sequence is shown in SEQ ID NO: 2.

[0120] Specific recognition component: The target guide RNA (crRNA) contained in the reaction system has a spacer region sequence that specifically recognizes miR-17, and its full-length nucleotide sequence is shown in SEQ ID NO: 3.

[0121] Signal amplification component: The reaction system contains a transcription-driven amplifier designed for miR-17. The amplifier is a DNA / RNA chimeric oligonucleotide containing a T7 promoter sequence, a DNA transcription template sequence complementary to miR-17, and an RNA cleavage site located in the hairpin loop, as shown in SEQ ID NO: 4.

[0122] Signal output component: The reaction system contains a universal fluorescent reporter probe, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0123] In another embodiment of the present invention, a nucleic acid detection method and reagent combination for the lung cancer-related biomarker miR-155 are provided:

[0124] Target sequence: The target nucleic acid is miR-155 (e.g., hsa-miR-155-5p), and its nucleotide sequence is shown in SEQ ID NO: 5.

[0125] Specific recognition component: The nucleotide sequence of the target guide RNA (crRNA) is shown in SEQ ID NO: 6.

[0126] Signal amplification component: The Amplifier sequence designed for miR-155 is shown in SEQ ID NO: 7.

[0127] Signal output component: The universal fluorescent reporter probe sequence is shown in SEQ ID NO: 1.

[0128] In one embodiment of the present invention, a nucleic acid detection method and reagent combination for the lung cancer-related biomarker miR-19b are provided:

[0129] Target sequence: The target nucleic acid is miR-19b (e.g., hsa-miR-19b-3p), and its nucleotide sequence is shown in SEQ ID NO: 8.

[0130] Specific recognition component: The nucleotide sequence of the target guide RNA (crRNA) is shown in SEQ ID NO: 9.

[0131] Signal amplification component: The Amplifier sequence designed for miR-19b is shown in SEQ ID NO: 10.

[0132] Signal output component: The universal fluorescent reporter probe sequence is shown in SEQ ID NO: 1.

[0133] The nucleic acid detection method (WASTON) described in the first aspect of this invention has significant advantages over existing technologies:

[0134] 1. High sensitivity without pre-amplification: This method overcomes the shortcomings of traditional CRISPR detection technologies (such as DETECTR and SHERLOCK) that rely on complex pre-amplification steps such as RPA or LAMP. By constructing a cascaded positive feedback loop of Cas13a and T7 polymerase, direct detection of low-abundance nucleic acid samples (detection limit as low as 1 aM) is achieved.

[0135] 2. High specificity: Thanks to the high fidelity of Cas13a and the key-locking design of Amplifier, this method has single-base resolution and can accurately distinguish highly homologous miRNA family members.

[0136] 3. Fast and convenient: The entire testing process is carried out under a constant temperature of 37℃, and the reaction time is only about 15-20 minutes.

[0137] 4. Clinical application prospects: Combined with a matching portable microfluidic chip, this method can be effectively applied to point-of-care testing (POCT) of microRNA (miRNA) biomarkers in clinical samples such as serum and exosomes, providing a low-cost and efficient new tool for early screening, subtyping diagnosis and efficacy monitoring of lung cancer.

[0138] Secondly, the present invention also provides a nucleic acid detection system comprising an amplifier and an independent fluorescent reporter probe.

[0139] 1. Regarding the amplifier, the amplifier is a DNA / RNA chimeric oligonucleotide, which itself does not contain fluorescent modification.

[0140] Structural features: The amplicon is a single-stranded nucleic acid sequence containing both DNA and RNA sequence regions. Through intramolecular base pairing, the template folds in its initial state to form a stem-loop structure or a similar secondary closed structure.

[0141] Functional regions: The DNA sequence region contains a T7 promoter sequence and a target transcription template sequence, wherein the T7 promoter sequence is located in the stem of the stem-loop structure; the RNA sequence region contains a nucleotide sequence (such as one containing a U sequence) that can be specifically recognized and cleaved by the Cas13a / crRNA complex, and this RNA sequence is located in the loop of the stem-loop structure.

[0142] Inhibition mechanism: In the initial state, the steric hindrance generated by the stem-loop structure effectively prevents the binding and recognition of T7 RNA polymerase to the T7 promoter sequence, thereby inhibiting the transcriptional response.

[0143] 2. Regarding the independent fluorescent reporter probe (Reporter): The fluorescent reporter probe is a single-stranded RNA (ssRNA) with fluorescent groups and fluorescence quenching groups modified at both ends, respectively.

[0144] Signal state: In the initial state of probe integrity, the fluorescence emitted by the fluorescent group is absorbed or suppressed by the nearby quenching group by the principle of fluorescence resonance energy transfer (FRET) or static quenching mechanism, resulting in a low background signal.

[0145] 3. Regarding the system's mechanism of action: When the system described in the second aspect of this invention is applied to nucleic acid detection, it utilizes the "collateral cleavage" activity of the activated Cas13a protein to simultaneously drive the signal amplification circuit and the signal output circuit.

[0146] Signal amplification loop (positive feedback loop): When the Cas13a protein is activated by the initial target, its trans-cleavage activity specifically cleaves the RNA sequence located in the loop of the amplicon, thereby removing steric hindrance and exposing the functional T7 promoter sequence located in the stem. The T7 RNA polymerase then binds to the exposed promoter and initiates transcription using DNA as a template, producing a large number of RNA replicas homologous to the initial target sequence. These newly generated replicas act as secondary activators, reactivating the free Cas13a protein in the system, forming an exponentially growing positive feedback amplification loop.

[0147] Signal output loop (real-time detection): The activated Cas13a protein (whether activated by the initial target or by newly generated RNA replicas) simultaneously and efficiently cleaves the free fluorescent reporter probe in the system. The breakage of the probe backbone causes the fluorescent group and quenching group to separate spatially, thereby releasing a significant fluorescent signal, achieving highly sensitive, real-time monitoring of the target nucleic acid.

[0148] Thirdly, the present invention also provides a reagent kit for nucleic acid detection, the reagent kit being based on a system design that decouples signal amplification and signal reporting functions.

[0149] 1. Core Specific Components of the Kit The kit includes the following two key nucleic acid elements:

[0150] Transcription-driven amplifier: The amplifier is a DNA / RNA chimeric oligonucleotide without fluorescent modification.

[0151] Structural features: It contains a T7 promoter sequence, a transcription template sequence, and an RNA cleavage site located inside the molecule. In its initial state, the Amplifier forms a stem-loop structure through intramolecular folding, using steric hindrance to shield the T7 promoter sequence and prevent non-specific binding of T7 RNA polymerase.

[0152] Function: Used to initiate T7 transcription via a "demasking" mechanism after Cas13a is activated, in order to generate a large number of target replicas.

[0153] Universal fluorescent reporter probe (Reporter): The Reporter is a single-stranded RNA (ssRNA, such as Poly-U sequence) with fluorescent groups and fluorescence quenching groups modified at both ends, respectively. It is used to respond to the trans-cleavage activity of Cas13a and generate a detectable fluorescent signal through the disruption of the fluorescence resonance energy transfer (FRET) effect.

[0154] 2. Packaging Format of the Reagent Kit (Preferred Embodiment) To enhance the long-term storage stability of the reagent and simplify field operation procedures, the reagent kit is preferably in the form of pre-filled lyophilized microspheres. The reagent kit comprises two independent components:

[0155] The first reagent component (e.g., enzyme-probe microspheres, Pellet 1) contains LwaCas13a protein, target guide RNA (crRNA), T7 RNA polymerase, ribonucleotide triphosphates (rNTPs), a universal fluorescent reporter probe, reaction buffer components (e.g., Tris-HCl, MgCl2, DTT), and a lyophilization protectant.

[0156] The second reagent component (e.g., template microspheres, Pellet 2) contains a transcription-driven amplifier designed for a specific target, an RNase inhibitor, and a lyophilization protectant.

[0157] 3. Technical effects and application scenarios of the reagent kit: The reagent kit has the following significant advantages:

[0158] Highly sensitive detection without pre-amplification: Through the WASTON cascade amplification mechanism, trace amounts of lung cancer-related microRNAs (such as miR-17, miR-155, and miR-19b) in serum samples can be detected at the aM level (Attomolar) without the need for PCR or LAMP pre-amplification.

[0159] Fast and portable: The entire testing process is carried out under constant temperature conditions (such as 37°C), and the reaction time is short (about 15-20 minutes).

[0160] Room temperature transport and POCT compatibility: The use of cryo-intervention embedding technology allows the core enzyme preparation to maintain high activity at room temperature, making it particularly suitable for point-of-care testing (POCT) in resource-scarce areas and large-scale early lung cancer screening.

[0161] Fourthly, the present invention also provides an integrated nucleic acid detection system, which consists of a manually driven centrifugal microfluidic chip.

[0162] 1. Regarding the sample reaction assembly (manually driven centrifugal microfluidic chip): The microfluidic chip adopts a spatial coding design, and the preferred material is polymethyl methacrylate (PMMA).

[0163] Structural Units: The chip integrates a sample application chamber, a waste liquid chamber, and multiple radially distributed independent reaction units. Each reaction unit contains pre-filled lyophilized reagent microspheres targeting different objectives.

[0164] Fluid control mechanism: The flow channel design of the chip combines centrifugal force drive and siphon valve control principle.

[0165] Centrifugal distribution: Under high-speed rotation, centrifugal force is used to overcome flow resistance and uniformly distribute the sample fluid to the temporary storage area of ​​each reaction unit.

[0166] Siphon injection: With the rotation stopped, the siphon valve is activated by capillary siphon action to drive fluid into the reaction chamber to reconstitute the lyophilized reagent.

[0167] Drive method: The chip is compatible with hand-powered centrifuges. Without relying on large electric centrifuges, users can generate sufficient centrifugal force simply by manually turning or rotating the chip to complete sample mixing, dispensing, and injection operations.

[0168] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0169] Example 1: Preparation of experimental materials and construction of lyophilized reagent microspheres

[0170] 1. Design and Synthesis of Oligonucleotide Sequences: The oligonucleotides involved in this invention include target guide RNA (crRNA), transcription-driven amplicon, and universal fluorescent reporter probe. All sequences were chemically synthesized by commercial biotechnology companies (such as Sangon Biotech) and purified to analytical grade by HPLC.

[0171] (1) Universal fluorescent reporter probe (Reporter): Designed as a single-stranded RNA sequence (Poly-U) with fluorescent and quenching groups modified at both ends. Its nucleotide sequence is 5'-6-FAM-rUrUrUrUrU-BHQ-1-3' (SEQ ID NO:1), that is, 6-carboxyfluorescein (6-FAM) is modified at the 5' end as a fluorescent group, Black Hole Quencher 1 (BHQ-1) is modified at the 3' end as a quenching group, and there are 5 ribonucleic acid (rU) linking sequences in the middle.

[0172] (2) Target guide RNA (crRNA): CrRNA sequences containing specific spacer regions are designed based on the sequence characteristics of target microRNAs.

[0173] The crRNA sequence targeting miR-17 is shown in SEQ ID NO:3;

[0174] The crRNA sequence targeting miR-155 is shown in SEQ ID NO:6;

[0175] The crRNA sequence targeting miR-19b is shown in SEQ ID NO:9.

[0176] (3) Amplifier: Designed as a DNA / RNA chimeric oligonucleotide. This sequence contains the T7 promoter sequence, the target complementary DNA transcription template sequence, and the RNA cleavage site located inside the molecule, which forms a stem-loop structure after annealing.

[0177] The Amplifier sequence for miR-17 is shown in SEQ ID NO:4;

[0178] The Amplifier sequence for miR-155 is shown in SEQ ID NO:7.

[0179] The Amplifier sequence for miR-19b is shown in SEQ ID NO:10;

[0180] (4) Target sequence:

[0181] The target nucleic acid is miR-17 (such as hsa-miR-17-5p), whose nucleotide sequence is shown in SEQ ID NO:2;

[0182] The target nucleic acid is miR-155 (e.g., hsa-miR-155-5p), whose nucleotide sequence is shown in SEQ ID NO:5;

[0183] The target nucleic acid is miR-19b (e.g., hsa-miR-19b-3p), whose nucleotide sequence is shown in SEQ ID NO:8.

[0184] The specific sequence information is shown in Table 1 below.

[0185] Table 1

[0186]

[0187] Note: In the sequence information in Table 1, the base U is displayed as T when the sequence listing is made. The actual sequence is based on the sequence in Table 1.

[0188] Figure 1 The schematic diagram of the nucleic acid detection method based on transcription-driven CRISPR / Cas13a cascade signal amplification (WASTON) provided by the present invention is shown, specifically including:

[0189] Design philosophy and operating logic of transcription-driven amplifiers

[0190] 1. Core Design Concept: "Molecular Conformational Lock" The Amplifier described in this invention is not a traditional double-stranded DNA substrate, but rather a smart molecular switch based on conformational regulation. Its core design concept lies in utilizing the secondary structure of nucleic acid molecules to construct a "metastable" silent mechanism.

[0191] Design objective: To solve the problem of "non-specific spontaneous transcription" that is common in traditional T7 transcriptional amplification.

[0192] Implementation: The Amplifier is designed to be in an "OFF" conformation in its natural state. Only under the action of a specific external trigger signal (i.e., the inverse cut of Cas13a) will an irreversible conformation collapse occur, converting it into an "ON" conformation.

[0193] 2. Structural Logic: DNA / RNA Chimera and Stem-Loop Structure To achieve the above concept, Amplifier is designed as a DNA / RNA chimeric oligonucleotide in its molecular structure. Its ingenuity lies in the spatial layout of the following three dimensions:

[0194] Chimeric component: The main body of the molecule is a DNA sequence (containing the T7 promoter and transcription template), but an RNA sequence (containing the Cas13a cleavage site) is embedded or linked at a key position.

[0195] Stem-loop folding: Through precise pairing of intramolecular bases, the Amplifier folds to form a compact stem-loop structure.

[0196] The stem is formed by the pairing of the T7 promoter sequence with its complementary sequence, but this pairing is designed to be restricted.

[0197] Loop: Composed of RNA sequences. This loop acts as a "lock," maintaining the rigidity and closure of the stem.

[0198] Steric hindrance: In the complete stem-loop state, the presence of the loop and the compact spatial folds constitute strong steric hindrance, which prevents the large T7 RNA polymerase from effectively binding to the promoter region, thus completely blocking the initiation of transcription at the physical level.

[0199] 3. Operational Logic: The cascaded amplifier's workflow, from "de-suppression" to "positive feedback," follows a strict logic gating mechanism:

[0200] Phase 1: De-repression / Unlocking. When the Cas13a protein is activated by the target, its non-specific RNase activity precisely "cleaves" the RNA lock in the amplifier loop, causing conformational disintegration of the amplifier. The T7 promoter is then exposed from the closed stem, completing the state switch from "OFF" to "ON".

[0201] The second stage: In situ transcription. T7 RNA polymerase rapidly recognizes the exposed functional promoter and uses the DNA sequence in the Amplifier as a template for transcription.

[0202] The third stage: Identity Copying & Feedback. This is the most crucial part of the Amplifier logic—sequence homology design. The Amplifier's DNA transcription template is designed to be complementary to the initial target (target miRNA). Therefore, the transcription product (RNA copy) is completely identical to the initial target in both sequence and function.

[0203] Logical closed loop: This means that each transcription product is a new "key". They do not depend on the initial sample, but continuously activate the remaining Cas13a in the system.

[0204] Exponential Explosion: This "product-as-activator" design constructs a self-driven exponential positive feedback loop within the reaction system, thereby amplifying the weak initial signal by millions of times in an extremely short time (about 15 minutes).

[0205] 4. Summary: In conclusion, the Amplifier of this invention achieves strict reaction control through a "stem-loop lock mechanism" and exponential signal amplification through "homologous transcription design." This three-in-one design logic is the fundamental reason why the WASTON platform achieves pre-amplification-free, aM-level sensitivity detection.

[0206] Preparation of Lyophilized Pellets: In order to achieve room temperature transport of reagents and integration with microfluidic chips, this invention adopts a stepwise lyophilization strategy to prepare the WASTON reaction system into two independent microsphere components.

[0207] (1) Preparation of the first component (Pellet 1, enzyme-probe microspheres): Prepare a mixture with the following components: LwaCas13a protein, crRNA, T7 RNA polymerase, rNTPs, Reporter and appropriate amount of freeze-drying protectant.

[0208] (2) Preparation of the second component (Pellet 2, template microspheres): Prepare a mixture with the following components: Amplifier for a specific target, RNase inhibitor and appropriate amount of lyophilization protectant.

[0209] Example 2: Pre-amplification-free nucleic acid detection process based on the Waston strategy

[0210] This embodiment provides a highly sensitive "one-step" nucleic acid detection method (WASTON Assay) that utilizes the cascaded positive feedback mechanism of the CRISPR / Cas13a system and the T7 transcription system to achieve exponential signal amplification under isothermal conditions.

[0211] 1. Preparation of the reaction system: A total volume of 20 μL was prepared. To balance specificity and amplification efficiency, the optimized final concentrations of each component are as follows:

[0212] Energy and substrate: 1.25 mM rNTPs

[0213] Enzymatic components: 200 nM LwaCas13a protein, 1.5 U / μL T7 RNA polymerase, 2 U / μL RNase inhibitor;

[0214] Functional nucleic acid components: 100 nM crRNA, 10 µM amplicon, 1000 nM universal fluorescent reporter probe.

[0215] Test sample: 0.2 μL of sample containing target RNA (target miRNA) or negative control.

[0216] 2. Detection steps (1) Preparation of premix: Under ice bath conditions, mix all biochemical reagents except the sample to be tested evenly to prepare the reaction premix (Master Mix). (2) Sample addition and start-up: Dispense 19.8 μL of the premix into the reaction tube or microfluidic chip reaction chamber, and add 0.2 μL of the sample to be tested. (3) Isothermal incubation: Place the reaction system in a 37℃ isothermal device for incubation in the dark. (4) Data acquisition: Use a fluorescence detector (FAM channel, Ex 490 nm / Em 520 nm) to acquire fluorescence signals in real time, with a sampling interval of 1 minute and a duration of 35 minutes.

[0217] 3. Reaction Principle and Mechanism Analysis. The detection mechanism of the method described in this invention is specifically divided into the following stages:

[0218] (1) Negative control (locked-in state): In the absence of target miRNA, the Cas13a protein is inactive. The amplifier (DNA / RNA chimera) in the system maintains a stable stem-loop structure through intramolecular base pairing. This structure effectively shields the T7 promoter sequence located in the stem using steric hindrance, preventing the binding of T7 RNA polymerase and thus inhibiting nonspecific transcription. At this time, the fluorescent reporter probe is not cleaved, and the background signal remains at the baseline level.

[0219] (2) Positive detection (unlocking and cascade amplification):

[0220] Primary activation: The target miRNA in the sample specifically binds to the Cas13a-crRNA complex, inducing a conformational change in the Cas13a protein and activating its trans-cleavage activity (Collateral Cleavage).

[0221] De-repression (promoter unlocking): Activated Cas13a specifically cleaves the RNA sequence in the loop of the amplifier. The break in the RNA strand causes thermodynamic instability and unfolding of the stem-loop structure of the amplifier, thereby exposing the functional double-stranded T7 promoter sequence.

[0222] Cyclic amplification (positive feedback): T7 RNA polymerase recognizes the exposed promoter and initiates transcription, producing a large number of RNA copies (activators) identical to the target sequence. These newly generated RNA copies act as secondary activators, further activating the remaining Cas13a protein, forming an exponential positive feedback loop where "product is activator".

[0223] Signal output: During this process, the universal fluorescent reporter probe in the Cas13a synchronous and efficient degradation system is activated in large quantities, causing the fluorescent group and quenching group to separate, releasing a high-intensity fluorescent signal, thus achieving highly sensitive detection of trace targets.

[0224] Example 3: Comparison and Optimization of Detection System Composition

[0225] To obtain optimal detection performance, this embodiment explores the key components of the WASTON system.

[0226] 1. Regarding the structural design of the signal amplification template (Amplifier), this invention does not simply combine T7 RNA polymerase amplification with CRISPR / Cas13a detection in parallel. Instead, it constructs an integrated blocking hybridization template comprising an RNA segment, a T7 promoter region, and a DNA template. This template is initially in a blocked conformation, inhibiting direct docking of T7 RNA polymerase. Only after the target RNA first activates the Cas13a / crRNA complex is the RNA segment in the Amplifier cleaved, exposing the corresponding promoter site and allowing T7 to initiate transcription, thereby generating target replicas and further activating subsequent Cas13a reactions, forming a positive feedback cascade amplification. This structure achieves a continuous coupling of "target recognition—template unlocking—transcriptional amplification—secondary Cas13 activation," which is the core of the Amplifier design in this invention.

[0227] To verify the rationality and superiority of the aforementioned Amplifier structural design, the applicant conducted targeted control experiments focusing on its key configurations, rather than simply performing a single feasibility verification. Figure 2 A, B). Specifically:

[0228] (1) The applicant further demonstrated the poor performance of other schemes through experiments showing the absence of key components. Under 1 pM RNAtarget conditions, when the system lacked ampliifier and / or T7 RNA polymerase, the fluorescence signal of the system decreased significantly due to the inability to achieve target RNA amplification; while when all key components were present, the signal reached its optimal level. This result indicates that the key components currently used in this invention, such as Cas13a, crRNA, ampliifier, and T7 RNA polymerase, have a synergistic effect, and the absence of key components will lead to a significant deterioration in the performance of the system. Figure 2 A, B).

[0229] (2) Experimental results show that when the matched target is replaced with a mismatched target, the Cas13a / crRNA complex cannot be effectively activated and cannot cleave the RNA segment in the amplifier, thus failing to effectively expose the target site of the subsequent T7 RNA polymerase. The system as a whole only produces a very low fluorescence signal. This result indicates that not all RNA sequences can achieve the amplification effect of this invention; the scheme under mismatch conditions is significantly less effective and can serve as a counter-control against the specific advantages of this invention. Figure 2 C).

[0230] (3) The applicant also verified the effect of amplifier configuration on system performance. The results showed that, under conditions without RNA addition, the annealed amplifier produced almost no detectable signal; however, if an unannealed amplifier was used, a significant non-specific background signal appeared. The paper analyzed that this was because, in the unannealed state, some amplifiers could be directly bound by T7 RNA polymerase and undergo non-specific transcription, leading to increased background. It can be seen that the unannealed amplifier is a structural state with poor performance in the research and development process, while the annealing blocking structure used in this invention can effectively suppress non-specific background and improve system specificity and signal-to-noise ratio. Figure 2 D).

[0231] Therefore, existing experimental data has proven that the current Amplifier structure of this application has better signal output and background control performance compared with key comparative schemes such as "non-cascaded amplification structure", "mismatched triggering structure" and "unannealed open structure".

[0232] Example 4: System optimization and performance verification of reaction conditions

[0233] To obtain the best detection performance, this embodiment optimized the key biochemical parameters in the WASTON system and verified the sensitivity and specificity of the method based on this.

[0234] 1. Optimization of amplicon structure and concentration

[0235] To address the issue of non-specific products generated during the T7 transcription reaction, the inventors focused on investigating the annealing status and working concentration of the amplifier.

[0236] Structural optimization: Experimental results show that the stem-loop structure formed by annealing the amplifier is key to reducing background signal. This structure effectively inhibits the non-specific binding of T7 RNA polymerase through steric hindrance.

[0237] Concentration optimization: Gradient testing of Amplifier concentrations (1 µM - 20 µM) was performed. Results showed ( Figure 3 The fluorescence signal was strongest when the Amplifier concentration was 10 µM. This concentration ensured sufficient transcription substrate while avoiding nonspecific spontaneous transcription caused by excessive concentration.

[0238] 2. Optimization of enzyme and probe concentrations: The concentrations of Cas13a protein, T7 RNA polymerase, and rNTPs were systematically optimized. Figure 3 The optimal reaction conditions were determined to be: 0.2 μM Cas13 protein, 1.5 U / μL T7 RNA polymerase, 2 U / μL LRNase inhibitor and 1.25 mM rNTPs.

[0239] In the above concentration exploration experiments, the applicant meticulously screened the amounts of key components such as Cas13a, T7 RNA polymerase, RNase inhibitor, rNTP, and amplifier, as well as parameters such as the ratio of Cas13a to crRNA and pre-incubation time. Ultimately, the optimal combination was determined to best distinguish positive / negative samples while balancing high signal and low background. Optimization results showed that the system performed best when Cas13a was 0.2 μM, T7 RNA polymerase was 1.5 U / μL, RNase inhibitor was 2 U / μL, rNTP was 1.25 mM, and amplifier was 10 μM; under these optimized conditions, the detection limit was approximately 100-fold higher than the initial system.

[0240] 3. Sensitivity (LOD) Test

[0241] Detection limit calculation: This invention compared the performance differences between the cascade amplification system and the conventional Cas13a-only detection system. The results showed that at 1 pM target RNA, the fluorescence signal of the Cas13a-only system was undetectable; furthermore, at concentrations of 1 nM and below, the signal generated by the Cas13a-only system was indistinguishable from the negative control, indicating that this type of scheme has insufficient sensitivity in low-abundance RNA detection scenarios. In contrast, this invention, by introducing an amplifier and T7 RNA polymerase, can form a positive feedback cascade amplification, obtaining clear and distinguishable detection signals even at lower concentrations; specifically, at 1 pM, the endpoint fluorescence signal of this invention's system was approximately 74.4 times that of the Cas13a-only system, indicating that this method can achieve single-molecule-level detection sensitivity without PCR pre-amplification. Figure 4 ).

[0242] 4. Specificity testing and "artificial mismatch" strategy

[0243] To distinguish miRNA family members with highly similar sequences (such as miR-17 and miR-106a, which differ by only one base), this embodiment introduces a "synthetic mismatch" strategy.

[0244] Design principle: An artificial mismatched base is introduced at a specific position in the spacer region of the crRNA targeting miR-17, named miR17 crRNA2. This design utilizes the characteristics of Cas13a recognition—the Cas13a / crRNA complex maintains high activity for a perfectly matched target (miR-17) (tolerating only one mismatch); however, for non-target sequences that already have natural mismatches (such as miR-106a), the introduced artificial mismatch creates a "double mismatch" effect, leading to a significant reduction in binding energy and thus a complete loss of activation ability.

[0245] The nucleotide sequence of miR17 crRNA2:

[0246] gauuuagacuaccccaaaaacgaaggggacuaaaacCUACCUGCACUGUAAGCAGUUUG (SEQ ID NO: 11. In the sequence listing, all U bases in this sequence are shown as T. The actual sequence is subject to the specification.)

[0247] Verification results: such as Figure 5As shown, after introducing artificial mismatches, the WASTON method exhibits extremely high specificity for the target miR-17; while for miR-106a (1 base difference), miR-20a and miR-20b (2 base differences), the fluorescence signals produced are all at background levels.

[0248] Anti-interference capability: such as Figure 5 As shown, even when the target miR-17 accounts for only 0.1% of the total RNA in complex samples with high background total RNA, this method can still accurately detect it, confirming its anti-interference ability in complex clinical samples.

[0249] Example 5: Construction of a manually driven centrifugal microfluidic chip

[0250] 1. For example Figure 6 As shown, the structure design of the hand-driven centrifugal microfluidic chip is designed to achieve point-of-care testing (POCT) without external power drive. This invention designs a "W-chip" based on centrifugal microfluidic technology.

[0251] Layered structure: The main body of the chip is made of polymethyl methacrylate (PMMA).

[0252] Functional Units: The chip is disk-shaped and integrates a sample chamber, a waste chamber, and multiple independent reaction units arranged radially.

[0253] Key fluid components: Each reaction unit includes a siphon valve. This valve design utilizes the counteracting mechanism of "centrifugal force blocking" and "capillary force conducting" to precisely control the start and stop of fluid flow, which is crucial for achieving manual actuation.

[0254] Spatial Coding: Different reaction units of the chip are pre-embedded with lyophilized reagent microspheres targeting different targets (such as miR-17, miR-155). The physical location of the reaction unit represents the type of target to be detected, thus enabling multiple detection in a single experiment.

[0255] Example 6: Clinical serum sample detection and performance evaluation based on a portable microfluidic system

[0256] This embodiment aims to verify the practical application performance of the WASTON nucleic acid detection system described in this invention in the clinical auxiliary diagnosis and early screening of lung cancer.

[0257] 1. Clinical Sample Collection and Experimental Grouping: With the approval of the ethics committee and the informed consent of the subjects, a clinical validation cohort was established.

[0258] Sample bank: Serum samples from 30 patients with pathologically confirmed non-small cell lung cancer (NSCLC) and 10 serum samples from healthy volunteers were collected as negative controls.

[0259] Sample processing: Total RNA was extracted from serum and subjected to parallel blind testing using the following two methods:

[0260] Example Method (WASTON Group): RNA samples were added to a hand-driven centrifugal microfluidic chip pre-embedded with lyophilization reagents. After manual injection, multiplex detection of miR-17, miR-155 and miR-19b was performed.

[0261] Comparative method (RT-qPCR group): The traditional real-time quantitative PCR method was used to detect the virus on a benchtop instrument (such as Bio-Rad CFX96) as the gold standard reference.

[0262] 2. Analysis of Test Results

[0263] (1) Expression differences and early detection capability: Detection results showed that ( Figure 7 The fluorescence signal intensity of the three target miRNAs in the serum of lung cancer patients was significantly higher than that in the healthy control group. Specificity advantage: The WASTON system was able to detect significantly elevated positive signals in all lung cancer samples. This confirms that the method has extremely high detection sensitivity, overcoming the bottleneck of low-abundance biomarkers being difficult to detect in the early stages of disease.

[0264] (2) Diagnostic efficacy assessment (ROC analysis): Figure 8 Assess diagnostic accuracy using receiver operating characteristic (ROC) curves:

[0265] Single-index detection: The area under the curve (AUC) of miR-17, miR-155, and miR-19b are all greater than 0.85;

[0266] Combined diagnosis: The three biomarkers were analyzed together using a logistic regression model, and the AUC value reached 0.955.

[0267] 3. Conclusion: Clinical validation data demonstrate that the WASTON-based nucleic acid detection system provided by this invention achieves multiplex detection of lung cancer serum biomarkers without the need for PCR pre-amplification and large-scale instruments. The detection method exhibits high sensitivity (aM level) and high specificity. Combined with its portable design featuring hand-operated and lyophilized reagents, it possesses practical value for large-scale lung cancer screening and point-of-care testing (POCT) in resource-scarce areas.

[0268] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An amplicon characterized in that, The amplicon includes a T7 promoter sequence, a transcription template sequence complementary to the target nucleic acid, and an RNA cleavage site.

2. The amplicon of claim 1, wherein, The amplicon has a stem-loop structure, with the stem containing the T7 promoter sequence and the loop containing the transcription template sequence and the RNA cleavage site; The RNA cleavage site is an RNA uracil residue.

3. A cascade signal amplification system based on transcription-driven CRISPR / Cas13a, characterized in that, include: The amplicon, Cas13a:crRNA complex, T7 RNA polymerase, and fluorescent reporter probe as described in claim 1 or 2; The Cas13a:crRNA complex comprises: Cas13 protein and crRNA targeting the nucleic acid; The fluorescent reporter probe can be cleaved by activated Cas13 protein.

4. The cascade signal amplification system of claim 3, wherein, The crRNA includes repeat sequences and spacer sequences; The repeat sequence binds specifically to the Cas13 protein; The spacer sequence specifically recognizes the target nucleic acid.

5. The cascade signal amplification system of claim 3 or 4, wherein, The Cas13 protein includes at least one of LbaCas13a, LbuCas13a, LwaCas13a, and PsmCas13b.

6. The cascade signal amplification system of claim 3, wherein, The target nucleic acid includes at least one of microRNA, mRNA, lncRNA, and circRNA.

7. The use of the amplifier according to claim 1 or 2 and / or the cascaded signal amplification system according to any one of claims 3 to 6 in the following ① and / or ②: ① Nucleic acid testing for non-diagnostic purposes; ② Prepare nucleic acid detection kits.

8. Use according to claim 7, characterized in that, The methods for nucleic acid testing for non-diagnostic purposes include: The sample to be tested is mixed with the cascaded signal amplification system, incubated, and fluorescent signals are collected to detect the target nucleic acid in the sample.

9. A nucleic acid detection kit, characterized in that, It includes the amplifier as described in claim 1 or 2 and / or the cascaded signal amplification system as described in any one of claims 3 to 6.

10. The reagent kit according to claim 9, characterized in that, It also includes rNTPs and RNase inhibitors; The kit includes 0.1–0.3 μM Cas13 protein, 1–2 U / μL T7 RNA polymerase, 2–6 U / μL RNase inhibitor, 1.25–3.75 mM rNTPs, 50–150 nM crRNA, 1–20 µM amplicon, and 800–1200 nM fluorescent reporter probe.

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