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CN-121975932-A - System and method for ultra-sensitive detection of acute myocardial infarction related microRNAs

CN121975932ACN 121975932 ACN121975932 ACN 121975932ACN-121975932-A

Abstract

The invention relates to a system and a method for ultra-sensitive detection of acute myocardial infarction related microRNAs. According to the method, the designed trans-cleavage activities of a circular RNA activator (CA-RNA) and Cas13a protein are utilized, the target microRNA is used for activating the Cas13a to cleave a continuous uracil (U) structure in the CA-RNA, so that the circular conformation of the CA-RNA is destroyed, a linear activator is released, and then multiple rounds of cascade signal amplification are triggered. And (3) dividing a reaction system into a large number of independent microchambers by combining a centrifugal digital microfluidic chip, and counting positive microchambers according to poisson distribution to realize accurate quantification of target microRNA. The system can complete detection within 15 minutes at 37 ℃ without reverse transcription and complex instruments, has the advantages of high sensitivity, strong specificity, good universality and the like, and is suitable for early clinical diagnosis of acute myocardial infarction, and the detection limit reaches the attomole level.

Inventors

  • QIN YULING
  • Pan Anli
  • WU LI

Assignees

  • 南通大学

Dates

Publication Date
20260505
Application Date
20260114

Claims (10)

  1. 1. A system for ultrasensitive detection of acute myocardial infarction-related micrornas, the system comprising: (a) A first crRNA that targets a target microRNA associated with acute myocardial infarction; (b) A second crRNA that targets CA-RNA; (c) Cas13a protein; (d) A CA-RNA, which is a circular RNA molecule comprising a continuous uracil (U) domain, the continuous U domain being the cleavage site of the CAs13a protein; (e) A reporter molecule capable of being cleaved by the trans-cleavage activity of the Cas13a protein, and (F) The centrifugal digital microfluidic chip is used for dividing a reaction system containing the components (a) - (e) into a plurality of independent microchambers for reaction and detection.
  2. 2. The system of claim 1, wherein the CA-RNA is prepared from single stranded RNA synthesis by a commercial circular RNA synthesis kit.
  3. 3. The system of claim 1, wherein the contiguous U domain in the CA-RNA comprises 3, 7, or 10 contiguous uracil (U) residues.
  4. 4. The system of claim 1, wherein the system is universal in that detection of other micrornas or nucleic acid targets is achieved by replacement of a spacer sequence in the first crRNA that is complementary to the target.
  5. 5. A method for detecting acute myocardial infarction related micrornas using the system of any one of claims 1-4, comprising the steps of: (i) Binding a target microRNA to the first crRNA, thereby activating trans-cleavage activity of Cas13a protein; (ii) The activated Cas13a protein cleaves the contiguous U domain in the CA-RNA, turning it into a linear activator; (iii) The linear activator further activates Cas13a protein, cleaves the reporter molecule to generate a detection signal, and simultaneously circularly cleaves more CA-RNAs, thereby triggering cascade signal amplification; (iv) Loading a liquid sample comprising the reaction components of steps (i) - (iii) into the centrifugal digital microfluidic chip, dividing the sample into a plurality of independent microchambers of the chip by centrifugal force; (v) After incubation, signals of each micro chamber are detected, and the number of positive micro chambers is counted according to the poisson distribution principle, so that quantification of target microRNA is achieved.
  6. 6. The method of claim 5, wherein the cascade of steps (i) - (iii) is performed in an optimized reaction system comprising 1000 nM reporter, 50 nM cas13a protein, 25 nM first crRNA, 5nM second crRNA, and 25 nM CA-RNA, and incubated at 37 ℃ for 15 minutes.
  7. 7. The method according to claim 5, wherein the chip-based operation in step (iv) is specifically performed by introducing the sample solution into the chip sample inlet, centrifuging at 1600 rpm for 1 minute, and then incubating the chip at 37 ℃ for 15 minutes.
  8. 8. The method of claim 5, wherein the statistical analysis method of step (v) comprises obtaining a global fluorescence image of the chip, discriminating positive droplets from background by software adjustment threshold, filtering droplets according to size and quantifying the number of effective droplets while rejecting false positive signals caused by non-droplet entities or weak fluorescent spots.
  9. 9. The method of claim 5, further comprising a labeled recovery test step to verify the actual detection performance of the method by adding a solution of a known concentration of the simulated microRNA to serum of a healthy person.
  10. 10. The method of claim 5, wherein the acute myocardial infarction-related microRNA is miR-208 or miR-499.

Description

System and method for ultra-sensitive detection of acute myocardial infarction related microRNAs Technical Field The invention relates to the technical field of medical examination and molecular diagnosis, in particular to a system and a method for ultra-sensitive detection of acute myocardial infarction related microRNAs. Background Acute Myocardial Infarction (AMI) is a severe manifestation of coronary artery disease, which can be initiated within the earliest 20 minutes after myocardial ischemia occurs and progresses rapidly within hours, resulting in massive myocardial cell necrosis. Epidemiological studies have clearly shown that AMI is a major cause of mortality in cardiovascular disease worldwide. Rapid and accurate diagnosis of AMI is critical for timely therapeutic measures to reduce mortality. Current clinical diagnosis AMI relies on comprehensive analysis of medical history assessment, physical examination, and blood cardiac biomarker detection. But electrocardiogram changes only occur in about 57% of AMI cases, limiting its independent diagnostic value. Although cardiac troponin is a gold standard biomarker for AMI diagnosis, its serum levels are usually elevated 3-4 hours after symptoms appear, and may also be elevated in other cardiac diseases, which limits its early diagnostic efficacy and specificity for AMI. Recently, micrornas (mirnas) have been attracting attention due to their stability, tissue specificity, and rapid entry into blood circulation after injury has occurred, and are considered as promising biomarkers. Studies have shown that specific mirnas associated with cardiac function may be significantly upregulated during acute myocardial infarction. However, detection of mirnas presents challenges due to their short sequence, low content in biological samples, and high sequence homology between different miRNA species. Traditional detection methods, such as Northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and microarray analysis, generally require complex instrumentation, involve multiple steps, and are time consuming, which limit their practical use in bedside or resource-limited scenarios. In contrast, CRISPR-Cas based systems have evolved into a powerful molecular diagnostic tool. In particular, RNA-targeting ribonuclease Cas13a exhibits strong non-specific cleavage activity on adjacent RNA sequences containing uracil (U) bases, making it suitable for sensitive and specific detection of RNA targets. However, the inherent sensitivity of Cas13a alone is often insufficient to perform trace level analysis. CRISPR-Cas self-sustained amplification strategies, while improving sensitivity, are still limited by increased background signal and non-specific cleavage (transcleavage cleavage) of the U-rich sequence by Cas13a, resulting in poor detection performance. In recent years, digital microfluidic chip technology provides a new idea for nucleic acid detection. The method divides the detection system into a plurality of independent micro-reaction units, realizes effective space isolation of single target molecules, fundamentally relieves the problem of nonspecific degradation caused by transcription cleavage activity of Cas13a in the traditional mixed reaction system, and remarkably improves the specificity and quantitative accuracy of detection. Therefore, the CRISPR-Cas amplification system with high specificity is combined with the digital microfluidic platform with high fidelity, and the bottleneck of the prior art in sensitivity, specificity and operability is hopeful to be broken through. Disclosure of Invention Technical problem The technical problem to be solved by the invention is to overcome the defects of insufficient sensitivity, complex operation, easy non-specific interference, difficult realization of rapid quantification and the like of the traditional acute myocardial infarction related microRNA detection method, and provide an in-vitro detection system and method which have the advantages of super sensitivity, high specificity, simple and rapid operation. Technical proposal The invention provides a system for ultra-sensitive detection of acute myocardial infarction related microRNA, which comprises: (a) A first crRNA that targets a target microRNA associated with acute myocardial infarction; (b) A second crRNA that targets CA-RNA; (c) Cas13a protein; (d) A CA-RNA, which is a circular RNA molecule comprising a continuous uracil (U) domain, the continuous U domain being the cleavage site of the CAs13a protein; (e) A reporter molecule capable of being cleaved by the trans-cleavage activity of the Cas13a protein, and (F) The centrifugal digital microfluidic chip is used for dividing a reaction system containing the components (a) - (e) into a plurality of independent microchambers for reaction and detection. Further, the CA-RNA is prepared from single-stranded RNA synthesis by a commercial circular RNA synthesis kit. Further, the con