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CN-122012686-A - Reagent and method for detecting instant nucleic acid by semi-quantitative one-pot method

CN122012686ACN 122012686 ACN122012686 ACN 122012686ACN-122012686-A

Abstract

The invention relates to the technical field of biology, in particular to a reagent and a detection method for detecting instant nucleic acid by a semi-quantitative one-pot method. The invention aims at one-pot method Recombinase Polymerase Amplification (RPA) and CRISPR detection (CRISPR-Dx) reaction, and a series of one-pot method RPA-CRISPR-Dx systems with different detection limits are constructed by redesigning RPA primers and screening CRISPR target PAM and combining the two. The detection array constructed based on the detection systems can output semi-quantitative results which are directly readable without calculation and processing, and a powerful tool is supplemented for improving the instant nucleic acid detection quantitative capability.

Inventors

  • YU HANQING
  • HAN JING
  • LIU DONGFENG
  • Cheng Zhouhua

Assignees

  • 中国科学技术大学

Dates

Publication Date
20260512
Application Date
20260303

Claims (10)

  1. 1. Reagents for semi-quantitative one-pot immediate nucleic acid detection, including RPA reagents, reverse transcriptase, amplification primers, lbCas a, crRNA and ssDNA reporter molecules; Wherein LbCas a has an amino acid sequence shown in SEQ ID NO. 17; The target PAM in the crRNA is TTTG, GTTC, TATC, CCCC, GTAG or ATTG.
  2. 2. The reagent of claim 1, wherein the crRNA has a sequence as set forth in any one of SEQ ID NOs 11 to 16.
  3. 3. The reagent of claim 1, wherein in the ssDNA reporter molecule, the single-stranded oligonucleotide is a random sequence with a length of 5-10 bp, The 5' -end modified fluorescent group is selected from FAM, HEX, TET, VIC, JOE, cy, cy5, ROX or TAMRA; the 3' -end modified quenching group is selected from BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, MGB or Eclipse.
  4. 4. The reagent of claim 1, wherein the RPA reagent comprises a recombinase, a ssDNA binding protein, a polymerase, dntps, and a buffer.
  5. 5. The reagent according to claim 1 or 4, wherein each reaction system comprises: 18. mu.L of RPA reagent, 1. Mu.L of reverse transcriptase, 1. Mu.M of RPA amplification primer, 50 nM LbCAs12a, 50 nM crRNA and 400 nM ssDNA reporter.
  6. 6. The reagent according to any one of claims 1 to 5, wherein the amplification primer comprises an upstream primer and a downstream primer, and the length of the upstream primer and the downstream primer is 20 to 30bp independently; CG% in the amplification primer sequence is 55% -80%.
  7. 7. The reagent according to claim 6, wherein the amplification primer is a primer targeting the PML:RARA fusion transcript bcr1 subtype or a primer targeting the PML:RARA fusion transcript bcr3 subtype; in the primer of the RARA fusion transcript bcr1 subtype, The upstream primer has any one of nucleic acid sequences shown in SEQ ID NO. 1-3; the downstream primer has any one of nucleic acid sequences shown in SEQ ID NO. 7-10; in the primer of the RARA fusion transcript bcr3 subtype, The upstream primer has any one of nucleic acid sequences shown in SEQ ID NO. 4-6; the downstream primer has any one of the nucleic acid sequences shown in SEQ ID NO. 7-10.
  8. 8. A method for one-pot real-time nucleic acid detection, comprising adding a sample to be detected to the reagent according to any one of claims 1 to 7, and detecting fluorescence intensity after incubation.
  9. 9. The method according to claim 8, wherein the incubation is at a temperature of 35-42 ℃ for a time of 30-60 min.
  10. 10. The method of claim 8 or 9, wherein the sample to be tested is an RNA sample.

Description

Reagent and method for detecting instant nucleic acid by semi-quantitative one-pot method Technical Field The invention relates to the technical field of biology, in particular to a reagent and a detection method for detecting instant nucleic acid by a semi-quantitative one-pot method. Background Nucleic acid detection is an important basic technology in the field of molecular biology, and is widely applied to a plurality of fields such as disease diagnosis, infectious disease monitoring, environment monitoring, food safety and the like. In order to effectively support clinical diagnosis and treatment decisions and public health management, the nucleic acid detection method not only needs to have higher sensitivity and accuracy, but also meets the requirements of high detection speed, simple and convenient operation flow, low equipment dependence and the like. However, the currently widely used real-time fluorescent quantitative polymerase chain reaction (qPCR) technology, although excellent in analysis performance, relies on a precise thermal cycle control system and a real-time optical signal acquisition device, and has the disadvantages of large equipment volume, high cost, relatively complex detection process, and difficulty in wide application in point-of-care (POC) and resource-limited environments. Therefore, improving the accessibility and applicability of detection methods while ensuring detection performance has become an important direction for the development of nucleic acid detection techniques. To reduce the reliance on complex instrumentation, a variety of isothermal reaction condition-based nucleic acid detection techniques have evolved successively, with CRISPR-Cas system-based nucleic acid detection methods receiving widespread attention for their highly programmable sequence recognition capability and excellent specificity. Under the guidance of the guide RNA, the Cas nuclease can specifically recognize and cut a target nucleic acid sequence, and part of Cas nuclease also shows non-specific side cutting activity after target recognition and can be used for amplifying detection signals. By combining CRISPR nucleic acid detection with isothermal nucleic acid amplification techniques, detection sensitivity can be further improved, making its analytical performance approach qPCR levels. To simplify the operation flow and reduce the risk of aerosol contamination, related art has gradually developed a one-pot detection mode that integrates a nucleic acid amplification reaction and CRISPR-mediated detection in the same reaction system. However, in one-pot detection systems, the nucleic acid amplification reaction and the Cas-mediated cleavage reaction are performed simultaneously in the same reaction environment, with a complex kinetic coupling relationship between the two. When the Cas-mediated cleavage reaction rate is high, the target nucleic acid may be consumed before it is sufficiently amplified, thereby affecting the detection sensitivity, whereas if the cleavage reaction rate is low, the signal generation may be delayed or background interference may be increased. Thus, the dynamic balance between the amplification reaction and the CRISPR cleavage reaction becomes a key factor affecting the performance of one-pot assays. Aiming at the problems, the prior art generally improves the detection performance by carrying out parameter optimization on a single detection reaction system, but the method always depends on maintaining reaction balance in a narrower parameter window, is sensitive to fluctuation of reaction conditions, and is difficult to achieve the combination of sensitivity, stability and repeatability. In addition, since the target amplification and cleavage reaction are performed simultaneously in one-pot detection, the generation of the detection signal is commonly affected by multiple factors, and it is difficult to establish a stable and repeatable quantitative relationship between the signal output and the initial concentration of the target nucleic acid. Therefore, the existing one-pot CRISPR nucleic acid detection method is mostly used for qualitative analysis. Although studies have been attempted to achieve quantitative detection by means of signal fitting, reaction time analysis, or a digital micro-reaction unit, related schemes generally rely on complex data processing algorithms or dedicated detection devices, significantly increasing system cost and operational complexity, and limiting their popularization and application in instant detection and low-resource application scenarios. Therefore, there is a need to develop a quantitative one-pot CRISPR-Dx method with low instrument requirements, wide dynamic range and convenient field use. Disclosure of Invention In view of the above, the technical problem to be solved by the present invention is to provide a reagent and a detection method for detecting a semi-quantitative one-pot method instant nucleic acid. The invention