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CN-121992078-A - Nucleic acid warm-expansion detection system, detection method and application thereof

CN121992078ACN 121992078 ACN121992078 ACN 121992078ACN-121992078-A

Abstract

The invention discloses a nucleic acid warm-expansion detection system, a detection method and application thereof, which combines an E.coli RNase HII with an RPA/RT-RPA technology and develops a multi-form, rapid and sensitive RH-RPA detection method. The RH-RPA method uses a long probe containing a single ribonucleotide as a substrate for RNase HII specific cleavage, and realizes fluorescence visualization. In addition, the long probe can be used as a forward primer after being cut and can be extended with a reverse primer with a biomarker to generate a product with double markers, and the product can be rapidly detected by a lateral flow chromatography method. Compared with the conventional RPA-exo and RPA-nfo methods, the method can realize the detection of a real-time fluorescence method and a lateral flow chromatography method at the same time under the low temperature condition, is quick, stable and accurate, improves the defects of the RPA probe method, and greatly improves the practical application performance of the RPA detection.

Inventors

  • ZHANG YONGYOU
  • JIN YUTING

Assignees

  • 厦门大学

Dates

Publication Date
20260508
Application Date
20241107

Claims (10)

  1. 1. A system for detecting the thermal expansion of nucleic acid is composed of RPA mix as the mixture needed by ordinary RPA reaction, RNase HII and probe, and features that the probe has its full sequence complementary to target DNA molecule to be detected and one or more RNA bases are embedded in it to divide the complementary region of probe into at least two segments, one segment of RNA base can be labeled with different recognition element, which is fluorescent group or antigen marker, another segment is correspondent to the quenching group of label or 3' end blocking group, and when said probe is cut from said RNA base by RNase HII, another segment is detached from template, and said fluorescent group generates detectable signal, or one segment with said antigen marker is used as new primer to generate double-chain DNA product with reverse primer with another antigen marker, and said two antigen markers are linked together.
  2. 2. The system for detecting nucleic acid amplification according to claim 1, wherein the length of the sequence fragment on one side of the RNA base is 30nt to 40nt, and the length of the sequence fragment on the other side is 3nt to 10nt.
  3. 3. A system for the detection of nucleic acid amplification according to claim 1, wherein the mixture RPA mix comprises: Tris-HCl 50-100mM;UvsX 400-800ng/μL;UvsY 30-100ng/μL; SSB 300-800ng/μL;BsuL 10-100ng/μL;CKM 50-150ng/μL; dNTPs 0.3-0.5mM;Primer-F/Primer-R 200-400nM; 50-150mM potassium acetate, 10-20mM magnesium acetate and 2-10mM ATP Creatine kinase 20-200 ng/. Mu.L, creatine phosphate sodium 10-50mM DTT 1-10mM, polyethylene glycol with molecular weight 8000-35000, 2.5% -10%.
  4. 4. A system for the detection of nucleic acid amplification according to claim 3, in which the final concentration of probe in the system is 50-200nM.
  5. 5. A system for detecting thermal amplification of nucleic acid according to claim 3, wherein the final concentration of RNase HII in the system is 0.5-40 ng/. Mu.L.
  6. 6. A method for detecting nucleic acid by warm expansion, comprising the following steps: providing a mixture RPA mix required by a conventional RPA reaction, and comprising RNase HII and a probe, wherein the whole sequence of the probe can be complementary to a target DNA molecule to be detected, one or more RNA bases are embedded in the probe region, the RNA bases divide the probe complementary region into at least two sections, one side section of each RNA base can be marked with different recognition elements, each recognition element is a fluorescent group or an antigenic marker, the other side section corresponds to a marker quenching group or a 3' -end blocking group, when the probe is cut from the RNA base by the RNase HII, the other side section is separated from a template, the fluorescent group generates a detectable signal, or one side section with the antigenic marker is used as a new primer and forms a double-chain DNA product with a reverse primer with the other antigenic marker, and the two antigenic markers are connected together; and secondly, performing amplification reaction at the temperature of 37-42 ℃, and judging whether a target has a target point or a target sequence by detecting a signal generated by the marker.
  7. 7. The method of claim 6, wherein the RNA has a sequence segment length of 30nt to 40nt on one side and a sequence segment length of 3nt to 10nt on the other side.
  8. 8. The method for detecting nucleic acid amplification by temperature change according to claim 6, wherein the mixture RPA mix comprises: Tris-HCl 50-100mM;UvsX 400-800ng/μL;UvsY 30-100ng/μL; SSB 300-800ng/μL;BsuL 10-100ng/μL;CKM 50-150ng/μL; dNTPs 0.3-0.5mM;Primer-F/Primer-R 200-400nM; 50-150mM potassium acetate, 10-20mM magnesium acetate and 2-10mM ATP Creatine kinase 20-200 ng/. Mu.L, creatine phosphate sodium 10-50mM DTT 1-10mM, polyethylene glycol with molecular weight 8000-35000, 2.5% -10%.
  9. 9. The method of claim 6, wherein the label comprises a fluorescent group and a quencher group, and the fluorescent group and the quencher group are located in sequence segments on both sides of the RNA base.
  10. 10. Use of a nucleic acid warm-expansion detection method according to any one of claims 6 to 9 for detecting target DNA.

Description

Nucleic acid warm-expansion detection system, detection method and application thereof Technical Field The invention relates to the technical field of biology, in particular to a nucleic acid warm-expansion detection system, a detection method and application thereof. Background In recent years, the isothermal nucleic acid detection technology has attracted attention because of the advantages of rapid and convenient reaction, simplicity and sensitivity, good specificity, strong multiplexing compatibility and the like, and has been widely applied to the fields of infectious disease detection, cancer diagnosis, food safety, environmental monitoring and the like. The Recombinant Polymerase Amplification (RPA) is a DNA isothermal amplification technology which can efficiently amplify at a low temperature of 37-42 ℃ by utilizing enzymes, crowding agents and activators, and is simple and rapid and does not depend on complex instruments and equipment. Compared to other isothermal amplifications, RPA can rapidly cleave dsDNA without the need for an initial denaturation step, and only one primer pair is needed per target, with the potential for multiplexing. Thus, RPA has been widely used for a variety of detection targets in the last twenty years, and new nucleic acid detection techniques based on RPA have also been proposed. The RPA real-time fluorescence detection method based on the exo probe comprises the steps of 1) identifying a target by using the exo probe, and generating a fluorescence signal when the probe is specifically combined with the target, wherein the fluorescence signal can be monitored in real time. 2) The RPA end-point detection method based on the nfo probe, after the nfo probe with antigen label is combined with a target, the nfo probe can be identified and cut by nfo enzyme, the cut probe is used as a new primer, and the new primer is extended together with an opposite RPA amplification primer with another antigen label at the 5' end, so that an amplicon with two antigen labels is obtained, and the amplicon can be visually detected through a lateral flow chromatography. 3) RPA real-time fluorescence detection method and end-point detection method based on CRISPR system, cas12 and Cas13 and their gRNA can specifically target cut amplicons, activate their trans-cutting activity, and efficiently cut ssDNA probes in the system, thereby generating fluorescence, and can be monitored in real time, and also can be used for visual end-point fluorescence detection under 365nm UV lamp. For RPA based on Exo probe, the method can not detect the end point fluorescence due to the digestion characteristic of Exo III, so that the real-time fluorescence detector still needs to be relied on in practical application, and the application scene is limited. For RPA based on nfo probe, this method is less efficient than detection of RPA based on exo probe, and is prone to falsely forming duplex between primer and probe under low temperature conditions, resulting in an increased risk of false positive of the test strip. For CRISPR-based RPA, poor compatibility of the two systems results in poor sensitivity, and multiple target detection can be realized through a complex strategy. Disclosure of Invention The invention mainly aims at providing a nucleic acid warm-expansion detection method. The technical scheme adopted for solving the technical problems is as follows: A system for detecting nucleic acid by thermal expansion, which comprises a mixture RPA mix required by a conventional RPA reaction, and comprises RNase HII and a probe, wherein the whole sequence of the probe can be complementary to a target DNA molecule to be detected, one or more RNA bases are embedded in the probe region, the RNA bases divide the probe complementary region into at least two sections, one section of each RNA base can be marked with different recognition elements, the recognition elements are fluorescent groups or antigenic markers, the other section corresponds to a marker quenching group or a 3' -end blocking group, when the probe is cut from the RNA bases by the RNase HII, the right section is separated from a template, the fluorescent groups generate a detectable signal, or one section with the antigenic markers serves as a new primer and forms a double-chain DNA product with a reverse primer with the other antigenic marker, and the two antigenic markers are connected together. In some embodiments, the length of the sequence fragment on one side of the RNA base is 30 nt-40 nt, and the length of the sequence fragment on the other side is 3 nt-10 nt. Further, the length of the sequence segment at the other side is 3 nt-10 nt. In some embodiments, the mixture RPA mix comprises: Tris-HCl 50-100mM, uvsX protein 400-800 ng/. Mu.L, uvsY protein 30-100 ng/. Mu.L; SSB (Single-stranded binding protein) 300-800 ng/. Mu.L, bsuL-100 ng/. Mu.L, CKM (creatine kinase) 50-150 ng/. Mu.L, dNTPs 0.3-0.5 mM, primer-F/Primer-R200-400 nM; 50-150mM potass