CN111676317A - Method for rapidly detecting SARS-CoV-2 based on DNA nano-support - Google Patents
Method for rapidly detecting SARS-CoV-2 based on DNA nano-support Download PDFInfo
- Publication number
- CN111676317A CN111676317A CN202010507344.7A CN202010507344A CN111676317A CN 111676317 A CN111676317 A CN 111676317A CN 202010507344 A CN202010507344 A CN 202010507344A CN 111676317 A CN111676317 A CN 111676317A
- Authority
- CN
- China
- Prior art keywords
- dna
- cov
- sars
- nanoscaffold
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 38
- 241001678559 COVID-19 virus Species 0.000 title claims abstract description 32
- 239000000523 sample Substances 0.000 claims abstract description 52
- 239000002062 molecular scaffold Substances 0.000 claims abstract description 47
- 238000001514 detection method Methods 0.000 claims abstract description 33
- 238000009396 hybridization Methods 0.000 claims abstract description 12
- 238000010791 quenching Methods 0.000 claims abstract description 11
- 230000003321 amplification Effects 0.000 claims abstract description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 8
- 238000001338 self-assembly Methods 0.000 claims abstract description 7
- 108020004414 DNA Proteins 0.000 claims description 66
- 239000002070 nanowire Substances 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 claims description 4
- 238000013461 design Methods 0.000 claims description 4
- 230000000977 initiatory effect Effects 0.000 claims description 4
- 230000000171 quenching effect Effects 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 3
- 108091081062 Repeated sequence (DNA) Proteins 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 abstract description 4
- 241000711573 Coronaviridae Species 0.000 abstract description 3
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 12
- 208000025721 COVID-19 Diseases 0.000 description 8
- 239000000872 buffer Substances 0.000 description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000013592 cell lysate Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 210000003296 saliva Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 108010052305 exodeoxyribonuclease III Proteins 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 108091062762 miR-21 stem-loop Proteins 0.000 description 3
- 108091041631 miR-21-1 stem-loop Proteins 0.000 description 3
- 108091044442 miR-21-2 stem-loop Proteins 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 238000010587 phase diagram Methods 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 230000001960 triggered effect Effects 0.000 description 3
- 108020004638 Circular DNA Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 108010007577 Exodeoxyribonuclease I Proteins 0.000 description 2
- 102100029075 Exonuclease 1 Human genes 0.000 description 2
- 238000010222 PCR analysis Methods 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 208000037847 SARS-CoV-2-infection Diseases 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000001506 fluorescence spectroscopy Methods 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 238000011896 sensitive detection Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OSBLTNPMIGYQGY-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;boric acid Chemical compound OB(O)O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O OSBLTNPMIGYQGY-UHFFFAOYSA-N 0.000 description 1
- 208000031504 Asymptomatic Infections Diseases 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- 239000008051 TBE buffer Substances 0.000 description 1
- 241000907316 Zika virus Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012456 homogeneous solution Substances 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 238000012933 kinetic analysis Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 230000004630 mental health Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 229910001868 water Inorganic materials 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for rapidly detecting SARS-CoV-2 based on DNA nano-bracket, comprising the following steps: (1) a DNA nano-scaffold is constructed through DNA chain self-assembly and a self-quenching probe H1, and target RNA triggers the quick hybridization of a free hairpin probe H2 and a hairpin probe H1 along the nano-scaffold, so that the amplification of signals is realized. The invention relates to a 2019-year global coronavirus disease (COVID-19) which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The invention has the advantages of high detection sensitivity, programmable sequence, high signal gain, high specificity, short reaction time (<10 minutes), low cost and simple operation.
Description
Technical Field
The invention relates to the technical field of genes, in particular to a method for rapidly detecting SARS-CoV-2 based on a DNA nano-bracket.
Background
In recent years, the increasing prevalence and outbreak of viral influenza has caused widespread concern worldwide, and large-scale epidemics caused by such new viruses (aids virus, SARS virus and middle east respiratory syndrome coronavirus, 2009 pandemic H1N1 virus, ebola virus, zika virus and, more recently, SARS-COV-2) have caused serious harm to the physical and mental health of people in the world. Almost all of these epidemic diseases are caused by the transmission of the original zoonotic animal to humans, either clinically apparent or insidiously to the susceptible population. Due to the lack of rapid, convenient and accurate molecular diagnostic techniques, public difficulties in management and control of epidemic spread are important.
Currently, the new coronavirus (SARS-CoV-2) pneumonia (COVID-19) epidemic is spreading widely in various countries all over the world. Due to the lack of efficient, rapid and accurate molecular detection technology, the diagnosis and treatment of patients with the novel coronavirus (SARS-CoV-2) pneumonia (COVID-19) still has a lot of difficulties, which seriously affects the speed of controlling and treating epidemic situations in all world boundaries.
The real-time fluorescent quantitative PCR (qRT-PCR) technology has been widely applied to the detection of SARS-COV-2 virus, and plays an important role in preventing and controlling the outbreak of COVID-19. In addition, qRT-PCR is also approved by the United states centers for disease control and prevention (CDC) for Emergency Use (EUA). However, the quantitative RT-PCR technique depends on expensive reagents and precise instruments, and is complicated in process and long in time, so that it cannot meet the current demand for detection of rapidly growing suspected cases and asymptomatic infected patients.
With the rapid development of the detection method of the IgM/IgG antibody, the IgM/IgG antibody is expected to become a novel method for rapidly detecting SARS-CoV-2. However, in the early stage of SARS-COV-2 infection, the detection of low abundance antibodies in a sample is likely to produce a false positive result. With the development of COVID-19 epidemic situation, asymptomatic infection and wide spread of epidemic situation, the number of people needing screening and detection is greatly increased. In order to cope with the outbreak of the global COVID-19 and accelerate the clinical diagnosis, the China department of science and technology (MOST) collected a COVID-19 rapid test scheme to the society in 2 months and 2 days in 2020, and the national medical products were subjected to examination and management (NMPA). There is an urgent need in the whole society for new diagnostic methods for rapid and efficient detection of SARS-CoV-2 infection to cope with the growing epidemic problem.
At present, a method for rapidly detecting SARS-CoV-2 based on DNA nano-scaffolds, which is simple in operation and ultra-fast, is lacked.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide the method for detecting SARS-CoV-2 based on the DNA nano-stent, which is simple and easy to operate and ultra-fast.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows: the invention relates to a method for rapidly detecting SARS-CoV-2 based on DNA nano-bracket, which is characterized by comprising the following steps:
(1) a DNA nano-scaffold is constructed through DNA chain self-assembly and a self-quenching probe H1, and target RNA triggers the quick hybridization of a free hairpin probe H2 and a hairpin probe H1 along the nano-scaffold, so that the amplification of signals is realized.
Further, in step (1), a nano-scaffold was constructed by self-assembling DNA hairpin probe H1 with DNA nanowires, wherein DNA nanowires containing repeated sequence fragments were generated by RCA reaction of Pre-cirDNA and Primer, and both ends of hairpin probe H1 were labeled with 5-carboxyfluorescein FAM dye and its fluorescence quenching group BHQ1 to form a self-quenched probe;
in step (2), based on H in the nanoscaffold when the target RNA is present1A coding design that will hybridize to one of the nanoscaffolds H1, allowing hairpin probe H1 to open and fluorescence to recover, thereby initiating cascade hybridization of hairpin probe H2 and hairpin probe H1 along the DNA nanoscaffold;
in step (3), one target RNA can immediately illuminate the entire nanoscaffold with a highly amplified signal gain.
Further, in the step (2), the Pre-cirDNA has a nucleotide sequence of SEQ ID No. 4.
Further, in the step (2), the Primer has a nucleotide sequence of SEQ ID No. 5.
Further, in step (2), the target RNA is the conserved region sequence of the artificially synthesized SARS-CoV-2 RNA.
Further, in step (2), the sequence of the conserved region of SARS-CoV-2RNA has the nucleotide sequence of SEQ ID No. 1.
Further, in the steps (1) to (3), the temperature is a room temperature environment of 15 ℃ to 35 ℃.
Further, in step (2), the cascade hybridization reaction is performed on a DNA nano-scaffold.
Has the advantages that: the invention has the advantages of high detection sensitivity, programmable sequence, high signal gain, high specificity, short reaction time (<10 minutes), low cost and simple operation.
Compared with the prior art, the invention has the following advantages:
(1) the present invention believes that the detection method based on NSHCR, or hopefully, provides a simple and fast alternative method for the traditional SARS-CoV-2qRT-PCR analysis. Is used for high-efficiency diagnosis and sensitive detection of SARS-CoV-2 RNA.
(2) The invention constructs a nucleic acid determination technology based on nano-scaffold hybrid chain reaction (NSHCR), which is used for high-efficiency diagnosis and sensitive detection of SARS-CoV-2 RNA. The method can realize rapid detection within 5 minutes, does not need a temperature change device, and can obtain a stronger signal result at room temperature by signal amplification.
(3) The experimental result shows that stronger fluorescence intensity can be observed under the target with the same concentration by virtue of the signal amplification function of the nanowire. The NSHCR method can significantly improve signal compared to conventional HCR. The invention also has better detection performance in cell lysate and saliva samples. Therefore, we believe that the NSHCR-based detection method is expected to provide a simple and rapid alternative to the traditional SARS-CoV-2qRT-PCR analysis.
Drawings
For ease of illustration, the invention is described in detail by the following specific embodiments and the accompanying drawings;
FIG. 1 is the experimental schematic diagram of the rapid detection of SARS-CoV-2RNA of the present invention.
FIG. 2 is an AFM phase diagram of the DNA nanoscaffold of the present invention.
FIG. 3 is an image of agarose gel (2%) electrophoresis of the DNA nanoscaffold self-assembly and NSHCR reaction of the present invention.
FIG. 4 is a graph of the feasibility of fluorescence spectroscopy analysis of the NSHCR and HCR methods of the invention in the presence and absence of target RNA.
FIG. 5 is a graph showing the effect of different separation distances on fluorescence intensity for the assembly of H1 probes of the present invention on nanowires.
FIG. 6 is a graph showing the change of fluorescence intensity with RCA time according to the present invention.
FIG. 7 is a graph of performance measurements of the NSHCR of the present invention at various temperatures.
FIG. 8 is a time-dependent fluorescence spectrum of the NSHCR of the present invention for 50nM target RNA. Data error bars represent mean ± SD (n ═ 3).
FIG. 9 is a graph of fluorescence spectra of the present invention in response to different concentrations of SARS-CoV-2 RNA.
Fig. 10 is a corresponding calibration graph of the present invention.
FIG. 11 shows the specificity of the method of the present invention for detecting SARS-CoV-2RNA (10nM) of interest using mutation probes (M1-M4 refer to mutation patterns of 1, 2, 3 and 4 bases; 100nM) and random sequence miR-21.
FIG. 12 is a graph showing the detection of different concentrations of targets in TM buffer, cell lysate and 10% saliva samples, respectively, according to the present invention. Data error bars represent mean ± SD (n ═ 3).
Detailed Description
The invention is further illustrated by the following examples. It should be understood that these examples are illustrative and exemplary of the present invention, and are not intended to limit the scope of the present invention in any way.
Example 1
The invention relates to a method for rapidly detecting SARS-CoV-2 based on DNA nano-bracket, comprising the following steps:
(1) a DNA nano-scaffold is constructed by self-assembly of a DNA chain and self-quenching of a probe H1, and free hairpin probe H is triggered by target RNA2Hairpin probe H1And (3) rapidly hybridizing along the nano-scaffold to realize signal amplification.
Example 2
The invention relates to a method for rapidly detecting SARS-CoV-2 based on DNA nano-bracket, comprising the following steps:
(1) by hairpin-probing DNA with probe H1Constructing a nano-scaffold by self-assembling with the DNA nano-wire, wherein the DNA nano-wire containing the repetitive sequence segment is generated by RCA reaction between Pre-cirDNA and Primer, and the two ends of the hairpin probe H1 are marked by 5-carboxyfluorescein FAM dye and a fluorescence quenching group BHQ1 thereof to form a self-quenching probe;
(2) based on H in the nanoscaffold when target RNA is present1A coding design that will hybridize to one of the nanoscaffolds H1, allowing hairpin probe H1 to open and fluorescence to recover, thereby initiating cascade hybridization of hairpin probe H2 and hairpin probe H1 along the DNA nanoscaffold; the Pre-cirDNA has the nucleotide sequence of SEQ ID No. 4. The Primer has a nucleotide sequence of SEQ ID No. 5. The target RNA is a conserved region sequence of artificially synthesized SARS-CoV-2 RNA. The sequence of the conserved region of SARS-CoV-2RNA has the nucleotide sequence of SEQ ID No. 1. The cascade hybridization reaction is carried out on a DNA nano-scaffold.
(3) One target RNA can immediately illuminate the entire nanoscaffold with a highly amplified signal gain. In the steps (1) to (3), the temperature is a room temperature environment of 15 ℃ to 35 ℃.
Results and discussion
FIG. 1 is the experimental schematic diagram of the rapid detection of SARS-CoV-2RNA of the present invention. First, a nanoscaffold was constructed by self-assembling DNA hairpin probes (H1) with DNA nanowires. Wherein the RCA reaction by Pre-cirDNA and Primer generates DNA nanowire containing repetitive sequence fragment, and the two ends of H1 are labeled with 5-carboxyfluorescein (FAM) dye and its fluorescence quenching group (BHQ1) to form self-quenching probe. Then, when the target RNA is present, based on the H1-encoding design in the nanoscaffold, it will hybridize to one of the H1 in the nanoscaffold, causing the H1 hairpin to open and fluorescence to recover, thereby initiating the cascade hybridization of H2 and H1 along the DNA nanoscaffold. Thus, one target RNA can immediately illuminate the entire nanoscaffold with a highly amplified signal gain.
FIG. 2 is an AFM phase diagram of the DNA nanoscaffold of the present invention.
FIG. 3 is an image of agarose gel (2%) electrophoresis of the DNA nanoscaffold self-assembly and NSHCR reaction of the present invention.
FIG. 4 is a graph of the feasibility of fluorescence spectroscopy analysis of the NSHCR and HCR methods of the invention in the presence and absence of target RNA.
Feasibility verification of SARS-CoV-2RNA triggering NSHCR response
First, the morphology of the DNA nano-scaffold of the present invention is a long-chain structure containing branches, as shown in AFM phase diagram, fig. 3, which is obtained by hybridization-binding of a large number of H1 probes to a long DNA chain of RCA product. In addition, as shown in FIG. 2, the data from the 2% agarose gel electrophoresis experiment showed that the mixture of H1 and H2 produced two bands in lane 2, indicating that the two probes are independently present in solution.
The addition of the nanowires formed by RCA to the mixture of H1 and H2 resulted in the band corresponding to H1 being almost disappeared and the band of the nanowires became brighter, demonstrating that successful assembly of H1 on the nanowires formed a nanoscaffold.
Finally, the NSHCR reaction triggered by the target RNA caused a decrease in H2, with the nanoscaffold band becoming bright (lane 5). At the same time, the results of the fluorescence spectrophotometer confirmed the feasibility of the method (FIG. 4). Without addition of target RNA, both H1 and the nanowires showed low background fluorescence (purple and blue lines). When the target is present, free H1 and H2 are able to hybridize to each other to trigger the HCR reaction, resulting in fluorescence recovery (green line). The experimental result shows that stronger fluorescence intensity can be observed under the condition of the target with the same concentration by virtue of the signal amplification function of the nanowire. Compared to conventional HCR, the NSHCR method can significantly improve the signal.
FIG. 5 is a graph of the effect of different separation distances on fluorescence intensity for the assembly of H1 probes of the invention on nanowires. FIG. 6 is a graph showing the change of fluorescence intensity with RCA time according to the present invention. FIG. 7 is a graph of performance measurements of the NSHCR of the present invention at various temperatures. All values chosen for the red circle are optimal conditions. All experiments were repeated three times to obtain an average.
Example 3
Optimizing the experimental conditions
In order to obtain better detection efficiency, some key factors in the experimental process are optimized. The first is the effect of the length of hybridization of H1 to DNA nanowires (foothold) on background signal. The invention optimizes the distance of the H1 probe on the nanowire, and as can be seen from FIG. 5, the probe has the strongest fluorescence signal at 25 base pairs. In addition, the time of the RCA reaction affects the length of the DNA scaffold produced, and further, the number of H1 probes assembled on the scaffold changes, ultimately affecting the sensitivity of the assay. As shown in FIG. 6, the signal intensity increased with the increase of the RCA time from 0 minute to 30 minutes, and decreased with the increase of the RCA time from 40 minutes to 100 minutes. Therefore, we chose 30 minutes as the optimal RCA reaction time. Finally, we explored the reproducibility of recognition of target RNA by the NSHCR system at different temperatures. As shown in fig. 7, it is shown that the fluorescence intensity is relatively stable in the range of 15-35 ℃, so compared to qRT-PCR, our method can still be performed efficiently at room temperature without the need for additional temperature variation equipment.
Example 4
Kinetic analysis of NSHCR accelerated reactions
Under optimal conditions, the entire course of the detection of the NSHCR reaction in homogeneous solution was monitored by a kinetic curve. As shown in fig. 8, in the absence of target RNA, the NSHCR detected almost no fluorescent signal. When the target RNA was added, the fluorescence intensity rapidly increased in the NSHRC method, reaching a peak at 5 min. These results indicate that the nanoscaffold can significantly shorten the reaction time, enabling rapid detection of 2019-cov. The reason for this may be that a large amount of H1 in the NSHCR hybridizes to the nanowire, increasing the local concentration of H1, thereby accelerating the probability of binding of H1 to H2.
FIG. 9 is a graph of fluorescence spectra of the present invention in response to different concentrations of SARS-CoV-2 RNA.
Fig. 10 is a corresponding calibration graph of the present invention.
Example 5
Analytical Performance of the NSHCR method
The signal amplification capability of the method in the quantitative analysis of the target RNA is verified through the analysis of fluorescence intensity data. As can be seen from FIG. 9, the fluorescence intensity gradually increased with the increase in the concentration of the target RNA. To obtain the relationship between fluorescence intensity and target RNA concentration, we further recorded the peak of fluorescence intensity at different target RNA concentrations. As shown in fig. 10, the fluorescence intensity in the range of 0.25nM to 8nM is linearly related to the RNA concentration, and the correlation equation is y 1020.5X +107.43(R2 0.9878). The limit of detection (LOD) is approximately 100 pM.
FIG. 11 shows the specificity of the method of the present invention for detecting SARS-CoV-2RNA (10nM) of interest using mutation probes (M1-M4 refer to 1, 2, 3 and 4 base mutations; 100nM) and the random sequence miR-21. FIG. 12 shows the detection of different concentrations of target in TM buffer, cell lysate and 10% saliva samples, respectively, according to the present invention. Data error bars represent mean ± SD (n ═ 3).
Example 6
Specificity and stability of the NSHCR method
The main difficulty of SARS-CoV-2RNA detection is the specificity of detection, so the specificity of the detection method is the key in accurately detecting target and avoiding false positive. Therefore, we used miR-21 and one to four mismatched base target RNA sequences as controls to validate the specificity of the method. As shown in FIG. 11, the fluorescence intensity of the sample decreased significantly (2.4-fold) when a sequence of mismatched bases was added. The fluorescence intensity gradually decreases with increasing number of mismatched bases. When 4 mismatched bases appear in the sequence, the detection result is consistent with the sample value of the random sequence. This result indicates that the method has the ability to specifically detect SARS-CoV-2 RNA. Furthermore, to explore the utility of this protocol, we added target standards to cell lysates and saliva samples for analysis. As can be seen from FIG. 12, the detection results tend to be stable under different environments and target concentrations, which indicates that the scheme has good repeatability and is expected to be expanded to clinical application.
Test example 1
Reagents and materials
The DNA nucleic acid sequences used in the experiments were purchased from Biolabs (Shanghai, China) and purified by HPLC. RNA sequences were synthesized by Takara Bio (Chinese Dalian). T4 DNA ligase, exonuclease I (Exo I), exonuclease III (ExoIII), phi29 DNA polymerase and RiboLock RNase inhibitors were purchased from Thermo Fisher Scientific (Watherman, USA). Deoxyribonucleoside 5' -triphosphate mixtures (dNTPs) were purchased from New England Biolabs (Beijing, China). Cell lysates were purchased from Sangon. The solutions used in all experiments were from ultrapure water purified using a Millipore system (>18.0M Ω). Unless otherwise indicated, all other chemicals were purchased from Sigma (st louis, missouri, usa).
Agarose gel electrophoresis analysis
A2% agarose gel electrophoresis was prepared using 1 XTBE buffer. The sample was prepared by mixing 1. mu.L of the nucleic acid sample, 1.5. mu.L of 6 Xbuffer, 1. mu.L of GelRed dye and 6.5. mu. L H2O, and then the mixture was left for 3 minutes and then injected into agarose gel electrophoresis. Agarose Gel electrophoresis was run in 1 × TBE buffer at 110V for 60 min and observed by Molecular Imager Gel Doc XR.
Preparation of DNA Long strands Using RCA
First, 10. mu.L of ultrapure water, 30. mu.L of DNA ligation buffer (2X), 10. mu.L of phosphorylated template (10. mu.M) and 50. mu.L of primer (10. mu.M) were mixed in a 100. mu.L EP tube to synthesize a circular DNA template. Heat at 95 ℃ for 5 minutes, then slowly cool the solution to room temperature. T4 DNA ligase (5. mu.L, 40,000U/mL) was added to the above solution and mixed well, and left at room temperature for 30 minutes. The linear nucleic acids were digested using exonuclease I and exonuclease III. Add 10 μ L of circular DNA template to 20 μ L of RCA reaction mixture, comprising: mu.L of Phi29 DNA polymerase (10X) reaction buffer, 4. mu.L of dNTPs (10mM), 2. mu.L of BSA (10X), 1. mu.L of DEPC, 1. mu.L of Phi29 DNA polymerase (1, 000U/ml), incubated at 35 ℃ for 40 minutes, and then heat-treated at 65 ℃ for 10 minutes to terminate the reaction. Finally, the concentration of RCA product treated with the PCR purification kit was measured by Thermo NanoDrop 2000.
Designing DNA nanoscaffolds for NSHCR
The DNA nano-scaffold consists of one long DNA single strand (RCA product) and some H1 probes. First, the long DNA strand and H1 probe were heated at 95 ℃ for 5 minutes, respectively, and then slowly cooled to room temperature (>30 minutes). DNA nanoscaffold substrates were generated by thoroughly mixing 25. mu. L H1 (10. mu.M), 25. mu.L of long DNA strands (0.6. mu.M) and 125. mu.L of Tris-MgCl2 buffer (500mM, pH 8.0) in a 200. mu.L centrifuge tube. The mixture was incubated at room temperature for 30 minutes and the H1 probe was hybridized well with the long DNA strand to synthesize a functional DNA nanoscaffold.
Target RNA triggered NSHCR response
First, a reaction mixture (containing 6. mu.L of DNA nanoscaffold (0.6. mu.M), 6. mu.L of target RNA, 1. mu. L H2 probe (1. mu.M) and 32. mu.L of Tris-MgCl2 buffer) in a volume of 60. mu.L was incubated in a thermocycler. The reaction mixture was transferred to a black 384-well microplate (Fluotrac 200, Greiner, Germany) and the fluorescence intensity between 450 and 650nm at 488nm excitation was obtained by means of a fluorescence microplate reader (BioTek Instrument, Winooski, VT, USA).
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification and equivalents thereof.
Sequence listing
<110> Nanjing university
<120> method for rapidly detecting SARS-CoV-2 based on DNA nano-bracket
<130>2020
<160>5
<170>SIPOSequenceListing 1.0
<210>1
<211>119
<212>DNA
<213> Artificial sequence (SARS-CoV-2 RNA conserved region)
<400>1
ccctgtgggt tttacactta aaaacacagt ctgtaccgtc tgcggtatgt ggaaaggtta 60
tggctgtagt tgtgatcaac tccgcgaacc catgcttcag tcagctgatg cacaatcgt 119
<210>2
<211>68
<212>DNA
<213> Artificial sequence (hairpin Probe H1)
<400>2
gagttgatca caactacagc catacaaagt agtctaggat tcggcgtgaa aaaaaactca 60
taccatat 68
<210>3
<211>72
<212>DNA
<213> Artificial sequence (hairpin Probe H2)
<400>3
tatggctgta gttgtgatca actccacgcc gaatcctaga ctactttgtt aacccacgcc 60
gaatcctaga ct 72
<210>4
<211>66
<212>DNA
<213> Artificial sequence (Pre-cirDNA)
<400>4
acctcatctc tcataccata taaatgcgct aggtatataa cctcatacca tattatacaa 60
cctact 66
<210>5
<211>22
<212>DNA
<213> Artificial sequence (Primer)
<400>5
agatgaggta gtaggttgta ta 22
Claims (8)
1. A method for rapidly detecting SARS-CoV-2 based on DNA nano-bracket is characterized by comprising the following steps:
(1) a DNA nano-scaffold is constructed through DNA chain self-assembly and a self-quenching probe H1, and target RNA triggers the quick hybridization of a free hairpin probe H2 and a hairpin probe H1 along the nano-scaffold, so that the amplification of signals is realized.
2. The method for rapid detection of SARS-CoV-2 based on DNA nanoscaffold according to claim 1, wherein:
in step (1), by hairpin-probing DNA with a probe H1Self-assembly with DNA nanowires to construct nanowiresA scaffold in which DNA nanowires containing repeated sequence fragments are generated by RCA reaction between Pre-cirDNA and Primer, and both ends of the hairpin probe H1 are labeled with 5-carboxyfluorescein FAM dye and its fluorescence quenching group BHQ1 to form a self-quenching probe;
in step (2), based on H in the nanoscaffold when the target RNA is present1A coding design that will hybridize to one of the nanoscaffolds H1, allowing hairpin probe H1 to open and fluorescence to recover, thereby initiating cascade hybridization of hairpin probe H2 and hairpin probe H1 along the DNA nanoscaffold;
in step (3), one target RNA can immediately illuminate the entire nanoscaffold with a highly amplified signal gain.
3. The method for rapid detection of SARS-CoV-2 based on DNA nanoscaffold according to claim 2, wherein: in step (2), the Pre-cirDNA has the nucleotide sequence of SEQ ID No. 4.
4. The method for rapid detection of SARS-CoV-2 based on DNA nanoscaffold according to claim 2, wherein: in the step (2), the Primer has a nucleotide sequence of SEQ ID No. 5.
5. The method for rapid detection of SARS-CoV-2 based on DNA nanoscaffold according to claim 1 or 2 or 3, wherein: in step (2), the target RNA is the conserved region sequence of the artificially synthesized SARS-CoV-2 RNA.
6. The method for rapid detection of SARS-CoV-2 based on DNA nanoscaffold according to claim 1 or 2 or 3, wherein: in step (2), the sequence of the conserved region of SARS-CoV-2RNA has the nucleotide sequence of SEQ ID No. 1.
7. The method for rapid detection of SARS-CoV-2 based on DNA nanoscaffold according to claim 1 or 2 or 3, wherein: in the steps (1) to (3), the temperature is a room temperature environment of 15 ℃ to 35 ℃.
8. The method for rapid detection of SARS-CoV-2 based on DNA nanoscaffold according to claim 1 or 2 or 3, wherein: in step (2), the cascade hybridization reaction is performed on a DNA nano-scaffold.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010507344.7A CN111676317B (en) | 2020-06-05 | 2020-06-05 | Method for rapidly detecting SARS-CoV-2 based on DNA nano-support |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010507344.7A CN111676317B (en) | 2020-06-05 | 2020-06-05 | Method for rapidly detecting SARS-CoV-2 based on DNA nano-support |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111676317A true CN111676317A (en) | 2020-09-18 |
| CN111676317B CN111676317B (en) | 2022-03-18 |
Family
ID=72454292
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010507344.7A Active CN111676317B (en) | 2020-06-05 | 2020-06-05 | Method for rapidly detecting SARS-CoV-2 based on DNA nano-support |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111676317B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112831599A (en) * | 2020-12-07 | 2021-05-25 | 郑州科蒂亚生物技术有限公司 | CoVID-19 virus detection kit based on signal amplification technology |
| CN114875178A (en) * | 2022-05-11 | 2022-08-09 | 山东大学 | SARS-CoV-2 detecting system and detecting method based on hybrid chain reaction |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154919A (en) * | 2020-03-04 | 2020-05-15 | 南京大学 | Novel coronavirus kit and method for detecting novel coronavirus nucleic acid by using single closed tube one-step method |
-
2020
- 2020-06-05 CN CN202010507344.7A patent/CN111676317B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154919A (en) * | 2020-03-04 | 2020-05-15 | 南京大学 | Novel coronavirus kit and method for detecting novel coronavirus nucleic acid by using single closed tube one-step method |
Non-Patent Citations (3)
| Title |
|---|
| JIAO J ET AL: "DNA nanoscaffold-based SARS-CoV-2 detection for COVID-19 diagnosis", 《BIOSENSORS AND BIOELECTRONICS》 * |
| MAGDIEL I.SETYAWATI ET AL: "Novel Theranostic DNA Nanoscaffolds for the Simultaneous Detection and Killing of Escherichia coli and Staphylococcus aureus", 《ACS APPL.MATER.INTERFACES》 * |
| 刘永新等: "杂交链式反应技术结合纳米材料用于核酸检测的研究进展", 《中国材料进展》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112831599A (en) * | 2020-12-07 | 2021-05-25 | 郑州科蒂亚生物技术有限公司 | CoVID-19 virus detection kit based on signal amplification technology |
| CN114875178A (en) * | 2022-05-11 | 2022-08-09 | 山东大学 | SARS-CoV-2 detecting system and detecting method based on hybrid chain reaction |
| CN114875178B (en) * | 2022-05-11 | 2024-04-12 | 山东大学 | SARS-CoV-2 detection system and method based on hybridization chain reaction |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111676317B (en) | 2022-03-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jiao et al. | DNA nanoscaffold-based SARS-CoV-2 detection for COVID-19 diagnosis | |
| CA2692633C (en) | Method for the simultaneous detection of multiple nucleic acid sequences in a sample | |
| CN111676317B (en) | Method for rapidly detecting SARS-CoV-2 based on DNA nano-support | |
| Ou et al. | Rapid and ultrasensitive detection of microRNA based on strand displacement amplification-mediated entropy-driven circuit reaction | |
| CN113652505B (en) | Method and kit for detecting novel coronavirus and VOC-202012/01 mutant strain thereof | |
| Yang et al. | A label-free fluorescent probe based on DNA-templated silver nanoclusters and exonuclease III-assisted recycling amplification detection of nucleic acid | |
| Hang et al. | Rapid and sensitive detection of Ebola RNA in an unamplified sample based on CRISPR-Cas13a and DNA roller machine | |
| Su et al. | Sensitive detection of hepatitis C virus using a catalytic hairpin assembly coupled with a lateral flow immunoassay test strip | |
| CN117265186B (en) | TaqMan fluorescent quantitative PCR primer group, kit and method for detecting pangolin east yang virus | |
| Ahn et al. | Zika virus lateral flow assays using reverse transcription-loop-mediated isothermal amplification | |
| Wang et al. | T4 DNA polymerase-assisted upgrade of a nicking/polymerization amplification strategy for ultrasensitive electrochemical detection of Watermelon mosaic virus | |
| CN112725539B (en) | RPA/Cas12a/IF kit for respiratory syncytial virus and detection method thereof | |
| Zheng et al. | A label-free signal amplification assay for DNA detection based on exonuclease III and nucleic acid dye SYBR Green I | |
| Zhou et al. | A dual fragment triggered DNA ladder nanostructure based on logic gate and dispersion-to-localization catalytic hairpin assembly for efficient fluorescence assay of SARS-CoV-2 and H1N1 | |
| Zeng et al. | Rapid RNA detection through intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction without amplification | |
| CN114875178B (en) | SARS-CoV-2 detection system and method based on hybridization chain reaction | |
| Chen et al. | Development of a loop-mediated isothermal amplification assay for the rapid detection of six common respiratory viruses | |
| CN113322308B (en) | Fluorescence sensor for detecting HBV (hepatitis B virus), and preparation and application thereof | |
| KR102131494B1 (en) | Detection of Target Nucleic Acid Sequences by PCR-coupled Rolling Circle Amplification | |
| Zhang et al. | A CRISPR/Cas12a-assisted array for Helicobacter pylori DNA analysis in saliva | |
| CN114592038A (en) | Fluorescent biosensing system for detecting Dam methyltransferase and construction and application thereof | |
| Li et al. | One-pot, ultrasensitive, and multiplex detection of SARS-CoV-2 genes utilizing self-priming hairpin-mediated isothermal amplification | |
| Li et al. | A functional RNA/DNA circuit for one-pot detection of SARS-CoV-2 RNA | |
| Su et al. | Water-soluble polythiophene-based colorimetry for the quick and accurate detection of SARS-CoV-2 RNA | |
| CN113151591A (en) | Nucleic acid composition and kit for detecting SARS-CoV-2 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |