CN112266985A - Anti-sense RNA probe for detecting coronavirus, preparation method thereof, kit and method for detecting coronavirus - Google Patents
Anti-sense RNA probe for detecting coronavirus, preparation method thereof, kit and method for detecting coronavirus Download PDFInfo
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Abstract
The invention discloses an antisense RNA probe for detecting coronavirus, a preparation method thereof, a kit for detecting coronavirus and a method, and belongs to the technical field of nucleic acid hybridization. In order to improve the accuracy and specificity of coronavirus detection, the method for detecting coronavirus provided by the invention comprises the steps of mixing a sample to be detected with proteinase K, rinsing, adding a prehybridization solution for incubation, adding a digoxin-modified antisense RNA probe for incubation, mixing with a blocking solution, adding a digoxin-modified antibody, mixing with an MABT solution containing lamb serum, adding an AP substrate staining buffer solution for incubation in a dark place at room temperature, and observing and photographing by using a microscope to obtain a result for detecting coronavirus. The method has the advantages of small detection sample amount, no need of extracting RNA in cells, capability of effectively avoiding the problem of false negative results caused by RNA loss in fluorescent quantitative PCR detection, high sensitivity and improvement on the accuracy of detection results.
Description
Technical Field
The invention belongs to the technical field of nucleic acid hybridization, and particularly relates to an antisense RNA probe for detecting coronavirus, a preparation method thereof, a kit for detecting coronavirus and a method.
Background
2019 novel coronavirus (SARS-CoV-2) has long latent period and strong infectivity, and mainly attacks the lung of an animal body to cause lung infection.
Currently, there are fluorescence quantitative PCR method and antibody detection method for identifying novel coronavirus, wherein the nucleic acid detection method is the gold standard for virus detection method, but there are still many problems in the existing methods, such as:
(1) the fluorescent quantitative PCR can identify nucleic acid in a suspected patient, the method needs to extract RNA in cells, then carry out reverse transcription, and then carry out PCR identification, the loss of the RNA is high in the experimental operation, and if the suspected patient is in the early stage of infection or the virus content in the body is low, the RNA is easily lost in the extraction process, and a false negative result is caused.
(2) Because the novel coronavirus is an RNA virus, the variation speed of the RNA virus is high, the variation can be accumulated continuously, the requirement of a fluorescent quantitative PCR experiment on a nucleic acid sequence is higher, particularly the requirement on a region covered by a PCR primer is severer, and if the nucleic acid in the region is mutated, the PCR amplification efficiency is reduced, even the nucleic acid cannot be amplified, so that a false negative result is generated.
(3) The fluorescent quantitative PCR experiment is not subjected to sequencing identification, and non-target fragments are possibly amplified, so that a false positive phenomenon occurs, and the detection result is inaccurate.
Therefore, the problem to be solved by those skilled in the art is how to provide a probe capable of effectively detecting SARS-CoV-2 and apply it to the detection of SARS-CoV-2.
Disclosure of Invention
The invention aims to improve the accuracy and specificity of SARS-CoV-2 coronavirus detection and solve the problem of low accuracy of SARS-CoV-2 coronavirus detection, and provides an antisense RNA probe for coronavirus detection, wherein the sequence of the antisense RNA probe is shown as SEQ ID NO. 4.
The invention also provides a preparation method of the antisense RNA probe, which comprises the following steps:
(1) designing a primer:
COVID-19-F1 has a sequence of 5'-GATCAAAACAACGTCGGCCC-3'; COVID-19-R1 has a sequence of 5'-GCGTAATACGACTCACTATAGGGTTCTGTCTCTGCGGTAAGGC-3';
(2) constructing a recombinant vector: inserting the base sequence of the region to be detected of the coronavirus SARS-CoV-2 into BamHI and XbaI in the plasmid pcDNA3.1-myc-HisA as shown in SEQ ID NO.1 to obtain a recombinant vector pcDNA3.1-myc-HisA-COVID-19;
(3) PCR amplification of the DNA sequence of the SARS-CoV-2 region to be tested: taking the recombinant vector obtained in the step (2) as a template, and amplifying the sequence by using the primer in the step (1) and utilizing a PCR (polymerase chain reaction) technology to obtain a DNA (deoxyribonucleic acid) sequence of a region to be detected of the coronavirus SARS-CoV-2;
(4) in vitro transcription to obtain antisense RNA probe: and (4) carrying out in-vitro transcription by taking the DNA fragment of the SARS-CoV-2 region to be detected of the coronavirus obtained in the step (3) as a template to obtain the antisense RNA probe.
The invention also provides a kit for detecting coronavirus, which comprises the digoxin-modified antisense RNA probe.
Further defined, the kit further comprises: prehybridization solution, blocking solution, AP substrate staining buffer, MABT solution, 50% formamide/2 XSSCT buffer, 0.2 XSSCT buffer, detection buffer, coronavirus SARS-CoV-2 negative control, and coronavirus SARS-CoV-2 positive control.
Further defined, the concentration of the digoxin-modified antisense RNA probe is 2 ng/. mu.L.
Further defined, the prehybridization solution comprises 50% formamide, 5 XSSCT, 50. mu.g/mL heparin, 5mM EDTA, 0.05mg/mL ribosomal RNA, 1M citric acid, and 0.1% Tween by volume; the blocking solution comprises 1% sheep serum, 2% blocking agent, 100mM maleic acid and 150mM NaCl; the AP substrate staining buffer solution comprises nitrotetrazolium blue chloride, 5-bromo-4-chloro-3-indolyl phosphate and levamisole; the MABT solution comprises maleic acid and NaCl; the detection buffer comprises 100mM NaCl and 50mM MgCl2100mM tris, 0.1% Tween-20 and 1mM levamisole.
Further defined, the coronary diseaseThe positive control of SARS virus-CoV-2 is 1X 105Hek293 cells harboring coronavirus SARS-CoV-2; the negative control of coronavirus SARS-CoV-2 is 1 × 105Hek293 cells not carrying the coronavirus SARS-CoV-2.
The invention also provides a method for detecting coronavirus, which is to detect coronavirus SARS-CoV-2 by using the kit for detecting coronavirus, and comprises the following steps:
1) hybridization pretreatment: adding protease K into a sample to be detected for incubation, then rinsing with a PBST solution, then fixing with paraformaldehyde, and then rinsing with the PBST solution;
2) pre-hybridization reaction: adding the pre-hybridization solution into the sample to be detected after pretreatment in the step 1), and incubating;
3) and (3) hybridization reaction: removing the pre-hybridization solution in the step 2), adding the pre-hybridization solution containing the digoxin-modified antisense RNA probe into a sample to be detected, and incubating;
4) and (3) post-hybridization treatment: removing the pre-hybridization solution in the step 3), rinsing the sample to be tested with 50% formamide/2 xSSCT buffer solution, 2 xSSCT buffer solution and 0.2 xSSCT buffer solution in sequence, mixing with blocking solution, culturing at room temperature, adding anti-digoxin antibody diluted by 1000-4000 times of the blocking solution, and incubating and culturing to obtain a hybridization sample;
5) and (3) color development reaction: adding the hybridization sample obtained in the step 4) into an MABT solution containing lamb serum with the final volume fraction of 1%, washing with the MABT solution, washing with a detection buffer solution, adding an AP substrate staining buffer solution for incubation in a dark place at room temperature, washing the hybridization sample with a PBS solution after the hybridization sample is stained, and observing and photographing with a microscope to obtain a result for detecting the coronavirus SARS-CoV-2.
Further defined, the incubation in step 2) is a reaction at 68 ℃ for 45 minutes to 240 minutes; the incubation in step 3) is a reaction at 68 ℃ for 10 hours; the incubation in step 4) is carried out at 4 ℃ for 10 hours, and the incubation time at room temperature is 45 minutes to 240 minutes.
Further defined, the room temperature is incubated in the dark for 30 minutes to 120 minutes, and the signal is detected every 15 minutes until the target signal appears.
Has the advantages that:
1. the problem that more and more false negative results are generated in the fluorescent quantitative PCR detection due to the rapid variation of the RNA virus is effectively avoided, and the accuracy of the detection result is improved;
2. the time cost for preparing the probe is saved to the maximum extent. And the target gene is obtained by PCR amplification, so that the method is stable, has low possibility of error, and is high in yield, rapid and efficient.
3. The in-situ hybridization technology is utilized to detect the sample to be detected, the required sample is small, RNA in cells does not need to be extracted, the loss of RNA is avoided, the problem of false negative result caused by the loss of RNA in the fluorescent quantitative PCR detection can be effectively avoided, the sensitivity is high, and the accuracy of the detection result is improved.
Drawings
FIG. 1 is a schematic diagram showing the cleavage site of recombinant plasmid pcDNA3.1-myc-HisA-COVID-19;
FIG. 2 is a recombinant plasmid verification electrophoresis diagram, wherein 1 is recombinant plasmid pcDNA3.1-myc-HisA-COVID-19(6695bp), 2 is enzyme-digested pcDNA3.1-myc-HisA partial vector (5432bp), virus gene fragment and partial vector (1263bp), and M is Marker;
FIG. 3 is an electrophoretogram of PCR amplification product gel after recovery and purification; wherein, 1 is a glue recovery product, and M is Marker;
FIG. 4 is an electrophoretogram of an antisense RNA probe; wherein, 1 is an antisense RNA probe, and M is Marker;
FIG. 5 is a diagram showing the results of antisense RNA probe detection, in which, panel A shows the results of in situ hybridization after the cell was transfected with pcDNA3.1-myc-HisA empty vector plasmid for 4 hours, and panel B shows the results of in situ hybridization after the cell was transfected with pcDNA3.1-myc-HisA-COVID-19 plasmid for 4 hours.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Experimental materials: hek293 cells are commercially available human embryonic kidney cells 293.
Description of abbreviations in the examples and related reagents formulation methods:
HYB: pre-hybridization solution, namely formamide with the volume ratio of 50 percent; 5 × SSCT; 50 μ g/mL heparin; 5mM EDTA; 0.05mg/mL ribosomal RNA; 920 μ L of 1M citric acid; 0.1% Tween.
MABT: 100mM maleic acid (maleic acid), 150mM nacl; 150mM NaCl, 0.1% Tween; the pH was 7.4.
EDTA: ethylene diamine tetraacetic acid.
PBST solution: phosphate Tween buffer, 0.1% Tween-20(V/V) was added to the PBS solution.
NBT: nitroblue tetrazolium chloride.
BCIP: 5-bromo-4-chloro-3-indolyl phosphate.
Blocking solution: 1% sheep serum, 2% blocker (Roche, REF:11096176001), dissolved in MABT.
50% formamide/2 × SSCT buffer: 50% formamide, 0.3M NaCl, 0.03M sodium citrate and 0.1% Tween 20.
2 × SSCT buffer: 0.3M NaCl, 0.03M sodium citrate and 0.1% Tween20 were dissolved in dH2O and the pH was adjusted to 7.0.
5 × SSCT: 0.75M NaCl; 0.075M sodium citrate, dissolved in dH2O, adjusted to pH 7.0.
0.2 × SSCT buffer: 0.03M NaCl, 0.003M sodium citrate and 0.1% Tween20, dissolved in ddH2In O, the pH was adjusted to 7.0.
Detection buffer solution: each 50mL of the solution contained 100mM NaCl, 50mM MgCl2100mM Tris-hydroxymethyl aminomethane, 0.1% V/V Tween-20, and 1mM levamisole.
AP substrate staining buffer: the volume was made up to 1mL from 3.5. mu.L of LNBT, 3.5. mu.L of LBCIP, 4. mu.L of levamisole and water.
Materials such as reagents or cloning vectors used in the examples are all common reagents in the field and can be purchased commercially, if not specified; the experimental methods not mentioned are all conventional experimental methods.
Example 1 preparation of antisense RNA Probe for detection of coronavirus
1. Designing a primer: according to the base sequence (Gene bank: MN988668.1) of the region to be detected of the coronavirus SARS-CoV-2, the base sequence of the region to be detected of the SARS-CoV-2 is shown as SEQ ID NO.1, and primers are designed by using Premier 5.0 software and BLAST analysis of NCBI to obtain primers COVID-19-F1 and COVID-19-R1;
the sequence of COVID-19-F is shown as SEQ ID NO.2, namely 5'-GATCAAAACAACGTCGGCCC-3';
the sequence of COVID-19-R1 is shown as SEQ ID NO.3, namely 5'-GCGTAATACGACTCACTATAGGGTTCTGTCTCTGCGGTAAGGC-3';
2. constructing a recombinant vector: the plasmid pcDNA3.1-myc-HisA is purchased from Beijing Ongzhi Biotechnology Limited, the organism synthesizes base sequence of region to be detected of coronavirus SARS-CoV-2 with BamHI and Xba I at two ends of the sequence, then the synthesized sequence is inserted between BamHI and Xba I in the plasmid pcDNA3.1-myc-HisA to obtain recombinant plasmid with DNA sequence of region to be detected of SARS-CoV-2, namely recombinant vector pcDNA3.1-myc-HisA-COVID-19, the vector diagram is shown in figure 1.
Verifying the construction of the recombinant vector, the obtained pcDNA3.1-myc-HisA-COVID-19 was digested with enzyme, the digestion result is shown in FIG. 2, lane 1 is the electrophoresis diagram of the recombinant plasmid pcDNA3.1-myc-HisA-COVID-19(6695bp in total length); lane 2 shows the results of the double digestion with BamHI and Xba I, the plasmid fragment of pcDNA3.1-myc-HisA is 5432bp, the size of the digestion product fragment is 1263bp, and the obtained digestion product is the DNA sequence to be tested for SARS-CoV-2.
PCR amplification of the DNA sequence of the SARS-CoV-2 region to be tested: and (2) amplifying by using the recombinant vector pcDNA3.1-myc-HisA-COVID-19 obtained in the step 2 as a template and using the upstream primer COVID-19-F1 and the downstream primer COVID-19-R1 described in the step 1 to obtain the DNA of the SARS-CoV-2 region to be detected, wherein the reaction system of PCR amplification is shown in Table 1:
TABLE 1 reaction System for PCR amplification
| pcDNA3.1-myc-HisA-COVID-19 | 1μL | |
| COVID-19-F1 | 3μL | |
| COVID-19-R1 | 3μL | |
| 5× | 10μL | |
| 2×PCRmix | 25μL | |
| ddH2O | 8μL | |
| Total volume | 50μL |
The reaction procedure for PCR amplification was: 5min at 98 ℃; 30 cycles of 98 ℃ for 30s, 59 ℃ for 30s and 72 ℃ for 30 s; 4min at 72 ℃.
The method comprises the following specific steps: amplifying the gel product by using pcDNA3.1-myc-HisA-COVID-19 as a template and COVID-19-F1 and COVID-19-R1 according to the system and the program, performing 2% agarose electrophoresis on the amplification product, recovering and purifying the amplification product at a cutting position of a reference DNA marker, and cutting a band at 1054nt on the gel product at the cutting position of the reference DNA marker, wherein the band is a required target product band; the cut band of the target product was recovered with a gel recovery kit to obtain a purified PCR amplification product, which was subjected to agarose gel electrophoresis, as shown in FIG. 3, and the concentration thereof (200 ng/. mu.L) was measured to obtain a DNA template.
4. In vitro transcription to obtain antisense RNA probe: and (3) carrying out in-vitro transcription by using the DNA template obtained in the step (3) to synthesize an antisense RNA probe with a digoxin label, wherein the in-vitro transcription system is shown in a table 2:
TABLE 2 in vitro transcription System
| 10×Buffer | 2μL |
| Digoxin-labeled rNTP mix (10mM) | 4μL |
| T7 RNA polymerase mix | 2μL |
| DNA template | 4μL |
| Nuclease-Free Water | 8μL |
| Total volume | 20μL |
The in vitro transcription method is as follows:
1) the in vitro transcription system was added to a 1.5mL EP tube (centrifuge tube), mixed well and then placed in a 37 ℃ water bath for 2 h.
2) After the water bath was completed, DNase was added for digestion for 15min, and then agarose electrophoresis was performed, as shown in FIG. 4, the size of RNA was only half the size of DNA marker used in the graph, since DNA was double-stranded and RNA was single-stranded, and digoxin label was incorporated into a part of U bases.
3) Purifying the successfully transcribed RNA by using an RNeasy Mini kit (RNA purification kit) to finally obtain an antisense RNA probe with a digoxin label, and storing the antisense RNA probe in an environment at the temperature of-80 ℃, wherein the nucleotide sequence of the antisense RNA probe is shown as SEQ ID NO. 4.
Example 2.
1. A kit for detecting novel coronavirus SARS-CoV-2, which consists of antisense RNA probe with digoxin label, prehybridization solution, blocking solution, AP substrate staining buffer solution, MABT solution, 50% formamide/2 XSSCT buffer solution, 0.2 XSSCT buffer solution, detection buffer solution, coronavirus SARS-CoV-2 negative control and coronavirus SARS-CoV-2 positive control, wherein the concentration of the digoxin modified antisense RNA probe is 2 ng/mu L; the prehybridization solution comprises 50% of formamide, 5 xSSCT, 50 mug/mL of heparin, 5mM of EDTA, 0.05mg/mL of ribosomal RNA, 1M of citric acid and 0.1% of Tween in volume ratio; the blocking solution comprises 1% sheep serum, 2% blocking agent, 100mM maleic acid and 150mM NaCl; the AP substrate staining buffer solution comprises nitrotetrazolium blue chloride, 5-bromo-4-chloro-3-indolyl phosphate and levamisole; the MABT solution comprises maleic acid and NaCl; the detection buffer comprises 100mM NaCl and 50mM MgCl2100mM trihydroxymethyl aminomethane, Tween-20 with volume fraction of 0.1% and 1mM levamisole; the positive control of coronavirus SARS-CoV-2 is 1 × 105Hek293 cells harboring coronavirus SARS-CoV-2; the negative control of coronavirus SARS-CoV-2 is 1 × 105Hek293 cells not carrying the coronavirus SARS-CoV-2.
2. The reagent kit for detecting the novel coronavirus SARS-CoV-2 is used for detecting the coronavirus SARS-CoV-2, and the specific steps are as follows:
1) hybridization pretreatment: adding protease K into a sample to be detected for incubation, then rinsing with a PBST solution, then fixing with paraformaldehyde, and then rinsing with the PBST solution;
2) pre-hybridization reaction: adding the pre-hybridization solution into the sample to be detected after pretreatment in the step 1), and incubating;
3) and (3) hybridization reaction: removing the pre-hybridization solution in the step 2), adding the pre-hybridization solution containing the digoxin-modified antisense RNA probe into a sample to be detected, and incubating;
4) and (3) post-hybridization treatment: removing the pre-hybridization solution in the step 3), rinsing the sample to be detected with 50% formamide/2 xSSCT buffer solution, 2 xSSCT buffer solution and 0.2 xSSCT buffer solution in sequence, mixing with the blocking solution, incubating at room temperature, and adding the anti-digoxin antibody diluted by 1000-4000 times of the blocking solution to obtain a hybrid sample;
5) and (3) color development reaction: adding the hybridization sample obtained in the step 4) into an MABT solution containing lamb serum with the final volume fraction of 1%, washing with the MABT solution, then washing with a detection buffer solution, adding an AP substrate staining buffer solution for incubation in a dark place at room temperature, washing the hybridization sample with a PBS solution after the hybridization sample is stained, and then observing and photographing with a microscope to obtain a result for detecting the coronavirus SARS-CoV-2.
The following experiments were used to verify the effect:
in order to demonstrate the method for detecting the novel coronavirus SARS-CoV-2 by the antisense RNA probe, the present example firstly prepares a sample to be detected carrying the nucleic acid sequence of the novel coronavirus SARS-CoV-2, which has the sequence shown in SEQ ID NO.1, and then utilizes the antisense RNA probe to detect, and the specific method is as follows:
recovery and passage of Hek293 cells
Resuscitation of Hek293 cells:
(1) taking out the frozen Hek293 cells from the liquid nitrogen, putting the cells into a constant-temperature water bath kettle at 37 ℃ for thawing for 1min, and shaking the frozen tubes without stopping;
(2) transferring the melted cell suspension into a preheated fresh DMEM complete culture solution, and lightly blowing and beating for 2 times;
(3) centrifuging at 1000rpm for 3min, and discarding the supernatant;
(4) 5ml of fresh DMEM complete medium was added and transferred to a new flask, placed at 37 ℃ and 5% CO2Culturing in an incubator;
(5) after 24 hours, the culture medium was replaced with a new one, and the subculture was continued.
Passage of Hek293 cells:
(1) when the cell growth confluency reaches 95% -100%, the old culture medium is sucked off, and PBS is added for washing once;
(2) adding trypsin, digesting for 30s, and sucking;
(3) adding preheated fresh DMEM to stop digestion, and repeatedly blowing and beating until the mixture is a single cell suspension;
(4) adding 3ml fresh DMEM medium into culture flask, adding 1ml cell suspension, and culturing at 37 deg.C with 5% CO2Cultured in an incubator.
3. Hek293 cells were transfected with pcDNA3.1-myc-HisA-COVID-19 plasmid.
1) Taking Hek293 cells in logarithmic phase, digesting with trypsin, adding a DMEM medium without double antibodies to dilute the cells, transferring the cells into a 24-pore plate with 1ml per pore, and replacing a new medium after culturing for 24 hours;
2) transfecting according to a Lipofectamine 2000Reagent kit, taking two 1.5ml centrifuge tubes, respectively adding 250 mu L of Opti-MEM culture medium, and adding pcDNA3.1-myc-HisA-COVID-19 plasmid (2-4 mu g) into one centrifuge tube; adding 6 μ L Lipofectamine 2000 to the other, and incubating at room temperature for 5 min;
3) mixing the two centrifuge tubes, incubating at room temperature for 20min to obtain plasmid-liposome mixture, and adding 1ml of DMEM medium without double antibody;
4) dropping the mixture into 24-pore plate, mixing, and adding 5% CO at 37 deg.C2The culture in the incubator to obtain Hek293 cells with coronavirus SARS-CoV-2.
Method for detecting novel coronavirus SARS-CoV-2
1. Hybridization pretreatment:
(1) culturing the cultured 1X 105200 μ L of proteinase K (10 ng/. mu.L) was added dropwise to Hek293 cells and incubated at 37 ℃ for 30 min;
(2) rinsing with PBST solution prepared with DEPC water for 3 times, each time for 5 min;
(3) fixing with 4% paraformaldehyde prepared with DEPC water for 20 min;
(4) rinsing with PBST solution prepared with DEPC water for 3 times, each time for 5 min;
2. pre-hybridization:
at 1X 105200 mu L of prehybridization solution is dripped into Hek293 cells, placed in a 24-hole culture dish, placed in a water bath kettle at 68 ℃ and incubated for 45-240 minutes to incubate for 60min to verify the experimental effect.
3. And (3) hybridization:
the prehybridization solution was aspirated, 200. mu.L of the hybridization solution (prehybridization solution containing 2ng/uL of antisense RNA to the digoxin marker prepared in example 1) was added dropwise, and the 24-well plate was incubated in a water bath at 68 ℃ for 10 hours.
4. And (3) post-hybridization treatment:
(1) sucking out the hybridization solution, adding 200 μ L of 50% formamide/2 × SSCT buffer solution preheated at 68 deg.C, and washing for 2 times, each time for 30 min;
(2) washing in preheated 2 × SSCT buffer solution at 68 deg.C for 1 time and 30 min;
(3) washing in preheated 0.2 × SSCT buffer solution at 68 deg.C for 2 times, each for 30 min;
(4) sucking out the liquid, dripping the blocking solution, and incubating at room temperature for 45-240 min to verify the experimental effect after incubating for 60 min.
(5) Digoxin antibody (Anti-Digoxigenin-AP, Fab fragments) diluted 1000-fold and 4000-fold with blocking solution was added and incubated at 4 ℃ for 10 hours. The experimental effect was verified with an antibody dilution factor of 3000.
5. Color development and photography:
(1) adding into MABT solution of 1% heat-treated lamb serum (heat treated lamb serum), and washing at room temperature for 25 min;
(2) washing with MABT for 3 times, each for 25 min;
(3) washing with detection buffer for 5min for 2 times;
(4) adding 200uLAP substrate staining buffer, incubating at room temperature for 30-120 min, observing the result every 15min, and wrapping with metal foil in dark until the target is colored.
(5) When the target coloring appeared, washing with PBS for 5min for 2 times to stop the reaction;
(6) and (5) observing by a microscope and taking a picture.
6. Results
The results are shown in FIG. 5A, which is a control transfected with pCNDA3.1myc-HisA empty vector plasmid, with no signal and very weak background; as shown in FIG. 5B, in situ hybridization results after 4 hours of cell transfection, the colored arrows indicate positive signals, and the dark color indicates a dark signal and a high RNA copy number; a light color indicates a light signal, indicating a low RNA copy number, and even a small RNA copy number can be detected. Colorless is a negative signal. As can be seen from the fact that the invention can detect the nucleic acid of the coronavirus SARS-CoV-2 at the single cell level, the sensitivity of the detection is improved.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
SEQUENCE LISTING
<110> Xidongyang
<120> antisense RNA probe for detecting coronavirus, preparation method thereof, kit and method for detecting coronavirus
Method of
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1269
<212> DNA
<213> SARS-CoV-2 region to be detected base sequence
<400> 1
ggatccatgt ctgataatgg accccaaaat cagcgaaatg caccccgcat tacgtttggt 60
ggaccctcag attcaactgg cagtaaccag aatggagaac gcagtggggc gcgatcaaaa 120
caacgtcggc cccaaggttt acccaataat actgcgtctt ggttcaccgc tctcactcaa 180
catggcaagg aagaccttaa attccctcga ggacaaggcg ttccaattaa caccaatagc 240
agtccagatg accaaattgg ctactaccga agagctacca gacgaattcg tggtggtgac 300
ggtaaaatga aagatctcag tccaagatgg tatttctact acctaggaac tgggccagaa 360
gctggacttc cctatggtgc taacaaagac ggcatcatat gggttgcaac tgagggagcc 420
ttgaatacac caaaagatca cattggcacc cgcaatcctg ctaacaatgc tgcaatcgtg 480
ctacaacttc ctcaaggaac aacattgcca aaaggcttct acgcagaagg gagcagaggc 540
ggcagtcaag cctcttctcg ttcctcatca cgtagtcgca acagttcaag aaattcaact 600
ccaggcagca gtaggggaac ttctcctgct agaatggctg gcaatggcgg tgatgctgct 660
cttgctttgc tgctgcttga cagattgaac cagcttgaga gcaaaatgtc tggtaaaggc 720
caacaacaac aaggccaaac tgtcactaag aaatctgctg ctgaggcttc taagaagcct 780
cggcaaaaac gtactgccac taaagcatac aatgtaacac aagctttcgg cagacgtggt 840
ccagaacaaa cccaaggaaa ttttggggac caggaactaa tcagacaagg aactgattac 900
aaacattggc cgcaaattgc acaatttgcc cccagcgctt cagcgttctt cggaatgtcg 960
cgcattggca tggaagtcac accttcggga acgtggttga cctacacagg tgccatcaaa 1020
ttggatgaca aagatccaaa tttcaaagat caagtcattt tgctgaataa gcatattgac 1080
gcatacaaaa cattcccacc aacagagcct aaaaaggaca aaaagaagaa ggctgatgaa 1140
actcaagcct taccgcagag acagaagaaa cagcaaactg tgactcttct tcctgctgca 1200
gatttggatg atttctccaa acaattgcaa caatccatga gcagtgctga ctcaactcag 1260
gcctctaga 1269
<210> 2
<211> 20
<212> DNA
<213> COVID-19-F
<400> 2
gatcaaaaca acgtcggccc 20
<210> 3
<211> 43
<212> DNA
<213> COVID-19-R1
<400> 3
gcgtaatacg actcactata gggttctgtc tctgcggtaa ggc 43
<210> 4
<211> 1054
<212> RNA
<213> antisense RNA Probe
<400> 4
uucugucucu gcgguaaggc uugaguuuca ucagccuucu ucuuuuuguc cuuuuuaggc 60
ucuguuggug ggaauguuuu guaugcguca auaugcuuau ucagcaaaau gacuugaucu 120
uugaaauuug gaucuuuguc auccaauuug auggcaccug uguaggucaa ccacguuccc 180
gaagguguga cuuccaugcc aaugcgcgac auuccgaaga acgcugaagc gcugggggca 240
aauugugcaa uuugcggcca auguuuguaa ucaguuccuu gucugauuag uuccuggucc 300
ccaaaauuuc cuuggguuug uucuggacca cgucugccga aagcuugugu uacauuguau 360
gcuuuagugg caguacguuu uugccgaggc uucuuagaag ccucagcagc agauuucuua 420
gugacaguuu ggccuuguug uuguuggccu uuaccagaca uuuugcucuc aagcugguuc 480
aaucugucaa gcagcagcaa agcaagagca gcaucaccgc cauugccagc cauucuagca 540
ggagaaguuc cccuacugcu gccuggaguu gaauuucuug aacuguugcg acuacgugau 600
gaggaacgag aagaggcuug acugccgccu cugcucccuu cugcguagaa gccuuuuggc 660
aauguuguuc cuugaggaag uuguagcacg auugcagcau uguuagcagg auugcgggug 720
ccaaugugau cuuuuggugu auucaaggcu cccucaguug caacccauau gaugccgucu 780
uuguuagcac cauagggaag uccagcuucu ggcccaguuc cuagguagua gaaauaccau 840
cuuggacuga gaucuuucau uuuaccguca ccaccacgaa uucgucuggu agcucuucgg 900
uaguagccaa uuuggucauc uggacugcua uugguguuaa uuggaacgcc uuguccucga 960
gggaauuuaa ggucuuccuu gccauguuga gugagagcgg ugaaccaaga cgcaguauua 1020
uuggguaaac cuuggggccg acguuguuuu gauc 1054
Claims (10)
1. An antisense RNA probe for detecting coronavirus, which is characterized in that the sequence of the antisense RNA probe is shown as SEQ ID NO. 4.
2. The method for preparing an RNA probe according to claim 1, comprising the following steps:
(1) designing a primer: COVID-19-F1 has a sequence of 5'-GATCAAAACAACGTCGGCCC-3'; COVID-19-R1 has a sequence of 5'-GCGTAATACGACTCACTATAGGGTTCTGTCTCTGCGGTAAGGC-3';
(2) constructing a recombinant vector: inserting the base sequence of the region to be detected of the coronavirus SARS-CoV-2 into BamHI and XbaI in the plasmid pcDNA3.1-myc-HisA as shown in SEQ ID NO.1 to obtain a recombinant vector pcDNA3.1-myc-HisA-COVID-19;
(3) PCR amplification of the DNA sequence of the SARS-CoV-2 region to be tested: taking the recombinant vector obtained in the step (2) as a template, and amplifying the sequence by using the primer in the step (1) and utilizing a PCR (polymerase chain reaction) technology to obtain a DNA (deoxyribonucleic acid) sequence of a region to be detected of the coronavirus SARS-CoV-2;
(4) in vitro transcription to obtain antisense RNA probe: and (4) carrying out in-vitro transcription by taking the DNA fragment of the SARS-CoV-2 region to be detected of the coronavirus obtained in the step (3) as a template to obtain the antisense RNA probe.
3. A kit for detecting coronaviruses, comprising the digoxin-modified antisense RNA probe of claim 1.
4. The kit of claim 3, further comprising: prehybridization solution, blocking solution, AP substrate staining buffer, MABT solution, 50% formamide/2 XSSCT buffer, 0.2 XSSCT buffer, detection buffer, coronavirus SARS-CoV-2 negative control, and coronavirus SARS-CoV-2 positive control.
5. The kit of claim 3, wherein the concentration of the digoxin-modified antisense RNA probe is 2ng/μ L.
6. The kit of claim 4, wherein the prehybridization solution comprises 50% formamide, 5 x SSCT, 50 μ g/mL heparin, 5mM EDTA, 0.05mg/mL ribosomal RNA, 1M citric acid, and 0.1% Tween by volume; the blocking solution comprises 1% sheep serum, 2% blocking agent, 100mM maleic acid and 150mM NaCl; the AP substrate staining buffer solution comprises nitrotetrazolium blue chloride, 5-bromo-4-chloro-3-indolyl phosphate and levamisole; the MABT solution comprises maleic acid and NaCl; the detection buffer comprises 100mM NaCl, 50mM MgCl2100mM tris, 0.1% Tween-20 and 1mM levamisole.
7. The kit of claim 4, wherein the positive control for coronavirus SARS-CoV-2 is 1 x 105Hek293 cells harboring coronavirus SARS-CoV-2; the negative control of coronavirus SARS-CoV-2 is 1 × 105Is not carriedHek293 cells of coronavirus SARS-CoV-2.
8. A method for detecting coronavirus, which is characterized in that the method is used for detecting coronavirus by using the kit of any one of claims 3-7, and comprises the following specific steps:
1) hybridization pretreatment: adding protease K into a sample to be detected for incubation, then rinsing with a PBST solution, then fixing with paraformaldehyde, and then rinsing with the PBST solution;
2) pre-hybridization reaction: adding the pre-hybridization solution into the sample to be detected after pretreatment in the step 1), and incubating;
3) and (3) hybridization reaction: removing the prehybridization solution in the step 2), adding the prehybridization solution containing the digoxin-modified antisense RNA probe in the claim 1 into a sample to be detected, and incubating;
4) and (3) post-hybridization treatment: removing the pre-hybridization solution in the step 3), rinsing the sample to be tested with 50% formamide/2 xSSCT buffer solution, 2 xSSCT buffer solution and 0.2 xSSCT buffer solution in sequence, mixing with blocking solution, culturing at room temperature, adding anti-digoxin antibody diluted by 1000-4000 times of the blocking solution, and incubating and culturing to obtain a hybridization sample;
5) and (3) color development reaction: adding the hybridization sample obtained in the step 4) into an MABT solution containing lamb serum with the final volume fraction of 1%, washing with the MABT solution, washing with a detection buffer solution, adding an AP substrate staining buffer solution for incubation in a dark place at room temperature, washing the hybridization sample with a PBS solution after the hybridization sample is stained, and observing and photographing with a microscope to obtain a result for detecting the coronavirus SARS-CoV-2.
9. The method according to claim 8, wherein the incubation in step 2) is carried out at 68 ℃ for 45 to 240 minutes; the incubation in step 3) is a reaction at 68 ℃ for 10 hours; the incubation in step 4) is carried out at 4 ℃ for 10 hours, and the incubation time at room temperature is 45 minutes to 240 minutes.
10. The method of claim 8, wherein the incubation is performed at room temperature in the dark for 30-120 minutes, and the signal is detected every 15 minutes until the target signal is present.
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