CN111518952A - Kit for nucleic acid-antibody dual detection of novel coronavirus and preparation method thereof - Google Patents
Kit for nucleic acid-antibody dual detection of novel coronavirus and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kit for nucleic acid-antibody dual detection of novel coronavirus and a preparation method thereof, belonging to the technical field of virus detection, wherein the kit comprises a nucleic acid detection system and an antibody detection test strip; the binding region of the nucleic acid detection test strip adsorbs the detection probe modified by the gold-coated silicon sphere nano particles; the detection area of the nucleic acid detection test strip comprises a detection line and a quality control line; and a capture probe is sprayed on the detection line, and a quality control probe is sprayed on the quality control line. The detection area of the antibody detection test strip comprises a first detection line, a second detection line and a quality control line; spraying a mixture of a novel coronavirus specific N protein and a novel coronavirus specific S protein on the first detection line; spraying a mixture of specific N protein and S protein of the novel coronavirus on the second detection line; and spraying a secondary antibody on the quality control line. The kit is simple to operate, the amplification result can be directly judged by naked eyes, and the omission factor is reduced.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a kit for nucleic acid-antibody dual detection of novel coronavirus and a preparation method thereof.
Background
Current detection reagents for novel coronavirus infections fall into two categories: one is direct detection of the virus and the other is indirect detection. The direct detection is the nucleic acid detection aiming at the virus widely applied in the laboratory at present, detects the existence of certain specific nucleic acid sequences in the novel coronavirus gene, and is the currently accepted gold standard method for diagnosing the novel coronavirus infection in the laboratory. Indirect detection reagents are classified into detection against virus antibodies and detection against virus antigens. The antibody detection reagent is used for detecting IgM or IgG antibodies which are produced by stimulating a human body after viruses enter the human body in serum, wherein the IgM antibodies appear earlier, and the IgG antibodies appear later. Virus antigen detection is primarily the detection of some proteins on the surface of the virus.
Most of the current novel coronavirus nucleic acid detection kits on the market adopt a real-time fluorescent quantitative PCR method, and the method is mature in virus detection, but has many limitations, such as: the requirements for sample collection and laboratories and the technical level requirements of laboratory personnel are high, and the detection time is long. From the perspective of feed-forward, the current detection kit for the novel coronavirus nucleic acid has high omission factor, and three to four samples are usually collected to confirm diagnosis. Single detection of antibodies against IgM or IgG is less specific and difficult to detect at the initial stage of infection.
Disclosure of Invention
In view of the above, the present invention aims to provide a nucleic acid-antibody dual detection kit for novel coronavirus and a preparation method thereof; the kit combines nucleic acid detection and antibody detection, greatly reduces the omission factor, is simple to operate, and can realize the rapid and accurate detection of the novel coronavirus.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a kit for double detection of novel coronavirus by nucleic acid-antibody, which comprises a nucleic acid detection system and an antibody detection test strip;
the nucleic acid detection system comprises a recombinase polymerase amplification detection system and a nucleic acid detection test strip; the recombinase polymerase amplification detection system comprises a primer group for amplifying a specific gene of the novel coronavirus; the upstream primer in the primer set comprises a first extension sequence, and the downstream primer in the primer set comprises a second extension sequence;
the binding area of the nucleic acid detection test strip adsorbs the gold-coated silicon sphere nano particles Si02@ Au modified detection probes; the detection probe is complementarily paired with the first extension sequence;
the detection area of the nucleic acid detection test strip comprises a detection line and a quality control line; a capture probe is sprayed on the detection line, and a quality control probe is sprayed on the quality control line; the capture probe is complementarily paired with the second extension sequence; the quality control probe and the detection probe are complementarily paired;
the detection area of the antibody detection test strip comprises a first detection line, a second detection line and a quality control line; spraying a mixture of a novel coronavirus specific N protein and a novel coronavirus specific S protein on the first detection line; spraying a mixture of specific N protein and S protein of the novel coronavirus on the second detection line; and spraying a secondary antibody on the quality control line.
Preferably, the novel coronavirus specific gene comprises a gene encoding ORF1ab and a gene encoding nucleocapsid protein N.
Preferably, the primer set comprises an upstream primer Orf1ab F and a downstream primer Orf1ab R; the nucleotide sequences of the upstream primer Orf1ab F and the downstream primer Orf1ab R are shown as SEQ ID No.1 and SEQ ID No. 2;
the primer group also comprises an upstream primer nucleocapsid protein F and a downstream primer nucleocapsid protein R; the nucleotide sequences of the upstream primer nucleocapsid protein F and the downstream primer nucleocapsid protein R are shown as SEQ ID No.6 and SEQ ID No. 7;
the detection probes comprise a first detection probe and a second detection probe; the nucleotide sequences of the first detection probe and the second detection probe are shown as SEQ ID No.3 and SEQ ID No. 8;
the detection lines comprise a first detection line and a second detection line; a first capture probe and a second capture probe are respectively sprayed on the first detection line and the second detection line; the nucleotide sequence of the first capture probe is shown as SEQ ID No.4, and the nucleotide sequence of the second capture probe is shown as SEQ ID No. 9; spraying a first quality control probe and a second quality control probe on the quality control line; the nucleotide sequence of the first quality control probe is shown as SEQ ID No.5, and the nucleotide sequence of the second quality control probe is shown as SEQ ID No. 10.
Preferably, the concentration of the mixture of the specific N protein and the specific S protein of the novel coronavirus, which is sprayed on the first detection line and the second detection line of the detection area of the antibody detection test strip, is (0.8-1.2) mg/mL; the mass ratio of the N protein to the S protein is 1: 1.
Preferably, the spraying amount of the mixture of the N protein and the S protein specific to the novel coronavirus is 0.8-1.2 mu L/cm.
Preferably, the concentration of the secondary antibody is (0.8-1.2) mg/mL, and the spraying amount of the secondary antibody is 0.8-1.2 muL/cm.
The invention provides a preparation method of the kit, which comprises the preparation of a nucleic acid detection test strip and the preparation of an antibody detection test strip;
the preparation method of the nucleic acid detection test strip comprises the following steps: gold-coated silicon ball nano particles Si02The detection probe modified by @ Au is adsorbed on the bonding pad, the capture probe is sprayed on the detection line of the detection area, and the quality control probe is sprayed on the quality control line of the detection area; then theSequentially overlapping the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on a bottom plate to obtain a nucleic acid detection test strip;
the preparation method of the antibody detection test strip comprises the following steps: adsorbing a gold-labeled antibody on a binding pad, spraying a mixture of a novel coronavirus specific N protein and a novel coronavirus specific S protein on a first detection line and a second detection line of a nitrocellulose membrane, and spraying a secondary antibody on a quality control line; and then the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped on the bottom plate to obtain the antibody detection test strip.
Preferably, the gold-coated silicon spherical nano particle Si02The preparation method of the @ Au modified detection probe comprises the following steps: gold-coated silicon ball nano particles Si02The @ Au solution is obtained by sequentially mixing and coupling dATP, SDS, NaCl and a detection probe.
Preferably, the coupling temperature is 58-62 ℃, and the coupling time is 2-4 h.
Preferably, the gold-coated silicon sphere nano-particle Si02The @ Au modified detection probe is stored in the nanoparticle storage solution.
The invention has the beneficial effects that: the nucleic acid-antibody double detection kit provided by the invention combines nucleic acid detection and antibody detection, greatly improves the detection rate, reduces false negative, reduces the omission factor and discovers suspicious cases as early as possible; the antibody detection test strip adopts a double-antigen mixing method, combines N and S proteins of the new coronavirus, and can enhance the sample detection rate of IgM and IgG, reduce cross reaction and increase specificity; the kit provided by the invention is used for detecting the novel coronavirus, and is simple and rapid to operate and accurate in detection result.
Drawings
FIG. 1 is a schematic diagram illustrating the principle of recombinase polymerase amplification in the present invention;
FIG. 2 is a schematic diagram of the detection principle of the nucleic acid detection test strip;
FIG. 3 shows the result of amplification of a specific gene of a novel coronavirus;
FIG. 4 shows the test result of the nucleic acid test strip;
FIG. 5 is a schematic diagram of the detection principle of the antibody detection test strip;
FIG. 6 is a photograph of the test result of the antibody test strip.
Detailed Description
The invention provides a kit for double detection of novel coronavirus by nucleic acid-antibody, which comprises a nucleic acid detection system and an antibody detection test strip;
the nucleic acid detection system comprises a recombinase polymerase amplification detection system and a nucleic acid detection test strip; the recombinase polymerase amplification detection system comprises a primer group for amplifying a specific gene of the novel coronavirus; the upstream primer in the primer set comprises a first extension sequence, and the downstream primer in the primer set comprises a second extension sequence;
the binding area of the nucleic acid detection test strip adsorbs the gold-coated silicon sphere nano particles Si02@ Au modified detection probes; the detection probe is complementarily paired with the first extension sequence;
the detection area of the nucleic acid detection test strip comprises a detection line and a quality control line; a capture probe is sprayed on the detection line, and a quality control probe is sprayed on the quality control line; the capture probe is complementarily paired with the second extension sequence; the quality control probe and the detection probe are complementarily paired;
the detection area of the antibody detection test strip comprises a first detection line, a second detection line and a quality control line; spraying a mixture of a novel coronavirus specific N protein and a novel coronavirus specific S protein on the first detection line; spraying a mixture of specific N protein and S protein of the novel coronavirus on the second detection line; and spraying a secondary antibody on the quality control line.
In the invention, the recombinase polymerase amplification detection system comprises a primer group for amplifying a specific gene of a novel coronavirus. In the present invention, the novel coronavirus specific gene preferably includes a gene encoding ORF1ab and/or a gene encoding nucleocapsid protein N. Preferably, the primer set comprises an upstream primer Orf1ab F and a downstream primer Orf1ab R; the nucleotide sequences of the upstream primer Orf1ab F and the downstream primer Orf1ab R are shown as SEQ ID No.1 and SEQ ID No. 2. In the invention, the primer group further comprises an upstream primer nucleocapsid protein F and a downstream primer nucleocapsid protein R; the nucleotide sequences of the upstream primer nucleocapsid protein F and the downstream primer nucleocapsid protein R are shown as SEQ ID No.6 and SEQ ID No. 7. In the present invention, the detection probe comprises a first detection probe and a second detection probe; the nucleotide sequences of the first detection probe and the second detection probe are shown as SEQ ID No.3 and SEQ ID No. 8; the detection lines comprise a first detection line and a second detection line; a first capture probe and a second capture probe are respectively sprayed on the first detection line and the second detection line; the nucleotide sequence of the first capture probe is shown as SEQ ID No.4, and the nucleotide sequence of the second capture probe is shown as SEQ ID No. 9; spraying a first quality control probe and a second quality control probe on the quality control line; the nucleotide sequence of the first quality control probe is shown as SEQ ID No.5, and the nucleotide sequence of the second quality control probe is shown as SEQ ID No. 10.
In the invention, the recombinase polymerase amplification detection system also comprises RPA reaction Mix and MgOAc. In the present invention, the source of the RPA reaction Mix is not particularly limited, and a commercially available RPA reaction Mix conventionally used in the art may be used, and in the present invention, the concentration of MgOAc is preferably 250 to 300mM, and more preferably 280 mM.
In the invention, the nucleic acid detection test strip comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped.
In the invention, the binding area of the test strip adsorbs the gold-coated silicon sphere nano particles Si02@ Au modified detection probes; the detection probe is complementarily paired with the first extension sequence; the detection area of the test strip comprises a detection line and a quality control line; a capture probe is sprayed on the detection line, and a quality control probe is sprayed on the quality control line; the capture probe is complementarily paired with the second extension sequence; and the quality control probe and the detection probe are complementarily paired.
In the invention, aiming at the gene coding ORF1ab, the nucleotide sequence of the detection probe is shown as SEQ ID No.3, and the specific steps are as follows: 5-AAAAAAAAAAAAAAA-3-SH of Orf1abdP, and the nucleotide sequence of the capture probe is shown in SEQ ID No.4, and the details are as follows: 5-GGGGGGGGGGGGGGG-3-Biotin; the nucleotide sequence of the quality control probe is shown as SEQ ID No.5, and specifically comprises the following steps: 5-TTTTTTTTTT-3-Biotin.
In the invention, when the gene for coding the nucleocapsid protein N is aimed at, the nucleotide sequence of the detection probe is shown as SEQ ID No.8, and the nucleotide sequence is as follows: 5-GAGAGCGGGTTCACG ttt-3-SH; the nucleotide sequence of the capture probe is shown as SEQ ID No.9, and specifically comprises the following steps: 5-CAGGAGAAGGGTTTT ttt-3-Biotin; the nucleotide sequence of the quality control probe is shown as SEQ ID No.10, and specifically comprises the following steps: 5-CGTGAACCCG-3-Biotin.
In the invention, the kit also comprises an antibody detection test strip. In the invention, the concentration of the mixture of the novel coronavirus specific N protein and the S protein sprayed on the first detection line and the second detection line of the detection area of the antibody detection test strip is preferably (0.8-1.2) mg/mL, more preferably 1.0mg/mL, and the mass ratio of the N protein to the S protein is preferably 1: 1. In the invention, the spraying amount of the mixture of the N protein and the S protein specific to the novel coronavirus is preferably 0.8-1.2 muL/cm, and more preferably 1.0 muL; in the present invention, the novel coronavirus specific N-protein and S-protein are preferably purchased from general biosystems (Anhui) Ltd. In the invention, the concentration of the secondary antibody is preferably (0.8-1.2) mg/mL, and more preferably 1.0 mg/mL; the spraying amount of the secondary antibody is preferably 0.8-1.2 mu L/cm, more preferably 1.0 mu L/cm, in the invention, the secondary antibody is preferably goat anti-rabbit IgG, the source of the secondary antibody is not particularly limited, and the secondary antibody can be a conventional commercial product in the field.
In the invention, the binding region of the antibody detection test strip adsorbs the gold-labeled antibody; the gold-labeled antibody preferably comprises gold-labeled rabbit anti-human IgM and gold-labeled rabbit anti-human IgG. In the present invention, the rabbit anti-human IgM and rabbit anti-human IgG are purchased from general biosystems (Anhui) Ltd.
The invention provides a preparation method of the kit, which comprises the preparation of a nucleic acid detection test strip and the preparation of an antibody detection test strip.
In the present invention, the test strip for detecting nucleic acidThe preparation method comprises the following steps: gold-coated silicon ball nano particles Si02The detection probe modified by @ Au is adsorbed on the bonding pad, the capture probe is sprayed on the detection line of the detection area, and the quality control probe is sprayed on the quality control line of the detection area; and then the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped on the bottom plate to obtain the nucleic acid detection test strip.
In the invention, the gold-coated silicon sphere nano-particle Si02The preparation method of the @ Au modified detection probe comprises the following steps: gold-coated silicon ball nano particles Si02The @ Au solution is obtained by sequentially mixing and coupling dATP, SDS, NaCl and a detection probe. In the invention, the coupling temperature is preferably 58-62 ℃, more preferably 60 ℃, and the coupling time is preferably 2-4 h, more preferably 3 h. In the invention, after the coupling, solid-liquid separation is preferably realized through centrifugation, and the collected solid-phase components are stored after being washed. In the invention, the rotation speed of the centrifugation is preferably 10000-14000 rpm, more preferably 12000rpm, the time of the centrifugation is preferably 8-12 min, more preferably 10min, the washing is preferably carried out by using a PBS buffer solution, and the number of times of the washing is preferably 2-4 times, more preferably 3 times. In the invention, the gold-coated silicon sphere nano-particle Si02The @ Au modified detection probe is preferably preserved in a nanoparticle storage solution; the gold-coated silicon spherical nano-particle Si02The preferred preservation temperature of the @ Au modified detection probe is 4 ℃; in the present invention, the nanoparticle storage solution uses water as a solvent, and preferably comprises the following components: 20mmol/L Na3PO4·12H2O, 5% BSA, 0.25% Tween 20, and 10% sucrose.
In the specific implementation process of the invention, the gold-coated silicon sphere nano-particles Si02Mixing the @ Au solution and dATP, and preferably oscillating; the gold-coated silicon spherical nano-particle Si02The volume ratio of the @ Au solution to the dATP is preferably (90-110): 1, and more preferably 100: 1; the concentration of dATP is preferably 0.8-1.2 mM, and more preferably 1 mM; the oscillation time is preferably 15-25 min, and more preferably 20 min. After the oscillation, mixing the oscillated liquid with SDS, and carrying out second oscillation; mass concentration of the SDSThe degree is preferably 0.8% to 1.2%, more preferably 1%; the SDS and the gold-coated silicon sphere nano-particle Si02The volume ratio of the @ Au solution is preferably (180-220): 3, and more preferably 200: 3; the time of the second oscillation is preferably 8-12 min, and more preferably 10 min. After the second oscillation, adding NaCl into the system, wherein the concentration of the NaCl is preferably 0.1-0.3M, and more preferably 0.2M; the NaCl and the gold-coated silicon sphere nano-particles Si02The volume ratio of the @ Au solution is preferably (18-22): 1, and more preferably 20: 1; in the invention, the adding speed of the NaCl is preferably 2 mu L every 2-3 min. In the present invention, after NaCl is added, a 1OD detection probe is preferably added to the system.
In the invention, the gold-coated silicon sphere nano-particle Si02The @ Au solution is preferably obtained by mixing and adsorbing the gold cluster solution and the silicon sphere solution; in the invention, the volume ratio of the gold cluster solution to the silicon sphere solution is preferably (3-5): 1, and more preferably 4: 1; the concentration of the silicon sphere liquid is preferably 2-3 mg/L, and more preferably 2.5 mg/L. In the present invention, the adsorption is preferably performed on a magnetic stirrer, the temperature of the adsorption is not particularly limited in the present invention, and the adsorption time is preferably 1 hour, and room temperature may be sufficient.
In the present invention, the preparation method of the silicon sphere liquid is preferably as follows: mixing and stirring absolute ethyl alcohol, deionized water and ammonia water, then mixing and reacting with tetraethoxysilane, carrying out solid-liquid separation, collecting solid phase components, and suspending the solid phase components in the deionized water or the ethanol to obtain silicon sphere liquid. In the invention, the volume ratio of the absolute ethyl alcohol to the deionized water to the ammonia water to the tetraethoxysilane is preferably 128:18:3: 3; the mixing and stirring temperature is preferably 28-32 ℃, and more preferably 30 ℃; the mixing and stirring time is preferably 8-12 min, and more preferably 10 min. In the invention, the mixing reaction time is preferably 1.5-2.5 h, and more preferably 2 h. In the invention, the solid-liquid separation is preferably centrifugation, the rotating speed is preferably 14000-16000 rpm, more preferably 15000rpm, and the centrifugation time is preferably 15-25 min, more preferably 20 min. In the present invention, the solid phase component is preferably washed and then resuspended, the washing is preferably performed by sequentially using ethanol and water, the number of times of ethanol washing is preferably 1 to 3 times, more preferably 2 times, and the number of times of water washing is preferably 1 to 3 times, more preferably 2 times. In the present invention, the ratio of the solid phase component to the resuspension water (or ethanol) is preferably 5g (45-55) mL, and more preferably 5g:50 mL.
In the present invention, the gold cluster solution is preferably prepared by the following method: adding HAuCl4And mixing the double distilled water and sodium citrate and then mixing with sodium borohydride to obtain the sodium hydrogen borate. In the present invention, the HAuCl is4The volume ratio of the double distilled water is preferably 1: 50; the HAuCl4The mass concentration of (b) is preferably 1%; in the present invention, the HAuCl is4The ratio of sodium citrate to sodium citrate is preferably 1mL: 0.03M; in the present invention, said sodium borohydride is reacted with HAuCl4Is preferably 1:1, and the concentration of the sodium borohydride is preferably 0.1M. In the present invention, the HAuCl is4The mixing time of the double distilled water and the sodium citrate is preferably 20 s.
The gold-coated silicon sphere nano-particle Si0 is prepared and obtained in the invention2After the detection probe modified by @ Au, the gold-coated silicon sphere nano-particle Si02The @ Au modified detection probe was sprayed (4. mu.L/strip) onto the conjugate pad.
In the invention, a capture probe is sprayed on a detection line of a detection area, and a quality control probe is sprayed on a quality control line of the detection area; and then the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped on the bottom plate to obtain the test strip.
In the present invention, the capture probe is preferably a biotin-or streptavidin-labeled capture probe. In the present invention, the method for preparing the capture probe preferably comprises the steps of: and mixing the capture probe marked by the biotin and streptavidin, and dialyzing to obtain the product. In the invention, the mixing time is preferably 50-70 min, and more preferably 1 h; the cut-off for the dialysis is preferably 30000; in the present invention, the dialysis is preferably performed in a dialysis tube; the invention is centrifuged after the dialysis, and the solution in the dialyzing tube after centrifugation is collected to obtain the capture probe. In the invention, the rotation speed of the centrifugation is preferably 5000-7000 rpm, more preferably 6000rpm, the time of the centrifugation is preferably 15-25 min, more preferably 20min, and the temperature of the centrifugation is preferably 3-5 ℃, more preferably 4 ℃; the centrifugation serves to remove unbound aptamer probes. In the present invention, the ratio of the biotin-labeled capture probe to streptavidin is preferably 50nM:0.5 mg.
In the invention, the spraying concentration of the capture probe and the quality control probe is preferably 1.2-1.8 muL/cm, and more preferably 1.5 muL/cm. The specific method of the spraying is not particularly limited in the present invention, and a conventional spraying method in the art can be adopted.
In the invention, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially lapped on a bottom plate to obtain the test strip. In the present invention, the material of the sample pad is preferably a glass fiber membrane. The sample pad has the function of slowing down the migration speed of a sample to be detected, is beneficial to the uniform distribution of liquid to be detected on the sample pad, and creates a precondition for better flowing to the combination pad. In the invention, the material of the bonding pad is preferably a glass fiber film; the bonding pad is used for adsorbing gold-coated silicon ball nanoparticles (Si 0)2@ Au), and uniformly conveying the sample solution to be detected to an NC membrane at a certain speed under the action of capillary adsorption force; the stability and the integrity of subsequent marker particles are maintained, the background signal is reduced, and the stability and the repeatability of detection are improved. In the present invention, the absorbent pad preferably uses absorbent paper having high absorption efficiency, large capacity and good stability. The absorption pad aims to promote the migration of liquid on the cross flow test strip to smoothly and completely reach the absorption pad, ensure that an object (liquid) to be detected can cross an NC membrane through the last step of siphoning action, improve the output value of a detection signal and reduce the signal-to-noise ratio.
In the practice of the present invention, it is preferred that the nitrocellulose membrane (NC membrane) is first adhered to the PVC base plate, then the conjugate pad is fixed in place and secured in overlap with the NC membrane, then the sample pad is adhered in place and secured in overlap with the conjugate pad, and finally the absorbent pad is adhered and secured in overlap with the NC membrane to ensure smooth liquid flow. In the present invention, the length of the overlap is preferably 2 mm.
In the invention, the preparation of the kit further comprises the preparation of an antibody detection test strip: the preparation method of the antibody detection test strip comprises the following steps: adsorbing a gold-labeled antibody on a binding pad, spraying a mixture of a novel coronavirus specific N protein and a novel coronavirus specific S protein on a first detection line and a second detection line of a nitrocellulose membrane, and spraying a secondary antibody on a quality control line; and then the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped on the bottom plate to obtain the antibody detection test strip.
In the present invention, the method for preparing the gold-labeled antibody preferably comprises the steps of: mixing and oscillating the nano gold solution and the antibody, then mixing the nano gold solution and BSA, carrying out solid-liquid separation, collecting solid phase components, washing and re-suspending to obtain the antibody. In the invention, the volume ratio of the nano gold solution to the antibody is (80-120): 1, and more preferably 100: 1; the concentration of the antibody is preferably 0.8-1.2 mg/mL, and more preferably 1.0 mg/mL; the particle size of the nano gold is preferably 15-20 nm. In the invention, the mixing oscillation is preferably performed on an oscillator, and the oscillation time is preferably 50-70 min, and more preferably 60 min. In the invention, the volume ratio of the BSA to the antibody is preferably (8-12) to 1, and more preferably 10 to 1; the mass concentration of BSA is preferably 8% to 12%, and more preferably 10%. After the BSA and the system are mixed, the system is vibrated, and the vibration time is preferably 25-35 min, and more preferably 30 min. In the invention, the solid-liquid separation is preferably centrifugation, and the rotation speed of the centrifugation is preferably 10000-14000 rpm, and more preferably 12000 rpm; the time for centrifugation is preferably 8-12 min, and more preferably 10 min. In the invention, the washing is performed by using a PBS buffer solution, and the number of times of washing is preferably 2-4 times, and more preferably 3 times. In the present invention, the gold-labeled antibody is preferably preserved in a nanoparticle storage solution; the storage temperature of the gold-labeled antibody is preferably 4 ℃; in the present invention, the nanoparticle storage solution uses water as a solvent, and preferably comprises the following components: 20mmol/L Na3PO4·12H2O, 5% BSA, 0.25% Tween 20, and 10% sucrose.
In the present invention, the material of the sample pad is preferably a glass fiber membrane. The sample pad has the function of slowing down the migration speed of a sample to be detected, is beneficial to the uniform distribution of liquid to be detected on the sample pad, and creates a precondition for better flowing to the combination pad. In the invention, the material of the bonding pad is preferably a glass fiber film; the bonding pad is used for adsorbing gold-coated silicon ball nanoparticles (Si 0)2@ Au), and uniformly conveying the sample solution to be detected to an NC membrane at a certain speed under the action of capillary adsorption force; the stability and the integrity of subsequent marker particles are maintained, the background signal is reduced, and the stability and the repeatability of detection are improved. In the present invention, the absorbent pad preferably uses absorbent paper having high absorption efficiency, large capacity and good stability. The absorption pad aims to promote the migration of liquid on the cross flow test strip to smoothly and completely reach the absorption pad, ensure that an object (liquid) to be detected can cross an NC membrane through the last step of siphoning action, improve the output value of a detection signal and reduce the signal-to-noise ratio.
In the practice of the present invention, it is preferred that the nitrocellulose membrane (NC membrane) is first adhered to the PVC base plate, then the conjugate pad is fixed in place and secured in overlap with the NC membrane, then the sample pad is adhered in place and secured in overlap with the conjugate pad, and finally the absorbent pad is adhered and secured in overlap with the NC membrane to ensure smooth liquid flow. In the present invention, the length of the overlap is preferably 2 mm.
In the present invention, the method of using the kit preferably comprises the steps of: s1) extracting the total RNA of the sample to be detected and then carrying out reverse transcription to obtain cDNA; s2) using the cDNA as a template, and using a recombinase polymerase amplification detection system in the kit to carry out RPA amplification to obtain an amplification product; s3) dripping the amplification product on a sample pad of the nucleic acid detection test strip, and observing the detection result; s4) adding the sample to be detected dropwise into the antibody detection test strip, and observing the result.
In the invention, when any one of the first detection line and the second detection line of the nucleic acid detection test strip is colored and the quality control line is colored, the fact that the sample to be detected contains the novel coronavirus is indicated, and when the first detection line and the second detection line of the nucleic acid detection test strip are not colored and the quality control line is colored, the fact that the sample to be detected contains the novel coronavirus is indicated; and when the quality control line of the test strip does not develop color, the test strip is invalid. The specific methods and parameters for extracting, reverse transcribing and amplifying the total RNA are not particularly limited in the present invention, and the methods and parameters conventional in the art may be used. In the present invention, the amplification product is preferably mixed with 1/4SSC buffer solution at a ratio of 1:9, and then dropped on the test strip sample pad, and the observation of the detection result is preferably performed 10min after the sample application.
In the invention, when any one of the first detection line and the second detection line of the antibody detection test strip develops color and the quality control line develops color, the fact that the sample to be detected contains the novel coronavirus is shown; the first detection line is colored, the second detection line is not colored to indicate early infection, the first detection line is not colored, and the second detection line is colored to indicate middle and later infection; when the first detection line and the second detection line of the antibody detection test strip do not develop color, and the quality control line develops color, the fact that the sample to be detected does not contain the novel coronavirus is shown; when the quality control line of the antibody detection test strip does not develop color, the test strip is invalid. In the invention, preferably, the sample to be detected and the sample buffer solution are mixed and then are dripped into the antibody detection test strip. In the present invention, the sample to be tested is preferably blood, and the sample buffer is preferably a PBS buffer containing 1% BSA; the volume ratio of the sample to be detected to the sample buffer solution is preferably 3: 5.
In the invention, a nucleic acid detection test strip and an antibody detection test strip are used for simultaneously detecting a sample to be detected, and when the detection result of the nucleic acid detection test strip or the antibody detection test strip is positive, the sample to be detected is considered to contain the novel coronavirus.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
SiO2Preparation of @ Au nano material:
firstly, preparing SiO with the diameter of 150nm2Silicon ballLiquid: 128mL of absolute ethanol, 18mL of deionized water and 3mL of ammonia water were mixed, and the mixture was stirred at 30 ℃ for 10 min. Then 3mL of tetraethyl orthosilicate TEOS is added, and the mixture is stirred and reacts for 2 hours. Centrifuging at 15000rpm for 20min, collecting precipitate, washing with ethanol twice, washing with water twice, centrifuging, and suspending the precipitate in 50mL deionized water or ethanol to obtain SiO2And (4) silicon ball liquid.
Preparing a gold cluster solution: 1mL of 1% HAuCl4+50mL of double distilled water +0.03M of sodium citrate, mixing for 20s, and +1mL of 0.1M of sodium borohydride.
Ratio of gold cluster liquid to silicon sphere liquid: 2mL (5mg) of silica sphere solution was slowly added dropwise to 8mL of gold clusters.
The specific adsorption conditions are as follows: stirring for 1h at room temperature on a magnetic stirrer.
Preparing the test strip:
(1) sample pad: the material is glass fiber film. The device has the function of slowing down the migration speed of a sample to be detected, is favorable for uniform distribution of liquid to be detected in a sample pad, and creates a precondition for better flowing to a combination pad.
(2) Combining the pads: a glass fiber membrane. The bonding pad is used for adsorbing gold-coated silicon sphere nano particles (Si 0)2@ Au), and uniformly conveying the sample solution to be detected to an NC membrane at a certain speed under the action of capillary adsorption force; the stability and the integrity of subsequent marker particles are maintained, the background signal is reduced, and the stability and the repeatability of detection are improved.
DNA Probe labeling Si02@ Au step: 1mL of 3-fold concentrated Si0 was taken2@ Au solution, 10. mu.L of 1mM dATP was added thereto, shaking was carried out at room temperature for 20min with a shaker, 15. mu.L of 1% SDS was then added thereto, after shaking culture was carried out for 10min, 50. mu.L of 0.2M NaCl was added thereto (2. mu.L per 2 to 3 min), 1OD detection probe was added thereto, reaction was carried out at 60 ℃ for 3 hours, centrifugation was carried out with a centrifuge (rotation speed 12,000rpm, 10min), the supernatant was removed, washing was carried out 3 times with PBS buffer (pH 7.2 to 7.4), and finally the precipitate was dissolved in 1mL of nanoparticle stock solution (20mmol/L Na)3PO4·12H2O, 5% BSA, 0.25% Tween 20, 10% sucrose), and storing the conjugate solution in a refrigerator at 4 ℃ for later use.
(3) NC membrane (Nitrocellulose membrane): comprises a detection line 1, a detection line 2 and a quality control line. The detection center region of the lateral flow strip was mainly concentrated on the nitrocellulose membrane. Generally, the detection result is measured by a detection line and a quality control line, and the color on the detection line can be subjected to semi-quantitative or quantitative and qualitative analysis.
The quality control line is used for verifying the detection effectiveness of the test strip. If the quality control line has no color, the test result of the test strip is not credible.
Quality control line spraying: 5-TTTTTTTTTT-3-Biotin and 5-CGTGAACCCG-3-Biotin
The spraying concentration is as follows: 1.5. mu.L/cm.
And (3) processing a capture probe: after incubation of 50nM biotin-labeled capture probe with 200. mu.L 2.5mg/ml streptavidin (Shanghai Biotech) in 0.01MPBS for 1h, the mixture was transferred to a dialysis tube (molecular weight cut-off 30000) and centrifuged (6000rpm, 20min, 4 ℃) in a refrigerated centrifuge to remove unbound aptamer probe. The above steps were repeated twice with PBS, and finally the solution trapped after centrifugation was collected and made up to 600. mu.L. And finally, spraying a mixture solution of biotin-labeled DNA and streptavidin on the test strip.
(4) An absorption pad: the absorbent paper with high absorption efficiency, large capacity and good stability is used. The absorption pad is used for promoting the migration of liquid on the cross flow test strip to smoothly and completely reach the absorption pad, ensuring that an object (liquid) to be detected can cross the NC membrane through the last step of siphoning action, improving the output value of a detection signal and reducing the signal-to-noise ratio.
The position relation is as follows: a nitrocellulose membrane (NC membrane) was first attached to the PVC base plate, then the conjugate pad was fixed in place and ensured to have a 2mm overlap with the NC membrane, then the sample pad was attached in place and ensured to have a 2mm overlap with the conjugate pad, and finally the absorbent pad was attached and ensured to have a 2mm overlap with the NC membrane to ensure smooth liquid flow.
The detection process of the kit comprises the following steps:
the first step is as follows: RPA reaction
41.5 mul of the solution A, 2 mul of the primers at the upstream and the downstream, 2 mul of the sample material to be detected, 2.5 mul of the solution B, and 37 ℃ are added into a reaction tube for reaction for 30 min.
Solution A is Mix of RPA reaction. The solution B is MgOAc with a concentration of 280 mM.
The template is artificially prepared PUC57 plasmid containing virus specific genes;
5-cccccccccc (first extension sequence) -C3-ccctgtgggttttacacttaaaaac-3
5-tttttttttt (second extension sequence) -C3-acgattgtgcatcagctgactgaag-3
Wherein C3 is not a base, the nucleotide sequence of the first extended sequence is shown in SEQ ID No.11,
the nucleotide sequence of the second extension sequence is shown as SEQ ID No. 12.
5-aaaacccttctcctg (third extension sequence) -C3-ggggaacttctcctgctagaat-3
5-cgtgaacccgctctc (fourth extension sequence) -C3-cagacattttgctctcaagctg-3
(primer 1, primer 2) and (primer 3, primer 4) were amplified separately.
Wherein C3 is not a base, the nucleotide sequence of the first extended sequence is shown in SEQ ID No.13,
the nucleotide sequence of the second extension sequence is shown as SEQ ID No. 14.
The product was subjected to polyacrylamide gel electrophoresis, and the results are shown in FIG. 3, with good amplification results and a single band at the amplification site.
The second step is that: test strip detection
And (3) taking 10 mu L of RPA amplification product, uniformly mixing with 90 mu L of 1/4SSC buffer solution, dropwise adding the mixture on a test strip sample pad, and observing the result after 10 min.
The test strip can specifically detect the new coronavirus nucleocapsid protein and the Orf1ab conserved sequence which are amplified isothermally, the result is shown in figure 4, and after RPA amplification is carried out by respectively taking artificially prepared plasmids with the new coronavirus nucleocapsid protein and the Orf1ab conserved sequence as templates, the test strip provided by the invention is adopted for detection, so that a positive strip appears in an amplification product.
The detection rate of the nucleic acid detection test strip reaches 100 percent by verifying 4 cases through positive samples in the Anhui disease control center by using the kit in the embodiment.
Antibody detection test paper strip
The main components and the detection principle of the test strip are shown in figure 5:
(1) sample pad: the material is glass fiber film. The device has the function of slowing down the migration speed of a sample to be detected, is favorable for uniform distribution of liquid to be detected in a sample pad, and creates a precondition for better flowing to a combination pad.
(2) Combining the pads: a glass fiber membrane. The combination pad is used for adsorbing the colloidal gold modified antibody and uniformly conveying the sample solution to be detected to an NC membrane at a certain speed under the action of capillary adsorption force; the stability and the integrity of subsequent marker particles are maintained, the background signal is reduced, and the stability and the repeatability of detection are improved.
Labeling the colloidal gold with an antibody: adding 10 mu L of 1mg/mL antibody (rabbit anti-human IgM and rabbit anti-human IgG purchased from general biological System (Anhui) Co., Ltd.) into 1mL of five-fold concentrated nanogold (15-20 nm) solution, shaking with a shaker at room temperature for 60min, adding 100 mu L of 10% BSA, shaking for 30min, centrifuging at 12,000rpm for 10min, removing supernatant, washing with PBS buffer (pH 7.2-7.4) for 3 times, and dissolving the precipitate in 1mL of nanoparticle stock solution (20mmol L-1 Na)3PO4·12H2O, 5% BSA, 0.25% Tween 20, 10% sucrose), stored at 4 ℃ in a refrigerator until use.
(3) NC membrane (Nitrocellulose membrane): comprises a first detection line, a second detection line 2 and a quality control line. The detection center region of the lateral flow strip was mainly concentrated on the nitrocellulose membrane. Generally, the detection result is measured by a detection line and a quality control line, and the color on the detection line can be subjected to semi-quantitative or quantitative and qualitative analysis.
The quality control line is used for verifying the detection effectiveness of the test strip. If the quality control line has no color, the test result of the test strip is not credible.
First detection line 1: a mixture of N and S proteins specific for the novel coronavirus (1 mg/mL); concentration: 1.0. mu.L/cm.
Second detection line 2: a mixture of N and S proteins specific for the novel coronavirus (1 mg/mL); concentration: 1.0. mu.L/cm.
Quality control line: secondary antibody (goat anti-rabbit IgG) concentration: 1.0. mu.L/cm
Protein sources: general biosystems (Anhui) Ltd
(4) An absorption pad: the absorbent paper with high absorption efficiency, large capacity and good stability is used. The absorption pad is used for promoting the migration of liquid on the cross flow test strip to smoothly and completely reach the absorption pad, ensuring that an object (liquid) to be detected can cross the NC membrane through the last step of siphoning action, improving the output value of a detection signal and reducing the signal-to-noise ratio.
The position relation is as follows: a nitrocellulose membrane (NC membrane) was first attached to the PVC base plate, then the conjugate pad was fixed in place and ensured to have a 2mm overlap with the NC membrane, then the sample pad was attached in place and ensured to have a 2mm overlap with the conjugate pad, and finally the absorbent pad was attached and ensured to have a 2mm overlap with the NC membrane to ensure smooth liquid flow.
The antibody detection test strip is used for detecting a sample to be detected, and partial detection results are shown in fig. 6.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (10)
1. A kit for nucleic acid-antibody dual detection of novel coronavirus is characterized by comprising a nucleic acid detection system and an antibody detection test strip;
the nucleic acid detection system comprises a recombinase polymerase amplification detection system and a nucleic acid detection test strip; the recombinase polymerase amplification detection system comprises a primer group for amplifying a specific gene of the novel coronavirus; the upstream primer in the primer set comprises a first extension sequence, and the downstream primer in the primer set comprises a second extension sequence;
the binding area of the nucleic acid detection test strip adsorbs the gold-coated silicon sphere nano particles Si02@ Au modified detection probes; the detection probe is complementarily paired with the first extension sequence;
the detection area of the nucleic acid detection test strip comprises a detection line and a quality control line; a capture probe is sprayed on the detection line, and a quality control probe is sprayed on the quality control line; the capture probe is complementarily paired with the second extension sequence; the quality control probe and the detection probe are complementarily paired;
the detection area of the antibody detection test strip comprises a first detection line, a second detection line and a quality control line; spraying a mixture of a novel coronavirus specific N protein and a novel coronavirus specific S protein on the first detection line; spraying a mixture of specific N protein and S protein of the novel coronavirus on the second detection line; and spraying a secondary antibody on the quality control line.
2. The kit according to claim 1, wherein the novel coronavirus specific genes comprise a gene encoding ORF1ab and a gene encoding nucleocapsid protein N.
3. The kit of claim 2, wherein the primer set comprises an upstream primer Orf1ab F and a downstream primer Orf1ab R; the nucleotide sequences of the upstream primer Orf1ab F and the downstream primer Orf1ab R are shown as SEQ ID No.1 and SEQ ID No. 2;
the primer group also comprises an upstream primer nucleocapsid protein F and a downstream primer nucleocapsid protein R; the nucleotide sequences of the upstream primer nucleocapsid protein F and the downstream primer nucleocapsid protein R are shown as SEQ ID No.6 and SEQ ID No. 7;
the detection probes comprise a first detection probe and a second detection probe; the nucleotide sequences of the first detection probe and the second detection probe are shown as SEQ ID No.3 and SEQ ID No. 8;
the detection lines comprise a first detection line and a second detection line; a first capture probe and a second capture probe are respectively sprayed on the first detection line and the second detection line; the nucleotide sequence of the first capture probe is shown as SEQ ID No.4, and the nucleotide sequence of the second capture probe is shown as SEQ ID No. 9; spraying a first quality control probe and a second quality control probe on the quality control line; the nucleotide sequence of the first quality control probe is shown as SEQ ID No.5, and the nucleotide sequence of the second quality control probe is shown as SEQ ID No. 10.
4. The kit according to claim 1, wherein the concentration of the mixture of the N protein and the S protein specific to the novel coronavirus sprayed on the first detection line and the second detection line of the detection zone of the antibody detection test strip is (0.8-1.2) mg/mL; the mass ratio of the N protein to the S protein is 1: 1.
5. The kit according to claim 4, wherein the amount of the mixture of N protein and S protein specific to the novel coronavirus to be sprayed is 0.8 to 1.2. mu.L/cm.
6. The kit according to claim 1, wherein the concentration of the secondary antibody is (0.8-1.2) mg/mL, and the spraying amount of the secondary antibody is 0.8-1.2 μ L/cm.
7. The preparation method of the kit of any one of claims 1 to 6, comprising the steps of preparing a nucleic acid detection test strip and preparing an antibody detection test strip;
the preparation method of the nucleic acid detection test strip comprises the following steps: gold-coated silicon ball nano particles Si02The detection probe modified by @ Au is adsorbed on the bonding pad, the capture probe is sprayed on the detection line of the detection area, and the quality control probe is sprayed on the quality control line of the detection area; then, sequentially overlapping the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on a bottom plate to obtain a nucleic acid detection test strip;
the preparation method of the antibody detection test strip comprises the following steps: adsorbing a gold-labeled antibody on a binding pad, spraying a mixture of a novel coronavirus specific N protein and a novel coronavirus specific S protein on a first detection line and a second detection line of a nitrocellulose membrane, and spraying a secondary antibody on a quality control line; and then the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially lapped on the bottom plate to obtain the antibody detection test strip.
8. The preparation method according to claim 7, wherein the gold-coated silicon sphere nanoparticles Si02The preparation method of the @ Au modified detection probe comprises the following steps: gold-coated silicon ball nano particles Si02The @ Au solution is obtained by sequentially mixing and coupling dATP, SDS, NaCl and a detection probe.
9. The method according to claim 8, wherein the temperature of the coupling is 58 to 62 ℃ and the time of the coupling is 2 to 4 hours.
10. The preparation method according to claim 8, wherein the gold-coated silicon sphere nanoparticles Si02The @ Au modified detection probe is stored in the nanoparticle storage solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023279703A1 (en) * | 2021-07-07 | 2023-01-12 | 英飞凌生物科技(江苏)有限公司 | Primer probe for detecting egfr gene l858r mutation, kit, and use thereof |
| WO2023284476A1 (en) * | 2021-07-12 | 2023-01-19 | 圣湘生物科技股份有限公司 | Novel sars-cov-2 antibody detection test strip |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013122453A1 (en) * | 2012-02-15 | 2013-08-22 | Universiti Sains Malaysia | Device and method for detection of nucleic acid(s) |
| CN110894217A (en) * | 2019-12-16 | 2020-03-20 | 中国农业大学 | Bovine coronavirus chimeric antigen and colloidal gold immunochromatography test paper card for detecting bovine coronavirus antibody |
| CN111089962A (en) * | 2020-03-25 | 2020-05-01 | 中山生物工程有限公司 | Colloidal gold kit for joint detection of novel coronavirus IgM/IgG antibody and preparation method thereof |
-
2020
- 2020-05-07 CN CN202010377660.7A patent/CN111518952A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013122453A1 (en) * | 2012-02-15 | 2013-08-22 | Universiti Sains Malaysia | Device and method for detection of nucleic acid(s) |
| CN110894217A (en) * | 2019-12-16 | 2020-03-20 | 中国农业大学 | Bovine coronavirus chimeric antigen and colloidal gold immunochromatography test paper card for detecting bovine coronavirus antibody |
| CN111089962A (en) * | 2020-03-25 | 2020-05-01 | 中山生物工程有限公司 | Colloidal gold kit for joint detection of novel coronavirus IgM/IgG antibody and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| MIRIAM JAUSET-RUBIO ET AL.: "Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay", 《SCIENTIFIC REPORTS》 * |
| XUN MAO ET AL.: "Disposable Nucleic Acid Biosensors Based on Gold Nanoparticle Probes and Lateral Flow Strip", 《ANAL. CHEM.》 * |
| 匡慧慧等: "新型冠状病毒实验室核酸检测方法及实践", 《中华医院感染学杂志》 * |
| 宋靓婧: "基于纳米金标记的沙门氏菌检测方法研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023279703A1 (en) * | 2021-07-07 | 2023-01-12 | 英飞凌生物科技(江苏)有限公司 | Primer probe for detecting egfr gene l858r mutation, kit, and use thereof |
| WO2023284476A1 (en) * | 2021-07-12 | 2023-01-19 | 圣湘生物科技股份有限公司 | Novel sars-cov-2 antibody detection test strip |
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