CN113189333A - Kit containing quantum dot immunofluorescence detection reagent strip and application of kit - Google Patents
Kit containing quantum dot immunofluorescence detection reagent strip and application of kit Download PDFInfo
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Abstract
The invention provides a quantum dot immunofluorescence detection reagent strip, which comprises a kit and application thereof, and relates to the technical field of biological detection. The reagent strip adopts a quantum dot immunochromatography technology, takes the novel coronavirus N protein as a detection antigen, can detect a biological sample of a suspected novel coronavirus infected person, is ultrasensitive to the detection of the novel coronavirus, and has an LOD value of 15TCID50/mL and a corresponding nucleic acid CT value of 34.25; through detection and judgment, when the CT is less than or equal to 35, the positive coincidence rate of the kit is 96.06%, the negative coincidence rate is 100.00%, and the total coincidence rate is 98.56%; when the CT of the sample is less than or equal to 38, the positive coincidence rate of the kit is 89.58%, the negative coincidence rate of the kit is 100.00%, and the total coincidence rate of the kit is 95.88%. The kit prepared by the reagent strip has the advantages of high specificity, no cross contamination and quantitative detection.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a quantum dot immunofluorescence detection reagent strip and a kit containing the same and application of the reagent strip.
Background
The existing nucleic acid detection and CT imaging are the main methods for screening the new coronavirus infected person. The nucleic acid detection has high false negative rate, high requirements on operators and field equipment and long detection time. CT examination of other viral pneumonia has the disadvantages of difficult differential diagnosis, easy cross infection and poor hygienic and economical operability. More importantly, the two methods can not realize rapid and large-scale field screening, and a rapid detection product with higher sensitivity, stronger reliability and more convenient operation is urgently needed to be developed at the present stage.
The existing clinical detection methods for novel coronavirus antibodies mainly comprise an enzyme-linked immunosorbent assay, a chemiluminescence assay and a colloidal gold assay. The enzyme-linked immunosorbent assay and the chemiluminescence assay can be used for quantitative detection, and are the main methods for antibody detection. The colloidal gold method is a point-of-care testing (POCT) method, has the advantages of rapidness, unlimited detection field and low requirement on operators, but the colloidal gold method can only carry out qualitative detection, and has poor sensitivity and specificity compared with a quantitative detection method.
At present, commercial ELISA, chemiluminescence and colloidal gold COVID-19 antibody diagnostic products at home and abroad mainly comprise a total antibody, IgM and IgG antibody combined detection reagent, however, the specificity of each detection method is 96.6-99.7%, the sensitivity is 66-97.8%, and the result shows that 0.3-3.4% of detected persons are wrongly diagnosed as new coronary pneumonia and 2.2-34% of new coronary pneumonia patients are missed. Meanwhile, the detection reagent for the conventional COVID-19 antibody has low early detection rate and cannot achieve the aim of precise epidemic prevention. In low prevalence populations, existing antibody tests may produce as many or even more false negative results as true positive results, resulting in unnecessary waste of manpower and material resources.
Therefore, the research and development of the quantum dot immunofluorescence detection kit for improving the early diagnosis rate and reducing the false positive rate have great clinical significance.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a quantum dot immunofluorescence detection reagent strip, which adopts a quantum dot immunochromatography technology, takes a novel coronavirus N protein as a detection antigen, uses quantum dot fluorescent microspheres to mark a novel coronavirus N protein antibody to form a quantum dot specific protein complex, and then detects a nasopharyngeal swab or an oropharyngeal swab of a patient.
The invention also provides a quantum dot immunofluorescence detection kit, which comprises the quantum dot immunofluorescence detection reagent strip.
The third purpose of the invention is to provide an application of the quantum dot immunofluorescence detection reagent strip or the quantum dot immunofluorescence detection kit in novel coronavirus detection.
The invention provides a quantum dot immunofluorescence detection reagent strip which comprises a bottom plate, wherein a sample pad, a quantum dot marker combination pad, a chromatography reaction membrane and absorbent paper are arranged on the bottom plate;
wherein, the quantum dot marker binding pad adsorbs a quantum dot specific protein compound, the quantum dot specific protein compound is mainly obtained by coupling quantum dots and specific protein, and the specific protein comprises novel coronavirus N protein;
the chromatography reaction membrane is provided with a detection line, and the detection line contains a novel coronavirus N protein antibody.
Furthermore, the chromatography reaction membrane is arranged in the middle of the bottom plate, the quantum dot marker combination pad and the sample pad are sequentially overlapped at one end of the chromatography reaction membrane, and the absorbent paper is overlapped at the other end of the chromatography reaction membrane.
Furthermore, the amino acid sequence of the novel coronavirus N protein is shown as Seq No. 01.
Furthermore, a quality control line is also arranged on the chromatography reaction membrane.
Further, the quality control line contains goat anti-rabbit IgG antibody.
Furthermore, the concentration of the novel coronavirus N protein antibody in the novel coronavirus antigen detection line is 0.2-2 mg/mL.
Furthermore, the concentration of the quantum dot specific protein complex in the quantum dot label binding pad is 0.001-0.01 mg/mL.
Further, the detection limit of the immunofluorescence detection reagent strip is 15TCID50/mL, and the CT value is 34.25.
The invention provides a quantum dot immunofluorescence detection kit, which comprises the quantum dot immunofluorescence detection reagent strip.
The invention provides an application of the quantum dot immunofluorescence detection reagent strip or the quantum dot immunofluorescence detection kit in novel coronavirus detection.
Compared with the prior art, the invention has the beneficial effects that:
the quantum dot immunofluorescence detection reagent strip comprises a bottom plate, wherein a sample pad, a quantum dot marker combination pad, a chromatography reaction membrane and absorbent paper are arranged on the bottom plate; wherein, the quantum dot marker binding pad adsorbs a quantum dot specific protein compound, the quantum dot specific protein compound is mainly obtained by coupling quantum dots and specific protein, and the specific protein comprises novel coronavirus N protein; the chromatography reaction membrane is provided with a detection line, and the detection line contains a novel coronavirus N protein antibody. The reagent strip adopts a quantum dot immunochromatography technology, takes the novel coronavirus N protein as a detection antigen, uses a quantum dot fluorescent microsphere to mark the novel coronavirus N protein antibody to form a quantum dot specific protein compound, and then detects the nasopharyngeal swab or the oropharyngeal swab of a patient. The reagent strip has the advantages of high sensitivity, high specificity and quantitative detection, and avoids the omission of the weak positive sample and the false positive sample false detection to the greatest extent. Meanwhile, the quantum dot immunochromatography method used by the invention is rapid in detection, results are obtained within 15 minutes, the exposure risk of medical workers can be reduced by using serum to detect samples, the field operation can be realized by simple training, and hundreds of samples can be detected by each portable fluorescence detector within 1 hour.
The quantum dot immunofluorescence detection kit provided by the invention comprises the quantum dot immunofluorescence detection reagent strip. The kit has the advantages of high sensitivity, high specificity and quantitative detection, can quickly detect the novel coronavirus antigen, and can avoid the omission of the weak positive sample and the false positive sample false detection to the maximum extent.
The quantum dot immunofluorescence detection reagent strip or the quantum dot immunofluorescence detection kit provided by the invention can be widely applied to detection of novel coronaviruses.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
Fig. 1 is a schematic structural diagram of the reagent strip for detecting N antigen of coronavirus provided in example 5 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. 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.
According to one aspect of the invention, the reagent strip for quantum dot immunofluorescence detection comprises a bottom plate, wherein a sample pad, a quantum dot marker combination pad, a chromatography reaction membrane and absorbent paper are arranged on the bottom plate;
wherein, the quantum dot marker binding pad adsorbs a quantum dot specific protein compound, the quantum dot specific protein compound is mainly obtained by coupling quantum dots and specific protein, and the specific protein comprises novel coronavirus N protein;
the chromatography reaction membrane is provided with a detection line, and the detection line contains a novel coronavirus N protein antibody.
The quantum dot immunofluorescence detection reagent strip comprises a bottom plate, wherein a sample pad, a quantum dot marker combination pad, a chromatography reaction membrane and absorbent paper are arranged on the bottom plate; wherein, the quantum dot marker binding pad adsorbs a quantum dot specific protein compound, the quantum dot specific protein compound is mainly obtained by coupling quantum dots and specific protein, and the specific protein comprises novel coronavirus N protein; the chromatography reaction membrane is provided with a detection line, and the detection line contains a novel coronavirus N protein antibody. The reagent strip adopts a quantum dot immunochromatography technology, takes the novel coronavirus N protein as a detection antigen, uses a quantum dot fluorescent microsphere to mark the novel coronavirus N protein antibody to form a quantum dot specific protein compound, and then detects the nasopharyngeal swab or the oropharyngeal swab of a patient. The reagent strip has the advantages of high sensitivity, high specificity and quantitative detection, and avoids the omission of the weak positive sample and the false positive sample false detection to the greatest extent. Meanwhile, the quantum dot immunochromatography method used by the invention is rapid in detection, results are obtained within 15 minutes, the exposure risk of medical workers can be reduced by using serum to detect samples, the field operation can be realized by simple training, and hundreds of samples can be detected by each portable fluorescence detector within 1 hour.
In a preferred embodiment of the present invention, the chromatography reaction membrane is disposed in the middle of the bottom plate, the quantum dot label binding pad and the sample pad are sequentially overlapped on one end of the chromatography reaction membrane, and the absorbent paper is overlapped on the other end of the chromatography reaction membrane.
In a preferred embodiment of the present invention, the amino acid sequence of the N protein of the novel coronavirus is shown as Seq No. 01.
The amino acid sequence of the novel coronavirus N protein is as follows:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA(Seq No.01)；
in a preferred embodiment of the present invention, the chromatography reaction membrane is further provided with a quality control line.
In the above preferred embodiment, the control line comprises goat anti-rabbit IgG antibodies.
In a preferred embodiment of the present invention, the concentration of the novel coronavirus N protein antibody in the novel coronavirus antigen detection line is 0.2-2 mg/mL.
Preferably, the novel crown protein specific antibody comprises any one of a novel crown protein specific antibody polyclonal antibody (murine source), a novel crown protein specific antibody monoclonal antibody (rabbit source), a novel crown protein specific antibody polyclonal antibody (rabbit source);
preferably, the influenza a virus N protein antibody comprises any one of an influenza a virus N protein antibody polyclonal antibody (murine source), an influenza a virus N protein antibody monoclonal antibody (rabbit source), and an influenza a virus N protein antibody (rabbit source);
preferably, the influenza b virus N protein antibody comprises any one of an influenza b virus N protein antibody polyclonal antibody (murine source), an influenza b virus N protein antibody monoclonal antibody (rabbit source), and an influenza b virus N protein antibody (rabbit source);
in the above preferred embodiment, the chromatography reaction membrane comprises any one of NC membrane, PVDF membrane, or nylon membrane;
in the above preferred embodiment, the chassis includes any one of a PVC chassis, a PC chassis, or a PS chassis;
in a preferred embodiment of the present invention, the release liner comprises any one of a glass fiber membrane, a polyester membrane, and a blood filtration membrane;
in a preferred embodiment of the present invention, the sample pad comprises any one of a glass fiber membrane, a polyester membrane, and a blood filtration membrane;
in a preferred embodiment of the present invention, the absorbent paper comprises one of thick filter paper, glass fiber, or blood filter.
In a preferred embodiment of the present invention, the concentration of the quantum dot specific protein complex in the quantum dot label binding pad is 0.001 to 0.01 mg/mL.
In a preferred embodiment of the invention, the immunofluorescence detection reagent strip has a detection limit of 15TCID50/mL and a CT value of 34.25.
According to one aspect of the invention, the quantum dot immunofluorescence detection kit comprises the quantum dot immunofluorescence detection reagent strip.
The quantum dot immunofluorescence detection kit provided by the invention comprises the quantum dot immunofluorescence detection reagent strip. The kit has the advantages of high sensitivity, high specificity and quantitative detection, can quickly detect the novel coronavirus antigen, and can avoid the omission of the weak positive sample and the false positive sample false detection to the maximum extent.
In a preferred embodiment of the present invention, the quantum dot immunofluorescence detection kit further comprises an ultraviolet irradiation device.
According to one aspect of the invention, the quantum dot immunofluorescence detection reagent strip or the quantum dot immunofluorescence detection kit is applied to novel coronavirus detection.
The quantum dot immunofluorescence detection reagent strip or the quantum dot immunofluorescence detection kit provided by the invention can be widely applied to detection of novel coronaviruses.
The technical solution of the present invention will be further described with reference to the following examples.
Example 1
Preparing an antibody:
(1) PCR amplifying the gene encoding the N protein of the novel coronavirus antigen by adopting a gene cloning technology;
(2) and transferring the gene of the novel coronavirus antigen N protein into escherichia coli to express so as to obtain the novel coronavirus recombinant antigen N protein.
(3) And preparing a novel coronavirus N protein antibody by using the novel coronavirus recombinant antigen N protein by adopting a conventional method in the field.
Methods for making such antibodies are exemplary as follows:
required reagents:
the mouse myeloma cell line SP2/0 is preserved by the company;
BALB/c mice are provided by Shanghai Slek animal center;
PEG1500, hypoxanthine, thymidine, aminopterin, DMSO were purchased from Sigma;
HRP was purchased from BBI solutions;
RPMI 1640 basal medium was purchased from Gibco;
fetal bovine serum was purchased from LONSA SCIENCE SRL company;
goat anti-mouse horseradish peroxidase, IgM, IgG1, IgG2a, IgG2b and IgG3 are products of Serotec corporation.
Preparing monoclonal antibody:
according to the conventional hybridoma preparation method, a female Balb/C mouse (Shanghai Slek animal center) with the age of 6 weeks is selected, the immunization target protein is injected subcutaneously, the immunization dose is 100 mu g/mouse, the equal amount of Freund's complete adjuvant is used for primary immunization, the equal amount of incomplete Freund's adjuvant is used for immune reinforcement, the immunity is strengthened for 4 times, the immunization interval is 2 weeks, and 100 mu g of protein is injected intravenously 3 days before fusion. Collecting spleen B cells of an immunized mouse and a mouse myeloma cell line (Sp2/0-Ag14, Sp2/0), performing cell fusion by using PEG1500, and performing ELISA detection, cloning operation, ascites induction and purification on formed hybridoma cells to obtain the monoclonal antibody. The monoclonal antibody is marked with horseradish peroxidase (HRP) by a modified sodium periodate method.
Example 2
Preparing a chromatography reaction membrane (a new coronavirus N protein antigen detection line):
attaching an NC membrane with the width of 25cm to the middle area of a PVC (polyvinyl chloride) bottom plate, diluting the novel coronavirus N protein antibody and the goat anti-rabbit IgG antibody obtained in the example 1 to 0.2-2 mg/mL by using PBS (phosphate buffer solution) diluent respectively, and scribing on a detection line area and a quality control line area corresponding to the NC membrane by using a gold spraying and membrane scribing instrument. Drying in an oven at 37 ℃ for 12-24h, and sealing for later use.
Example 3
Preparing quantum dot-marker protein complex (specific protein is novel coronavirus N protein antibody):
(1) adding 200 mu l of quantum dots into 800 mu l of MES (pH 6) buffer solution, adding 40-160 mu g of EDC and 10-40 mu g of NHS for activation, and activating at 37 ℃ for 15-60 min. The activated solution was centrifuged under certain conditions and the supernatant removed with a pipette.
(2) And (3) re-dissolving the precipitate obtained in the step (1) after centrifugation by using 1000 mu l of 10-50 mM MES (pH 6) buffer solution, and if re-dissolving is not complete, using ultrasonic to assist re-dissolving. Centrifuging the redissolved solution under certain conditions, removing supernatant by using a pipette gun, redissolving the precipitate by 1000 mu l of 10-50 mM MES (pH 6) buffer solution, and if the redissolution is not complete, using ultrasonic to assist the redissolution.
(3) Adding 20-100 μ l of novel coronavirus N protein antibody diluted to 1mg/ml with 10-50 mM MES (pH 6) buffer solution into the re-dissolved solution, and coupling at 37 deg.C for 60-300 min.
(4) After the coupling is finished, 100. mu.l of 20-100 mM Tris-HCl diluent (pH8.0, containing 100-500 mM glycine + 10% BSA) is added for blocking, and the blocking is carried out at 37 ℃ for 30-120 min.
(5) Centrifuging the sealed solution under certain conditions, and removing the supernatant by using a pipette. And (3) redissolving the precipitate by using 500-1000 mu l of Tris-HCl diluent, and if the redissolution is not complete, using ultrasonic to assist the redissolution, continuing to centrifuge under certain conditions, and removing the supernatant by using a pipette gun (the step can be omitted). And (3) carrying out redissolution precipitation by using 500-.
Preparing a quantum dot marker binding pad (the specific protein is a novel coronavirus N protein antibody):
diluting the prepared quantum dot-labeled protein complex by using 20-100 mM Tris-HCl diluent (pH8.0, containing 0.5% BSA and 2% trehalose) for 3-30 times according to requirements, spraying dots on the treated release pad by using a film-scribing metal spraying instrument, and drying at 37 ℃ for 12-24 hours to obtain the quantum dot label binding pad. Or diluting the prepared quantum dot-labeled protein complex by 20-100 mM Tris-HCl diluent (pH8.0, containing 0.5% BSA and 2% trehalose) by 20-100 times according to requirements, spraying the diluted complex on an untreated release pad by using a liquid transfer gun, uniformly coating the release pad by using a coating rod, finally putting the release pad into a freeze dryer for freeze drying, and sealing the release pad for later use after the program is run.
Example 4
Preparing a sample pad:
uniformly coating 20-100 mM Tris-HCl diluent (pH8.0, containing 0.5% BSA, 0.5% PVP and 0.5% TWEEN-20) on an untreated release pad by using a liquid transfer gun and a coating rod, drying at 37 ℃ for 12-24h, and sealing for later use.
Example 5
As shown in figure 1, the preparation method of the novel reagent strip for detecting the coronavirus N antigen comprises the following steps:
the chromatography reaction membrane containing the new coronavirus N protein antigen detection line prepared in the example 2, the quantum dot marker binding pad prepared in the example 3, the sample pad prepared in the example 4 and the absorbent paper are attached to a PVC (polyvinyl chloride) backboard in a lap joint manner;
the sample pad and the quantum dot marker combination pad are sequentially lapped at one end of the chromatographic reaction membrane, the absorbent paper is lapped at the other end of the chromatographic reaction membrane, and then the parameters of the slitter are adjusted to slit the test paper strips into test paper strips with the width of 3-4 mm.
Example 6
A quantum dot immunofluorescence detection kit, the kit comprising: example 5 novel reagent strip for detecting coronavirus antigen, the specific protein of which is novel coronavirus N protein antibody;
and, an ultraviolet irradiation device.
Test example 1
The quantum dot immunofluorescence detection kit prepared in the embodiment 6 is selected for LOD (low of detection) determination, and the specific experimental method is as follows:
(1) the minimum detection limit of the novel coronavirus (SARSCoV-2) antigen rapid detection kit (fluorescence method) is established by a limiting dilution method. The concentration of the virus sample is 3.0 multiplied by 105TCID50/mL。
(2) The concentration is 3.0 multiplied by 105TCID50/mL of the initial virus sample is added into the mixed nasopharyngeal swab sample matrix to select a batch of antigen reagent, 100ul of diluted sample is added into the quantum dot immunofluorescence detection kit well prepared in example 6, each gradient sample is repeated for 3 persons, and after 15min, the dry immunofluorescence instrument is used for reading.
(3) And selecting the concentration range between 3 positive results of the 3 samples of the last gradient dilution and 3 negative results of the 3 samples of the first gradient dilution.
(4) And performing 2-time gradient dilution test on the concentrations of 3 positive results of the 3 samples subjected to the last gradient dilution. The last dilution showed a concentration that was 100% positive and was confirmed to be LOD, and 20 more identical replicates were performed.
The results of the experiments are shown in the following table:
therefore, the LOD of the quantum dot immunofluorescence detection kit (fluorescence method) prepared in the embodiment 6 is 15TCID50/mL, corresponds to Daan nucleic acid detection CT34.25, and has very good detection sensitivity.
Test example 2
The quantum dot immunofluorescence detection kit prepared in the embodiment 6 of the application is subjected to effect evaluation, and the specific method is as follows:
(1) and sample treatment:
A. throat swab samples: fully mixing virus preservation solution containing a throat swab sample with detection buffer solution in a ratio of 1: 1;
B. sputum sample: centrifuging the sputum sample at a high speed to remove coarse particles, and fully mixing the centrifuged supernatant with a buffer solution in a ratio of 1: 1;
C. alveolar lavage fluid: mixing the alveolar lavage fluid with the buffer solution in a ratio of 1: 1;
D. stool sample: taking 100mg of a fecal sample, adding 1mL of physiological saline, uniformly mixing, centrifuging, taking supernatant, and mixing with a buffer solution in a ratio of 1: 1;
(2) and detecting a reagent plate:
and (3) taking 100 mu L of the sample which is prepared in the previous step and is treated by the buffer solution, dropwise adding the sample to a reagent plate, inserting the test card into a fluorescence immunoassay analyzer in the indicated direction after 15min, selecting the test item of the corresponding batch of the reagent, and clicking 'quick test'.
(3) And (3) detecting nucleic acid:
the detection was carried out with reference to the instructions of the novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR method).
Specific positive test results:
the diagnosis standard and reagent sensitivity of clinical laboratory in hospital are used as reference, and the diagnosis is negative according to the CT value of fluorescent PCR being more than 40 and no amplification curve. In the following, a graded statistical evaluation will be made in accordance with the Ct value results of PCR.
157 clinical specimens from new coronary patients, 137 sputum samples, 15 throat swab samples, 3 alveolar lavage samples, 2 stool samples.
(I), wherein the CT of the sample is less than or equal to 35, and the total number of the samples is 127, wherein 112 sputum samples, 11 pharyngeal swab samples, 3 alveolar lavage fluid samples and 1 feces sample.
As can be seen from the above table, the positive coincidence rate of the quantum dot immunofluorescence detection kit prepared in example 6 of the present application is as follows: 96.06 percent; negative coincidence rate: 100.00 percent; the total coincidence rate is as follows: 98.56% (97.31% -99.81%); kappa number is: 0.969.
and (II) the CT of the sample is less than or equal to 38, and 144 samples are counted, wherein 126 samples of sputum, 13 samples of throat swabs, 3 samples of alveolar lavage fluid and 2 samples of feces.
As can be seen from the above table, the positive coincidence rate of the quantum dot immunofluorescence detection kit prepared in example 6 of the present application is as follows: 89.58 percent; negative coincidence rate: 100.00 percent; the total coincidence rate is as follows: 95.88% (93.84% -97.92%); kappa number is: 0.912.
(III) the detection rate of the nucleic acid low copy sample (CT is more than 35 and less than or equal to 38):
in 17 cases, 7 positive samples were detected in total by the PCR positive sample plate, and the positive detection rate was 41.18%.
From the above experimental results, the quantum dot immunofluorescence detection kit and the fluorescence PCR detection result prepared in the embodiment 6 of the present application are used as references, and in all the types of the samples to be evaluated:
and when the CT of the sample is less than or equal to 35, evaluating the positive coincidence rate of the reagent (immunochromatography): 96.06%, negative coincidence: 100.00%, total compliance: 98.56 percent; and when the sample CT is less than or equal to 38, evaluating the positive coincidence rate of the reagent (immunochromatography): 89.58%, negative match rate: 100.00%, total compliance: 95.88 percent. Therefore, when the CT value of the sputum sample and the pharyngeal swab sample is less than or equal to 35, the evaluation reagent has higher detection rate. The number of alveolar lavage fluid and fecal samples tested was less than 20, and the test data was only for reference and was not statistically significant.
The raw test data of the positive samples in this example are as follows:
the raw test data for the negative samples of this example are as follows:
test example 3
The kit of embodiment 6 of the present application is specifically verified, and the specific experimental method is as follows:
(1) various pathogen cultures (see table below) and a certain amount of normal human throat swab samples were collected;
(2) the following pathogens were diluted to the corresponding concentrations with delayed swab samples from normal persons (bacteria, yeast, mycoplasma, chlamydia all diluted to 1.0X 106CFU/mL; the viruses were all diluted to 1.0X 105pFU/mL or 1.0X 105TCID 50/mL; flushing the nasal cavity with normal saline, collecting the flushing liquid, and mixing with normal nasopharyngeal swab sample at a ratio of 1: 1);
(3) selecting three batches of reagents, adding 100ul of samples into a sample adding hole, detecting 1 person in each batch of samples, and reading by using a dry immunofluorescence instrument after 15 min;
the experimental results are as follows:
the clinical detection of 27 kinds of pathogens and human nasal cavity washing liquid has no cross reaction with the novel coronavirus antigen. The research reagent and the contrast reagent are proved to have extremely strong consistency on the novel coronavirus antigen under the condition that the interference sample exists.
Experimental example 4 interference experiment:
the experimental steps are as follows:
(1) collecting various endogenous substances and normal human throat swab samples;
(2) diluting the following table interferents to corresponding concentrations by using normal human nasopharynx swab samples;
(3) selecting three batches of reagents, adding 100ul of samples into a sample adding hole, detecting 1 person in each batch of samples, and reading by using a dry immunofluorescence instrument after 15 min;
the experimental results are as follows:
finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Hangzhou Baojin Biotechnology Co., Ltd
<120> reagent kit containing quantum dot immunofluorescence detection reagent strip and application thereof
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 419
<212> PRT
<213> novel coronavirus N protein
<400> 1
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala
Claims (10)
1. The quantum dot immunofluorescence detection reagent strip is characterized by comprising a bottom plate, wherein a sample pad, a quantum dot marker combination pad, a chromatography reaction membrane and absorbent paper are arranged on the bottom plate;
the quantum dot marker binding pad is adsorbed with a quantum dot specific protein compound, the quantum dot specific protein compound is mainly obtained by coupling quantum dots and specific protein, and the specific protein is novel coronavirus N protein;
the chromatography reaction membrane is provided with a detection line, and the detection line contains a novel coronavirus N protein antibody.
2. The quantum dot immunofluorescence detection reagent strip according to claim 1, wherein the chromatography reaction membrane is arranged in the middle of the bottom plate, the quantum dot marker binding pad and the sample pad are sequentially overlapped on one end of the chromatography reaction membrane, and the absorbent paper is overlapped on the other end of the chromatography reaction membrane.
3. The quantum dot immunofluorescence detection reagent strip according to claim 1, wherein the amino acid sequence of the novel coronavirus N protein is shown as Seq No. 01.
4. The quantum dot immunofluorescence detection reagent strip according to claim 1, wherein a quality control line is further arranged on the chromatography reaction membrane.
5. The quantum dot immunofluorescence detection reagent strip according to claim 4, wherein the quality control line contains goat anti-rabbit IgG antibody.
6. The quantum dot immunofluorescence detection reagent strip according to claim 1, wherein the concentration of the novel coronavirus N protein antibody in the detection line is 0.2-2 mg/mL.
7. The quantum dot immunofluorescence detection reagent strip according to claim 1, wherein the concentration of the quantum dot specific protein complex in the quantum dot label binding pad is 0.001-0.01 mg/mL.
8. The quantum dot immunofluorescence detection reagent strip according to claim 1, wherein the immunofluorescence detection reagent strip has a detection limit of 15TCID50/mL and a CT value of 34.25.
9. A quantum dot immunofluorescence detection kit, which is characterized by comprising the quantum dot immunofluorescence detection reagent strip of any one of claims 1 to 8.
10. The application of the quantum dot immunofluorescence detection reagent strip according to any one of claims 1 to 8 or the quantum dot immunofluorescence detection kit according to claim 9 in novel coronavirus detection.
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| CN114264819A (en) * | 2021-12-08 | 2022-04-01 | 上海理工大学 | Quantum dot nanosphere immunochromatography test strip for rapidly detecting new coronavirus |
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