WO2021185034A1 - 新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒及制备方法及检测方法 - Google Patents

新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒及制备方法及检测方法 Download PDF

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WO2021185034A1
WO2021185034A1 PCT/CN2021/077652 CN2021077652W WO2021185034A1 WO 2021185034 A1 WO2021185034 A1 WO 2021185034A1 CN 2021077652 W CN2021077652 W CN 2021077652W WO 2021185034 A1 WO2021185034 A1 WO 2021185034A1
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covid
solution
nucleic acid
positive
probe
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PCT/CN2021/077652
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French (fr)
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雷洋
张捷李
张利伟
黄认训
白佳委
乐宜萃
胡啸
汪大明
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安邦（厦门）生物科技有限公司
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Priority to US17/618,958 priority Critical patent/US20230221304A1/en
Priority to EP21772078.8A priority patent/EP3957755A4/en
Publication of WO2021185034A1 publication Critical patent/WO2021185034A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of nucleic acid detection, in particular to a novel coronavirus nucleic acid rapid hybridization capture immunofluorescence detection kit and a preparation method of the kit.
  • coronavirus COVID-19 belongs to the ⁇ genus of coronaviruses. It has an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140nm. Its genetic characteristics are obviously different from SARSr-CoV and MERSr-CoV. , And the bat SARS-like coronavirus (bat-SL-CoVZC45) homology is over 85%.
  • New coronavirus-infected pneumonia is inflammation of the lungs caused by new coronavirus infection.
  • the disease has now been included in the "People's Republic of China's Infectious Disease Prevention and Control Law" as a Class B infectious disease, and the prevention and control of Class A infectious diseases have been adopted. measure. With the spread of the epidemic, there is an urgent need for a test reagent that can accurately and quickly diagnose the new coronavirus.
  • New coronavirus COVID-19 nucleic acid detection kit fluorescent PCR method from Huada Biotechnology (Wuhan) Co., Ltd. (National Machinery Note 20203400060);
  • the above-mentioned new coronavirus COVID-19 nucleic acid detection kits mainly use the fluorescent PCR nucleic acid detection method.
  • the detection principle is to add a fluorescent reporter group and a fluorescent quenching group to the PCR reaction system. As the PCR reaction proceeds, expand The continuous accumulation of increased products leads to the continuous accumulation of fluorescent signals.
  • Real-time monitoring of the fluorescent signals and obtains the Ct (Threshold cycle) value of the unknown sample.
  • the Ct value refers to the minimum number of cycles required to generate a detectable fluorescent signal. The number of cycles corresponding to the inflection point where the fluorescence signal starts from the background and enters the exponential growth phase during the PCR cycle.
  • the Ct values of standard samples of different concentrations are usually used to generate the standard curve, and the initial template amount of the unknown sample is automatically calculated from the standard curve.
  • the above-mentioned fluorescent PCR nucleic acid detection method is accurate and reproducible, it requires a professional PCR laboratory, a dedicated PCR instrument, a specially trained technician to operate it, and nucleic acid extraction and purification of the sample. Processing requires cold chain transportation below -20°C, and the detection time is up to 1-3 hours, the detection efficiency is low, and the detection process has nucleic acid amplification, which can easily cause laboratory positive product contamination, and many other problems, which are inconvenient to use.
  • the present invention provides a novel coronavirus nucleic acid rapid hybridization capture immunofluorescence detection kit, preparation method and detection method, which not only requires shorter detection time, but also does not require professional technicians to operate, and does not require supporting laboratories and special instruments. , It is more convenient and quick to use.
  • One of the objectives of the present invention is to provide a novel coronavirus nucleic acid rapid hybridization capture immunofluorescence detection kit, the kit includes a COVID-19 reaction solution, the COVID-19 reaction solution is composed of COVID-19 fluorescent markers and COVID-19 probe solution is prepared; the COVID-19 probe solution includes: ORF1ab section probe, N section probe, E section probe, and the ORF1ab section probe is used to detect COVID- 19 open reading coding frame lab, the N-segment probe is used to detect the COVID-19 envelope protein gene, and the E-segment probe is used to detect the COVID-19 nucleocapsid protein gene.
  • sequence of the ORF1ab segment probe is:
  • the sequence of the N segment probe is:
  • the sequence of the E segment probe is:
  • the COVID-19 fluorescent label is made by coupling a fluorescent substance and a COVID-19 labeling material;
  • the COVID-19 labeling material is a COVID-19 antigen or antibody;
  • the fluorescent substance is any one of the following: FITC Fluorescein, fluorescent microspheres, fluorescent particles, biofluorescein.
  • the kit also includes a COVID-19 negative control substance
  • the negative control substance includes one or more of the following control substances: a physiological saline control substance, a purified aqueous control substance, and a non-COVID-19 pathogen control substance.
  • Products, pseudoviruses that do not contain the target sequence of COVID-19 are not contain the target sequence of COVID-19.
  • the negative control substances include N1 ⁇ N17: N1 ⁇ N2 are normal saline control substances, N3 ⁇ N4 are purified water-based control substances, N5 ⁇ N13 are human throat swab samples, and N14 ⁇ N17 are samples that do not contain COVID-19.
  • Target sequence pseudovirus wherein, in the human throat swab samples, COVID-19 is negative, and the non-COVID-19 pathogen control substance is positive; the pseudovirus that does not contain the COVID-19 target sequence is coronavirus Subtype, N14 is positive for human coronavirus 229E N-segment, N15 is positive for human coronavirus NL63 N-segment, N16 is positive for human coronavirus OC43 N-segment, and N17 is positive for human coronavirus HKU1 N-segment.
  • the kit also includes a COVID-19 positive control substance, which is a pseudovirus containing a COVID-19 target sequence;
  • the positive control substance includes P1, P2, P3, where P1 is COVID-19 19 N segment is positive, P2 is COVID-19 E segment is positive, P3 is COVID-19 ORF 1ab segment is positive; the concentration of the positive control substance is 3000TU/mL ⁇ 5%.
  • the kit further includes a precision control substance and a detection limit control substance
  • the precision control substance includes J1, J2, J3, J1 ⁇ J2 are pseudoviruses containing the target sequence of COVID-19, all of which are COVID- 19 ORF 1ab segment is positive, the concentrations are 2000TU/mL ⁇ 5% and 5000TU/mL ⁇ 5%, J3 is a mixed human negative throat swab sample, and COVID-19 is negative
  • the detection limit control substance includes L1, L2 , L3, L1 is positive for COVID-19 N segment, L2 is positive for COVID-19 E segment, L3 is positive for COVID-19 ORF 1ab, the concentration of the detection limit reference substance is 1000TU/mL ⁇ 5%.
  • the kit further includes a COVID-19 test strip, a COVID-19 reconstituted solution, and a COVID-19 sample preservation solution.
  • the second objective of the present invention is to provide a method for preparing the novel coronavirus nucleic acid rapid hybridization capture immunofluorescence detection kit as described in any one of the above, which includes the preparation of a COVID-19 fluorescent marker, and the steps are as follows:
  • Activation add 1% fluorescent microsphere solution at a ratio of 1.0 mg/mL ⁇ 5%, and EDC solution at a ratio of 0.6 mg/mL ⁇ 5% into the prepared 0.05M ⁇ 5% borate buffer and mix well. Rotate on a rotary mixer for more than 20 minutes, centrifuge at 15000-16000rpm for more than 30 minutes after activation, remove the supernatant, and resuspend in 0.05M ⁇ 5% boric acid buffer and mix.
  • Coupling Add COVID-19 antibody at 0.2 mg/mL to the activated fluorescent microsphere solution, mix well, and place on a rotary mixer to rotate for more than 2 hours to obtain a fluorescent microsphere labeled conjugate solution ;
  • Blocking Add 10% BSA solution to the fluorescent microsphere labeled conjugate solution at a ratio of 0.1ml/mL ⁇ 5%, mix well, and place on a rotary mixer to rotate for 12-16 hours.
  • Centrifugal resuspension Centrifuge the fluorescent microsphere labeled conjugate solution at 15000 ⁇ 16000rpm, remove the supernatant, wash with an equal volume of 0.05M ⁇ 5% borate buffer, and finally dilute the precipitate with an equal volume of the supernatant solution of the label Resuspend the solution to prepare a fluorescent marker for COVID-19.
  • the steps are as follows:
  • Probe solution preparation Dilute the COVID-19 probe with nucleic acid dissolving solution to 10 ⁇ M ⁇ 5% to obtain the COVID-19 probe solution;
  • Preparation of the reaction solution mix the diluted COVID-19 fluorescent marker and the COVID-19 probe solution at a volume ratio of 1:1 to obtain a COVID-19 reaction solution;
  • the reaction solution is dividedly packed: the COVID-19 reaction solution is dividedly packed in a reaction tube, and dried for 6 to 8 hours under the conditions of a temperature of 18-28°C and a humidity of ⁇ 30%.
  • the third objective of the present invention is to provide the detection method of the novel coronavirus nucleic acid rapid detection kit as described in any one of the above, which includes the following steps:
  • the novel coronavirus nucleic acid rapid hybridization capture immunofluorescence detection kit of the present invention uses hybrid capture immunofluorescence analysis, referred to as HC-IFA in English, is a method of acquiring target nucleic acid fragments in the sample to be tested by hybridization capture, and identifying it through fluorescence signals.
  • the present invention covers nucleic acid detection, reaches molecular level detection sensitivity, and realizes fluorescence recognition of nucleic acid detection; compared with general fluorescent PCR and sequencing detection, the signal intensity is stronger and the specificity is better.
  • Shorter detection time no need for professional technical personnel to operate, no need for refrigeration for transportation and storage, no temperature change during the reaction process, no need for supporting laboratories and supporting PCR equipment, single detection can be carried out, detection time is 30 minutes per person, detection time Short, one-hour detection throughput is 100 to 200 servings.
  • Figure 1 is a schematic diagram of the preparation process of a novel coronavirus nucleic acid rapid hybridization capture immunofluorescence detection kit of the present invention.
  • Figure 2 shows the S9.6 protein model information used in the specific detection process of a novel coronavirus nucleic acid rapid hybridization capture immunofluorescence detection kit of the present invention.
  • kits includes a COVID-19 reaction solution, a COVID-19 re-solution, a COVID-19 negative control substance, a COVID-19 positive control substance, COVID-19 sample preservation solution, each liquid is separately packed in reaction tubes or standable tubes, and also includes COVID-19 test strips, aluminum foil bag cards, kit labels, kit instructions, etc.
  • the COVID-19 reaction solution is prepared from a COVID-19 fluorescent marker and a COVID-19 probe solution;
  • the COVID-19 probe solution includes: ORF1ab section probe, N section probe, E section
  • the ORF1ab segment probe is used to detect the COVID-19 open reading coding frame lab, the N segment probe is used to detect the COVID-19 envelope protein gene, and the E segment probe is used to detect COVID-19 nucleocapsid protein gene.
  • sequence of the ORF1ab segment probe is:
  • the sequence of the N segment probe is:
  • the sequence of the E segment probe is:
  • the COVID-19 fluorescent label is made by coupling a fluorescent substance and a COVID-19 labeling material; the COVID-19 labeling material is made of COVID-19 antibody; the fluorescent substance is made of any of the following: FITC fluorescein, fluorescent micro Balls, fluorescent particles, biofluorescein, and other substances that can emit fluorescence are not limited to this.
  • the COVID-19 negative control substance includes one or more of the following control substances: a physiological saline control substance, a purified aqueous control substance, a non-COVID-19 pathogen control substance, and a pseudovirus that does not contain a COVID-19 target sequence.
  • the negative control substances include N1 ⁇ N17: N1 ⁇ N2 are normal saline control substances, N3 ⁇ N4 are purified aqueous control substances, N5 ⁇ N13 are human throat swab samples, and N14 ⁇ N17 are those that do not contain COVID-19.
  • Target sequence pseudovirus wherein, in the human throat swab samples, COVID-19 is negative, and the non-COVID-19 pathogen control substance is positive; the pseudovirus that does not contain the COVID-19 target sequence is coronavirus Subtype, N14 is positive for human coronavirus 229E N-segment, N15 is positive for human coronavirus NL63 N-segment, N16 is positive for human coronavirus OC43 N-segment, and N17 is positive for human coronavirus HKU1 N-segment.
  • the COVID-19 positive control substance is a pseudovirus containing the target sequence of COVID-19; the positive control substance includes P1, P2, P3, where P1 is positive for COVID-19 N segment, and P2 is COVID-19 E area Segment positive, P3 is positive for COVID-19 ORF 1ab segment; the concentration of the positive control substance is 3000TU/mL ⁇ 5%.
  • the precision reference materials include J1, J2, and J3.
  • J1 ⁇ J2 are pseudoviruses containing COVID-19 target sequences, all of which are positive for the COVID-19 ORF 1ab segment, and the concentrations are 2000TU/mL ⁇ 5% and 5000TU/mL, respectively.
  • mL ⁇ 5% J3 is a mixed human negative throat swab sample, and COVID-19 is negative;
  • the detection limit control substance includes L1, L2, L3, L1 is COVID-19 N segment positive, L2 is COVID-19 E The segment is positive, L3 is positive for the COVID-19 ORF 1ab segment, and the concentration of the detection limit reference substance is 1000TU/m ⁇ 5%L. See Table 1 below for details.
  • the test strip can adopt chromatography or percolation method.
  • the test strip adopts an immunofluorescence chromatography test strip, which includes an absorbent pad arranged on one end region of the test strip, and The sample application area on the other end area of the test strip, and the nitrocellulose membrane (NC membrane) arranged between the absorbent pad and the sample application area, and the nitrocellulose membrane is arranged on the T-line detection Area.
  • the treated glass fiber mat, the treated nitrocellulose membrane, and the water absorbent pad are sequentially overlapped on the PVC bottom plate, and cut into test strips with a set width to obtain the test strip of the present invention.
  • this embodiment provides a method for preparing the novel coronavirus nucleic acid rapid hybridization capture immunofluorescence detection kit as described in any one of the above, which includes:
  • Activation add 1% fluorescent microsphere solution at a ratio of 1.0 mg/mL ⁇ 5%, and EDC solution at a ratio of 0.6 mg/mL ⁇ 5% into the prepared 0.05M ⁇ 5% borate buffer and mix well. Rotate on a rotary mixer for more than 20 minutes, centrifuge at 15000-16000rpm for more than 30 minutes after activation, remove the supernatant, and resuspend in 0.05M ⁇ 5% boric acid buffer and mix.
  • Coupling Add COVID-19 antibody to the activated fluorescent microsphere solution in an amount of 0.2 mg/mL ⁇ 5%, mix well, and place on a rotary mixer to spin for more than 2 hours to obtain fluorescent microsphere label Conjugate solution
  • Blocking Add 10% BSA solution to the fluorescent microsphere labeled conjugate solution at a ratio of 0.1ml/mL ⁇ 5%, mix well, and place on a rotary mixer to rotate for 12-16 hours.
  • Centrifugation and resuspension Centrifuge the fluorescent microsphere labeled conjugate solution at 15000 ⁇ 16000rpm, remove the supernatant, wash twice with an equal volume of 0.05M ⁇ 5% borate buffer (pH 8.0), and finally precipitate with the supernatant solution, etc. Resuspend a volume of the label diluent to prepare a COVID-19 fluorescent label.
  • Probe solution preparation Dilute the COVID-19 probe with nucleic acid dissolving solution to 10 ⁇ M ⁇ 5% to obtain the COVID-19 probe solution;
  • Preparation of the reaction solution mix the diluted COVID-19 fluorescent marker and the COVID-19 probe solution at a volume ratio of 1:1 to obtain a COVID-19 reaction solution;
  • Reaction liquid aliquot the COVID-19 reaction solution is aliquoted into a reaction tube, 4 ⁇ L/tube, and dried for 6-8 hours under the conditions of a temperature of 18-28°C and a humidity of ⁇ 30%.
  • the coating concentration of T-line is 0.5mg/mL ⁇ 5%, and the coating concentration of C-line is 1.0mg/mL ⁇ 5%;
  • Spray volume is 1.0 ⁇ L/cm ⁇ 5%, rail speed: 100mm/s ⁇ 5%, nozzle interval: 6mm ⁇ 5%;
  • Drying The temperature is 18-28°C, the humidity is ⁇ 30%, and the drying time: 16h-20h.
  • the amount of compound solution added to the reaction tube is 85 ⁇ L ⁇ 5%
  • the sample volume is 20 ⁇ L ⁇ 5%
  • the nucleic acid hybridization reaction conditions are 37°C, and the reaction time is more than 15 minutes;
  • the reagent detection reaction time is more than 15 minutes.
  • the intermediate preparations involved in the reaction process of the preparation method of this embodiment are all freeze-dried products, and only need to prepare the working solution, without dilution and other complicated operations;
  • the freeze-drying treatment method is: freeze-drying at -30°C for 5 hours, and then freeze-drying for 12 hours. During the drying process, the temperature is gradually increased to -10°C, and the heating rate is 5°C/3h, and finally freeze-dried at -10°C for 7-19h hours.
  • the third embodiment (detection method):
  • This embodiment also provides the detection method of the novel coronavirus nucleic acid rapid hybridization capture immunofluorescence detection kit as described in any one of the above, which includes the following steps:
  • test liquid to the COVID-19 test strip for fluorescent signal identification; in this embodiment, the test liquid is added to the sample application area of the test strip, and after 90 seconds, it is added and rinsed from the sample area again After waiting for 10 minutes, perform fluorescence detection on the detection area of the test strip, and read the detection result.
  • the identification instrument can use a general fluorescence reader, such as the portable immunofluorescence analyzer produced by Suzhou Hemai Technology.
  • reaction tube contains the powder after the reaction liquid is dried, and the powder is defined as the reaction liquid powder.
  • the preparation of the reaction liquid powder can refer to the following:
  • Probe solution preparation Dilute the COVID-19 probe with nucleic acid dissolving solution to 10 ⁇ M ⁇ 5% to obtain the COVID-19 probe solution; preparation of the reaction solution: Combine the diluted COVID-19 fluorescent marker and the COVID-19 The probe solution was mixed in a volume ratio of 1:1 to obtain the COVID-19 reaction solution; the reaction solution was divided into a reaction tube, 4 ⁇ L/tube, and the temperature was 18-28°C, Dry for 6-8 hours under the condition of humidity ⁇ 30%.
  • the reaction liquid powder contains the probe solution and the protein labeled with a fluorescent signal.
  • the protein labeled with fluorescent signal please refer to the following:
  • Activation add 1% fluorescent microsphere solution at a ratio of 1.0 mg/mL ⁇ 5%, and EDC solution at a ratio of 0.6 mg/mL ⁇ 5% into the prepared 0.05M ⁇ 5% borate buffer and mix well. Rotate on a rotary mixer for more than 20 minutes, centrifuge at 15000-16000rpm for more than 30 minutes after activation, remove the supernatant, and resuspend in 0.05M ⁇ 5% boric acid buffer and mix.
  • Coupling Add COVID-19 antibody to the activated fluorescent microsphere solution in an amount of 0.2 mg/mL ⁇ 5%, mix well, and place on a rotary mixer to spin for more than 2 hours to obtain fluorescent microsphere label Conjugate solution
  • Blocking Add 10% BSA solution to the fluorescent microsphere labeled conjugate solution at a ratio of 0.1ml/mL ⁇ 5%, mix well, and place on a rotary mixer to rotate for 12-16 hours.
  • Centrifugation and resuspension Centrifuge the fluorescent microsphere labeled conjugate solution at 15000 ⁇ 16000rpm, remove the supernatant, wash twice with an equal volume of 0.05M ⁇ 5% borate buffer (pH 8.0), and finally precipitate with the supernatant solution, etc. Resuspend a volume of the label diluent to prepare a COVID-19 fluorescent label.
  • the fluorescent signal used is from fluorescent microspheres, and the protein is S9.6 protein produced by Merck. Please refer to Figure 2 for the specific model.
  • the powder of the reaction solution is reconstituted to form a liquid phase environment.
  • the reconstituted solution of the reaction solution is insufficient, the reconstituted solution can be appropriately added, and the added amount is 85 ⁇ L ⁇ 5%.
  • the reconstituted solution is generally physiological saline or physiological saline with added bactericidal components.
  • DNA recognizes, hybridizes, captures, and binds to target RNA fragments into a DNA-RNA complex
  • Protein 1 specifically recognizes and binds to DNA-RNA complex to form a DNA-RNA-protein-fluorescence signal complex , Positioning this complex as a reaction complex.
  • the reaction complex Take out the reaction complex, add it to the detection reagent card, perform chromatography on the test strip under the action of the auxiliary liquid, and chromatograph to the T line, which is coated with S9.6 protein produced by Merck.
  • S9.6 protein produced by Merck can specifically recognize and bind to the DNA-RNA complex Therefore, the DNA-RNA-protein-fluorescence signal complex in the reaction complex can be fixed on the T line to form a "protein-DNA-RNA-protein-fluorescence signal" and a fluorescent band is formed on the T line.
  • the fluorescent signal can be detected by the instrument on the T line.
  • the C line is coated with rabbit polyclonal antibody.
  • the rabbit polyclonal antibody can non-specifically bind to the S9.6 protein produced by Merck. Therefore, under the premise of excess, it must be theoretically certain A certain amount of proteins connected with fluorescent signals will be captured to form a fluorescent band, which can be detected and identified by the instrument.
  • the above-mentioned T-line markers and C-line markers are exactly the S9.6 protein produced by Merck coated on the T-line and the rabbit polyclonal antibody coated on the C-line. All proteins are unlabeled. Fluorescence signal.
  • the main research methods of each performance are combined with the actual domestic evaluation methods, including the product's positive reference product compliance rate, negative reference product compliance rate, minimum detection limit, precision, interference experiment, cross-reaction and other projects. Experimental test.
  • the sample and other material information used in the analysis performance evaluation test is as follows:
  • the new coronavirus 2019-nCoV nucleic acid assay kit (hybridization capture immunofluorescence method) was used to detect 8 positive reference products (P1-P8), and 19 negative reference products (N1-N19) were tested once each.
  • 3 segments (including 2019-CoV N segment, 2019-nCoV E segment, 2019-nCoV ORF 1ab segment) pseudoviruses were serially diluted with mixed negative samples as diluents, diluted to 5000 copies/mL, 2500 copies/ mL, 1000copies/mL, 800copies/mL, 500copies/mL, 250copies/mL, 100copies/mL.
  • the new coronavirus 2019-nCoV nucleic acid determination kit hybridization capture immunofluorescence method was used to determine samples (pseudovirus samples, throat swab samples, sputum samples), and each sample was tested 20 times to calculate the positive detection rate.
  • the lowest concentration with a positive detection rate ⁇ 95% is the lowest detection limit of the segment.
  • the concentration of each sample of the three batches of reagents is 1000 copies/mL
  • the positive detection rate is ⁇ 95%. Therefore, the minimum detection limit of this product is 1000 copies/mL.
  • the products are respectively measured for precision reference products, and the coefficient of variation CV is not higher than 10%, and the coefficient of variation between batches is not higher than 10%.
  • Hemoglobin interference sample sample 0.072mL+0.008mL 20g/L hemoglobin
  • Mucin interference sample sample 0.072mL+0.008mL 200mg/mL mucin
  • the basic sample is directly used for detection, and the detection result is the basic value.
  • Basic sample Basic sample 0.09mL+0.01mL normal saline
  • Virus cross sample sample 0.072mL+0.008mL 106pfu/mL
  • test results of the cross-sample prepared by the above sample and the corresponding basic sample are consistent.
  • the ratio of the cross-sample detection value to the basic sample detection value is between 0.9 and 1.1, and the common pathogenic microorganisms in different regions are positive, 2019-
  • the test results of nCoV-negative samples are all negative, indicating that there is no crossover phenomenon between this reagent and the following common respiratory pathogens.
  • DNA extract 2 DNA extract 3 concentration 95 ⁇ g/mL 90 ⁇ g/mL 102 ⁇ g/mL volume 0.1mL 0.1mL 0.1mL 0.1mL
  • test results of the cross-sample prepared by the above sample and the corresponding basic sample are consistent, and the ratio of the cross-sample detection value to the basic sample detection value is between 0.9 and 1.1, indicating that there is no crossover phenomenon between this reagent and human genomic DNA .

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Abstract

本发明公开了一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒及其制备方法及检测方法，所述试剂盒包括COVID-19反应液，所述COVID-19反应液由COVID-19荧光标记物与COVID-19探针溶液配制而成；所述COVID-19探针溶液包括：ORF1ab区段探针、N区段探针、E区段探针，所述ORF1ab区段探针用于检测COVID-19开放阅读编码框lab，所述N区段探针用于检测COVID-19囊膜蛋白基因，所述E区段探针用于检测COVID-19核壳蛋白基因。

Description

新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒及制备方法及检测方法 技术领域
本发明涉及核酸检测技术领域，特别是一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒及该试剂盒的制备方法。
背景技术
新型冠状病毒COVID-19属于β属的冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60-140nm，其基因特征与SARSr-CoV和MERSr-CoV有明显区别，与蝙蝠SARS样冠状病毒(bat-SL-CoVZC45)同源性达85％以上。
新型冠状病毒感染的肺炎是由新型冠状病毒感染导致的肺部炎症，现已将该病纳入《中华人民共和国传染病防治法》规定的乙类传染病，并采取甲类传染病的预防、控制措施。随着疫情的蔓延，急需一种能够准确快速诊断新型冠状病毒的检测试剂。
目前已经有6家企业取得了新型冠状病毒COVID-19核酸检测试剂的注册文号：
1)圣湘生物科技股份有限公司的新型冠状病毒COVID-19核酸检测试剂盒(荧光PCR法)(国械注准20203400064)；
2)中山大学达安基因股份有限公司的新型冠状病毒COVID-19核酸检测试剂盒(荧光PCR法)(国械注准20203400063)；
3)上海捷诺生物科技有限公司的新型冠状病毒COVID-19核酸检测试剂盒(荧光PCR法)(国械注准20203400058)；
4)上海之江生物科技股份有限公司的新型冠状病毒COVID-19核酸检测试剂盒(荧光PCR法)(国械注准20203400057)；
5)华大生物科技(武汉)有限公司的新型冠状病毒COVID-19核酸检测试剂盒(荧光PCR法)(国械注准20203400060)；
6)华大生物科技(武汉)有限公司的新型冠状病毒COVID-19核酸检测试剂盒(联合探针锚定聚合测序法)(国械注准20203400059)。
上述各新型冠状病毒COVID-19核酸检测试剂盒主要是采用荧光PCR核酸检测方法，检测原理是通过在PCR反应体系中加入荧光报告基团和荧光淬灭基团，随着PCR反应的进行，扩增产物不断积累，导致荧光信号不断积累，对荧光信号进行实时监测并得到未知样品的Ct(Threshold cycle)值，Ct值是指产生可被检测到的荧光信号所需的最小循环数，是在PCR循环过程中荧光信号由本底开始进入指数增长阶段的拐点所对应的循环次数。通常用不同浓度的标准样品的Ct值来产生标准曲线，从标准曲线中自动地计算出未知样本的初始模板量。
上述荧光PCR核酸检测方法虽然定量准确、重现性好，但是，该方法需要专业的PCR实验室、专用的PCR仪器，需要专门经过培训的技术人员进行操作，还需要对样本进行核酸提取和纯化处理，需要-20℃以下的冷链运输，检测时间长达1-3小时，检测效率低，并且检测过程有核酸扩增，容易引起实验室阳性产物污染，等诸多问题，使用不便。
发明内容
本发明为解决上述问题，提供了一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒及制备方法及检测方法，不仅检测时间更短，而且无需专业技术人员操作，无需配套实验室和专用仪器，使用更加方便快捷。
本发明的目的之一，在于提供一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒，所述试剂盒包括COVID-19反应液，所述COVID-19反应液由COVID-19荧光标记物与COVID-19探针溶液配制而成；所述COVID-19探针溶液包括：ORF1ab区段探针、N区段探针、E区段探针，所述ORF1ab区段探针用于检测COVID-19开放阅读编码框lab，所述N区段探针用于检测COVID-19囊膜蛋白基因，所述E区段探针用于检测COVID-19核壳蛋白基因。
优选的，所述ORF1ab区段探针的序列为：
Figure PCTCN2021077652-appb-000001
所述N区段探针的序列为：
Figure PCTCN2021077652-appb-000002
所述E区段探针的序列为：
Figure PCTCN2021077652-appb-000003
优选的，所述COVID-19荧光标记物采用荧光物质与COVID-19标记原料耦合制成；所述COVID-19标记原料采用COVID-19抗原或抗体；所述荧光物质采用以下任一种：FITC荧光素、荧光微球、荧光颗粒、生物荧光素。
优选的，所述试剂盒还包括COVID-19阴性对照品，所述阴性对照品包括以下对照品中的一种或多种：生理盐水性对照品、纯化水性对照品、非COVID-19病原体对照品、未含有COVID-19靶序列的假病毒。
进一步的，所述阴性对照品包括N1～N17：N1～N2为生理盐水性对照品，N3～N4为纯化水性对照品，N5～N13为人咽拭子样本，N14～N17为未含有COVID-19靶序列的假病毒；其中，所述人咽拭子样本中，COVID-19均为阴性，非COVID-19病原体对照品为阳性；所述未含有COVID-19靶序列的假病毒为冠状病毒的亚型，N14为人冠状病毒229E N区段阳性，N15为人冠状病毒NL63 N区段阳性，N16为人冠状病毒OC43 N区段阳性，N17为人冠状病毒HKU1 N区段阳性。
优选的，所述试剂盒还包括COVID-19阳性对照品，所述阳性对照品为含有COVID-19靶序列的假病毒；所述阳性对照品包括P1、P2、P3，其中，P1为COVID-19 N区段阳性、P2为COVID-19 E区段阳性、P3为COVID-19 ORF 1ab区段阳性；所述阳性对照品的浓度为3000TU/mL±5％。
优选的，所述试剂盒还包括精密度对照品和检测限对照品，所述精密度对照品包括J1、J2、J3，J1～J2为含有COVID-19靶序列的假病毒，均为COVID-19 ORF 1ab区段阳性，浓度分别为2000TU/mL±5％和5000TU/mL±5％，J3为混合人阴性咽拭子样本，且COVID-19阴性；所述检测限对照品包括L1、L2、L3，L1为COVID-19 N区段阳性、L2为COVID-19 E区段阳性、L3为COVID-19 ORF 1ab区段阳性，所述检测限对照品的浓度为1000TU/mL±5％。
优选的，所述试剂盒还包括COVID-19检测条、COVID-19复溶液、COVID-19样品保存液。
本发明的目的之二在于，提供如上任一项所述的新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒的制备方法，其包括COVID-19荧光标记物的制备，步骤如下：
活化：将1％荧光微球溶液以1.0mg/mL±5％的比例，EDC溶液以0.6mg/mL±5％的比例加入配制好的0.05M±5％硼酸盐缓冲液混匀，置于旋转混匀器上旋转20分钟以上，活化后以15000～16000rpm离心30分钟以上，去上清，并用0.05M±5％硼酸缓冲液重悬，混匀。
偶联：向所述活化完的荧光微球溶液中按0.2mg/mL的量加入COVID-19抗体，混匀，置于旋转混匀器上旋转2小时以上，得到荧光微球标记结合物溶液；
封闭：按0.1ml/mL±5％比例将10％BSA溶液加入到荧光微球标记结合物溶液，混匀，置于旋转混匀器上旋转12～16小时。
离心重悬：将荧光微球标记结合物溶液以15000～16000rpm离心，去上清，用等体积0.05M±5％硼酸盐缓冲液洗涤，最后沉淀用上清溶液等体积量的标记物稀释液重悬，制备成COVID-19荧光标记物。
优选的，包括COVID-19反应液的制备，步骤如下：
荧光标记物配制：将所述COVID-19荧光标记物按照比例用标记物稀释液稀释，所述比例为：稀释液:T线标记物:C线标记物＝17:2:1；
探针溶液配制：将COVID-19探针用核酸溶解液稀释至10μM±5％，得到COVID-19探针溶液；
反应液配制：将稀释后的COVID-19荧光标记物和所述COVID-19探针溶液按1:1的体积比混合，得到COVID-19反应液；
反应液分装：将所述COVID-19反应液分装在反应管，并在温度18～28℃、湿度≤30％条件下干燥6～8小时。
优选的，还包括COVID-19阳性对照品的制备步骤：用阳性对照品稀释液将人工合成的COVID-19  RNA稀释到2000TU/mL±5％；将稀释后的阳性对照品分装在可立管，并在温度18～28℃、湿度≤30％条件下干燥6～8小时。
本发明的目的之三在于，提供如上任一项所述的新型冠状病毒核酸快速检测试剂盒的检测方法，其包括以下步骤：
通过杂交捕获获取待测样本中的目标核酸片段；
将所述目标核酸片段与所述COVID-19反应液进行核酸杂交反应，得到待测液；
将所述待测液加入COVID-19检测条中进行荧光信号识别。
本发明的有益效果是：
本发明的新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒运用杂交捕获免疫荧光分析法，英文简称HC-IFA，是一种通过杂交捕获获取待测样本中的目标核酸片段，通过荧光信号识别，对样本中目标核酸片段的数量进行定性、半定量、定量判断的方法。相比一般的抗原抗体免疫检测，本发明涵盖了核酸检测，达到了分子水平的检测灵敏度，实现了核酸检测荧光识别；相比一般的荧光PCR和测序检测，信号强度更强，特异性更好，检测时间更短，无需专业技术人员操作，运输和保存无需冷藏，反应过程中无变温环节，无需配套实验室和配套PCR仪器，可进行单人份检测，检测时间单人份30min，检测时间短，一小时检测通量100人份～200人份。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本发明的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1为本发明一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒的制备工艺流程简图。
图2为本发明一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒的具体检测过程所用S9.6蛋白型号信息。
具体实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚、明白，以下结合附图及实施例对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
第一实施例(试剂盒)：
本实施例的一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒，所述试剂盒包括COVID-19反应液、COVID-19复溶液、COVID-19阴性对照品、COVID-19阳性对照品、COVID-19样本保存液，各液体分别分装在反应管或可立管中，还包括COVID-19检测条，以及铝箔袋卡、试剂盒盒签、试剂盒说明书等。
所述COVID-19反应液由COVID-19荧光标记物与COVID-19探针溶液配制而成；所述COVID-19探针溶液包括：ORF1ab区段探针、N区段探针、E区段探针，所述ORF1ab区段探针用于检测COVID-19开放阅读编码框lab，所述N区段探针用于检测COVID-19囊膜蛋白基因，所述E区段探针用于检测COVID-19核壳蛋白基因。
其中，所述ORF1ab区段探针的序列为：
Figure PCTCN2021077652-appb-000004
所述N区段探针的序列为：
Figure PCTCN2021077652-appb-000005
所述E区段探针的序列为：
Figure PCTCN2021077652-appb-000006
所述COVID-19荧光标记物采用荧光物质与COVID-19标记原料耦合制成；所述COVID-19标记原料采用COVID-19抗体；所述荧光物质采用以下任一种：FITC荧光素、荧光微球、荧光颗粒、生物荧光素，以及其他可发出荧光的物质，不以此为限。
所述COVID-19阴性对照品包括以下对照品中的一种或多种：生理盐水性对照品、纯化水性对照品、非COVID-19病原体对照品、未含有COVID-19靶序列的假病毒。具体的，所述阴性对照品包括N1～N17： N1～N2为生理盐水性对照品，N3～N4为纯化水性对照品，N5～N13为人咽拭子样本，N14～N17为未含有COVID-19靶序列的假病毒；其中，所述人咽拭子样本中，COVID-19均为阴性，非COVID-19病原体对照品为阳性；所述未含有COVID-19靶序列的假病毒为冠状病毒的亚型，N14为人冠状病毒229E N区段阳性，N15为人冠状病毒NL63 N区段阳性，N16为人冠状病毒OC43 N区段阳性，N17为人冠状病毒HKU1 N区段阳性。
所述COVID-19阳性对照品为含有COVID-19靶序列的假病毒；所述阳性对照品包括P1、P2、P3，其中，P1为COVID-19 N区段阳性、P2为COVID-19 E区段阳性、P3为COVID-19 ORF 1ab区段阳性；所述阳性对照品的浓度为3000TU/mL±5％。
所述精密度对照品包括J1、J2、J3，J1～J2为含有COVID-19靶序列的假病毒，均为COVID-19 ORF 1ab区段阳性，浓度分别为2000TU/mL±5％和5000TU/mL±5％，J3为混合人阴性咽拭子样本，且COVID-19阴性；所述检测限对照品包括L1、L2、L3，L1为COVID-19 N区段阳性、L2为COVID-19 E区段阳性、L3为COVID-19 ORF 1ab区段阳性，所述检测限对照品的浓度为1000TU/m±5％L。详见以下表1。
表1-参考品组成表
Figure PCTCN2021077652-appb-000007
Figure PCTCN2021077652-appb-000008
所述检测条可采用层析法或渗滤法，本实施例中，所述检测条采用免疫荧光层析试条，其包括设置于所述检测条一个端部区域上的吸水垫、设置于所述检测条另一个端部区域上的加样区，以及设置在所述吸水垫和所述加样区之间的硝酸纤维膜(NC膜)，所述硝酸纤维膜上设置在T线检测区。将处理过的玻纤垫、处理过的硝酸纤维膜、以及吸水垫依序搭接在PVC底板上，切割成设定宽度的试纸条，即得本实用新型的检测条。
第二实施例(制备方法)：
如图1所示，本实施例提供如上任一项所述的新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒的制备方法，其包括：
(1)COVID-19荧光标记物的制备，步骤如下：
活化：将1％荧光微球溶液以1.0mg/mL±5％的比例，EDC溶液以0.6mg/mL±5％的比例加入配制好的0.05M±5％硼酸盐缓冲液混匀，置于旋转混匀器上旋转20分钟以上，活化后以15000～16000rpm离心30分钟以上，去上清，并用0.05M±5％硼酸缓冲液重悬，混匀。
偶联：向所述活化完的荧光微球溶液中按0.2mg/mL±5％的量加入COVID-19抗体，混匀，置于旋转混匀器上旋转2小时以上，得到荧光微球标记结合物溶液；
封闭：按0.1ml/mL±5％比例将10％BSA溶液加入到荧光微球标记结合物溶液，混匀，置于旋转混匀器上旋转12～16小时。
离心重悬：将荧光微球标记结合物溶液以15000～16000rpm离心，去上清，用等体积0.05M±5％硼酸盐缓冲液(PH 8.0)洗涤两次，最后沉淀用上清溶液等体积量的标记物稀释液重悬，制备成COVID-19荧光标记物。
(2)COVID-19反应液的制备，步骤如下：
荧光标记物配制：将所述COVID-19荧光标记物按照比例用标记物稀释液稀释，所述比例为：稀释液:T线标记物:C线标记物＝17:2:1；
探针溶液配制：将COVID-19探针用核酸溶解液稀释至10μM±5％，得到COVID-19探针溶液；
反应液配制：将稀释后的COVID-19荧光标记物和所述COVID-19探针溶液按1:1的体积比混合，得到COVID-19反应液；
反应液分装：将所述COVID-19反应液分装在反应管，4μL/管，并在温度18～28℃、湿度≤30％条件下干燥6～8小时。
(3)COVID-19阳性对照品的制备步骤：用阳性对照品稀释液将人工合成的COVID-19 RNA稀释到2000TU/mL±5％；将稀释后的阳性对照品分装在可立管，5μL/管，并在温度18～28℃、湿度≤30％条件下干燥6～8小时。
(4)COVID-19检测条的包被条件：
T线包被浓度为0.5mg/mL±5％、C线包被浓度为1.0mg/mL±5％；
喷量为1.0μL/cm±5％、导轨速度：100mm/s±5％、喷嘴间隔：6mm±5％；
干燥：温度为18～28℃、湿度为≤30％、干燥时间：16h-20h。
(5)检测试剂的检测条件：
反应管复溶液加入量为85μL±5％；
样本取样量为20μL±5％；
吸取100μL±5％混匀后的样本，加入到检测卡的加样孔中；
核酸杂交反应条件为37℃，反应时间为15分钟以上；
试剂检测反应时间为15分钟以上。
(6)冻干工艺：
本实施例的制备方法的反应过程中涉及的中间制剂均为冻干品，仅需配制工作液，无稀释等复杂操作；冻干处理方式为：在-30℃冻干5h，后续的12h冻干过程中逐步升温至-10℃，升温速率为5℃/3h，最后在-10℃冻干7-19h小时。
第三实施例(检测方法)：
本实施例还提供如上任一项所述的新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒的检测方法，其包括以下步骤：
通过杂交捕获获取待测样本中的目标核酸片段；
将所述目标核酸片段与所述COVID-19反应液进行核酸杂交反应，得到待测液；
将所述待测液加入COVID-19检测条中进行荧光信号识别；本实施例中，是将所述待测液加入所述检测条的加样区，90s后，从加样区再次加入冲洗液100ul，等待10分钟后，对所述检测条的检测区进行荧光检测，读取检测结果。
检测结果的判断如下:
使用480nm的激发光进行照射，识别520nm的荧光发出信号，存在荧光发出信号的情况判读为阳性，反之则为阴性。根据需要可以根据发出信号的强弱来判读反应的强弱，识别仪器运用一般的荧光读数仪即可，例如苏州和迈科技生产的便携式免疫荧光分析仪。
需要说明的是，本说明书中的各个实施例均采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似的部分互相参见即可。对于制备方法和检测方法实施例而言，由于其与试剂盒实施例基本相似，所以描述的比较简单，相关之处参见试剂盒实施例的部分说明即可。
本发明的基于核酸杂交捕获的免疫荧光分析法的新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒及检测方法，与现有的荧光PCR核酸检测方法的效果比对具体如下：
表2-技术效果对比图
Figure PCTCN2021077652-appb-000009
本发明具体检测过程说明：
(1)采取咽拭子样本，放入样本保存管(样本保存管有保存液)，获得保存液与样本的混合物， 并将这个混合物定义为检测样本。
(2)取适量的检测样本放入反应管，反应管中有反应液干燥处理后的粉末，并将粉末定义为反应液粉末。反应液粉末的制作可参考如下：
探针溶液配制：将COVID-19探针用核酸溶解液稀释至10μM±5％，得到COVID-19探针溶液；反应液配制：将稀释后的COVID-19荧光标记物和所述COVID-19探针溶液按1:1的体积比混合，得到COVID-19反应液；反应液分装：将所述COVID-19反应液分装在反应管，4μL/管，并在温度18～28℃、湿度≤30％条件下干燥6～8小时。反应液粉末中包含了探针溶液与标记了荧光信号的蛋白。标记了荧光信号的蛋白的制作过程可参考如下：
活化：将1％荧光微球溶液以1.0mg/mL±5％的比例，EDC溶液以0.6mg/mL±5％的比例加入配制好的0.05M±5％硼酸盐缓冲液混匀，置于旋转混匀器上旋转20分钟以上，活化后以15000～16000rpm离心30分钟以上，去上清，并用0.05M±5％硼酸缓冲液重悬，混匀。
偶联：向所述活化完的荧光微球溶液中按0.2mg/mL±5％的量加入COVID-19抗体，混匀，置于旋转混匀器上旋转2小时以上，得到荧光微球标记结合物溶液；
封闭：按0.1ml/mL±5％比例将10％BSA溶液加入到荧光微球标记结合物溶液，混匀，置于旋转混匀器上旋转12～16小时。
离心重悬：将荧光微球标记结合物溶液以15000～16000rpm离心，去上清，用等体积0.05M±5％硼酸盐缓冲液(PH 8.0)洗涤两次，最后沉淀用上清溶液等体积量的标记物稀释液重悬，制备成COVID-19荧光标记物。
其中所使用的荧光信号来自荧光微球，蛋白为默克生产的S9.6蛋白，具体型号请参考图2。
(3)当检测样本进入反应管后，在检测样本中保存液的作用下，反应液粉末复溶，形成液相环境。当反应液复溶液体不足时，可适当加入复溶液，加入量为85μL±5％，复溶液一般为生理盐水或者加了杀菌成分的生理盐水。在反应管中混匀后，将反应管放置用加热器加热到37±2摄氏度，在这个液相环境中，进行了两个部分的反应，分别是：
a.DNA对目标RNA片段的识别，杂交，捕获，结合成DNA-RNA复合物；b.蛋白1对DNA-RNA复合物的特异性识别并结合，形成DNA-RNA-蛋白-荧光信号复合物，将此复合物定位为反应复合物。
(4)将反应复合物取出，加入到检测试剂卡上，在辅助液的作用下在试条上进行层析，层析到T线，T线包被有默克生产的S9.6蛋白。当检测样本中有COVID-19病毒时，便会在反应复合物中存在DNA-RNA-蛋白-荧光信号复合物，因为默克生产的S9.6蛋白可以特异性的识别并结合DNA-RNA复合物，因此可将反应复合物中的DNA-RNA-蛋白-荧光信号复合物固定在T线上，形成“蛋白-DNA-RNA-蛋白-荧光信号”，在T线上形成荧光带，于是在T线上可以通过仪器检测到荧光信号。反应复合物经过T线后继续层析会经过C线，C线包被有兔多抗，兔多抗可以非特异性结合默克生产的S9.6蛋白，所以在过量的前提下，理论上一定会捕获一定量的联接有荧光信号的蛋白，形成荧光带，可以通过仪器检测识别。以上提及的T线标记物与C线标记物准确来说是包被在T线上的默克生产的S9.6蛋白和包被在C线上的兔多抗，所有的蛋白均未标记荧光信号。
本发明具体实验数据内容：
第一部分 实验准备
每项性能的主要研究方法均采用国内实际采用的评价方法相结合的方法，包括产品的阳性参考品符合率、阴性参考品符合率、最低检测限、精密度、干扰实验、交叉反应等项目完成实验测试。
表3-实验所需物料信息
序号 名称 批号/型号 状态
1 新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法) /  
2 免疫分析仪    
3 2019-nCoV参考品   /
分析性能评估试验所用样本及其他物料信息如下表：
表4-分析性能评估试验所用样本及其他物料信息
Figure PCTCN2021077652-appb-000010
第二部分 阳/阴性参考品符合率
1、标准要求
1.1阳性参考品符合率
检测13份阳性参考品(P1-P13)，包括2019-CoV N区段、2019-nCoV E区段、2019-nCoV ORF 1ab区段阳性及痰液、咽拭子液阳性样本各5例，结果均应为阳性。
1.2阴性参考品符合率
检测19份阴性参考品(N1-N19)，结果均应为阴性。
表5-19份阴性参考品(N1-N19)信息
Figure PCTCN2021077652-appb-000011
Figure PCTCN2021077652-appb-000012
2、方法
用新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法)检测8份阳性参考品(P1-P8)，检测19份阴性参考品(N1-N19)，各检测1次。
3、结果
表6-试剂阳/阴性参考品符合率结果
Figure PCTCN2021077652-appb-000013
Figure PCTCN2021077652-appb-000014
4、结论
三批试剂的13例阳性参考品、19例阴性参考品检测结果均符合拟定的标准。
第三部分 最低检测限的确定
1、目的
确定新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法)的最低检测限。
2、物料信息
表7-确定最低检测限实验所需物料信息
Figure PCTCN2021077652-appb-000015
3、建立
3.1假病毒样本制备
3个区段(包括2019-CoV N区段、2019-nCoV E区段、2019-nCoV ORF 1ab区段)假病毒分别用混合阴性样本作为稀释液进行梯度稀释，稀释成5000copies/mL、2500copies/mL、1000copies/mL、800copies/mL、500copies/mL、250copies/mL、100copies/mL。
3.3样本测定
用新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法)进行样本(假病毒样本、咽拭子样本、痰液样本)测定，每个样本各测定20次，计算阳性检出率。
3.4确定
阳性检出率≥95％的最低浓度为该区段的最低检测限。
3.5样本检测
表8-各稀释度样本测定结果
Figure PCTCN2021077652-appb-000016
Figure PCTCN2021077652-appb-000017
上述结果显示，试剂检测最低检测限浓度1000copies/mL水平的样本，其阳性检出率均≥95％。
6、结论
综上所述，三批试剂各样本浓度为1000copies/mL时，阳性检出率≥95％，因此，该产品的最低检测限为1000copies/mL。
第四部分 精密度测试
1、标准要求
(1)批内精密度变异系数CV：≤10％，且结果均一致；
(2)批间精密度变异系数CV：≤10％，且结果均一致。
(3)中间精密度变异系数CV：≤10％，且结果均一致。
2、物料信息
表9-确定精密度实验所需物料信息
Figure PCTCN2021077652-appb-000018
Figure PCTCN2021077652-appb-000019
3、方法
3.1精密度参考品测定
取三批新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法)，分别用3份精密度参考品进行检测
得到20组数据，进行精密度分析；
4、结果
4.1精密度参考品结果
表10-三批试剂验证精密度参考品J1测定结果
Figure PCTCN2021077652-appb-000020
Figure PCTCN2021077652-appb-000021
Figure PCTCN2021077652-appb-000022
Figure PCTCN2021077652-appb-000023
4.2 5、结论
产品分别测定精密度参考品，变异系数CV不高于10％，批间变异系数CV不高于10％。
第五部分 内源性干扰实验
1、目的
分析常见样本中的内源性干扰物质血红蛋白、粘蛋白对新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法)检测结果的影响，评估产品的抗干扰性能。
2、物料信息
表11-内源性干扰试验所需物料信息
Figure PCTCN2021077652-appb-000024
3、方法
3.1选择咽拭子
3.2基础样本制备
基础样本：临床样本0.072mL+0.008mL生理盐水
3.2干扰样本制备
在上述样本中分别添加下列干扰物，制备对应的干扰样本：
血红蛋白干扰样本：样本0.072mL+0.008mL 20g/L血红蛋白
样本0.072mL+0.008mL10g/L血红蛋白
样本0.072mL+0.008mL 5g/L血红蛋白
粘蛋白干扰样本：样本0.072mL+0.008mL 200mg/mL粘蛋白
样本0.072mL+0.008mL100mg/mL粘蛋白
样本0.072mL+0.008mL50mg/mL粘蛋白
3.3测定
取新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法)分别检测上述制备样本，计算干扰样本检测值与基础样本检测值的比值。
4、结果
4.1咽拭子样本干扰结果
4.1.2血红蛋白干扰检测结果
表12-三批试剂咽拭子阴性样本干扰试验(干扰物：血红蛋白)检测结果
Figure PCTCN2021077652-appb-000025
Figure PCTCN2021077652-appb-000026
5、结论
上述样本制备的干扰样本和相应的基础样本检测结果均一致，表明样本中浓度含量为2g/L的血红蛋白和20mg/mL的粘蛋白对检测无影响。
第六部分 交叉反应实验
1、目的
分析常见呼吸道病原体对新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法)检测结果的影响，评估产品的特异性。
2、物料信息
表13-交叉反应实验所需物料信息
Figure PCTCN2021077652-appb-000027
3、方法
3.1.2基础样本制备
基础样本直接用于检测，检测结果为基础值。
基础样本：基础样本0.09mL+0.01mL生理盐水
本，即制备每种病原微生物交叉的阴性、临界阳性样本各2例。
4)交叉样本制备方法
病毒交叉样本：样本0.072mL+0.008mL 106pfu/mL
细菌、支原体、衣原体交叉样本：样本0.072mL+0.008mL 107cfu/mL
表14-各病原微生物信息
Figure PCTCN2021077652-appb-000028
Figure PCTCN2021077652-appb-000029
Figure PCTCN2021077652-appb-000030
Figure PCTCN2021077652-appb-000031
3.1.4测定
取新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法)分别检测上述制备样本)，计算交叉样本检测值与基础样本检测值的比值。
表15-试剂交叉试验检测结果
Figure PCTCN2021077652-appb-000032
Figure PCTCN2021077652-appb-000033
5、结论
综上所述，上述样本制备的交叉样本和相应的基础样本检测结果均一致，交叉样本检测值与基础样本检测值的比值在0.9～1.1之间，并且不同地区常见病原微生物阳性有，2019-nCoV阴性样本检测结果均为阴性，表明本试剂与下列常见呼吸道病原体不存在交叉现象。
表16-与2019-nCoV阴性样本不存在交叉现象的常见呼吸道病原体
Figure PCTCN2021077652-appb-000034
Figure PCTCN2021077652-appb-000035
第七部分 人类基因组DNA交叉反应实验
1、目的
分析人类基因组DNA对新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法)检测结果的影响，评估产品的特异性。
2、物料信息
表17-人类基因组DNA交叉反应实验所需物料信息
Figure PCTCN2021077652-appb-000036
3、方法
3.1人类基因组DNA来源、制备、定值
取3例来源不同的全血样本，各1mL用天根生化科技(北京)有限公司的血液基因组DNA提取系统(0.1-20mL)试剂盒进行DNA提取，提取的DNA用紫外分光光度计进行浓度测试。
3.3样本制备
基础样本：基础样本0.072mL+0.008mL生理盐水
交叉样本：样本0.072mL+0.008mL DNA提取液
3.4测定
取三批新型冠状病毒2019-nCoV核酸测定试剂盒(杂交捕获免疫荧光法)分别检测上述制备样本)，计算交叉样本检测值与基础样本检测值的比值。
4、结果
4.1 DNA提取液浓度
表18-DNA提取液浓度
  DNA提取液1 DNA提取液2 DNA提取液3
浓度 95μg/mL 90μg/mL 102μg/mL
体积 0.1mL 0.1mL 0.1mL
4.2咽拭子样本交叉结果
表19-试剂交叉试验检测结果(临界阳性咽拭子样本1)
Figure PCTCN2021077652-appb-000037
Figure PCTCN2021077652-appb-000038
5、结论
综上所述，上述样本制备的交叉样本和相应的基础样本检测结果均一致，交叉样本检测值与基础样本检测值的比值在0.9～1.1之间，表明本试剂与人类基因组DNA不存在交叉现象。
上述说明示出并描述了本发明的优选实施例，应当理解本发明并非局限于本文所披露的形式，不应看作是对其他实施例的排除，而可用于各种其他组合、修改和环境，并能够在本文发明构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本发明的精神和范围，则都应在本发明所附权利要求的保护范围内。

Claims (12)

  1. 一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒，其特征在于，所述试剂盒包括COVID-19反应液，所述COVID-19反应液由COVID-19荧光标记物与COVID-19探针溶液配制而成；所述COVID-19探针溶液包括：ORF1ab区段探针、N区段探针、E区段探针，所述ORF1ab区段探针用于检测COVID-19开放阅读编码框lab，所述N区段探针用于检测COVID-19囊膜蛋白基因，所述E区段探针用于检测COVID-19核壳蛋白基因。
  2. 根据权利要求1所述的一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒，其特征在于：
    所述ORF1ab区段探针的序列为：
    Aacgattgtgcatcagctgactgaagcatgggttcgcggagttgatcacaactacagccataacctttccacataccgcagac；
    所述N区段探针的序列为：
    Agagcagcatcaccgccattgccagccattctagcaggagaagttc；
    所述E区段探针的序列为：
    aaggatggctagtgtaactagcaagaataccacgaaagcaagaaaaa。
  3. 根据权利要求1所述的一种新型冠状病毒核酸快速检测试剂盒，其特征在于，所述COVID-19荧光标记物采用荧光物质与COVID-19标记原料耦合制成；所述COVID-19标记原料采用COVID-19抗原或抗体；所述荧光物质采用以下任一种：FITC荧光素、荧光微球、荧光颗粒、生物荧光素。
  4. 根据权利要求1所述的一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒，其特征在于，所述试剂盒还包括COVID-19阴性对照品，所述阴性对照品包括以下对照品中的一种或多种：生理盐水性对照品、纯化水性对照品、非COVID-19病原体对照品、未含有COVID-19靶序列的假病毒。
  5. 根据权利要求4所述的一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒，其特征在于，所述阴性对照品包括N1～N17：N1～N2为生理盐水性对照品，N3～N4为纯化水性对照品，N5～N13为人咽拭子样本，N14～N17为未含有COVID-19靶序列的假病毒；其中，所述人咽拭子样本中，COVID-19均为阴性，非COVID-19病原体对照品为阳性；所述未含有COVID-19靶序列的假病毒为冠状病毒的亚型，N14为人冠状病毒229E N区段阳性，N15为人冠状病毒NL63 N区段阳性，N16为人冠状病毒OC43 N区段阳性，N17为人冠状病毒HKU1 N区段阳性。
  6. 根据权利要求1所述的一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒，其特征在于，所述试剂盒还包括COVID-19阳性对照品，所述阳性对照品为含有COVID-19靶序列的假病毒；所述阳性对照品包括P1、P2、P3，其中，P1为COVID-19 N区段阳性、P2为COVID-19 E区段阳性、P3为COVID-19 ORF 1ab区段阳性；所述阳性对照品的浓度为3000 TU/mL±5％。
  7. 根据权利要求1所述的一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒，其特征在于，所述试剂盒还包括精密度对照品和检测限对照品，所述精密度对照品包括J1、J2、J3，J1～J2为含有COVID-19靶序列的假病毒，均为COVID-19 ORF 1ab区段阳性，浓度分别为2000TU/mL±5％和5000TU/mL±5％，J3为混合人阴性咽拭子样本，且COVID-19阴性；所述检测限对照品包括L1、L2、L3，L1为COVID-19 N区段阳性、L2为COVID-19 E区段阳性、L3为COVID-19 ORF 1ab区段阳性，所述检测限对照品的浓度为1000 TU/mL±5％。
  8. 根据权利要求1所述的一种新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒，其特征在于，所述试剂盒还包括COVID-19检测条、COVID-19复溶液、COVID-19样品保存液。
  9. 如权利要求1至8任一项所述的新型冠状病毒核酸快速杂交捕获免疫荧光检测试剂盒的制备方法，其特征在于，包括COVID-19荧光标记物的制备，步骤如下：
    活化：将1％荧光微球溶液以1.0mg/mL±5％的比例，EDC溶液以0.6mg/mL±5％的比例加入配制好的0.05M±5％硼酸盐缓冲液混匀，置于旋转混匀器上旋转20分钟以上，活化后以15000～16000rpm离心30分钟以上，去上清，并用0.05M±5％硼酸缓冲液重悬，混匀；
    偶联：向所述活化完的荧光微球溶液中按0.2mg/mL±5％的量加入COVID-19抗体，混匀，置于旋转混匀器上旋转2小时以上，得到荧光微球标记结合物溶液；
    封闭：按0.1ml/mL±5％比例将10％BSA溶液加入到荧光微球标记结合物溶液，混匀，置于旋转混匀器上旋转12～16小时；
    离心重悬：将荧光微球标记结合物溶液以15000～16000rpm离心，去上清，用等体积0.05M±5％硼酸盐缓冲液洗涤，最后沉淀用上清溶液等体积量的标记物稀释液重悬，制备成COVID-19荧光标记物。
  10. 根据权利要求9所述的的制备方法，其特征在于，包括COVID-19反应液的制备，步骤如下：
    荧光标记物配制：将所述COVID-19荧光标记物按照比例用标记物稀释液稀释，所述比例为：稀释液:T线标记物:C线标记物＝17:2:1；
    探针溶液配制：将COVID-19探针用核酸溶解液稀释至10μM±5％，得到COVID-19探针溶液；
    反应液配制：将稀释后的COVID-19荧光标记物和所述COVID-19探针溶液按1:1的体积比混合，得到COVID-19反应液；
    反应液分装：将所述COVID-19反应液分装在反应管，并在温度18～28℃、湿度≤30％条件下干燥6～8小时。
  11. 根据权利要求9所述的制备方法，其特征在于，还包括COVID-19阳性对照品的制备步骤：用阳性对照品稀释液将人工合成的COVID-19 RNA稀释到2000TU/mL±5％；将稀释后的阳性对照品分装在可立管，并在温度18～28℃、湿度≤30％条件下干燥6～8小时。
  12. 如权利要求1至8任一项所述的新型冠状病毒核酸快速检测试剂盒核酸快速杂交捕获免疫荧光检测试剂盒的检测方法，其特征在于，包括以下步骤：
    通过杂交捕获获取待测样本中的目标核酸片段；
    将所述目标核酸片段与所述COVID-19反应液进行核酸杂交反应，得到待测液；
    将所述待测液加入COVID-19检测条中进行荧光信号识别。
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