CN113493857A - Coronavirus fluorescence detector, coronavirus fluorescence detection kit and coronavirus fluorescence detection method - Google Patents
Coronavirus fluorescence detector, coronavirus fluorescence detection kit and coronavirus fluorescence detection method Download PDFInfo
- Publication number
- CN113493857A CN113493857A CN202010261223.9A CN202010261223A CN113493857A CN 113493857 A CN113493857 A CN 113493857A CN 202010261223 A CN202010261223 A CN 202010261223A CN 113493857 A CN113493857 A CN 113493857A
- Authority
- CN
- China
- Prior art keywords
- detection
- coronavirus
- optical filter
- fluorescence
- image acquisition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000001917 fluorescence detection Methods 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 137
- 230000003287 optical effect Effects 0.000 claims abstract description 52
- 230000005284 excitation Effects 0.000 claims abstract description 21
- 230000003321 amplification Effects 0.000 claims description 34
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 34
- 239000000523 sample Substances 0.000 claims description 33
- 102000039446 nucleic acids Human genes 0.000 claims description 23
- 108020004707 nucleic acids Proteins 0.000 claims description 23
- 150000007523 nucleic acids Chemical class 0.000 claims description 23
- 238000003752 polymerase chain reaction Methods 0.000 claims description 23
- 239000013612 plasmid Substances 0.000 claims description 20
- 239000007853 buffer solution Substances 0.000 claims description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 19
- 102100034343 Integrase Human genes 0.000 claims description 16
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 239000000975 dye Substances 0.000 claims description 12
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 12
- 229920002477 rna polymer Polymers 0.000 claims description 11
- 239000000411 inducer Substances 0.000 claims description 10
- 108020004414 DNA Proteins 0.000 claims description 9
- 102000053602 DNA Human genes 0.000 claims description 9
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 9
- 239000013558 reference substance Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 7
- 241001678559 COVID-19 virus Species 0.000 claims description 6
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 238000003259 recombinant expression Methods 0.000 claims description 6
- 239000008213 purified water Substances 0.000 claims description 5
- 238000010791 quenching Methods 0.000 claims description 5
- 230000000171 quenching effect Effects 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 101710141454 Nucleoprotein Proteins 0.000 claims description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 4
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 claims description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 3
- 229960000956 coumarin Drugs 0.000 claims description 3
- 235000001671 coumarin Nutrition 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 239000001022 rhodamine dye Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- 102100031673 Corneodesmosin Human genes 0.000 claims description 2
- 101710139375 Corneodesmosin Proteins 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
- 102000006382 Ribonucleases Human genes 0.000 claims description 2
- 108010083644 Ribonucleases Proteins 0.000 claims description 2
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims description 2
- 108700010904 coronavirus proteins Proteins 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
- 239000008223 sterile water Substances 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
- 229960000281 trometamol Drugs 0.000 claims description 2
- 239000000987 azo dye Substances 0.000 claims 3
- 239000012266 salt solution Substances 0.000 claims 2
- 239000000337 buffer salt Substances 0.000 claims 1
- 239000008363 phosphate buffer Substances 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 2
- 230000010460 detection of virus Effects 0.000 abstract 1
- 238000010438 heat treatment Methods 0.000 description 29
- 241000700605 Viruses Species 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 108091028664 Ribonucleotide Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 239000013074 reference sample Substances 0.000 description 5
- 239000002336 ribonucleotide Substances 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical group [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007975 buffered saline Substances 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 229910052744 lithium Inorganic materials 0.000 description 2
- 238000007726 management method Methods 0.000 description 2
- 229910001453 nickel ion Inorganic materials 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 1
- GZEYLLPOQRZUDF-UHFFFAOYSA-N 7-(dimethylamino)-4-methylchromen-2-one Chemical compound CC1=CC(=O)OC2=CC(N(C)C)=CC=C21 GZEYLLPOQRZUDF-UHFFFAOYSA-N 0.000 description 1
- 101000786194 Acinetobacter calcoaceticus Uncharacterized protein in PQQ-III 3'region Proteins 0.000 description 1
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N Cadaverine Natural products NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 101000618322 Enterobacteria phage T4 Uncharacterized 10.3 kDa protein in Gp46-Gp47 intergenic region Proteins 0.000 description 1
- UYTPUPDQBNUYGX-UHFFFAOYSA-N Guanine Natural products O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 1
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Natural products O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- GLNDAGDHSLMOKX-UHFFFAOYSA-N coumarin 120 Chemical compound C1=C(N)C=CC2=C1OC(=O)C=C2C GLNDAGDHSLMOKX-UHFFFAOYSA-N 0.000 description 1
- AFYCEAFSNDLKSX-UHFFFAOYSA-N coumarin 460 Chemical compound CC1=CC(=O)OC2=CC(N(CC)CC)=CC=C21 AFYCEAFSNDLKSX-UHFFFAOYSA-N 0.000 description 1
- UIMOXRDVWDLOHW-UHFFFAOYSA-N coumarin 481 Chemical compound FC(F)(F)C1=CC(=O)OC2=CC(N(CC)CC)=CC=C21 UIMOXRDVWDLOHW-UHFFFAOYSA-N 0.000 description 1
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Natural products NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000013500 data storage Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 102100035859 eIF5-mimic protein 2 Human genes 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- WGTODYJZXSJIAG-UHFFFAOYSA-N tetramethylrhodamine chloride Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C(O)=O WGTODYJZXSJIAG-UHFFFAOYSA-N 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The application belongs to the technical field of chemical detection, and relates to a coronavirus fluorescence detection instrument, a kit and a coronavirus fluorescence detection method, which are used for solving the problems that a coronavirus detection instrument is expensive, large in size, time-consuming in analysis, complex in operation, incapable of meeting the requirements of convenient and rapid detection of viruses and the like. The detector includes: a housing with an upper end opened; the detection clamping groove is arranged in the shell and used for fixing the detection card; the light source is positioned on a first optical filter at the light emergent side of the light source, the light source and the first optical filter form an excitation light path, and the detection card is positioned at the light emergent side of the first optical filter; the image acquisition equipment is positioned on a second optical filter at the light inlet side of the image acquisition equipment, and the image acquisition equipment and the second optical filter form an acquisition light path; and the detection chip is connected with the image acquisition equipment and is used for detecting and analyzing the image of the detection card. The device is simple to use, convenient to carry and low in cost, and can be applied to rapid and convenient detection of coronavirus.
Description
Technical Field
The application relates to the technical field of chemical detection, and particularly discloses a coronavirus fluorescence detector, a coronavirus fluorescence detection kit and a coronavirus fluorescence detection method.
Background
The new coronavirus pneumonia caused by 2019 new coronavirus poses a significant and urgent threat to global public health. The virus belongs to the genus of coronavirus, is an RNA virus which is mainly transmitted through respiratory tract, has extremely high infectivity, is mainly transmitted through droplets and contact, has higher pneumonia probability of infected people, can cause acute respiratory distress syndrome and serious respiratory complications in a few patients, even leads to death, and is listed as one of the most serious viruses harming human beings by the world health organization. Therefore, the proper detection technology and method are selected to accurately and quickly identify the etiology, and the method plays a key role in improving the diagnosis and treatment efficiency of diseases and restraining the outbreak of infectious diseases.
In the related technology, the detection of coronavirus by adopting fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction), a technology combining Reverse Transcription of RNA and Polymerase Chain amplification of cDNA, is still the gold standard for confirmation, such as the confirmation of 2019 novel coronavirus, however, the method depends on a real-time fluorescent quantitative PCR instrument with extremely high precision, needs the operation of professional technicians, and has the disadvantages of expensive instrument and equipment, large volume, long analysis and detection time and complicated operation, thus obviously failing to meet the requirement of on-site rapid detection. Therefore, it is necessary to develop a portable detection device to provide technical support for on-site rapid early warning and detection of coronavirus.
Disclosure of Invention
According to one aspect of the application, a coronavirus fluorescence detection instrument, a coronavirus fluorescence detection kit and a coronavirus fluorescence detection method are provided, so that the problem that the instrument cannot meet the requirement for conveniently and rapidly detecting viruses due to the fact that a related coronavirus detection instrument is expensive, large in size, time-consuming in analysis and complex in operation is solved.
According to a first aspect of the present application, there is provided a coronavirus fluorescence detector, the detector comprising:
a housing with an upper end opened;
the detection clamping groove is arranged in the shell and used for fixing the detection card;
the light source and the first optical filter form an excitation light path, and the detection card is positioned on the light emergent side of the first optical filter;
the image acquisition equipment and the second optical filter form an acquisition light path and are used for acquiring a detection card image after fluorescence is excited;
and the detection chip is arranged in the shell, is connected with the image acquisition equipment and is used for detecting and analyzing the detection card image acquired by the image acquisition equipment.
Optionally, the detection card is provided with a plurality of detection areas, and a gap is formed between the detection areas and is used for respectively bearing the objects to be detected;
the detector comprises a plurality of excitation light paths which are arranged in parallel, and each excitation light path corresponds to each detection area.
Optionally, the apparatus further comprises:
the circuit board is arranged in the shell and connected with the light source and the image acquisition equipment, and the detection chip is arranged on the circuit board;
the battery is arranged in the shell, is connected with the circuit board and is used for supplying power to the light source and the image acquisition equipment through the circuit board;
and the upper cover body is buckled at the opening end of the shell.
Optionally, the apparatus further comprises:
the display screen is arranged on the upper cover body and used for protecting the display panel;
and the display panel corresponding to the display screen is arranged in the shell and used for displaying the detection and analysis result obtained by the detection chip.
Optionally, the battery is a lithium battery or an alkaline battery.
Optionally, the wavelength range of the light source is 300-700 nm;
the lower limit of the wavelength of the light source is independently selected from 300nm, 470nm, 550nm, 650nm and 680 nm;
the upper limit of the wavelength of the light source is independently selected from 470nm, 560nm, 640nm, 690nm and 700 nm;
optionally, the wavelength range of the first optical filter is 300-700 nm;
the lower limit of the wavelength of the first optical filter is independently selected from 300nm, 470nm, 550nm, 650nm and 680 nm;
the upper limit of the wavelength of the first light-emitting filter is independently selected from 470nm, 560nm, 640nm, 690nm and 700 nm;
optionally, the light source is detachably connected to the detector, and the first optical filter is detachably connected to the detector.
Optionally, the light source is a light emitting diode.
Optionally, the resolution of the image acquisition device is 1 to 1000 million pixels (pixels);
the lower limit of the resolution of the image acquisition equipment is independently selected from 1 ten thousand pixels, 400 ten thousand pixels, 600 ten thousand pixels, 800 ten thousand pixels and 900 ten thousand pixels;
the upper limit of the resolution of the image acquisition equipment is independently selected from 300 ten thousand pixels, 500 ten thousand pixels, 700 ten thousand pixels, 900 ten thousand pixels and 1000 ten thousand pixels;
optionally, the volume of the image acquisition equipment is 0.1-125 cm3；
Optionally, the image acquisition device is a camera.
Optionally, the wavelength range of the second optical filter is 300-700 nm;
the lower limit of the wavelength of the second optical filter is independently selected from 300nm, 530nm, 550nm, 600 nm and 650 nm;
the upper limit of the wavelength of the second optical filter is independently selected from 400nm, 550nm, 650nm, 680nm and 700 nm;
optionally, the image acquisition device is further provided with a memory;
optionally, the image acquisition device is detachably connected to the detector, and the second optical filter is detachably connected to the detector.
According to a second aspect of the present application, there is provided a fluorescent detection kit for detecting coronavirus, for use with the fluorescent detection apparatus for coronavirus, the kit comprising three sets of specific primer pair and primer probe, inorganic buffer, rna reverse transcriptase, dna polymerase, positive reference plasmid, and negative reference;
the pH value of the inorganic buffer solution is 6.0-8.0;
the positive reference plasmid is a positive reference plasmid of three different gene segments in a coronavirus protein gene;
the negative reference substance is any one of purified water, deionized water or sterile water without the ribonuclease.
The lower limit of the pH value of the inorganic buffer solution is independently selected from 6.0, 6.5, 7.3, 7.5 and 7.9;
the upper limit of the pH value of the inorganic buffer solution is independently selected from 6.5, 7.0, 7.5, 7.8 and 8.0;
optionally, the 5 'end of the primer probe is labeled with a fluorescence quenching group, and the 3' end of the primer probe is labeled with a fluorescence group;
optionally, the quenching group is any one of 4- [4- (dimethylamino) phenylazo ] benzoic acid (Dabcyl), azo-based dyes of black hole quencher paraazo dye 1(BHQ1) and black hole quencher paraazo dye 2(BHQ 2);
optionally, the fluorescent group is selected from any one of coumarin dyes, fluorescein dyes, BODIPY dyes and rhodamine dyes;
the coumarin dye comprises 7-amino-4-methylcoumarin, 7-dimethylamino-4-methylcoumarin, 7-diethylamino-4-methylcoumarin, and 7-diethylamino-4-trifluoromethylcoumarin;
fluorescein dyes include fluorescein isothiocyanate, 5-fluorescein isothiocyanate cadaverine, 6-carboxy-2 ',4,7', 7-tetrachlorofluorescein succinimidyl ester;
the boranopyrrole dyes include 1,3,5, 7-tetramethyl-8-phenyl-4, 4-difluorodiazabiobutane, 1,3,5, 7-tetramethyl-4, 4-difluorodiazabiobutane, 1,3,5,7, 8-pentamethyl-4, 4-difluoro-4-bora-3 a,4 a-diaza-s-indacene, 4, 4-difluoro-1, 3,5, 7-tetramethyl-4-bora-3 a,4 a-diaza-sym-indacene;
the rhodamine dye comprises 6-carboxyl-2 ',4,7', 7-tetrachlorofluorescein succinimidyl ester, 6-carboxyl rhodamine 6G and tetramethyl rhodamine.
Optionally, the RNA reverse transcriptase and the DNA polymerase are respectively subjected to recombinant expression and purification;
cloning a gene sequence of ribonucleic acid reverse transcriptase or deoxyribonucleic acid polymerase into an escherichia coli expression vector for recombinant expression, collecting bacteria containing recombinant proteins, and suspending the bacteria in an inorganic buffer solution for purification;
the conditions for recombinant expression are:
the expression temperature is 30-37 ℃;
the lower limit of the expression temperature is independently selected from 30 ℃, 32 ℃, 34 ℃, 35 ℃ and 36 ℃;
the upper limit of the expression temperature is independently selected from the group consisting of 31 ℃, 33 ℃, 35 ℃, 36 ℃ and 37 ℃.
Optionally, the cell optical density is 0.3-1.0;
the lower limit of the optical density of the cells is independently selected from 0.3, 0.4, 0.6, 0.8, 0.9;
the upper limit of the optical density of the cells is independently selected from 0.4, 0.5, 0.7, 0.9, 1.0.
Optionally, the inducer adopts any one of isopropyl thiogalactoside, lactose and arabinose;
optionally, if the recombinant expression is performed without adopting an inducer inducing mode, a heating inducing mode can also be adopted;
specifically, after the expression temperature is 30-37 ℃, the expression system is heated to 37-45 ℃, and then induction is realized.
Preferably, the method is carried out by using an inducer, wherein the inducer is isopropyl thiogalactoside.
Optionally, the concentration of the inducer is 0.5-1.0 mM;
the lower limit of the concentration of the inducer is independently selected from the group consisting of 0.5mM, 0.6mM, 0.7mM, 0.8mM, and 0.9 mM;
the upper limit of the concentration of the inducer is independently selected from the group consisting of 0.6mM, 0.65mM, 0.75mM, 0.85mM, and 1.0 mM.
Optionally, the expression time is 4-6 h;
the expression time is independently selected from 4h, 4.5h, 5h, 5.5h and 6 h.
Optionally, the buffer solution is at least one solution of phosphate buffered saline solution, tromethamine buffered saline solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffered saline solution, imidazole solution and sodium chloride solution;
the inorganic buffer solution contains four nucleotides, namely adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide and uracil ribonucleotide.
Preferably, the buffer solution is phosphate buffered saline.
Optionally, the coronavirus is a 2019 novel coronavirus, and the three specific primers are sequences artificially synthesized according to corresponding gene sequences of an N protein, an S protein and an ORF lab interval of the 2019 novel coronavirus respectively.
According to a third aspect of the present application, there is provided a fluorescent detection method for coronavirus, which is applied to any one of the fluorescent detection kit for coronavirus and a fluorescent detector for coronavirus used in combination, wherein the detection step comprises:
(1) extracting nucleic acid of a sample;
(2) preparing a reaction system by using a specific primer pair, a primer probe, an inorganic buffer solution, ribonucleic acid reverse transcriptase, deoxyribonucleic acid polymerase and the sample nucleic acid obtained in the step (1);
(3) respectively taking the positive reference plasmid and the negative reference substance, and configuring a reaction system which is the same as the sample nucleic acid;
(4) respectively carrying out polymerase chain reaction on the three reaction systems obtained in the steps (2) to (3) to obtain an amplification mixed solution after reaction;
(5) respectively carrying out fluorescence detection on the amplification mixed liquor obtained in the step (4) by using the coronavirus fluorescence detector, and analyzing and judging according to a detection result;
preferably, the concentration of the ribonucleic acid reverse transcriptase during the detection is: 1-1000U/. mu.L; the concentration of the DNA polymerase is as follows: 0.1 to 100U/. mu.L.
The lower limit of the concentration of the ribonucleic acid reverse transcriptase is independently selected from 200U/mu L, 400U/mu L, 600U/mu L, 800U/mu L and 900U/mu L;
the upper limit of the concentration of the ribonucleic acid reverse transcriptase is independently selected from the group consisting of 300U/. mu.L, 500U/. mu.L, 700U/. mu.L, 900U/. mu.L, and 1000U/. mu.L.
The lower limit of the concentration of the deoxyribonucleic acid polymerase is independently selected from 20U/mu L, 40U/mu L, 45U/mu L, 60U/mu L and 80U/mu L;
the upper limit of the concentration of the deoxyribonucleic acid polymerase is independently selected from 25U/mu L, 45U/mu L, 50U/mu L, 70U/mu L and 100U/mu L.
The beneficial effect of this application includes:
according to the coronavirus fluorescence detector and the kit matched with the same, a sample to be detected is dripped onto a detection card, a light source emits light with a specific waveband through a first optical filter and then irradiates the light with the specific waveband to a detection area to be detected in the detection card, an image of the detection card after fluorescence is excited is collected through image collection equipment, and the image is processed through a detection chip to obtain a detection result; the equipment is simple to use, convenient to carry and low in cost, and can meet the requirement of on-site rapid detection of the coronavirus;
further, the detection card can be equipped with a plurality of detection regions, and the detector can include the excitation light path of a plurality of parallel settings, and every excites the light path and corresponds with every detection region, can carry out the multiunit simultaneously and detect according to the actual demand that detects, improves detection efficiency, sparingly detects latency.
Drawings
FIG. 1 is a diagram of a hardware circuit connection of the circuit board of FIG. 1 according to the present application;
FIG. 2 is a schematic diagram of a portable fluorescence detector for coronavirus according to the present application;
FIG. 3 is a graph showing the linear relationship of fluorescein measurement by the detector according to example 6 of the present application.
List of parts and reference numerals:
1, a shell; 2, detecting the card slot;
3, detecting the card; 4, a light source;
5 a first optical filter; 6, a camera;
7 a second optical filter; 8 circuit boards;
9, an upper cover body; 10 a display screen;
11 display panel corresponding to the display screen; 12 batteries;
201 a first detection zone; 202 second detection zone.
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples.
Unless otherwise specified, the raw materials in the examples were purchased commercially and used without treatment; the used instruments and equipment adopt the use parameters recommended by manufacturers.
In the examples, the commercial kit used was a nucleic acid detection kit for a new coronavirus (ORF1ab/N gene) 2019 from shanghai berjie medical science and technology ltd;
in the examples, the polymerase chain reaction apparatus used was the model number, ibdennexus GX 2;
in the embodiment, a fluorescence spectrum detector of Hitachi F-4600 model is adopted to detect fluorescein with unknown concentration as a contrast test;
in the examples, the synthesis of specific primers was carried out using an automatic nucleic acid synthesizer model OligoMaker 48.
According to one aspect of the present application, there is provided a coronavirus fluorescence detector, comprising:
a housing with an upper end opened;
the detection clamping groove is arranged in the shell and used for fixing the detection card;
the light source and the first optical filter form an excitation light path, and the detection card is positioned on the light emergent side of the first optical filter;
the image acquisition equipment and the second optical filter form an acquisition light path and are used for acquiring a detection card image after fluorescence is excited;
and the detection chip is arranged in the shell, is connected with the image acquisition equipment and is used for detecting and analyzing the detection card image acquired by the image acquisition equipment.
Alternatively, the light source may be a light emitting diode;
optionally, the image acquisition device may be a camera.
Optionally, the detection card is provided with a plurality of detection areas, and gaps exist among the detection areas and are used for respectively bearing the objects to be detected;
optionally, the detector may comprise one or more excitation light paths arranged in parallel;
in some possible embodiments, the detecting apparatus may include an excitation light path, in which case, the detecting card is provided with a detecting region for carrying the detecting object, the detecting region is located on the light-emitting side of the first filter in the excitation light path, and the light emitted through the first filter in the excitation light path irradiates the detecting region;
in some possible embodiments, the detector may comprise a plurality of excitation light paths arranged in parallel, e.g. 2, 3, 4, etc.; under this circumstances, each detection region is located the light-emitting side of the first light filter in the excitation light path respectively, and every detection region corresponds with every laser light path, on the light irradiation of the first light filter outgoing of each excitation light path corresponds to the detection region to realize the detection to a plurality of samples simultaneously, improve detection efficiency.
Alternatively, the test card may be a filter paper sheet with a test zone in the middle.
Alternatively, the detection card slot may be located on the side of the housing; optionally, the detection card slot may also be located at an inner position of the housing, which is not particularly required by the present application.
Alternatively, the detection card slot may be inserted into the lower housing from the side of the housing; alternatively, the detection card slot may be inserted into the housing from an upper end opening of the housing, and this application is not particularly limited thereto.
Optionally, the coronavirus fluorescence detector of the present application further comprises:
the circuit board is arranged in the shell and connected with the light source and the image acquisition equipment, and the detection chip is arranged on the circuit board; the battery is arranged in the shell, is connected with the circuit board and is used for supplying power to the light source and the image acquisition equipment through the circuit board;
optionally, the coronavirus fluorescence detector of the present application further comprises: the upper cover body is buckled at the opening end of the shell;
the display screen is arranged on the upper cover body and used for protecting the display panel; and the display panel corresponding to the display screen is connected with the circuit board and arranged in the shell, and the battery is also used for supplying power to the display screen through the circuit board.
In some possible embodiments, as shown in fig. 1, a hardware circuit connection diagram of a circuit board according to an exemplary embodiment of the present application is shown, as shown in fig. 1, a detection chip may be a 32-bit single chip microcomputer STM32MCU (STM32Micro Controller Unit), an image capture device (such as a camera) and a display screen are respectively connected to the single chip microcomputer STM32MCU through an RGB interface (red, green, and blue channels), the single chip microcomputer STM32MCU stores and displays data captured by the camera on the display screen through the RGB interface, and a battery is connected to a power management module on the circuit board and supplies power to other electronic components based on the circuit board, including the display screen, a light source, and the image capture device. Meanwhile, the single chip microcomputer STM32MCU further connects a USB (Universal Serial Bus) chip through a Serial port to realize USB external communication, performs data storage through a memory FLAHS, an EEPROM, and the like, and realizes external network connection through a GPRS (general packet radio service), a GPS (Global Positioning System), and the like.
Alternatively, the battery is a lithium battery or an alkaline battery, which is not particularly limited in this application.
Optionally, the first filter is a narrowband filter;
optionally, the wavelength range of the light source is 300-700 nm; the wavelength range of the first optical filter is 300-700 nm;
the light source and/or the first optical filter are/is detachably connected with the detector and used for replacement, and the light source with the required wavelength and the corresponding first optical filter can be replaced according to actual detection requirements.
Optionally, the resolution of the image acquisition device is 1 to 1000 million pixels (pixels); the volume of the image acquisition equipment is 0.1-125; the image acquisition equipment is also provided with a memory for storing data after the camera converts the acquired image of the detection card from an optical signal to an electric signal.
Optionally, the wavelength range of the second optical filter is 300-700 nm;
optionally, a narrowband filter of the second filter.
Optionally, the image acquisition device and/or the second optical filter are detachably connected to the detector for replacement, and the second optical filter with a required wavelength can be replaced according to actual detection requirements.
Fig. 2 shows a coronavirus fluorescence detector according to an exemplary embodiment of the present application, and as shown in fig. 2, the coronavirus fluorescence detector includes a housing 1 with an upper end opened, a detection card slot 2, a detection card 3 fixed in the detection card slot 2, a light source 4, a first optical filter 5 located on a light outgoing side of the light source 4 (the light source 4 and the first optical filter form an excitation light path), a camera 6, a second optical filter 7 located on a light incoming side of the camera (the camera 6 and the second optical filter form a collection light path), and a circuit board 8 (including a detection chip); the detector comprises two excitation light paths, wherein each excitation light path corresponds to each detection area (a first detection area 201 and a second detection area respectively) on the detection card 3;
with continued reference to fig. 2, the detector further includes an upper cover 9 to be fastened to the opening end of the housing 1; the display screen 10 is arranged on the shell 1, and the display panel 11 corresponding to the display screen is connected with the circuit board; the camera 6, the display screen 10, the corresponding panel 11, and the battery 12 are respectively connected to the circuit board 8 through wires, wherein the camera 6 and the display screen 10 are connected to a detection chip (not identified in fig. 2) in the circuit board 8, and the battery 12 is connected to a power management module (not identified in fig. 2) in the circuit board 8.
The working process of the coronavirus fluorescence detector of the present application is described below with reference to fig. 2:
firstly, a negative sample is dripped on a first detection area 201 on a detection card 3 to be used as a reference point, and an amplification mixed solution of a nucleic acid sample is dripped on a second detection area 202 to be used as a detection point;
then, the detection card 3 is inserted into the detection card slot 2; wherein, optionally, the detection card slot 2 can be inserted into the housing 1 from the side of the housing 1; optionally, the detection card slot 2 may be inserted into the housing 1 or removed from a slot provided at an upper opening end of the housing 1, and the application includes, but is not limited to, the above manner of inserting the detection card slot 2 into the housing 1;
then, a light source (e.g., a light emitting diode) 4 in the excitation light path is used as an excitation light source to emit light of a specific waveband, which passes through a corresponding first optical filter 5 and irradiates the light to a first detection area 201 and a second detection area 202 on the detection card 3 respectively;
finally, the image of the detection card after the fluorescence is excited is collected by the camera 6 after passing through the second optical filter 7; the camera 6 converts the acquired image into digital information and transmits the digital information to a detection chip (singlechip) on the circuit board 8, and the singlechip calculates and processes the extracted digital signal according to a set algorithm and transmits a fluorescence detection result, a virus detection result and other final results to the display screen 10 for display. The camera 6 and the detection chip (singlechip), and the detection chip (singlechip) and the display screen 10 transmit information in a sixteen-bit bus parallel transmission mode.
Before the working process of the detector, the positive reference sample and the negative reference sample are subjected to the above detection process under the same conditions, and the read fluorescence change value is used as a virus threshold; and comparing the fluorescence change values obtained by the nucleic acid sample and the negative reference sample with the virus threshold value to obtain final detection results such as virus detection.
The coronavirus fluorescence detector provided by the application is convenient to use, simple to operate, convenient to carry, and low in cost, can meet the on-site rapid detection of coronaviruses, and realizes rapid detection and early warning of the coronaviruses.
According to an aspect of the present application, a kit used with the above-mentioned fluorescent detector for coronavirus and a fluorescent detection method for coronavirus using the same are provided, and the following embodiments are combined to describe the kit used with the above-mentioned fluorescent detector for coronavirus and the fluorescent detection method for coronavirus using the same.
EXAMPLE 1 preparation of fluorescent assay kit for coronavirus
(1) Synthesis of primer probes
Three independent primer probes for identifying gene sequences of different regions of the coronavirus are adopted, the 5 'end of each primer probe is labeled with a fluorescence quenching group, the 3' end of each primer probe is labeled with a fluorescence group, wherein the quenching group adopts BHQ1, and the fluorescence group adopts 5-carboxyfluorescein (FAM);
(2) recombination and purification of RNA reverse transcriptase and thermostable DNA polymerase
10ng (1. mu.L, 10 ng/. mu.L) of E.coli expression vector pET-28b plasmid containing the gene sequence of RNA reverse transcriptase or 10ng (1. mu.L, 10 ng/. mu.L) of thermostable DNA polymerase was transformed into E.coli. Adding 0.6mM isopropyl thiogalactoside inducer at 30 deg.C and cell optical density of 0.4, co-expressing for 6h, and collecting recombinant protein-containing fine powderBacteria suspended in inorganic buffer solution, wherein the inorganic buffer solution comprises 137mM NaCl,2.7mM KCl and 10mM Na2HPO4,2mM KH2PO4The pH value is 7.8; finally, protein purification was performed by ammonium sulfate salt cut experiments and nickel ion binding chromatography columns. Ammonium sulfate salt cutting experiment includes dissolving solid ammonium sulfate in cell lysate containing target protein gradually to separate out target protein and centrifuging; then the salt concentration is diluted by dialysis to resuspend the target protein, and the primary purification step of salt cutting is completed. Loading the salt cut product on a nickel ion binding chromatographic column through a rapid protein liquid chromatograph, and eluting high-purity target protein through imidazole solution with gradient concentration to complete the whole purification process.
(3) Synthesis of specific primers
Setting a primer sequence according to the specific gene sequence of the N protein and ORF interval of the novel coronavirus, and synthesizing a primer by using an automatic nucleic acid synthesizer; wherein, the primer sequence of the 2019 novel coronavirus is as follows:
the sequence of N1-F is GACCCCAAAATCAGCGAAAT;
the sequence of N1-R is CAGATTCAACTGGCAGTAACCAGA;
the sequence of N3-F is GGGAGCCTTGAATACACCAAAA;
the sequence of N3-R is CAATGCTGCAATCGTGCTACA;
the ORF-F sequence is ACTTCTTTTTCTTGCTTTCGTGGT;
the ORF-R sequence is GATTGTGTGCGTACTGCTGC.
Specific primers were synthesized by a nucleic acid amplifier. The PCR polymerase amplification procedure was: heating at 50 deg.C for 10min, then heating at 95 deg.C for 5min, and then performing 40 rounds of polymerase chain reaction; the conditions for each round of polymerase chain reaction were: heating at 95 deg.C for 20s, followed by heating at 55 deg.C for 60 s.
(4) Inorganic buffer solution
The composition of the 10-fold concentrated inorganic buffer was (500mM Tris-HCl, pH 7.3,75mM KCl, 30mM MgCl)250mM DTT (bis-p-chlorophenyl trichloroethane).
(5) Positive reference plasmid and negative reference
Taking three independent positive reference plasmids containing different gene segments in the protein gene sequence of the novel coronavirus, specifically three independent positive reference plasmids of the gene segments corresponding to the N protein ORF interval of the novel coronavirus; the negative reference is selected from purified water without ribonucleotides.
Example 2
This example 2 is a kit prepared based on example 1 and the coronavirus fluorescence detector of the present application, and the coronavirus fluorescence detection method is specifically described.
(1) Preparation of amplification mixture for polymerase chain reaction amplification
2. mu.L of the fluorescence-activated primer probe synthesized in example 1, 2. mu.L of a 2.5mM aqueous solution containing four kinds of nucleotides, 2. mu.L of a 100U/. mu.L ribonucleic acid reverse transcriptase, 0.5. mu.L of a 5U/. mu.L thermostable deoxyribonucleic acid polymerase, 2.5. mu.L of two kinds of 5. mu.M nucleic acid primers, and 10. mu.M inorganic salt buffer solution, 5. mu.L, were mixed and stirred in 30.5. mu.L ultrapure water to obtain an amplification mixture (hereinafter referred to as amplification mixture) for polymerase chain reaction amplification;
(2) reaction system for nucleic acid sample
Taking 10 mu L of purified nucleic acid sample of a patient with unknown concentration, adding the sample into 40 mu L of amplification mixed solution prepared in the step (1), centrifuging, and adding the sample into a polymerase chain reaction instrument for temperature gradient amplification; wherein the amplification procedure is as follows: heating at 50 deg.C for 10min, then heating at 95 deg.C for 5min, and then performing 40 rounds of polymerase chain reaction; the conditions for each round of polymerase chain reaction were: heating at 95 deg.C for 20s, and heating at 55 deg.C for 60 s;
(3) reaction system of positive reference sample
Taking 10 mu L of positive reference plasmid with the concentration of 1 ng/. mu.L, adding the positive reference plasmid into 40 mu L of amplification mixed solution prepared in the step (1), centrifuging, and adding the positive reference plasmid into a polymerase chain reaction instrument for temperature gradient amplification; wherein the amplification procedure is as follows: heating at 50 deg.C for 10min, then heating at 95 deg.C for 5min, and then performing 40 rounds of polymerase chain reaction; the conditions for each round of polymerase chain reaction were: heating at 95 deg.C for 20s, and heating at 55 deg.C for 60 s;
(4) reaction system of negative reference substance
Adding 10 mu L of purified water into 40 mu L of amplification mixed solution prepared in the step (1), centrifuging, and adding into a polymerase chain reaction instrument for temperature gradient amplification; wherein the amplification procedure is as follows: heating at 50 deg.C for 10min, then heating at 95 deg.C for 5min, and then performing 40 rounds of polymerase chain reaction; the conditions for each round of polymerase chain reaction were: heating at 95 deg.C for 20s, and heating at 55 deg.C for 60 s;
(5) spotting the nucleic acid sample, the positive reference plasmid and the negative reference substance obtained by amplification in the steps (2) to (4), respectively taking 1uL through a pipette, and dropwise adding the 1uL to different areas on a detection card of a detector to perform fluorescence detection; the camera pixels of the detector adopt 200 ten thousand pixels, the wavelength of a light source is 470nm, the wavelength of a first optical filter is 470nm, and the wavelength of a second optical filter is 550 nm.
Example 3
This example 3 is a kit prepared based on example 1 and the coronavirus fluorescence detector of the present application, and the coronavirus fluorescence detection method is specifically described.
(1) Preparation of amplification mixture for polymerase chain reaction amplification
2. mu.L of the fluorescence-activated primer probe synthesized in example 1, 2. mu.L of a 2.5mM aqueous solution containing four kinds of nucleotides, 2. mu.L of a 100U/. mu.L ribonucleic acid reverse transcriptase, 0.5. mu.L of a 5U/. mu.L thermostable deoxyribonucleic acid polymerase, 2.5. mu.L of two kinds of 5. mu.M nucleic acid primers, and 10. mu.M inorganic salt buffer solution, 5. mu.L, were mixed and stirred in 30.5. mu.L ultrapure water to obtain an amplification mixture (hereinafter referred to as amplification mixture) for polymerase chain reaction amplification;
(2) reaction system for nucleic acid sample
Adding 10 mu L of patient purified nucleic acid sample with unknown concentration into 40 mu L of amplification mixed solution prepared in the step (1), centrifuging, and adding into a polymerase chain reaction instrument for temperature gradient amplification; wherein the amplification procedure is as follows: heating at 50 deg.C for 10min, then heating at 95 deg.C for 10min, and then performing 30 rounds of polymerase chain reaction; the conditions for each round of polymerase chain reaction were: heating at 95 deg.C for 5s, and heating at 55 deg.C for 40 s;
(3) reaction system of positive reference sample
Taking 10 mu L of positive reference plasmid with the concentration of 1 ng/. mu.L, adding the positive reference plasmid into 40 mu L of amplification mixed solution prepared in the step (1), centrifuging, and adding the positive reference plasmid into a polymerase chain reaction instrument for temperature gradient amplification; wherein the amplification procedure is as follows: heating at 55 deg.C for 5min, then heating at 95 deg.C for 10min, and then performing 30 rounds of polymerase chain reaction; the conditions for each round of polymerase chain reaction were: heating at 95 deg.C for 5s, and heating at 55 deg.C for 40 s;
(4) reaction system of negative reference substance
Adding 10 mu L of purified water into 40 mu L of amplification mixed solution prepared in the step (1), centrifuging, and adding into a polymerase chain reaction instrument for temperature gradient amplification; wherein the amplification procedure is as follows: heating at 55 deg.C for 5min, then heating at 95 deg.C for 10min, and then performing 30 rounds of polymerase chain reaction; the conditions for each round of polymerase chain reaction were: heating at 95 deg.C for 5s, and heating at 55 deg.C for 40 s;
(5) spotting the nucleic acid sample, the positive reference plasmid and the negative reference substance obtained by amplification in the steps (2) to (4), respectively taking 1uL through a pipette, and dropwise adding the 1uL to different areas on a detection card of a detector to perform fluorescence detection; the pixel of the camera of the detector adopts 500 ten thousand pixels, the wavelength of a light source is 470nm, the wavelength of a first optical filter is 470nm, and the wavelength of a second optical filter is 550 nm.
(6) After the spotting detection is carried out on the steps (2) to (4), the obtained fluorescence values are 78742 and 65721 respectively, and then the obtained virus threshold value is 13021; for nucleic acid samples, the fluorescence value obtained was 29735, and by comparison with this viral threshold, 29735>13021 was positive.
Example 4
Example 4 the detection apparatus, reagents and procedures were the same as those of example 3 except that the wavelength of the light-emitting secondary light was 365nm, the wavelength of the first filter was 365nm, and the wavelength of the second filter was 530 nm.
Example 5
In this example 5, the detection of the coronavirus detector of the present application and the quantitative detection of the positive reference gene in the commercial kit are compared, and as shown in table 1, the detection results of the coronavirus detector of the present application and the fluorescence real-time PCR instrument used for clinical nucleic acid diagnosis are compared.
TABLE 1
As can be seen from Table 1, the coronavirus detection apparatus of the present application has a minimum detection limit of the same order of magnitude as that of the conventional fluorescent real-time PCR apparatus for clinical nucleic acid diagnosis, has a similar minimum detection limit, and requires less amplification time.
Example 6
In this example 6, the detection effect of the coronavirus fluorescence detector shown in FIG. 2 was examined.
(1) Taking five detection cards, and respectively dropwise adding fluorescein standard samples with the concentration of 1uL being 0.26 ng/mu L, 0.8 ng/mu L, 1.3 ng/mu L, 1.95 ng/mu L and 2.6 ng/mu L;
(2) sequentially inserting five detection cards into a detection card slot 2 in the coronavirus fluorescence detector shown in fig. 2, reading a fluorescence signal value of a detection internal indicator through a display screen 10, and subtracting an initial fluorescence value of the detection card from a fluorescence signal value obtained after dropwise adding a fluorescein standard sample, so as to obtain a fluorescence signal change value of the indicator;
the results show that the test card has a very good linear relationship to fluorescein at concentrations between 0.26 and 2.6 ng/. mu.L, where R is20.9959, the linear equation is y 27.824x +9.1469 (see fig. 3), where R2As a fitting coefficient, as shown in FIG. 3, x represents the concentration of fluorescein, and y represents the color taking of the detection card before and after the fluorescein is added, and the color is recorded into the single chip microcomputer.
(3) The method comprises the steps of dropwise adding the fluorescein to be detected with unknown concentration into a detection point, placing a detection card into an instrument, detecting, and determining the concentration of the fluorescein to be 0.8 ng/mu L, wherein the result is consistent with the fluorescence spectrum detection result.
Although the present application has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application.
Claims (10)
1. A coronavirus fluorescence detector, comprising:
a housing with an upper end opened;
the detection clamping groove is arranged in the shell and used for fixing the detection card;
the light source and the first optical filter form an excitation light path, and the detection card is positioned on the light emergent side of the first optical filter;
the image acquisition equipment and the second optical filter form an acquisition light path and are used for acquiring a detection card image after fluorescence is excited;
and the detection chip is arranged in the shell, is connected with the image acquisition equipment and is used for detecting and analyzing the detection card image acquired by the image acquisition equipment.
2. The detecting instrument according to claim 1, wherein the detecting card is provided with a plurality of detecting areas, and a gap is formed between the detecting areas for respectively carrying the objects to be detected;
the detector comprises a plurality of excitation light paths which are arranged in parallel, and each excitation light path corresponds to each detection area.
3. The meter of claim 1, further comprising:
the circuit board is arranged in the shell and connected with the light source and the image acquisition equipment, and the detection chip is arranged on the circuit board;
the battery is arranged in the shell, is connected with the circuit board and is used for supplying power to the light source and the image acquisition equipment through the circuit board;
and the upper cover body is buckled at the opening end of the shell.
4. The meter of any one of claims 1 to 3, wherein the light source has a wavelength in the range 300 to 700 nm;
the first optical filter is a narrow-band optical filter, and the wavelength range of the first optical filter is 300-700 nm.
5. The detector of any one of claims 1 to 3, wherein the image capturing device has a resolution of 1 to 1000 million pixels;
the second optical filter is a narrow-band optical filter, and the wavelength range of the second optical filter is 300-700 nm.
6. A coronavirus fluorescence detection kit used in combination with the coronavirus fluorescence detector of any one of claims 1-5 for detecting coronavirus, wherein the kit comprises three groups of specific primer pairs and primer probes, inorganic buffer solution, ribonucleic acid reverse transcriptase, deoxyribonucleic acid polymerase, positive reference plasmid and negative reference substance;
the pH value of the inorganic buffer solution is 6.0-8.0;
the positive reference plasmid is a positive reference plasmid of three different gene segments in a coronavirus protein gene;
the negative reference substance is at least one of purified water, deionized water or sterile water without the ribonuclease.
7. The kit of claim 6, wherein the 5 'end of the primer probe is labeled with a fluorescence quencher and the 3' end of the primer probe is labeled with a fluorophore;
wherein the quenching group is selected from any one of 4- [4- (dimethylamino) phenylazo ] benzoic acid, azo dyes of a black hole quencher pair azo dye 1 and a black hole quencher pair azo dye 2;
the fluorescent group is selected from any one of coumarin dyes, fluorescein dyes, BODIPY dyes and rhodamine dyes.
8. The kit according to claim 6, wherein the RNA reverse transcriptase and the DNA polymerase are recombinantly expressed and purified, respectively;
cloning a gene sequence of ribonucleic acid reverse transcriptase or deoxyribonucleic acid polymerase into an escherichia coli expression vector for recombinant expression, collecting bacteria containing recombinant proteins, and suspending the bacteria in an inorganic buffer solution for purification;
the conditions for recombinant expression were:
the expression temperature is 30-37 ℃;
the optical density of the cells is 0.3-1.0;
the inducer is any one of isopropyl thiogalactoside, lactose and arabinose;
the concentration of the inducer is 0.5-1.0 mM;
the expression time is 4-6 h;
the buffer solution is any one of phosphate buffer salt solution, tromethamine buffer salt solution, 4-hydroxyethyl piperazine ethanesulfonic acid buffer salt solution, imidazole solution and sodium chloride solution.
9. The kit according to claim 6, wherein the coronavirus is a 2019 novel coronavirus, and the three specific primers are sequences artificially synthesized according to gene sequences corresponding to the N protein, S protein and ORFlab intervals of the 2019 novel coronavirus respectively.
10. A coronavirus fluorescence detection method applied to the coronavirus fluorescence detection kit of claim 6 and a coronaviruse fluorescence detector matched with the coronavirus fluorescence detection kit for use is characterized in that the detection step comprises the following steps:
(1) extracting nucleic acid of a sample;
(2) preparing a reaction system by using a specific primer pair, a primer probe, an inorganic buffer solution, ribonucleic acid reverse transcriptase, deoxyribonucleic acid polymerase and the sample nucleic acid obtained in the step (1);
(3) respectively taking the positive reference plasmid and the negative reference substance, and configuring a reaction system which is the same as the sample nucleic acid;
(4) respectively carrying out polymerase chain reaction on the three reaction systems obtained in the steps (2) to (3) to obtain an amplification mixed solution after reaction;
(5) respectively carrying out fluorescence detection on the amplification mixed liquor obtained in the step (4) by using the coronavirus fluorescence detector, and analyzing and judging according to a detection result;
preferably, the concentration of the ribonucleic acid reverse transcriptase during the detection is: 1-1000U/. mu.L; the concentration of the DNA polymerase is as follows: 0.1 to 100U/. mu.L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010261223.9A CN113493857A (en) | 2020-04-03 | 2020-04-03 | Coronavirus fluorescence detector, coronavirus fluorescence detection kit and coronavirus fluorescence detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010261223.9A CN113493857A (en) | 2020-04-03 | 2020-04-03 | Coronavirus fluorescence detector, coronavirus fluorescence detection kit and coronavirus fluorescence detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113493857A true CN113493857A (en) | 2021-10-12 |
Family
ID=77995316
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010261223.9A Pending CN113493857A (en) | 2020-04-03 | 2020-04-03 | Coronavirus fluorescence detector, coronavirus fluorescence detection kit and coronavirus fluorescence detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113493857A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030151735A1 (en) * | 1999-11-04 | 2003-08-14 | Martin Blumenfeld | Scanning of biological samples |
| WO2004109266A1 (en) * | 2003-06-10 | 2004-12-16 | Digital Bio Technology | Dna detector |
| US20080013092A1 (en) * | 2006-05-19 | 2008-01-17 | George Maltezos | Fluorescence detector, filter device and related methods |
-
2020
- 2020-04-03 CN CN202010261223.9A patent/CN113493857A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030151735A1 (en) * | 1999-11-04 | 2003-08-14 | Martin Blumenfeld | Scanning of biological samples |
| WO2004109266A1 (en) * | 2003-06-10 | 2004-12-16 | Digital Bio Technology | Dna detector |
| US20080013092A1 (en) * | 2006-05-19 | 2008-01-17 | George Maltezos | Fluorescence detector, filter device and related methods |
Non-Patent Citations (2)
| Title |
|---|
| BD: ""BD MAX™全自动核酸提取及荧光PCR分析系统", 《仪器网》 * |
| 美通社: "BD全力投身抗击新冠病毒肺炎（COVID-19）全球战役", 《美通社》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20230265495A1 (en) | Analytic Device | |
| US10928321B2 (en) | Portable device for detecting molecule(s) | |
| CN101375150A (en) | Device and methods for quantifying analytes | |
| CN1985171A (en) | Fluorescence resonance energy transfer enzyme substrates | |
| US20140158541A1 (en) | Transilluminator Base and Scanner for Imaging Fluorescent Gels | |
| CN113493857A (en) | Coronavirus fluorescence detector, coronavirus fluorescence detection kit and coronavirus fluorescence detection method | |
| CN109797097B (en) | Portable instrument for top-illuminated nucleic acid isothermal amplification detection | |
| Cao et al. | Non-nucleic acid extraction and ultra-sensitive detection of African swine fever virus via CRISPR/Cas12a | |
| CN109557060B (en) | Based on NH2Method for visually detecting alkaline phosphatase activity in serum by using (E) -Cu-MOF | |
| Portugal et al. | High frequency of multiple HPV types detection in Fanconi anemia patients oral swabs | |
| US9464329B2 (en) | Portable systems and methods for amplifying nucleotides and detecting nucleotide sequences | |
| WO2014058064A1 (en) | Inspection system and inspection method | |
| BR112017020259B1 (en) | MODULAR TEST DEVICE FOR ANALYZING BIOLOGICAL SAMPLES | |
| CN113433119A (en) | Nucleic acid qualitative detection device and method for realizing visual display of result and application | |
| CN212955181U (en) | Digital PCR kit for detecting novel coronavirus nucleic acid | |
| CN215517478U (en) | On-site detection system for LAMP or RT-LAMP and portable device box | |
| US20220389524A1 (en) | Method for detection of nucleic acid sequences from biological samples of medical, agricultural and biotechnological interest and apparatus thereof | |
| WO2023140500A1 (en) | Test device and method for determining presence or absence of infection by using color variations of reagent | |
| CN110195128B (en) | Nucleotide sequence of skin type HPV typing detection primer based on constant temperature nucleic acid amplification technology and application | |
| JPH0484751A (en) | Gene detecting method | |
| US20240120087A1 (en) | Method and apparatus for efficiently transmitting nucleic acid amplification result data made by measuring instrument | |
| US20230109581A1 (en) | Color reading for diagnostic tests | |
| CN116710755A (en) | LAMP assay devices and methods using RNA/DNA isothermal amplification for pathogen identification | |
| CN105018642A (en) | Visual gene chip and kit used for detecting porcine epidemic encephalitis b viruses and/or porcine reproductive and respiratory syndrome viruses | |
| US20190376124A1 (en) | Apparatus and method for specific detection and quantization of nucleic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211012 |
|
| RJ01 | Rejection of invention patent application after publication |