CN113846094A - LAMP visual detection method and solid system of novel coronavirus - Google Patents
LAMP visual detection method and solid system of novel coronavirus Download PDFInfo
- Publication number
- CN113846094A CN113846094A CN202111188085.7A CN202111188085A CN113846094A CN 113846094 A CN113846094 A CN 113846094A CN 202111188085 A CN202111188085 A CN 202111188085A CN 113846094 A CN113846094 A CN 113846094A
- Authority
- CN
- China
- Prior art keywords
- primer
- solid
- sequence
- novel coronavirus
- kit
- 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 31
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 239000007787 solid Substances 0.000 title claims abstract description 19
- 230000000007 visual effect Effects 0.000 title abstract description 14
- 229920000936 Agarose Polymers 0.000 claims abstract description 26
- 239000007793 ph indicator Substances 0.000 claims abstract description 6
- 238000002844 melting Methods 0.000 claims description 17
- 230000008018 melting Effects 0.000 claims description 14
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical group Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 claims description 12
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 9
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 9
- 102100034343 Integrase Human genes 0.000 claims description 9
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 9
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 8
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 8
- 229960003237 betaine Drugs 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 7
- 230000004544 DNA amplification Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 abstract description 42
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 241000700605 Viruses Species 0.000 abstract description 5
- 239000000443 aerosol Substances 0.000 abstract description 3
- 238000007397 LAMP assay Methods 0.000 description 26
- 230000003321 amplification Effects 0.000 description 26
- 238000003199 nucleic acid amplification method Methods 0.000 description 26
- 102000004190 Enzymes Human genes 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 14
- 238000000034 method Methods 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 10
- 238000011901 isothermal amplification Methods 0.000 description 10
- 150000007523 nucleic acids Chemical class 0.000 description 9
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 239000013642 negative control Substances 0.000 description 7
- 108020004707 nucleic acids Proteins 0.000 description 7
- 102000039446 nucleic acids Human genes 0.000 description 7
- 239000012085 test solution Substances 0.000 description 7
- 208000002979 Influenza in Birds Diseases 0.000 description 6
- 206010064097 avian influenza Diseases 0.000 description 6
- 241001112090 Pseudovirus Species 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 241001678559 COVID-19 virus Species 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000012800 visualization Methods 0.000 description 3
- 241000710827 Dengue virus 1 Species 0.000 description 2
- 241000710815 Dengue virus 2 Species 0.000 description 2
- 241000710872 Dengue virus 3 Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 101150023114 RNA1 gene Proteins 0.000 description 2
- 101150084101 RNA2 gene Proteins 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 101100353432 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PRP2 gene Proteins 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005096 rolling process Methods 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000011179 visual inspection Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 241000008904 Betacoronavirus Species 0.000 description 1
- 208000001528 Coronaviridae Infections Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 208000028399 Critical Illness Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108050009160 DNA polymerase 1 Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 206010028748 Nasal obstruction Diseases 0.000 description 1
- 206010038687 Respiratory distress Diseases 0.000 description 1
- 206010039101 Rhinorrhoea Diseases 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- 101001024637 Severe acute respiratory syndrome coronavirus 2 Nucleoprotein Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 208000003152 Yellow Fever Diseases 0.000 description 1
- 241000710772 Yellow fever virus Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 208000017574 dry cough Diseases 0.000 description 1
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 208000010753 nasal discharge Diseases 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 229940051021 yellow-fever virus Drugs 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a LAMP visual detection method of a novel coronavirus and a solid-state system. The invention provides a primer group, which consists of 6 primers and is respectively shown as a sequence 1 to a sequence 6. The primer group is based on LAMP. The invention also discloses a solid-state system which comprises the primer group, the low-melting-point agarose and the pH indicator. The solid state system is a solid visual LAMP system. The invention also provides an LAMP visual kit for detecting the novel coronavirus, which can realize convenient transportation, avoid aerosol pollution and realize the detection of the novel coronavirus, has the advantages of high sensitivity and strong specificity, has simple reaction conditions, can realize rapid detection in a region with poor conditions, and is very suitable for the rapid virus detection in the basic level field.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a LAMP visual detection method and a solid system of a novel coronavirus.
Background
The novel coronavirus (SARS-CoV-2) belongs to the family Coronaviridae, the subfamily Coronaviridae, the genus Betacoronavirus, the species SARS coronavirus. Is first found in human in 2019. The novel coronavirus infection can cause people to generate heat, dry cough, general weakness, dyspnea and the like, some patients can also have symptoms of nasal obstruction, watery nasal discharge, abdominal pain, diarrhea and the like, and critically ill patients can also have respiratory distress and the like. There are also patients who have no clinical symptoms after infection. Rapid and accurate window detection is considered to be an effective way to prevent its spread, and among them, nucleic acid detection plays a key role in epidemic prevention and control because it is more sensitive and becomes the most effective infection screening means. The fluorescence quantitative PCR detection method is considered as the first choice method for the confirmed diagnosis of the virus and is most widely used, but the rapid detection in time in poor areas, rural basic levels and remote mountainous areas is limited due to the expensive instrument price, professional operation steps and complex judgment standards.
The loop-mediated isothermal amplification (LAMP) technology does not need expensive amplification instruments, is simple to operate, and can make result judgment simpler and more intuitive by matching with a visual detection method.
Disclosure of Invention
The invention aims to provide a LAMP visual detection method and a solid system for a novel coronavirus.
LAMP: loop-mediated isothermal amplification.
The invention provides a primer group, which consists of a primer N-1-F3, a primer N-1-B3, a primer N-1-Floop, a primer N-1-Bloop, a primer N-1-FIP and a primer N-1-BIP; the primer N-1-F3 is shown as a sequence 1 in a sequence table, the primer N-1-B3 is shown as a sequence 2 in the sequence table, the primer N-1-Floop is shown as a sequence 3 in the sequence table, the primer N-1-Bloop is shown as a sequence 4 in the sequence table, the primer N-1-FIP is shown as a sequence 5 in the sequence table, and the N-1-BIP is shown as a sequence 6 in the sequence table. The primers are all single-stranded DNA molecules.
The primer group is based on LAMP.
The primer group can be used for preparing a kit for detecting the novel coronavirus.
The primer set can be used for detecting novel coronavirus.
The invention also protects the application of the primer group in preparing a kit for detecting the novel coronavirus.
The invention also provides a kit for detecting the novel coronavirus, which comprises the primer group.
The invention also discloses a solid-state system which comprises the primer group, the low-melting-point agarose and the pH indicator. The solid state system is a solid visual LAMP system.
Specifically, the pH indicator is neutral red.
Specifically, the melting temperature of the low-melting-point agarose is below 65 ℃.
Specifically, the solid stating system comprises: primer N-1-F3, primer N-1-B3, primer N-1-Floop, primer N-1-Bloop, primer N-1-FIP, primer N-1-BIP, (NH)4)2SO4、MgSO4KCl, neutral red, dNTPs,
20. Betaine, low melting point agarose.
Specifically, the solid-stating system is obtained by drying and cooling a reaction precursor system.
The drying conditions may specifically be: 80 ℃ for 10 minutes.
The cooling may specifically be: and (5) cooling at room temperature.
Specifically, in the system before the reaction, the concentration of the low-melting-point agarose is 0.1-0.1g/100 ml.
Specifically, in the system before the reaction, the concentration of the low-melting-point agarose is 0.5g/100 ml.
Specifically, the concentration of neutral red in the system before the reaction is 40-60 mu M.
Specifically, the concentration of neutral red in the system before the reaction is 50 μ M.
Specifically, in the system before reaction, the concentration of each primer is as follows: 30.1-0.2 muM of primer N-1-F, 30.1-0.2 muM of primer N-1-B, 0.2-0.6 muM of primer N-1-Floop, 0.2-0.6 muM of primer N-1-Bloop, 1-2 muM of primer N-1-FIP and 1-2 muM of primer N-1-BIP.
Specifically, in the system before reaction, the concentration of each primer is as follows: 30.16 mu M of primer N-1-F, 30.16 mu M of primer N-1-B, 0.4 mu M of primer N-1-Floop, 0.4 mu M of primer N-1-Bloop, 1.6 mu M of primer N-1-FIP and 1.6 mu M of primer N-1-BIP.
Specifically, the system before reaction comprises: primer N-1-F3, primer N-1-B3, primer N-1-Floop, primer N-1-Bloop, primer N-1-FIP, primer N-1-BIP, (NH)4)2SO4、MgSO4KCl, neutral red, dNTPs,
20. Betaine, low melting point agarose.
Specifically, the composition of the system before reaction is as follows: 30.15-0.17. mu.M of primer N-1-F, 30.15-0.17. mu.M of primer N-1-B, 0.3-0.5. mu.M of primer N-1-Floop, 0.3-0.5. mu.M of primer N-1-Bloop, 1.5-1.7. mu.M of primer N-1-FIP, 1.5-1.7. mu.M of primer N-1-BIP, (NH)4)2SO4 9-11mM、MgSO47-9mM, KCl 48-52mM, neutral red 48-52 mu M, dNTPs 1.3.3-1.5 mM,
200.08-0.12% (volume ratio), betaine 0.4-0.6M, low melting point agarose 0.4-0.6g/100ml, and water in balance.
Specifically, the composition of the system before reaction is as follows: 30.16. mu.M of primer N-1-F, 30.16. mu.M of primer N-1-B, 0.4. mu.M of primer N-1-Floop, 0.4. mu.M of primer N-1-Bloop, 1.6. mu.M of primer N-1-FIP, 1.6. mu.M of primer N-1-BIP, (NH)4)2SO4 10mM、MgSO48mM, KCl 50mM, neutral red 50 mu M, dNTPs 1.4.4 mM,
200.1% (volume ratio), betaine 0.5M, low-melting agarose 0.5g/100ml, and water in balance.
The pH value of the system before the reaction is 7-8.
Specifically, the pH of the system before the reaction is 7.5.
The invention also provides a kit for detecting the novel coronavirus, which comprises any one of the solid stating systems. The kit is a solid visual LAMP kit.
The kit also includes a DNA polymerase and an AMV reverse transcriptase.
The kit is provided with a reaction tube, and any solid stating system is arranged in the reaction tube.
Specifically, the preparation method of the reaction tube comprises the following steps: and (3) placing the system before reaction in an oven at 80 ℃ for 10 minutes, taking out, turning upside down, mixing uniformly, cooling at room temperature for 5 minutes, subpackaging into PCR tubes (17 mu l each), and standing at room temperature until the gel is solidified, wherein the PCR tubes are reaction tubes.
The kit comprises an enzyme tube, wherein DNA polymerase and AMV reverse transcriptase are contained in the enzyme tube.
The proportion of the DNA polymerase and the AMV reverse transcriptase is 720U: 100U.
Specifically, the preparation method of the enzyme tube comprises the following steps: a PCR tube was added with 90. mu.l of DNA polymerase (enzyme content: 720U) and 10. mu.l of AMV reverse transcriptase (enzyme content: 100U), and the PCR tube was referred to as an enzyme tube.
The invention also protects the application of the low-melting-point agarose in preparing a solid-state system.
Specifically, the melting temperature of the low-melting-point agarose is below 65 ℃.
The solid-stating system is obtained by carrying out solid-stating on a DNA amplification system in the prior art.
The solid stating is achieved with low melting point agarose.
Specifically, the low melting point agarose is used at a concentration of 0.4-0.6g/100 ml.
Specifically, the low melting point agarose is used at a concentration of 0.5g/100 ml.
DNA amplification systems of the prior art include, but are not limited to: a loop-mediated isothermal amplification (LAMP) system, a rolling circle nucleic acid amplification (RCA) system, a real-time fluorescent isothermal amplification (SAT) system, a Recombinase Polymerase Amplification (RPA) system, a Strand Displacement Amplification (SDA) system, an enzymatic isothermal amplification (ERA) system, a nucleic acid sequence-dependent amplification and unzipping enzyme amplification system, an isothermal amplification system, a fluorescent quantitative PCR amplification system, a PCR amplification system and a microfluidic chip amplification system.
The invention also protects a solid-stating system which is obtained by carrying out solid-stating on the DNA amplification system in the prior art by using the low-melting-point agarose.
Specifically, the melting temperature of the low-melting-point agarose is below 65 ℃.
Specifically, the low melting point agarose is used at a concentration of 0.4-0.6g/100 ml.
Specifically, the low melting point agarose is used at a concentration of 0.5g/100 ml.
DNA amplification systems of the prior art include, but are not limited to: a loop-mediated isothermal amplification (LAMP) system, a rolling circle nucleic acid amplification (RCA) system, a real-time fluorescent isothermal amplification (SAT) system, a Recombinase Polymerase Amplification (RPA) system, a Strand Displacement Amplification (SDA) system, an enzymatic isothermal amplification (ERA) system, a nucleic acid sequence-dependent amplification and unzipping enzyme amplification system, an isothermal amplification system, a fluorescent quantitative PCR amplification system, a PCR amplification system and a microfluidic chip amplification system.
The inventor firstly determines that the N gene of the novel coronavirus is used as a target gene, then selects a conserved region in the N gene as a target segment, and then obtains an LAMP primer group with the best effect through a preliminary experiment.
Further, the inventors of the present invention have established a LAMP visualization kit for detection of a novel coronavirus, which has a reaction tube, and the contents of which are solidified both before and after the reaction and are only non-solidified (liquefied at the reaction temperature) during the reaction, thereby making it possible to realize convenient transportation and to avoid aerosol contamination. The kit provided by the invention can realize the detection of novel coronavirus, has the advantages of high sensitivity and strong specificity, has simple reaction conditions (constant temperature amplification), can realize the rapid detection in a region with poor conditions, and is very suitable for the rapid virus detection in the basic level field.
Drawings
Fig. 1 is a graph showing the results of visual inspection in example 4.
Fig. 2 is a graph showing the result of visual inspection in example 5.
FIG. 3 is an electrophoretogram in example 5.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Bst2.0WarmStart DNA polymerase: NEB corporation, USA, catalog number M0538L. AMV reverse transcriptase: promega corporation, USA, catalog No. M5108. Unless otherwise stated, the quantitative tests in the following examples were performed in triplicate, and the results were averaged.
Novel coronavirus N gene standard plasmid (2019-nCoV N): biometrics (Shanghai) Ltd. Novel coronavirus in vitro transcription RNA standard substance: shanghai city, institute for metrological testing technology, product number GBW (E) 091111. Novel coronavirus pseudovirus (pseudovirus SARS-CoV-2-N): biometrics (Shanghai) Inc., catalog No. M591009.
Example 1 design and preparation of primers
And (3) carrying out sequence comparison on a great number of novel coronavirus sequences in the existing database, and finally determining that the N gene is used as a target gene. On the basis of determining a target gene, a large number of LAMP primer groups are designed, and the performances of the primer groups, including amplification efficiency, specificity, sensitivity and the like, are compared. Finally, the primer group with the best effect, namely the primer group N-1, is screened.
A large number of primer groups with poor effects are involved in the screening process, and a part of primer groups are listed as control primer groups, namely a primer group N-2, a primer group N-3, a primer group N-4 and a primer group N-5.
The individual primers and primer sequences of the individual primer sets are shown in Table 1. Each primer of each primer set was prepared.
TABLE 1
Example 2 comparison of amplification efficiency of primer set
The test primer group is respectively a primer group N-1, a primer group N-2, a primer group N-3, a primer group N-4 or a primer group N-5.
Composition of the reaction system: 10 × Isothermal Amplification buffer, 8U/. mu.l Bst2.0WarmStart DNA polymerase, 10U/. mu.l AMV reverse transcriptase, 5M betaine aqueous solution, 10mM dNTP solution, Eva green (20 ×) solution, 100mM MgSO4Aqueous solution, template DNA, primers of the primer set to be tested, and the balance of water. Template DNA: a standard plasmid of N gene of coronavirus. 10 × Isothermal Amplification buffer: NEB corporation, USA, catalog number B0537S.
Each reaction system was 20. mu.l. Each reaction system contained 10 × Isothermal Amplification buffer 2 μ l, 8U/. mu.l Bst2.0WarmStart DNA polymerase 1 μ l, 10U/. mu.l AMV reverse transcriptase 0.1 μ l, 5M betaine aqueous solution 2.4 μ l, 10mM dNTP solution 2.8 μ l, Eva green (20 ×) solution 1 μ l, 100mM MgSO 2. mu.l4Aqueous solution 1.2. mu.l. The amount of template DNA in each reaction system was 0.0067 ng. In each reaction system, the concentrations of the primer F3 and the primer B3 were 0.16. mu.M, the concentrations of the primer Floop and the primer Bloop were 0.4. mu.M, and the concentrations of the primer FIP and the primer BIP were 1.6. mu.M.
The fluorescent quantitative PCR instrument is model 480 II of Roche. Reaction procedure: amplifying for 60 minutes at 65 ℃; the fluorescence channel was set to Sybr green, and fluorescence signals were detected once per minute.
The results are shown in Table 2. The amplification efficiency of the primer group N-1 is highest.
TABLE 2Ct value data
| Primer set N-1 | Primer set N-2 | Primer set N-3 | Primer set N-4 | Primer set N-5 | |
| Repetition of 1 | 11.44 | No numerical value | No numerical value | No numerical value | 21.36 |
| Repetition 2 | 11.44 | No numerical value | No numerical value | No numerical value | 20.94 |
| Repetition of 3 | 11.87 | No numerical value | No numerical value | No numerical value | 19.87 |
Example 3 preparation of solid State visualization LAMP kit
Preparation of kit
1. Preparing a system before reaction.
Composition of the system before reaction (pH 7.5): contains 30.16 mu M of primer N-1-F, 30.16 mu M of primer N-1-B, 0.4 mu M of primer N-1-Floop, 0.4 mu M of primer N-1-Bloop, 1.6 mu M of primer N-1-FIP, 1.6 mu M of primer N-1-BIP, (NH)4)2SO410mM、MgSO48mM, KCl 50mM, neutral red 50 mu M, dNTPs 1.4.4 mM,
200.1% (volume ratio), betaine 0.5M, low-melting agarose 0.5g/100ml, and water in balance. dNTPs 1.4mM means that the concentrations of dATP, dTTP, dCTP and dGTP are all 1.4 mM.
N-1-F3 (SEQ ID NO: 1): TGTAACACAAGCTTTCGGCAGAC, respectively;
N-1-B3 (SEQ ID NO: 2): GATCTTTGAAATTTGGATCTTTGTCA, respectively;
N-1-Floop (sequence 3): AGTTCCTTGTCTGATTAGTTCCTGG, respectively;
N-1-Bloop (sequence 4): CACCTTCGGGAACGTGGTTG, respectively;
N-1-FIP (SEQ ID NO: 5): CGCTGGGGGCAAATTGTGCTTTTGTCCAGAACAAACCCAAGGAAAT, respectively;
N-1-BIP (SEQ ID NO: 6): CTTCAGCGTTCTTCGGAATGTCGTTTTAATTTGATGGCACCTGTGTAGGTC are provided.
Neutral red: sigma, USA, catalog number N4638.
Low melting agarose (low gel temperature agarose, CAS 39346-81-1): sigma, USA, catalog number A9414.
2. And (2) placing the system before reaction prepared in the step (1) in an oven at 80 ℃ for 10 minutes, taking out, turning upside down, mixing uniformly, cooling at room temperature for 5 minutes, subpackaging into PCR tubes (17 mu l per tube), and standing at room temperature until gel is solidified (about half an hour), wherein the PCR tubes are reaction tubes.
3. Taking a new PCR tube, and adding 90 mu l of Bst2.0WarmStart DNA polymerase (enzyme content is 720U) and 10 mu l of AMV reverse transcriptase (enzyme content is 100U), wherein the PCR tube is the PCR tube filled with RT-LAMP enzyme, and is called an enzyme tube for short.
4. Assembly of the kit
The kit comprises a reaction tube and an enzyme tube which are independently packaged.
Second, using method of kit
The reaction tube in the kit was taken, and 1. mu.l of the solution in the enzyme tube and 2. mu.l of the test solution were added, followed by reaction at 65 ℃ for 1 hour. And (5) judging a result: if the contents of the reaction tube show yellow/yellowish color, the result is negative, and the test solution does not contain the novel coronavirus nucleic acid; if the contents of the reaction tube show a red/pink color, the result is positive and the test solution contains the novel coronavirus nucleic acid.
Third, the working principle of the kit
Neutral red is a weakly basic pH indicator with a color change range of pH6.4 (red) to pH 8.0 (yellow). If the test solution has a target fragment of the LAMP primer group as a template, LAMP amplification can be realized, and in the LAMP amplification process, one hydrogen ion is released by DNA polymerase every time one base is extended, so that the pH value of the system is gradually reduced, and the system is displayed in red/pink. If the test solution does not have the target fragment of the LAMP primer group, the LAMP amplification cannot be realized due to the lack of the template, the pH value of the system is not reduced, and the system is yellow/light yellow.
Fourthly, the advantages of the kit
Visual reading of results can be achieved without the need for expensive instrumentation.
The reaction conditions are simple (isothermal amplification), and an expensive temperature-variable amplification instrument is not needed.
The system before the reaction and the system after the reaction are both solid, thereby being convenient for transportation and avoiding aerosol pollution.
Example 4 sensitivity detection
Taking novel coronavirus N gene in vitro transcription RNA standard substance, using ddH2Diluting with O to obtain concentrations of 1 × 103copies/μl、1×102copies/μl、1×101copies/μl、1×100copies/μ l of standard solution. Will ddH2O as a negative control.
Firstly, the kit prepared in example 3 is adopted for detection
The instrument used was a constant temperature visual amplification instrument (model MLB-H01, Michelobo medical science and technology, Beijing).
The test solution is a standard solution or a negative control solution.
The procedure was performed according to the method of use of the kit.
The results are shown in FIG. 1 and Table 3. In fig. 1: corresponding to the standard solutions from high concentration to low concentration from left to right, and negative control solution on the rightmost side; each row represents a set of iterations.
TABLE 3
Secondly, detecting by adopting a commercial kit
The commercially available kit is a novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) produced by the Daan gene. The test solution is a standard solution or a negative control solution. According to the kit instruction.
The results are shown in Table 4.
TABLE 4
The results of example 4 show that the sensitivity of the solid-state visualization LAMP kit is consistent with that of the fluorescent quantitative PCR kit, and the sensitivity can reach 1 × 101copies/μl。
Example 5 specific detection
Supplying a sample book: novel coronavirus N gene standard plasmid and novel coronavirus N baseRNA standard substance, novel coronavirus pseudovirus, dengue virus type 1 RNA, dengue virus type 2 RNA, dengue virus type 3 RNA, yellow fever RNA, avian influenza H1N1 virus RNA, avian influenza N3N2 virus RNA, avian influenza H7N9 virus RNA, healthy human saliva RNA1, healthy human saliva RNA2, healthy human oral swab RNA1, healthy human oral swab RNA2, negative control 1 and negative control 2 are transcribed in vitro. Dengue virus type 1 RNA, dengue virus type 2 RNA, dengue virus type 3 RNA, yellow fever virus RNA, avian influenza H1N1 virus RNA, avian influenza N3N2 virus RNA, avian influenza H7N9 virus RNA are all provided by Zhejiang entry and exit inspection and quarantine bureau. Negative control was ddH2O。
Firstly, the kit prepared in example 3 is adopted for detection
The instrument used was a constant temperature visual amplification instrument (model MLB-H01, Michelobo medical science and technology, Beijing).
The procedure was performed according to the method of use of the kit.
The results are shown in FIG. 2 and Table 5. In fig. 2, 16 reaction tubes are from left to right and from top to bottom in each repetition, which sequentially correspond to 16 test samples in the sequence of the text description of the test samples.
TABLE 5
The reaction product was removed and subjected to 1.5% agarose gel electrophoresis, the electrophoresis chart of which is shown in FIG. 3. Of the 16 samples tested, only 3 samples (novel coronavirus N gene standard plasmid, novel coronavirus N gene in vitro transcription RNA standard substance, and novel coronavirus pseudovirus) showed LAMP bands with ladder-like characteristics.
Secondly, detecting by adopting a commercial kit
The commercially available kit is a novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) produced by the Daan gene. According to the kit instruction.
The Ct value of the novel coronavirus N gene standard plasmid is 26.27, the Ct value of the novel coronavirus N gene in-vitro transcription RNA standard substance is 26.5, the Ct value of the novel coronavirus pseudovirus is 27.17, and other samples for sample do not show an amplification curve.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> Beijing Meilaibo medical science and technology Co., Ltd
<120> LAMP visual detection method of novel coronavirus and solid-state system
<130> GNCYX212533
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgtaacacaa gctttcggca gac 23
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gatctttgaa atttggatct ttgtca 26
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
agttccttgt ctgattagtt cctgg 25
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
caccttcggg aacgtggttg 20
<210> 5
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cgctgggggc aaattgtgct tttgtccaga acaaacccaa ggaaat 46
<210> 6
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cttcagcgtt cttcggaatg tcgttttaat ttgatggcac ctgtgtaggt c 51
Claims (10)
1. The primer group consists of a primer N-1-F3, a primer N-1-B3, a primer N-1-Floop, a primer N-1-Bloop, a primer N-1-FIP and a primer N-1-BIP; the primer N-1-F3 is shown as a sequence 1 in a sequence table, the primer N-1-B3 is shown as a sequence 2 in the sequence table, the primer N-1-Floop is shown as a sequence 3 in the sequence table, the primer N-1-Bloop is shown as a sequence 4 in the sequence table, the primer N-1-FIP is shown as a sequence 5 in the sequence table, and the N-1-BIP is shown as a sequence 6 in the sequence table.
2. Use of the primer set of claim 1 for the preparation of a kit for the detection of a novel coronavirus.
3. A kit for detecting a novel coronavirus comprising the primer set of claim 1.
4. A solid stating system comprising the primer set of claim 1, a low melting agarose, and a pH indicator.
5. The solid stating system of claim 4, wherein: the pH indicator is neutral red.
6. The solid stating system of claim 5, wherein: the solid stating system comprises: primer N-1-F3, primer N-1-B3, primer N-1-Floop, primer N-1-Bloop, primer N-1-FIP, primer N-1-BIP, (NH)4)2SO4、MgSO4KCl, neutral red, dNTPs,
20. Betaine, low melting point agarose.
7. A kit for detecting a novel coronavirus comprising the solid-stating system of any one of claims 4 to 6.
8. The kit of claim 7, wherein: the kit also includes a DNA polymerase and an AMV reverse transcriptase.
9. The application of the low-melting-point agarose in preparing a solid-state system; the solid-state system is obtained by carrying out solid-state treatment on a DNA amplification system by using low-melting-point agarose.
10. A solid-state system is obtained by solid-state DNA amplification system with low-melting point agarose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111188085.7A CN113846094A (en) | 2021-10-12 | 2021-10-12 | LAMP visual detection method and solid system of novel coronavirus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111188085.7A CN113846094A (en) | 2021-10-12 | 2021-10-12 | LAMP visual detection method and solid system of novel coronavirus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113846094A true CN113846094A (en) | 2021-12-28 |
Family
ID=78978037
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111188085.7A Pending CN113846094A (en) | 2021-10-12 | 2021-10-12 | LAMP visual detection method and solid system of novel coronavirus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113846094A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525951A (en) * | 2013-10-24 | 2014-01-22 | 中国水产科学研究院黑龙江水产研究所 | LAMP (Loop-Mediated Isothermal Amplification) primer used for detecting infectious haematopoietic necrosis virus and application thereof |
| CN108588196A (en) * | 2018-04-23 | 2018-09-28 | 北京中能通达科技发展中心（有限合伙） | A method of prevent PCR from forming Aerosol Pollution |
| CN112176104A (en) * | 2020-10-09 | 2021-01-05 | 中国农业科学院兰州兽医研究所 | Visual LAMP (loop-mediated isothermal amplification) detection kit for porcine circovirus type 3 |
| CN112226538A (en) * | 2020-10-21 | 2021-01-15 | 江苏默乐生物科技股份有限公司 | Primer-probe combination, kit and method for detecting novel coronavirus |
-
2021
- 2021-10-12 CN CN202111188085.7A patent/CN113846094A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525951A (en) * | 2013-10-24 | 2014-01-22 | 中国水产科学研究院黑龙江水产研究所 | LAMP (Loop-Mediated Isothermal Amplification) primer used for detecting infectious haematopoietic necrosis virus and application thereof |
| CN108588196A (en) * | 2018-04-23 | 2018-09-28 | 北京中能通达科技发展中心（有限合伙） | A method of prevent PCR from forming Aerosol Pollution |
| CN112176104A (en) * | 2020-10-09 | 2021-01-05 | 中国农业科学院兰州兽医研究所 | Visual LAMP (loop-mediated isothermal amplification) detection kit for porcine circovirus type 3 |
| CN112226538A (en) * | 2020-10-21 | 2021-01-15 | 江苏默乐生物科技股份有限公司 | Primer-probe combination, kit and method for detecting novel coronavirus |
Non-Patent Citations (1)
| Title |
|---|
| 国家知识产权局学术委员会: "产业专利分析报告，第81册，应用与及时检测关键技术", 31 July 2021, 知识产权出版社, pages: 7 - 2 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111394511B (en) | 2019 novel coronavirus detection primer group, probe group and detection kit | |
| CN106947838B (en) | African swine fever virus non-structural gene real-time fluorescence LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method | |
| CN112080585B (en) | Novel coronavirus (SARS-CoV-2) rapid detection kit and method thereof | |
| CN112266986B (en) | Virus nucleic acid extraction or preservation reagent, primer probe combination, virus amplification reagent, kit and application thereof | |
| CN111690776A (en) | Primer, probe, reagent, method and kit for quickly detecting novel coronavirus SARS-CoV-2 at normal temperature and isothermal | |
| CN111218529B (en) | Primer composition, kit and method for detecting novel coronaviruses | |
| CN112553373A (en) | Kit and detection method for quickly detecting novel coronavirus 2019-nCoV nucleic acid | |
| CN108977580A (en) | A kind of kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation | |
| CN112239794B (en) | Primer pair, probe and kit for detecting novel coronavirus SARS-CoV-2 and application thereof | |
| EP4006167A1 (en) | Composition for improving qpcr test performance, reaction liquid, use, and method | |
| CN112280879A (en) | RPA primer and kit for rapidly detecting citrus yellow shoot Asian species, detection method and application thereof | |
| CN110982940A (en) | Composition and kit for detecting nucleic acid of measles virus, rubella virus and mumps virus based on melting curve | |
| CN108070636A (en) | A kind of processing method and kit of fluorescent PCR amplified sample | |
| CN113512548A (en) | Novel coronavirus detection kit based on CRISPR-Cas12a system and application thereof | |
| CN112831605A (en) | Multienzyme isothermal amplification detection kit and application thereof | |
| KR20190026403A (en) | Primers for Analyzing Porcine Epidemic Diarrhea Virus Genotype and Uses Thereof | |
| CN113846094A (en) | LAMP visual detection method and solid system of novel coronavirus | |
| CN111394518A (en) | Method and kit for in-vitro amplification detection of new coronavirus nucleic acid | |
| CN113278736B (en) | Reagents and methods for qualitative detection of bovine herpesvirus type I | |
| CN111926109A (en) | African swine fever virus fluorescence thermal convection PCR amplification primer pair, probe primer and prepared kit | |
| CN113151599A (en) | Primer group, reagent, kit and detection method for detecting novel coronavirus | |
| CN115029345A (en) | Nucleic acid detection kit based on CRISPR and application thereof | |
| CN111893213A (en) | Primer for rapid screening and identification of novel coronavirus, kit and application | |
| CN115989323A (en) | Method for detecting SARS-CoV-2 infection | |
| CN113151591A (en) | Nucleic acid composition and kit for detecting SARS-CoV-2 |
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 |