CN111763768A - COVID-19 rapid detection color development indication kit - Google Patents
COVID-19 rapid detection color development indication kit Download PDFInfo
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- CN111763768A CN111763768A CN202010619218.0A CN202010619218A CN111763768A CN 111763768 A CN111763768 A CN 111763768A CN 202010619218 A CN202010619218 A CN 202010619218A CN 111763768 A CN111763768 A CN 111763768A
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Abstract
The invention relates to the technical field of molecular biology and discloses a COVID-19 rapid detection color development indication kit, and a using method of the kit comprises the following steps: s1, thawing the reagent, respectively and uniformly mixing the detection color indicator, the detection reaction solution and the LAMP primer group according to a certain proportion, S2, respectively adding the nucleic acid sample to be detected, the positive reference substance and the negative reference substance into the mixed solution prepared by S1 according to a certain proportion, uniformly mixing and centrifuging, S3, incubating the reaction at 65 ℃ for 30 minutes, S4, observing in an environment with sufficient natural light after the reaction is finished, and directly judging the result by naked eyes; the indication kit is used for rapidly detecting the COVID-19 virus N gene based on the LAMP amplification technology, efficient specific strand displacement reaction is carried out under the constant temperature condition, the characteristics of high temperature control precision requirement and complex and time-consuming process of the traditional PCR reaction are overcome, and meanwhile, the indication kit is simple and convenient to operate and low in requirement on experiment operators.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a COVID-19 rapid detection color development indication kit.
Background
Molecular diagnosis based on nucleic acid amplification is to detect the existence of endogenous (genetic or variant) or exogenous (pathogen) target genes by primer-mediated specific amplification of the target genes, thereby providing information and decision basis for diagnosis and treatment of diseases. The main application scenes of the kit include diagnosis of infectious diseases, blood screening, early auxiliary diagnosis of tumors, molecular typing of tumors, diagnosis of genetic diseases, prenatal diagnosis, tissue typing and the like. Among them, molecular diagnostics based on nucleic acid amplification can shorten the "window" of diagnosis in the diagnosis of infectious diseases and blood screening, and can quantitatively detect pathogens. Compared with the traditional immunodiagnostic mode, the method has irreplaceable advantages.
Among the detection methods, Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification technique reported by Notomi et al in 1998. This method relies on 4 primers (2 outer primers and 2 inner primers) recognizing 6 specific fragments of conserved sequence DNA and one strand displacement DNA polymerase (Bst DNA polymerase). The amplification of the gene and the detection of the product in the LAMP detection system can be completed in one step, the amplification efficiency is high, 10^9 to 10^10 times can be amplified in 30 to 60min, and the specificity is high. The principle of LAMP technology is that DNA can be in dynamic equilibrium at about 65 deg.C, and at this temperature, DNA synthesis is continuously self-circulated by means of a strand displacement DNA polymerase using 4 specific primers. The final product of the amplification is a mixture of DNAs with different stem-loop structures and different lengths, and the product DNA is an alternating inverted repeat of the amplification targeting sequence.
The LAMP amplification system is combined with an acid-base indicator, the color difference between the positive product and the negative product after reaction is obvious, the result can be directly judged by observation, the LAMP amplification system has the advantages of easy result judgment, simple and convenient operation, high sensitivity and the like, the whole reaction process only needs 65 ℃ for 30min, the detection speed is high, the reaction time is short, no complex temperature control equipment is needed, and the LAMP amplification system is suitable for rapid detection for on-site instant screening of COVID-19 epidemic situation.
Disclosure of Invention
Technical problem to be solved
Aiming at the problems in the prior art, the invention can solve the problems of low detection lower limit, complex and time-consuming operation process, complex laboratory instrument and equipment and serious aerosol pollution of the existing novel coronavirus molecule nucleic acid detection technology, simplifies the detection process while not needing complex temperature control equipment and instruments, directly judges and reads the result through the color change of a product after reaction, and only can a sample enter, thereby effectively preventing pollution. The method is suitable for virus molecule diagnosis and screening in any scene.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: a COVID-19 rapid detection color indicator kit comprises a detection color indicator, a detection reaction solution, a positive reference substance, a negative reference substance and an LAMP primer group.
Preferably, the detection color indicator comprises 0.5-2.3 mM dNTPs, 2-12 mM MgSO4, 10-25 mM Tris-HCl, 5-15 mM KCl, 8-16 mM (NH4) SO4, 0.1-0.3% Triton X-100, 0.5-1.5M betaine and an acid-base indicator.
Preferably, the detection reaction solution contains 6-10U Bst DNA Polymerase, 16-25U Thermostable V reverse transcriptase, DNA Polymerase, 20U AMV reverse transcriptase, 6-10U RNase inhibitor and 18-26% trehalose.
Preferably, the positive control is an artificially synthesized plasmid corresponding to the COVID-19 specific gene N.
Preferably, the negative control substance is an artificially synthesized plasmid corresponding to a non-COVID-19 specific gene N.
Preferably, the LAMP primer group comprises LAMP primer groups corresponding to specific gene N of the COVID-19 virus, 0.1-0.3 mu M primer group F3/B3, 0.8-3.2 mu M primer group FIP/BIP and 0.2-0.6 mu M primer group loopF/loopR.
Preferably, the LAMP primer group consists of an outer primer group F3/B3, an inner primer group FIP/BIP and a loop primer group LoopF/LoopR,
the sequence of F3 is: 5'-GCTGCTGAGGCTTCTAAG-3', respectively;
the sequence of B3 is: 5'-GCGTCAATATGCTTATTCAGC-3', respectively;
the FIP sequence is: 5'-GCGGCCAATGTTTGTAATCAGTAGACGTGGTCCAGAACAA-3', respectively;
the BIP sequence is: 5'-TCAGCGTTCTTCGGAATGTCGCTGTGTAGGTCAACCACG-3', respectively;
the LoopF sequence is: 5'-CCTTGTCTGATTAGTTCCTGGT-3', respectively;
the loopR sequence is: 5'-CAGCGCTTCAGCGTTCTTCG-3' are provided.
Preferably, the use method of the kit comprises the following steps:
s1, thawing the reagent, and respectively and uniformly mixing the detection color indicator, the detection reaction solution and the LAMP primer group according to a certain proportion.
S2, adding the nucleic acid sample to be detected, the positive control substance and the negative control substance into the mixed solution prepared in the S1 according to a certain proportion, mixing uniformly and centrifuging.
S3, the reaction was incubated at 65 ℃ for 30 minutes.
And S4, observing in an environment with sufficient natural light after the reaction is finished, and directly judging the result by naked eyes.
Preferably, in step S3, the incubation process may use a temperature control device, a constant temperature incubator at 65 ℃, a thermal cycler, a water bath, or the like.
(III) advantageous effects
The invention provides a COVID-19 rapid detection color development indicating kit, which has the following beneficial effects:
in the invention, the indication kit is used for rapidly detecting the COVID-19 virus N gene based on the LAMP amplification technology, and carrying out efficient specific strand displacement reaction under the constant temperature condition, so that the characteristics of high temperature control precision requirement and complex and time-consuming process of the traditional PCR reaction are overcome, meanwhile, the indication kit is simple and convenient to operate, has low requirement on experimental operators and low single reaction cost, can be used for rapidly screening samples in a task scene, only needs 30 minutes in the whole process, is short in detection flow, high in speed and efficiency, has obvious color difference of detection results, can be directly interpreted by naked eyes, is convenient to observe, does not need to be uncapped for detection again, avoids aerosol pollution caused by uncapping detection amplification products after reaction, and reduces the risk of pollution in the detection process.
Drawings
FIG. 1 is a graph comparing the concentration of COVID-19RNA molecules of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1, the present invention provides a technical solution: a COVID-19 rapid detection color development indicating kit comprises a detection color development indicator, a detection reaction solution, a positive reference substance, a negative reference substance and an LAMP primer group.
Preferably, the detection color indicator comprises 0.5-2.3 mM dNTPs, 2-12 mM MgSO4, 10-25 mM Tris-HCl, 5-15 mM KCl, 8-16 mM (NH4) SO4, 0.1-0.3% Triton X-100, 0.5-1.5M betaine and an acid-base indicator.
Preferably, the detection reaction solution contains 6-10U Bst DNA Polymerase, 16-25U Thermostable V reverse transcriptase, DNA Polymerase, 20U AMV reverse transcriptase, 6-10U RNase inhibitor and 18-26% trehalose.
Preferably, the positive control is an artificially synthesized plasmid corresponding to COVID-19 specific gene N.
Preferably, the negative control is an artificially synthesized plasmid corresponding to the non-COVID-19 specific gene N.
Preferably, the LAMP primer group comprises LAMP primer group corresponding to specific gene N of COVID-19 virus, 0.1-0.3 mu M primer group F3/B3, 0.8-3.2 mu M primer group FIP/BIP and 0.2-0.6 mu M primer group loopF/loopR.
Preferably, the LAMP primer group consists of an outer primer group F3/B3, an inner primer group FIP/BIP and a loop primer group LoopF/LoopR,
the sequence of F3 is: 5'-GCTGCTGAGGCTTCTAAG-3', respectively;
the sequence of B3 is: 5'-GCGTCAATATGCTTATTCAGC-3', respectively;
the FIP sequence is: 5'-GCGGCCAATGTTTGTAATCAGTAGACGTGGTCCAGAACAA-3', respectively;
the BIP sequence is: 5'-TCAGCGTTCTTCGGAATGTCGCTGTGTAGGTCAACCACG-3', respectively;
the LoopF sequence is: 5'-CCTTGTCTGATTAGTTCCTGGT-3', respectively;
the loopR sequence is: 5'-CAGCGCTTCAGCGTTCTTCG-3' are provided.
Preferably, the method for using the kit comprises the following steps:
s1, thawing the reagent, and respectively and uniformly mixing the detection color indicator, the detection reaction solution and the LAMP primer group according to a certain proportion.
S2, adding the nucleic acid sample to be detected, the positive control substance and the negative control substance into the mixed solution prepared in the S1 according to a certain proportion, mixing uniformly and centrifuging.
S3, the reaction was incubated at 65 ℃ for 30 minutes.
And S4, observing in an environment with sufficient natural light after the reaction is finished, and directly judging the result by naked eyes.
Preferably, in step S3, the incubation process may use a temperature control device, a constant temperature incubator at 65 ℃, a thermal cycler, a water bath, etc.
In this embodiment, the detection of the RNA molecule of the COVID-19 sample comprises the following steps:
thawing the reagent, respectively and uniformly mixing the detection color indicator, the detection reaction solution and the LAMP primer group according to a certain proportion, respectively adding 5.5 mu l of samples with different concentrations of COVID-19 virus RNA molecules and negative reference substances into a detection system, uniformly mixing, placing a reaction tube at 65 ℃ for reaction for 30 minutes, and directly judging the result after the reaction is finished, wherein the detection system is as follows:
| reagent | Dosage of |
| Detection color reagent | 10μL |
| Detection reaction solution | 2.5μL |
| Specific primer pair | 2μL |
| Template to be detected | 5.5μL |
And after the reaction is finished, observing in an environment with sufficient natural light, directly judging the result by naked eyes, and observing the color of the solution in each reaction tube, wherein red without color change is negative, and yellow is positive.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A COVID-19 rapid detection color development indication kit is characterized in that: the indication kit comprises a detection color indicator, a detection reaction solution, a positive reference substance, a negative reference substance and an LAMP primer group.
2. The kit for rapid detection of COVID-19 chromogenic indicator according to claim 1, wherein: the detection color indicator comprises 0.5-2.3 mM dNTPs, 2-12 mM MgSO4, 10-25 mM Tris-HCl, 5-15 mM KCl, 8-16 mM (NH4) SO4, 0.1-0.3% Triton X-100, 0.5-1.5M betaine and an acid-base indicator.
3. The kit for rapid detection of COVID-19 chromogenic indicator according to claim 1, wherein: the detection reaction solution comprises 6-10U Bst DNA polymerase, 16-25U Thermostable V reverse transcriptase, DNApolymerase, 20U AMV reverse transcriptase, 6-10U RNase inhibitor and 18-26% trehalose.
4. The kit for rapid detection of COVID-19 chromogenic indicator according to claim 1, wherein: the positive control substance is an artificially synthesized plasmid corresponding to the specific gene N of COVID-19.
5. The kit for rapid detection of COVID-19 chromogenic indicator according to claim 1, wherein: the negative control substance is an artificially synthesized plasmid corresponding to a non-COVID-19 specific gene N.
6. The kit for rapid detection of COVID-19 chromogenic indicator according to claim 1, wherein: the LAMP primer group comprises a LAMP primer group corresponding to specific gene N of the COVID-19 virus, a 0.1-0.3 mu M primer group F3/B3, a 0.8-3.2 mu M primer group FIP/BIP and a 0.2-0.6 mu M primer group loopF/loopR.
7. The kit for rapid detection of COVID-19 chromogenic indicator according to claim 1, wherein: the LAMP primer group consists of an outer primer group F3/B3, an inner primer group FIP/BIP and a loop primer group loopF/loopR,
the sequence of F3 is: 5'-GCTGCTGAGGCTTCTAAG-3', respectively;
the sequence of B3 is: 5'-GCGTCAATATGCTTATTCAGC-3', respectively;
the FIP sequence is: 5'-GCGGCCAATGTTTGTAATCAGTAGACGTGGTCCAGAACAA-3', respectively;
the BIP sequence is: 5'-TCAGCGTTCTTCGGAATGTCGCTGTGTAGGTCAACCACG-3', respectively;
the LoopF sequence is: 5'-CCTTGTCTGATTAGTTCCTGGT-3', respectively;
the loopR sequence is: 5'-CAGCGCTTCAGCGTTCTTCG-3' are provided.
8. The kit for rapid detection of COVID-19 chromogenic indicator according to claim 1, wherein: the use method of the kit comprises the following steps:
s1, thawing the reagent, and respectively and uniformly mixing the detection color indicator, the detection reaction solution and the LAMP primer group according to a certain proportion.
S2, adding the nucleic acid sample to be detected, the positive control substance and the negative control substance into the mixed solution prepared in the S1 according to a certain proportion, mixing uniformly and centrifuging.
S3, the reaction was incubated at 65 ℃ for 30 minutes.
And S4, observing in an environment with sufficient natural light after the reaction is finished, and directly judging the result by naked eyes.
9. The kit for rapid detection of COVID-19 chromogenic indicator according to claim 8, wherein: in step S3, the temperature control device used in the incubation process may be a constant temperature incubator at 65 ℃, a thermal cycler, a water bath, or the like.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112646932A (en) * | 2021-01-08 | 2021-04-13 | 湖南超亟检测技术有限责任公司 | Primer group and kit for one-step visual detection of novel coronavirus nucleic acid |
| CN112961943A (en) * | 2021-04-30 | 2021-06-15 | 广州普世利华科技有限公司 | Primer probe combination product for detecting SARS-CoV-2 |
| CN114752657A (en) * | 2022-05-05 | 2022-07-15 | 中山大学 | Polydisperse liquid drop digital nucleic acid detection method and application thereof |
| WO2022226870A1 (en) * | 2021-04-29 | 2022-11-03 | 中国科学院大学宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature conditions, kit, and application |
| EP4215903A1 (en) * | 2022-01-21 | 2023-07-26 | 1Drop Inc. | Test apparatus and method for determining whether infection is present using ratio of color change of reagent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111154922A (en) * | 2020-04-07 | 2020-05-15 | 广东环凯生物科技有限公司 | SARS-CoV-2 dry powder LAMP rapid detection kit |
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2020
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111154922A (en) * | 2020-04-07 | 2020-05-15 | 广东环凯生物科技有限公司 | SARS-CoV-2 dry powder LAMP rapid detection kit |
Non-Patent Citations (2)
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| MOHAMED EL-THOLOTH ET AL.: "A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry", 《CHEMRXIV》 * |
| WEIHUA YANG ET AL.: "Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method", 《CHEMRXIV》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112646932A (en) * | 2021-01-08 | 2021-04-13 | 湖南超亟检测技术有限责任公司 | Primer group and kit for one-step visual detection of novel coronavirus nucleic acid |
| CN112646932B (en) * | 2021-01-08 | 2022-06-07 | 湖南超亟检测技术有限责任公司 | Primer group and kit for one-step visual detection of novel coronavirus nucleic acid |
| WO2022226870A1 (en) * | 2021-04-29 | 2022-11-03 | 中国科学院大学宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature conditions, kit, and application |
| CN112961943A (en) * | 2021-04-30 | 2021-06-15 | 广州普世利华科技有限公司 | Primer probe combination product for detecting SARS-CoV-2 |
| EP4215903A1 (en) * | 2022-01-21 | 2023-07-26 | 1Drop Inc. | Test apparatus and method for determining whether infection is present using ratio of color change of reagent |
| CN114752657A (en) * | 2022-05-05 | 2022-07-15 | 中山大学 | Polydisperse liquid drop digital nucleic acid detection method and application thereof |
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