CN111270020A - Nucleic acid detection reagent and application thereof in novel coronavirus detection - Google Patents
Nucleic acid detection reagent and application thereof in novel coronavirus detection Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, in particular to a nucleic acid detection reagent and application thereof in novel coronavirus detection. The invention provides a reagent for nucleic acid amplification, which comprises Bst polymerase, AMV reverse transcriptase, dye and buffer; the reagent utilizes the change of pH values before and after the amplification reaction, so that the amplification result can be distinguished by naked eyes, the use of a complex instrument is avoided, and the detection efficiency is improved. In addition, in the nucleic acid detection reagent, the nucleic acid extraction reagent can complete the nucleic acid extraction of the sample under the condition of no professional equipment, and the rapid, accurate and visual detection of the novel coronavirus can be realized by combining the LAMP primer provided by the invention.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a nucleic acid detection reagent and application thereof in novel coronavirus detection.
Background
The novel coronavirus (SARS-COV-2), cough or sneeze from the airways produced by the main transmission of respiratory droplets (similar to the influenza in the course of time). The time between exposure and onset of symptoms is typically 5 days, but may range from 2 to 14 days. Symptoms may include fever, cough, diarrhea and shortness of breath. Complications may include pneumonia and acute respiratory distress syndrome. There is currently no vaccine or antiviral treatment, but research is still in progress. The purpose is to manage symptoms and support therapy. The suggested precautions include washing hands, keeping a distance from the patient, and monitoring and self-isolating for two weeks if the individual suspects that he is infected.
The main diagnostic method for the novel coronavirus (SARS-COV-2) is PCR method, which is the most approved detection method so far. However, the current PCR method requires a relatively complex instrument, and most of the result analysis also requires computer-aided analysis. Therefore, the existing detection method has the defects of complex operation steps, high requirements on personnel skills, incapability of visualizing results and the like.
In order to further improve the efficiency and accuracy of detection, the art still searches for a detection method capable of detecting novel coronavirus more rapidly and accurately.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a nucleic acid detection reagent and its application in the detection of novel coronavirus, the reagent can perform detection at a constant temperature, and the amplification result can be identified by naked eyes.
The present invention provides a nucleic acid amplification reagent comprising: bst polymerase, AMV reverse transcriptase, dye and buffer;
the dye is selected from: cresol red, neutral red, 3-nitrophenol, m-cresol purple, naphthol peptide or methylene blue;
the buffer comprises water and: KCl, MgSO4Tween 20 and dNTPs.
Under the action of DNA polymerase, one H is released for each nucleotide bound+The pH of the system will change. If no amplification occurs, no H + is released and the pH of the system will not change. Therefore, the addition of a suitable indicator to the system,the color of the system in which the amplification reaction occurred changed, while the color of the system in which no amplification occurred did not change. Therefore, the method provided by the invention can realize the visual visualization of the reaction result.
In some embodiments, the dye is cresol red, neutral red, a combination of cresol red and methylene blue, or a combination of neutral red and methylene blue.
In the present invention, the nucleic acid amplification reagent includes water and the following concentrations:
in the present invention, the nucleic acid amplification reagent includes water and the following concentrations:
in some embodiments, the nucleic acid amplification reagents of the invention comprise water and the following concentrations:
in some embodiments, the nucleic acid amplification reagents of the invention comprise water and the following concentrations:
in some embodiments, the nucleic acid amplification reagents of the invention comprise water and the following concentrations:
in some embodiments, the nucleic acid amplification reagents of the invention comprise water and the following concentrations:
the nucleic acid amplification reagent provided by the invention is suitable for amplification of LAMP technology, and combines a loop-mediated isothermal amplification (LAMP) technology, a microfluidic chip technology, a nucleic acid purification technology, a pH dye color change principle and other technologies to establish the nucleic acid amplification reagent; the reagent is used for amplification, and naked eye interpretation of results is realized according to whether the color of the obtained product changes.
In the nucleic acid reagent, the concentration of each component is a working concentration, and the concentration of each component can be 2-100 times of the working concentration in the processes of storage and transportation. The components may be present independently or in admixture, for example, the nucleic acid reagent includes three independently present reagents, an enzyme, a buffer, and a dye.
The invention also provides a nucleic acid detection reagent combination, which comprises: a nucleic acid extraction reagent, the nucleic acid amplification reagent and the LAMP amplification primer.
In the present invention, the nucleic acid extraction reagent comprises: sample treatment liquid, lysis solution, cleaning solution and eluent; wherein:
the sample treatment liquid: comprises water and 0.1mmol/L dithiothreitol, 0.2mmol/L sodium chloride, 10mmol/L potassium chloride, 1mmol/L disodium hydrogen phosphate and 10mmol/L potassium dihydrogen phosphate, and the pH value of the solution is 7.5 +/-0.2.
The lysis solution: including water and 1mmol/L Tris-Hcl (pH 8.0), 0.5mmol/L EDTA (pH 8.0), 10% SDS and 0.1mmol/L NaCl.
The cleaning solution: including water, 80 vol% absolute ethanol, and 0.5M EDTA (pH 8.0).
The eluent is as follows: nuclease-free water.
The eluent is nuclease-free water.
In the present invention, the nucleic acid extraction reagent further comprises 95% ethanol.
The invention also provides a nucleic acid detection method, which comprises the steps of extracting nucleic acid of a sample, amplifying by using the nucleic acid amplification reagent and the LAMP primer, and judging whether target nucleic acid is contained according to the color of an amplification product.
The step of extracting nucleic acid comprises: cracking, adsorbing, cleaning and eluting.
The nucleic acid amplification reagent or the nucleic acid detection reagent combination is applied to the preparation of a novel coronavirus detection kit.
The invention also provides a novel coronavirus detection kit, which comprises the nucleic acid detection reagent combination; the LAMP amplification primer comprises:
amplification primers for the S gene:
as shown in SEQ ID NO: 1, the outer primer S-F3,
as shown in SEQ ID NO: 2, the outer primer S-B3,
as shown in SEQ ID NO: 3, an inner primer S-FIP,
as shown in SEQ ID NO: 4, an inner primer S-BIP shown in the specification,
as shown in SEQ ID NO: 5, a loop primer S-LF shown in the specification,
as shown in SEQ ID NO: 6, a loop primer S-LB shown in the specification,
amplification primers for the N gene:
as shown in SEQ ID NO: 7, the outer primer N-F3,
as shown in SEQ ID NO: 8, the outer primer N-B3 shown in the figure,
as shown in SEQ ID NO: 9 is shown as the inner primer N-FIP,
as shown in SEQ ID NO: 10, the inner primer N-BIP shown in the specification,
as shown in SEQ ID NO: 11, the loop primer N-LF shown in the figure,
as shown in SEQ ID NO: 12, and the loop primer N-LB shown in the specification.
In the kit, the nucleic acid amplification kit further comprises: and extracting consumables. The extraction consumable comprises a sterile suction pipe filled with pickaxe beads and a nucleic acid enricher. The nucleic acid amplification kit may further include a positive control and a negative control. The positive control is a plasmid of a novel coronavirus S gene segment and/or a segment of a novel coronavirus N gene.
The present invention also provides a method for detecting a novel coronavirus, which is not of diagnostic interest, comprising:
after the nucleic acid of the sample is extracted, a nucleic acid amplification reagent and an LAMP primer are used for amplification, and whether the novel coronavirus is contained or not is judged according to the color of an amplification product.
The system for amplification comprises:
sample nucleic acid 100. mu.L, the nucleic acid amplification reagent 75. mu.L, 0.3. mu.L of each 0.3mM outer primer, 9.6. mu.L of each 2.4mM inner primer, and 1. mu.L of each 1mM loop primer, and water to make up 200. mu.L.
The amplification condition is constant temperature of 60-70 ℃ for 50 min. In a specific embodiment, the temperature of the amplification is 65 ℃.
The sample of the present invention comprises: sputum, pharyngeal swabs, nasal swabs, or washing fluids on the surface of an object.
The judgment comprises the following steps:
the dye in the nucleic acid amplification reagent is cresol red, the amplification product is yellow, and the result is positive; the amplification product is not yellow, and the result is negative;
the dye in the nucleic acid amplification reagent is neutral red, the amplification product is purple red, and the result is positive; the amplification product is not purple red, and the result is negative;
dyes in the nucleic acid amplification reagent are cresol red and methylene blue, an amplification product is green, and the result is positive; the amplification product is not green, and the result is negative;
the dyes in the nucleic acid amplification reagent are neutral red and methylene blue, the amplification product is purple, and the result is positive; the amplification product was not purple and the result was negative
The invention provides a reagent for nucleic acid amplification, which comprises Bst polymerase, AMV reverse transcriptase, dye and buffer; the reagent utilizes the change of pH values before and after the amplification reaction, so that the amplification result can be distinguished by naked eyes, the use of a complex instrument is avoided, and the detection efficiency is improved. In addition, in the nucleic acid detection reagent, the nucleic acid extraction reagent can complete the nucleic acid extraction of the sample under the condition of no professional equipment, and the rapid, accurate and visual detection of the novel coronavirus can be realized by combining the LAMP primer provided by the invention.
Drawings
FIG. 1a is a simplified extraction method of SARS-cov-2 as a novel coronavirus (SARS-cov-2) nucleic acid in a household self-test with a 1ml pipette pre-loaded with pickaxel beads, which assists in the lysis of sputum;
FIG. 1b is a simplified nucleic acid enrichment device for household self-testing of coronavirus (SARS-cov-2) nucleic acid, which is used with 1ml or 5ml screw piston injector for nucleic acid enrichment in sample;
FIG. 1c is a simplified method for home-use self-test of the nucleic acid of a novel coronavirus (SARS-cov-2), the extracted viral nucleic acid RNA, PCR test results; extracting coronavirus 229E with original concentration, 10-fold dilution, 100-fold dilution and 1000-fold dilution by using the method, amplifying by using corresponding PCR primers, and inspecting extraction efficiency;
FIG. 1d shows the results of PCR detection of viral RNA from QIAGEN RNA Kit, a conventional nucleic acid extraction Kit from purification column (coronavirus 229E was extracted at original concentration, diluted 10-fold, 100-fold and 1000-fold, and amplified using the corresponding PCR primers to examine the efficiency of extraction);
FIG. 2a is a diagram showing the result of amplification of cresol red dye in a simple visualized isothermal amplification reagent for home-use self-test of novel coronavirus (SARS-cov-2) nucleic acid (wherein magenta is negative and bright yellow is positive);
FIG. 2b is a diagram showing the result of the amplification of neutral red dye in a simple visualized isothermal amplification reagent for home-use self-test of a novel coronavirus (SARS-cov-2) nucleic acid (wherein, orange is negative and purple is positive);
FIG. 2c is a diagram showing the results of the amplification of cresol red + methylene blue dye in a simple visual isothermal amplification reagent for home-use self-test of a novel coronavirus (SARS-cov-2) nucleic acid (wherein black-purple is negative and bright-green is positive);
FIG. 2d is a graph showing the result of the amplification of neutral red and methylene blue dyes in a simple visual isothermal amplification reagent for home-use self-test of a novel coronavirus (SARS-cov-2) (wherein, greenish grass is negative and purple is positive);
FIG. 3a is a diagram showing the result of amplifying plasmid DNA containing "S gene fragment of novel coronavirus (SARS-cov-2)" by using the home-use self-test S gene LAMP primer for novel coronavirus (SARS-cov-2) nucleic acid (wherein, the concentration of template DNA is 1000 copies/. mu.1);
FIG. 3b is a diagram showing the result of amplifying plasmid DNA containing "S gene fragment of novel coronavirus (SARS-cov-2)" by using the home-use self-test S gene LAMP primer for novel coronavirus (SARS-cov-2) nucleic acid (wherein, the concentration of template DNA is 100 copies/. mu.l);
FIG. 3c is a diagram showing the result of amplification of plasmid DNA containing "S gene fragment of novel coronavirus (SARS-cov-2)" by using the LAMP primer for home-use self-test of novel coronavirus (SARS-cov-2) nucleic acid (wherein, the concentration of template DNA is 10 copies/. mu.l);
FIG. 3d is a diagram showing the result of "plasmid DNA containing N gene fragment of novel coronavirus (SARS-cov-2)" amplified by N gene LAMP primer for home-use self-test of novel coronavirus (SARS-cov-2) nucleic acid (wherein, the concentration of template DNA is 1000 copies/. mu.l);
FIG. 3e is a diagram showing the result of "plasmid DNA containing N gene fragment of novel coronavirus (SARS-cov-2)" amplified by N gene LAMP primer for home-use self-test of novel coronavirus (SARS-cov-2) nucleic acid (wherein, the concentration of template DNA is 100 copies/. mu.l);
FIG. 3f is a diagram showing the result of "plasmid DNA containing N gene fragment of novel coronavirus (SARS-cov-2)" amplified by N gene LAMP primer for home-use self-test of novel coronavirus (SARS-cov-2) nucleic acid (wherein, the concentration of template DNA is 10 copies/. mu.l);
FIG. 4a is a negative control of loop-mediated isothermal amplification reaction using the LAMP detection primer set for the S gene of the present invention, and the specificity of the primers themselves is examined (wherein the negative control template includes coronavirus 22E, coronavirus OC43, influenza H1, and nuclease-free water);
FIG. 4b is a negative control of the LAMP detection primer set for loop-mediated isothermal amplification reaction, which is used for examining the specificity of the primers (wherein, the negative control template comprises coronavirus 22E, coronavirus OC43, influenza H1 and nuclease-free water);
FIG. 5a is a real sample detection result of the LAMP detection primer set for loop-mediated isothermal amplification reaction (wherein the real sample is from the center for controlling diseases in great-interest areas in Beijing City);
FIG. 5b is a real sample detection result of the LAMP detection primer set for loop-mediated isothermal amplification reaction (wherein the real sample is from the center for controlling diseases in great-interest areas in Beijing City);
FIG. 5-c-1, FIG. 5-c-2, FIG. 5-c-3, FIG. 5-c-4 and FIG. 5-c-5 show the real sample detection results of the approved PCR products in China (wherein the real sample is from the great-interest center in Beijing City);
FIG. 6 shows the results of the detection of a sample on the surface of an object;
FIG. 7 shows an alignment of the sequences.
Detailed Description
The invention provides a nucleic acid detection reagent and application thereof in novel coronavirus detection, and a person skilled in the art can appropriately modify process parameters for realization by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention provides a simple nucleic acid extraction method, which solves the problem that the common extraction method depends on instruments; provides a simple visual nucleic acid amplification system based on pH change, and solves the problem of dependence on expensive detection instruments. The LAMP detection primer aiming at the S gene and the N gene of the novel coronavirus (SARS-COV-2) is provided, and the nucleic acid amplification reagent and the detection kit of the novel coronavirus can finish the nucleic acid extraction of a sample and the nucleic acid detection of the sample under the condition of no professional equipment and provide a direction for the nucleic acid detection to enter the ordinary life by performing the early sequence comparison analysis, the later real clinical sample test and the performance comparison with the market evidence PCR product.
In the kit provided by the invention, 1-6 primer sequences in 12 primer sequences are combined into a loop-mediated isothermal amplification (LAMP) primer group of an S gene, 7-12 primer sequences are combined into a loop-mediated isothermal amplification (LAMP) primer group of an N gene, 6 primers need to work in coordination, and the detection target is novel coronavirus (SARS-COV-2) (the same species with high similarity such as SARS, SARS-like and MARS can be completely distinguished).
The detection gene aimed by the primer provided by the application is the S gene and the N gene of the novel coronavirus, the total length of the S gene is about 3822bp, and the design region of the primer is between 1942-2721bp of the gene; the total length of the N gene is about 1260bp, and the design region of the primer is between 800-1260bp of the gene. Wherein, SEQ ID NO: the nucleotide sequence shown in 13 is a partial sequence of the target sequence S gene, and 6 primers for detection are designed in the region. SEQ ID NO: 14 is a partial sequence of the target sequence N gene, and 6 primers for detection were designed in the following regions.
In the detection kit for the novel coronavirus (SARS-COV-2), the extraction method of nucleic acid is simpler and more convenient, and the nucleic acid extracted by the simplified extraction method provided by the invention is used as a template for loop-mediated isothermal amplification (LAMP).
The amplification reagent provided by the invention can enable the detection result to observe the color change before and after the reaction by naked eyes, thereby judging the result. In addition, in the method, a fluorescent probe and a fluorescent dye are not needed, and the judgment of the detection result is carried out in a color change mode generated by the change of the pH value.
In the novel loop-mediated isothermal amplification detection kit for the coronavirus (SARS-COV-2), a nucleic acid extraction part comprises: a sputum box; a throat swab; 1ml of treatment solution; 1ml sterile suction pipe with pickaxe beads in advance; 1ml of lysate; 1ml of absolute ethyl alcohol; 3ml of cleaning solution; 100 μ l of the eluate; 5ml screw piston injector; 1ml screw piston injector; a nucleic acid enricher. Further, the extraction conditions for the simplified nucleic acid extraction are as follows: keeping the temperature at 56-65 ℃ for 10-15 min.
Further, in the novel coronavirus (SARS-COV-2) loop-mediated isothermal amplification detection kit, the amplification detection part comprises: 0.3. mu.L each of 0.3mM of outer primer F3(S2 and N1) and outer primer B3(S2 and N1); 2.4mM of inner primer FIP (S2 and N1) and inner primer BIP (S2 and N1) each 9.6. mu.L; 1. mu.L of each of 1mM loop primer LF (S2 and N1) and loop primer LB (S2 and N1); visual constant temperature buffer solution 65 μ L; 10. mu.l of enzyme reaction solution (containing Bst enzyme and AMV enzyme); 100. mu.L of template DNA was added to 200. mu.L of sterilized water. Further, the pH dye in the visualization reaction buffer was a mixture of cresol red, neutral red and methylene blue. Further, the detection reaction conditions of the loop-mediated isothermal amplification are as follows: keeping the temperature of 60-70 ℃ for 50 min.
The invention is further illustrated by the following examples:
example 1
1. Preparation of the kit
1.1 preparation of nucleic acid extraction reagent:
the sample treatment liquid: comprises water and 0.1mmol/L dithiothreitol, 0.2mmol/L sodium chloride, 10mmol/L potassium chloride, 1mmol/L disodium hydrogen phosphate and 10mmol/L potassium dihydrogen phosphate, and the pH value of the solution is 7.5 +/-0.2.
The lysis solution: including water and 1mmol/L Tris-Hcl (pH 8.0), 0.5mmol/L EDTA (pH 8.0), 10% SDS and 0.1mmol/L NaCl.
The cleaning solution: including water, 80 vol% absolute ethanol, and 0.5M EDTA (pH 8.0).
The eluent is as follows: nuclease-free water.
1.2 consumable preparation:
extracting consumables: adding 0.6mm pickaxe beads into a 1ml sterile suction pipe; sealing the pipe orifice with sealing film and storing for use. When the sputum is liquefied, the sputum sample mixed with the treatment fluid in advance is repeatedly blown and sucked through the suction force of the suction pipe, and the sputum sample is liquefied from two aspects of a chemical method and a physical method by combining the friction force of the pickaxe beads.
A nucleic acid enricher: and (3) placing the round silica gel mold into a clamping shell of the enricher, wherein the middle position of the clamping shell of the enricher is a clamping groove, and placing the cut silica gel mold into the clamping groove to form the nucleic acid enricher.
5ml and 1ml screw piston injectors are consumables in the extraction kit and are purchasing consumables.
2. The extraction method of the sample nucleic acid comprises the following steps:
2.1, the sample is sputum:
2.1.1 sputum needs to be liquefied before cracking, the sputum needs to be firstly visually observed, and if the saliva accounts for the most part, the specimen needs to be collected again. Adding 1-2 times of the volume of the sputum treatment fluid into the sputum collector, fully oscillating and mixing uniformly, and standing at room temperature to fully liquefy the sputum treatment fluid (generally, no obvious solid matter exists after 20-30 minutes, no tow phenomenon exists when the sputum treatment fluid is sucked out, and if the sputum treatment fluid is not completely liquefied, a small amount of the sputum treatment fluid can be properly added until the sputum treatment fluid is completely liquefied).
2.1.2 lysis of samples: taking 1mL of the liquid sample subjected to the pretreatment into a 5mL centrifuge tube, adding 0.6mL of lysate, repeatedly sucking and pumping the lysate and uniformly mixing the lysate and the lysate, and then putting the 5mL centrifuge tube into boiling water for incubating for 10 minutes without turning the 5mL centrifuge tube upside down so as to prevent the liquid from leaking.
2.1.3 adsorption of nucleic acids: after the sample is cooled to room temperature, 1.4mL of 95% ethanol is added and mixed uniformly, the cracked sample is slowly sucked by a disposable nucleic acid extraction device, and the whole sample is sucked into a 3mL syringe and then slowly discarded (without repeated push-suction). Note that: the nucleic acid concentrator was not removable from the front end of the 3mL syringe before use.
2.1.4 washing nucleic acids: 2mL of the washing solution was taken into a 2mL centrifuge tube, and the whole solution was slowly aspirated by a disposable nucleic acid extraction apparatus, and then slowly discarded (without repeated aspiration). Note that: before the operation, the cleaning solution was confirmed to have been added with a corresponding volume of 95% ethanol.
2.1.5 cleaning of residual liquid: the syringe piston was slowly pulled and aspirated 5 times to remove the residual liquid sufficiently.
2.1.6 syringe replacement: the 3mL syringe connected to the nucleic acid concentrator was removed and replaced with a 1mL syringe.
2.1.7 elution of nucleic acids: the eluent was slowly drawn up using a 1mL syringe. And slowly pushing and sucking for 1 time, standing for 1 minute by using a 1mL syringe, and then pushing the eluted nucleic acid out to a detection tube, or pushing the eluted nucleic acid out to a 0.6mL centrifugal tube for storage at the temperature of-20 ℃ for subsequent detection.
2.2, the sample is a throat swab or a nasal swab:
throat swab: after sampling the throat swab, the swab was eluted in 1mL of the swab treatment solution, and after several times of agitation, the swab was discarded and the eluate was ready for use. The extraction process is the same as 2.1.2-2.1.7.
2.3, the sample is the rinsing liquid on the surface of the article:
rinsing liquor on the surface of animal tissue: sucking 1ml of 0.9% physiological saline with a sterile pipette, repeatedly rinsing for 10 times to obtain an animal tissue surface suspected to contain the novel coronavirus (SARS-COV-2), and rinsing the rinsing solution for later use. The extraction process is the same as 2.1.2-2.1.7.
Rinsing liquor on the metal surface: sucking 1ml of 0.9% physiological saline with a sterile pipette, repeatedly rinsing for 10 times to obtain a metal surface suspected to contain the novel coronavirus (SARS-COV-2), and rinsing the liquid for later use. The extraction process is the same as 2.1.2-2.1.7.
3. And (3) verifying the extraction effect:
as a control, coronavirus 229E was extracted using a currently commercially available purification column extraction Kit (QIAGEN RNA Kit).
The extraction method refers to the description, and the virus stock solution of coronavirus 229E is diluted 10 times, 100 times and 1000 times before extraction.
The extracted nucleic acids were examined using PCR primers and a PCR system, and repeated twice at each concentration.
The amplification primers are as follows:
229E-F：5’-TGGCACAGGACCCCATAAAG-3’；
229E-R：5’-CAACCCAGACGACACCTTCA-3’。
the amplification results are shown in FIG. 1c and FIG. 1 d. FIGS. 1c and 1d show the results of experiments using the extraction method of the kit in comparison with the conventional extraction method using a purification column (QIAGEN RNAKit). The method comprises the steps of extracting nucleic acid RNA of the coronavirus 229E by respectively using two extraction methods (an original sample of the new coronavirus is strictly controlled at present, so that 229E which belongs to the same RNA virus but has low toxicity is used as a research object for extracting nucleic acid), extracting viruses at original concentration, diluting 10 times, diluting 100 times and diluting 1000 times, amplifying the nucleic acid RNA extracted at each concentration by using PCR primers of the coronavirus 229E, carrying out two-time repeated detection at each concentration, and examining the extraction difference. There are four curves in both FIG. 1c and FIG. 1d (i.e., original concentration extracted versus ten fold dilution of virus), indicating that both replicates are positive, indicating that the simplified extraction method of the present invention is consistent and stable when the same amount of RNA virus is extracted using the conventional extraction method on a purification column.
Example 2
1. Preparation of visualized isothermal amplification reagent
The enzyme reaction solution of the visual amplification reagent comprises:
6 μ L of Bst polymerase 8U/. mu.L;
4 μ L of 10U/. mu.L AMV reverse transcriptase;
1.6 μ L of 50mM visualization dye;
the buffer solution comprises: 10 μ L of 1M KCl; 4 μ L of 0.4M MgSO4(ii) a 2 μ L of 10% tween 20; 11.2. mu.L of 25mM dNTPs; 36.2 μ L of sterile water.
Experiments were divided into four groups according to the type of visualization dye selected:
group 1: cresol Red 50mM
Group 2: neutral Red 50mM
Group 3: cresol Red 30mM + methylene blue 20mM
Group 4: neutral Red 30mM + methylene blue 20mM
2. Design of LAMP primers
According to the Sequence of the novel coronavirus SARS-COV-2 standard strain (Reference _ Sequence _ NC _045512) recorded on GenBank and 35 gene sequences recorded on GISAID (collection time is up to 20200130, see figure 7 in particular), Sequence alignment is carried out (full-length sequences of N gene and S gene are aligned), conserved regions (which can effectively distinguish homologous coronaviruses with SARS, SARS-like and MARS isogeny higher than 70%) are screened, and 16 pairs of 20 primers are artificially designed and compared and screened. The sequence status of the alignment collected for the new coronavirus SARS-COV-2 (see FIG. 7 where the first is the standard sequence from NCBI; the subsequent sequences are all derived from GISAID)
The primer sequences used were synthesized by the firm Biotech, China. For the target sequence SEQ ID NO: 13, 6 primers for detecting S gene of novel coronavirus (SARS-cov-2) are designed, including two inner primers (FIP and BIP) and two outer primers (F3 and B3) and two loop primers (LF and LB), aiming at the target sequence SEQ ID NO: 14 6 primers for detecting N gene of novel coronavirus (SARS-cov-2) were designed, including two inner primers (FIP and BIP) and two outer primers (F3 and B3) and two loop primers (LF and LB), and the nucleotide sequences of the obtained primer sets are shown in Table 1.
TABLE 1 LAMP primer set for detection of novel coronavirus (SARS-cov-2)
3. Sample amplification
The nucleic acid sample obtained in example 1 was tested using the 4 sets of amplification reagents described above, using the nucleic acid of coronavirus 229E as a positive sample and the nuclease-free water as a negative sample,
the specific configuration of the visualized isothermal amplification detection system is as follows: 0.3. mu.L of each of 0.3mM of the outer primers F3 and B3; 2.4mM of inner primers FIP and BIP, each 9.6. mu.L; 1. mu.L each of 1mM loop primers LF and LB; visualization buffer 65 μ L (with dye); mu.l of the enzyme mixture (containing Bst and AMV) 100. mu.L of the template DNA (prepared in example 1) was added to a 200. mu.L system with sterilized purified water.
The isothermal amplification detection reaction conditions are as follows: keeping the temperature at 65 ℃ for 50 min.
In table 1 above, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 to form a primer group, which is called S2 group loop-mediated isothermal amplification primer group for short. In table 1 above, SEQ id no: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 form a primer group, which is called N1 group loop-mediated isothermal amplification primer group for short.
The amplification results are shown in FIG. 2:
FIG. 2a illustrates the positive and negative amplification results when plasmid DNA containing S/N sequences is used as a template (the template concentration is 100 copies/. mu.l, and the LAMP primer is the S/N detection primer) using cresol red as a visual isothermal amplification reagent prepared using a detection dye. The positive color after the reaction is bright yellow, and the negative color is magenta.
FIG. 2b illustrates the positive and negative amplification results when plasmid DNA containing S/N sequences is used as template (template concentration 100 copies/. mu.l, LAMP primer used is S/N detection primer) using visualized isothermal amplification reagents prepared using neutral red as detection dye. The positive color after the reaction is purple red, and the negative color is orange yellow.
FIG. 2c illustrates the positive and negative amplification results when plasmid DNA containing S/N sequences is used as template (template concentration 100 copies/. mu.l, LAMP primers used are S/N detection primers) using visualized isothermal amplification reagents prepared using cresol red + methylene blue as detection dye. The positive color after reaction is bright green, and the negative color is black purple.
FIG. 2d illustrates the positive and negative amplification results when plasmid DNA containing S/N sequences is used as template (template concentration 100 copies/. mu.l, LAMP primer used as S/N detection primer) using visualized isothermal amplification reagents prepared using neutral Red + methylene blue as detection dye. The positive color after reaction is purple, and the negative color is grass green.
Example 3
1. Examination of sensitivity of primers based on the set of loop-mediated isothermal amplification primers S2 and N1 obtained in the set 3 of example 2, the reaction system and the detection method, the concentrations of plasmid nucleic acids of S gene and N gene were: the nucleic acids of 1000 copies/. mu.l, 100 copies/. mu.l and 10 copies/. mu.l are subjected to the loop-mediated isothermal amplification detection, and the detection results are shown in 3a, 3b, 3c, 3d, 3e and 3 f.
Verification of primer specificity
The loop-mediated isothermal amplification detection was performed on 3 other viruses and nuclease-free water according to the loop-mediated isothermal amplification primer set, reaction system and detection method of the S2 and N1 groups obtained in group 3 of example 2. The results are shown in Table 2.
Amplifying by using a vacuum cup or a water bath kettle or other equipment capable of maintaining for 1-2h within a certain temperature range, wherein the reaction time is 50min, and observing the color change after the reaction is finished. The negative control test is performed by using sterilized purified water, and the result of the negative control test is purple, as shown in fig. 4a and 4 b. (1) If the green color appears, judging that the test result is positive; (2) if purple color (unchanged color) appears, the test result is judged to be negative.
For the other non-novel coronavirus SARS-COV-2, no detection (color change) was observed within 50 minutes (min) of the reaction, and a negative reaction was observed. The detection results in Table 2 show that the loop-mediated isothermal amplification primer group for detecting the novel coronavirus SARS-COV-2 provided by the invention has strong specificity.
TABLE 2 Experimental strains and isothermal amplification test results
Note: the ATCC is the American ATCC deposit management center.
The plasmid construction methods described in the table include: based on the published gene sequence of the novel coronavirus SARS-COV-2 (GenBank: NC-045512), Shanghai' S company was assigned to synthesize conserved sequences of the N gene and the S gene (the conserved sequence is a sequence with high conservation degree obtained by comparing NC-045512 with another 35 sequences), and the conserved sequences were cloned into a Puc-57 vector to form plasmids of Puc-57-N and Puc-57-S.
EXAMPLE 4 determination of authentic samples
1. The above system and primers were used to perform amplification detection on 5 authentic samples provided by great disease control, the 5 samples being confirmed by PCR as shown in FIG. 5c and containing the novel coronavirus (SARS-cov-2). Because of the limited supply of viral nucleic acid (30. mu.l) for great disease control, the final volume of the reaction using the above system, the amount of primers, was reduced from 200. mu.l to 10. mu.l for ease of verification. The detection results are shown in FIG. 5a, FIG. 5b, and FIG. 5 c.
2. Examination and verification of sample nucleic acid extraction and detection performance of instrument surface
Respectively taking 200 mu l of plasmid bacterial liquid with original concentration and containing the S gene of the novel coronavirus and 200 mu 1 of plasmid bacterial liquid with original concentration and containing the N gene of the novel coronavirus, and evenly dividing the plasmid bacterial liquid into sterile metal tablespoons for natural air drying. So as to simulate the attachment of virus to devices such as door handles in natural environment. The nucleic acid extraction was then performed according to the extraction method provided in example 1. LAMP primers for detecting S genes and N genes of the novel coronavirus are respectively used, the visual constant-temperature amplification detection method is used for amplification detection, and the result is shown in figure 6. (from left to right, the positive control of the S gene, the positive control of the N gene, the two repetitions of the S gene plasmid bacterial liquid extracted from the surface of the metal spoon, the two repetitions of the N gene plasmid bacterial liquid extracted from the surface of the metal spoon, and the two repetitions of the nuclease-free water are performed in sequence)
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
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Claims (10)
1. A nucleic acid amplification reagent, comprising: bst polymerase, AMV reverse transcriptase, dye and buffer;
the dye is selected from: cresol red, neutral red, 3-nitrophenol, m-cresol purple, naphthol peptide or methylene blue;
the buffer comprises water and: KCl, MgSO4Tween 20 and dNTPs.
2. The nucleic acid amplification reagent according to claim 1, comprising water and the following concentrations:
3. a nucleic acid detection reagent combination comprising: a nucleic acid extraction reagent, the nucleic acid amplification reagent according to claim 1 or 2, and a LAMP amplification primer.
4. The reagent combination according to claim 3, wherein the nucleic acid extraction reagent comprises: sample treatment liquid, lysis solution, cleaning solution and eluent; wherein:
the sample treatment liquid: comprises water and 0.1mmol/L dithiothreitol, 0.2mmol/L sodium chloride, 10mmol/L potassium chloride, 1mmol/L disodium hydrogen phosphate and 10mmol/L potassium dihydrogen phosphate, and the pH value of the solution is 7.5 +/-0.2;
the lysis solution: including water and 1mmol/L Tris-HCl (pH 8.0), 0.5mmol/L EDTA (pH 8.0), 10% SDS, and 0.1mmol/L NaCl;
the cleaning solution: comprising water, 80 vol% absolute ethanol and 0.5mmol/L EDTA (pH 8.0);
the eluent is as follows: nuclease-free water.
5. A method for detecting a nucleic acid, which comprises extracting a nucleic acid from a sample, amplifying the nucleic acid with the nucleic acid amplification reagent according to claim 3 or 4 and a LAMP primer, and determining whether or not the target nucleic acid is contained based on the color of the amplification product.
6. Use of the nucleic acid amplification reagent according to claim 1 or 2, or the nucleic acid detection reagent combination according to claim 3 or 4, for the preparation of a novel coronavirus detection kit.
7. A novel coronavirus detection kit comprising the nucleic acid detection reagent combination according to claim 3 or 4; the LAMP amplification primer comprises:
amplification primers for the S gene:
as shown in SEQ ID NO: 1, the outer primer S-F3,
as shown in SEQ ID NO: 2, the outer primer S-B3,
as shown in SEQ ID NO: 3, an inner primer S-FIP,
as shown in SEQ ID NO: 4, an inner primer S-BIP shown in the specification,
as shown in SEQ ID NO: 5, a loop primer S-LF shown in the specification,
as shown in SEQ ID NO: 6, a loop primer S-LB shown in the specification,
amplification primers for the N gene:
as shown in SEQ ID NO: 7, the outer primer N-F3,
as shown in SEQ ID NO: 8, the outer primer N-B3 shown in the figure,
as shown in SEQ ID NO: 9 is shown as the inner primer N-FIP,
as shown in SEQ ID NO: 10, the inner primer N-BIP shown in the specification,
as shown in SEQ ID NO: 11, the loop primer N-LF shown in the figure,
as shown in SEQ ID NO: 12, and the loop primer N-LB shown in the specification.
8. A method for detecting a novel coronavirus for non-diagnostic purposes, comprising:
after the nucleic acid of the sample is extracted, a nucleic acid amplification reagent and an LAMP primer are used for amplification, and whether the novel coronavirus is contained or not is judged according to the color of an amplification product.
9. The method of claim 8,
the amplification condition is constant temperature of 60-70 ℃ for 50 min;
the system for amplification comprises: sample nucleic acid 100. mu.L, nucleic acid amplification reagent according to claim 1 or 2 75. mu.L, outer primers 0.3. mu.L each, inner primers 2.4 mmol/L9.6. mu.L each, loop primers 1 mmol/L1. mu.L each, and water to make up to 200. mu.L.
10. The method of claim 8, wherein the determining comprises:
the dye in the nucleic acid amplification reagent is cresol red, the amplification product is yellow, and the result is positive; the amplification product is not yellow, and the result is negative;
the dye in the nucleic acid amplification reagent is neutral red, the amplification product is purple red, and the result is positive; the amplification product is not purple red, and the result is negative;
dyes in the nucleic acid amplification reagent are cresol red and methylene blue, an amplification product is green, and the result is positive; the amplification product is not green, and the result is negative;
the dyes in the nucleic acid amplification reagent are neutral red and methylene blue, the amplification product is purple, and the result is positive; the amplification product was not purple and the result was negative.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111926112A (en) * | 2020-07-01 | 2020-11-13 | 上海速创诊断产品有限公司 | Primer composition, kit and method for detecting SARS-CoV-2 nucleic acid |
| CN111996238A (en) * | 2020-08-12 | 2020-11-27 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | New virus trace detection method using electrochemical luminescence scanning imaging system |
| CN112063758A (en) * | 2020-09-29 | 2020-12-11 | 吉派德生物医药科技(河北)有限公司 | LAMP primer group and kit for detecting SARS-CoV-2 and use method thereof |
| CN112301161A (en) * | 2020-08-18 | 2021-02-02 | 上海纳米技术及应用国家工程研究中心有限公司 | Primer group and kit for detecting novel coronavirus |
| CN112779355A (en) * | 2021-01-06 | 2021-05-11 | 常州市疾病预防控制中心 | Self-heating amplification detection device and preparation method and use method thereof |
| CN113493862A (en) * | 2021-05-10 | 2021-10-12 | 江西省科学院微生物研究所 | Visualized LAMP synchronous detection kit and detection method for SARS-CoV-2 |
| WO2023057799A1 (en) * | 2021-10-08 | 2023-04-13 | National Cheng Kung University | Methods and kits for detecting severe acute respiratory syndrome coronavirus 2 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018648A (en) * | 2015-08-03 | 2015-11-04 | 博奥生物集团有限公司 | Kit for detecting respiratory viruses and application thereof |
| CN106868220A (en) * | 2017-03-31 | 2017-06-20 | 中国科学院过程工程研究所 | A kind of LAMP primer group and kit for expanding MERS CoV |
-
2020
- 2020-04-07 CN CN202010267141.5A patent/CN111270020B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018648A (en) * | 2015-08-03 | 2015-11-04 | 博奥生物集团有限公司 | Kit for detecting respiratory viruses and application thereof |
| CN106868220A (en) * | 2017-03-31 | 2017-06-20 | 中国科学院过程工程研究所 | A kind of LAMP primer group and kit for expanding MERS CoV |
Non-Patent Citations (4)
| Title |
|---|
| RENFEI LU ET AL.: "Development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2", 《VIROLOGICA SINICA》 * |
| YAN DU ET AL.: "A sweet spot for molecular diagnostics:coupling isothermal amplification and strand exchange circuits to glucometers", 《SCI REP》 * |
| YINHUA ZHANG ET AL.: "Rapid molecular detection of SARS-CoV-2(COVID-19) virus RNA using colorimetric LAMP", 《MEDRXIV》 * |
| 叶迅等: "严重急性呼吸道综合征冠状病毒疫苗研究现状", 《生物化学与生物物理进展》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111926112A (en) * | 2020-07-01 | 2020-11-13 | 上海速创诊断产品有限公司 | Primer composition, kit and method for detecting SARS-CoV-2 nucleic acid |
| CN111996238A (en) * | 2020-08-12 | 2020-11-27 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | New virus trace detection method using electrochemical luminescence scanning imaging system |
| CN111996238B (en) * | 2020-08-12 | 2024-04-05 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | New virus trace detection method using electrochemiluminescence scanning imaging system |
| CN112301161A (en) * | 2020-08-18 | 2021-02-02 | 上海纳米技术及应用国家工程研究中心有限公司 | Primer group and kit for detecting novel coronavirus |
| CN112063758A (en) * | 2020-09-29 | 2020-12-11 | 吉派德生物医药科技(河北)有限公司 | LAMP primer group and kit for detecting SARS-CoV-2 and use method thereof |
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| WO2023057799A1 (en) * | 2021-10-08 | 2023-04-13 | National Cheng Kung University | Methods and kits for detecting severe acute respiratory syndrome coronavirus 2 |
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