CN113564282A - Visual virus detection method integrating nucleic acid extraction and LAMP amplification - Google Patents
Visual virus detection method integrating nucleic acid extraction and LAMP amplification Download PDFInfo
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Abstract
The invention discloses a visual virus detection method integrating nucleic acid extraction and LAMP amplification. The method comprises the following steps: releasing nucleic acid from a sample to be detected in a high-concentration guanidine salt solution and capturing the nucleic acid by silicon hydroxyl magnetic beads; adding a reagent for LAMP into the silicon hydroxyl magnetic beads which capture the virus nucleic acid to obtain an LAMP system; performing LAMP reaction, and judging whether the sample to be detected contains viruses or not according to the color change of the solution. The method has the following advantages: the nucleic acid adsorbed by the magnetic beads does not need to be eluted, the LAMP mixed solution can be directly added for amplification, and meanwhile, the detection result can be visually read only by heating at constant temperature for 30min by virtue of phenol red color development; the whole detection process is simple, convenient and quick to operate, and the nucleic acid extraction and result reading can be realized within 35 min; the detection sensitivity is high. The invention has important application value for detecting viruses, especially viruses with strong infectivity and/or potential danger.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for visually detecting viruses by integrating nucleic acid extraction and LAMP amplification.
Background
Early screening and detection of infectious disease viruses is important in dealing with outbreaks of infectious diseases. The earlier the early detection is performed, the more beneficial the control and treatment at the later stage. Therefore, it is important to establish a rapid, convenient, accurate and highly sensitive method for detecting viruses.
Viral nucleic acid detection is primarily involved in three main processes: (1) extracting and purifying nucleic acid; (2) amplifying nucleic acid; (3) and detecting an amplification product. For nucleic acid extraction and purification, the lysis solution is mainly used to destroy virus particles to release nucleic acid, and then the nucleic acid is adsorbed by silica gel or surface modified magnetic beads to purify the nucleic acid. However, nucleic acid extraction and purification often involve many steps and are time-consuming and labor-consuming. Therefore, it is necessary to develop a cheap, simple and fast method for extracting viral nucleic acid, which not only reduces the investment of manpower and material resources, but also is compatible with the subsequent nucleic acid amplification and detection.
Currently, the global gold standard for viral nucleic acid detection is real-time fluorescent quantitative polymerase chain amplification (RT-PCR), which is the first choice for viral detection due to its sensitive detection limit and mature application, but it also has certain limitations, such as the need of expensive instruments, professional technicians, high requirement for purity of viral nucleic acid to be detected, long detection time, high cost, etc. Compared with the temperature cycle amplification technology of PCR, the isothermal amplification technology is more convenient to operate, saves time and labor and is a good choice for quickly detecting viruses. Among the isothermal amplification technologies, the loop-mediated isothermal amplification technology (LAMP) has the advantages of strong specificity, high sensitivity, high amplification speed, convenient reading of detection results and strong tolerance to external interferents, so that the LAMP has great application potential in the aspect of virus nucleic acid detection.
Disclosure of Invention
The invention aims to detect viruses simply, quickly and sensitively.
The invention firstly protects a visual virus detection method integrating nucleic acid extraction and LAMP amplification, which sequentially comprises the following steps:
(1) releasing nucleic acid from a sample to be detected in a high-concentration guanidine salt solution and capturing the nucleic acid by silicon hydroxyl magnetic beads;
(2) adding a reagent for LAMP into the silicon hydroxyl magnetic beads which capture the virus nucleic acid to obtain an LAMP system;
the reagent for LAMP comprises a reaction solution for LAMP, an LAMP primer group for virus detection and an acid-base indicator;
(3) LAMP reaction was performed, after which the solution was observed for color change: if the color of the solution changes, the sample to be detected contains viruses, otherwise, the sample to be detected does not contain viruses.
The step (1) can comprise the following steps in sequence:
(1-1) uniformly mixing the lysis solution and the silicon hydroxyl magnetic beads, wherein the volume mass ratio of the lysis solution to the silicon hydroxyl magnetic beads is 1ml, namely (0.8-1.2) mg (such as 1ml, 0.8-1.0) mg, 1ml, 1.0-1.2) mg, 1ml, 0.8mg, 1ml, 1.0mg or 1ml, 1.2 mg);
(1-2) adding a sample to be detected with the same volume as the lysate, uniformly mixing, and standing for more than 2min (such as 2min, 3min and 4 min);
(1-3) capturing magnetic beads by magnetic separation;
(1-4) washing the magnetic beads with an ethanol solution, followed by drying.
In the step (1-1), the lysis solution and the silicon hydroxyl magnetic beads are uniformly mixed, and specifically, 500. mu.L of lysis solution and 50. mu.L of silicon hydroxyl magnetic bead solution with the concentration of 25mg/mL are mixed.
The lysate may have a solute and its concentration of 4-6M (e.g., 4-5M, 5-6M, 4M, 5M, or 6M) guanidine hydrochloride, 1-3M (e.g., 1-2M, 2-3M, 1M, 2M, or 3M) NaCl, 2.0-3.0mM (e.g., 2.0-2.5mM, 2.5-3.0mM, 2.0mM, 2.5mM, or 3.0mM) EDTA, and 18-22mM (e.g., 18-20mM, 20-22mM, 18mM, 20mM, or 22mM) Tris, a solvent may be ultrapure water, and a pH of 4.8-5.2 (e.g., 4.8-5.0, 5.0-5.2, 4.8, 5.0, or 5.2).
In the step (2), the reagent for LAMP may specifically comprise a reaction solution for LAMP, an LAMP primer group for virus detection, and an acid-base indicator.
The acid-base indicator may be a phenol red solution.
When the acid-base indicator is phenol red solution, if the color of the solution in the step (3) is changed into yellow, the sample to be detected contains viruses; if the solution color in the step (3) keeps red, the sample to be tested does not contain the virus.
The phenol red solution may be specifically a 0.4% (m/v) phenol red solution.
In the step (1-4), the ethanol solution may be an 80% (v/v) ethanol aqueous solution. The washing of the magnetic beads with the ethanol solution can be carried out by washing the magnetic beads with 500. mu.L of 80% (v/v) ethanol aqueous solution more than 1 time (e.g., 1 time, 2 times, 3 times). The drying may be room temperature drying.
In the step (3), the LAMP reaction is carried out with parameters of 63-67 ℃ (such as 63-65 ℃, 65-67 ℃, 63 ℃, 65 ℃ or 67 ℃) and heat preservation for 20-40min (such as 20-30min, 30-40min, 20min, 30min or 40 min).
In any of the above methods, the virus may be SARS-CoV-2 or shrimp virus.
Any of the above lysates also falls within the scope of the present invention.
The application of any lysate in the visual detection of the virus integrating nucleic acid extraction and LAMP amplification also belongs to the protection scope of the invention.
In the above application, the virus may be SARS-CoV-2 or shrimp virus.
Any of the above SARS-CoV-2 can be a SARS-CoV-2 pseudovirus.
Any of the shrimp viruses described above may be wenzho shrimp virus 8.
The visual virus detection method integrating nucleic acid extraction and LAMP amplification provided by the invention has the following advantages: 1) the silicon hydroxyl magnetic beads can realize nucleic acid enrichment on a large-volume sample within 3-5 min; 2) the nucleic acid adsorbed by the magnetic beads does not need to be eluted, the LAMP mixed solution can be directly added for amplification, and meanwhile, the detection result can be visually read only by heating at constant temperature for 30min by virtue of phenol red color development; 3) the whole detection process is simple, convenient and quick to operate, and the nucleic acid extraction and result reading can be realized within 35 min; 4) the method does not depend on professional instruments and equipment, has low requirements on detection environment and has reliable results; 5) the detection sensitivity is high (the sensitivity for detecting SARS-CoV-2 pseudovirus reaches 200 copies/ml, and the sensitivity for detecting Wenzhou shrimp virus8 reaches 250 copies/ml); 6) can avoid cross infection possibly caused by traditional detection and can be used for convenient or household virus detection. The method provided by the invention has important application value for detecting viruses, especially viruses with stronger infectivity and/or potential danger (such as SARS-CoV-2 and shrimp viruses).
Drawings
FIG. 1 is a flow chart of a visual virus detection method integrating nucleic acid extraction and LAMP amplification.
FIG. 2 shows the sensitivity of Wenzhou shrimp virus detection using the method established in example 1.
FIG. 3 shows the sensitivity of SARS-CoV-2 pseudovirus detection using the method established in example 1.
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.
Wenzhou shrimp viruses 8 (described in Shi, M., Lin, XD., Tian, JH.et al.refining the invertebrate RNA virosphere 540, 539-543 (2016). https:// doi.org/10.1038/nature 20167; NCBI Reference Sequence: NC-032852.1.) were obtained by the inventors from in vivo detection of shrimp seedlings using high throughput sequencing techniques.
SARS-CoV-2 pseudovirus was purchased from the national institute of metrology and research, accession number BIM-RM 5203.
A LAMP primer group for detecting Wenzhou shrimp virus8 is designed and synthesized according to the nucleotide sequence of Wenzhou shrimp virus8, and specifically consists of WF3, WB3, WFIP, WBIP, WLF and WLB 6 primers. The nucleotide sequence of each primer is as follows:
WF3：5’-CACTCTCGCATCATGACCAT-3’(SEQ ID NO:1)；
WB3：5’-TGGATGAGTCTCGCGATGA-3’(SEQ ID NO:2)；
WFIP：5’-CTTATGGGAACGGTCCCGCGTTTCAATCCTGAGTGGGCAA-3’(SEQ ID NO:3)；
WBIP：5’-AATTCGCTCTCTGCTCCTTCGGCGTAGTCGAGGGGCTTCT-3’(SEQ ID NO:4)；
WLF：5’-GCAGTCGGCGACGTACT-3’(SEQ ID NO:5)；
WLB：5’-AACGGCCAACGAGATAGTC-3’(SEQ ID NO:6)。
according to the nucleotide sequence of SARS-CoV-2 pseudovirus, the LAMP primer group for detecting SARS-CoV-2 pseudovirus is designed and synthesized, and is composed of SF3, SB3, SFIP, SBIP, SLF and SLB 6 primers. The nucleotide sequence of each primer is as follows:
SF3：5’-CGGTGGACAAATTGTCAC-3’(SEQ ID NO:7)；
SB3：5’-CTTCTCTGGATTTAACACACTT-3’(SEQ ID NO:8)；
SLF：5’-TTACAAGCTTAAAGAATGTCTGAACACT-3’(SEQ ID NO:9)；
SLB：5’-TTGAATTTAGGTGAAACATTTGTCACG-3’(SEQ ID NO:10)；
SFIP：5’-TCAGCACACAAAGCCAAAAATTTATCTGTGCAAAGGAAATTAAGGAG-3’(SEQ ID NO:11)；
SBIP：5’-TATTGGTGGAGCTAAACTTAAAGCCCTGTACAATCCCTTTGAGTG-3’(SEQ ID NO:12)。
the solute of the lysis solution and the concentration thereof are 5M guanidine hydrochloride, 2M NaCl, 2.5mM EDTA and 20mM Tris, the solvent is ultrapure water, and the pH value is 5.
Example 1 establishment of a method for visual detection of viruses integrating nucleic acid extraction and LAMP amplification
Through a large number of experiments, the inventor establishes a visual virus detection method integrating nucleic acid extraction and LAMP amplification. The method comprises the following specific steps:
firstly, releasing nucleic acid from virus in high-concentration guanidine salt solution and capturing the virus by using silicon hydroxyl magnetic beads
1. Mixing
An EP tube was used, and 200. mu.L of the lysate and 20. mu.L of a 25mg/mL solution of silica-based hydroxo magnetic beads (BioMag) were added thereto and mixed well.
2. Cracking
Add 200. mu.L of the sample to be tested, mix well, and let stand at room temperature for 3min (for lysis).
3. Capture
The magnetic beads in the solution were separated by magnetic separation.
4. Washing machine
Washing the magnetic beads for 2 times; each time, 500. mu.L of 80% (v/v) aqueous ethanol was used for washing.
5. Drying
The EP tube was opened with its lid open and the beads were dried at room temperature.
Second, LAMP reaction
Adding a reagent (comprising LAMP reaction liquid, an LAMP primer group for detecting viruses and an acid-base indicator) for LAMP into the silicon hydroxyl magnetic beads with the captured virus nucleic acids, preserving the heat at 65 ℃ for 30min, observing the color change of the solution, wherein if the color of the solution changes, the sample to be detected contains the viruses, otherwise, the sample to be detected does not contain the viruses.
The acid-base indicator may be a 0.4% (m/v) phenol red solution. When the acid-base indicator is 0.4% (m/v) phenol red solution, after the LAMP reaction is finished, if the solution color is changed into yellow, the sample to be detected contains viruses; if the solution color remains red, it indicates that the test sample does not contain virus.
The flow chart of the visual virus detection method integrating nucleic acid extraction and LAMP amplification is shown in figure 1.
Example 2 sensitivity of Wenzhou shrimp virus8 detection Using the method established in example 1
First, the sensitivity of Wenzhou shrimp virus8 was examined by the method established in example 1
1. Each of 18 ribozyme-free EP tubes (1.5 mL) was loaded with 200. mu.L of lysis buffer and 20. mu.L of 25mg/mL silica-based hydroxMag solution (BioMag).
2. Taking 18 EP tubes completing the step 1, and dividing the tubes into 8 groups, wherein the 1 st to 7 th groups comprise 2 tubes and the 8 th group comprises 4 tubes. The following operations are carried out:
group 1: 200. mu.L of virus solution (containing Wenzhou shrimp virus 8. about.10) was added to each tube7Number of copies);
group 2: 200. mu.L of virus solution (containing Wenzhou shrimp virus 8. about.10) was added to each tube6Number of copies);
group 3: 200. mu.L of virus solution (containing Wenzhou shrimp virus 8. about.10) was added to each tube5Number of copies);
group 4: 200. mu.L of virus solution (containing Wenzhou shrimp virus 8. about.10) was added to each tube4Number of copies);
group 5: 200. mu.L of virus solution (containing Wenzhou shrimp virus 8. about.10) was added to each tube3Number of copies);
group 6: 200. mu.L of virus solution (containing Wenzhou shrimp virus 8. about.10) was added to each tube2Number of copies);
group 7: 200. mu.L of virus solution (containing Wenzhou shrimp virus 8. about.10) was added to each tube1Number of copies);
group 8 (negative control): adding 200 mul of deionized water into each tube;
then, the upper covers of all the EP tubes are tightly covered, the solution is evenly mixed by left and right shaking, and the mixture is kept stand for 3min at room temperature.
3. And (3) after the step 2 is finished, respectively separating the magnetic beads in the solution by magnetic separation, and simultaneously discarding the supernatant.
4. After the step 3 is completed, washing the magnetic beads for 2 times respectively; each time, 500. mu.L of 80% (v/v) aqueous ethanol was used for washing.
5. After completion of step 4, the top lid of the EP tube was opened and the beads were dried at room temperature.
6. After completion of step 5, 25. mu.L of NEB WarmStart colorimetric LAMP2 × Master Mix, 5. mu.L of Primer Mix (the Primer Mix is a mixture of WF3, WB3, WFIP, WBIP, WLF, and WLB, where WF3 and WB3 are present at 2. mu.M in the Primer Mix, WFIP and WBIP are present at 16. mu.M in the Primer Mix, and WLF and WLB are present at 4. mu.M in the Primer Mix), 1. mu.L of 0.4% (M/v) phenol red solution, and 19. mu.L of ribozyme-free water were added to the EP tubes, respectively. Finally, 100. mu.L of mineral oil was added and the lid was closed.
7. After step 6 was completed, the EP tubes were each placed at 65 ℃ for 30min, the solution color change was observed, and the following determinations were made:
if the solution color turns yellow, the corresponding copy number in the reaction system can be detected;
if the solution color remains red, it indicates that the corresponding copy number in the reaction system cannot be detected.
The results are shown in A in FIG. 2 (NTC is negative control, the remaining numbers are copy number of virus): the color of the solution in the EP tubes from the 1 st group to the 5 th group turns yellow, and the color of the solution in the EP tubes from the 6 th group to the 8 th group remains red. The results show that the sensitivity of Wenzhou shrimp virus8 detection by the method established in example 1 reaches 103Number of copies/ml.
Second, the sensitivity of Wenzhou shrimp virus8 was optimized after the amount of sample and lysate added for the method set up in example 1
1. Each of 16 ribozyme-free EP tubes (1.5 mL) was loaded with 500. mu.L of lysis buffer and 20. mu.L of 25mg/mL silica-based hydroxMag solution (BioMag).
2. The 16 EP tubes that completed step 1 were taken and divided into 4 groups of 4 tubes each. The following operations are carried out:
group 1: add 500. mu.L of virus solution (containing Wenzhou shrimp virus 8. about.5X 10) to each tube2Number of copies);
group 2: mu.L of virus solution (containing Wenzhou shrimp virus 8. about.2.5X 10) was added to each tube2Number of copies);
group 3: add 500. mu.L of virus solution (containing Wenzhou shrimp virus 8. about.1.2X 10) to each tube2Number of copies);
group 4 (negative control): adding 500 mu L of deionized water into each tube;
then, the upper covers of all the EP tubes are tightly covered, the solution is evenly mixed by left and right shaking, and the mixture is kept stand for 3min at room temperature.
3. And (3) after the step 2 is finished, respectively separating the magnetic beads in the solution by magnetic separation, and simultaneously discarding the supernatant.
4. After the step 3 is completed, washing the magnetic beads for 2 times respectively; each time, 500. mu.L of 80% (v/v) aqueous ethanol was used for washing.
5. After completion of step 4, the top lid of the EP tube was opened and the beads were dried at room temperature.
6. After completion of step 5, 25. mu.L of NEB WarmStart colorimetric LAMP2 × Master Mix, 5. mu.L of Primer Mix (the Primer Mix is a mixture of WF3, WB3, WFIP, WBIP, WLF, and WLB, where WF3 and WB3 are present at 2. mu.M in the Primer Mix, WFIP and WBIP are present at 16. mu.M in the Primer Mix, and WLF and WLB are present at 4. mu.M in the Primer Mix), 1. mu.L of 0.4% (M/v) phenol red solution, and 19. mu.L of ribozyme-free water were added to the EP tubes, respectively. Finally 50. mu.L of mineral oil was added and the lid was closed.
7. After step 6 was completed, the EP tubes were each placed at 65 ℃ for 30min, the solution color change was observed, and the following determinations were made:
if the solution color turns yellow, the corresponding copy number in the reaction system can be detected;
if the solution color remains red, it indicates that the corresponding copy number in the reaction system cannot be detected.
The results are shown in B in FIG. 2 (NTC is negative control, the remaining numbers are the copy number of the virus): the color of the solution in the EP tubes of both group 1 and group 2 turned yellow, the color of the solution in 3 EP tubes of 4 EP tubes of group 3 turned yellow, the color of the solution in 1 EP tube remained red, and the color of the solution in the EP tubes of group 4 remained red. The results show that when Wenzhou shrimp virus8 was detected using the method established in example 1, the volume of the sample was increased from 200. mu.L to 500. mu.L and the volume of the lysate was increased from 200. mu.L to 500. mu.L, the sensitivity of the detection reached 250 copies/ml.
Accordingly, the sample volume and the lysate volume in the method established in example 1 were each optimized to be 500. mu.L from 200. mu.L.
Example 3 sensitivity of detection of SARS-CoV-2 pseudovirus by the optimized method
1. Each of 12 ribozyme-free EP tubes (1.5 mL) was loaded with 500. mu.L of lysis buffer and 20. mu.L of 25mg/mL silica-based hydroxMag solution (BioMag).
2. The 12 EP tubes that completed step 1 were taken and divided into 6 groups of 2 tubes each. The following operations are carried out:
group 1: add 500. mu.L of virus solution (containing about 1600 copies of SARS-CoV-2 pseudovirus) per tube;
group 2: add 500. mu.L of virus solution (containing SARS-CoV-2 pseudovirus about 800 copies) per tube;
group 3: add 500. mu.L of virus solution (containing SARS-CoV-2 pseudovirus about 400 copies) per tube;
group 4: add 500. mu.L of virus solution (containing SARS-CoV-2 pseudovirus about 200 copies) per tube;
group 5: add 500. mu.L of virus solution (containing SARS-CoV-2 pseudovirus about 100 copies) per tube;
group 6 (negative control): adding 500 mu L of deionized water into each tube;
then, the upper covers of all the EP tubes are tightly covered, the solution is evenly mixed by left and right shaking, and the mixture is kept stand for 3min at room temperature.
3. And (3) after the step 2 is finished, respectively separating the magnetic beads in the solution by magnetic separation, and simultaneously discarding the supernatant.
4. After the step 3 is completed, washing the magnetic beads for 2 times respectively; each time, 500. mu.L of 80% (v/v) aqueous ethanol was used for washing.
5. After completion of step 4, the top lid of the EP tube was opened and the beads were dried at room temperature.
6. After completion of step 5, 25. mu.L of NEB WarmStart colorimetric LAMP2 × Master Mix, 5. mu.L of Primer Mix (Primer Mix made by mixing SF3, SB3, SFIP, SBIP, SLF, and SLB, where SF3 and SB3 are 2. mu.M in Primer Mix, SFIP and SBIP are 16. mu.M in Primer Mix, and SLF and SLB are 4. mu.M in Primer Mix), 1. mu.L of 0.4% (M/v) phenol red solution, and 19. mu.L of ribozyme-free water were added to the EP tube, respectively. Finally 50. mu.L of mineral oil was added and the lid was closed.
7. After step 6 was completed, the EP tubes were each placed at 65 ℃ for 30min, the solution color change was observed, and the following determinations were made:
if the solution color turns yellow, the corresponding copy number in the reaction system can be detected;
if the solution color remains red, it indicates that the corresponding copy number in the reaction system cannot be detected.
The results are shown in FIG. 3(NTC is negative control, the remaining numbers are the copy number of the virus): the color of the solution in the EP tubes of the 1 st group and the 4 th group is changed into yellow, the color of the solution in the 1 st EP tube of the 5 th group is changed into yellow, the color of the solution in the 1 st EP tube is kept red, and the color of the solution in the EP tube of the 6 th group is kept red. The result shows that the sensitivity of detecting SARS-CoV-2 pseudovirus after the method established in example 1 is optimized reaches 200 copies/ml.
The results show that the optimized virus visualized detection method integrating nucleic acid extraction and LAMP amplification is adopted to detect SARS-CoV-2 pseudovirus and shrimp virus respectively, and the method has the advantages of simple operation, short time consumption and high detection sensitivity. The method provided by the invention has important application value for virus detection.
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.
<110> Beijing university of chemical industry
<120> visual virus detection method integrating nucleic acid extraction and LAMP amplification
<160> 12
<170> PatentIn version 3.5
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Claims (10)
1. A visual virus detection method integrating nucleic acid extraction and LAMP amplification sequentially comprises the following steps:
(1) releasing nucleic acid from a sample to be detected in a high-concentration guanidine salt solution and capturing the nucleic acid by silicon hydroxyl magnetic beads;
(2) adding a reagent for LAMP into the silicon hydroxyl magnetic beads which capture the virus nucleic acid to obtain an LAMP system;
the reagent for LAMP comprises a reaction solution for LAMP, an LAMP primer group for virus detection and an acid-base indicator;
(3) LAMP reaction was performed, after which the solution was observed for color change: if the color of the solution changes, the sample to be detected contains viruses, otherwise, the sample to be detected does not contain viruses.
2. The method of claim 1, wherein: the step (1) comprises the following steps in sequence:
(1-1) uniformly mixing lysis solution and silicon hydroxyl magnetic beads, wherein the volume mass ratio of the lysis solution to the silicon hydroxyl magnetic beads is 1ml (0.8-1.2) mg;
(1-2) adding a sample to be detected with the same volume as the lysate, uniformly mixing, and standing for more than 2 min;
(1-3) capturing magnetic beads by magnetic separation;
(1-4) washing the magnetic beads with an ethanol solution, followed by drying.
3. The method according to claim 1 or 2, characterized in that: the solute and its concentration of the lysis solution are 4-6M guanidine hydrochloride, 1-3M NaCl, 2.0-3.0M EDTA and 18-22M Tris, the solvent is ultrapure water, and the pH value is 4.8-5.2.
4. The method of claim 1, wherein: in the step (2), the acid-base indicator is phenol red solution.
5. The method of claim 4, wherein: when the acid-base indicator is phenol red solution, if the color of the solution in the step (3) is changed into yellow, the sample to be detected contains viruses; if the solution color in the step (3) keeps red, the sample to be tested does not contain the virus.
6. The method of claim 1, wherein: in the step (3), the parameters for LAMP reaction are that the temperature is kept for 20-40min at 63-67 ℃.
7. The method of claim 1, wherein: the virus is SARS-CoV-2 or shrimp virus.
8. A lysate as claimed in claim 3.
9. The use of the lysate of claim 8 in the visual detection of viruses that integrates nucleic acid extraction and LAMP amplification.
10. Use according to claim 9, characterized in that: the virus is SARS-CoV-2 or shrimp virus.
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| CN112063697B (en) * | 2020-10-13 | 2023-04-28 | 北京化工大学 | Pathogen detection method based on one-step extraction of nucleic acid by combining LAMP in-situ amplification of glass material |
| CN113151397B (en) * | 2021-02-03 | 2023-06-23 | 广东粤港澳大湾区国家纳米科技创新研究院 | Nucleic acid extraction kit for extracting virus sample based on magnetic bead method |
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| CN114058613B (en) * | 2021-11-18 | 2023-10-27 | 广州血液中心(中国医学科学院输血研究所广州分所、广州器官移植配型中心) | Large-volume high-sensitivity nucleic acid extraction method |
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