CN117385001A - Method for concentrating and enriching pathogens in water body, product and application - Google Patents
Method for concentrating and enriching pathogens in water body, product and application Download PDFInfo
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Abstract
The invention discloses a method for concentrating and enriching pathogens in water, a product and application thereof. The method of the invention provides buffer A, buffer B, buffer C, buffer D and buffer E, and provides filtering parameters and centrifugation parameters. The method is simple to operate, the reagent is nontoxic and harmless, and the purity of the extracted pathogen nucleic acid is high. The nucleic acid extracted by the method can be directly used for real-time fluorescence quantitative PCR detection and other conventional molecular biological operations.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for concentrating and enriching pathogens in a water body, a product and application thereof.
Background
In the flow of the novel coronavirus covd-19, diagnosis and case management are performed by detecting SARS-CoV-2 in nasopharyngeal swabs in a traditional epidemiological manner in China, which takes a lot of manpower and material resources, and sampling the whole population in this manner is largely impractical, resulting in missing positive cases. In addition, traditional epidemiology cannot timely and comprehensively monitor the epidemic situation of other pathogens in the crowd, and usually monitors after symptoms appear, and outbreaks of some pathogens cannot be early warned.
Thus, epidemiology of wastewater provides another solution, most pathogens can be discharged through body fluid, urine or feces, such as new coronavirus, hepatitis A virus, norovirus, etc., which can generally survive in the wastewater for 3-7 days, and the outbreak of certain specific pathogens in the region can be monitored by collecting water, concentrating and extracting nucleic acid of the pathogens in the water, and detecting by using fluorescent quantitative PCR technology.
The detection of pathogens from water mainly requires concentration and extraction of nucleic acids of the pathogens, followed by fluorescent quantitative PCR or other means. The method for concentrating nucleic acid in water is mainly centrifugal ultrafiltration, aluminium salt coagulating sedimentation and polyethylene glycol sedimentation. The centrifugal ultrafiltration method is simple to operate, but has a high detection limit (100 copies/mL). The detection limit of the aluminum salt coagulating sedimentation method is low (10 copies/mL), but the operation is very complex and complicated, and a large amount of harmful substances can be released in the process of preparing solution reagents by using medicines (anhydrous aluminum chloride), and hydrochloric acid and sodium hydroxide solution used in the process of regulating the pH are corrosive and have great harm to human bodies. The polyethylene glycol precipitation method is convenient to operate, can be completed by only a centrifugal machine, has a low detection limit (10 copies/mL), can only complete 2 samples at a time by one centrifugal machine, and cannot complete detection of a large number of samples in time in a general laboratory, and has low efficiency. In addition, the concentrated solutions obtained by the methods also need to be subjected to a nucleic acid extraction step, so that the efficiency is relatively low and the cost is high.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the following technical scheme:
the invention provides a product for concentrating and enriching pathogens in a water body; the product comprises a buffer solution A, a buffer solution B, a buffer solution C, a buffer solution D and a buffer solution E; the buffer solution A contains sodium citrate, sodium chloride, disodium ethylenediamine tetraacetate and tris (hydroxymethyl) aminomethane, the buffer solution B contains ethanol, the buffer solution C contains sodium chloride, ethanol and tris (hydroxymethyl) aminomethane, the buffer solution D contains ethanol, disodium ethylenediamine tetraacetate and tris (hydroxymethyl) aminomethane, and the buffer solution E contains tris (hydroxymethyl) aminomethane and disodium ethylenediamine tetraacetate.
Further, the product comprises a kit, a nucleic acid membrane strip, chromatographic test paper and a system.
In certain specific embodiments, the product is a kit.
Further, the concentration range of each component in the buffer solution A is 1-5M sodium citrate, 0.1-0.5M sodium chloride, 0.5-5 mM disodium ethylenediamine tetraacetate, 0.1-1 mM tris-hydroxymethyl-aminomethane, the concentration range of ethanol in the buffer solution B is 50-80%, the concentration range of each component in the buffer solution C is 1-5M sodium chloride, 10-40% ethanol, 0.5-2.5M tris-hydroxymethyl-aminomethane, the concentration range of each component in the buffer solution D is 50-80% ethanol, 0.5-2.5M disodium ethylenediamine tetraacetate, 0.5-2.5M tris-hydroxymethyl-aminomethane, the concentration range of each component in the buffer solution E is 10-50 mM tris-hydroxymethyl-aminomethane, and 1-20 mM disodium ethylenediamine tetraacetate.
In certain specific embodiments, the concentration of component sodium citrate in buffer A ranges from 1M, 2M, 3M, 4M, 5M, the concentration of component sodium chloride in buffer A ranges from 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, the concentration of component disodium ethylenediamine tetraacetate in buffer A ranges from 0.5mM, 1.0mM, 1.5mM, 2.0mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM, and the concentration of component tris (hydroxymethyl) aminomethane in buffer A ranges from 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM.
In certain specific embodiments, the concentration of ethanol in buffer B ranges from 50%, 55%, 60%, 65%, 70%, 75%, 80%.
In certain specific embodiments, the concentration of component sodium chloride in buffer C ranges from 1M, 2M, 3M, 4M, 5M, the concentration of component ethanol in buffer C ranges from 10%, 15%, 20%, 25%, 30%, 35%, 40%, and the concentration of component tris in buffer C ranges from 0.5M, 1.0M, 1.5M, 2.0M, 2.5M.
In certain specific embodiments, the concentration of component ethanol in buffer D ranges from 50%, 55%, 60%, 65%, 70%, 75%, 80%, the concentration of component disodium edetate in buffer D ranges from 0.5M, 1.0M, 1.5M, 2.0M, 2.5M, and the concentration of component tris-hydroxymethyl-aminomethane in buffer D ranges from 0.5M, 1.0M, 1.5M, 2.0M, 2.5M.
In certain specific embodiments, the concentration of component tris (hydroxymethyl) aminomethane in buffer E ranges from 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, and the concentration of component disodium ethylenediamine tetraacetate in buffer E ranges from 1.0mM, 1.5mM, 2.0mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM, 5.5mM, 6.0mM, 6.5mM, 7.0mM, 7.5mM, 8.0mM, 8.5mM, 9.0mM, 9.5mM, 10.0mM, 10.5mM, 11.0mM, 11.5mM, 12.0mM, 12.5mM, 13.0mM, 13.5mM, 14.5mM, 15.0mM, 15.5mM, 16.0mM, 16.5mM, 17.0mM, 18.0mM, 19.5mM, and 19.0 mM.
Further, the concentration of each component in the buffer A was 2.0M sodium citrate, 0.4M sodium chloride, 5mM disodium edetate, 0.6mM tris, the concentration of ethanol in the buffer B was 75%, the concentration of each component in the buffer C was 5M sodium chloride, 20% ethanol, 1M tris, the concentration of each component in the buffer D was 80% ethanol, 0.5M disodium edetate, 1M tris, and the concentration of each component in the buffer E was 25mM tris, 2.5mM disodium edetate.
In certain specific embodiments, the buffer a can be used to disrupt the protein structure of a pathogen, allowing it to release nucleic acids and stabilize the nucleic acids against degradation by other substances in the body of water for a short period of time.
In certain specific embodiments, the buffer B is capable of settling the pathogen-released nucleic acid, and is capable of being adsorbed by a silica gel adsorption column.
In certain specific embodiments, the buffer C is capable of purifying nucleic acids and the high salt environment is capable of removing contaminating proteins.
In certain embodiments, the buffer D is capable of removing a high salt environment such that nucleic acids are not affected during PCR.
In certain specific embodiments, the buffer E is capable of eluting nucleic acids into a liquid for subsequent molecular biological detection.
The invention provides a method for concentrating and enriching pathogens in water, which comprises the following steps:
1) Taking an original water body sample, filtering, adding the buffer solution A, uniformly mixing and standing;
2) Adding the buffer solution B into the sample obtained in the step 1), and uniformly mixing;
3) The silica gel adsorption column is connected into a vacuum device, and the sample in the step 2) is added into the silica gel adsorption column to carry out vacuum pumping operation;
4) Adding the buffer solution C into the silica gel adsorption column in the step 3), and vacuumizing;
5) Adding the buffer solution D into the silica gel adsorption column in the step 4), and vacuumizing;
6) Adding the buffer solution D into the silica gel adsorption column in the step 5), and vacuumizing;
7) Adding the buffer solution E into the silica gel adsorption column in the step 6), standing, centrifuging and collecting filtrate.
Further, in the method, the volume ratio of the original water sample to the buffer solution A is 2:1, the volume ratio of the original water sample to the buffer solution B is 1:1, the volume ratio of the original water sample to the buffer solution C is 2:1, the volume ratio of the original water sample to the buffer solution D in the step 5) is 1:1, the volume ratio of the original water sample to the buffer solution D in the step 6) is 1:1, and the volume ratio of the original water sample to the buffer solution E is 200:1.
Further, the pathogen includes bacteria, viruses, chlamydia, rickettsia, mycoplasma, spirochetes, fungi, or parasites.
The term "virus" as used herein refers to a non-cellular organism that is tiny, simple in structure, contains only one nucleic acid (DNA or RNA), and must be parasitic and replicated in living cells. Viruses are not only classified into plant viruses, animal viruses and bacterial viruses. The structure is also divided into: single-stranded RNA viruses, double-stranded RNA viruses, single-stranded DNA viruses and double-stranded DNA viruses. The barlmo virus classification system classifies viruses into 7 classes: double-stranded DNA viruses; single-stranded DNA viruses; pseudoretroviruses (double-stranded DNA retroviruses); double-stranded RNA viruses; positive strand RNA viruses; negative strand RNA viruses; retrovirus (single-stranded RNA retrovirus). In certain specific embodiments, the pathogen is a novel coronavirus, hepatitis b virus, or norovirus.
Further, the pathogens include aquatic pathogens that survive in an aqueous environment for a long period of time, and the disinfection system is unable to completely eliminate, including salmonella, shigella, escherichia coli, vibrio cholerae, new coronavirus, hepatitis b virus, or norovirus.
In certain embodiments, the "bacteria" refers specifically to gram positive bacteria, gram negative bacteria, or acid fast organisms. Gram-positive bacteria can be identified as retaining crystal violet staining used in the gram staining method of bacterial identification and thus appear violet under the microscope. Gram-negative bacteria do not retain crystal violet, thereby making positive identification possible. In other words, the term 'bacterium' is herein applicable to bacteria having a thicker peptidoglycan layer in the cell wall outside the cell membrane (gram positive), and to bacteria having a thin peptidoglycan layer in the cell wall sandwiched between the inner cytoplasmic cell membrane and the outer bacterial membrane (gram negative).
Further, the water body comprises natural water body, artificial constructed water body and artificial polluted water body.
The present invention relates to concentration and enrichment of pathogens in a body of water, wherein the term "body of water" refers to any body of water, typically artificially constructed on land or floating structures, including ponds, lagoons, reservoirs, lakes, waterscapes, artificial ponds, artificial lakes, floating lagoons, etc., or naturally occurring polluted or non-polluted aqueous environments, including rivers, lakes, oceans, groundwater, etc., and the invention also includes water environments that are artificially constructed under various conditions, including the addition of special extracts to the body of water to be extracted.
Further, the standing time in the step 1) is 10min.
Further, the standing time in the step 7) is 5min, the centrifugal force is 8000Xg, the centrifugal time is 1min, and the centrifugal temperature is 25 ℃.
Further, filtration in step 1) uses a 0.7 μm PVDF filter.
The invention provides a device for concentrating and enriching pathogens of water, which comprises the method, wherein the method is automatically executed by a computer storage program.
Further, the apparatus includes:
operation 1: taking an original water body sample, filtering, adding the buffer solution A, uniformly mixing and standing;
operation 2: adding the buffer solution B into the sample obtained in the operation 1, and uniformly mixing;
operation 3: the silica gel adsorption column is connected into a vacuum device, and the sample in the operation 2 is added into the silica gel adsorption column to carry out vacuum pumping operation;
operation 4: adding the buffer solution C into the silica gel adsorption column in the operation 3, and vacuumizing;
operation 5: adding the buffer solution D into the silica gel adsorption column in the operation 4, and vacuumizing;
operation 6: adding the buffer solution D into the silica gel adsorption column in the operation 5, and vacuumizing;
operation 7: adding the buffer solution E into the silica gel adsorption column in the operation 6, standing, centrifuging and collecting filtrate.
Further, the apparatus includes a single or multiple processors, and a memory including a single or multiple computer programs that when executed by the single or multiple processors perform the operations of operations 1-7 described above.
The term "means" in the present invention is not limited to one or a specific number of physical objects. As used herein, a device may be any medical or electronic component having a plurality of components that may implement at least some portions of the present disclosure. Although the following description and examples use the term "device" to describe certain aspects of the disclosure, the term "device" is not limited to a particular configuration, type, or number of objects.
The term "processor" or "memory" in this disclosure includes computing devices having one processor or one memory as well as devices having multiple processors or multiple memories that may be used to perform some or all of the described steps. A "processor" may include more than one processor, for example, a multiple core design or multiple processors each having a multiple core design.
The invention provides application of a combination of sodium citrate, sodium chloride, disodium ethylenediamine tetraacetate and tris (hydroxymethyl) aminomethane, a combination of ethanol, sodium chloride, ethanol and tris (hydroxymethyl) aminomethane, a combination of ethanol, disodium ethylenediamine tetraacetate and tris (hydroxymethyl) aminomethane, and a combination of tris (hydroxymethyl) aminomethane and disodium ethylenediamine tetraacetate in preparing a product for concentrating and enriching pathogens in a water body.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Unless otherwise indicated, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"). Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. For the purposes of the description and appended claims, unless otherwise indicated, all numbers expressing quantities, amounts, percentages and so forth, are to be understood as being modified in all instances by the term "about". Moreover, all ranges include any combination of the disclosed maximum and minimum points, and include any intermediate ranges therein, which may or may not be specifically enumerated herein.
The invention has the beneficial effects that:
the reagent used in the method is nontoxic and harmless, has low cost and simple operation, can be used for preparing 24 samples at one time, and only needs 1h for obtaining the nucleic acid sample. The obtained concentrated solution is nucleic acid of pathogen, and can be directly subjected to real-time fluorescent quantitative PCR or other biological detection. The detection limit of the method is lower and can reach 5copies/mL.
Drawings
FIG. 1 is a graph of detection of a novel crown N target after concentration of a pathogen by a kit and method of the invention;
FIG. 2 is a graph of detection of a novel crown ORF1ab target after concentration of a pathogen using the kit and method of the invention;
FIG. 3 is a graph showing the detection of hepatitis B virus after concentration of pathogens using the kit and method of the present invention;
FIG. 4 is a graph of norovirus detection after concentration of pathogens using the kit and method of the present invention;
FIG. 5 is a graph of detection of a novel coronavirus N target after concentration of pathogens using the kit and method of the present invention;
FIG. 6 is a graph of detection of a novel coronavirus N target by an aluminum salt coagulation precipitation method.
Detailed Description
The invention provides a method for concentrating and extracting pathogens from a water body sample, and a person skilled in the art can properly improve the process parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein 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.
Example 1 preparation of buffer
Buffer a: the buffer solution A used in this case may be a mixed solution of 1-5M sodium citrate, 0.1-0.5M sodium chloride, 0.5-5 mM disodium ethylenediamine tetraacetate, and 0.1-1 mM tris (hydroxymethyl) aminomethane, and the concentration of the buffer solution A used in this case may be 2.0M sodium citrate, 0.4M sodium chloride, 5mM disodium ethylenediamine tetraacetate, or 0.6mM tris (hydroxymethyl) aminomethane.
Buffer B:75% ethanol.
Buffer C:5M sodium chloride, 20% ethanol, 1M tris.
Buffer D:80% ethanol, 0.5M disodium edetate, 1M tris (hydroxymethyl) aminomethane.
Buffer E:25mM tris (hydroxymethyl) aminomethane, 2.5mM disodium ethylenediamine tetraacetate.
Example 2 concentrated extraction of New coronavirus nucleic acid in Water sample
1. Experimental method
1) The new coronavirus is added into 10mL river water sample without the new coronavirus, the final concentration is 20copies/mL, 10copies/mL, 5copies/mL, and the mixture is uniformly mixed.
2) The method of the invention is used for nucleic acid concentration and extraction, and the extraction steps are as follows:
a) 10mL of water body sample is obtained into a centrifuge tube, a 0.7 mu m PVDF filter membrane is used and is connected into a syringe, and sewage is poured into the syringe for filtration. 5mL of buffer A was added to the centrifuge tube.
b) Adding 10mL of buffer solution B into the sewage, uniformly mixing, and precipitating nucleic acid;
c) And (3) connecting the silica gel adsorption column into a vacuum pump, pouring the sample into the silica gel adsorption column, and vacuumizing.
d) 5mL of buffer C was added to the silica gel adsorption column, and the silica gel adsorption column was evacuated.
e) 10mL of buffer D was added to the silica gel adsorption column, and the silica gel adsorption column was evacuated.
f) 10mL of buffer D was added to the silica gel adsorption column, and the silica gel adsorption column was evacuated.
g) 50. Mu.L of buffer E was added to a silica gel adsorption column, allowed to stand for 5 minutes, centrifuged at 8000Xg at 25℃for 1 minute, and the filtrate was collected and transferred to a 1.5mL centrifuge tube.
3) Using a novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) (Shanghai Berjie medical science and technology Co., ltd.) and using a real-time fluorescence quantitative PCR instrument (ABI)5 real-time fluorescent quantitative PCR instrument).
2. Experimental results
The experimental results are shown in table 1, fig. 1 and fig. 2, and the results show that the concentration and enrichment experiment of the novel coronavirus in the water sample can be performed by using the buffer solution A-E and the method for concentrating and enriching the water body, so that the positive of the novel coronavirus N target can be detected, the sensitivity can reach 5copies/mL, and the positive of the novel coronavirus ORF1ab target can be detected, and the sensitivity can reach 5copies/mL.
TABLE 1
| New crown concentration (copies/mL) | N target Ct mean | ORF1ab target Ct mean |
| 20 | 32.605 | 32.262 |
| 10 | 34.667 | 33.511 |
| 5 | 36.372 | 35.428 |
Example 3 concentrated extraction of hepatitis B Virus from Water samples
1. Experimental method
1) The hepatitis B pseudovirus is added into 10mL of groundwater sample without hepatitis B virus, the final concentration is 20copies/mL, 10copies/mL, 5copies/mL, and the mixture is mixed evenly.
2) The method of the invention is used for nucleic acid concentration and extraction, and the extraction steps are as follows:
a) 10mL of water body sample is obtained into a centrifuge tube, a 0.7 mu m PVDF filter membrane is used and is connected into a syringe, and sewage is poured into the syringe for filtration. 5mL of buffer A was added to the centrifuge tube.
b) Adding 10mL of buffer solution B into the sewage, uniformly mixing, and precipitating nucleic acid;
c) And (3) connecting the silica gel adsorption column into a vacuum pump, pouring the sample into the silica gel adsorption column, and vacuumizing.
d) 5mL of buffer C was added to the silica gel adsorption column, and the silica gel adsorption column was evacuated.
e) 10mL of buffer D was added to the silica gel adsorption column, and the silica gel adsorption column was evacuated.
f) 10mL of buffer D was added to the silica gel adsorption column, and the silica gel adsorption column was evacuated.
g) 50. Mu.L of buffer E was added to a silica gel adsorption column, allowed to stand for 5 minutes, centrifuged at 8000Xg at 25℃for 1 minute, and the filtrate was collected and transferred to a 1.5mL centrifuge tube.
3) Using a hepatitis B virus nucleic acid assay kit (fluorescent PCR method) (Shanghai's river Biotech Co., ltd.) and using a real-time fluorescent quantitative PCR instrument (ABI5 real-time fluorescent quantitative PCR instrument).
2. Experimental results
The experimental results are shown in Table 2 and FIG. 3, and the results show that the concentration and enrichment experiment of the hepatitis B virus in the water sample by using the buffer solutions A-E and the method for concentrating and enriching the water body can detect the positive of the hepatitis B virus, and the sensitivity can reach 5copies/mL.
TABLE 2
Example 4 concentrated extraction of norovirus from Water samples
1. Experimental method
1) The method is used for explaining the concentration and extraction experiment of the norovirus in the water body sample, and the collected sewage in different sewage plants is used for carrying out concentration and extraction detection on the norovirus.
2) The method of the invention is used for nucleic acid concentration and extraction, and the extraction steps are as follows:
a) And (3) uniformly mixing 10mL of sewage collected in a domestic sewage plant in an urban area into a centrifuge tube, numbering, using a 0.7 mu m PVDF filter membrane, accessing the filter membrane into a syringe, and pouring the sewage into the syringe for filtering. 5mL of buffer A was added to the centrifuge tube.
b) Adding 10mL of buffer solution B into the sewage, uniformly mixing, and precipitating nucleic acid;
c) And (3) connecting the silica gel adsorption column into a vacuum pump, pouring the sample into the silica gel adsorption column, and vacuumizing.
d) 5mL of buffer C was added to the silica gel adsorption column, and the silica gel adsorption column was evacuated.
e) 10mL of buffer D was added to the silica gel adsorption column, and the silica gel adsorption column was evacuated.
f) 10mL of buffer D was added to the silica gel adsorption column, and the silica gel adsorption column was evacuated.
g) 50. Mu.L of buffer E was added to a silica gel adsorption column, allowed to stand for 5 minutes, centrifuged at 8000Xg at 25℃for 1 minute, and the filtrate was collected and transferred to a 1.5mL centrifuge tube.
3) The detection was performed using a norovirus nucleic acid assay kit (fluorescent PCR method) (Shanghai 'S river Biotechnology Co., ltd.) and using a real-time fluorescent quantitative PCR instrument (Shanghai' S Marble SLAN96S real-time fluorescent quantitative PCR instrument).
2. Experimental results
The experimental results are shown in Table 3 and FIG. 4, and it is clear from the results that the norovirus positive can be detected by performing the concentration and enrichment experiment of the norovirus in the sewage by using the buffers A-E and the method for concentrating and enriching the water body.
TABLE 3 Table 3
| Sewage numbering | Ct mean |
| 1 | 29.63 |
| 2 | 33.68 |
| 3 | 33.70 |
| 4 | 41.19 |
| 5 | 34.32 |
| 6 | 31.69 |
| 7 | 29.97 |
| 8 | 34.42 |
Example 5 concentrating enrichment Capacity of aluminium salt coagulation precipitation method compared with the method of the present description
1. Experimental method
1) The novel coronavirus is added into 10mL of sewage without the novel coronavirus, and the final concentration is 50copies/mL, 20copies/mL, 10copies/mL and 5copies/mL, and the method is operated according to the operation steps by using the aluminum salt coagulation sedimentation method after being uniformly mixed.
2) 200 mu L of the concentrated solution obtained by the aluminum salt coagulating sedimentation method is added into a nucleic acid extraction kit, and the mixture is put into a nucleic acid extractor for extraction, and the final elution volume is about 50 mu L. The nucleic acid extraction reagent and the instrument used are GeneRotex 96 and matched reagents thereof.
3) Using a novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) (Shanghai Berjie medical science and technology Co., ltd.) and using a real-time fluorescence quantitative PCR instrument (ABI)5 real-time fluorescent quantitative PCR instrument).
2. Experimental results
As shown in Table 4, FIG. 5 and FIG. 6, the experimental results show that the experimental method and the kit used in the invention have lower Ct value compared with the Ct value obtained by the aluminum salt coagulating sedimentation method, the kit and the method can still detect positive when the concentration of the novel coronavirus is 5copies/mL, and the aluminum salt coagulating sedimentation method can not detect positive when the concentration of the novel coronavirus is 5copies/mL, which indicates that the concentration and enrichment effects of the kit and the method provided by the invention are better than those of the aluminum salt coagulating sedimentation method.
TABLE 4 Table 4
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Claims (10)
1. A product for concentrating and enriching pathogens in a body of water; the product comprises a buffer solution A, a buffer solution B, a buffer solution C, a buffer solution D and a buffer solution E; the buffer solution A contains sodium citrate, sodium chloride, disodium ethylenediamine tetraacetate and tris (hydroxymethyl) aminomethane, the buffer solution B contains ethanol, the buffer solution C contains sodium chloride, ethanol and tris (hydroxymethyl) aminomethane, the buffer solution D contains ethanol, disodium ethylenediamine tetraacetate and tris (hydroxymethyl) aminomethane, and the buffer solution E contains tris (hydroxymethyl) aminomethane and disodium ethylenediamine tetraacetate.
2. The product according to claim 1, wherein the concentration of each component in buffer a ranges from 1 to 5M sodium citrate, from 0.1 to 0.5M sodium chloride, from 0.5 to 5mM disodium edetate, from 0.1 to 1mM tris-hydroxymethyl-aminomethane, the concentration of ethanol in buffer B ranges from 50% to 80%, the concentration of each component in buffer C ranges from 1 to 5M sodium chloride, from 10% to 40% ethanol, from 0.5 to 2.5M tris-hydroxymethyl-aminomethane, the concentration of each component in buffer D ranges from 50% to 80% ethanol, from 0.5 to 2.5M disodium edetate, from 0.5 to 2.5M tris-hydroxymethyl-aminomethane, the concentration of each component in buffer E ranges from 10 to 50mM tris-hydroxymethyl-aminomethane, and from 1 to 20mM disodium edetate.
3. The product according to claim 2, wherein the concentration of each component in buffer a is 2.0M sodium citrate, 0.4M sodium chloride, 5mM disodium edetate, 0.6mM tris, wherein the concentration of ethanol in buffer B is 75%, wherein the concentration of each component in buffer C is 5M sodium chloride, 20% ethanol, 1M tris, wherein the concentration of each component in buffer D is 80% ethanol, 0.5M disodium edetate, 1M tris, and wherein the concentration of each component in buffer E is 25mM tris, 2.5mM disodium edetate.
4. A method of concentrating and enriching pathogens in a body of water, the method comprising the steps of:
1) Taking an original water body sample, filtering, adding the buffer solution A according to any one of claims 1-3, uniformly mixing and standing;
2) Adding the buffer solution B according to any one of claims 1-3 into the sample obtained in the step 1), and uniformly mixing;
3) The silica gel adsorption column is connected into a vacuum device, and the sample in the step 2) is added into the silica gel adsorption column to carry out vacuum pumping operation;
4) Adding the buffer solution C in any one of claims 1-3 into the silica gel adsorption column in the step 3), and vacuumizing;
5) Adding the buffer solution D as set forth in any one of claims 1-3 into the silica gel adsorption column in the step 4), and vacuumizing;
6) Adding the buffer solution D as claimed in any one of claims 1 to 3 into the silica gel adsorption column in the step 5), and vacuumizing;
7) Adding the buffer solution E according to any one of claims 1-3 into the silica gel adsorption column in the step 6), standing, centrifuging, and collecting filtrate.
5. The method of claim 4, wherein the volume ratio of the original water sample to buffer a in the method is 2:1, the volume ratio of the original water sample to buffer B is 1:1, the volume ratio of the original water sample to buffer C is 2:1, the volume ratio of the original water sample to buffer D in step 5) is 1:1, the volume ratio of the original water sample to buffer D in step 6) is 1:1, and the volume ratio of the original water sample to buffer E is 200:1.
6. The method of claim 4, wherein the pathogen comprises a bacterium, a virus, a chlamydia, a rickettsia, a mycoplasma, a spirochete, a fungus, or a parasite.
7. The method according to claim 4, wherein the standing time in the step 1) is 10min.
8. The method according to claim 4, wherein the standing time in the step 7) is 5min, the centrifugal force is 8000xg, the centrifugal time is 1min, and the centrifugal temperature is 25 ℃.
9. An apparatus for concentrating and enriching pathogens in a body of water, the apparatus comprising the method of any one of claims 4 to 8, the method being automatically performed by a computer stored program.
10. The use of a combination of sodium citrate, sodium chloride, disodium edetate, tris (hydroxymethyl) aminomethane, a combination of ethanol, sodium chloride, ethanol, tris (hydroxymethyl) aminomethane, a combination of ethanol, disodium edetate, tris (hydroxymethyl) aminomethane, and a combination of tris (hydroxymethyl) aminomethane, disodium edetate for the preparation of a product enriched in pathogens in a body of water.
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