CN111792712A - Method for inactivating enterovirus in water and application - Google Patents
Method for inactivating enterovirus in water and application Download PDFInfo
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- CN111792712A CN111792712A CN202010799475.7A CN202010799475A CN111792712A CN 111792712 A CN111792712 A CN 111792712A CN 202010799475 A CN202010799475 A CN 202010799475A CN 111792712 A CN111792712 A CN 111792712A
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/50—Treatment of water, waste water, or sewage by addition or application of a germicide or by oligodynamic treatment
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/002—Grey water, e.g. from clothes washers, showers or dishwashers
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/04—Disinfection
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- Life Sciences & Earth Sciences (AREA)
- Hydrology & Water Resources (AREA)
- Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a method for inactivating enterovirus in water and application thereof, belonging to the technical field of water purification treatment; the method comprises the following steps: adding 5-10 mg/L of peroxyacetic acid into water to be treated, and inactivating the enterovirus. The peracetic acid adopted by the invention can release nascent oxygen, and the nascent oxygen destroys virus genes through redox reaction and enables protein of the virus to be coagulated and denatured, thereby killing the virus. The method of the invention can overcome the problems of carcinogenesis, teratogenesis, mutagenesis and the like caused by inactivating viruses by using a chlorine-containing disinfectant, and can effectively kill the enterovirus in water.
Description
Technical Field
The invention relates to the technical field of water purification treatment, in particular to a method for inactivating enterovirus in water and application thereof.
Background
Along with the development of society, the urban sewage recycling technology is gradually popularized, and meanwhile, the safety problem of recycled water gradually draws attention of people. The urban sewage usually contains pathogenic microorganisms with strong pathogenicity and easy infection, and if the pathogenic microorganisms are not completely inactivated in the sewage disinfection process, the reclaimed water recycled as the urban sewage brings huge risks and threats to public health. In urban sewage, enteroviruses are widely present. At present, chlorine-containing disinfectants are mostly adopted in the traditional method for killing the enteronitis viruses in the regenerated water, and have the defects of carcinogenesis, teratogenesis, mutagenesis and the like.
Disclosure of Invention
The invention aims to provide a method for inactivating enterovirus in water and application thereof, and the method can overcome the problems of carcinogenesis, teratogenesis, mutation and the like caused by inactivating virus by using a chlorine-containing disinfectant and can effectively kill the enterovirus in water.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for inactivating enterovirus in water, which comprises the following steps:
after the pH value of the water to be treated is adjusted to 4.5-8, 5-10 mg/L peroxyacetic acid is added into the water to be treated, and the enterovirus inactivation is carried out.
Preferably, the aquatic enterovirus comprises rotavirus and/or norovirus.
Preferably, the inactivation time is 1-35 min.
Preferably, the inactivation is accompanied by stirring; the rotating speed of the stirring is 200-300 rpm.
Preferably, the method further comprises the following steps after the enterovirus is inactivated: adjusting the pH value of the inactivated water body to 4.5-8, and adding a sodium thiosulfate-catalase solution to quench the inactivation reaction.
Preferably, the sodium thiosulfate-catalase solution takes ultrapure water as a solvent, and comprises catalase with the concentration of 1.5-1.8 mg/50mL and sodium thiosulfate with the concentration of 25-35 mg/50 mL.
Preferably, the volume ratio of the inactivated water body of the sodium thiosulfate-catalase solution is 1: 1.
preferably, the water to be treated comprises municipal sewage.
The invention also provides application of the method in sewage treatment or water purification.
The invention has the beneficial effects that: the invention provides a method for inactivating enterovirus in water, which comprises the following steps: after the pH value of the water to be treated is adjusted to 4.5-8, 5-10 mg/L peroxyacetic acid is added into the water to be treated, and the enterovirus inactivation is carried out. In the present invention, peracetic acid can release nascent oxygen, which in turn destroys viral genes by redox reactions and denatures viral proteins by coagulation. The peroxyacetic acid is decomposed into acetic acid and oxygen, no disinfection by-products are generated, the water quality safety is guaranteed, and the harm to the human health can be reduced. Compared with the traditional chlorine disinfectant, the peracetic acid has less odor, and reduces the stimulation and the uncomfortable feeling to the human body; compared with an ozone disinfectant, the preparation is simple and easy to obtain; the decomposition rate of the peracetic acid is lower than 1% in each day under the condition of being less than or equal to 40 ℃, and the peracetic acid can play a role in a longer time and has continuous sterilization capability compared with an ultraviolet disinfection method.
In addition, the method for inactivating the enterovirus by the peroxyacetic acid supplements the systematic research of the domestic peroxyacetic acid on the enterovirus disinfection, and has important significance for debugging the dosage of the water disinfectant and the water environment condition in urban recycling. The method is economical and practical, and the existing process is not required to be added or upgraded and modified. Does not need a large amount of medicament, is easy to operate in the preparation of peroxyacetic acid, and has no complex process of the traditional ozone and chlorine dioxide preparation. Therefore, the method provided by the invention is simple to operate, and is economical and practical.
Drawings
FIG. 1 shows the inactivation of porcine rotavirus by peracetic acid at pH 7.8 (PAA: 5mg/L) in example 1 of the present invention;
FIG. 2 shows the inactivation of Dulan virus by peracetic acid at pH 7.8 (PAA: 10mg/L) in example 2 of the present invention;
FIG. 3 shows the inactivation of porcine rotavirus by peracetic acid at pH 5.4 (PAA: 10mg/L) in example 3 of the present invention;
FIG. 4 shows the inactivation of Dulan virus by peracetic acid under the condition of pH 4.5 (PAA: 10mg/L) in example 4 of the present invention.
Detailed Description
The invention provides a method for inactivating enterovirus in water, which comprises the following steps: after the pH value of water to be treated is adjusted to 4.5-8, 5-10 mg/L of peracetic acid (PAA) is added into the water to be treated, and enterovirus inactivation is carried out; the pH value of the water to be treated is preferably adjusted to 7.8; the agent for adjusting the pH of the water to be treated is preferably a phosphate buffer solution.
In the invention, the concentration of 5-10 mg/L can achieve good disinfection effect under the condition of controlling the amount of PAA; the water to be treated preferably comprises municipal sewage; co in the water to be treated2+Concentration of (2) is less than or equal to 10-6mol/L; the enterovirus preferably comprises rotavirus and/or norovirus; the rotavirus preferably comprises a porcine rotavirus.
In the invention, the addition amount of the peroxyacetic acid is preferably 8 mg/L; preferably, the peroxyacetic acid is dissolved in a phosphate buffer solution to obtain a peroxyacetic acid phosphate buffer solution, and then the peroxyacetic acid phosphate buffer solution is added into water; the pH value of the phosphate buffer solution is preferably 5, 7 or 8; the concentration of the phosphate buffer solution is preferably 10 mM; the concentration of the peroxyacetic acid phosphate buffer solution is preferably 20 mg/L; in the specific implementation process of the invention, the concentration of the peroxyacetic acid is detected by a K-7913 peroxyacetic acid kit of Vacu-vitamins company, and the concentration range detected by the kit is 0-5 mg/L.
In the invention, the inactivation time is preferably 1-35 min, more preferably 5-30 min, and most preferably 10-20 min; preferably with agitation during said inactivation; the rotation speed of the stirring is preferably 200-300 rpm, and more preferably 250 rpm.
In the present invention, peroxyacetic acid causes the destruction of genetic material by destroying the protein coat of rotaviruses and dualan viruses, resulting in the entry of peroxyacetic acid into the virus, thereby rendering the virus non-infectious.
After the enterovirus is inactivated, the method preferably further comprises the steps of adjusting the pH value of the inactivated solution to 4.5-8, and adding a sodium thiosulfate-catalase solution to quench the inactivation reaction. In the invention, the pH value after adjustment is preferably 6-7; the sodium thiosulfate-catalase solution preferably takes ultrapure water as a solvent, and comprises catalase with the concentration of 1.5-1.8 mg/50mL and sodium thiosulfate with the concentration of 25-35 mg/50 mL; the concentration of the sodium thiosulfate is preferably 30mg/50 mL. In the present invention, the volume ratio of the inactivated water body of the sodium thiosulfate-catalase solution is preferably 1: 1. in the present invention, sodium thiosulfate quenches peracetic acid to produce acetic acid, and catalase is used to quench a small amount of hydrogen peroxide contained in peracetic acid.
After quenching and inactivating reaction, the invention also comprises diluting the reaction solution after quenching and inactivating reaction until the concentration of the virus in water is 101～102pfu/mL to obtain a diluent, and performing a virus plaque counting experiment on the diluent; the specific process of the virus plaque counting experiment is not particularly limited, and the conventional virus plaque counting experiment process in the field is taken as a standard; rotavirus was counted after 3 days of culture and norovirus was counted after 2 days of culture.
In the present invention, rotavirus or norovirus needs to be counted in time. If the virus plaque counting experiment cannot be carried out in time after sampling, the virus plaque counting experiment is preferably placed in a refrigerator at the temperature of minus 80 ℃ for storage to be tested.
The invention also provides application of the method in sewage treatment or water purification.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1) Solution preparation
Firstly, the handle is provided with a handle 105The pH of the water to be treated of PFU/mL porcine rotavirus (ATCC-VR-892) is adjusted to 7.8, and the water with the adjusted pH is placed on a magnetic stirrer and stirred at a speed of 200-300 rpm.
2) Disinfection process
Preparing peroxyacetic acid solution with the concentration of 20mg/L by using phosphate buffer solution with the pH value of 8. Then adding the prepared peroxyacetic acid solution into the water obtained in the step 1), wherein the ratio of the prepared peroxyacetic acid solution is as follows: 1, water to be treated: 3, so that the initial concentration of peroxyacetic acid reached 5 mg/L. After 5min and 10min of reaction, samples were taken, and 0.5mL of sodium thiosulfate-Catalase solution (1.6 mg of Catalase (Catalase) and 30mg of sodium thiosulfate were weighed, dissolved in 50mL of ultrapure water, and added with 0.5mL of quencher in a 1.7mL sterile centrifuge tube before inactivation test) was added to quench and stop the reaction. And (3) performing a virus plaque counting experiment after diluting the sample, and counting the experiment result after 3 days.
The specific flow of the virus plaque counting experiment is as follows:
(1) preparation work:
a) inoculating the virus suspension in the cell bottle into a sterile 6-hole cell culture plate, wherein each hole is 2mL, and culturing for 24h to ensure that the cell coverage rate reaches 95% to form a monolayer cell membrane;
b) diluting a virus sample to be detected to a proper concentration by using a 10-fold gradient of MEM culture solution without FBS;
c) semi-solid nutritional cover solutions were prepared according to table 1.
TABLE 1 semi-solid nutritional cover solutions
(2) The plaque counting method comprises the following experimental steps:
a) washing a monolayer of cell membranes in 6-well cell plates 1 time using MEM without FBS;
b) slowly adding 450 mu L of diluted virus sample, and gently shaking to uniformly distribute the virus sample on cell membranes;
c) the 6-well cell plate added with the virus sample is placed at 37 ℃ and 5% CO2The cell plate is gently shaken every 10min during the culture for 1h so that the virus can fully and uniformly contact the cell membrane;
d) adding 2.5mL of the semi-solid nutrient covering liquid, and placing in a refrigerator at 4 ℃ for 20min to cool and solidify as soon as possible;
e) after the covering liquid is solidified, 5 percent CO is added at 37 DEG C2Culturing for 3d in a constant-temperature incubator;
f) adding 2mL of 10% formaldehyde fixing solution into the cultured cell plate to fix the cells, and standing at 25 ℃ for 3 h;
g) pouring off the agar covering liquid, and adding 4-5 drops of 0.05% crystal violet dye liquid into the holes to dye for 20 min;
h) excess crystal violet in 6-well cell plates was washed with deionized water and counted:
the virus concentration is the number of counts × dilution factor.
The results are shown in FIG. 1. The result shows that when the Ct value reaches 18 mg-min/L, the inactivation logarithm of the peroxyacetic acid to the porcine rotavirus is 0.32, and the corresponding inactivation rate is 52.1 percent; when the Ct value reaches 39 mg-min/L, the inactivation logarithm is 1.53, and the corresponding inactivation rate is 97.0%.
Example 2
1) Solution preparation
Firstly, the handle is provided with a handle 105PFU/mL Dulan virus (ATCC-VR-1027, Dulan virus and norovirus structure similar, so with Dulan virus to show the norovirus inactivation performance) to treat water pH to 7.8, the adjusted water pH on a magnetic stirrer, in 200 ~ 300rpm speed stirring.
2) Disinfection process
Preparing peroxyacetic acid solution with the concentration of 20mg/L by using phosphate buffer solution with the pH value of 8. Then adding the prepared peroxyacetic acid solution into the water obtained in the step 1), wherein the ratio of the prepared peroxyacetic acid solution is as follows: 1, water to be treated: 1, so that the initial concentration of peroxyacetic acid reached 10 mg/L. Reactions were stopped by sampling after 4, 8, 12 and 16min and immediately quenching with 0.5mL each of sodium thiosulfate-catalase solution. And (5) performing a virus plaque counting experiment after diluting the sample, and counting the experiment result after 2 days. The results are shown in FIG. 2. The result shows that when the Ct value reaches 88 mg-min/L, the inactivation logarithm of the peroxyacetic acid to the Dulan virus is 3.64, and the corresponding inactivation rate is 99.9%; when the Ct value reaches 122 mg-min/L, the inactivation logarithm is 3.74, and the corresponding inactivation rate is 99.9%.
Example 3
1) Solution preparation
Firstly, the handle is provided with a handle 105The pH value of water to be treated of PFU/mL porcine rotavirus is adjusted to 5.4, and the water with the adjusted pH value is placed on a magnetic stirrer and stirred at the speed of 200-300 rpm.
2) Disinfection process
Preparing peroxyacetic acid solution with the concentration of 20mg/L by using phosphate buffer solution with the pH value of 5. Then adding the prepared peroxyacetic acid solution into the water obtained in the step 1), wherein the ratio of the prepared peroxyacetic acid solution is as follows: 1, water to be treated: 1, so that the initial concentration of peroxyacetic acid reached 10 mg/L. Samples were taken after 1, 2, 4 and 8min of reaction and quenched immediately by adding 0.5mL of sodium thiosulfate-catalase solution, respectively. And (3) performing a virus plaque counting experiment after diluting the sample, and counting the experiment result after 3 days. The results are shown in FIG. 3. The result shows that when the Ct value reaches 31 mg-min/L, the inactivation logarithm of the peroxyacetic acid to the porcine rotavirus is 2.96, and the corresponding inactivation rate is 99.89%; when the Ct value reaches 63 mg/min/L, the inactivation logarithm is 5.51, and the corresponding inactivation rate is 99.999%.
Example 4
1) Solution preparation
Firstly, the handle is provided with a handle 105The pH value of water to be treated of PFU/mL Dulan virus is adjusted to 4.5, and the water with the adjusted pH value is placed on a magnetic stirrer and stirred at the speed of 200-300 rpm.
2) Disinfection process
Preparing peroxyacetic acid solution with the concentration of 20mg/L by using phosphate buffer solution with the pH value of 5. Then adding the prepared peroxyacetic acid solution into the water obtained in the step 1), wherein the ratio of the prepared peroxyacetic acid solution is as follows: 1, water to be treated: 1, so that the initial concentration of peroxyacetic acid reached 10 mg/L. Reactions 4, 8, 16, 24 and 35min later were sampled and quenched by adding 0.5mL of sodium thiosulfate-catalase solution immediately. And (5) performing a virus plaque counting experiment after diluting the sample, and counting the experiment result after 2 days. The results are shown in FIG. 4. The result shows that when the Ct value reaches 234 mg-min/L, the inactivation logarithm of the peroxyacetic acid to the Dulan virus is 3.00, and the corresponding inactivation rate is 99.9%; when the Ct value reaches 378 mg-min/L, the inactivation logarithm is 3.70, and the corresponding inactivation rate is 99.98%.
Example 5
1) Solution preparation
Firstly, the handle is provided with a handle 105The pH value of water to be treated of PFU/mL porcine rotavirus is adjusted to 7.8, and the water with the adjusted pH value is placed on a magnetic stirrer and stirred at the speed of 200-300 rpm.
2) Disinfection process
Preparing peroxyacetic acid solution with the concentration of 20mg/L by using phosphate buffer solution with the pH value of 8. Then adding the prepared peroxyacetic acid solution into the water obtained in the step (1), wherein the ratio of the prepared peroxyacetic acid solution is as follows: 1, water to be treated: 1, so that the initial concentration of peroxyacetic acid reached 10 mg/L. After 16min of reaction, samples were taken and quenched by adding 0.5mL each of sodium thiosulfate-catalase solution immediately. And carrying out two-step RT-qPCR detection.
(1) Experimental reagent
OSU Reverse Transcription (RT) primers: VP7-R, qPCR primer VP7-R, VP7-F
TV Reverse Transcription (RT) primers: VP2-R, qPCR primers NSP-R1, NSP1-F1
TABLE 4.1 primer List
(2) Experimental procedure
After Ribonucleic Acid (RNA) is reverse-transcribed into deoxyribonucleic Acid (DNA), i.e., complementary DNA (cDNA), by a reverse transcriptase according to a base complementary pairing method, a polymerase chain reaction is used to amplify a large amount of DNA corresponding to the cDNA. And finally, representing the DNA concentration by using a quantitative polymerase chain reaction (qPCR) through a fluorescent dye to obtain a linear relation, and then quantitatively measuring the fluorescence intensity characteristic concentration of the amplified DNA.
The results show that: the inactivation mechanism of the porcine rotavirus and the duran virus by the peroxyacetic acid is similar, namely, the protein shells of the two viruses are damaged, so that the peroxyacetic acid enters the viruses to damage genetic materials, and the viruses lose infectivity.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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<212>DNA
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<400>3
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<212>DNA
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<400>4
<210>5
<211>21
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>5
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<213> Artificial sequence (Artificial sequence)
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Claims (9)
1. A method of inactivating an enterovirus in water, comprising the steps of:
after the pH value of the water to be treated is adjusted to 4.5-8, 5-10 mg/L peroxyacetic acid is added into the water to be treated, and the enterovirus inactivation is carried out.
2. The method of claim 1, wherein the aquatic enterovirus comprises rotavirus and/or norovirus.
3. The method according to claim 1, wherein the inactivation time is 1-35 min.
4. The method of claim 1, wherein the inactivation is accompanied by agitation; the rotating speed of the stirring is 200-300 rpm.
5. The method of claim 1, wherein after the inactivation of the enterovirus, further comprising: adjusting the pH value of the inactivated water body to 4.5-8, and adding a sodium thiosulfate-catalase solution to quench the inactivation reaction.
6. The method according to claim 5, wherein the sodium thiosulfate-catalase solution uses ultrapure water as a solvent, and comprises catalase at a concentration of 1.5-1.8 mg/50mL and sodium thiosulfate at a concentration of 25-35 mg/50 mL.
7. The method of claim 5, wherein the volume ratio of the inactivated water body of the sodium thiosulfate-catalase solution is 1: 1.
8. the method of claim 1, wherein the water to be treated comprises municipal sewage.
9. Use of the method of any one of claims 1 to 8 for sewage treatment or water purification.
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Cited By (1)
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| CN114920345A (en) * | 2022-05-11 | 2022-08-19 | 同济大学 | Peroxyacetic acid reinforced disinfection method |
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