CN102260751A - Water virus enrichment method in water sample in large volume - Google Patents
Water virus enrichment method in water sample in large volume Download PDFInfo
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- CN102260751A CN102260751A CN2011102045183A CN201110204518A CN102260751A CN 102260751 A CN102260751 A CN 102260751A CN 2011102045183 A CN2011102045183 A CN 2011102045183A CN 201110204518 A CN201110204518 A CN 201110204518A CN 102260751 A CN102260751 A CN 102260751A
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Abstract
The invention belongs to the technical field of environmental monitoring, in particular to relating to a water virus enrichment method in a water sample in large volume. The method comprises the following steps of: sterilizing a virus acquisition device; eliminating residual disinfectants in the water sample; filtering the water sample with a negative pressure by using a sterilized filtration system; placing a filter element enriching viruses in water into beef extract eluent with PH (Potential of Hydrogen)of 9.5 for infiltrating; centrifuging the eluent with a centrifuge, separating out bacteria, collecting supernatant, adding PEG (Polyethylene Glycol) 8000 solution, centrifuging with the centrifuge again, discarding the supernatant and reserving precipitates arranged at bottom of a centrifuge tube; adding PBS(Phosphate Buffer Solution) into the precipitates; and obtaining re-suspended virus concentrated solution. The method provided by the invention has the advantages of convenience for operation, simple process and not high requirements on equipment and is suitable for condensing the water sample in large volume into concentrated solution of a small sample at a work field for facilitating the subsequent laboratory detection.
Description
Technical field
The invention belongs to the environmental monitoring technology field, be specifically related to filter in a kind of large volume water sample the method for virus in the enrichment water body.
Background technology
The biological safety problem of tap water is that drinking water safety ensures one of most crucial problem, but present numerous scholars' research concentrates on the correlative study to bacterium substantially, and less relatively for the research of the virus in the tap water.And that virus exists in water is quite general, and the disease caused by infectious water poison is quantitatively based on enterovirus, but harm is bigger, and what paid close attention to for edema poison worker is infectious hepatitis virus.The water-borne transmission of infectious hepatitis virus, existing both at home and abroad repeatedly example and report.For example the India's New Delhi infectious hepatitis that takes place in the period of the 1955-1956 one is very popular, and 3000 cases are arranged a middle of the month, through investigation be by the drinking water source be subjected to severe contamination and chlorine disinfectant insufficient due to; The non-A non-B hepatitis that the Xinjiang of China south takes place since 1986 is very popular, and involves 14 counties and cities, the people surplus in the of 120,000 that falls ill, and reason is that drinking water pollution causes; Shanghai hepatitis A in 1988 is popular, and to reach 4 months also be to be polluted by water indirectly to cause.Virus is not the intestinal tract normal flora clump, only exists only in infected individuals, therefore, discharges from ight soil with the quantity more much lower than colibacillus usually.Because of disease caused by infectious water poison concentration is generally lower, about 10
3PFU/100L, particularly sterilization back water need just can detect after a large amount of water samples concentrate, and simple, quick, the responsive concentration process reliably of therefore seeking a large amount of water samples of check is the important topic of disease caused by infectious water poison method of inspection research.
Concentrating of disease caused by infectious water poison is different with bacterium.Concentrating of bacterium can rely on mechanical detention fully on film, and virus is very little because of particle diameter, much more general based on adsorb, be separated, effects such as centrifugation by force, electrophoresis and immunochemistry, basic skills can be divided into two classes: (1) absorb-elute method.Comprise film absorption method, precipitable salt, ferric oxide, polyelectrolyte adsorption method, aluminium hydroxide and plant flocculation absorption method and glass powder absorption method, phase separation method etc.(2) be characterized as basic method with viral physics, as ultracentrifugation, reverse osmosis method, electroosmose process and dehydration dialysis method etc.It is generally acknowledged that in aforesaid method ultracentrifugation, electrophoresis and electroosmose process etc. not only need the certain device condition, and the inspection water yield is limited, it is feasible being used for viral reconcentration as supplementary means, but in the check of concentrating of a large amount of disease caused by infectious water poison not seen widespread use.Polymkeric substance two phase separation methods are applicable to the virus in quantitative testing or the separation a small amount of muddy water.Although the film absorption method can be used to check bulky water sample, also there are problems such as complicated operation, cost height, easy obstruction, be difficult to be applied in the viral enrichment of drinking water source ground and water factory.
The absorb-elute Master utilizes various sorbent materials to come viral adsorption, with certain eluent it is eluted again, and the enrichment that is widely used in virus in the water surrounding detects and remove all kinds of pathogenic agent in the environmental wastewater.Wherein, the film absorption method is because of it has the higher viral rate of recovery, required device is simple, treatment capacity is big etc., and obtains using comparatively widely, and successful Application is removed virus etc. in drinking water disinfection, protein soln.
The film absorption method mainly utilizes virus under electrochemical reaction surface of filter medium to be had the method that the principle of reversibility adsorptive power concentrates.Schedule of operation reaches the purpose that concentrates, with a small amount of eluent elution of virus is reclaimed then for the disease caused by infectious water poison being adsorbed under certain condition on the filter material.Filter material commonly used is the cruel or glass fiber filter of Mierocrystalline cellulose, and because of the difference of the organic binder resin that adopts, filter material can be divided into the surface and have anionic charge or positive charge two kinds.Anionic charge filter material viral adsorption needs tested water is transferred to pH3.0 ~ 3.5, promptly reaches below the virus protein iso-electric point, and adds a small amount of polyvalent cation, as Al
3+And Mg
2+Change Deng promoting the virus surface electric charge can produce the good adsorption effect.And the positive charge filter material can not need to regulate water sample pH and add the multivalence salt at the effective viral adsorption of pH scope widely.Not only easy and simple to handle, and can obtain and the comparable recovering effect of anionic charge filter material.
In the film absorption method process, the suspended substance in the water sample easily blocks filter core, has limited the water sample amount that detects and may disturb elution process; In addition, the organism in the water may with the absorption of competing property of virus, active adsorption and wash-out that can viral interference.
The wash-out of viral adsorption, normally original position is filtered the purpose that a small amount of elutriant reaches wash-out by filter.Elutriant commonly used is the glycine solution of subalkaline (pH9.0) beef infusion broth, nutrient broth or more alkaline (pH10.5 or 11.0).General beef infusion broth or nutrient broth elute effect are better, detect but be used for a large amount of disease caused by infectious water poison, and be relatively more difficult when needing reconcentration.And the glycine elution efficiency is generally lower, and particularly under stronger alkaline condition, virus is stable inadequately.For this reason, the someone proposes to make beef infusion broth produce the method for virus in the elementary elutriant of flocculation reconcentration with reduction.
Therefore at the water sample of comparatively large vol, set up effectively, the concentrating of disease caused by infectious water poison, elution process efficiently, Causative virus in the tap water is detected fast and effectively, understand virus pollution situation in drinking water source ground and the water factory, for ensureing drinking water safety, guaranteeing that human health has important effect.
Summary of the invention
The object of the present invention is to provide a kind of easy, filter in the enrichment water body method of virus fast so that can detect the Causative virus in the tap water fast and effectively, understand virus pollution situation in drinking water source ground and the water factory, the guarantee drinking water safety.
The method of virus in the sampling enrichment water body provided by the invention need not water sample is regulated pre-treatments such as pH value, pre-filtering, only needs to remove sterilizing agents such as chlorine remaining in the water sample, chloramines, to improve the accuracy of subsequent detection; This method can be used for the big and water yield of water sample amount change greatly the waterworks water inlet and water outlet in the detection of virus, the variation of water sample amount is bigger, the 500L from the 50L of the water sample of intaking to the water outlet water sample is all applicable; This method is all carried out the UV sterilization to the virus collection device, has improved the accuracy of viral enrichment and detection.
Before the virus, earlier to the gathering device disinfection, the virus collection device comprises filter, sample bucket, silicone rubber tube, centrifuge tube etc., uses the UV(ultraviolet ray in sampling, enrichment water body) sterilize more than 24 hours.
The method of virus in the sampling enrichment water body provided by the invention, concrete steps are:
(1) gathers water sample, and water sample is carried out pre-treatment, eliminate sterilizing agent remaining in the water sample
Gather water sample, the water sample amount: former water is 50 ~ 100 L, and filter back water or water factory's water outlet are 250 ~ 500 L; Carry out pre-treatment to adopting the water sample that comes, promptly remove sterilizing agents such as chlorine, chloramines, ozone with S-WAT, to remove the influence of sterilizing agent to virus, the amount of the S-WAT that is added is determined according to the amount of sterilizing agent remaining in the water; When the suspended particulate of big particle diameter in the water body is too much, need to adopt the gauze of sterilizing to carry out filtration treatment earlier;
(2) filter
By peristaltic pump, with the speed extraction actual water sample of 4 ~ 7 L/min, utilize filtering system, the water sample so that negative pressure leaching is handled through step 1 is enriched in the virus in the water sample on the filter core of filtering system, and wherein filter core is the blend fiber ester millipore filtration of positive charge;
(3) wash-out virus
With enrichment the filter core of disease caused by infectious water poison from filter, take out, put into pH and be 8.5 ~ 10 200 ~ 600 mL beef extract elutriants and soak 1 ~ 5 min, take out then, the beef extract elutriant of putting into pH again and be 8.5 ~ 10 200 ~ 400 mL soaks 2 ~ 6 min, discard filter core, merge twice elutriant.Wherein in the beef extract elutriant, the concentration of extractum carnis be 1.0 ~ 3.0% (M/V, mg/mL), the concentration of glycine is 0.02 ~ 0.06 M;
(4) elutriant concentrates
The rapid elutriant that obtains of previous step is placed the cryogenic freezing whizzer, at 4 ℃, centrifugal 10 ~ 20 min of 3000 ~ 5000 r/min, bacterium is separated, collect supernatant liquor, add biological flocculant PEG 8000 solution, again behind 4 ℃, centrifugal 30 ~ 60 min of 6000 ~ 10000 r/min, abandoning supernatant keeps the throw out of centrifuge tube bottom.Wherein biological flocculant PEG8000 solution is 26% ~ 36% PEG, the NaCl of 0.6 ~ 1.0mol/L;
(5) resuspended concentrated solution
The PBS(Phosphate Buffered Saline that in the rapid throw out that obtains of previous step, adds 1 ~ 6 mL, 0.1 ~ 0.4 mol/L, phosphate buffered saline buffer, pH 6.8 ~ 7.6) solution, fully dissolve centrifuge tube bottom settlings thing, obtain the concentrated solution of resuspended disease caused by infectious water poison.
Then, adopt existing method to measure the virus titer of this sample, the water sample amount that extracts according to reality is calculated the virus titer in the actual water.
Advantage of the present invention:
The filter core that the present invention utilizes heavy body filters, wash-out, concentrates the enrichment of virus in the water body of realizing big water sample amount (50 ~ 500 L) the virus in the water body; Present method does not need water sample is carried out pre-treatment, do not need to regulate the pH value, and a spot of suspended substance can not exert an influence to the enrichment of virus yet in the water; Present method has been carried out the UV sterilization to the virus collection device, has improved the accuracy of follow-up viral enrichment and detection.The sensitivity that numerous advantages can make follow-up virus detect improves greatly, thereby has realized the effective monitoring to virus in the water body.With respect to traditional viral enriching method, this method is simple to operate, step is succinct, equipment requirements is not high and save time, and is convenient to apply in basic units such as water factory and waterhead areas.
Description of drawings
Fig. 1 is the example structure synoptic diagram of the present invention's virus enrichment integral method.
Fig. 2 is the workflow block diagram of the viral enriching method of Fig. 1 embodiment.
Number in the figure: the 1-sample bucket, the 2-agitator, the big flow peristaltic pump of 3-, the 4-strainer, the filter core in the 5-filter, 6-hold the container of elutriant, 7-whizzer, 8-sample bottle.
Embodiment
The invention is further illustrated by the following examples.
Before the virus, earlier to the gathering device disinfection, the virus collection device comprises filter, sample bucket, silicone rubber tube, centrifuge tube etc., uses the UV(ultraviolet ray in sampling, enrichment water body) sterilize more than 24 hours;
The method of virus in the enrichment water body provided by the invention, concrete steps are:
1, gathers water sample, and water sample is carried out pre-treatment, eliminate sterilizing agent remaining in the water sample
Gather water sample, the water sample amount: former water is 50 ~ 100 L, and filter back water or water factory's water outlet are 250 ~ 500 L; Carry out pre-treatment to adopting the water sample that comes, remove sterilizing agents such as chlorine, chloramines, ozone with S-WAT, to remove the influence of sterilizing agent virus;
2, filter
By powerful peristaltic pump, speed with 4 ~ 7 L/min extracts actual water sample (former water 50 ~ 100 L, filter back water and water factory's water outlet 250 ~ 500 L), utilize filtering system, with the water of negative pressure leaching through step 1 pre-treatment, virus in the water is enriched on the filter core of filtering system, wherein filter core is the blend fiber ester millipore filtration of positive charge;
3, wash-out virus
With enrichment the filter core of disease caused by infectious water poison from filter, take out, put into pH and be 9.5 400 mL beef extract elutriants and soak 2 min, take out the beef extract elutriant of putting into 350 mL again then and soak 4 min, discard filter core, merge twice elutriant.Wherein in the beef extract elutriant, the concentration of extractum carnis be 1.5% (M/V, mg/mL), the concentration of glycine is 0.05 M;
4, elutriant concentrates
The rapid elutriant that obtains of previous step is placed the cryogenic freezing whizzer, at 4 ℃, centrifugal 15 min of 4000 r/min, bacterium is separated, collect supernatant liquor, add biological flocculant PEG 8000 solution, again behind 4 ℃, centrifugal 40 min of 7000 r/min, abandoning supernatant keeps the throw out of centrifuge tube bottom.Wherein biological flocculant PEG8000 solution is 32% PEG, the NaCl of 0.8mol/L;
5, resuspended concentrated solution
The PBS(Phosphate Buffered Saline that in the rapid throw out that obtains of previous step, adds 5 mL, 0.2 mol/L, phosphate buffered saline buffer, pH 7.2) solution, and fully dissolve centrifuge tube bottom settlings thing, obtain the concentrated solution of resuspended disease caused by infectious water poison.
Then, adopt existing method to measure the virus titer of this sample, the water sample amount that extracts according to reality is calculated the virus titer in the actual water.
With method enrichment of the present invention southern china city A and the former water of two waterworkss of B and the sample of each processing unit water outlet detect, thereby there is situation in investigation virus in each process section water outlet, determine the feasibility and the practicality of present method.Wherein, A water factory is a domestic Large Water Works that designs voluntarily, builds, and the Jia He upstream is taken from the water source, its, treatment capacity was 1,600,000 tons every day, treatment scheme is traditional handling technology of water supply, and its treatment process is simple, is traditional coagulating sedimentation, sand filtration and sterilization.Its sterilization process is a chloramines disinfection, guarantees that the water outlet chlorine residue is 1.5 ~ 2.0 mg/L.
B water factory takes from the second river in the water source, and its, treatment capacity was 40,000 tons every day.Because little pollution level is heavier in the former water, B water factory treatment scheme is different with traditional technology, has increased processing units such as Biological Pretreatment, biological activated carbon, and its sterilization process is a chloramines disinfection, guarantees that the water outlet chlorine residue is 1.5 ~ 2.0 mg/L.
With MS2 phage (F-specific phage) is the indicator of enterovirus, is subjected to the degree of virus pollution on the whole with Φ X174 phage (somatocyte phage) reflection.Detect after the enrichment virus, the results are shown in Table 1 and 2.
The former water of table 1 A water factory and each process section virus detect situation
| Each process section | MS2 tire (PFU/100L) | Φ X174 tire (PFU/100L) |
| Former water | 2.77×10 4 | 7.72×10 4 |
| Coagulating sedimentation | 2.45×10 1 | 1.23×10 1 |
| Sand filtration | 0 | 0 |
| Water outlet | 0 | 0 |
As shown in Table 1, MS2 that detects in the former water of A water factory and Φ X174 phage are respectively 2.77 * 10
4With 7.72 * 10
4PFU/100L.The result that detects of the former water of A water factory is equivalent to the MS2 of 277 PFU/L and the Φ X174 of 772 PFU/L, the possibility that exists enterovirus to pollute in this former water, and will there be certain risk in the processing as if without strictness to HUMAN HEALTH.In addition, A water factory is behind coagulating sedimentation, and after sand filtration and final outflow water, two kinds of phages all do not detect, and can reach the drinking water treatment requirement of USEPA again.
Table 2 has been listed seven process sections of B water factory and has been detected viral result.Can see, detect MS2 in the former water and Φ X174 phage is respectively 6.65 * 10
4PFU/100L and 1.09 * 10
5PFU/100L, with the former water phage content of A water factory in same level, difference is that Φ X174 is than the high order of magnitude of MS2 in this former water, therefore also may there be harmful virus in this former water.Biological Pretreatment is not removed phage, tiring of two kinds of phages raises on the contrary, this is because there is the bacterium that comprises intestinal bacteria in a large number in the active sludge in the biological pre-treatment process, intestinal bacteria are as the host of two kinds of phages, make and itself have MS2 and Φ X174 phage in the Biological Pretreatment unit, therefore in treating water, introduced more phages, made it all reach 10
4Level.This also remote effect to the treatment effect of coagulating sedimentation.And BAC process is not obvious to the removal effect of phage, and in addition, the growth of bacterium also may be introduced phage in the biological activated carbon.But all do not detect through the back two kinds of phages of sterilization, after this sand filtration and water outlet do not detect yet.
The former water of table 2 B water factory and each process section virus detect situation
| Each process section | MS2 tire (PFU/100L) | Φ X174 tire (PFU/100L) |
| Former water | 6.65×10 4 | 1.09×10 5 |
| Biological Pretreatment | 2.03×10 5 | 3.22×10 5 |
| Coagulating sedimentation | 1.04×10 3 | 6.32×10 2 |
| BAC | 8.96×10 2 | 5.04×10 2 |
| Sterilization | 0 | 0 |
| Sand filtration | 0 | 0 |
| Water outlet | 0 | 0 |
Claims (5)
1. the method for virus in the enrichment water body is characterized in that concrete steps are:
(1) gathers water sample, and water sample is carried out pre-treatment, eliminate sterilizing agent remaining in the water sample;
(2) filter
By peristaltic pump, with the speed extraction actual water sample of 4 ~ 7 L/min, utilize filtering system, the water sample so that negative pressure leaching is handled through step 1 is enriched in the virus in the water sample on the filter core of filtering system, and wherein filter core is the blend fiber ester millipore filtration of positive charge;
(3) wash-out virus
With enrichment the filter core of disease caused by infectious water poison from filter, take out, put into pH and be 8.5 ~ 10 200 ~ 600 mL beef extract elutriants and soak 1 ~ 5 min, take out then, the beef extract elutriant of putting into pH again and be 8.5 ~ 10 200 ~ 400 mL soaks 2 ~ 6 min, discard filter core, merge twice elutriant; Wherein in the beef extract elutriant, the concentration of extractum carnis is 1.0 ~ 3.0% (mg/mL), and the concentration of glycine is 0.02 ~ 0.06 M;
(4) elutriant concentrates
The rapid elutriant that obtains of previous step is placed the cryogenic freezing whizzer, at 4 ℃, centrifugal 10 ~ 20 min of 3000 ~ 5000 r/min, bacterium is separated, collect supernatant liquor, add biological flocculant PEG 8000 solution, again behind 4 ℃, centrifugal 30 ~ 60 min of 6000 ~ 10000 r/min, abandoning supernatant keeps the throw out of centrifuge tube bottom; Wherein biological flocculant PEG8000 solution is 26% ~ 36% PEG, the NaCl of 0.6 ~ 1.0mol/L;
(5) resuspended concentrated solution
Add the phosphate buffered saline buffer of 1 ~ 6 mL, 0.1 ~ 0.4 mol/L in the rapid throw out that obtains of previous step, the pH 6.8 ~ 7.6 of this phosphate buffered saline buffer fully dissolves centrifuge tube bottom settlings thing, obtains the concentrated solution of resuspended disease caused by infectious water poison;
Then, measure the virus titer of this sample, the water sample amount that extracts according to reality is calculated the virus titer in the actual water.
2. the method for virus in the enrichment water body according to claim 1 is characterized in that using ultraviolet disinfection more than 24 hours to the virus collection device in advance.
3. the method for virus in the enrichment water body according to claim 1 and 2, it is characterized in that the middle amount of gathering water sample of step (1): former water is 50 ~ 100 L, filter back water or water factory's water outlet are 250 ~ 500 L; Remove chlorine, chloramines, ozonization agent to adopting the water sample that comes with S-WAT.
4. the method for virus in the enrichment water body according to claim 1 and 2 is characterized in that adopting the gauze of sterilizing to carry out filtration treatment earlier when the suspended particulate of big particle diameter in the water body is too much.
5. the method for virus in the enrichment water body according to claim 1 and 2, it is characterized in that: the filtering system that is adopted in the step (2) is selected the filtering system of U.S. NanoCeram company for use.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111850171A (en) * | 2020-08-03 | 2020-10-30 | 浙江大学 | Method for detecting lentivirus in deep sea sediment sample |
| CN112626167A (en) * | 2020-12-18 | 2021-04-09 | 中国科学院生态环境研究中心 | Method for enriching and detecting viruses in water |
| CN114085829A (en) * | 2021-11-18 | 2022-02-25 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Efficient enrichment method for viruses in environmental medium |
-
2011
- 2011-07-21 CN CN2011102045183A patent/CN102260751A/en active Pending
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| GIBBONS: "Concentration of adenoviruses and norovirus from seawater with argonide nanoceram cartridge filters: Methond effectiveness and occurrence in southern California recreational waters", 《北卡罗来纳大学毕业论文数据库》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111850171A (en) * | 2020-08-03 | 2020-10-30 | 浙江大学 | Method for detecting lentivirus in deep sea sediment sample |
| CN112626167A (en) * | 2020-12-18 | 2021-04-09 | 中国科学院生态环境研究中心 | Method for enriching and detecting viruses in water |
| CN114085829A (en) * | 2021-11-18 | 2022-02-25 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Efficient enrichment method for viruses in environmental medium |
| CN114085829B (en) * | 2021-11-18 | 2023-08-11 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Efficient enrichment method for viruses in environmental medium |
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Application publication date: 20111130 |