CN101430281A - Active detection method for concealed spore egg vesicle and Giardia sporocyst in drinking water - Google Patents
Active detection method for concealed spore egg vesicle and Giardia sporocyst in drinking water Download PDFInfo
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- CN101430281A CN101430281A CNA2008102097275A CN200810209727A CN101430281A CN 101430281 A CN101430281 A CN 101430281A CN A2008102097275 A CNA2008102097275 A CN A2008102097275A CN 200810209727 A CN200810209727 A CN 200810209727A CN 101430281 A CN101430281 A CN 101430281A
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Abstract
The invention relates to a method for detecting the activity of cryptosporidium cyst and giardia lamblia sporocyst in drinking water. The method solves the problem that the existing method for detecting the cryptosporidium cyst and the giardia lamblia sporocyst in the drinking water has high cost and low precision and can not confirm the activity of the cryptosporidium cyst and the giardia lamblia sporocyst. The method comprises the steps as follows: 1, water sample pretreatment; 2, elution; 3, condensation; 4 the purification of the cryptosporidium cyst and the giardia lamblia sporocyst; 5, dying; 6, microscopical examination for observing the cryptosporidium cyst and the giardia lamblia sporocyst; and the detection of the cryptosporidium cyst and the giardia lamblia sporocyst in the drinking water is realized. The method does not require complex instrument and equipment; the method is simple and easy for grasping, thereby reducing the investment on the equipment greatly and reducing the detection cost; and the method can detect whether the cryptosporidium cyst and the giardia lamblia sporocyst have the activity or not and has the high precision of detection result.
Description
Technical field
The present invention relates to the detection method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in a kind of potable water.
Background technology
Because Cryptosporidium and giardia lamblia are to the pollution of domestic water and potable water and cause the outbreak of epidemic of associated conditions in developed countries such as Great Britain and Americas, and the health to the public has constituted serious threat, and Cryptosporidium egg capsule and giardia lamblia sporangiocyst (being called for short " two worms ") have also become Jie's water that receives much attention and propagated one of bio-hazard factor.In " drinking water sanitary standard " of the new promulgation of China, Cryptosporidium and giardia lamblia have been stipulated its limit value as the unconventional index of water quality, but drinking water treatment technology and source quality and water supply quality present situation according to present China, exist the potential risk of " two worms " outburst very high, therefore from ensureing the consideration of drinking water safety angle, the detection method of studying two worms has very important and very urgent realistic meaning.
" two worms " detection method commonly used at present has U.S. EPA 1623 methods, grain count method and acid dyeing method etc.U.S. EPA 1623 methods are that current application is the widest, develop the most ripe method, also recommended to use by China simultaneously, this method is carried out the fluorescence microscopy then with egg capsule and sporangiocyst in the immunomagnetic beads collection water sample, has very high specificity, but the shortcoming of this method is to judge the activity of egg capsule and sporangiocyst, so also just can't weigh drinking water treatment technology to its killing effect with it, and the experiment material costliness, cost is very high, promotes to get up to acquire a certain degree of difficulty; The U.S. adopts in a lot of laboratories the grain count method to replace the EPA1623 method to be used for the detection of water sample, this method automaticity height, can be used for online detection, but be subject to interference with the approximate particle of " two worms " particle diameter, error is bigger, lack certain degree of accuracy, and can't distinguish the life or death of " two worms " equally; The acid dyeing method is simple and easy to do, and the result is obvious, does not need complicated instrument and equipment, and recall rate is also higher, but equally also can't detect the activity of " two worms ", is applicable to epidemiology survey and is not suitable for evaluation to the drinking water technology treatment effect.
Summary of the invention
The present invention for the detection method cost height that solves Cryptosporidium egg capsule and giardia lamblia sporangiocyst in the existing potable water, degree of accuracy is low and can't determine the Cryptosporidium egg capsule and the problem of giardia lamblia sporangiocyst activity, and provides the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in a kind of potable water.
The activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst carries out according to following steps in the potable water of the present invention: one, earlier with 200～500 purpose screen filtration water samples, be that the filter membrane of 1.5～3.5 μ m carries out suction filtration with the aperture then; Two, the filter membrane behind the step 1 suction filtration being placed beaker, is that 7.2 buffer solution cleans with 5～10mL, pH value, collects the attachment under cleaning; Three, the attachment that step 2 is collected is positioned in the centrifuge tube, is centrifugal 10～20min under the condition of 2000～4000r/min at rotating speed, abandons supernatant; Four, carrying out sucrose gradient centrifugation, is centrifugal 5～15min under the condition of 1500～2500r/min at rotating speed, draws the middle layer between the two-layer liquid level in the centrifuge tube then, puts in the centrifuge tube of 1.5mL; Wherein sucrose solution from top to bottom mass concentration be followed successively by 12%～16% and 20%～25%, two-layer up and down sucrose solution volume is identical; Five, in the centrifuge tube of the 1.5mL of step 4, add 100 μ L, the pH value is 7.2 buffer solution, 10 μ L, mass concentration are that the DAPI solution of 2g/L and PI solution that 10 μ L, mass concentration are 2g/L mix, incubation 2h under 37 ℃ of conditions is that 2000～4000r/min, centrifugation time are centrifugal 9～11 μ L that are concentrated under the condition of 5～15min at rotating speed then; Six, with the centrifugal sample drop that concentrates 9～11 μ L obtain in the step 5 on blood counting chamber, be under 330～385nm burst of ultraviolel light source and wavelength is to carry out fluorescence microscope under the green glow excitation source of 520～550nm at wavelength; Promptly realized detection to Cryptosporidium egg capsule in the potable water and giardia lamblia sporangiocyst.
The inventive method is as follows to the advantage of the activity test method of Cryptosporidium egg capsule in the potable water and giardia lamblia sporangiocyst:
1, the inventive method can detect the Cryptosporidium egg capsule and the giardia lamblia sporangiocyst has non-activity.The present invention observes the egg capsule or the sporangiocyst of the fluorescence that sends shiny red by fluorescent microscope under green fluorescence, be the egg capsule or the sporangiocyst of the PI positive, and not luminous be the egg capsule or the sporangiocyst of PI feminine gender; Under uv excitation light, observe egg capsule or the sporangiocyst that sends sky blue fluorescence by fluorescence microscope, be the egg capsule or the sporangiocyst individuality of the DAPI positive, and not luminous be the egg capsule or the sporangiocyst individuality of DAPI feminine gender; Wherein, the individuality of the positive PI feminine gender of DAPI is to have active Cryptosporidium egg capsule and giardia lamblia sporangiocyst individuality, the individuality of the negative PI positive of DAPI be the Cryptosporidium egg capsule and the giardia lamblia sporangiocyst individuality of death, thereby determines the Cryptosporidium egg capsule and the giardia lamblia sporangiocyst has non-activity.Detect by method of the present invention that Cryptosporidium egg capsule and giardia lamblia sporangiocyst have non-activity in the tap water, grasp in the tap water treatment process the removal and the deactivation situation of " two worms " thereby more help the waterworks, further reduced and have active " two worms " individual probability that in potable water, exists.
2, the inventive method degree of accuracy height.Method of the present invention is observed Cryptosporidium egg capsule size and is 4～6 μ m under fluorescent microscope, rounded or oval, as seen inside have zygoblast; Giardia lamblia sporangiocyst size is 8～12 μ m, ovalize, in the nuclear of 2～4 dark colors is arranged; The inventive method adopts DAPI solution and PI solution as coloring agent, can clearly observe Cryptosporidium egg capsule and giardia lamblia sporangiocyst size, form; Method of the present invention is under fluorescent microscope, can see the egg capsule and the sporangiocyst that are colored clearly, other impurity that do not contain DNA then can't be colored, just can not send fluorescence excitation yet, and the interference of the biosome in other water also can be got rid of by the morphological feature of " two worms ", testing result degree of accuracy height.
3, method of the present invention is low to the detection cost of Cryptosporidium egg capsule in the potable water and giardia lamblia sporangiocyst.Detection method of the present invention does not need complicated instrument and equipment, and method is simple to be grasped easily, has reduced the input of equipment greatly, has reduced the detection cost.
Embodiment
Embodiment one: the detection method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst is carried out according to following steps in a kind of potable water of present embodiment: one, earlier with 200～500 purpose screen filtration water samples, be that the filter membrane of 1.5～3.5 μ m carries out suction filtration with the aperture then; Two, the filter membrane behind the step 1 suction filtration being placed beaker, is that 7.2 buffer solution cleans with 5～10mL, pH value, collects the attachment under cleaning; Three, the attachment that step 2 is collected is positioned in the centrifuge tube, is centrifugal 10～20min under the condition of 2000～4000r/min at rotating speed, abandons supernatant; Four, carrying out sucrose gradient centrifugation, is centrifugal 5～15min under the condition of 1500～2500r/min at rotating speed, draws the middle layer between the two-layer liquid level in the centrifuge tube then, puts in the centrifuge tube of 1.5mL; Wherein sucrose solution from top to bottom mass concentration be followed successively by 12%～16% and 20%～25%, two-layer up and down sucrose solution volume is identical; Five, in the centrifuge tube of the 1.5mL of step 4, add 100 μ L, the pH value is 7.2 buffer solution, 10 μ L, mass concentration are that the DAPI solution of 2g/L and PI solution that 10 μ L, mass concentration are 2g/L mix, incubation 2h under 37 ℃ of conditions is that 2000～4000r/min, centrifugation time are centrifugal 9～11 μ L that are concentrated under the condition of 5～15min at rotating speed then; Six, with the centrifugal sample drop that concentrates 9～11 μ L obtain in the step 5 on blood counting chamber, be under 330～385nm burst of ultraviolel light source and wavelength is to carry out fluorescence microscope under the green glow excitation source of 520～550nm at wavelength; Promptly realized detection to Cryptosporidium egg capsule in the potable water and giardia lamblia sporangiocyst.
The temperature that the used water sample of present embodiment is positioned in 24h below 4 ℃ keeps in Dark Place.
Present embodiment can be observed at microscopically: Cryptosporidium egg capsule size is 4～6 μ m, and is rounded or oval, and as seen inside have zygoblast; Giardia lamblia sporangiocyst size is 8～12 μ m, ovalize, in the nuclear of 2～4 dark colors is arranged.
Present embodiment is observed the egg capsule or the sporangiocyst of the fluorescence that sends shiny red by fluorescent microscope under green fluorescence, be the egg capsule or the sporangiocyst of the PI positive, and not luminous be the egg capsule or the sporangiocyst of PI feminine gender; Under uv excitation light, observe egg capsule or the sporangiocyst that sends sky blue fluorescence by fluorescence microscope, be the egg capsule or the sporangiocyst individuality of the DAPI positive, and not luminous be the egg capsule or the sporangiocyst individuality of DAPI feminine gender; Wherein, the individuality of the positive PI feminine gender of DAPI is to have active Cryptosporidium egg capsule and giardia lamblia sporangiocyst individuality, and the individuality of the negative PI positive of DAPI is dead Cryptosporidium egg capsule and giardia lamblia sporangiocyst individuality.
Embodiment two: present embodiment and embodiment one are different be in the step 1 earlier with 300～400 purpose screen filtration water samples, be that the filter membrane of 2～3 μ m carries out suction filtration with the aperture then.Other step and parameter are identical with embodiment one.
Embodiment three: present embodiment and embodiment one are different be in the step 1 earlier with 300 purpose screen filtration water samples, be that the filter membrane of 2～3 μ m carries out suction filtration with the aperture then.Other step and parameter are identical with embodiment one.
Embodiment four: present embodiment and embodiment one are different is that the pH value is that 7.2 buffer solution is phosphate buffer in the step 2.Other step and parameter are identical with embodiment one.
Embodiment five: present embodiment and embodiment one are different is that the attachment in the step 3 step 2 collected is centrifugal 15min under the condition of 3000r/min at rotating speed.Other step and parameter are identical with embodiment one.
Embodiment six: present embodiment and embodiment one are different is to be centrifugal 10min under the condition of 2000r/min at rotating speed in the step 4.Other step and parameter are identical with embodiment one.
Embodiment seven: present embodiment and embodiment one are different be in the step 4 sucrose solution from top to bottom mass concentration be followed successively by 13%～15% and 21%～24%.Other step and parameter are identical with embodiment one.
Embodiment eight: present embodiment and embodiment one are different be in the step 4 sucrose solution from top to bottom mass concentration be followed successively by 15% and 24%.Other step and parameter are identical with embodiment one.
Embodiment nine: present embodiment and embodiment one are different be in the step 5 be under the 3000r/min condition at rotating speed, centrifugation time is that the centrifugal of 10min concentrates.Other step and parameter are identical with embodiment one.
Embodiment ten: that present embodiment and embodiment one are different is the centrifugal 10 μ L that are concentrated in the step 5.Other step and parameter are identical with embodiment one.
Embodiment 11: the detection method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst is carried out according to following steps in a kind of potable water of present embodiment: one, earlier with 300 purpose screen filtration water samples, be that the filter membrane of 3 μ m carries out suction filtration with the aperture then; Two, the filter membrane behind the step 1 suction filtration is placed beaker, clean, collect the attachment under cleaning with the buffer solution of 10mL, pH 7.2; Three, the attachment that step 2 is collected is positioned in the centrifuge tube, is centrifugal 15min under the condition of 3000r/min at rotating speed then, abandons supernatant; Four, carrying out sucrose gradient centrifugation, is centrifugal 10min under the condition of 2000r/min at rotating speed, draws the middle layer between the two-layer liquid level in the centrifuge tube then, puts in the centrifuge tube of 1.5mL; Wherein sucrose solution from top to bottom mass concentration be followed successively by 15% and 24%, two-layer up and down sucrose solution volume is identical; Five, in the centrifuge tube of the 1.5mL of step 4, add 100 μ L, the pH value is 7.2 buffer solution, 10 μ L, mass concentration are that the DAPI of 2g/L and PI that 10 μ L, mass concentration are 2g/L mix, incubation 2h under 37 ℃ of conditions is that 3000r/min, centrifugation time are centrifugal 9～11 μ L that are concentrated under the condition of 10min at rotating speed; Six, the sample drop after getting that step 5 is centrifugal and concentrating is on blood counting chamber, is under the 330nm burst of ultraviolel light source and wavelength is to carry out the fluorescence microscope counting under the green glow excitation source of 530nm at wavelength; Promptly realized detection to Cryptosporidium egg capsule in the potable water and giardia lamblia sporangiocyst.
Get distilled water and compare experiment as water sample, wherein, first group: get 10L distilled water, in water sample, add 50 Cryptosporidium egg capsules and 50 giardia lamblia sporangiocysts, activity test method with the specific embodiment of the present invention 11 detects the water sample that adds Cryptosporidium egg capsule and giardia lamblia sporangiocyst then, second group: get 10L distilled water, in water sample, add 50 Cryptosporidium egg capsules and 50 giardia lamblia sporangiocysts, with existing U.S. EPA 1623 methods the water sample that adds Cryptosporidium egg capsule and giardia lamblia sporangiocyst is detected then; The 3rd group: get 10L distilled water, in water sample, add 50 Cryptosporidium egg capsules and 50 giardia lamblia sporangiocysts, with the grain count method water sample that adds Cryptosporidium egg capsule and giardia lamblia sporangiocyst is detected then; The 4th group: get 10L distilled water, in water sample, add 50 Cryptosporidium egg capsules and 50 giardia lamblia sporangiocysts, with the acid dyeing method water sample that adds Cryptosporidium egg capsule and giardia lamblia sporangiocyst is detected then.This testing result of four groups is shown in table 1,2, and wherein table 1 is the testing result of Cryptosporidium egg capsule, and table 2 is the testing result of giardia lamblia sporangiocyst.
The testing result of table 1 Cryptosporidium egg capsule
The testing result of table 2 giardia lamblia sporangiocyst
As can be seen from Table 1, activity detection of the present invention is 15 to the number that detects of Cryptosporidium egg capsule, the live body number is 11, recall rate is 30%, as can be seen from Table 2, activity detection of the present invention is 13 to the number that detects of giardia lamblia sporangiocyst, the live body number is 8, recall rate is 26%, activity detection of the present invention and existing U.S. EPA 1623 methods, grain count method and acid dyeing method are compared, and the number that method of the present invention detects Cryptosporidium egg capsule and giardia lamblia sporangiocyst is many, the recall rate height, and can determine that detected Cryptosporidium egg capsule and giardia lamblia sporangiocyst have non-activity, testing result degree of accuracy height, method of the present invention do not use complicated instrument and equipment in testing process, method is simple to be grasped easily, reduce the input of equipment greatly, reduced the detection cost.
Claims (10)
1, the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in the potable water, it is characterized in that the detection method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst is carried out according to following steps in the potable water:, be that the filter membrane of 1.5～3.5 μ m carries out suction filtration with the aperture then one, earlier with 200～500 purpose screen filtration water samples; Two, the filter membrane behind the step 1 suction filtration being placed beaker, is that 7.2 buffer solution cleans with 5～10mL, pH value, collects the attachment under cleaning; Three, the attachment that step 2 is collected is positioned in the centrifuge tube, is centrifugal 10～20min under the condition of 2000～4000r/min at rotating speed, abandons supernatant; Four, carrying out sucrose gradient centrifugation, is centrifugal 5～15min under the condition of 1500～2500r/min at rotating speed, draws the middle layer between the two-layer liquid level in the centrifuge tube then, puts in the centrifuge tube of 1.5mL; Wherein sucrose solution from top to bottom mass concentration be followed successively by 12%～16% and 20%～25%, two-layer up and down sucrose solution volume is identical; Five, in the centrifuge tube of the 1.5mL of step 4, add 100 μ L, the pH value is 7.2 buffer solution, 10 μ L, mass concentration are that the DAPI solution of 2g/L and PI solution that 10 μ L, mass concentration are 2g/L mix, incubation 2h under 37 ℃ of conditions is that 2000～4000r/min, centrifugation time are centrifugal 9～11 μ L that are concentrated under the condition of 5～15min at rotating speed then; Six, with the centrifugal sample drop that concentrates 9～11 μ L obtain in the step 5 on blood counting chamber, be under 330～385nm burst of ultraviolel light source and wavelength is to carry out fluorescence microscope under the green glow excitation source of 520～550nm at wavelength; Promptly realized detection to Cryptosporidium egg capsule in the potable water and giardia lamblia sporangiocyst.
2, the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in the potable water according to claim 1 is characterized in that in the step 1 earlier with 300～400 purpose screen filtration water samples, is that the filter membrane of 2～3 μ m carries out suction filtration with the aperture then.
3, the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in the potable water according to claim 1 is characterized in that in the step 1 earlier with 300 purpose screen filtration water samples, is that the filter membrane of 3 μ m carries out suction filtration with the aperture then.
4, the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in the potable water according to claim 1 and 2 is characterized in that pH value in the step 2 is that 7.2 buffer solution is phosphate buffer.
5, the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in the potable water according to claim 4 is characterized in that in the step 3 that with the attachment that step 2 is collected be centrifugal 15min under the condition of 3000r/min at rotating speed.
6, the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in the potable water according to claim 5 is characterized in that in the step 4 that at rotating speed be centrifugal 10min under the condition of 2000r/min.
7, according to the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in claim 1,2, the 5 or 6 described potable water, it is characterized in that in the step 4 sucrose solution from top to bottom mass concentration be followed successively by 13%～15% and 21%～24%.
8, the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in the potable water according to claim 7, it is characterized in that in the step 4 sucrose solution from top to bottom mass concentration be followed successively by 15% and 24%.
9, the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in the potable water according to claim 8 is characterized in that in the step 5 at rotating speed being that 3000r/min, centrifugation time are centrifugal concentrating under the condition of 10min.
10, the activity test method of Cryptosporidium egg capsule and giardia lamblia sporangiocyst in the potable water according to claim 9, the centrifugal 10 μ L that are concentrated in its characterization step five.
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| CN102191307A (en) * | 2010-03-11 | 2011-09-21 | 中国科学院城市环境研究所 | Method for enriching Cryptosporidium parvum oocysts and Giardia Lamblia sporocysts in water |
| CN101665820B (en) * | 2009-09-15 | 2012-03-21 | 上海交通大学 | Filtration and concentration method for improving detection recovery rate of cryptosporidium and giardia |
| CN104726337A (en) * | 2013-12-20 | 2015-06-24 | 中国科学院生态环境研究中心 | Method for enriching and purifying cryptosporidium and giardia in alga-containing water, and method for determining content of cryptosporidium and giardia |
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| CN101665820B (en) * | 2009-09-15 | 2012-03-21 | 上海交通大学 | Filtration and concentration method for improving detection recovery rate of cryptosporidium and giardia |
| CN102191307A (en) * | 2010-03-11 | 2011-09-21 | 中国科学院城市环境研究所 | Method for enriching Cryptosporidium parvum oocysts and Giardia Lamblia sporocysts in water |
| CN104726337A (en) * | 2013-12-20 | 2015-06-24 | 中国科学院生态环境研究中心 | Method for enriching and purifying cryptosporidium and giardia in alga-containing water, and method for determining content of cryptosporidium and giardia |
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| JP2020114243A (en) * | 2014-04-03 | 2020-07-30 | インターベット インターナショナル ベー． フェー． | Method of purifying coccidial oocysts from animal feces, system suitable for applying the method, and oocysts obtained therewith |
| CN107271413A (en) * | 2017-06-07 | 2017-10-20 | 暨南大学 | Improve Cryptosporidium and giardia lamblia detects the eluent and elution process of the rate of recovery |
| CN107271413B (en) * | 2017-06-07 | 2020-04-14 | 暨南大学 | Eluent for improving detection recovery rate of cryptosporidium and giardia and elution method |
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