CN112730414A - Method for efficiently detecting giardia and cryptosporidium in water - Google Patents
Method for efficiently detecting giardia and cryptosporidium in water Download PDFInfo
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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Abstract
The invention discloses a method for efficiently detecting giardia and cryptosporidium in water, belonging to the technical field of water quality detection, and the method for efficiently detecting the giardia and the cryptosporidium in the water comprises the following steps: sampling, randomly taking city water, filtering, elutriating, and elutriating the filtered water; concentrating, namely concentrating the water sample enrichment elutriation liquid; separating, namely separating the precipitate obtained by concentration; dyeing; microscopic examination; calculation and reporting of results; and (5) quality control, and performing error analysis on the calculation result.
Description
Technical Field
The invention belongs to the technical field of water quality detection, and particularly relates to a method for efficiently detecting giardia and cryptosporidium in water.
Background
Giardia (Giardia) is a pathogenic protozoan parasite, of which there are two major species, g. Infection of humans with giardia cysts (cysts) can lead to giardiasis (giardiasis), which typically manifests as acute giardiasis symptoms 5-6 days after infection, lasting for 1-3 weeks, including: diarrhea, abdominal pain, swelling, nausea, vomiting, and the like; chronic giardiasis symptoms include recurrent attacks, nutrient malabsorption, and may lead to debilitation.
Cryptosporidium (Cryptosporidium) is a protozoan parasite found in water and mainly consists of 6 species: C. parvum, c.baileyi, c.meleagris, c.muris, c.serpenti, c.nasorums, wherein c.parvum (cryptosporidium parvum) is currently known as the only cryptosporidium infecting humans. Infection with cryptosporidium oocysts (oocysts) can cause cryptosporidiosis, with the common symptom of watery diarrhea, which can be accompanied by dehydration, weight loss, abdominal pain, fever, nausea, and vomiting. The duration of general symptoms is short (1-2 weeks) for immunocompromised persons; the symptoms are serious and sometimes even life-threatening for people with low immunity.
The World Health Organization (WHO) fourth edition of the drinking water quality criteria introduces the harm and transmission routes of the two worms in detail, but does not list recommended limit values. WHO also ranks cryptosporidiosis as one of the suspected indicators of aids, and does not list the clear "two worms" detection limits. The detection limit value of 'two worms' is definitely specified in the 2006 edition drinking water quality standard in China to be less than 1/10L, and the standard addition recovery rate of the method is not less than 10%.
Various methods for detecting giardia and cryptosporidium have been established. The methods have the advantages of strong specificity, high sensitivity, good stability and simple operation. Mainly comprises the following steps: 1) an immunofluorescence method: the method has high sensitivity, specificity and repeatability, is beneficial to the separation and identification of the antigen and the antibody, and can be used for exploring the distribution of the antigen and the formation part of the antibody so as to research the essence of immune response and the pathological mechanism of parasite immunity. 2) Immunoblotting techniques: the method is a method combining polyacrylamide gel electrophoresis and immunoblotting technology. Can be used for detecting cryptosporidium and giardia, diagnosing cryptosporidium and giardiasis and analyzing specific antigen and antibody. The technology can separate the cryptosporidium oocyst and giardia sporangium antigens with high resolution, high sensitivity and high specificity, and is favorable for improving the detection rate of the two worms and the immunological diagnosis effect of the two worm diseases. 3) The PCR method comprises the following steps: strong specificity and high sensitivity. The method does not depend on human subjective factors, can simultaneously process samples in batches, selects different primers, can accurately identify the species of giardia and cryptosporidium, can distinguish cyst from cyst death and activity, and is a research hotspot in the field at present. 4) Enzyme-linked immunosorbent assay (ELISA): has high specificity, sensitivity, stability and repeatability. Can be used for detecting drinking water, sewage, serum, etc. The Homoexuan and the like establish an indirect ELISA method by using CP15/60 recombinant protein as a coating antigen, and the specificity is better. In the united states, ELISA kits have been widely used in clinical examinations. However, the above method is expensive. In addition, the dyeing method is to use organic dye to carry out fluorescence dyeing marking on the oocysts and the cysts for microscopic examination, and the dye is easy to decompose when exposed to light and is not beneficial to long-term storage of samples.
Disclosure of Invention
The invention aims to provide a method for efficiently detecting giardia and cryptosporidium in water, which has the advantages of greatly reduced detection cost, high recovery rate, quick and simple pretreatment and capability of simultaneously detecting a plurality of 'two-worm' samples.
In order to achieve the purpose, the invention provides the following technical scheme: a method for efficiently detecting Giardia and Cryptosporidium in water comprises the following steps:
a. sampling, and immediately taking city water and filtering;
b. elutriation, wherein the filtered water is elutriated;
c. concentrating, namely concentrating the water sample enrichment elutriation liquid;
d. separating, namely separating the precipitate obtained by concentration;
e. dyeing;
f. microscopic examination;
i. calculation and reporting of results;
j. and (5) quality control, and performing error analysis on the calculation result.
Preferably, the sampling comprises the steps of:
A. during sampling, the disposable filter element is arranged in the filter, and the filter is connected with the sampling connecting device;
B. and adjusting the flow rate of the peristaltic pump to be about 2L/min, and beginning to filter the water sample.
Preferably, the elutriation comprises the steps of:
A. after the water sample is filtered, the filter is arranged on the rapid elutriation device, and an elutriation inductor is arranged;
B. placing a 500mL centrifuge tube with a conical bottom on a collecting bracket, and starting automatic elutriation;
C. and after the elutriation is finished, taking the centrifugal tube down from the quick elutriation device, and closing the air compressor and the quick elutriation device.
Preferably, the concentration comprises the steps of:
A. centrifuging 2000 g of a 500mL centrifuge tube filled with the water sample enrichment elutriation solution for 15 min. Slow deceleration is required to avoid agitating the precipitate;
B. after centrifugation, 10mL of precipitate remained and the supernatant was carefully discarded.
Preferably, the separation comprises the steps of:
A. fully whirling, oscillating and mixing the centrifugal precipitate uniformly, and evenly distributing the centrifugal precipitate into 2 conical centrifugal tubes with the volume of 15 mL;
B. respectively washing 500mL centrifuge tubes with 1mL PBS solution for 4 times, and averagely distributing the washed PBST solution into the 2 centrifuge tubes with 15 mL;
C. adding 1mL of 10% formaldehyde solution and 5mL of ethyl acetate into each 15mL tube, and fully shaking up;
D. centrifuging for 10min, separating the centrifuge tube mixture into two layers, carefully discarding the supernatant, and leaving the lower layer precipitate for use.
Preferably, the dyeing comprises the following steps:
A. placing a PTFE membrane with the aperture of 1 μm or 3 μm and the diameter of 25 mm on a stainless steel filtering bracket, installing a filter cartridge, and opening a vacuum pump;
B. dripping 3mL of PBS buffer solution on the filter membrane, sucking the PBS buffer solution, and keeping a certain liquid level on the filter membrane without draining;
C. carefully pouring or dripping the enrichment in a 15mL centrifuge tube on a filter membrane, and performing suction filtration;
D. washing the centrifuge tube with 10mL of PBST solution for 2 times, pouring the cleaning solution into the filter tube and filtering;
E. cleaning the wall of the filter cylinder by using 10mL of PBST solution, sucking the cleaning solution and closing a suction filter valve;
F. dropping 500 mu L of 1% BSA right above the filter membrane, keeping for 2min, and sucking 1% BSA after 2 min;
G. transferring the microporous filter membrane onto a glass slide, dropwise adding 50 mu L of fluorescent staining agent onto the filter membrane, and standing for 30 min in a dark place;
H. adding 50 μ L DAPI working solution on the filter membrane, standing in dark for 10 min;
I. transferring the filter membrane to a suction filtration bracket, and sucking a fluorescent staining solution and a DAPI working solution;
J. eluting the filter membrane by using 3ml of PBST buffer solution, and sucking the PBST buffer solution;
K. dropping 1 drop of DABCO-glycerol on the glass slide, translating the filter membrane to the glass slide, dropping a drop of DABCO-glycerol solution on the filter membrane, and covering the glass slide.
Preferably, the microscopic examination comprises the following steps:
A. and (5) opening a microscope and a mercury lamp before microscopic examination, and preheating for 15 min.
B. Observing by using a 20-time objective lens in a 4FITC mode, switching to a DAPI mode after finding a target object, observing by using a 40-time objective lens, and observing an internal structure in a 100-time objective DIC mode when FITC and DAPI staining characteristics accord with the staining characteristics to further verify that the whole membrane is counted.
Preferably, the calculation and reporting of the result comprises:
number of spore (egg) sacs per liter of sample
Y=(X×V)/(V1×V2) （1）
In the formula:
y: the number of cysts or oocysts per liter of water; x: counting the number of cysts or oocysts obtained by a microscope;
v: volume of resuspension after centrifugal concentration, unit mL; v1: counting the volume of the sample in mL;
v2: the volume of the filtered water sample, in units of L.
Calculation of relative standard deviation
RSD=Y/X
Y2=∑(Xi-X)2/(N-1) （2）
X=∑Xi/N
In the formula: RSD: relative standard deviation; xi: the number of the recovery by adding the mark each time; n: the number of times of detection.
Detection limit
D=V/(V1×V2) （3）
In the formula: d: limit of detection per liter of cysts or oocysts; v: volume resuspended after concentration, Unit mL
V1: counting the volume of the sample in mL; v2: the volume of the water sample was filtered during concentration, in units of L.
Preferably, the quality control comprises:
negative control: A. adding 10L of pure water into a clean sample bottle;
B. through the detection steps of sample enrichment, elution, centrifugation, formaldehyde-ethyl acetate centrifugal purification, immunofluorescence staining, microscopic examination counting and the like, cryptosporidium oocysts and giardia cysts cannot be detected on a PTFE membrane during microscopic examination.
Positive control: A. adding 100-500 giardia cysts and cryptosporidium oocysts into a sample bottle filled with 10L of pure water, and fully and uniformly mixing;
B. enriching a sample by using an elutriation filter element, after the enrichment is finished, washing the same sample bottle by using 6L of pure water for 3 times, and enriching the eluted pure water in the same filter element;
C. the initial recovery rate of the cryptosporidium oocysts is 24-100%, and the relative standard deviation is less than 55%; the initial recovery of giardia cysts should range from 24% to 100% and the relative standard deviation should be less than 49%. Outside this range, all equipment and reagents were checked and each experimental step was checked for errors.
Actual sample standard adding recovery rate
The operation steps are as follows:
A. taking 3 parts of actual water sample, each 10L;
B. filtering and concentrating 1 part of 10L water sample, separating and purifying, dyeing and microscopic counting, and recording the number of cryptosporidium and giardia in the water sample.
C. Respectively adding 100-500 cryptosporidium and 100-500 giardia in other 2 water samples, filtering, concentrating, separating, purifying, dyeing and counting by microscopic examination to the added standard water sample, and recording the number of the cryptosporidium and the giardia in the added standard water sample.
D. Calculating the average recovery rate and relative standard deviation, wherein the recovery rate of the cryptosporidium is 13-111%, the relative standard deviation is less than 61%, the recovery rate of the giardia is 15-118%, and the relative standard deviation is less than 30%.
Calculating the background standard addition recovery rate:
R=(NSP-NS)/T
in the formula: r: recovery rate;
NSP: adding the number of the detected two insects in a standard water sample;
and NS: the number of "two worms" detected in the unstained water sample.
In the formula: x (mean): average detection number of the two worms;
MS: adding a standard to an actual water sample to detect the number of the two insects;
MSD: and repeating the number of the detected two insects in the actual water sample labeling.
In the formula: RPD: relative deviation of twice standard addition recovery rates;
NMS: the number of the detected two insects is marked in the first water sample;
NMSD: and the number of the detected two insects is marked on the water sample for the second time.
The invention has the beneficial effects that: the method uses ethyl acetate and formaldehyde to purify the two insects by centrifugation, and replaces the most expensive separation and purification reagent in the method of the United states EPA 1623: magnetic beads are subjected to immune separation, so that the detection cost is greatly reduced; the method has the advantages of high recovery rate and quick and simple pretreatment, can simultaneously detect a plurality of 'two-worm' samples, can be used for detecting giardia and cryptosporidium in drinking water and source water, and provides a basis for daily monitoring of health risk evaluation of the giardia and the cryptosporidium in the drinking water and the source water.
Detailed Description
In order to further understand the contents, features and effects of the present invention, the following examples are given and detailed below.
The method for efficiently detecting Giardia lamblia and Cryptosporidium in water according to the embodiment of the present invention will be described in detail below.
The method for efficiently detecting giardia and cryptosporidium in water comprises the following steps:
a. sampling, randomly taking urban water and filtering;
b. elutriation, wherein the filtered water is elutriated;
c. concentrating, namely concentrating the water sample enrichment elutriation liquid;
d. separating, namely separating the precipitate obtained by concentration;
e. dyeing;
f. microscopic examination;
i. calculation and reporting of results;
j. and (5) quality control, and performing error analysis on the calculation result.
The following test reagents and test equipment were used:
tween80 (Tween 80, CAS: 9005-65-6), analytically pure.
Tween20 (Tween 20, CAS: 9005-64-5), analytically pure.
50g/L sodium hypochlorite solution, analytically pure.
Elutriation buffer: 900 mL of pure water was charged with 1.44g of Na2HPO4, 0.24g of KH2PO4, 0.2g of KCl, and 8g of NaCl. Stirring to completely dissolve, adding 0.1 mL of Tween20, stirring for 10min, diluting with pure water to 1000mL, and making the components analytically pure.
0.1mol/L hydrochloric acid solution, analytically pure.
0.1mol/L sodium hydroxide solution, analytically pure.
DABCO (1, 4-diazobicyclo [2,2,2] octane, CAS: 280-57-9) -glycerol: weighing 12.6g of glycerol, heating to 60-70 ℃, adding 0.2g of DABCO, and stirring for dissolving. The product is stored for 12 months at room temperature and analyzed.
1% BSA (bovine serum albumin, CAS: 9048-46-8): 1 g of BSA was weighed, dissolved in 100 mL of purified water, filtered through a 0.45 μm filter and analyzed for purity.
PBS solution (phosphate buffer): each 1000mL PBS solution contained: 8.0 g NaCl, 2.9 g Na2HPO4.12H2O, 0.2g KCl, 0.2g KH2PO 4. Adjusting the pH value to 7.2-7.4 by using hydrochloric acid or sodium hydroxide. The components are kept at 4 ℃ for 1 week and are all analytically pure.
PBST solution: 0.01mL of Tween80 was added to 100 mL of PBS solution to obtain PBST solution. Storing at room temperature for 1 month.
The monoclonal fluorescent antibody-isosulfocyanic fluorescein staining kit for resisting giardia and cryptosporidium is stored at 4 deg.c.
Giardia, cryptosporidium standards.
Methanol (chromatographically pure).
DAPI stock solution: 2 mg of DAPI (4, 6-diamino-2-phenylindole, CAS: 28718-90-3) was dissolved in 1mL of methanol and stored at 4 ℃ in the dark for 15 days, DAPI: and (5) analyzing and purifying.
DAPI working solution: 50 μ L of DAPI stock was added to 50 mL PBS buffer. Prepared immediately before use, and stored at 4 ℃ in dark.
Ethyl acetate, analytically pure.
10% formaldehyde solution, analytically pure.
Instrumentation and equipment
Sampling equipment: a fast elutriation filter and a matched fast connecting device; quickly elutriating the filter element; a peristaltic pump (the flow rate reaches 0.3-2.0L/min); plastic sampling barrels (10L or 20L).
Elutriation/concentration/purification equipment: a fast elutriator; a centrifuge (centrifugal force can reach 2500g, and 500mL and 15mL centrifuge tubes are equipped); air compressor (pressure above 0.4MPa, 15L compressed air). A ten-thousandth electronic balance; glass pipettes or pasteur pipettes; conical centrifuge tube: 500mL and 15 mL; a pH meter; a vortex oscillator; a magnetic stirrer; a glass vacuum filtration device; a vacuum pump; a glass slide and a cover slip; a blood cell counter; a constant temperature incubator at 37 ℃; fluorescence microscopy: the device is provided with a blue filter of 450 nm to 480 nm, an ultraviolet filter of 330 nm to 385 nm and an objective lens of 20, 40 and 100 times. (ii) a PTFE (polytetrafluoroethylene) membranes with pore sizes of 1 μm and 3 μm and a diameter of 25 mm.
Examples
Sampling:
A. during sampling, the disposable filter element is arranged in the filter, and the filter is connected with the sampling connecting device;
B. and adjusting the flow rate of the peristaltic pump to be about 2L/min, and beginning to filter the water sample.
Elutriation:
A. after the water sample is filtered, the filter is arranged on the rapid elutriation device, and an elutriation inductor is arranged;
B. placing a 500mL centrifuge tube with a conical bottom on a collecting bracket, and starting automatic elutriation;
C. and after the elutriation is finished, taking the centrifugal tube down from the quick elutriation device, and closing the air compressor and the quick elutriation device.
Concentration:
A. centrifuging 2000 g of a 500mL centrifuge tube filled with the water sample enrichment elutriation solution for 15 min. Slow deceleration is required to avoid agitating the precipitate;
B. after centrifugation, 10mL of precipitate remained and the supernatant was carefully discarded.
Separation:
A. fully whirling, oscillating and mixing the centrifugal precipitate uniformly, and evenly distributing the centrifugal precipitate into 2 conical centrifugal tubes with the volume of 15 mL;
B. respectively washing 500mL centrifuge tubes with 1mL PBS solution for 4 times, and averagely distributing the washed PBST solution into the 2 centrifuge tubes with 15 mL;
C. adding 1mL of 10% formaldehyde solution and 5mL of ethyl acetate into each 15mL tube, and fully shaking up;
D. centrifuging for 10min, separating the centrifuge tube mixture into two layers, carefully discarding the supernatant, and leaving the lower layer precipitate for use.
Dyeing:
A. placing a PTFE membrane with the aperture of 1 μm or 3 μm and the diameter of 25 mm on a stainless steel filtering bracket, installing a filter cartridge, and opening a vacuum pump;
B. dripping 3mL of PBS buffer solution on the filter membrane, sucking the PBS buffer solution, and keeping a certain liquid level on the filter membrane without draining;
C. carefully pouring or dripping the enrichment in a 15mL centrifuge tube on a filter membrane, and performing suction filtration;
D. washing the centrifuge tube with 10mL of PBST solution for 2 times, pouring the cleaning solution into the filter tube and filtering;
E. cleaning the wall of the filter cylinder by using 10mL of PBST solution, sucking the cleaning solution and closing a suction filter valve;
F. dropping 500 mu L of 1% BSA right above the filter membrane, keeping for 2min, and sucking 1% BSA after 2 min;
G. transferring the microporous filter membrane onto a glass slide, dropwise adding 50 mu L of fluorescent staining agent onto the filter membrane, and standing for 30 min in a dark place;
H. adding 50 μ L DAPI working solution on the filter membrane, standing in dark for 10 min;
I. transferring the filter membrane to a suction filtration bracket, and sucking a fluorescent staining solution and a DAPI working solution;
J. eluting the filter membrane by using 3ml of PBST buffer solution, and sucking the PBST buffer solution;
K. dropping 1 drop of DABCO-glycerol on the glass slide, translating the filter membrane to the glass slide, dropping a drop of DABCO-glycerol solution on the filter membrane, and covering the glass slide.
Microscopic examination:
A. and (5) opening a microscope and a mercury lamp before microscopic examination, and preheating for 15 min.
B. Observing by using a 20-time objective lens in a 4FITC mode, switching to a DAPI mode after finding a target object, observing by using a 40-time objective lens, and observing an internal structure in a 100-time objective DIC mode when FITC and DAPI staining characteristics accord with the staining characteristics to further verify that the whole membrane is counted.
Calculation and reporting of results:
number of spore (egg) sacs per liter of sample
Y=(X×V)/(V1×V2) （1）
In the formula:
y: the number of cysts or oocysts per liter of water; x: counting the number of cysts or oocysts obtained by a microscope;
v: volume of resuspension after centrifugal concentration, unit mL; v1: counting the volume of the sample in mL;
v2: the volume of the filtered water sample, in units of L.
Calculation of relative standard deviation
RSD=Y/X
Y2=∑(Xi-X)2/(N-1) （2）
X=∑Xi/N
In the formula: RSD: relative standard deviation; xi: the number of the recovery by adding the mark each time; n: the number of times of detection.
Detection limit
D=V/(V1×V2) （3）
In the formula: d: limit of detection per liter of cysts or oocysts; v: volume resuspended after concentration, Unit mL
V1: counting the volume of the sample in mL; v2: the volume of the water sample was filtered during concentration, in units of L.
Quality control:
negative control: A. adding 10L of pure water into a clean sample bottle;
B. through the detection steps of sample enrichment, elution, centrifugation, formaldehyde-ethyl acetate centrifugal purification, immunofluorescence staining, microscopic examination counting and the like, cryptosporidium oocysts and giardia cysts cannot be detected on a PTFE membrane during microscopic examination.
Positive control: A. adding 100-500 giardia cysts and cryptosporidium oocysts into a sample bottle filled with 10L of pure water, and fully and uniformly mixing;
B. enriching a sample by using an elutriation filter element, after the enrichment is finished, washing the same sample bottle by using 6L of pure water for 3 times, and enriching the eluted pure water in the same filter element;
C. the initial recovery rate of the cryptosporidium oocysts is 24-100%, and the relative standard deviation is less than 55%; the initial recovery of giardia cysts should range from 24% to 100% and the relative standard deviation should be less than 49%. Outside this range, all equipment and reagents were checked and each experimental step was checked for errors.
Actual sample standard adding recovery rate
The operation steps are as follows:
A. taking 3 parts of actual water sample, each 10L;
B. filtering and concentrating 1 part of 10L water sample, separating and purifying, dyeing and microscopic counting, and recording the number of cryptosporidium and giardia in the water sample.
C. Respectively adding 100-500 cryptosporidium and 100-500 giardia in other 2 water samples, filtering, concentrating, separating, purifying, dyeing and counting by microscopic examination to the added standard water sample, and recording the number of the cryptosporidium and the giardia in the added standard water sample.
D. Calculating the average recovery rate and relative standard deviation, wherein the recovery rate of the cryptosporidium is 13-111%, the relative standard deviation is less than 61%, the recovery rate of the giardia is 15-118%, and the relative standard deviation is less than 30%.
Calculating the background standard addition recovery rate:
R=(NSP-NS)/T
in the formula: r: recovery rate;
NSP: adding the number of the detected two insects in a standard water sample;
and NS: the number of "two worms" detected in the unstained water sample.
In the formula: x (mean): average detection number of the two worms;
MS: adding a standard to an actual water sample to detect the number of the two insects;
MSD: and repeating the number of the detected two insects in the actual water sample labeling.
In the formula: RPD: relative deviation of twice standard addition recovery rates;
NMS: the number of the detected two insects is marked in the first water sample;
NMSD: and the number of the detected two insects is marked on the water sample for the second time.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A method for efficiently detecting Giardia and Cryptosporidium in water is characterized by comprising the following steps:
sampling, randomly taking urban water and filtering;
elutriation, wherein the filtered water is elutriated;
concentrating, namely concentrating the water sample enrichment elutriation liquid;
separating, namely separating the precipitate obtained by concentration;
dyeing;
microscopic examination;
calculation and reporting of results;
and (5) quality control, and performing error analysis on the calculation result.
2. The method for detecting giardia and cryptosporidium in water with high efficiency according to claim 1, wherein the sampling comprises the following steps:
A. during sampling, the disposable filter element is arranged in the filter, and the filter is connected with the sampling connecting device;
B. and adjusting the flow rate of the peristaltic pump to be about 2L/min, and beginning to filter the water sample.
3. The method for efficient detection of giardia and cryptosporidium in water according to claim 1, wherein the elutriation comprises the steps of:
A. after the water sample is filtered, the filter is arranged on the rapid elutriation device, and an elutriation sensor is arranged;
B. placing a 500mL centrifuge tube with a conical bottom on a collecting bracket, and starting automatic elutriation;
C. and after the elutriation is finished, taking the centrifugal tube down from the quick elutriation device, and closing the air compressor and the quick elutriation device.
4. The method for detecting giardia and cryptosporidium in water with high efficiency according to claim 1, wherein the concentration comprises the following steps:
A. centrifuging 2000 g of a 500mL centrifuge tube filled with the water sample enrichment elutriation liquid for 15 min;
slow deceleration is required to avoid agitating the precipitate;
B. after centrifugation, 10mL of precipitate remained and the supernatant was carefully discarded.
5. The method for detecting giardia and cryptosporidium in water with high efficiency according to claim 1, wherein the separation comprises the following steps:
A. fully whirling, oscillating and mixing the centrifugal precipitate uniformly, and evenly distributing the centrifugal precipitate into 2 conical centrifugal tubes with the volume of 15 mL;
B. respectively washing 500mL centrifuge tubes with 1mL PBS solution for 4 times, and averagely distributing the washed PBST solution into the 2 centrifuge tubes with 15 mL;
C. adding 1mL of 10% formaldehyde solution and 5mL of ethyl acetate into each 15mL tube, and fully shaking up;
D. centrifuging for 10min, separating the centrifuge tube mixture into two layers, carefully discarding the supernatant, and leaving the lower layer precipitate for use.
6. The method for detecting giardia and cryptosporidium in water with high efficiency according to claim 1, wherein the staining comprises the steps of:
A. placing a PTFE membrane with the aperture of 1 μm or 3 μm and the diameter of 25 mm on a stainless steel filtering bracket, installing a filter cartridge, and opening a vacuum pump;
B. dripping 3mL of PBS buffer solution on the filter membrane, sucking the PBS buffer solution, and keeping a certain liquid level on the filter membrane without draining;
C. carefully pouring or dripping the enrichment in a 15mL centrifuge tube on a filter membrane, and performing suction filtration;
D. washing the centrifuge tube with 10mL of PBST solution for 2 times, pouring the cleaning solution into the filter tube and filtering;
E. cleaning the wall of the filter cylinder by using 10mL of PBST solution, sucking the cleaning solution and closing a suction filter valve;
F. dropping 500 mu L of 1% BSA right above the filter membrane, keeping for 2min, and sucking 1% BSA after 2 min;
G. transferring the microporous filter membrane onto a glass slide, dropwise adding 50 mu L of fluorescent staining agent onto the filter membrane, and standing for 30 min in a dark place;
H. adding 50 μ L DAPI working solution on the filter membrane, standing in dark for 10 min;
I. transferring the filter membrane to a suction filtration bracket, and sucking a fluorescent staining solution and a DAPI working solution;
J. eluting the filter membrane by using 3ml of PBST buffer solution, and sucking the PBST buffer solution;
K. dropping 1 drop of DABCO-glycerol on the glass slide, translating the filter membrane to the glass slide, dropping a drop of DABCO-glycerol solution on the filter membrane, and covering the glass slide.
7. The method for detecting giardia and cryptosporidium in water with high efficiency according to claim 1, wherein the microscopic examination comprises the following steps:
A. opening a microscope and a mercury lamp before microscopic examination, and preheating for 15 min;
B. observing by using a 20-time objective lens in an FITC mode, switching to a DAPI mode after finding a target object, observing by using a 40-time objective lens, and observing an internal structure in a DIC mode of a 100-time objective lens when FITC and DAPI staining characteristics accord with the staining characteristics to further verify that the whole membrane is counted.
8. The method for detecting giardia and cryptosporidium in water with high efficiency as claimed in claim 1, wherein the calculation and reporting of the results comprises:
number of spore (egg) sacs per liter of sample
Y=(X×V)/(V1×V2) （1）
In the formula:
y: the number of cysts or oocysts per liter of water; x: counting the number of cysts or oocysts obtained by a microscope;
v: volume of resuspension after centrifugal concentration, unit mL; v1: counting the volume of the sample in mL;
v2: filtering the volume of the water sample in unit L;
calculation of relative standard deviation
RSD=Y/X
Y2=∑(Xi-X)2/(N-1) （2）
X=∑Xi/N
In the formula: RSD: relative standard deviation; xi: the number of the recovery by adding the mark each time; n: the number of times of detection;
detection limit
D=V/(V1×V2) （3）
In the formula: d: limit of detection per liter of cysts or oocysts; v: volume resuspended after concentration, Unit mL
V1: counting the volume of the sample in mL; v2: the volume of the water sample was filtered during concentration, in units of L.
9. The method for efficiently detecting giardia and cryptosporidium in water according to claim 1, wherein the quality control comprises:
negative control: A. adding 10L of pure water into a clean sample bottle;
B. through the detection steps of sample enrichment, elution, centrifugation, formaldehyde-ethyl acetate centrifugal purification, immunofluorescence staining, microscopic examination counting and the like, cryptosporidium oocysts and giardia cysts cannot be detected on a PTFE membrane during microscopic examination;
positive control: A. adding 100-500 giardia cysts and cryptosporidium oocysts into a sample bottle filled with 10L of pure water, and fully and uniformly mixing;
B. enriching a sample by using an elutriation filter element, after the enrichment is finished, washing the same sample bottle by using 6L of pure water for 3 times, and enriching the eluted pure water in the same filter element;
C. the initial recovery rate of the cryptosporidium oocysts is 24-100%, and the relative standard deviation is less than 55%; the initial recovery rate of the giardia cysts ranges from 24% to 100%, and the relative standard deviation is less than 49%;
if the concentration is not within the range, all equipment and reagents need to be checked, and whether each experimental step is wrong or not needs to be checked;
actual sample standard adding recovery rate
The operation steps are as follows:
A. taking 3 parts of actual water sample, each 10L;
B. filtering and concentrating 1 part of 10L water sample, separating and purifying, dyeing and performing microscopic examination and counting, and recording the number of cryptosporidium and giardia in the water sample;
C. respectively adding 100-500 cryptosporidium and 100-500 giardia in other 2 water samples, filtering, concentrating, separating, purifying, dyeing and counting by microscopic examination to the added standard water sample, and recording the number of the cryptosporidium and the giardia in the added standard water sample;
D. calculating the average recovery rate and relative standard deviation, wherein the recovery rate of cryptosporidium is 13-111%, the relative standard deviation is less than 61%, the recovery rate of giardia is 15-118%, and the relative standard deviation is less than 30%;
calculating the background standard addition recovery rate:
R=(NSP-NS)/T
in the formula: r: recovery rate;
NSP: adding the number of the detected two insects in a standard water sample;
and NS: the number of two worms detected in the water sample without the addition of the standard;
in the formula: x (mean): average detection number of the two worms;
MS: adding a standard to an actual water sample to detect the number of the two insects;
MSD: repeating the number of the two worms detected by adding the standard to the actual water sample;
in the formula: RPD: relative deviation of twice standard addition recovery rates;
NMS: the number of the detected two insects is marked in the first water sample;
NMSD: and the number of the detected two insects is marked on the water sample for the second time.
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