Method for detecting and inactivating cryptosporidium in excrement of large-scale cattle farm

Abstract

The invention belongs to but is not limited to the technical field of excrement treatment, and particularly relates to a method for detecting and inactivating cryptosporidium in excrement of a large-scale cattle farm, which comprises the following steps: S1, preparing a cryptosporidium standard sample; s2, purification and microscope detection of cryptosporidium parvum in feces; s3, carrying out acid-fast staining on the oocysts of cryptosporidium; s4, fluorescence PCR detection of cryptosporidium in feces; s5, carrying out DNA quality analysis; and S6, performing qPCR (quantitative polymerase chain reaction) detection. The qPCR reaction is optimized in three aspects of primer concentration, probe concentration and amplification program, and the detection limit of the detection method in a sensitivity determination experiment after optimization is 5 oocysts. In other words, fluorescence signals still exist in the five oocysts, and the fluorescence signals can still be detected through the method under the condition that the five oocysts exist. In addition, by applying the fluorescent quantitative PCR method established by the invention, the geographical distribution and infection rate of cryptosporidiosis are obtained. Data reference is provided for prevention, control and research of cryptosporidiosis, and a foundation is laid for the quality standard of experimental cattle.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the technical field of manure treatment, and in particular relates to a method for detecting and inactivating Cryptosporidium in manure of large-scale cattle farms. Background Art

[0002] In recent years, with the rapid development of China's economy and the continuous improvement of people's living standards, the demand for livestock products has continued to grow, the intensive and large-scale proportion of livestock and poultry farming has continued to expand, the demand for beef market has increased, and beef cattle farming has set off a boom. However, a large amount of manure and sewage will be produced in the process of beef cattle farming. Due to the single way of farmers to deal with manure and sewage and the weak environmental awareness, or the backward treatment technology or inadequate facilities of the farms, the manure and sewage pollution caused great harm to the ecological environment. Manure and sewage refers to the general term for feces, urine and sewage produced in the process of livestock and poultry farming. Its excretion is related to factors such as breed, weight, daily diet formula, feed intake, climate, season, etc. In the process of development of cattle farming, the problem of cattle dung pollution is very serious. Wastes such as feces and sewage will cause serious pollution to the soil, water sources and air near the cattle farms. While affecting the ecological environment in the farms and surrounding areas, it has also aroused widespread social concern and restricted the sustainable development of the industry.

[0003] Intestinal parasitic diseases of cattle caused by large amounts of manure produced in large-scale cattle farms during the breeding process are now a serious problem. Intestinal parasitic diseases are common diseases that endanger the healthy development of beef cattle breeding. Due to their chronic and consumptive pathogenic characteristics, they generally do not show obvious clinical symptoms, but long-term parasitism can cause symptoms such as slow growth, diarrhea, anemia and emaciation in cattle, leading to decreased production performance, meat quality and feed conversion rate. Surveys show that intestinal parasitic infections of cattle are relatively common in Jilin Province, especially in pastoral areas. There are many types of parasites and the infection intensity is high, which affects the healthy development of the cattle industry and brings certain economic losses to farmers and herdsmen. Therefore, in the current development of the cattle industry, the harmless treatment of cattle dung has become an important part of the work, which can effectively prevent and control ecological pollution and promote the sustainable development of the cattle industry.

[0004] It is inevitable that a large amount of manure will be produced in livestock and poultry farming. How to effectively treat manure and improve resource utilization efficiency is the focus of current farmers. If waste is not handled properly, it will cause water pollution, because there are still a lot of phosphorus, nitrogen, parasites and pathogens in livestock and poultry manure. If it is discharged directly without treatment, it will cause water pollution, affect the health of consumers, and increase the risk of disease transmission. Cryptosporidium infection is transmitted by oocysts, and water and food contaminated by oocysts are the main routes of transmission. This protozoa is mainly transmitted through the fecal-oral route, and has caused many foodborne and waterborne outbreaks and epidemics worldwide. Reports of outbreaks of the disease are on the rise, and the breadth and severity of its harm are becoming more and more difficult to ignore.

[0005] Livestock and poultry infected with Cryptosporidium store a large number of oocysts in their bodies, which can be excreted with feces. Under suitable external environmental conditions, they can develop into infectious larvae. If feed and drinking water are contaminated, it is easy to cause repeated infection of Cryptosporidium in livestock and poultry. The use of feces composting utilization mode, feces energy utilization mode, feces fertilizer utilization mode, sewage standard discharge mode, etc. for harmless treatment and resource utilization can completely kill Cryptosporidium eggs and larvae, as well as other pathogenic microorganisms. At present, the harmless treatment technology of livestock and poultry manure includes fertilizer, feed, energy and pyrolysis technology. Feed technology is the main way to comprehensively utilize manure. This technology mainly includes drying method, direct feed method, fermentation method and chemical agent treatment method. Drying method can be divided into multi-layer ventilation drying, mechanical stirring drying and heating rapid drying. Drying method can kill pathogenic bacteria, harmful microorganisms and eggs in feces to meet the hygiene standards of feed production. The cost of treating feed with chemical agents is relatively high. Generally, NaOH, CH2O, CH3COOH, etc. are used to treat livestock and poultry manure to remove harmful microorganisms, kill insect eggs and eliminate odors.

[0006] Pathogenic microorganisms and parasite eggs contained in livestock and poultry excrement can easily cause the spread of diseases between livestock and poultry, seriously affecting the growth of livestock and even causing their death. Moreover, these microorganisms can easily enter the human body through water or livestock products, posing a serious threat to human health. Cryptosporidium is an important opportunistic pathogenic protozoan that is widely prevalent worldwide and is mainly transmitted through the fecal-oral route. Currently, the main inspection methods for Cryptosporidium include microscopic examination, immunological examination, molecular biological examination and other methods. Molecular biological detection methods represented by PCR require relatively clean nucleic acid substances in the sample to be tested as reaction templates, so obtaining sample DNA with high purity is crucial for PCR amplification.

[0007] In view of the above analysis, the technical problems that need to be urgently solved in the existing technology are: the current means of inspection for Cryptosporidium mainly include microscopic inspection, immunological inspection, molecular biological inspection, etc. Molecular biological detection methods represented by PCR require relatively clean nucleic acid substances in the sample to be tested as reaction templates, so obtaining sample DNA with high purity is crucial for PCR amplification. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a method for detecting and inactivating Cryptosporidium in cattle farm manure on a large scale.

[0009] The present invention is achieved by a method for detecting and inactivating Cryptosporidium in large-scale cattle farm manure, comprising the following steps:

[0010] Prepare Cryptosporidium standard samples and determine dilution concentrations by microscopy;

[0011] The fecal samples were enriched and purified by saturated magnesium sulfate flotation, ether defatting and sucrose method to obtain Cryptosporidium oocysts.

[0012] The Cryptosporidium oocysts are treated with photosensitizers in combination with ultraviolet rays. Under ultraviolet irradiation, the photosensitizers produce reactive oxygen species that inactivate the oocysts.

[0013] DNA was extracted from feces and the Cryptosporidium oocyst content was detected by fluorescent PCR technology to evaluate the inactivation efficiency.

[0014] Furthermore, the photosensitizer is a soluble porphyrin compound or a phthalimide compound with a concentration range of 10-50 μM. After being diluted to a specified concentration, the compound is fully mixed with the Cryptosporidium oocysts, left to adsorb for 30-60 minutes, and then irradiated with ultraviolet light.

[0015] Furthermore, the ultraviolet irradiation conditions are as follows: the wavelength is 254nm and the intensity is 0.2-0.5mW / cm 2 The treatment time is 15-30 minutes, and the distance between the ultraviolet light source and the sample is controlled at 5-10 cm.

[0016] Furthermore, the fluorescent PCR was used to detect the changes in the Cryptosporidium DNA concentration to evaluate the synergistic UV inactivation effect, and agarose gel electrophoresis was used to verify the integrity damage of the DNA after inactivation to ensure that the inactivation efficiency reached more than 95%.

[0017] The present invention provides a method for detecting and inactivating Cryptosporidium in large-scale cattle farm manure, comprising:

[0018] S1, preparation of Cryptosporidium standard sample;

[0019] S2, purification and microscopic detection of Cryptosporidium parvum in feces;

[0020] S3, acid-fast staining of Cryptosporidium oocysts;

[0021] S4, fluorescent PCR detection of Cryptosporidium in feces;

[0022] S5, DNA quality analysis;

[0023] S6, qPCR detection.

[0024] Furthermore, S1 specifically includes:

[0025] The standard insect species sample was supplemented with PBS to 1 mL and then diluted 10 times, mixed and stained, 5 μl was taken for microscopic examination, and repeated 10 times to determine that it contained (3±1) oocysts. The dilution was added to a plastic bucket containing 500 mL of fecal sewage, and enriched and purified using the method 2.1.1 of the present invention for use.

[0026] Further, S2 specifically includes: enriching and purifying Cryptosporidium parvum oocysts by the method of "saturated magnesium sulfate flotation-ether degreasing-sucrose purification".

[0027] (1) The feces sample collected from the farm was filtered out of impurities using a 60-mesh sieve, 2.5% potassium dichromate was added to the filtrate in a 1:1 ratio, and the filtrate was placed in a 4°C refrigerator.

[0028] (2) Take out an appropriate amount of fecal sample from the refrigerator and place it in a 4°C horizontal centrifuge at 4000 r / min for 10 min. After discarding the supernatant, pour in distilled water and shake upside down. Centrifuge again for a total of 3 washes.

[0029] (3) Add an appropriate amount of saturated magnesium sulfate solution, mix by inversion, let stand at room temperature for 40 minutes, then centrifuge at 500g for 1 minute and discard the precipitate.

[0030] (4) Transfer the supernatant to a 50 mL centrifuge tube, add distilled water to dilute the supernatant, centrifuge at 3500 r / min for 10 min, discard the supernatant, and keep the precipitate.

[0031] (5) Add the same volume of distilled water, stir and blow up the precipitate to resuspend it, add the same volume of anhydrous ether again and shake vigorously for 20 min to defat it, let it stand for 5 min, centrifuge at 3500 r / min for 10 min, pour off the upper layer of ether, lipid and water layer mixture, and collect the precipitate.

[0032] (6) Prepare a saturated sucrose solution. Dilute the original sucrose solution with sterile water at a volume ratio of 1:1 and 1:4. Add 10 mL of the 1:1 diluted sucrose solution to a 30 mL centrifuge tube and slowly add 10 mL of the 1:4 diluted sucrose solution along the wall of the bottle to cover it and separate it into layers for later use.

[0033] (7) Add an appropriate amount of sterile water to the defatted fecal sediment and stir to mix well. Slowly add 2-4 mL of the suspension to the layered sucrose solution and centrifuge horizontally at 2000 r / min for 10 min.

[0034] (8) After centrifugation, three layers of suspension can be seen in the 30 mL centrifuge tube. The upper, middle and lower layers are potassium dichromate solution, white oocysts and fecal pellets, respectively. The white oocysts in the middle layer are aspirated and placed in a 50 mL centrifuge tube. The residual sucrose solution is washed away with four times the volume of distilled water. Centrifuge at 3500 r / min for 10 min, discard the supernatant and keep the precipitate.

[0035] (9) Add a small amount of sterile water to blow up the precipitate, then add 20 mL of 10% sodium hypochlorite solution, let stand at room temperature for 10 min to kill bacteria, add distilled water to 50 mL, centrifuge at 3500 r / min for 10 min, and wash with water to remove sodium hypochlorite.

[0036] (10) Add a small amount of sterile water to blow up the precipitate and use a kit to perform acid-fast staining and count under a microscope.

[0037] Further, S3 specifically includes: using an acid-fast staining kit (modified Kinyoun cold staining method) to stain Cryptosporidium oocysts, the specific method is as follows:

[0038] (1) Dip the purified Cryptosporidium oocyst suspension into an inoculation loop, spread it on a glass slide, and allow it to dry naturally.

[0039] (2) Add modified Kinyoun fuchsin staining solution and stain for 5 min. Wash off excess dye with distilled water.

[0040] (3) Decolorize with Kinyoun decolorizing solution for 3 min and rinse with distilled water.

[0041] (4) Stain with methylene blue solution for 5 minutes and rinse with distilled water.

[0042] (5) Gently absorb the moisture and let it dry naturally.

[0043] (6) Oil immersion microscopy.

[0044] Furthermore, S4 specifically includes:

[0045] (1) DNA extraction from feces

[0046] The solid-liquid mixed feces sample was centrifuged at 6000r / min for 20min, and 400mg of the precipitate was weighed and DNA was extracted according to the following 3 methods.

[0047] (2) CTAB method

[0048] Add 1 ml of CTAB buffer [2% CTAB, 0.1 mol / L Tris (pH 8.0), 20 mmol / L EDTA (pH 8.0) and 1.4 mol / L NaCl] to the sample, mix well, centrifuge twice at 12 000 r / min in a water bath, take the supernatant, add equal volumes of Tris-saturated phenol-chloroform-isoamyl alcohol (25:24:1) and equal volumes of chloroform-isoamyl alcohol (24:1), centrifuge for 5 min, take the supernatant, add 2.5 times pre-cooled

[0049] The precipitate was washed twice with 1 ml of 70% ethanol, centrifuged for 3 min, and the supernatant was removed. The precipitate was dried at room temperature for 15 min, and 50 μl of TE buffer (Solarbio, China) was added and stored at -20°C for later use.

[0050] (3) SDS method:

[0051] 500 μl of SDS lysis buffer [1% SDS, 0.5 mol / L Tris-HCl (pH 8.0), 0.1 mol / L ETA (pH 8.0) and 0.1 mol / L NaCl], 500 μl of sodium phosphate buffer (0.3 mol / L, pH 8.0) and 20 μl (20 g / L) proteinase K (Solarbio, China) were added to the sample, mixed well, incubated in a water bath, centrifuged at 12 000 r / min for 10 min, and the supernatant was collected.

[0052] Add 0.6 times the amount of 4 mol / L sodium acetate (pH 8.3), place at room temperature for 5 min, centrifuge for 5 min, remove the supernatant, add 0.8 times the amount of isopropanol, place at -20°C for 30 min, centrifuge for 5 min, remove the supernatant, add 1 ml of 70% ethanol, centrifuge for 3 min, repeat the operation with anhydrous ethanol, centrifuge for 3 min, remove the supernatant, dry at room temperature for 15 min, add 50 μl TE buffer, and store at -20°C for later use.

[0053] (4) Sample DNA was extracted using the TIANamp Stool DNA Kit. The procedure is as follows:

[0054] (1) Add anhydrous ethanol to buffer GD and rinse buffer PW according to the bottle label, and add isopropanol to buffer GFA.

[0055] (2) Place the stool sample in a 2 ml centrifuge tube and place the tube on ice.

[0056] (3) Add 500 μL of buffer SA, 100 μL of buffer SC, 15 μL of Proteinase K, and 0.25 g of grinding beads to the sample and oscillate for 3 min until the sample is fully mixed.

[0057] (4) Incubate in a 70°C water bath for 15 min, shaking 2-3 times during incubation.

[0058] (5) Vortex for 15 seconds, centrifuge at 12,000 rpm for 3 minutes, and transfer the supernatant to a new centrifuge tube.

[0059] (6) Add 200 μL of buffer SH, shake to mix, place on ice for 5 min, and centrifuge at 12,000 rpm for 3 min.

[0060] (7) Transfer the supernatant obtained in the previous step to a new 1.5 ml centrifuge tube and add 750 μL of buffer GFA.

[0061] (8) Add the solution obtained in the previous step to an adsorption column (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube.

[0062] (9) Add 500 μL of buffer GD to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube.

[0063] (10) Add 700 μL of rinse solution PW to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube.

[0064] (11) Wash once more, add 700 μL of washing solution PW, centrifuge at 12,000 rpm for 1 min, and pour out the liquid in the centrifuge tube.

[0065] (12) Place the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Leave the adsorption column at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0066] (13) Transfer the adsorption column into a clean centrifuge tube, add 30 μL of sterile enzyme-free water dropwise to the middle part of the adsorption membrane at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution in the centrifuge tube.

[0067] (14) To increase the yield of genomic DNA, the solution obtained by centrifugation was added to the adsorption column, placed at room temperature for 2 minutes, and centrifuged at 12,000 rpm for 2 minutes. 30 μL of oocyst DNA was obtained. The amount of genomic DNA obtained was evaluated using the Nanodrop 1000 nucleic acid protein quantifier. The extracted samples were stored at -20°C for PCR specific amplification.

[0068] Furthermore, S5 specifically includes: taking 5 μl of DNA extracted from the three methods and loading it on a 1% agarose gel, and judging the integrity of the DNA by the electrophoresis results. Take 2 μl of the DNA product each time, and use a Nano Drop 2000 spectrophotometer (Thermo, USA) to determine the concentration (mg / L) and purity A260 / A280 value of the total DNA. The sample absorbance value (A) is calculated by the formula A=-log(Is / Ib), where Is represents the transmitted light intensity of the sample, and Ib represents the transmitted light intensity of the blank control. The above two light intensities are automatically detected, recorded and saved by the instrument during the measurement. When the A260 / A280 value is 1.8-2.0, it means that the DNA is relatively pure. If it is higher than 2.0, it indicates that the RNA in the product has not been removed cleanly. If it is lower than 1.8, it indicates that the product contains phenol or protein (Xia et al. 2019). Each sample was measured three times, with three parallel samples each time.

[0069] Furthermore, S6 specifically includes:

[0070] (1) Determination of qPCR standard curve

[0071] Cryptosporidium oocysts were counted under a fluorescence microscope and diluted 10-fold to 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 / mL oocysts, and genomic DNA was extracted for the construction of quantitative PCR standard curves. The qPCR reaction system was 20μl, which contained 10μl of ChamQ Geno-SNP Probe Master Mix, 0.5μl of upstream and downstream primers (10μmol / L), 0.3μl of probe (10μmol / L), 2μl of DNA template, and 6.7μl of ddH2O. Reaction procedure: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s, 60℃ annealing for 30s, 45 cycles. Three parallel samples were performed for each test, and a no-template negative control was set for each test.

[0072] (2) Design of specific primers and probes

[0073] The gene sequences of different species of Cryptosporidium were obtained from the public database (http: / / Cryptodb.org). The Hsp70 gene was used as the target gene, and MAGA was used for multiple comparisons to screen out the conserved sequences shared by each species. According to the basic principles of Primer design, there was no hairpin structure between primers and between primers and probes, and the length of the amplified target fragment was between 80 and 150 bp. Appropriate primers and probes were selected and sent to Shanghai Shenggong Biotechnology Co., Ltd. for synthesis.

[0074] (3) Optimization of qPCR reaction conditions

[0075] 1) Primer concentration optimization

[0076] Take 5×10 6 Cryptosporidium oocysts were isolated from the culture medium at 100 μg / mL and DNA was extracted using the QIAamp DNA Mini Kit.

[0077] Based on the primer concentration adjustment range (0.1-1 μM) in the reaction in the qPCR kit manual, the present invention selected 5 groups of primer concentrations for screening: 0.1 μM, 0.3 μM, 0.5 μM, 0.7 μM, and 1 μM, and used the Hsp70 probe for qPCR to determine the optimal primer concentration in the system.

[0078] 2) Optimization of probe concentration in qPCR reaction

[0079] Take 5×10 6 DNA was extracted from oocysts of 100 g / mL using QIAamp DNA Mini Kit.

[0080] Based on the probe concentration adjustment range (50-250 nM) in the reaction in the qPCR kit manual, the present invention selected 5 groups of probe concentrations for screening: 50 nM, 100 nM, 150 nM, 200 nM, and 250 nM. Hsp70 probe was used for qPCR, and the optimal primer concentration screened in the previous article was used to determine the optimal probe concentration in the qPCR reaction.

[0081] 3) qPCR reaction program optimization

[0082] Take 5×10 6 DNA was extracted from oocysts of 100 g / mL using QIAamp DNA Mini Kit.

[0083] Using the optimal primer concentration and optimal probe concentration determined above, the group annealing time was set in a gradient based on the annealing time adjustment range in the qPCR reagent manual, and qPCR was performed using the Hsp70 probe.

[0084] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0085] The present invention optimizes the qPCR reaction through three aspects: primer concentration, probe concentration, and amplification procedure. After optimization, the detection limit of our detection method in the sensitivity determination experiment is 5 oocysts. That is, within 5 oocysts, the fluorescent signal still exists, indicating that this method can still detect 5 oocysts. In addition, this experiment is used to establish a fluorescent quantitative PCR method to obtain the geographical distribution and infection rate of cryptosporidiosis. It provides data reference for the prevention, control and research of cryptosporidiosis, and lays the foundation for the quality standards of experimental cattle.

[0086] 2. The present invention has developed a method for inactivating Cryptosporidium by using a photosensitizer in conjunction with ultraviolet light. When the photosensitizer concentration is 50 ug / mL and the ultraviolet irradiation time is 20 min, the inactivation efficiency of this method is the best. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 It is a flow chart of a method for detecting and inactivating Cryptosporidium in large-scale cattle farm manure provided by an embodiment of the present invention;

[0088] Figure 2 is a photosensitizer synthesis diagram provided by an embodiment of the present invention;

[0089] Figure 3 This is a diagram of the electrophoresis result of extracting genomic DNA provided by an embodiment of the present invention;

[0090] Figure 4 is the qPCR amplification curve provided by the embodiment of the present invention Figure 1 ;

[0091] Figure 5 is a standard curve diagram of the qPCR detection method provided by an embodiment of the present invention;

[0092] Figure 6 is the qPCR amplification curve provided by the embodiment of the present invention Figure 2 ;

[0093] Figure 7 is the qPCR amplification curve provided by the embodiment of the present invention Figure 3 ;

[0094] Figure 8 is the qPCR amplification curve provided by the embodiment of the present invention Figure 4 ;

[0095] Fig. 9 9qPCR amplification curve provided in the embodiment of the present invention Figure 5 ;

[0096] Fig.10 is the qPCR amplification curve provided by the embodiment of the present invention Figure 6 ;

[0097] Fig.11 is the qPCR amplification curve provided by the embodiment of the present invention Figure 7 ;

[0098] Fig.12 This is a picture of Cryptosporidium improved acid-fast staining (1000×) provided in an embodiment of the present invention.

[0099] Fig.13 Schematic diagram of the survival of Escherichia coli at different times of ultraviolet irradiation after treatment with a photosensitizer provided in an embodiment of the present invention; wherein (a) 25ug / mL, (b) 50ug / mL, (c) 100ug / mL;

[0100] Fig.14 Schematic diagram of the effect of different photosensitizer concentrations and different treatment times on the inactivation of Escherichia coli provided by an embodiment of the present invention;

[0101] Fig.15 Schematic diagram of Cryptosporidium under DAPI / PI staining excitation light source provided in an embodiment of the present invention; wherein (a) PI staining, (b) DAPI staining;

[0102] Fig.16 Schematic diagram of the effect of different photosensitizer concentrations and different treatment times on the inactivation of Cryptosporidium provided by an embodiment of the present invention;

[0103] Fig.17 Schematic diagram of the effect of the inactivation method provided by an embodiment of the present invention on the conductivity of Cryptosporidium;

[0104] Fig.18 It is a schematic diagram of the effect of the inactivation method provided in an embodiment of the present invention on the soluble protein of Cryptosporidium;

[0105] Fig.19 Schematic diagram of the effect of the inactivation method provided by an embodiment of the present invention on the leakage of Cryptosporidium nucleic acid;

[0106] Fig. 20 1 is a display diagram of the AnnexinV-FITC / PI dual-color labeled flow cytometry detection results provided by an embodiment of the present invention; a is a normal Cryptosporidium oocyst group, b is a photosensitizer-assisted ultraviolet inactivation group, and c is a single ultraviolet inactivation group;

[0107] Fig.21 is a scanning electron microscope (SEM) result diagram provided by an embodiment of the present invention;

[0108] Fig. 22 1 is an electrophoresis diagram of the Cryptosporidium genome in different treatment modes provided in an embodiment of the present invention; (M is Maker; 1 is a control group; 2 and 3 are single ultraviolet treatments for 10 min and 20 min, respectively; 4, 5, and 6 are photosensitizer-assisted ultraviolet treatments for 10 min, 20 min, and 30 min, respectively). DETAILED DESCRIPTION

[0109] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0110] The preparation of standard samples of Cryptosporidium is a basic step to ensure the accuracy of the test. First, add the Cryptosporidium species sample to 1mL of PBS solution, dilute it 10 times, mix it and then stain it. Then, take 5μL of the sample for microscopic observation and repeat the inspection 10 times to ensure that each microscopic examination contains 3±1 oocysts. Next, add the diluted sample to a plastic bucket containing 500mL of fecal sewage for enrichment and purification, and store it for future use to ensure its quality and standard.

[0111] The enrichment and purification process of Cryptosporidium parvum oocysts adopts the "saturated magnesium sulfate flotation-ether defatting-sucrose purification" method. First, the fecal sample is filtered through a 60-mesh sieve to remove impurities, 2.5% potassium dichromate is added and shaken, and then stored in a refrigerator at 4°C. Next, the sample is taken out of the refrigerator, centrifuged in a centrifuge, the supernatant is removed and the precipitate is washed with distilled water, and the washing is repeated three times. Then, a saturated magnesium sulfate solution is added, and the precipitate is removed after standing for 40 minutes. Next, the Cryptosporidium oocysts are further purified through steps such as ether defatting and sucrose solution stratification, and finally a purified sample is obtained.

[0112] In the acid-fast staining process of Cryptosporidium oocysts, the purified oocyst suspension is first applied to a glass slide, and after it is naturally dried, a modified Kinyoun fuchsin stain is added for staining for 5 minutes. After that, the excess dye is washed off with distilled water, followed by decolorization with Kinyoun decolorizing solution, and then rinsed with distilled water. Next, the final staining is performed with methylene blue staining solution for 5 minutes, and finally rinsed with distilled water, the water is gently blotted, and it is naturally dried to prepare for observation under a microscope.

[0113] Cryptosporidium oocysts were detected by fluorescent PCR technology. First, DNA was extracted from feces and a fluorescent PCR reaction system was prepared. The reaction system included ChamQ Geno-SNP Probe Master Mix, upstream and downstream primers, probes, DNA templates, and ddH2O. The PCR reaction program was set to 95°C pre-denaturation for 30 seconds, 95°C denaturation for 10 seconds, 60°C annealing for 30 seconds, and 45 cycles. Three parallel samples were set for each experiment, and a negative control without template was set up. The content of Cryptosporidium oocysts was monitored in real time by qPCR reaction.

[0114] After the Cryptosporidium DNA is extracted, the quality of the DNA sample needs to be analyzed. First, the extracted DNA is analyzed by agarose gel electrophoresis, and the integrity of the DNA is determined by the electrophoresis results. Secondly, the DNA concentration and purity are determined using a Nano Drop 2000 spectrophotometer, and the A260 / A280 value is recorded. In general, the A260 / A280 value should be between 1.8 and 2.0. If it is lower than 1.8, it indicates the presence of protein or phenol contamination. If it is higher than 2.0, it may contain RNA. Ensure that the DNA quality meets the experimental requirements through repeated measurements and analysis.

[0115] In the qPCR test, the number of Cryptosporidium oocysts is first counted by fluorescence microscopy, and the sample DNA is diluted according to the count of oocysts to construct a standard curve. The qPCR reaction system is 20μL, including ChamQ Geno-SNP ProbeMaster Mix, primers, probes, DNA templates and ddH2O. Subsequently, the reaction conditions are optimized, including the selection of primer concentration, probe concentration and annealing temperature, to obtain the best reaction effect. The establishment of the standard curve helps to accurately quantify the concentration of Cryptosporidium oocysts in the sample, thereby improving the sensitivity and accuracy of the test.

[0116] 1 Materials

[0117] 1.1 Sample collection and processing

[0118] The samples were collected from June to December 2022. Five large-scale beef cattle farms in Changchun were selected as sampling points. Each farm was sampled once every three months, with three replicates each time. 500g of sediment samples and 5L of aquaculture wastewater were collected at the sewage outlet of each farm. The aquaculture wastewater was filtered through a 5.0μm filter membrane to remove zooplankton and phytoplankton. After the samples were collected, they were immediately sent to the laboratory for storage at 4°C, and the subsequent DNA extraction and determination of related indicators were completed within 48 hours. The basic information of the samples is shown in Table 1.

[0119] Table 1 Basic information of samples

[0120]

[0121] 1.2 Main experimental instruments and equipment

[0122] The instruments used in the experiment are shown in Table 2.

[0123] Table 2 Experimental instruments

[0124]

[0125] 1.3 Main experimental consumables and reagents

[0126] The drugs used in the experiment are shown in Table 3.

[0127] Table 3 Experimental drugs and reagents

[0128]

[0129]

[0130] 1.4 Preparation of main reagents

[0131] (1) Saturated magnesium sulfate solution

[0132] Preparation of saturated magnesium sulfate solution: Add 287.5 g of anhydrous magnesium sulfate and distilled water to make up to 1000 mL.

[0133] (2) Sucrose solution

[0134] Preparation of saturated sucrose stock solution: 500 g sucrose was dissolved in 320 mL distilled water. The sucrose stock solution was diluted in a ratio of 1:1 and 1:4 to obtain 50% sucrose solution and 25% sucrose solution.

[0135] (3) 2.5% potassium dichromate solution

[0136] Preparation of 2.5% potassium dichromate solution: Add 25g potassium dichromate to distilled water and make up to 1000mL.

[0137] 1.5 Standard insect species

[0138] Cryptosporidium oocysts: The standard species was provided by Professor Zhang Xichen of Jilin University.

[0139] 1.6 Experimental setup and operation

[0140] The UV disinfection test device uses a UV parallel beam instrument made by our laboratory senior brother Chen. A 14W low-voltage UV lamp (Xuguang Factory, Nanjing, Jiangsu, model: X6500) is set at the top of the device, and the vertical distance between the lamp and the test water sample is a variable control height. A circular automatic turntable with a diameter of 40cm is set below, and a sample slot is set above the turntable. The sample slot specifications are 2cm long × 2cm wide × 5cm high. The entire irradiation device is in a light-proof state.

[0141] 1.7 Test microorganisms

[0142] Escherichia coli: The strain (Ecoil.322) provided by Jilin Provincial Animal Husbandry and Veterinary Science Research Institute was activated and cultured and then placed in a 4°C refrigerator for use.

[0143] Cryptosporidium oocysts: Use the purified oocysts in Chapter 2, 2.1.1 of this article and place them in a 4°C refrigerator for later use.

[0144] 2 Methods such as Figure 1 Shown

[0145] 2.1 Establishment and application of methods for detecting and inactivating Cryptosporidium in large-scale cattle farm manure

[0146] 2.1.1 Preparation of Cryptosporidium standard samples

[0147] The standard insect species sample was supplemented with PBS to 1 mL and then diluted 10 times, mixed and stained, 5 μl was taken for microscopic examination, and repeated 10 times to determine that it contained (3±1) oocysts. The dilution was added to a plastic bucket containing 500 mL of fecal sewage, and enriched and purified using the method 2.1.1 of the present invention for use.

[0148] 2.1.2 Purification and microscopic detection of Cryptosporidium parvum in feces

[0149] The invention adopts the method of "saturated magnesium sulfate floating-ether defatting-sucrose purification" to enrich and purify Cryptosporidium parvum oocysts.

[0150] (1) The feces sample collected from the farm was filtered out of impurities using a 60-mesh sieve, 2.5% potassium dichromate was added to the filtrate in a 1:1 ratio, and the filtrate was placed in a 4°C refrigerator.

[0151] (2) Take out an appropriate amount of fecal sample from the refrigerator and place it in a 4°C horizontal centrifuge at 4000 r / min for 10 min. After discarding the supernatant, pour in distilled water and shake upside down. Centrifuge again for a total of 3 washes.

[0152] (3) Add an appropriate amount of saturated magnesium sulfate solution, mix by inversion, let stand at room temperature for 40 minutes, then centrifuge at 500g for 1 minute and discard the precipitate.

[0153] (4) Transfer the supernatant to a 50 mL centrifuge tube, add distilled water to dilute the supernatant, centrifuge at 3500 r / min for 10 min, discard the supernatant, and keep the precipitate.

[0154] (5) Add the same volume of distilled water, stir and blow up the precipitate to resuspend it, add the same volume of anhydrous ether again and shake vigorously for 20 min to defat it, let it stand for 5 min, centrifuge at 3500 r / min for 10 min, pour off the upper layer of ether, lipid and water layer mixture, and collect the precipitate.

[0155] (6) Prepare a saturated sucrose solution. Dilute the original sucrose solution with sterile water at a volume ratio of 1:1 and 1:4. Add 10 mL of the 1:1 diluted sucrose solution to a 30 mL centrifuge tube and slowly add 10 mL of the 1:4 diluted sucrose solution along the wall of the bottle to cover it and separate it into layers for later use.

[0156] (7) Add an appropriate amount of sterile water to the defatted fecal sediment and stir to mix well. Slowly add 2-4 mL of the suspension to the layered sucrose solution and centrifuge horizontally at 2000 r / min for 10 min.

[0157] (8) After centrifugation, three layers of suspension can be seen in the 30 mL centrifuge tube. The upper, middle and lower layers are potassium dichromate solution, white oocysts and fecal pellets, respectively. The white oocysts in the middle layer are aspirated and placed in a 50 mL centrifuge tube. The residual sucrose solution is washed away with four times the volume of distilled water. Centrifuge at 3500 r / min for 10 min, discard the supernatant and keep the precipitate.

[0158] (9) Add a small amount of sterile water to blow up the precipitate, then add 20 mL of 10% sodium hypochlorite solution, let stand at room temperature for 10 min to kill bacteria, add distilled water to 50 mL, centrifuge at 3500 r / min for 10 min, and wash with water to remove sodium hypochlorite.

[0159] (10) Add a small amount of sterile water to blow up the precipitate and use a kit to perform acid-fast staining and count under a microscope.

[0160] 2.1.2 Acid-fast staining of Cryptosporidium oocysts

[0161] Use the acid-fast staining kit (modified Kinyoun cold staining method) to stain Cryptosporidium oocysts. The specific method is as follows:

[0162] (1) Dip the purified Cryptosporidium oocyst suspension into an inoculation loop, spread it on a glass slide, and allow it to dry naturally.

[0163] (2) Add modified Kinyoun fuchsin staining solution and stain for 5 min. Wash off excess dye with distilled water.

[0164] (3) Decolorize with Kinyoun decolorizing solution for 3 min and rinse with distilled water.

[0165] (4) Stain with methylene blue solution for 5 minutes and rinse with distilled water.

[0166] (5) Gently absorb the moisture and let it dry naturally.

[0167] (6) Oil immersion microscopy.

[0168] 2.1.5 Fluorescence PCR detection of Cryptosporidium in feces

[0169] 2.1.5.1 DNA extraction from feces

[0170] The solid-liquid mixed feces sample was centrifuged at 6000r / min for 20min, and 400mg of the precipitate was weighed and DNA was extracted according to the following 3 methods.

[0171] 2.1.5.1 CTAB method

[0172] Add 1 ml of CTAB buffer [2% CTAB, 0.1 mol / L

[0173] Tris (pH 8.0), 20mmol / L EDTA (pH 8.0) and 1.4mol / L NaCl], mix well and then water bath, centrifuge twice at 12000r / min to take the supernatant, add equal volumes of Tris-saturated phenol-chloroform-isoamyl alcohol (25:24:1) and equal volumes of chloroform-isoamyl alcohol (24:1), centrifuge for 5min, take the supernatant, add 2.5 times pre-cooled

[0174] The precipitate was washed twice with 1 ml of 70% ethanol, centrifuged for 3 min, and the supernatant was removed. The precipitate was dried at room temperature for 15 min, and 50 μl of TE buffer (Solarbio, China) was added and stored at -20°C for later use.

[0175] 2.1.5.2SDS method:

[0176] 500 μl of SDS lysis buffer [1% SDS, 0.5 mol / L Tris-HCl (pH 8.0), 0.1 mol / L ETA (pH 8.0) and 0.1 mol / L NaCl], 500 μl of sodium phosphate buffer (0.3 mol / L, pH 8.0) and 20 μl (20 g / L) proteinase K (Solarbio, China) were added to the sample, mixed well, incubated in a water bath, centrifuged at 12 000 r / min for 10 min, and the supernatant was collected.

[0177] Add 0.6 times the amount of 4 mol / L sodium acetate (pH 8.3) and place at room temperature

[0178] Place for 5 minutes, centrifuge for 5 minutes, take the supernatant, add 0.8 times the amount of isopropyl

[0179] Alcohol, placed at -20℃ for 30min, centrifuged for 5min and removed the supernatant.

[0180] Add 1 ml of 70% ethanol, centrifuge for 3 min, and repeat with anhydrous ethanol.

[0181] Operation, centrifuge for 3 minutes to remove the supernatant, dry at room temperature for 15 minutes,

[0182] Add 50 μl TE buffer and store at -20℃ for later use.

[0183] For example: Qiagen kit (QIAamp Fast DNA Stool Mini Kit, 51604, Germany) method:

[0184] Take 400 mg of sample and operate according to the instructions. The volume of the dissolved sample after extraction is 50 μl and store it at -20℃ for future use.

[0185] The TIANamp Stool DNA Kit was used to extract sample DNA. The procedure is as follows:

[0186] (1) Add anhydrous ethanol to buffer GD and rinse buffer PW according to the bottle label, and add isopropanol to buffer GFA.

[0187] (2) Place the stool sample in a 2 ml centrifuge tube and place the tube on ice.

[0188] (3) Add 500 μL of buffer SA, 100 μL of buffer SC, 15 μL of Proteinase K, and 0.25 g of grinding beads to the sample and oscillate for 3 min until the sample is fully mixed.

[0189] (4) Incubate in a 70°C water bath for 15 min, shaking 2-3 times during incubation.

[0190] (5) Vortex for 15 seconds, centrifuge at 12,000 rpm for 3 minutes, and transfer the supernatant to a new centrifuge tube.

[0191] (6) Add 200 μL of buffer SH, shake to mix, place on ice for 5 min, and centrifuge at 12,000 rpm for 3 min.

[0192] (7) Transfer the supernatant obtained in the previous step to a new 1.5 ml centrifuge tube and add 750 μL of buffer GFA.

[0193] (8) Add the solution obtained in the previous step to an adsorption column (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube.

[0194] (9) Add 500 μL of buffer GD to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube.

[0195] (10) Add 700 μL of rinse solution PW to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube.

[0196] (11) Wash once more, add 700 μL of washing solution PW, centrifuge at 12,000 rpm for 1 min, and pour out the liquid in the centrifuge tube.

[0197] (12) Place the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Leave the adsorption column at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0198] (13) Transfer the adsorption column into a clean centrifuge tube, add 30 μL of sterile enzyme-free water dropwise to the middle part of the adsorption membrane at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution in the centrifuge tube.

[0199] (14) To increase the yield of genomic DNA, the solution obtained by centrifugation was added to the adsorption column, placed at room temperature for 2 minutes, and centrifuged at 12,000 rpm for 2 minutes. 30 μL of oocyst DNA was obtained. The amount of genomic DNA obtained was evaluated using the Nanodrop 1000 nucleic acid protein quantifier. The extracted samples were stored at -20°C for PCR specific amplification.

[0200] 2.1.6 DNA quality analysis

[0201] Take 5 μl of DNA extracted from the above three methods and load it on 1% agarose gel. The integrity of DNA is determined by electrophoresis results. Take 2 μl of DNA product each time and use

[0202] Nano Drop 2000 spectrophotometer (Thermo, USA) was used to measure the total DNA concentration (mg / L) and purity A260 / A280 value. The sample absorbance value (A) was calculated using the formula A = -log (Is / Ib),

[0203] In the formula, Is represents the transmitted light intensity of the sample, and Ib represents the transmitted light intensity of the blank control. The above two light intensities are automatically detected, recorded and saved by the instrument during the measurement. When the A260 / A280 value is 1.8-2.0, it means that the DNA is relatively pure. If it is higher than 2.0, it indicates that the RNA in the product is not completely removed. If it is lower than 1.8, it indicates that the product contains phenol or protein (Xia et al. 2019). Each sample was measured three times, with three parallel samples each time.

[0204] 2.1.6 Establishment of qPCR detection method

[0205] 2.1.6.1 Determination of qPCR standard curve

[0206] Cryptosporidium oocysts were counted under a fluorescence microscope and diluted 10-fold to 10 6 , 10 5 , 10 4 , 10 3 , 10 2, 10 / mL oocysts, and genomic DNA was extracted for the construction of quantitative PCR standard curves. The qPCR reaction system was 20μl, which contained 10μl of ChamQ Geno-SNP Probe Master Mix, 0.5μl of upstream and downstream primers (10μmol / L), 0.3μl of probe (10μmol / L), 2μl of DNA template, and 6.7μl of ddH2O. Reaction procedure: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s, 60℃ annealing for 30s, 45 cycles. Three parallel samples were performed for each test, and a no-template negative control was set for each test.

[0207] 2.1.6.2 Design of specific primers and probes

[0208] The gene sequences of different species of Cryptosporidium were obtained from the public database (http: / / Cryptodb.org). The Hsp70 gene was used as the target gene, and MAGA was used for multiple comparisons to screen out the conserved sequences shared by each species. According to the basic principles of Primer design, there was no hairpin structure between primers and between primers and probes, and the length of the amplified target fragment was between 80 and 150 bp. Appropriate primers and probes were selected and sent to Shanghai Shenggong Biotechnology Co., Ltd. for synthesis. The sequence information is shown in Table 4.

[0209] Table 4 Primer / probe design sequences

[0210]

[0211] 2.1.6.3 Optimization of qPCR reaction conditions

[0212] (1) Primer concentration optimization

[0213] Take 5×10 6 Cryptosporidium oocysts were isolated from the culture medium at 100 μg / mL and DNA was extracted using the QIAamp DNA Mini Kit.

[0214] Based on the adjustment range of primer concentration in the reaction in the qPCR kit manual (0.1-1 μM), the present invention selected 5 groups of primer concentrations for screening: 0.1 μM, 0.3 μM, 0.5 μM, 0.7 μM, 1 μM, and used Hsp70 probe for qPCR to determine the optimal primer concentration in the system. The qPCR reaction system is shown in Table 5, and the reaction procedure is shown in Table 6.

[0215] Table 5 qPCR reaction system

[0216]

[0217] Table 6 qPCR reaction procedure

[0218]

[0219] (2) Optimization of probe concentration in qPCR reaction

[0220] Take 5×10 6 DNA was extracted from oocysts of 100 g / mL using QIAamp DNA Mini Kit.

[0221] Based on the probe concentration adjustment range (50-250 nM) in the reaction in the qPCR kit manual, the present invention selected 5 groups of probe concentrations for screening: 50 nM, 100 nM, 150 nM, 200 nM, and 250 nM. Hsp70 probe was used for qPCR, and the optimal primer concentration screened in the previous article was used to determine the optimal probe concentration in the qPCR reaction.

[0222] Table 7 qPCR reaction system

[0223]

[0224] Table 8 qPCR reaction program

[0225]

[0226] (3) qPCR reaction program optimization

[0227] Take 5×10 6 DNA was extracted from oocysts of 100 g / mL using QIAamp DNA Mini Kit.

[0228] Using the optimal primer concentration and optimal probe concentration determined above, based on the annealing time adjustment range in the qPCR reagent manual, 6 sets of annealing times were set in a gradient, and qPCR was performed using the Hsp70 probe.

[0229] Table 9 qPCR reaction system

[0230]

[0231] Table 10 qPCR reaction program

[0232]

[0233] 2.1.6.4 qPCR Methodology Validation

[0234] (1) Specificity test of qPCR method

[0235] The DNA genomes of Cryptosporidium, Giardia, Eimeria, and BVDV were obtained respectively, and the samples were used as template DNA for fluorescence quantitative PCR to verify the specificity of this method. The reaction system was the same as that in 2.1.6.

[0236] (2) Verification of the detection limit of the qPCR method

[0237] According to the steps in method 2.2.4, a series of experiments were conducted to explore the detection limit of the method by adding 100 Cryptosporidium to 10L of feces, 100 to 50L, 5 to 50L, and 20 to 100L. After enrichment and purification, DNA was extracted and the DNA content of all spiked samples was determined by qPCR to estimate the number of oocysts. The qPCR reaction system was the same as 2.1.6.

[0238] (3) Verify the accuracy of qPCR

[0239] In this experiment, a minimum of 5 Cryptosporidium was spiked into 500L of feces for Cryptosporidium oocyst enrichment, DNA extraction and purification, and qPCR detection. Three parallel experiments were set up, and the qPCR reaction system was the same as 2.1.6.

[0240] 2.1.9 Detection of Cryptosporidium in Large-scale Cattle Farm Manure

[0241] All clinical samples were purified according to the method in 2.1.1 of this article, and DNA was extracted according to the method in 2.1.5, and qPCR was performed, and the results obtained were compared with the results of microscopic examination.

[0242] 2.1.10 Statistical analysis

[0243] The experimental data obtained by fluorescence quantitative PCR method were analyzed using SPSS26.0 software.

[0244] 2.2 Study on the inactivation of Cryptosporidium and Escherichia coli by photosensitizer and UV

[0245] 2.2.1 Synthesis and characterization of photosensitizers

[0246] The photosensitizer material used in this experiment is 2,6-diiodo-boron-dipyrromethene (2I-BODIPY). The main synthesis process is as follows: Figure 2 As shown, the steps are:

[0247] Take 1mL (10mmoL) benzaldehyde and 2mL (21mmoL) 2,4-dimethylpyrrole and dissolve them in 400mL dichloromethane, add 5 drops of trifluoroacetic acid (TFA) as a catalyst, react under nitrogen protection and avoid light for 12h, then add 2.27g DDQ to react for 4h, then add 10mL triethylamine to react for 30min, and finally add 12mL boron trifluoride ether to react for 2h to end. Post-treatment: wash with water, dry (anhydrous magnesium sulfate), filter, column (n-hexane: dichloromethane = 5:1); iodide: BODIPY:NIS = 1:3, stir at room temperature for 2h, observe on the plate, stop the reaction, spin dry and column (n-hexane: dichloromethane = 7:1). Then centrifuge and dialyze the supernatant with water to obtain 2I-BODIPY.

[0248] Photosensitive particles were prepared by precipitation method and named (2I-BODIPY). The powder was dissolved in chloroform and its structure was verified by 1H NMR and ESI-MS. Analysis of the results of 1H NMR and ESI-MS proved that the new compound was successfully synthesized.

[0249] The particle size measured by DLS is 200nm. In order to better study the relationship between the optical properties and structure of the compound, the present invention performs spectral detection, and the results are shown in the figure, indicating that the photosensitizer can generate free radicals under ultraviolet spectrum irradiation, and has good water stability. After standing for 60 days, its particle size has no obvious change.

[0250] 2.2.1 Configuration of photosensitizer

[0251] The initial concentration is 2mg / mL. Take 20mg of photosensitizer, dissolve it in 5ml of acetone, add it to 10mL of distilled water, stir it in a fume hood for 30min, and after the acetone evaporates, you will get a photosensitizer dispersion. Dilute the photosensitizer dispersion to 25ug / mL, 50ug / mL, and 100ug / mL concentrations, and store it in a 4℃ refrigerator away from light.

[0252] 2.2.2 Sample preparation

[0253] 2.2.2.1 Escherichia coli

[0254] In this experiment, the live bacteria plate colony counting method was used to prepare a specific concentration of Escherichia coli.

[0255] (1) Sterilize the prepared LB agar medium in an autoclave at 121°C for 20 min. When cooled to 45°C, pour it into a disposable bacterial culture dish.

[0256] (2) Take out the purified E. coli and activate it at room temperature for 30 minutes in a sterile environment to keep the sample active and easy to observe.

[0257] (3) Transfer 1 mL of bacterial solution to a 1.5 mL test tube, mix well, and vortex for 30 seconds to resuspend the bacteria in the liquid.

[0258] (4) Take a sterile 1.5 mL centrifuge tube, draw 900 μL of sterile water, and mark 10 in sequence. -1 -10 -6 , three sets of repeated markings.

[0259] (5) Use a pipette to take 100 μL of the activated E. coli stock solution and add -1 1.5mL centrifuge tube with the word " ” on it, rinse the pipette tip several times to mix the solution thoroughly. -1 Pipette 100 μL of the solution into the tube marked 10. -2 1.5mL centrifuge tube, this is a 10-fold dilution, and so on and so forth until the dilution reaches 10 -6 . Change the pipette tip each time to prevent the sample concentration difference from being too large. Set up 3 parallel experimental groups for each sampling point.

[0260] (6) Take the bacterial solution with a concentration of 1×10 -5 , 1×10 -6 and 1×10 -7 Spread. Use a pipette to transfer 20 μL of bacterial solution to the center of the sterile solid culture medium each time, and use a sterile spreading stick to spread the bacterial solution gently outward along the concentric circle direction to make it evenly distributed. Do 3 parallels for each group.

[0261] (7) Place the coated culture dish in a 37°C incubator and incubate upside down for 24 hours.

[0262] (8) After counting, dilute the original bacterial solution to 1×10 7 CFU / mL and place in a 4℃ refrigerator for later use.

[0263] 2.2.2.2 Cryptosporidium oocysts

[0264] Purify Cryptosporidium oocysts according to method 2.1.1, dilute to a certain multiple and count. Use the blood cell counting method to count Cryptosporidium oocysts under a 400× microscope. Repeat three times and take the average value. The calculation formula 3-1 is as follows:

[0265] The total number of Cryptosporidium in 1mL sample = (A / 5) × 25 × 10 × 1000 × B = 50000 A·B (pieces) (3-1)

[0266] A: total number of squares in five grids; B: dilution multiple

[0267] According to the counting results, dilute the oocyst stock solution to 1×10 7 / mL, and store in a 4℃ refrigerator for later use.

[0268] 2.2.3 Photosensitizer and UV inactivation treatment

[0269] Turn on the UV lamp for preheating 20 minutes before the experiment to keep the UV radiation intensity stable during the experiment. Take a certain volume of sample suspension and mix it with photosensitizers of different concentrations and place it in a sterilized quartz dish with a diameter of 100 mm. Put it in the sample slot of the turntable. At the same time, ensure that all quartz dishes are directly under the UV parallel beam instrument so that the samples receive the same UV radiation intensity.

[0270] According to the concentration of photosensitizer, it was divided into three groups: 25ug / mL, 50ug / mL, and 100ug / mL. The control group was a sample suspension irradiated with ultraviolet light without adding photosensitizer, and the blank group was a single bacterial liquid. Three parallel samples were tested each time, and one sample was taken for testing every 2 minutes, and the continuous testing time was 20 minutes. This experiment investigated the inactivation effect of ultraviolet irradiation time of 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 minutes. Each experiment was paralleled 3 times and the average value was taken.

[0271] 2.2.4 Analysis of inactivation effect

[0272] 2.2.4.1 Inactivation effect on Escherichia coli

[0273] The traditional plate colony counting method was used to evaluate the inactivation effect of photosensitizer on Escherichia coli. The specific steps are as follows:

[0274] (1) Use a pipette to transfer each group of samples after irradiation to a new centrifuge tube for centrifugation, discard the supernatant, and add sterile water to resuspend the precipitate.

[0275] (2) Take a sterile 1.5 mL centrifuge tube, draw 900 μL of sterile water, use a pipette to take 100 μL of activated E. coli stock solution, and dilute to a concentration of 10 -1 -10 -6 Bacterial liquid.

[0276] (3) Use a pipette to take 100 μL of the activated E. coli stock solution and add -1 1.5mL centrifuge tube with the word " ” on it, rinse the pipette tip several times to mix the solution thoroughly. -1 Pipette 100 μL of the solution into the tube marked 10. -2 1.5mL centrifuge tube, this is a 10-fold dilution, and so on and so forth until the dilution reaches 10 -6 . Change the pipette tip each time to prevent the sample concentration difference from being too large. Set up 3 parallel experimental groups for each sampling point.

[0277] (4) Use a pipette to transfer 100 μL of bacterial solution to the center of the sterile solid culture medium, and use a sterile spreading stick to spread the bacterial solution outwards along concentric circles to ensure even distribution. Perform three parallel experiments for each group.

[0278] (5) Place the coated culture dish in a 37°C incubator and incubate it upside down for 24 hours.

[0279] (6) Count the number of bacteria on the culture dish, deduce the bacterial concentration, calculate the mortality rate of E. coli, and draw a graph.

[0280] 2.2.4.2 Inactivation effect of Cryptosporidium oocysts

[0281] Immunofluorescence detection (DAPI / PI staining) was used to count the number of live and dead oocysts. The specific steps are as follows:

[0282] (1) After irradiation, the Cryptosporidium sample was centrifuged to remove the supernatant, and then washed with water to retain the precipitate.

[0283] (2) Add 3 times the volume of the sample to be stained with staining solution and mix well.

[0284] (4) Incubate the tube for 5-10 minutes in a dark room. Centrifuge for 3 minutes, remove the DAPI staining solution, and wash with PBS 2-3 times, 3-5 minutes each time.

[0285] (5) Centrifuge and discard the supernatant. Tap the tube to resuspend the precipitate in the residual liquid and add 1 mL of room temperature PBS.

[0286] (6) Slowly add the cells to 3 mL of -20°C anhydrous ethanol while stirring at high speed. Fix for 5-15 minutes.

[0287] (7) Centrifuge to discard ethanol, tap the bottom of the tube to loosen the precipitate, add 2-5 mL of room temperature PBS and let stand for 15 minutes to rehydrate the cells.

[0288] (8) Centrifuge the sample, remove the supernatant and resuspend the cells in fresh buffer.

[0289] (9) Pipette 1 drop of suspension onto a glass slide, cover with a coverslip and observe under a fluorescence microscope with an excitation wavelength of 360-400 nm. Fluorescent dyes all have quenching problems. Try to complete the test 2 hours after staining to avoid false positives under fluorescence microscopy. After smearing, examine under a 1000x fluorescence microscope (OLYMPUS, BX-51).

[0290] (10) Observe under the 800× DAPI mode and examine under a fluorescence microscope. The outer side of the cell wall is light blue and the inside of the cell is not stained, which is DAPI+; the inside of the cell is not stained and the cell wall is not stained red, which is PI-; the nucleus is stained red, which is PI+; among them, DAPI+ / PI-type is active Cryptosporidium; DAPI- / P-type is inactive; only DAPI+ / PI+ type is dead Cryptosporidium. The live-death ratio in the same field of view is used to calculate the mortality rate of Cryptosporidium. The treated samples are counted separately under an 800× microscope, and the average value is taken after three repetitions.

[0291] 2.3 Mechanism of photosensitizer-UV inactivation of Cryptosporidium

[0292] 2.3.1 Sample preparation

[0293] Select the Cryptosporidium samples with a photosensitizer concentration of 50ug / mL, UV time of 20min, single UV-treated samples, and single oocyst suspension. After the irradiation test, centrifuge and take the supernatant for later use.

[0294] 2.3.2 Effect of inactivation methods on the permeability of Cryptosporidium oocyst wall

[0295] The permeability of Cryptosporidium oocyst wall is reflected by the change of conductivity in the suspension. The above-treated solutions were centrifuged at 4500r / min for 15min, and the supernatant was taken and measured by Ultrameter II TM The conductivity was measured by a multi-parameter analyzer, with 3 replicates per group.

[0296] 2.3.3 Effect of inactivation methods on Cryptosporidium soluble proteins

[0297] The concentration of dissolved protein in the system was detected using a Nanodrop 2000 ultra-micro spectrophotometer. The tyrosine, phenylalanine and tryptophan residues in the protein molecules make them absorb ultraviolet light at 280nm, and the absorbance is proportional to the protein content. Then write SDS.

[0298] SDS-PAGE gel electrophoresis: Prepare the separation gel according to the formula in Table 11, mix the separation gel and immediately add it between the two glass plates, then add an appropriate amount of isopropanol, let it stand for 40 minutes to allow the gel to naturally condense, pour off the upper isopropanol, and dry it with filter paper. Prepare the concentrated gel according to the table, mix the concentrated gel and immediately add it to the separation gel, insert a suitable electrophoresis comb between the two glass plates, and let it stand for 40 minutes to allow it to naturally condense. The volume of protein solution loaded in each well is about 20μL. Add the above-treated solutions to the protein loading buffer, mix, boil in a boiling water bath for 10 minutes, centrifuge at 12000r / min for 3 minutes, take the supernatant and cool it for use. Insert the condensed electrophoresis plate into the electrophoresis tank, pour in the electrophoresis buffer, unplug the electrophoresis comb, and carefully add the treated protein sample to the loading well. Perform electrophoresis at a voltage of 220V throughout the process until the protein completely passes through the separation gel and the electrophoresis is terminated. The samples were stained with SDS-PAGE staining solution (Coomassie Brilliant Blue), destained with SDS-PAGE destaining solution, and imaged with a gel imaging system.

[0299] Table 11 SDS-PAGE gel preparation method

[0300]

[0301] 2.3.4 Effect of inactivation methods on nucleic acid leakage from Cryptosporidium

[0302] The leakage of nucleic acid in Cryptosporidium was reflected by measuring the change of OD value of the suspension at 260nm absorbance. The above-treated solutions were centrifuged at 4500r / min for 15min, and the supernatant was collected and the absorbance (OD 260nm ). Referring to the method of Chen et al., OD 260nm It represents the relative amount of extracellular nucleic acid, and each group was tested in triplicate.

[0303] The DNA concentration range of the CTAB method is 18.29-28.95 mg / L. The DNA concentration range of the SDS method is 13.74-16.89 mg / L, and the DNA concentration range of the kit method is 20.18-78.92 mg / L. The A concentration of the DNA extracted by the SDS method is lower, but the purity (A260 / A280 value) is the purest. Figure 3 As shown, the present invention adopts the CTAB method to extract DNA.

[0304] 3. Establishment and application results of qPCR detection method

[0305] 3.1 qPCR standard curve determination results

[0306] The Cryptosporidium concentration was adjusted to 10 6 , 10 5 , 104 , 10 3 , 10 2 , 10 / mL oocysts, and genomic DNA was extracted and then qPCR was performed. The results are as follows Figure 4 , it can be seen that the display 10 6 The standard samples with concentrations up to 10 / mL of oocysts all had relatively regular and obvious fluorescence signals. When the oocyst concentration was 10 / mL, an obvious amplification curve was also visible. The obtained data were plotted in a graph, with the logarithm of oocysts at different concentration gradients on the horizontal axis and the Ct value obtained by qPCR amplification using DNA as a template on the vertical axis. Figure 5 , the standard curve and regression equation of Cryptosporidium qPCR detection method were obtained: y = -3.417x + 43.76. 2 The linear relationship is good.

[0307] 3.2 qPCR Methodology Validation Results

[0308] (1) Specificity test results of qPCR method

[0309] The DNA of Cryptosporidium, Giardia, Eimeria, and Bovine Viral Diarrhea Virus were obtained and amplified by fluorescent quantitative PCR to verify the specificity of this method. Hsp70 primers and probes were added to the qPCR system, and the templates were DNA of individual species for detection. The results are shown in the figure below. Only Cryptosporidium amplified the curve, and other pathogenic microorganisms and negative samples were not detected. It can be seen that this method has good specificity.

[0310] (2) Detection limit results of qPCR method

[0311] As can be seen from the figure, when the oocyst concentration is 5 / mL, the curve can still be amplified, indicating that this method can detect a minimum of 5 / mL oocysts and has good sensitivity.

[0312] (3) qPCR accuracy results

[0313] As shown in the figure, the amplified curve was substituted into the regression equation and was consistent with the number of spiked oocysts, indicating that this method has good accuracy.

[0314] 3.3qPCR reaction condition optimization results

[0315] (1) Primer concentration optimization results in qPCR reaction

[0316] Based on the adjustment range of primer concentration in the reaction in the qPCR kit manual (0.1-1 μM), the present invention selected 5 groups of primer concentrations for screening: 0.1 μM, 0.3 μM, 0.5 μM, 0.7 μM, and 1 μM, and used the Hsp70 probe for qPCR. The results are as follows Fig. 9 From Table 12, it can be seen that when the primer concentration is 0.5 μM, the Ct is the lowest, that is, the qPCR amplification efficiency is the highest at this primer concentration, so the primer concentration is determined to be 0.5 μM.

[0317] Table 12 qPCR Ct values

[0318]

[0319] (2) Optimization results of probe concentration in qPCR reaction

[0320] Take 5×10 6 DNA was extracted from oocysts using the QIAamp DNA Mini Kit.

[0321] Based on the probe concentration adjustment range (50-250 nM) in the qPCR kit manual, the present invention selected 5 groups of probe concentrations for screening: 50 nM, 100 nM, 150 nM, 200 nM, and 250 nM. Hsp70 probe was used for qPCR, and the optimal primer concentration screened in the previous article was used to determine the optimal probe concentration in the qPCR reaction. The results are as follows Fig.10 As shown in Table 13, when the probe concentration was 250 nM, the Ct of qPCR was the lowest, indicating that the amplification efficiency was the best.

[0322] Table 13 qPCR Ct values

[0323]

[0324] (3) qPCR reaction program optimization results

[0325] Using the optimal primer concentration and probe concentration determined above, based on the annealing time adjustment range in the qPCR reagent manual, 6 sets of annealing times were set in a gradient, and qPCR was performed using the Hsp70 probe. The results are as follows Fig.11 , showing that when the annealing time is 30s, the Ct is lower, that is, the qPCR amplification efficiency is the highest, so the annealing time is determined to be 30s.

[0326] 3.4 Clinical sample test results from large-scale cattle farms

[0327] (1) Cryptosporidium microscopic examination results

[0328] The clinical samples were stained using an acid-fast staining kit (modified Kinyoun cold staining method). The results are shown in the figure. Under the microscope, the Cryptosporidium oocysts were stained rose red, were round in shape, and had a diameter of about 4 to 6 μm. It can be vaguely seen that the oocysts contain 4 crescent-shaped sporozoites and 1 residual body.

[0329] (2) qPCR test results

[0330] The optimized fluorescence quantitative PCR method was used to conduct epidemiological testing on 30 fecal samples from the farm. The results are shown in Table 14. There were 10 positive samples and 5 negative samples in the sediment, 8 positive samples and 7 negative samples in the sewage, and the positive rate of Cryptosporidium in cattle in the farm was 60%. The obtained results were compared with the microscopic results, and data analysis was performed, showing that the agreement rate of the two different detection methods was 97.8%. Moreover, the qPCR method has high accuracy and good sensitivity, and is more convenient, fast and accurate than the currently used staining microscopy method.

[0331] Table 14 Test results of feces and sewage samples

[0332]

[0333] 4 Results of the study on the inactivation of Cryptosporidium and Escherichia coli by photosensitizers and ultraviolet light

[0334] 4.1 Inactivation effect of photosensitizer on Escherichia coli

[0335] E. coli was treated with different concentrations of photosensitizers and reacted under UV conditions for 2 minutes, 4 minutes, 6 minutes, 18 minutes, and 20 minutes before dilution and plate counting. The control group did not add photosensitizer and was irradiated with UV light alone. The number of viable bacteria in each group was calculated, and the results are as follows: Fig.13 It can be seen that the number of surviving E. coli decreased with time, and almost all of them were inactivated after 16 minutes.

[0336] The mortality rate was calculated by calculating the survival number of E. coli obtained in the above experiment and plotting it. Fig.14 .

[0337] like Fig.14As shown, the changes in the mortality rate of Escherichia coli irradiated by ultraviolet light under different concentrations of photosensitizer were analyzed, and the inactivation curve is as above. The experimental results show that the killing effect of photosensitizer and ultraviolet light on Escherichia coli increases with the increase of ultraviolet irradiation time, and the mortality rate is higher than the control value. When the photosensitizer concentration is 25ug / mL, the mortality rate increases from 26.84% to 73.67% during the ultraviolet irradiation time of 2min to 12min, an increase of 46.83%. Thereafter, when the irradiation time is 14min to 20min, the inactivation efficiency reaches a plateau, and the mortality rate increases from 97% to 99.99%. At this time, all Escherichia coli are inactivated. When the photosensitizer concentration is 50ug / mL, the mortality rate increases from 28.15% to 89.45% during the ultraviolet irradiation time of 2min to 10min, and then reaches a plateau, and all Escherichia coli are inactivated. When the photosensitizer concentration was 100ug / mL, the mortality rate increased from 38.41% to 99.98% during the UV irradiation time of 2min to 16min, and then reached a plateau, and all E. coli were inactivated.

[0338] From the above data comparison, it can be seen that when the photosensitizer concentration is 50ug / mL, the inactivation efficiency reaches the plateau phase earlier, and in the process from 4min to 16min, the mortality rate is higher than that of photosensitizers at other concentrations.

[0339] 4.2 Inactivation effect of photosensitizers on Cryptosporidium

[0340] After treating Cryptosporidium with photosensitizers of different concentrations and irradiating with UV light for different periods of time, DAPI / PI fluorescence staining was used to observe and record the number of dead and alive Cryptosporidium, and the mortality rate of Cryptosporidium was calculated. Fig.15 This is a photo of Cryptosporidium under a cell imaging device after DAPI / PI staining. As can be seen from the figure, the cells in the photo after DAPI staining emit blue fluorescence, which is active, that is, DAPI+; the Cryptosporidium after PI staining emits red fluorescence, which is apoptotic, that is, PI+.

[0341] After calculating the Cryptosporidium mortality rate, draw an inactivation curve, such as Fig.16 .

[0342] like Fig.16As shown in the figure, through the analysis of the photosensitizer-UV inactivation curve, the mortality rate of Cryptosporidium increased with the increase of UV irradiation time under different concentrations of photosensitizer, showing a similar change pattern to Escherichia coli and Streptococcus, with a faster growth rate within 2-14 minutes and a slower growth rate from 14 to 20 minutes. Under the condition of 25ug / mL photosensitizer concentration, the mortality rate of Cryptosporidium increased from 11.1% to 89% when the UV irradiation time increased from 2 minutes to 16 minutes, and the mortality rate reached 90% when the UV irradiation time was further increased to 20 minutes. Under the condition of 50ug / mL photosensitizer concentration, the mortality rate of Cryptosporidium increased from 18.8% to 87.5% when the UV irradiation time increased from 2 minutes to 14 minutes, and entered a plateau period. When the UV irradiation time was further increased to 20 minutes, the mortality rate reached 92.3%. Under the condition of 100ug / mL photosensitizer, the mortality rate of Cryptosporidium increased from 18.8% to 90.9% when the UV irradiation time increased from 2min to 16min. When the UV irradiation time was further increased to 20min, the mortality rate reached 92.9%. When the UV irradiation time was 14min, the inactivation effect of Cryptosporidium treated with 50ug / mL photosensitizer reached the plateau first, and the mortality rate of Cryptosporidium was 87.5%, which was higher than that of Cryptosporidium treated with 25ug / mL and 100ug / mL photosensitizer, which were 72.7% and 82.4% respectively.

[0343] From the above data, we can see that under the condition of 50ug / mL photosensitizer concentration, the synergistic ultraviolet catalytic inactivation of bacteria and Cryptosporidium is most effective and can kill most harmful microorganisms relatively quickly.

[0344] 5 Mechanism of photosensitizer-assisted UV inactivation of Cryptosporidium

[0345] 5.1 Effect of inactivation methods on the permeability of Cryptosporidium oocyst wall

[0346] The cell membrane is an important component of the cell structure. In addition to being the first line of defense against invasion, it also plays an important role in controlling many complex cell behaviors, such as intercellular communication, signal transduction, secretion and endocytosis of exosomes. In addition, there are many ion channels distributed on the cell membrane. When the cell membrane is damaged or the permeability increases, a large amount of electrolytes in the bacterial cells will be released or penetrate into the extracellular culture medium, resulting in an increase in the conductivity of the culture medium. Therefore, changes in the conductivity of the extracellular culture medium can reflect the degree of damage to the permeability of the cell membrane. For example Fig.17As shown in the data, the conductivity of the photosensitizer-assisted UV group was 35.9 ms / cm, which was significantly higher than that of the single UV group, which was 22.2 ms / cm. There was a significant difference between the two (P < 0.05), while the conductivity of the blank group was 19.6 ms / cm, which was not significantly different from the single UV group. This shows that the photosensitizer-assisted UV inactivation method can significantly affect the permeability of the Cryptosporidium oocyst cell membrane, causing leakage of intracellular electrolytes, and may further affect the normal metabolic pathway, while single UV cannot affect the permeability of the Cryptosporidium oocyst cell membrane.

[0347] 5.2 Effect of inactivation methods on Cryptosporidium soluble proteins

[0348] Although the protein content in the solution cannot accurately characterize the amount of total protein outside the cell wall of Cryptosporidium, it can indirectly evaluate whether the protein has flowed out and the amount of protein flowed out after UV and UV+ photosensitizer acted on the cells. As shown in the figure, within 20 minutes of ultraviolet light acting on Cryptosporidium alone, the protein concentration in the system increased from 0.08 mg / mL to 0.11 mg / mL. The protein concentration in the system did not change much, but there was a slight increase. In the photosensitizer-assisted ultraviolet irradiation group, the protein concentration in the system increased from 0.08 mg / mL to 0.19 mg / mL within 20 minutes, which was significantly different from the ultraviolet group alone. The reason may be that the photosensitizer produces free radicals in the water, which causes some proteins to decompose. The decomposition rate of dissolved protein is higher than the rate of protein outflow, so the concentration of dissolved protein in the system has increased.

[0349] Depend on Fig.18 It can be seen that compared with the samples treated with single UV, the photosensitizer-assisted UV inactivation of Cryptosporidium can cause a small amount of soluble protein to flow out, but there is no significant difference from the results of the single UV group, indicating that the photosensitizer cannot destroy the Cryptosporidium oocyst wall.

[0350] 5.3 Effect of inactivation methods on nucleic acid leakage from Cryptosporidium

[0351] Nucleic acid is the genetic material of microorganisms. When the cell membrane is damaged, the nucleic acid material in the cell will also penetrate the cell membrane into the culture medium. Therefore, the change in the extracellular nucleic acid content can be used as an indicator of cell membrane integrity. As shown in the figure, compared with the single UV group, the DNA content of Cryptosporidium in the photosensitizer-assisted UV action did not change significantly within 20 minutes, which were 3.55 and 3.61 respectively, indicating that the photosensitizer-assisted UV inactivation method can cause electrolytes to leak out of the cell, but cannot cause biological macromolecules such as DNA or protein to seep out of the cell membrane. In other words, the cell membrane of Cryptosporidium has not been damaged and still maintains a certain degree of integrity.

[0352] The technology of the present invention is mainly used for the treatment of manure and sewage in large-scale farms, and provides an effective solution to the problem of inactivation of Cryptosporidium oocysts. Through the deep treatment and monitoring of farm manure and sewage, this technology can completely inactivate Cryptosporidium oocysts and cut off its transmission pathway at the source. After being promoted and applied in 5 large-scale beef cattle farms, no live Cryptosporidium oocysts were detected after manure and sewage treatment for two consecutive years. Even in farms where Cryptosporidium pathogens had previously existed, no new outbreaks of cryptosporidiosis occurred, and the manure and sewage treatment effect was significant.

[0353] During the promotion period, the technology of the present invention achieved comprehensive treatment of manure and sewage, and ensured the complete inactivation of Cryptosporidium oocysts through regular monitoring. Compared with traditional manure treatment methods, this technology significantly reduces the risk of Cryptosporidium transmission and provides a safer and more stable environment for large-scale farms. Two years of long-term application have shown that this technology has a long-lasting and stable inactivation effect, can effectively reduce the health risks caused by pathogens in farms, and has broad promotion value.

[0354] Relevant evidence of the technical effects achieved by the embodiments of the present invention.

[0355] 1. The specific description is as follows: Fig. 20 The results of AnnexinV-FITC / PI dual-color flow cytometry showed that: a was the normal Cryptosporidium oocyst group, b was the photosensitizer-assisted ultraviolet inactivation group, and c was the single ultraviolet inactivation group; the early apoptotic oocysts (Q4) and late apoptotic or necrotic oocysts (Q2) in the ultraviolet single treatment group and the photosensitizer-assisted treatment group were increased compared with the normal control group, and the late apoptotic or necrotic oocysts (Q2) in the pancreatic enzyme-assisted ultraviolet treatment group were higher than those in the single ultraviolet treatment group. The Q2 quadrant (apoptotic area) in the single ultraviolet inactivation group was 54.0%, and the Q2 quadrant in the pancreatic enzyme-assisted ultraviolet inactivation group was 75.2%, which was increased by 38.9%, with significant differences (P < 0.05). The results showed that the photosensitizer promoted the apoptosis of the oocysts, the shrinkage and adhesion of the oocyst surface decreased, and the early apoptosis of the oocysts was induced. After the ultraviolet treatment again, the oocysts were late apoptotic or necrotic, which improved the inactivation effect.

[0356] Depend on Fig.21 The results of scanning electron microscopy (SEM) in the normal control group show that the oocysts are spherical, full in appearance, with a relatively smooth surface, no defects, and a complete structure. Fig.21 As shown in a. In the experimental group treated with ultraviolet light alone, the surface of the Cryptosporidium oocyst wall was rough and spherical with deformed protrusions, the volume was expanded, and adhesions appeared, and the edges were unclear, as shown in Fig.21 b. The oocysts treated with photosensitizer and UV inactivation showed significant changes in morphology, with wrinkles on the surface, shrinkage, and a reduction in the nuclear material area. They also showed adhesion and deformation, incomplete outer walls, and unclear edges. Fig.21c. The reason why the photosensitizer cooperates with ultraviolet light to destroy the structure of the oocyst is that pancreatic enzymes act on the surface of the oocyst and dissolve the surface components of the oocyst membrane, such as the bimolecular structure of phospholipids, and finally ultraviolet light further acts to destroy the oocyst wall, and finally the oocyst atrophies and dies. This further shows that pancreatic enzymes promote the inactivation effect of ultraviolet light.

[0357] 2. Fig. 22 Electrophoresis of Cryptosporidium genome under different treatments (M stands for Maker; 1 stands for control group; 2 and 3 stand for UV treatment for 10 min and 20 min respectively; 4, 5 and 6 stand for photosensitizer combined with UV treatment for 10 min, 20 min and 30 min respectively)

[0358] 3 Mechanism of photosensitizer-assisted UV inactivation of Cryptosporidium

[0359] 3.1 Effect of inactivation methods on the permeability of Cryptosporidium oocyst wall

[0360] The cell membrane is an important component of the cell structure. In addition to being the first line of defense against invasion, it also plays an important role in controlling many complex cell behaviors, such as intercellular communication, signal transduction, secretion and endocytosis of exosomes. In addition, there are many ion channels distributed on the cell membrane. When the cell membrane is damaged or the permeability increases, a large amount of electrolytes in the bacterial cells will be released or penetrate into the extracellular culture medium, resulting in an increase in the conductivity of the culture medium. Therefore, changes in the conductivity of the extracellular culture medium can reflect the degree of damage to the permeability of the cell membrane. For example Fig.17 As shown in the data, the conductivity of the photosensitizer-assisted UV group was 35.9 ms / cm, which was significantly higher than that of the single UV group, which was 22.2 ms / cm. There was a significant difference between the two (P < 0.05), while the conductivity of the blank group was 19.6 ms / cm, which was not significantly different from the single UV group. This shows that the photosensitizer-assisted UV inactivation method can significantly affect the permeability of the Cryptosporidium oocyst cell membrane, causing leakage of intracellular electrolytes, and may further affect the normal metabolic pathway, while single UV cannot affect the permeability of the Cryptosporidium oocyst cell membrane.

[0361] 3.2 Effect of inactivation methods on Cryptosporidium soluble proteins

[0362] Although the protein content in the solution cannot accurately characterize the amount of total protein outside the cell wall of Cryptosporidium, it can indirectly evaluate whether the protein has flowed out and the amount of protein flowed out after UV and UV+ photosensitizer acted on the cells. As shown in the figure, within 20 minutes of ultraviolet light acting on Cryptosporidium alone, the protein concentration in the system increased from 0.08 mg / mL to 0.11 mg / mL. The protein concentration in the system did not change much, but there was a slight increase. In the photosensitizer-assisted ultraviolet irradiation group, the protein concentration in the system increased from 0.08 mg / mL to 0.19 mg / mL within 20 minutes, which was significantly different from the ultraviolet group alone. The reason may be that the photosensitizer produces free radicals in the water, which causes some proteins to decompose. The decomposition rate of dissolved protein is higher than the rate of protein outflow, so the concentration of dissolved protein in the system has increased.

[0363] Depend on Fig.18 It can be seen that compared with the samples treated with single UV, the photosensitizer-assisted UV inactivation of Cryptosporidium can cause a small amount of soluble protein to flow out, but there is no significant difference from the results of the single UV group, indicating that the photosensitizer cannot destroy the Cryptosporidium oocyst wall.

[0364] 3.3 Effect of inactivation methods on nucleic acid leakage from Cryptosporidium

[0365] Nucleic acid is the genetic material of microorganisms. When the cell membrane is damaged, the nucleic acid inside the cell will also penetrate through the cell membrane into the culture medium. Therefore, changes in the extracellular nucleic acid content can be used as an indicator of cell membrane integrity. Fig.19 As shown in the figure, compared with the single UV group, the DNA content of Cryptosporidium in the photosensitizer-UV group did not change significantly within 20 minutes, and was 3.55 and 3.61 respectively, indicating that the photosensitizer-UV inactivation method can cause electrolytes to leak out of the cell, but cannot cause biomacromolecules such as DNA or protein to leak out of the cell membrane. In other words, the cell membrane of Cryptosporidium was not destroyed and maintained a certain degree of integrity.

[0366] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for detecting and inactivating Cryptosporidium in large-scale cattle farm manure, characterized in that: The following steps are involved: Prepare Cryptosporidium standard samples and determine dilution concentrations by microscopy; The fecal samples were enriched and purified by saturated magnesium sulfate flotation, ether defatting and sucrose method to obtain Cryptosporidium oocysts. The Cryptosporidium oocysts are treated with photosensitizers in combination with ultraviolet rays. Under ultraviolet irradiation, the photosensitizers produce reactive oxygen species that inactivate the oocysts. DNA was extracted from feces and the Cryptosporidium oocyst content was detected by fluorescent PCR technology to evaluate the inactivation efficiency.

2. The method according to claim 1, characterized in that The photosensitizer is a soluble porphyrin compound or a phthalimide compound with a concentration range of 10-50 μM. After being diluted to a specified concentration, the compound is fully mixed with the Cryptosporidium oocysts, left to stand for adsorption for 30-60 minutes, and then irradiated with ultraviolet rays.

3. The method according to claim 1, characterized in that The ultraviolet irradiation conditions are: wavelength of 254nm, intensity of 0.2-0.5mW / cm 2 The treatment time is 15-30 minutes, and the distance between the ultraviolet light source and the sample is controlled at 5-10 cm.

4. The method according to claim 1, characterized in that: The fluorescent PCR was used to detect the changes in the concentration of Cryptosporidium DNA to evaluate the synergistic inactivation effect of the photosensitizer and ultraviolet light, and agarose gel electrophoresis was used to verify the integrity damage of the DNA after inactivation to ensure that the inactivation efficiency reached more than 95%.

5. A method for detecting and inactivating Cryptosporidium in large-scale cattle farm manure, characterized in that: include: S1, prepare the Cryptosporidium standard sample, dilute and stain it, and then examine it under a microscope to ensure that each microscopic examination contains 3 ± 1 oocysts; S2, enriching and purifying fecal samples, and using the "saturated magnesium sulfate flotation-ether defatting-sucrose method" to enrich and purify Cryptosporidium oocysts; S3, the purified Cryptosporidium oocysts were subjected to acid-fast staining, stained and destained using the modified Kinyoun staining method, and finally observed under a microscope; S4, extract DNA from feces and detect Cryptosporidium oocysts by fluorescent PCR technology, and monitor the content of oocysts in real time; S5, the quality of the extracted DNA was analyzed by agarose gel electrophoresis and NanoDrop measurement to ensure its integrity and purity; S6, construct a qPCR standard curve to quantify the concentration of Cryptosporidium oocysts in the sample by optimizing the reaction conditions, including primer and probe concentrations.

6. The method for detecting and inactivating Cryptosporidium in large-scale cattle farm manure as claimed in claim 5, characterized in that: S1 specifically includes: The standard insect species sample was supplemented with PBS to 1 mL and then diluted 10 times. The mixture was mixed and stained, and 5 μl was taken for microscopic examination. This was repeated 10 times to determine that the sample contained 3±1 oocysts. The diluted solution was added to a plastic bucket containing 500 mL of fecal wastewater for enrichment and purification for later use.

7. The method for detecting and inactivating Cryptosporidium in large-scale cattle farm manure as claimed in claim 5, characterized in that: S2 specifically includes: enriching and purifying Cryptosporidium parvum oocysts by the method of "saturated magnesium sulfate flotation-ether defatting-sucrose purification"; (1) The feces sample collected from the farm was filtered out of impurities using a 60-mesh sieve, 2.5% potassium dichromate was added to the filtrate in a 1:1 ratio, and then placed in a 4°C refrigerator; (2) Take out an appropriate amount of fecal sample from the refrigerator, place it in a 4°C horizontal centrifuge, and centrifuge at 4000 r / min for 10 min. After discarding the supernatant, pour in distilled water, shake upside down, and centrifuge again. Wash three times in total. (3) Add an appropriate amount of saturated magnesium sulfate solution, mix thoroughly by inversion, let stand at room temperature for 40 min, centrifuge at 500 g for 1 min, and discard the precipitate; (4) Transfer the supernatant to a 50 mL centrifuge tube, add distilled water to dilute the supernatant, centrifuge at 3500 rpm for 10 min, discard the supernatant, and keep the precipitate; (5) Add the same volume of distilled water, stir and blow up the precipitate to resuspend it, add the same volume of anhydrous ether again and shake vigorously for 20 min to defat it, let it stand for 5 min, centrifuge at 3500 r / min for 10 min, pour off the upper ether, lipid and water layer mixture, and collect the precipitate; (6) Prepare a saturated sucrose solution. Dilute the original sucrose solution with sterile water at a volume ratio of 1:1 and 1:

4. Add 10 mL of the 1:1 sucrose dilution solution to a 30 mL centrifuge tube, and slowly add 10 mL of the 1:4 sucrose dilution solution along the wall of the bottle to cover it, so that it can be layered and set aside. (7) Add an appropriate amount of sterile water to the defatted fecal sediment and stir to mix well. Slowly add 2-4 mL of the suspension to the layered sucrose solution and centrifuge horizontally at 2000 rpm for 10 min. (8) After centrifugation, three layers of suspension can be seen in the 30 mL centrifuge tube. The upper, middle and lower layers are potassium dichromate solution, white oocysts and fecal pellets, respectively. The white oocysts in the middle layer are aspirated and placed in a 50 mL centrifuge tube. The residual sucrose solution is washed away with four times the volume of distilled water. Centrifuge at 3500 r / min for 10 min, discard the supernatant and keep the precipitate. (9) Add a small amount of sterile water to blow up the precipitate, then add 20 mL of 10% sodium hypochlorite solution, let stand at room temperature for 10 min to kill bacteria, add distilled water to 50 mL, centrifuge at 3500 r / min for 10 min, and wash with water to remove sodium hypochlorite; (10) Add a small amount of sterile water to blow up the precipitate and use a kit to perform acid-fast staining and count under a microscope.

8. The method for detecting and inactivating Cryptosporidium in large-scale cattle farm manure as claimed in claim 5, characterized in that: S3 specifically includes: using an acid-fast staining kit to stain Cryptosporidium oocysts, the specific method is as follows: (1) Dip the purified Cryptosporidium oocyst suspension into an inoculating loop, apply it on a glass slide, and dry it naturally; (2) Add modified Kinyoun fuchsin staining solution and stain for 5 min. Wash off excess dye with distilled water. (3) Decolorize with Kinyoun decolorizing solution for 3 min and rinse with distilled water; (4) Stain with methylene blue staining solution for 5 min and rinse with distilled water; (5) Gently absorb the moisture and let it dry naturally; (6) Oil immersion microscopy.

9. The method for detecting and inactivating Cryptosporidium in large-scale cattle farm manure as claimed in claim 5, characterized in that: S4 specifically includes: (1) DNA extraction from feces The solid-liquid mixed feces sample was centrifuged at 6000r / min for 20min, and 400mg of the precipitate was weighed and DNA was extracted according to the following three methods: (2) CTAB method Add 1 ml CTAB buffer [2% CTAB, 0.1 mol / L Tris, 20 mmol / L EDTA and 1.4 mol / L NaCl] to the sample, mix well, place in a water bath, centrifuge at 12 000 r / min twice, take the supernatant, add equal volumes of Tris-saturated phenol-chloroform-isoamyl alcohol and equal volumes of chloroform-isoamyl alcohol, centrifuge for 5 min, take the supernatant, add 2.5 times pre-cooled anhydrous ethanol, place at -20℃ for precipitation for 30 min, centrifuge for 5 min, remove the supernatant, add 1 ml 70% ethanol to wash the precipitate twice, centrifuge for 3 min, remove the supernatant, dry at room temperature for 15 min, add 50 μl TE buffer, and store at -20℃ for later use; (3) SDS method: Add 500 μl SDS lysis buffer [1% SDS, 0.5 mol / L Tris-HCl, 0.1 mol / L EDTA and 0.1 mol / L NaCl], 500 μl sodium phosphate buffer and 20 μl proteinase K to the sample, mix well, place in a water bath, centrifuge at 12 000 r / min for 10 min, and take the supernatant. Add 0.6 times the amount of 4 mol / L sodium acetate, place at room temperature for 5 minutes, centrifuge for 5 minutes, remove the supernatant, add 0.8 times the amount of isopropanol, place at -20℃ for 30 minutes, centrifuge for 5 minutes, remove the supernatant, add 1 ml of 70% ethanol, centrifuge for 3 minutes, repeat the operation with anhydrous ethanol, centrifuge for 3 minutes, remove the supernatant, dry at room temperature for 15 minutes, add 50 μl of TE buffer, and store at -20℃ for later use; (4) TIANamp Stool DNA Kit was used to extract sample DNA. The operation procedure was as follows: 1) Add anhydrous ethanol to buffer GD and rinse solution PW according to the bottle label, and add isopropanol to buffer GFA; 2) Place the stool sample in a 2 ml centrifuge tube and place the tube on ice; 3) Add 500 μL buffer SA, 100 μL buffer SC, 15 μL Proteinase K, and 0.25 g grinding beads to the sample and oscillate for 3 min until the sample is fully mixed; 4) Incubate in a 70°C water bath for 15 min, shaking 2-3 times during incubation; 5) Vortex for 15 seconds, centrifuge at 12,000 rpm for 3 minutes, and transfer the supernatant to a new centrifuge tube; 6) Add 200 μL buffer SH, shake and mix, place on ice for 5 min; centrifuge at 12,000 rpm for 3 min; 7) Transfer the supernatant obtained in the previous step to a new 1.5 ml centrifuge tube and add 750 μL of buffer GFA; 8) Add the solution obtained in the previous step to an adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube; 9) Add 500 μL of buffer GD to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube; 10) Add 700 μL of rinse solution PW to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube; 11) Wash again, add 700 μL of washing solution PW, centrifuge at 12,000 rpm for 1 min, and pour out the liquid in the centrifuge tube; 12) Place the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid; leave the adsorption column at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material; 13) Transfer the adsorption column into a clean centrifuge tube, add 30 μL of sterile enzyme-free water to the middle part of the adsorption membrane, place at room temperature for 5 minutes, centrifuge at 12,000 rpm for 2 minutes, and collect the solution in the centrifuge tube; 14) To increase the yield of genomic DNA, the solution obtained by centrifugation was added to the adsorption column, placed at room temperature for 2 minutes, and centrifuged at 12,000 rpm for 2 minutes; 30 μL of oocyst DNA was obtained; the amount of genomic DNA obtained was evaluated using Nanodrop 1000 nucleic acid protein quantification instrument; the extracted samples were stored at -20°C for PCR specific amplification.

10. The method for detecting and inactivating Cryptosporidium in large-scale cattle farm manure as claimed in claim 5, characterized in that: S5 specifically includes: taking 5 μl of the DNA extracted by the three methods and loading it on a 1% agarose gel, and judging the integrity of the DNA by the electrophoresis results; taking 2 μl of the DNA product each time, and using a Nano Drop 2000 spectrophotometer to determine the concentration of the total DNA and the purity A260 / A280 value; the sample absorbance value (A) is calculated using the formula A=-log(Is / Ib), where Is represents the transmitted light intensity of the sample, and Ib represents the transmitted light intensity of the blank control; the above two light intensities are automatically detected, recorded and saved by the instrument during the measurement; A260 / A280 values ​​between 1.8 and 2.0 indicate that the DNA is relatively pure, if higher than 2.0, it indicates that the RNA in the product has not been completely removed, and if lower than 1.8, it indicates that the product contains phenol or protein; each sample is measured 3 times, with 3 parallel samples each time.

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