Method for isolation, purification, staining and counting of Enterocytozoon hepatopenaei

Abstract

The present invention discloses a method for the isolation, purification, staining and counting of Enterocytozoon hepatopenaei. The method comprises the following steps: sample preparation: dissecting the hepatopancreas and intestinal tissues of prawns infected with Enterocytozoon hepatopenaei, preparing a tissue homogenate and diluting it with sterile physiological saline to obtain a diluted homogenate; crude extraction: using differential centrifugation to prepare a crude extract of Enterocytozoon hepatopenaei from the obtained diluted homogenate; the washing medium used in the crude extraction step includes glycerol; purification: using discontinuous sucrose density gradient centrifugation to separate and purify the crude extract of Enterocytozoon hepatopenaei obtained in step S2 to obtain a purified suspension of Enterocytozoon hepatopenaei; staining: staining the Enterocytozoon hepatopenaei in the obtained purified suspension of Enterocytozoon hepatopenaei with a fluorescent brightener; counting: filtering the stained Enterocytozoon hepatopenaei onto a black nuclear pore filter membrane and observing and counting under a high-power fluorescence microscope. The method has the advantages of simple operation, low cost, high pathogen purity, strong specificity of the staining agent and accurate counting.

Description

Technical Field

[0001] The present invention relates to the technical field of parasite research, and specifically relates to a method for the isolation, purification, staining and counting of Enterocytozoon hepatopenaei Background Art

[0002] Enterocytozoon hepatopenaei (EHP) belongs to the family Microsporidae and the genus Enterocytozoon. It is a highly contagious intracellular parasitic microsporidium. It was first isolated and named in the epithelial cells of the hepatic pancreatic tubules of farmed Penaeus monodon with slow growth in Thailand in 2009. In recent years, it has spread and prevailed in major shrimp farming countries such as Thailand, India, Vietnam, Indonesia and China, mainly infecting farmed shrimps such as Penaeus vannamei and Penaeus monodon. This pathogen can multiply in large numbers in the digestive system cells of shrimps, absorb nutrients, thus affecting the normal digestive and absorption functions of the hepatopancreas and intestines, and can cause extremely slow or stagnant growth of shrimps, seriously affecting the yield and economic value of shrimps. It is one of the important diseases endangering the global shrimp farming industry.

[0003] EHP belongs to intracellular parasitic microsporidia, has strong transmission ability, and is difficult to prevent. There are no effective treatment measures and drugs. So far, the research on the life history, pathogenic mechanism and factors leading to the outbreak of this disease of EHP at home and abroad is almost blank. Therefore, it is extremely difficult to carry out research on prevention and control technologies for this pathogen. The main reason is that Enterocytozoon hepatopenaei is extremely small, with a particle size of only about 1 μm, and it cannot be proliferated in vitro, resulting in the inability to obtain a large amount of highly pure pathogens required for research, seriously affecting the process of in-depth research on this pathogen. Therefore, many researchers have tried to isolate highly pure Enterocytozoon hepatopenaei from diseased shrimps. However, the existing methods for the isolation and purification of Enterocytozoon hepatopenaei have disadvantages such as lack of technical details, poor repeatability, low purity of the obtained pathogens, and high impurity content. At the same time, the existing research methods also lack specific staining for spore-forming pathogens and relatively accurate counting methods, and can neither effectively distinguish impurities from Enterocytozoon hepatopenaei under the microscope nor evaluate the effect and quantity of the extraction method. Therefore, it is urgent to establish a method for the isolation, purification, staining and counting of Enterocytozoon hepatopenaei to solve the above problems, so as to accelerate the research on EHP prevention and control technologies. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a method for the isolation, purification, staining and counting of Enterocytozoon hepatopenaei. This method can efficiently isolate and extract highly pure Enterocytozoon hepatopenaei of shrimps, and can quickly and effectively distinguish Enterocytozoon hepatopenaei of shrimps from other microorganisms, thus conveniently and accurately realizing the counting of Enterocytozoon hepatopenaei of shrimps.

[0005] To achieve the above object, the present invention discloses a method for the isolation, purification, staining and counting of Enterocytozoon hepatopenaei, comprising the following steps:

[0006] Sample preparation: Dissect the hepatopancreas and intestinal tissues of shrimp infected with Enterocytozoon hepatopenaei at low temperature, prepare a tissue homogenate and dilute it with sterile physiological saline to obtain a diluted homogenate;

[0007] Crude extraction: Use differential centrifugation to prepare a crude extract of Enterocytozoon hepatopenaei from the obtained diluted homogenate; the washing medium used in the crude extraction step includes glycerol;

[0008] Purification: Use discontinuous sucrose density gradient centrifugation to separate and purify the crude extract of Enterocytozoon hepatopenaei obtained in step S2 to obtain a purified suspension of Enterocytozoon hepatopenaei;

[0009] Staining and counting: Stain Enterocytozoon hepatopenaei in the obtained purified suspension of Enterocytozoon hepatopenaei with a fluorescent brightener, filter the stained Enterocytozoon hepatopenaei onto a black nuclear pore filter membrane, and observe and count under a high-power fluorescence microscope.

[0010] Preferably, the sample preparation step includes: Dissect the hepatopancreas and intestinal tissues of shrimp severely infected with Enterocytozoon hepatopenaei at low temperature, grind them with a tissue homogenizer or a glass homogenizer until there are no obvious tissue blocks, extract 5 - 10 ml of the tissue homogenate and dilute it to 30 - 35 ml with sterile physiological saline to obtain a diluted homogenate.

[0011] Preferably, the crude extraction includes the following steps:

[0012] S2: Centrifuge the diluted homogenate at 4°C and 600 r / min for 10 min, aspirate the supernatant for standby; add sterile physiological saline to the precipitate to 15 - 20 ml and resuspend;

[0013] S3: Centrifuge the resuspended liquid obtained in step S2 at 4°C and 600 r / min for 10 min, aspirate the supernatant, and mix it with the supernatant obtained in step S2 to obtain a mixed supernatant;

[0014] S4: Add glycerol to the mixed supernatant obtained in step S3, mix glycerol and the supernatant in a volume ratio of 1:3, centrifuge at 4°C and 8000 r / min for 30 min, discard the supernatant; wash the precipitate three times by centrifugation at 4°C and 6000 r / min for 10 min with 20 - 25 ml of sterile physiological saline;

[0015] S5: Resuspend the precipitate obtained in step S4 with 3 - 5 ml of sterile physiological saline to obtain a crude extract of Enterocytozoon hepatopenaei.

[0016] Preferably, the purification includes the following steps:

[0017] S6: In the order of increasing sucrose solution concentration, use a lengthened needle to inject into the bottom of the centrifuge tube one by one. The injection volume of each concentration of sucrose solution is 1.5 - 2 ml;

[0018] S7: Slowly add 1.5 ml of the crude extract of Enterocytozoon hepatopenaei to the top of the sucrose gradient liquid surface using a pipette, ensuring that the sample does not mix with the top - layer sucrose solution and the interface is clearly visible;

[0019] S8: Use a horizontal rotor to centrifuge the sucrose density gradient centrifugation liquid prepared in step S7 at 4 °C and 2000 r / min for 30 min;

[0020] S9: Use a syringe to aspirate the white band at the gradient boundary from top to bottom into a centrifuge tube, centrifuge at 4 °C and 6000 r / min for 10 min, discard the supernatant, and centrifuge and wash the obtained precipitate three times with 5 - 10 ml of sterile physiological saline under the condition of centrifuging at 4 °C and 6000 r / min for 10 min to obtain a purified suspension of Enterocytozoon hepatopenaei.

[0021] Preferably, in step S6, the injection speed of the sucrose solution is 1 ml / min, and the total time used in step S6 ≤ 15 min; five concentrations of sucrose solutions are used in step S6, and their concentrations are 20%, 25%, 30%, 35%, and 40% respectively; the preparation methods of each concentration of sucrose solution are as follows:

[0022] 20% sucrose solution: Weigh 20 g of sucrose and make up the volume to 100 ml with deionized water;

[0023] 25% sucrose solution: Weigh 25 g of sucrose and make up the volume to 100 ml with deionized water;

[0024] 30% sucrose solution: Weigh 30 g of sucrose and make up the volume to 100 ml with deionized water;

[0025] 35% sucrose solution: Weigh 35 g of sucrose and make up the volume to 100 ml with deionized water;

[0026] 40% sucrose solution: Weigh 40 g of sucrose and make up the volume to 100 ml with deionized water.

[0027] Preferably, step S9 is to use a syringe to aspirate the third milky - white band from top to bottom into a centrifuge tube, centrifuge at 4 °C and 6000 r / min for 10 min, discard the supernatant, and centrifuge and wash the precipitate three times with sterile physiological saline under the condition of centrifuging at 4 °C and 6000 r / min for 10 min to obtain a purified suspension of Enterocytozoon hepatopenaei.

[0028] Preferably, the method of adding the crude extract of Enterocytozoon hepatopenaei in step S7 is as follows: at the top of the liquid surface with a gradient paved, place the pipette tip close to the inner wall of the centrifuge tube, making an angle of 80-90° between the tip and the tube wall, and add 1.5 ml of the crude extract at a rate of 1 ml / min, ensuring that the sample does not mix with the topmost sucrose solution and the liquid interface is clearly visible.

[0029] Preferably, the fluorescent brightener is Calcofluor White M2R staining solution. The preparation method of Calcofluor White M2R staining solution is as follows: weigh 1 g of Calcofluor White M2R staining agent and dissolve it in 100 ml of TBS buffer with a pH value of 7.2 to make a concentration of 1% (W / V), then add 0.1 g of Evan's blue dye and store it in the dark at 4 °C; before use, centrifuge it at 12,000 rpm / min for 5 min and take the supernatant for use.

[0030] Preferably, the counting includes the following steps:

[0031] 1) Take a 0.22 μm black nuclear pore filter membrane and rinse it clean with sterile water; place the smooth side of the black nuclear pore filter membrane facing up and flat on the bottom of the filter, ensuring that there are no air bubbles above and below the black nuclear pore filter membrane, then install the filter on the horizontal table and clamp it with a clip to make the filter in a horizontal state;

[0032] 2) Pre-add 5 ml of sterile water into the filter cylinder, suck the suspension of Enterocytozoon hepatopenaei and inject it into the filter, and at the same time add 0.2 ml of the Calcofluor White M2R staining solution, mix well, stain for 1 min, and filter under negative pressure until the black nuclear pore filter membrane is just moist;

[0033] 3) Remove the filter, gently lift the black nuclear pore filter membrane with forceps, transfer it to a clean glass slide, drop 1 drop of non-fluorescent immersion oil on the black nuclear pore filter membrane, cover it with a cover glass, and observe it under the high-power objective of a fluorescence microscope. Oval bright blue worms can be observed under the ultraviolet light channel of the fluorescence microscope, and count the oval bright blue worms, and randomly calculate the number of worms in at least 10 fields of view;

[0034] 4) Calculate the worm concentration of the sample.

[0035] Preferably, the calculation formula in step 4) is BN = Na × S / (Sf × V); where BN is the worm concentration of the sample, in units of number / ml, Na is the average number of worms in each field of view, in units of number, S is the actual filtration area of the filter membrane, in units of mm 2 , Sf is the area of the microscope field of view, in units of mm 2 , V is the volume of the filtered sample, in units of ml.

[0036] Compared with the prior art, the advantages and positive effects of the present invention are as follows: A method for the isolation, purification, staining and counting of Enterocytozoon hepatopenaei is provided. This method has the advantages of high purity, strong staining specificity and accurate counting, and can provide technical support for cracking the whole genome sequence of Enterocytozoon hepatopenaei, deeply studying the pathogenic mechanism of Enterocytozoon hepatopenaei, establishing effective prevention and control technologies, etc., and has high scientific and practical value. Specifically:

[0037] (1) The present invention establishes a method for the isolation and purification of Enterocytozoon hepatopenaei by combining differential centrifugation and sucrose density gradient centrifugation. The purity can reach more than 99%, and it is inexpensive and easy to operate.

[0038] (2) In the crude extraction step of the present invention, glycerol is used as a washing medium, which can remove most of the tissue fragments and pigments with small density and other impurities, and obtain a relatively pure crude extract.

[0039] (3) The Calcofluor White M2R fluorescent dye prepared in the preferred scheme of the present invention can specifically stain Enterocytozoon hepatopenaei, and can quickly and effectively distinguish Enterocytozoon hepatopenaei from other microorganisms, solving the problems that it is difficult to observe and easy to confuse due to the small size of the spore parasite, and also providing a rapid staining and identification method for Enterocytozoon hepatopenaei in actual aquaculture production work.

[0040] (4) In the preferred scheme of the present invention, a set of accurate counting methods for Enterocytozoon hepatopenaei is established by using a suction filtration device and a specific staining technique, realizing the direct counting of Enterocytozoon hepatopenaei. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0042] Figure 1 It is a photo of the sucrose density gradient centrifugation stratification state of Enterocytozoon hepatopenaei in penaeid shrimp for this embodiment;

[0043] Figure 2 It is a microscope photo of the sample of Enterocytozoon hepatopenaei in penaeid shrimp stained with Calcofluor White M2R for this embodiment;

[0044] Figure 3 It is a comparison chart of the staining effects of Enterocytozoon hepatopenaei in penaeid shrimp with Calcofluor White M2R and DAPI staining solutions. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] The present invention can be better understood according to the following embodiments. However, those skilled in the art can easily understand that the specific structures, process conditions and their results described in the embodiments are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0046] A method for the isolation, purification, staining and counting of Enterocytozoon hepatopenaei includes the following steps:

[0047] Sample preparation: Dissect the hepatopancreas and intestinal tissues of shrimp infected with Enterocytozoon hepatopenaei, make them into tissue homogenate and dilute it with sterile physiological saline to obtain a diluted homogenate.

[0048] Crude extraction: Use differential centrifugation to make the obtained diluted homogenate into a crude extract of Enterocytozoon hepatopenaei; the washing medium used in the crude extraction step includes glycerol.

[0049] Purification: Use discontinuous sucrose density gradient centrifugation to separate and purify the crude extract of Enterocytozoon hepatopenaei obtained in step S2 to obtain a purified suspension of Enterocytozoon hepatopenaei.

[0050] Staining and counting: Use fluorescent brightener to stain Enterocytozoon hepatopenaei in the obtained purified suspension of Enterocytozoon hepatopenaei, filter the stained Enterocytozoon hepatopenaei onto a black nuclear pore filter membrane, and observe and count under a high-power fluorescence microscope.

[0051] The above method establishes a method for the isolation and purification of Enterocytozoon hepatopenaei by combining differential centrifugation and sucrose density gradient centrifugation. The purity can reach more than 99%, and it is low in price and simple to operate. Among them, glycerol is used as the washing medium in the crude extraction step, which can remove most of the tissue fragments and pigments and other impurities with small density, and obtain a relatively pure crude extract. Staining the obtained purified Enterocytozoon hepatopenaei with fluorescent brightener can effectively label Enterocytozoon hepatopenaei, thus facilitating the rapid observation and counting of Enterocytozoon hepatopenaei using a fluorescence microscope.

[0052] Among them, the sample preparation step includes: Dissect the hepatopancreas and intestinal tissues of shrimp severely infected with Enterocytozoon hepatopenaei at low temperature, grind them with a tissue homogenizer or a glass homogenizer until there are no obvious tissue blocks, extract 5-10 ml of tissue homogenate and dilute it to 30-35 ml with sterile physiological saline to obtain a diluted homogenate.

[0053] Among them, the crude extraction step includes the following steps:

[0054] S2: Centrifuge the diluted homogenate at 4°C and 600 r / min for 10 min, aspirate the supernatant for standby; add sterile physiological saline to the precipitate to 15-20 ml and resuspend it.

[0055] S3: Centrifuge the resuspended liquid obtained in step S2 at 4°C and 600 r / min for 10 min, aspirate the supernatant, and mix it with the supernatant obtained in step S2 to obtain a mixed supernatant;

[0056] S4: Add glycerol to the mixed supernatant obtained in step S3. To ensure the washing effect, glycerol and the supernatant are mixed at a volume ratio of 1:3, centrifuge at 4°C and 8000 r / min for 30 min, and discard the supernatant; the precipitate is centrifuged and washed three times with 20 - 25 ml of sterile normal saline under the condition of centrifuging at 4°C and 6000 r / min for 10 min;

[0057] S5: Resuspend the precipitate obtained in step S4 with 3 - 5 ml of sterile normal saline to obtain a crude extract of Hepatocyclus.

[0058] Among them, the purification steps include the following steps:

[0059] S6: In the order of increasing sucrose solution concentration, use a lengthened needle to inject into the bottom of the centrifuge tube in turn, and the injection volume of each concentration of sucrose solution is 1.5 - 2 ml;

[0060] S7: Slowly add 1.5 ml of the crude extract of Hepatocyclus to the top of the sucrose gradient liquid surface using a pipette, ensure that the sample does not mix with the top - layer sucrose solution, and the interface is clearly visible;

[0061] S8: Centrifuge the sucrose density gradient centrifugation liquid prepared in step S7 at 4°C and 2000 r / min for 30 min using a horizontal rotor;

[0062] S9: Use a syringe to aspirate the white band at the gradient boundary from top to bottom into a centrifuge tube, centrifuge at 4°C and 6000 r / min for 10 min, discard the supernatant, and the obtained precipitate is centrifuged and washed three times with 5 - 10 ml of sterile normal saline under the condition of centrifuging at 4°C and 6000 r / min for 10 min to obtain a purified suspension of Hepatocyclus.

[0063] Specifically, in the following examples, the injection speed of the sucrose solution in step S6 is 1 ml / min, and the total time used in step S6 ≤ 15 min. Each time when preparing the sucrose solution, 0.1 ml of liquid is left in the syringe to avoid injecting air bubbles.

[0064] Specifically, in the following examples, five concentrations of sucrose solutions are used in step S6, and their concentrations are 20%, 25%, 30%, 35%, and 40% respectively; the preparation methods of each concentration of sucrose solution are as follows:

[0065] 20% sucrose solution: Weigh 20 g of sucrose and make up the volume to 100 ml with deionized water;

[0066] 25% Sucrose Solution: Weigh 25 g of sucrose and make up the volume to 100 ml with deionized water;

[0067] 30% Sucrose Solution: Weigh 30 g of sucrose and make up the volume to 100 ml with deionized water;

[0068] 35% Sucrose Solution: Weigh 35 g of sucrose and make up the volume to 100 ml with deionized water;

[0069] 40% Sucrose Solution: Weigh 40 g of sucrose and make up the volume to 100 ml with deionized water.

[0070] Specifically, in the following examples, the method of adding the crude extract of Enterocytozoon hepatopenaei in step S7 is as follows: at the top of the liquid surface with a gradient paved, place the pipette tip close to the inner wall of the centrifuge tube, making an angle of 80 - 90° between the tip and the tube wall, and add 1.5 ml of the crude extract at a speed of 1 ml / min, ensuring that the sample does not mix with the topmost sucrose solution and the liquid interface is clearly visible.

[0071] Specifically, in the collection process of step S9, a 2.5 ml syringe equipped with an 8 cm extended needle is used to sequentially aspirate the white band containing Enterocytozoon hepatopenaei from top to bottom and place it in a centrifuge tube.

[0072] Specifically, to ensure the staining effect, the following examples use Calcofluor White M2R staining solution for staining. Its preparation method is as follows: Weigh 1 g of Calcofluor White M2R stain (Sigma, 910090) and dissolve it in 100 ml of TBS buffer with a pH value of 7.2 to make the concentration 1% (W / V). Then add 0.1 g of Evan’s blue dye (Sigma, E2129) and store it in the dark at 4°C. Before use, centrifuge it at 12000 rpm / min for 5 min and take the supernatant for use. The above Calcofluor White M2R staining solution can specifically stain Enterocytozoon hepatopenaei in shrimp, can quickly and effectively distinguish this pathogen from other microorganisms, solves the problem that it is difficult to observe and easy to confuse due to the tiny size of the spore, and also provides a rapid staining and identification method for Enterocytozoon hepatopenaei in actual aquaculture production work.

[0073] Specifically, to achieve rapid staining and accurate counting, the staining and counting method used in the following examples includes the following steps:

[0074] 1) Take a 0.22 μm black nuclear pore filter membrane and rinse it thoroughly with sterile water; Place the smooth side of the black nuclear pore filter membrane facing up and flat against the bottom of the filter, ensuring that there are no air bubbles above and below the black nuclear pore filter membrane. Then install the filter on the horizontal table and clamp it with a clip to make the filter in a horizontal state;

[0075] 2) Pre-add 5 ml of sterile water into the filter cartridge in advance, aspirate the Enterocytozoon hepatopenaei suspension and inject it into the filter, and at the same time add 0.2 ml of the Calcofluor White M2R staining solution, mix well, stain for 1 min, and filter under negative pressure until the black nuclear pore filter membrane is just moist;

[0076] 3) Remove the filter, gently lift the black nuclear pore filter membrane with forceps, transfer it to a clean glass slide, drop 1 drop of non-fluorescent immersion oil on the black nuclear pore filter membrane, cover it with a cover glass, and observe under the high-power lens of a fluorescence microscope. Oval bright blue worms can be observed under the ultraviolet light channel of the fluorescence microscope. Count the oval bright blue worms, and randomly calculate the number of worms in at least 10 fields of view; the number in each field of view is preferably about 50 - 80. Prepare a blank slide, and no more than 1 worm should appear in each field of view.

[0077] 4) Calculate the worm concentration of the sample. The calculation formula is BN = Na × S / (Sf × V); where BN is the worm concentration in the sample, in units of number / ml, Na is the average number of worms in each field of view, in units of number, S is the actual filtration area of the filter membrane, in units of mm 2 , Sf is the area of the microscope field of view, in units of mm 2 , and V is the volume of the filtered sample, in units of ml.

[0078] Example 1

[0079] A method for the isolation, purification, staining and counting of Enterocytozoon hepatopenaei, comprising the following steps:

[0080] S1: Dissect the hepatopancreas and intestinal tissues of penaeid shrimp severely infected with Enterocytozoon hepatopenaei at low temperature, grind them with a tissue homogenizer or a glass homogenizer until there are no obvious tissue blocks, and aspirate 5 ml of the tissue homogenate and dilute it to 30 ml with sterile physiological saline to obtain a diluted homogenate;

[0081] S2: Centrifuge the diluted homogenate at 4°C and 600 r / min for 10 min, aspirate the supernatant for standby; add sterile physiological saline to the precipitate to 15 ml and resuspend it;

[0082] S3: Centrifuge the resuspended liquid obtained in step S2 at 4°C and 600 r / min for 10 min, aspirate the supernatant, and mix it with the supernatant obtained in step S2 to obtain a mixed supernatant;

[0083] S4: Add glycerol to the mixed supernatant obtained in step S3, and the ratio of glycerol to the supernatant is 1:3 (V / V). Centrifuge at 4°C and 8000 r / min for 30 min, discard the supernatant; resuspend the precipitate with 25 ml of sterile physiological saline, centrifuge at 4°C and 6000 r / min for 10 min, discard the supernatant, and repeat the above sterile physiological saline centrifugation and washing operation three times;

[0084] S5: resuspending the precipitate obtained in step S4 with 3 ml of sterile physiological saline to obtain a crude extract of hepatocellular carcinoma;

[0085] S6: using sucrose solutions with concentrations of 20%, 25%, 30%, 35%, and 40% to separate and purify hepato-enteric parasites by discontinuous sucrose density gradient, laying out discontinuous sucrose density gradient centrifuge liquid in the order of sucrose solution concentration from small to large, using an extended needle to sequentially inject into the bottom of the centrifuge tube, and the injection volume of each concentration of sucrose solution is 1.5 ml; when laying out each concentration of sucrose solution, 0.1 ml of liquid is left in the syringe to avoid injecting bubbles; the speed of injecting sucrose solution is 1 ml / min, and the whole process is controlled within 15 min;

[0086] S7: Use a pipette to slowly add 1.5 ml of the crude extract of shrimp hepatobiliary cysts to the top of the sucrose gradient liquid surface, place the pipette tip close to the inner wall of the centrifuge tube, make the pipette tip and the tube wall at an angle of 80-90 degrees, and add 1.5 ml of the crude extract at a rate of 1 ml / min to ensure that the sample is immiscible with the top layer of sucrose solution and the liquid surface is clearly visible;

[0087] S8: Centrifuge the prepared sucrose density gradient centrifuge solution in a horizontal rotor at 4°C, 2000 r / min for 30 min and then take it out. Figure 1 The results shown show that a total of 5 bands were separated, among which 1 is the first layer, where a large amount of impurities and a small amount of shrimp hepatobiliary parasites can be seen under the microscope; 2 is the second layer, where a certain amount of impurities and a small amount of hepatobiliary parasites can be seen under the microscope; 3 is the third layer, where high-purity hepatobiliary parasites and no impurities can be seen under the microscope; 4 is the fourth layer, where a small amount of hepatobiliary parasites and a small amount of impurities can be seen under the microscope; 5 is the fifth layer, where a small amount of impurities can be seen under the microscope; it can be seen that the parasites separated from the third layer have the highest purity and the largest number;

[0088] S9: Use a 2.5 ml syringe with an 8 cm extended needle to absorb the third milky white band from top to bottom into a centrifuge tube, centrifuge at 4°C, 6000 r / min for 10 min, discard the supernatant, and suspend the precipitate with 5 ml sterile saline, centrifuge at 4°C, 6000 r / min for 10 min, discard the supernatant, and repeat the above sterile saline centrifugation and washing operation three times to obtain a purified shrimp hepatocystis suspension;

[0089] S10: Take a 0.22-μm black nuclear pore filter membrane and rinse it thoroughly with sterile water. Place the black nuclear pore filter membrane with its smooth side facing up flat on the bottom of the filter, ensuring no air bubbles remain above or below the black nuclear pore filter membrane. Then, install the filter on a horizontal tabletop and clamp it tightly with a clip to keep the filter in a horizontal state. Pre-add 5 ml of sterile water into the filter cartridge, suck the suspension of Hepatocystis and inject it into the filter. At the same time, add 0.2 ml of the Calcofluor White M2R staining solution, mix well, and stain for 1 minute. Filter under negative pressure until the black nuclear pore filter membrane is just moist. Remove the filter, gently lift the black nuclear pore filter membrane with forceps, transfer it to a clean glass slide, place 1 drop of non-fluorescent immersion oil on the black nuclear pore filter membrane, cover it with a coverslip, and move it to the high-power objective of a fluorescence microscope. Observe under the ultraviolet light channel to see oval bright blue worms (the staining effect is as shown in Figure 2 ). Count the oval bright blue worms, randomly calculate the number of worms in 10 fields of view, and the calculation results are shown in Table 1. Prepare blank slides according to the above method to ensure that no more than 1 worm appears in each field of view, so as to exclude the interference of impurity fluorescence signals and ensure the accuracy of counting. Substitute the corresponding data into the formula BN = Na × S / (Sf × V) to calculate the concentration of worms in the sample (worms / ml), where Na represents the average number of worms in each field of view, which is 65.7, S is the actual filtration area of the filter membrane, which is 186.26 mm 2 , Sf is the area of the microscope field of view, which is 0.233 mm 2 , and V is the volume of the filtered sample, which is 0.01 ml. Calculate that BN is 5.25 × 10 6 worms / ml.

[0090] Table 1

[0091]

[0092] Example 2

[0093] After thoroughly grinding the hepatopancreas and intestinal tissues of 5 g of shrimp severely infected with Enterocytozoon hepatopenaei until there were no obvious tissue masses, add sterile physiological saline to dilute to 30 ml, centrifuge at 4°C and 600 r / min for 10 min, and aspirate the supernatant; add sterile physiological saline to the precipitate to 30 ml again, resuspend and repeat the above centrifugation operation, aspirate the supernatant, mix the two supernatants, and divide them into 2 equal parts on average. One part is added with glycerol, and the ratio of glycerol to the supernatant is 1:3 (V / V). The other part is not treated. At the same time, take it out after centrifuging at 4°C and 8000 r / min for 30 min, discard the supernatant, resuspend the obtained precipitate with 25 ml of sterile physiological saline respectively, centrifuge at 4°C and 6000 r / min for 10 min, discard the supernatant, repeat this operation 3 times, and finally resuspend the obtained precipitate with 3 ml of sterile physiological saline to obtain two crude extracts of Enterocytozoon hepatopenaei. By comparing the two samples, it can be seen that the sample added with glycerol is bright orange-yellow in color and has no obvious impurities, while the sample without glycerol is dull light yellow in color and has more impurities. That is, a large amount of pigments and impurities are separated from the sample with glycerol as the washing medium, and less impurities and pigments are separated without adding glycerol.

[0094] From the above experimental results, it can be seen that the density of the glycerol solution at an appropriate concentration is slightly lower than that of Enterocytozoon hepatopenaei and slightly higher than that of impurities. Using glycerol as a medium during differential centrifugation can remove most of the hepatopancreas tissue fragments and pigments of shrimp, and does not affect the collection of the parasite. In addition, glycerol itself has no toxicity and has no effect on Enterocytozoon hepatopenaei. Glycerol can be removed by repeated washing and centrifugation, and has no effect on subsequent experiments.

[0095] Example 3

[0096] To verify the staining specificity of the Calcofluor White M2R staining solution prepared in this example for Enterocytozoon hepatopenaei, the following experiment was designed: Use Calcofluor White M2R staining solution and DAPI fluorescent staining agent (Sigma, D9542) as staining agents to stain a mixed solution of a purified solution of Enterocytozoon hepatopenaei and a pure culture solution of Vibrio harveyi (rod-shaped bacteria, with obvious morphological differences from Enterocytozoon hepatopenaei). The staining method is as follows: Aspirate 10 μL of the purified Enterocytozoon hepatopenaei suspension in step S9 of Example 1 onto a glass slide, mix evenly with an equal volume of the staining agent, incubate at room temperature for 1 min and then prepare a slide, and observe the staining results under a fluorescence microscope. As Figure 3 shown, where Figure a is the staining result of Calcofluor White M2R; Figure b is the staining result of DAPI. In Figure b, A is Enterocytozoon hepatopenaei and B is Vibrio harveyi.

[0097] The experimental results show that: the Calcofluor White M2 staining solution prepared in the present invention only stains Enterocytozoon hepatopenaei, while the conventional dye DAPI not only stains Enterocytozoon hepatopenaei, but also stains Vibrio harveyi. That is, the Calcofluor White M2R staining solution prepared in the present invention can specifically bind to the chitin component in the spore wall of Enterocytozoon hepatopenaei, and can quickly and effectively distinguish this pathogen from tissue debris, other pathogenic bacteria, and virus polyhedra, etc., with strong specificity.

[0098] Example 4

[0099] Using the methods involved in S1 to S9 in Example 1, isolate and purify Enterocytozoon hepatopenaei from 3 g of hepatopancreas and intestinal tissues of penaeid shrimp severely infected with Enterocytozoon hepatopenaei. Resuspend the obtained purified worms in 3 ml of sterile physiological saline. Pipette 10 μl of the resuspended solution onto a glass slide, add an equal volume of DAPI staining agent, mix well quickly and thoroughly, and then observe under a fluorescence microscope. Randomly select 10 fields of view, record the number of worms and the number of impurities in each field of view respectively. Through the calculation formula: purity of Enterocytozoon hepatopenaei = total number of Enterocytozoon hepatopenaei / (total number of Enterocytozoon hepatopenaei + total number of impurities) × 100%, calculate the purity of Enterocytozoon hepatopenaei purified by this method. The experimental results are shown in Table 2.

[0100] Table 2

[0101]

[0102] In the above experiment, DAPI was used as the staining agent, which can stain both the worms and the impurities at the same time, and can better observe whether the impurities are completely removed. From the experimental results, we can calculate that the method for isolating and purifying Enterocytozoon hepatopenaei involved in the present invention can obtain Enterocytozoon hepatopenaei with a purity greater than 99%.

[0103] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for the isolation, purification, staining and counting of Enterocytozoon hepatopenaei, characterized in that, It includes the following steps: Sample preparation: Dissect the hepatopancreas and intestinal tissues of shrimp infected with Enterocytozoon hepatopenaei, make them into tissue homogenate, and dilute it with sterile saline to obtain a diluted homogenate. Crude extraction: Use differential centrifugation to make the obtained diluted homogenate into a crude extract of Enterocytozoon hepatopenaei; the washing medium used in the crude extraction step includes glycerol. Purification: Use discontinuous sucrose density gradient centrifugation to separate and purify the crude extract of Enterocytozoon hepatopenaei obtained in step S2 to obtain a purified suspension of Enterocytozoon hepatopenaei. Staining and counting: Stain Enterocytozoon hepatopenaei in the obtained purified suspension of Enterocytozoon hepatopenaei with fluorescent brightener, filter the stained Enterocytozoon hepatopenaei onto a black nuclear pore filter membrane, and observe and count under a high-power fluorescence microscope.

2. The method for isolation, purification, staining and counting of Enterocytozoon hepatopenaei according to claim 1, wherein The sample preparation step includes: Dissect the hepatopancreas and intestinal tissues of shrimp severely infected with Enterocytozoon hepatopenaei at low temperature, grind them with a tissue homogenizer or a glass homogenizer until there are no obvious tissue lumps, extract 5 - 10 ml of tissue homogenate and dilute it to 30 - 35 ml with sterile saline to obtain a diluted homogenate.

3. The method for isolation, purification, staining and counting of Enterocytozoon hepatopenaei according to claim 2, characterized in that, The crude extraction includes the following steps: S2: Centrifuge the diluted homogenate at 4°C and 600 r / min for 10 min, aspirate the supernatant for standby; add sterile saline to the precipitate to 15 - 20 ml and resuspend it. S3: Centrifuge the resuspended liquid obtained in step S2 at 4°C and 600 r / min for 10 min, aspirate the supernatant, and mix it with the supernatant obtained in step S2 to obtain a mixed supernatant. S4: Add glycerol to the mixed supernatant obtained in step S3, mix glycerol and the supernatant in a volume ratio of 1:3, centrifuge at 4°C and 8000 r / min for 30 min, and discard the supernatant; wash the precipitate three times by centrifugation with 20 - 25 ml of sterile saline at 4°C and 6000 r / min for 10 min. S5: Resuspend the precipitate obtained in step S4 with 3 - 5 ml of sterile saline to obtain a crude extract of Enterocytozoon hepatopenaei.

4. The method for isolation, purification, staining and counting of Enterocytozoon hepatopenaei according to claim 3, wherein, The purification includes the following steps: S6: Inject into the bottom of the centrifuge tube successively in ascending order of sucrose solution concentration using a lengthened needle, and the injection volume of each concentration of sucrose solution is 1.5 - 2 ml. S7: Slowly add 1.5 ml of the crude extract of Enterocytozoon hepatopenaei to the top of the sucrose gradient liquid surface using a pipette, ensure that the sample does not mix with the topmost sucrose solution, and the interface is clearly visible. S8: Centrifuge the sucrose density gradient centrifugation liquid prepared in step S7 at 4°C and 2000 r / min for 30 min using a horizontal rotor. S9: Use a syringe to aspirate the white band at the gradient boundary from top to bottom into a centrifuge tube, centrifuge at 4°C and 6000 r / min for 10 min, discard the supernatant, and wash the obtained precipitate three times by centrifugation with 5 - 10 ml of sterile saline at 4°C and 6000 r / min for 10 min to obtain a purified suspension of Enterocytozoon hepatopenaei.

5. The method for isolation, purification, staining and counting of Enterocytozoon hepatopenaei according to claim 4, wherein The injection speed of the sucrose solution in step S6 is 1 ml / min, and the total time used in step S6 ≤ 15 min.

6. The method for isolation, purification, staining and counting of Enterocytozoon hepatopenaei according to claim 5, wherein Five concentrations of sucrose solutions are used in step S6, and their concentrations are 20%, 25%, 30%, 35%, and 40% respectively; the preparation methods of each concentration of sucrose solution are as follows: 20% Sucrose Solution: Weigh 20 g of sucrose and make up the volume to 100 ml with deionized water; 25% Sucrose Solution: Weigh 25 g of sucrose and make up the volume to 100 ml with deionized water; 30% Sucrose Solution: Weigh 30 g of sucrose and make up the volume to 100 ml with deionized water; 35% Sucrose Solution: Weigh 35 g of sucrose and make up the volume to 100 ml with deionized water; 40% Sucrose Solution: Weigh 40 g of sucrose and make up the volume to 100 ml with deionized water.

7. The method for isolation, purification, staining and counting of Enterocytozoon hepatopenaei according to claim 6, wherein Step S9 is to aspirate the third milky white band from top to bottom with a syringe into a centrifuge tube, centrifuge at 6000 r / min at 4 °C for 10 min, discard the supernatant, and wash the precipitate three times by centrifugation at 6000 r / min at 4 °C with sterile normal saline to obtain a purified suspension of Hepatocystis.

8. The method for isolation, purification, staining and counting of Enterocytozoon hepatopenaei according to claim 5, wherein The method of adding the crude extract of Hepatocystis in step S7 is to place the pipette tip close to the inner wall of the centrifuge tube at the top of the liquid surface with a prepared gradient, make the pipette tip form an angle of 80 - 90° with the tube wall, and add 1.5 ml of the crude extract at a speed of 1 ml / min to ensure that the sample does not mix with the topmost sucrose solution and the interface is clearly visible.

9. The method for isolation, purification, staining and counting of Enterocytozoon hepatopenaei according to claim 1, characterized in that, The fluorescent brightener is Calcofluor White M2R staining solution. The preparation method of the Calcofluor White M2R staining solution is as follows: Weigh 1 g of Calcofluor White M2R staining agent and dissolve it in 100 ml of TBS buffer with a pH value of 7.2 to make the concentration 1% (W / V), then add 0.1 g of Evan’s blue dye and store it in the dark at 4 °C; before use, centrifuge at 12000 rpm / min for 5 min and take the supernatant for use.

10. The method for isolation, purification, staining and counting of Enterocytozoon hepatopenaei according to claim 9, wherein, The counting includes the following steps: 1) Take a 0.22 - μm black nuclear pore filter membrane and rinse it thoroughly with sterile water; place the black nuclear pore filter membrane with the smooth side up and flat on the bottom of the filter, ensuring that there are no air bubbles above and below the black nuclear pore filter membrane. Then install the filter on a horizontal table and clamp it with a clip to make the filter in a horizontal state; 2) Pre - add 5 ml of sterile water into the filter cylinder, aspirate the suspension of Hepatocystis and inject it into the filter, and at the same time add 0.2 ml of the Calcofluor White M2R staining solution, mix well, stain for 1 min, and filter under negative pressure until the black nuclear pore filter membrane is just moist; 3) Remove the filter, gently lift the black nuclear pore filter membrane with forceps, transfer it to a clean glass slide, drop 1 drop of non - fluorescent immersion oil on the black nuclear pore filter membrane, cover it with a cover glass, and observe it under the high - power lens of a fluorescence microscope. Oval bright blue worms can be observed under the ultraviolet light channel of the fluorescence microscope. Count the oval bright blue worms, and randomly calculate the number of worms in at least 10 fields of view; 4) Calculate the concentration of worms in the sample. The calculation formula is BN = Na × S / (Sf × V); where BN is the concentration of worms in the sample, in units of number / ml, Na is the average number of worms in each field of view, in units of number, S is the actual filtration area of the filter membrane, in units of mm 2 , Sf is the area of the microscope field of view, in units of mm 2 , and V is the volume of the filtered sample, in units of ml.

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