A method for separating, purifying and collecting planarian eggs

Abstract

The application discloses a planarian egg separation, purification and collection method, which can effectively remove other small organisms in water bodies, realize purification and collection of target planarian eggs, and make the collected planarian eggs available for molecular operations such as 18s and 28s sequence amplification and whole genome sequencing and gene sequencing experiments, and not be interfered by other aquatic organisms. The method comprises the following steps: collecting original planarian eggs after culture; continuously screening and removing 95% of impure planarian eggs; removing 99.5% of the impure planarian eggs by air suspension and retaining target planarian eggs by using sintered mesh pore interception; dissolving impure planarian bodies by air suspension of a chemical reagent aqueous solution; washing with clean water; collecting planarian eggs again after screening and removing impurities; manually sorting and collecting under a microscope to remove crystal small fragments. The application can realize large-batch separation and clean collection of planarian eggs, is convenient to operate, and the planarian eggs after clean collection still have biological activity, and the extracted genomic DNA meets the requirements of subsequent molecular operations and gene sequencing.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of egg separation, and particularly relates to a planarian egg separation, purification and collection method. BACKGROUND

[0002] Planarians are a class of animals belonging to Platyhelminthes, usually having a flat body similar to a worm, and being the earliest animals with three germ layers and two-sided symmetry in the history of species system evolution. Planarians are often selected as model organisms for stem cell, anti-aging and biological adhesion researches due to their strong tissue repair and regeneration abilities after excision. According to their habitats, planarians can be divided into three categories: marine planarians, freshwater planarians and terrestrial planarians. Unlike planarians in water environment, terrestrial planarians have evolved a set of effective hunting skills to adapt to the terrestrial environment, making them natural predators of small invertebrates on land or in soil, and providing good biological materials for pest control.

[0003] At present, researches on planarians are mostly focused on the design of field worm capture and collection devices, planarian chromosome specimen preparation, the design of a planarian moving rate measuring device, the construction of a gene deletion model in planarians by using gene interference, and the like. However, there is no report on the separation, purification and collection of planarian eggs, especially small terrestrial planarian eggs. In addition, most insects (arthropods) lay eggs on the surface of plant tissues, and most insect egg collection devices and methods are not suitable for planarian eggs cultured in water. Moreover, the egg collection method in fish culture is not suitable for planarian egg collection because the size of planarian eggs is quite different from that of fish eggs, and the large-scale high-airflow flushing environment in a culture pond can easily damage planarian eggs.

[0004] In nature, planarians reproduce and are cultured indoors in water derived from original mountain spring water or stored mountain spring water, which contains small organisms such as algae, oxygen-producing bacteria and trace mineral elements such as calcium and selenium that are beneficial to planarians, and this natural ecological microenvironment is most conducive to the growth and breeding of planarians. At the same time, some other small aquatic organisms harmful to planarians, such as rotifers and flagellates, exist in mountain spring water. During the process of culturing planarians using mountain spring water, the harmful organisms will reproduce and produce a certain amount of harmful egg masses within 3-5 months, which will destroy the production process and environmental stability of planarian eggs on the one hand, and the mixed sample of planarian eggs will seriously affect the quality of genomic DNA and cause great difficulty in subsequent on-machine analysis, resulting in that researchers cannot obtain high-purity planarian eggs, thereby delaying the research process such as nucleic acid extraction, molecular sequence analysis and editing. SUMMARY

[0005] This section is intended to introduce some aspects of one or more embodiments of the present application, which are described below. This section is not intended to limit the scope or the patentability of the embodiments.

[0006] In view of the above and / or other problems existing in the prior art, the present application is proposed.

[0007] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art, and to provide a method for separating, purifying and collecting planarian eggs.

[0008] To solve the above technical problems, the present application provides the following technical solutions: a method for separating, purifying and collecting planarian eggs, comprising: removing impurities from raw worm eggs and collecting raw worm egg liquid; continuously screening the raw worm egg liquid to screen out 95% of the impure worm eggs; removing 99.5% of the impure worm eggs by air suspension, and using a sintered mesh to trap target planarian eggs; dissolving the impure worm bodies by air suspension of a chemical reagent aqueous solution, washing and screening the worm eggs again after washing and screening; manually sorting and collecting under a microscope to remove crystal fragments, and obtaining purified planarian eggs.

[0009] As a preferred solution of the method of the present application, wherein: the removing impurities from raw worm eggs comprises filtering the raw worm eggs through a 350-mesh screen, washing the collected worm eggs with double distilled water after filtering out small particle impurities.

[0010] As a preferred solution of the method of the present application, wherein: the continuously screening the raw worm egg liquid comprises continuously passing the raw worm egg liquid through a 150-mesh and a 200-mesh screen, collecting planarian eggs on the screen, and discarding the impure worm eggs in the filtrate, thereby removing 95% of the impure worm eggs.

[0011] As a preferred solution of the method of the present application, wherein: the removing 99.5% of the impure worm eggs by air suspension comprises collecting the treated worm eggs in a glass container with a 120-mesh sintered mesh face, making the edge strictly adhere to the inner wall of the container, and connecting an air pump to the bottom layer of the worm egg liquid for air suspension; the sintered mesh traps planarian eggs of a target pore size, thereby removing 99.5% of the final impure worm eggs.

[0012] As a preferred solution of the method of the present application, wherein: the chemical reagent aqueous solution comprises a urea solution and a hydroxylamine hydrochloride solution, and the chemical reagent treatment time is 16-18 hours.

[0013] As a preferred solution of the method of the present application, wherein: the concentration of the urea solution is 6-10% W / W.

[0014] As a preferred solution of the method, the concentration of the hydroxylamine hydrochloride solution is 5-8% W / W.

[0015] As a preferred solution of the method, the water washing and the impurity removal by sieving are followed by collecting the worm eggs again, wherein the filter screen is 350 mesh, and the worm eggs are collected after being washed by spraying with double distilled water.

[0016] As a preferred solution of the method, the manual sorting and collecting under the microscope include manual hand shaking and vortexing to gather the worm eggs, and the worm eggs are collected after the removal of the tiny crystal fragments by using the sterilized insect dissecting needle.

[0017] As a preferred solution of the method, the original worm eggs include the nematode eating planarian worm eggs.

[0018] The present application has the following advantages:

[0019] (1) The present application establishes a set of planarian worm egg separation, purification and collection techniques for the first time, including the physical sieving and impurity removal of the remaining mixed worm eggs in the original worm eggs, the chemical reagent digestion of the remaining mixed worm bodies in the worm egg mixture, the washing of the worm eggs after the digestion of the impurities, the manual microscope examination, separation and impurity removal of the worm eggs, and the concentrated collection of the worm eggs. The present application combines the physical sieving and chemical digestion methods for the first time, and can remove more than 99.5% of the impurities in the planarian worm egg stock solution. These operation methods are simple, time-saving and labor-saving, and are effective, and can meet the requirements of single clean worm eggs for subsequent molecular operations and gene sequencing experiments.

[0020] (2) The present application uses a screen and a sintered mesh for the impurity removal of the mixed worm eggs, and removes the mixed worm eggs and retains the target particle size and relatively clean planarian worm eggs by different pore sizes and air suspension.

[0021] (3) The reagent urea used in the method of the present application is relatively mild to the planarian worm eggs. The gradient concentration test treatment determines that 6%-10% and 5%-8% are the effective concentration ranges of urea and hydroxylamine hydrochloride for impurity digestion, respectively. And the planarian worm eggs still have biological activity after being treated with urea below 10% concentration and hydroxylamine hydrochloride below 8% concentration for two days, and can successfully hatch into larvae.

[0022] (4) After the planarian worm eggs treated by the present application are manually sorted and collected, the genomic DNA of 10,000 worm eggs is extracted by using a reagent kit. The gel electrophoresis result shows that the genomic DNA of the worm eggs treated by the present application still maintains integrity, has sufficient DNA concentration and good quality, and completely meets the requirements of subsequent molecular operations and gene sequencing experiments for the crude sample DNA of the worm eggs. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings. Among them:

[0024] Figure 1 The figure shows the size of nematode worm eggs of the present application.

[0025] Figure 2 The figure shows the untreated worm egg stock solution in the embodiment of the present application.

[0026] Figure 3 The figure shows the cleaning and impurity removal device for air suspension and embedding worm eggs in the sintered mesh surface in the embodiment of the present application.

[0027] Figure 4 The figure shows the appearance characteristics of worm eggs treated by 8% urea, 5% hydroxylamine hydrochloride and 3% urea combined with 2% hydroxylamine hydrochloride in the embodiment of the present application.

[0028] Figure 5 The figure shows the clean worm eggs after artificial sorting in the embodiment of the present application.

[0029] Figure 6 The figure shows the single clean worm eggs after treatment, separation and collection by the present application in the embodiment of the present application.

[0030] Figure 7 The figure shows the integrity of worm egg genomic DNA after gel electrophoresis verification treatment in the embodiment of the present application.

[0031] Figure 8 The figure shows the worm egg genomic DNA electropherogram that does not meet the standard detected by Beijing Baimaikesi Biological Technology Co., Ltd. in the embodiment of the present application.

[0032] Figure 9 The figure shows the worm egg genomic DNA electropherogram that meets the standard and can be used for subsequent machine sequencing analysis detected by Beijing Baimaikesi Biological Technology Co., Ltd. in the embodiment 1 of the present application. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail in combination with the description of the embodiments.

[0034] In the following description, a lot of specific details are set forth in order to provide a thorough understanding of the present application, however, the present application can be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the concept of the present application, therefore, the present application is not limited to the specific embodiments disclosed below.

[0035] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an embodiment that is independent of or mutually exclusive with other embodiments.

[0036] The present application first establishes a set of nematophagous planarian egg separation, purification and collection technology, and successfully separates and collects nearly 200,000 single clean worm eggs.

[0037] The nematophagous planarian eggs in the embodiments of the present application are the worm eggs of the planarian discovered by the research group in the natural environment, which can directionally prey on root-knot nematodes, especially have strong predatory capacity on southern root-knot nematodes. The research group discovered the nematophagous planarian in a tobacco planting field in Shizixiang, Pingba District, Anshun City, Guizhou Province in spring 2019 when carrying out soil resource investigation. The planarian was fed with various types of nematodes, and its predatory capacity was observed. It was found through research that after feeding southern root-knot nematodes, it was found that one planarian adult could prey on 7-15 southern root-knot nematodes in 24 hours, the body length of the larvae was 200-300 μm, and the body size of the adult could reach 1 mm, and the planarian could survive in various environments such as water, humid soil and mud. The size chart of the nematophagous planarian eggs of the present application is shown in Figure 1 .

[0038] The related parameters of the air pump equipment in the present application are shown in Table 1.

[0039] Table 1

[0040]

[0041] In the embodiments of the present application, the DNA sample extraction and preparation required for three-generation sequencing of the planarian cultured in the laboratory by Beijing Baimaikes Technology Co., Ltd. are as follows:

[0042] I. Sample preparation:

[0043] 1. After the worm eggs treated and collected according to the scheme of the present application embodiment 1, the worm eggs were rapidly transferred to a-80℃ refrigerator for preservation after being rapidly frozen in liquid nitrogen for 5 min.

[0044] 2. Contact the project officer of Beijing Baimaikes Technology Co., Ltd. to send the sample, and send the sample to the whole genome sequencing department of the company in Huangdao District, Qingdao City for subsequent DNA extraction and testing

[0045] II. Sample extraction (SDS method)

[0046] 1. Reagents used: SDS solution chloroform / isopentanol (24:1), NaCl solution 75% ethanol Rnase, water

[0047] 2. Experimental steps:

[0048] 1.1. Grind the tissue sample in liquid nitrogen and divide into centrifuge tubes;

[0049] 1.2. Add SDS solution, water bath;

[0050] 1.3. Add NaCl solution, stand for a while and then centrifuge;

[0051] 1.4. Add chloroform / isopentanol (24:1) to the supernatant and centrifuge;

[0052] 1.5. Add isopropyl alcohol to the supernatant and centrifuge, discard the supernatant;

[0053] 1.6. Add 75% ethanol, centrifuge, discard the supernatant;

[0054] 1.7. Air dry, add TE to dissolve the DNA, and store in a -20°C refrigerator.

[0055] 3. Column purification

[0056] 3.1. Purification instruments and equipment: centrifuge, grinder, water bath, vortex mixer, vortex shaker, calibrated pipettor, 1.5 ml centrifuge tube, 15 ml centrifuge tube, 50 ml centrifuge tube.

[0057] 3.2. Purification reagents (supplies): Buffer QBT, Buffer QC, Buffer QF, Buffer G2, Buffer TE

[0058] 3..3. Purification environment: no special requirements, room temperature

[0059] Operation steps

[0060] 3.2. Operation steps

[0061] 3.2.1. Lysis: add lysis solution to nucleic acids, then add l RNase A and proteinase K, mix well by inverting, and lyse at 50°C

[0062] 3.2.2. Lysis collection 5000x g, 4°C centrifuge for 10 min, transfer the supernatant to a new 15 ml or 50 ml centrifuge tube, and stand at room temperature for a few minutes to balance the lysis solution to room

[0063] 3.2.3. Column

[0064] 3.2.3.1 Equilibrate the 20G column with Buffer QBT, let the liquid flow out naturally by gravity, discard the waste liquid;

[0065] After centrifugation, transfer the supernatant of the lysate into the column, let it flow out naturally by gravity, if the liquid flow rate is lower than 2 drops / min, 3.2.3.2 wash the column with Buffer QC, let the liquid flow out naturally by gravity, discard the waste liquid

[0066] 3.2.3.3 Elute the DNA with Buffer QF into 1.5ml centrifuge tubes, about

[0067] 3.2.3.4 Add isopropanol, gently invert and mix 10-20 times, stand at room temperature for 15min;

[0068] 3.2.3.5 12000g, 4℃ centrifuge for 20min, discard the supernatant;

[0069] 3.2.3.6 Wash the precipitate with 1ml 75% ethanol, gently invert several times, then centrifuge at 12000rpm for 5min, discard the supernatant;

[0070] 3.2.3.7 Repeat the 75% ethanol washing once, finally centrifuge for a moment, remove the liquid completely;

[0071] 3.2.3.8 Dry at room temperature until the DNA edge is translucent, add an appropriate amount of TE to dissolve the DNA, and incubate at 50℃ metal bath for 1-2h, gently shake several times during the incubation to promote dissolution;

[0072] 3.2.3.9 Store the extracted DNA at 4℃ for testing (<1 week), and store at -20℃ for long-term preservation.

[0073] III. Index detection

[0074] Detection method: Use Nanodrop (manufacturer: Thermo Fisher Scientific, model NANODROP2000), Qubit (manufacturer: invitrogen, model: QubitTM3Flurometer) to detect the concentration of the extracted nucleic acid, and use agarose gel electrophoresis (electrophoresis instrument manufacturer: Tanon, model EPS600; electrophoresis tank manufacturer: Tiangen Biochemical Technology (Beijing) Co., Ltd., model: HE-120) to detect the integrity.

[0075] The qualified index is shown in Table 2.

[0076] Table 2

[0077]

[0078] m: total mass of total quantity of DNA;

[0079] c: concentration of Qubit detection concentration of DNA solution;

[0080] N / Q: ratio of Nanodrop detection concentration to Qubit detection concentration;

[0081] Size: fragment size, refers to the main band size of DNA molecule fragments;

[0082] OD260 / 280: ratio of OD260 and OD280 in Nanodrop detection, one of the indicators reflecting DNA purity;

[0083] OD260 / 230: ratio of OD260 and OD230 in Nanodrop detection, one of the indicators reflecting DNA purity.

[0084] Example 1

[0085] The present embodiment provides a method for separating, purifying and collecting nematode planarian eggs, the main steps of which are:

[0086] (1) Collect the original eggs of laboratory culture by filtering through a 350-mesh screen, and then spray the eggs collected on the 350-mesh screen with double distilled water after removing small-particle impurities by filtration;

[0087] Figure 2 (Left) shows the untreated egg stock solution.

[0088] (2) Continuously pass the egg liquid collected in step (1) through a 150-mesh and a 200-mesh screen, and collect the eggs on the screen.

[0089] (3) Place a 120-mesh (pore size 125 μm) sintered screen in a glass cylindrical container at 1 / 2 volume, make its edge strictly fit the inner wall of the container, add clean water to 2 / 3 volume of the container, add the sieved eggs collected in step (2) to the clean water, and perform aeration suspension by connecting an air pump to the bottom layer of the egg liquid, so that the water body flows, the air flow size is adjusted to circulate suspension, until the target planarian eggs (about 150 μm long and about 120 μm wide) are embedded in the pores of the 120-mesh sintered screen, the planarian eggs are trapped through the screen pores, and the rest of the mixed insect eggs are precipitated at the bottom of the container dish;

[0090] Remove the sintered screen and place it in sterile water, gently shake it to elute the planarian eggs in the sterile water, then collect the egg liquid in a centrifuge tube and centrifuge at 200 rpm to precipitate and aggregate the eggs. This step can remove 99.5% of the mixed insect eggs;

[0091] Figure 3 The application discloses a cleaning and impurity removing device for aerating suspension and embedding planarian eggs by using a sintered mesh.

[0092] (4) The effective concentration range of urea for dissolving impurities is determined as 6% to 10% through gradient concentration test treatment, and the planarian eggs still have biological activity and can successfully hatch into larvae after being transferred to clean water after being treated by 10% urea for two days, so 8% (W / W) urea solution is selected for dissolving impurities in the collected egg liquid in step (3);

[0093] The specific process is as follows:

[0094] A water breeding aerating pump with a 35W power, equipped with a lithium battery and four air outlets is used for suspension treatment, so that the urea and impurities are fully mixed and contacted, and the impurities are maximally dissolved.

[0095] The external appearance of the planarian eggs and impurities after treatment by different concentration gradient urea is shown in Table 3.

[0096] Table 3

[0097]

[0098]

[0099] It can be seen that the planarian eggs treated by 8% urea have better external characteristics;

[0100] Meanwhile, the external characteristics of the planarian eggs treated by 8% urea for 18h are shown in Figure 4 .

[0101] (5) After the planarian eggs are treated by urea for 18h, the planarian eggs in the liquid are settled, the upper liquid is pumped out, and the planarian eggs are suspended in double-distilled water treated by high temperature and high pressure for washing, and the washing is repeated for 10 to 15 times, so as to remove residual urea.

[0102] The external appearance of the planarian eggs and impurities after treatment by 8% urea for different time is shown in Table 4.

[0103] Table 4

[0104]

[0105]

[0106]

[0107] (6) The planarian eggs washed by double-distilled water are filtered through a 350-mesh screen, small-particle impurities are filtered out, and then the planarian eggs are sprayed by double-distilled water and collected in a disposable plastic lunch box.

[0108] (7) Put no more than 30ml of the egg liquid into a 9cm disposable plastic culture dish, then manually shake and swirl the eggs under a microscope to gather them, and then remove the impurities and fragments with a sterilized insect dissection needle and collect the eggs, and then store them in a -80°C refrigerator for use.

[0109] Figure 5 (left) and Figure 6 (left) respectively show the clean eggs after manual sorting and the single clean eggs after being treated and separated by the method of the application.

[0110] (8) Extract the genomic DNA of 10,000 single clean eggs collected by the Omega Mollusc DNA Kit, and the specific steps are as follows:

[0111] 1) Grind the egg sample in liquid nitrogen with a mortar and pestle, and transfer the ground powder sample to a 1.5mL microcentrifuge tube;

[0112] 2) Add 350μl BufferML1 and 25μl ProteinaseK, vortex to mix, and incubate at 60°C for at least 30min until the sample is completely dissolved;

[0113] 3) Add 350μl of a chloroform:isopropyl alcohol (24:1) mixture, vortex to mix, centrifuge at 10,000xg for 2min at room temperature, and carefully transfer the supernatant to a new 1.5mL centrifuge tube;

[0114] 4) Add 5μl of RNaseA, stand at room temperature for 10-30min, and continue with the following steps;

[0115] 5) Measure the volume of the solution in the previous step, add an equal volume of BufferMBL, vortex to mix at maximum speed for 15s, and incubate at 70°C for 10min;

[0116] 6) Add 0.5 times the volume of anhydrous ethanol, vortex to mix at maximum speed for 15s;

[0117] 7) Put the column into a 2mL collection tube, transfer 750μl of the mixture in step 6 to the column, centrifuge at 10,000xg for 1min at room temperature, and discard the filtrate;

[0118] 8) Repeat step 7) until all the mixture has been transferred through the column;

[0119] 9) Put the column back into a 2mL collection tube, add 500μl of HB Buffer, centrifuge at 10,000xg for 30s, and discard the filtrate;

[0120] 10) Insert the column into a 2mL collection tube, add 700μl DNA Wash Buffer, centrifuge at 10,000xg for 1min, and discard the filtrate;

[0121] 11) Repeat step 10;

[0122] 12) Insert the column into a 2 mL collection tube and centrifuge the empty column at 15,000 mL for 2 min at room temperature.

[0123] 13) will Insert the column into a new 1.5 mL centrifuge tube, add 40 μl of double-distilled water preheated at 60–70 °C, and centrifuge at 10,000 x g for 1 min to elute the DNA.

[0124] 14) Repeat step 13, wash off the DNA with fresh 40 μl of double-distilled water, and freeze the sample at -20°C if it is not to be detected by electrophoresis immediately.

[0125] Then, gel electrophoresis was performed according to the electrophoresis parameters in Table 4 to detect the integrity, concentration, and quality of the genomic DNA of the insect eggs treated with the technology of this invention.

[0126] Table 5

[0127]

[0128] See gel electrophoresis for verification of genomic DNA integrity in insect eggs treated with 8% urea. Figure 7 (Left) Display.

[0129] See the electrophoresis image of the genomic DNA of the insect eggs detected by Beijing Biomarker Biotechnology Co., Ltd. Figure 9 See the electrophoresis image of the genomic DNA of the non-compliant eggs. Figure 8 .

[0130] Example 2

[0131] (1) The original insect eggs cultured in the laboratory were collected by filtering through a 350-mesh screen. After filtering to remove small-diameter impurities, the insect eggs that had gathered on the 350-mesh screen were collected by spraying with double-distilled water.

[0132] Figure 2 (Middle) shows the untreated original solution of insect eggs.

[0133] (2) Pass the liquid containing insect eggs collected in step (1) through 150 mesh and 200 mesh screens continuously. Large-diameter impurities are left on the screens. Collect the insect eggs in the filtrate.

[0134] (3) In the glass cylindrical container 1 / 2 volume, 120 mesh (125 μm aperture) sintered screen is placed with its edges strictly adhering to the inner wall of the container, and clean water is added to 2 / 3 volume of the container. The screened worm eggs in step (2) are added to the clean water, and the worm egg liquid bottom layer is connected to the air pump for air suspension. The water body is made to flow, and the air flow size is adjusted for circulation suspension until the target planarian worm eggs (about 150 μm long and about 110 μm wide) are embedded in the 120 mesh sintered screen aperture. The planarian worm eggs are trapped through the screen aperture, and the remaining miscellaneous worm eggs are precipitated at the bottom of the container dish.

[0135] The sintered screen is taken out and placed in sterile water and gently shaken to elute the planarian worm eggs in the sterile water. The worm egg liquid is collected in a centrifuge tube and centrifuged at 200 rpm to precipitate and aggregate the worm eggs. This step can remove 99.5% of the miscellaneous worm eggs. Figure 3 A cleaning and impurity removal device for air suspension and embedding planarian worm eggs using a sintered screen is shown.

[0136] (4) The gradient concentration test treatment determines that 5% to 8% is the effective concentration range of hydroxylamine hydrochloride for impurity digestion. After the planarian worm eggs are treated with hydroxylamine hydrochloride at a concentration of 8% or less for one day, the external color and morphology of the worm eggs are restored to the same as before treatment. Therefore, 5% (W / W) hydroxylamine hydrochloride aqueous solution is used to digest the impurity worm bodies in the worm egg liquid collected in step 3. A 35W power air pump for water culture with four air outlets equipped with lithium batteries is used for suspension treatment to make hydroxylamine hydrochloride and impurities fully mixed and contacted to maximize impurity digestion.

[0137] The external appearance of worm eggs and impurities after treatment with different concentrations of hydroxylamine hydrochloride is shown in Table 6.

[0138] Table 6

[0139]

[0140]

[0141] The external characteristics of worm eggs after treatment with 5% hydroxylamine hydrochloride are shown in Figure 4 (Middle).

[0142] (5) After 18h of hydroxylamine hydrochloride digestion, the worm eggs in the liquid are settled, the upper liquid is removed, and double distilled water treated by high temperature and high pressure is injected for suspension washing. The washing is repeated 10-15 times to remove residual hydroxylamine hydrochloride.

[0143] The external appearance of worm eggs and impurities after treatment with 5% hydroxylamine hydrochloride for different times is shown in Table 7.

[0144] Table 7

[0145]

[0146]

[0147] (6) The insect eggs washed with double distilled water are filtered through a 350 mesh screen to remove small impurities, and then the insect eggs are sprayed with double distilled water and collected in a disposable plastic lunch box.

[0148] (7) The insect eggs are collected in a 9 cm disposable plastic petri dish, and then the insect eggs are manually agitated under a microscope to gather the insect eggs, and then the impurities are removed using a sterilized insect dissection needle, and the insect eggs are collected and stored in a -80°C refrigerator.

[0149] Figure 5 (Chinese) and Figure 6 (Chinese) show clean insect eggs after manual sorting and single clean insect eggs after treatment and separation according to the present application.

[0150] (8) The collected 10,000 single clean insect eggs are used to extract the insect egg genomic DNA using an Omega Mollusc DNA Kit, and the specific steps are as follows:

[0151] 1) The insect egg sample is ground in liquid nitrogen using a mortar and pestle, and the ground powder sample is transferred to a 1.5 mL microcentrifuge tube;

[0152] 2) 350 μl of Buffer MBL and 25 μl of Proteinase K are added, and the mixture is vortexed and incubated at 60°C for at least 30 min until the sample is completely dissolved;

[0153] 3) 350 μl of chloroform:isopropyl alcohol (24:1) mixture is added, and the mixture is vortexed and centrifuged at 10,000 xg for 2 min at room temperature, and the supernatant is carefully transferred to a new 1.5 mL centrifuge tube;

[0154] 4) 5 μl of RNase A is added, and the mixture is incubated at room temperature for 10-30 min, and the following steps are continued;

[0155] 5) The volume of the solution in the previous step is measured, and an equal volume of Buffer MBL is added, and the mixture is vortexed at maximum speed for 15 s, and incubated at 70°C for 10 min;

[0156] 6) 0.5 times the volume of anhydrous ethanol is added, and the mixture is vortexed at maximum speed for 15 s;

[0157] 7) The column is inserted into a 2 mL collection tube, and 750 μl of the mixture in step 6 is transferred to the column, and centrifuged at 10,000 xg for 1 min at room temperature, and the filtrate is discarded;

[0158] 8) Repeat step 7 until all the mixture has been transferred through the column;

[0159] 9) Return the column to the 2mL collection tube, add 500μl of HB Buffer, centrifuge at 10,000xg for 30s, and discard the filtrate;

[0160] 10) Insert the column into a 2mL collection tube, add 700μl DNA Wash Buffer, centrifuge at 10,000xg for 1min, and discard the filtrate;

[0161] 11) Repeat step 10;

[0162] 12) Insert the column into a 2 mL collection tube and centrifuge the empty column at 15,000 mL for 2 min at room temperature.

[0163] 13) Insert the column into a new 1.5 mL centrifuge tube, add 40 μl of double-distilled water preheated at 60–70 °C, and centrifuge at 10,000 x g for 1 min to elute the DNA.

[0164] 14) Repeat step 13, wash off the DNA with fresh 40 μl of double-distilled water, and freeze the sample at -20°C if it is not to be detected by electrophoresis immediately.

[0165] Then, gel electrophoresis was performed according to the electrophoresis parameters in Table 8 to detect the integrity, concentration, and quality of the genomic DNA of the insect eggs treated with the technology of this invention.

[0166] Table 8

[0167]

[0168]

[0169] Figure 7 (Right) shows gel electrophoresis verification of the genomic DNA integrity of insect eggs after treatment with 5% hydroxylamine hydrochloride.

[0170] Example 3

[0171] (1) The original insect eggs cultured in the laboratory were collected by filtering through a 350-mesh screen. After filtering to remove small-diameter impurities, the insect eggs that had gathered on the 350-mesh screen were collected by spraying with double-distilled water.

[0172] Figure 2 (Right) Shows untreated insect egg stock solution.

[0173] (2) Collect the eggs in step (1) and continuously pass them through 150 mesh and 200 mesh screens, leaving large-sized impurities on the screens, and collecting the eggs in the filtrate.

[0174] (3) Place a 120 mesh (125 μm pore size) sintered screen in a glass cylindrical container at 1 / 2 volume, with the edges of the screen tightly fitted to the inner wall of the container, add clean water to 2 / 3 volume, and add the eggs collected in step (2) to the clean water. Connect an air pump to the bottom of the egg liquid to suspend the eggs by air, and adjust the air flow to circulate the suspension until the target planarian eggs (about 150 μm long and about 110 μm wide) are embedded in the pores of the 120 mesh sintered screen. The planarian eggs are trapped by the screen pores, and the remaining impurity eggs are deposited at the bottom of the container.

[0175] Remove the sintered screen and place it in sterile water, and gently shake it to elute the planarian eggs into the sterile water. Collect the egg liquid in a centrifuge tube and centrifuge it at 200 rpm to make the eggs precipitate and aggregate. This step can remove 99.5% of the impurity eggs. Figure 3 A device for cleaning and removing impurities by air suspension and embedding planarian eggs with a sintered screen is shown.

[0176] (4) After gradient concentration test treatment, it is determined that the combination of 3% urea and 2% hydroxylamine hydrochloride has the best effect on dissolving impurities, and after one day of treatment at this concentration, the color and morphology of the planarian eggs return to the same as before treatment. Therefore, a combination of 3% urea and 2% (W / W) hydroxylamine hydrochloride aqueous solution is used to dissolve the impurity bodies in the egg liquid collected in step 3, and a 35W power air pump with four air outlets is used to suspend the treatment, so that the hydroxylamine hydrochloride and the impurities are fully mixed and contacted to maximize the dissolution of impurities.

[0177] The external appearance of the eggs and impurities after treatment with different concentrations of urea and hydroxylamine hydrochloride combination is shown in Table 9.

[0178] Table 9

[0179]

[0180]

[0181]

[0182] The external appearance of the eggs after treatment with 3% urea and 2% hydroxylamine hydrochloride combination is shown in Figure 4 (right).

[0183] (5) After digesting 3% urea and 2% hydroxylamine hydrochloride for 6 hours, wait for the insect eggs in the liquid to settle, remove the upper layer of liquid, and inject double-distilled water that has been treated with high temperature and high pressure for suspension washing. Repeat the washing 10 to 15 times to remove residual hydroxylamine hydrochloride.

[0184] The external appearance of insect eggs and impurities after treatment with a combination of 3% urea and 2% hydroxylamine hydrochloride for different durations is shown in Table 8.

[0185] Table 8

[0186]

[0187]

[0188] (6) After washing the insect eggs with double-distilled water, filter them through a 350-mesh screen to remove small-diameter impurities, then spray the insect eggs with double-distilled water and collect them in a disposable plastic lunch box.

[0189] (7) Put no more than 30 ml of insect egg liquid into a 9 cm disposable plastic culture dish, and then manually shake and vortex the insect eggs under a microscope. Then, use a sterilized insect dissection needle to remove impurities and fragments and collect the insect eggs together. Store in a -80℃ refrigerator for later use.

[0190] Figure 5 (right) and Figure 6 (Right) Shows clean insect eggs after manual sorting and single clean insect eggs after being processed, separated and collected by the invented technology.

[0191] (8) Genomic DNA was extracted from 10,000 collected single clean insect eggs using the Omega Mollusc DNA Kit. The specific steps are as follows:

[0192] 1) Grind the insect egg sample in liquid nitrogen using a mortar and pestle, and transfer the ground powder sample to a 1.5 mL microcentrifuge tube;

[0193] 2) Add 350 μl Buffer ML1 and 25 μl Proteinase K, vortex to mix, and incubate at 60 °C for at least 30 min until the sample is completely dissolved;

[0194] 3) Add 350 μl of chloroform:isoamyl alcohol (24:1) mixture, vortex to mix, centrifuge at 10,000 x g for 2 min at room temperature, and carefully transfer the supernatant to a new 1.5 mL centrifuge tube;

[0195] 4) Add 5 μl of RNase A, let stand at room temperature for 10–30 min, and continue with the following steps;

[0196] 5) Measure the volume of the solution from the previous step, add an equal volume of Buffer MBL, vortex at maximum speed for 15 s, and incubate at 70°C for 10 min;

[0197] 6) Add 0.5 volume of absolute ethanol, vortex at maximum speed for 15 s;

[0198] 7) Put the column into a 2 mL collection tube, transfer 750 μl of the mixture from step 6 into the column, centrifuge at 10,000 x g for 1 min at room temperature, and discard the filtrate;

[0199] 8) Repeat step 7 until all the mixture has been transferred through the column;

[0200] 9) Put the column back into a 2 mL collection tube, add 500 μl of HB Buffer, centrifuge at 10,000 x g for 30 s, and discard the filtrate;

[0201] 10) Put the column into a 2 mL collection tube, add 700 μl of DNA Wash Buffer, centrifuge at 10,000 x g for 1 min, and discard the filtrate;

[0202] 11) Repeat step 10;

[0203] 12) Put the column into a 2 mL collection tube, centrifuge at 15,000 x g for 2 min at room temperature;

[0204] 13) Put the column into a new 1.5 mL centrifuge tube, add 40 μl of double-distilled water preheated to 60-70°C, and centrifuge at 10,000 x g for 1 min to elute the DNA;

[0205] 14) Repeat step 13 using a new 40 μl of double-distilled water to elute the DNA, and store the sample at -20°C if not immediately analyzed by electrophoresis.

[0206] The integrity and concentration of the genomic DNA of the insect eggs treated by the present technology are then analyzed by gel electrophoresis according to the electrophoresis parameters in Table 10.

[0207] Table 10

[0208]

[0209] Figure 7 (Right) shows the gel electrophoresis verifying the integrity of the genomic DNA of the insect eggs treated by the combination of 3% urea + 2% hydroxylamine hydrochloride.

[0210] Cumulative daily hatching rate of the worm eggs of the worm eggs after the treatment of the embodiments 1-3 and the original worm eggs in the clean water, see the following table.

[0211] Table 11

[0212]

[0213] Embodiment 4

[0214] (1) The unqualified reasons of the genomic DNA detected by Beijing Baimaikes Technology Co., Ltd. and the specific circumstances of the improper operation in the process of the worm egg separation, purification and collection are shown in Table 12.

[0215] Table 12

[0216]

[0217] (2) The quality parameters, concentration and results of the worm egg genomic DNA detected by Beijing Baimaikes Technology Co., Ltd. and meeting the subsequent machine sequencing analysis are shown in Table 13.

[0218] Table 13

[0219]

[0220] It can be seen that the planaria worm egg sample treated by the method of the present application can extract genomic DNA normally, and the total amount and quality of the DNA extracted from a large amount of planaria worm eggs can meet the experimental requirements of subsequent whole genome sequencing and molecular sequence operation, etc., and an effective planaria worm egg separation, purification and collection technical scheme is provided.

[0221] The first step of separating and collecting planarian eggs is to remove the small size of mixed eggs of other insects by different mesh size filter screen, which can remove 95% of the mixed eggs of other insects. Then, a 120 mesh sintered screen is placed at 1 / 2 volume of the glass cylindrical container, and the edge is strictly attached to the inner wall of the container. The water is added to 2 / 3 volume of the container, and the screened and collected eggs are added to the water. The air pump is connected to the bottom layer of the egg liquid to suspend the eggs by air. The planarian eggs are trapped by the mesh, and the remaining mixed eggs of other insects are precipitated at the bottom of the container. This step can remove 99.5% of the final mixed eggs of other insects. Because most of the mixed eggs of other insects are between 40-60 μm in size, a small part of the mixed eggs of other insects are larger than 120 μm, and the planarian eggs are oval in shape and range from 110-170 μm in size. The continuous passage of 150 mesh (pore size 106 μm) and 200 mesh (pore size 74 μm) screens can effectively filter and remove about 95% of the smaller size mixed eggs of other insects. Then, the 120 mesh (pore size 125 μm) sintered screen is suspended by air to trap the target size planarian eggs on the screen, and the remaining mixed eggs of other insects with a size larger than 120 μm are left in the water layer at the bottom. Finally, the remaining 4.5% of the mixed eggs of other insects can be removed. The second step of separating and collecting planarian eggs is to remove the water mites, rotifers, ciliates, nematodes and other mixed insects adhered around the eggs by using the chemical properties of urea or hydroxylamine. This step can remove 90%-99% of other mixed insects. Because the outer structure of water mites, rotifers, ciliates, nematodes and other impurities is mainly composed of proteins, urea can combine with the polar groups and hydrogen bonds in the protein molecules, thereby destroying the spatial structure and hydration layer of the protein molecules, reducing the attractive force between the protein molecules, and making the protein molecules become relaxed and soft. Urea can also form a hydration layer in the protein molecules, so that water molecules more easily enter the internal space of the protein molecules, dissolve the hydrogen bonds and polar residues therein, destroy the outer structure of these impurities, and achieve the purpose of eliminating biological activity. The reducing property of hydroxylamine hydrochloride reacts with the oxidized metal ions on the outer structure proteins of these impurities, reducing them to a lower oxidation state, destroying the outer structure, and breaking the internal and external acid-base balance of these organisms in the weakly acidic water environment, finally achieving the purpose of eliminating biological activity.

[0222] The outer membrane structure of the planarian egg is composed of basic proteins, polyphenols and phenol oxidase system. These particles are released from the yolk cells and form quinone substances on the inner membrane surface, which then form cocoon-like shells of scleroprotein. The phenolic substances on the outer membrane surface of the egg are weakly acidic, and the hydrochloric acid hydroxylamine solution is also weakly acidic, and does not react with the phenolic substances. However, the polyphenol oxidase system combines with hydrochloric acid hydroxylamine, so that the phenolic substances on the outer surface of the egg are not oxidized to quinones. Therefore, the redox process between polyphenol oxidase and hydrochloric acid hydroxylamine will cause the outer structure of the egg to be temporarily "defective". However, the continuous oxygen supply process will accelerate the supply of oxygen to the synthesis and renewal of internal substances and the division activity of the egg, so that after the egg is mixed with hydrochloric acid hydroxylamine for a period of time, the renewal speed of internal phenolic substances and polyphenol oxidase system exceeds the consumption speed of the reaction with hydrochloric acid hydroxylamine, and the outer structure of the egg gradually increases, and then restores the original round state. The treated eggs still have the biological activity of hatching into larvae.

[0223] At the same time, when selecting reagents, the following chemical reagents cannot be used for the digestion of miscellaneous worms: ethanol, methanol, acetone, chloroform, monoammonium phosphate and diammonium phosphate. Organic reagents such as ethanol have strong protein solubility, which can cause planarian eggs to be stimulated and deformed when the miscellaneous worm bodies are lysed. In addition, some intact planarian eggs treated with organic reagents cannot hatch into larvae, i.e. lose the biological activity of the eggs. Monoammonium phosphate and diammonium phosphate have a slow effect, and planarian eggs are easily hatched into larvae at room temperature, and the miscellaneous worm bodies still have biological activity, so the digestion of miscellaneous worm bodies is weak. Therefore, through the screening of various chemical reagents, urea and hydrochloric acid hydroxylamine are finally determined as the digestion reagents for miscellaneous worm bodies.

[0224] The third step of separating, purifying and collecting planarian eggs is to remove the remaining 1% impurity crystal fragments based on the removal of more than 99% impurities in the previous two steps, so as to achieve the final purpose of cleaning and purifying the eggs. Meanwhile, in the process of chemical digestion, the application preferably uses 3% urea + 2% hydroxylamine hydrochloride combination treatment to achieve good impurity removal effect while reducing the treatment time, and to achieve good egg hatching rate. There may be a synergistic effect between 3% urea and 2% hydroxylamine hydrochloride to increase the treatment effect.

[0225] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limited. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the present application.

Claims

1. A method for separating, purifying and collecting planarian eggs, characterized by: The application relates to a method for purifying planarian eggs. The original planarian eggs are filtered through a 350-mesh screen, impurities with small particle sizes are filtered out, the original planarian egg liquid is collected after being sprayed with double-distilled water; The original planarian egg liquid is continuously filtered through a 150-mesh screen and a 200-mesh screen, planarian eggs are collected on the screens, discarded miscellaneous planarian eggs are in the filtrate, and 95% of the miscellaneous planarian eggs are removed; The treated planarian eggs are collected in a glass container provided with a 120-mesh sintered screen surface, the edges of the planarian eggs are strictly attached to the inner wall of the container, the planarian egg liquid is suspended by connecting an air pump for aeration, the target planarian eggs are intercepted through the screen surface pores, 99.5% of the miscellaneous planarian eggs are removed, and the target planarian eggs are intercepted by the sintered screen surface pores; The planarian eggs are collected again after being washed with clean water and screened to remove impurities; The planarian eggs are manually sorted and collected under a microscope to remove crystal small fragments, and purified planarian eggs are obtained. The chemical reagent aqueous solution is a 3% W / W urea and 2% W / W hydroxylamine hydrochloride combined aqueous solution.

2. The method of claim 1, wherein: The planarian eggs are collected again after being washed with clean water and screened to remove impurities, wherein the filtering screen is a 350-mesh screen, and the original planarian egg liquid is sprayed with double-distilled water after small-particle-size impurities are filtered out.

3. The method of claim 1, wherein: The manual sorting and collecting under the microscope include manual manual oscillation vortex aggregation of the planarian eggs and collection of the planarian eggs after microcrystal fragments are removed by using disinfected insect dissecting needles.

4. The method of any one of claims 1 to 3, characterized in that: The original planarian eggs include food nematode planarian eggs.

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