A simple method for breaking the cell wall of Protoptera

Abstract

The present invention discloses a simple method for breaking the wall of Prototheca, specifically, adding Prototheca of different species into a chemical lysis solution, and oscillating and mixing at 60-80°C to obtain a broken wall Prototheca sample, which is convenient for extracting the genomes of common clinical Prototheca such as Prototheca wickerhamii, Prototheca ciferrii and Prototheca bovis. The present invention is easy to operate, does not require special equipment, can efficiently break the wall of Prototheca, improves the efficiency of Prototheca genome extraction, and provides high-quality genome samples for subsequent molecular biology research. The present invention provides a technical basis for rapid molecular diagnosis and kit development of Prototheca disease, and also provides a new idea for obtaining the Prototheca genome.

Description

Technical Field

[0001] The invention belongs to the field of algae biotechnology, and in particular relates to a simple method for breaking the cell wall of Prochlorococcus. Background Art

[0002] Prototheca disease is a relatively rare disease caused by the invasion of the conditionally pathogenic algae Prototheca, which can cause skin infections, bloodstream infections, nervous system infections, etc. There are currently 18 species in the genus Prototheca, the more common of which are Prototheca wickerhamii, Prototheca ciferrii, and Prototheca bovis. After cultivation, the appearance of Prototheca and yeast is basically similar. It is difficult to accurately identify them to species using traditional microscopy, culture, and mass spectrometry methods. In addition, due to the lack of clinical specific symptoms, the misdiagnosis and missed diagnosis rates of Prototheca disease are high in clinical practice. Timely and accurate identification of different species of Prototheca is not only of great significance in the study of the epidemiology and pathogenic mechanisms of infection, but also of great significance to improving the diagnosis level of rare diseases in my country.

[0003] At present, different species of Prototheca can be identified to the species level through molecular biology sequencing technology. The first step in molecular biology research on Prototheca is the extraction and purification of nucleic acids. The key problem is that the cell wall structure of Prototheca is special, which makes the cell wall breaking process very difficult. Sporopollenin contained in the cell wall of Prototheca is a stable biopolymer that makes it highly resistant to mechanical pressure, physical and chemical factors, and enzyme degradation. It is difficult to break the cell wall, which makes it difficult to release genomic nucleic acids from the cell.

[0004] At present, there are few studies on the wall breaking methods of Prototheca, and the commonly used wall breaking methods include: mechanical homogenization, mechanical grinding, ultrasonic wall breaking, enzyme digestion wall breaking, repeated freezing and thawing, steam swelling, etc. These methods mostly require special instruments, such as homogenizers, grinders, ultrasonic devices, or high temperature, high pressure, and ultra-low temperature process conditions, which lead to high wall breaking costs, complex operations, and are difficult to carry out routinely in the laboratory. For example: the patent document with application publication number CN116987595A discloses a composite wall breaking technology that combines mechanical wall breaking and ultrasonic wall breaking; the patent document with application publication number CN104371926A discloses a method for breaking the wall of microalgae, that is, breaking the wall of acidic microalgae culture fluid under hydrothermal conditions and stirring conditions; the patent document with application publication number CN110628634A discloses a method for breaking the wall of Chlorella using a colloid mill assisted enzyme method; the patent document with application publication number CN11507 Patent document No. 4250A discloses an efficient method for breaking the cell wall of Chlorella, which specifically comprises the following steps: centrifuging and concentrating Chlorella cultured for 7 to 10 days, treating the collected algae liquid with cellulase, freezing the enzyme-treated mixture completely in liquid nitrogen or at -80°C, taking it out and thawing it in a 37°C water bath, repeating this freezing and thawing step 2 to 5 times, and then subjecting the algae liquid to ultrasonic disruption to obtain the cell wall-broken algae liquid; Patent document No. CN116024090A discloses a method for breaking the cell wall of Chlorella powder by steam thermal expansion.

[0005] Therefore, developing a method for breaking the cell wall of Chlorella that is simple to operate, mild in conditions, rapid and effective is of great significance for the rapid molecular diagnosis and reagent development of Chlorella disease. Summary of the invention

[0006] In order to solve the above technical problems, efficiently break the cell wall of Prototheca, thereby improving the efficiency of molecular biological identification of Prototheca, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides a simple method for breaking the cell wall of Prototheca, the main process of which is: adding Prototheca into a chemical lysis solution, and oscillating and mixing at 60-80°C.

[0008] Preferably, the instrument for the oscillation mixing treatment includes but is not limited to a conventional constant temperature oscillator or a constant temperature mixer, and any instrument that can achieve mechanical mixing can be used.

[0009] Preferably, the temperature of the oscillating mixing treatment is 70-80°C, for example, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C.

[0010] Preferably, the oscillation mixing treatment time is 20 to 40 minutes, for example: 20 minutes, 22 minutes, 25 minutes, 27 minutes, 30 minutes, 32 minutes, 35 minutes, 37 minutes, 40 minutes. More preferably, it is 25 to 35 minutes.

[0011] Preferably, the rotation speed of the oscillating mixing is ≥1500 rpm, for example: 1500 rpm, 1600 rpm, 1700 rpm, 1800 rpm, 1900 rpm, 2000 rpm, 2200 rpm, 2500 rpm, 3000 rpm, 3500 rpm.

[0012] Preferably, glass beads are added to the cell wall breaking reaction system before the oscillation mixing.

[0013] Preferably, the particle size of the glass beads is 0.3-0.7 mm, for example, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, and more preferably 0.4-0.6 mm.

[0014] Preferably, the chemical lysis solution is prepared from guanidine thiocyanate and EDTA.

[0015] Preferably, the concentration of guanidine thiocyanate in the chemical lysis solution is 3-8 mol / L, for example: 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L.

[0016] Preferably, the concentration of EDTA in the chemical lysis solution is 20-30 mM, for example: 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM.

[0017] Preferably, the Prototheca is first configured into a suspension, and the turbidity of the suspension is 1-5 MCF, for example: 1 MCF, 2 MCF, 3 MCF, 4 MCF, 5 MCF.

[0018] Preferably, the volume ratio of the suspension of Prototheca to the chemical lysis solution is 1:1-3, for example: 1:1, 1:2, 1:3.

[0019] In a specific embodiment of the present invention, the common clinical Prototheca wickerhamii, Prototheca ciferrii and Prototheca bovis are prepared into a suspension with a turbidity of 1 to 3 MCF, added to a sample tube, and then 0.4 to 0.6 mm glass beads are added. Then an equal volume of chemical lysis solution (3 to 5 mol / L guanidine thiocyanate, 20 to 30 mM EDTA) is added to the sample tube, and then the sample tube is placed on a constant temperature mixer, the shaking speed is set to 1500 to 3000 rpm, the temperature is 70 to 80°C, and the wall is broken for 25 to 35 minutes to obtain a Prototheca sample after wall breaking.

[0020] In a second aspect, the present invention provides an optimized molecular biology research method for the genome of Prototheca, which comprises the steps of treating Prototheca using the cell wall breaking method described in the first aspect.

[0021] In a third aspect, the present invention provides application of the method described in the first aspect in preparing a Prototheca extract.

[0022] Beneficial effects of the present invention:

[0023] The present invention can efficiently break the cell wall of Prototheca without special equipment, thereby improving the efficiency of molecular biological identification of Prototheca; the present invention provides a technical basis for rapid molecular diagnosis and reagent development of Prototheca disease, and also provides a new idea for breaking the cell wall of algae. DETAILED DESCRIPTION

[0024] The technical scheme of the present invention is clearly and completely described below in conjunction with the embodiments. The described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0025] It should be noted that, unless otherwise specified, the experimental methods and reagents used in the embodiments of the present invention are conventional experimental methods and reagents in the art.

[0026] Reagents and instruments used in the experiment:

[0027] Nucleic acid extraction reagent (Silidi Biomedical Technology Co., Ltd., REF: 3000000000358), dsDNA detection kit (Qubit TM 1X dsDNA HS, REF: Q33231), constant temperature mixer (Eppendorf ThermoMixer), biological sample homogenizer (Bioprep-6).

[0028] Example 1 Chemical pyrolysis + heating + mechanical oscillation

[0029] Prepare the freshly cultured Prototheca into a Prototheca suspension with a turbidity of 2MCF, take 200 μL of the suspension into a sample tube, add an appropriate amount of 0.5 mm glass beads, and then add 200 μL of chemical lysis solution (5 mol / L guanidine thiocyanate, 20 mM EDTA), then put the sample tube on a constant temperature mixer, set the shaking speed to 1500 rpm, the temperature to 70°C, break the wall for 30 minutes, and then take the supernatant after the glass beads settle for later use.

[0030] Example 2 Chemical pyrolysis + heating + mechanical oscillation

[0031] Prepare the freshly cultured Prototheca into a Prototheca suspension with a turbidity of 2MCF, take 200 μL of the suspension into a sample tube, add an appropriate amount of 0.5 mm glass beads, and then add 200 μL of chemical lysis solution (5 mol / L guanidine thiocyanate, 25 mM EDTA), then put the sample tube on a constant temperature mixer, set the shaking speed to 2000 rpm, the temperature to 75 ° C, break the wall for 30 minutes, and then take the supernatant after the glass beads settle for later use.

[0032] Example 3 Chemical pyrolysis + heating + mechanical oscillation

[0033] Prepare the freshly cultured Prototheca into a Prototheca suspension with a turbidity of 2MCF, take 200 μL of the suspension into a sample tube, add an appropriate amount of 0.5 mm glass beads, and then add 200 μL of chemical lysis solution (5 mol / L guanidine thiocyanate, 30 mM EDTA), then put the sample tube on a constant temperature mixer, set the shaking speed to 3000 rpm, the temperature to 80°C, break the wall for 30 minutes, and then take the supernatant after the glass beads settle for later use.

[0034] Comparative Example 1 Heating and Wall Breaking

[0035] Prepare the freshly cultured Prototheca into a Prototheca suspension with a turbidity of 2 MCF, take 200 μL of the suspension into a sample tube, put the mixed sample tube on a metal bath, heat at 75°C to break the wall for 30 minutes and set aside.

[0036] Comparative Example 2 Chemical pyrolysis method

[0037] Prepare the freshly cultured Prototheca into a Prototheca suspension with a turbidity of 2 MCF. Take 200 μL of the suspension into a sample tube, add 200 μL of chemical lysis solution (5 mol / L guanidine thiocyanate, 25 mM EDTA), shake and mix, put the sample tube on a metal bath, set the temperature to 75°C, and break the wall for 30 minutes for use.

[0038] Comparative Example 3 Mixed Enzyme Digestion and Cell Wall Breaking

[0039] The freshly cultured Prototheca was prepared into a Prototheca suspension with a turbidity of 2 MCF. 200 μL of the suspension was taken into a sample tube, and 50 μL of mixed enzyme digestion solution (10 mg / mL Lysozyme, 10 mg / mL Zymolyase, 1 mM DTT, 30% glycerol, 0.1% Tween-20, 10 mM Tris-HCl (pH 6.8)) was added. After gently shaking to mix, the sample tube was placed on a metal bath, the enzyme reaction temperature was set to 45°C, and the cell wall was broken for 30 minutes for use.

[0040] Comparative Example 4: Homogenizer wall breaking

[0041] Prepare the freshly cultured Prototheca into a Prototheca suspension with a turbidity of 2MCF. Take 200 μL of the suspension in a sample tube, add an appropriate amount of glass beads with a particle size of about 0.5 mm, 200 μL of chemical lysis solution (5 mol / L guanidine thiocyanate, 25 mM EDTA), and then put the sample tube on the homogenizer, set the shaking speed to 4000 rpm, the cycle time to 30 seconds / interval time to 30 seconds, the number of cycles to 10, and after the wall breaking is completed, wait for the glass beads to settle and take the supernatant for use.

[0042] Detection of Broken Cell Samples of Prototheca

[0043] The cell wall-breaking samples of Prototheca in Examples 1 to 3 and Comparative Examples 1 to 4 were taken, and genomic DNA was extracted using a nucleic acid extraction reagent. The concentration of gDNA in the extracted product was measured by Qubit 4.0 to detect the cell wall breaking performance of each method. The specific process is as follows:

[0044] Preparation of Prototheca suspension: Samples 1, 2 and 3 are P.wickerhamii, Samples 4, 5 and 6 are P.ciferrii, and Samples 7 and 8 are P.bovis. After each bacterial suspension sample is mixed, it is divided into 7 tubes according to 200 μL per tube to ensure that the starting sample volume of each method is consistent. A total of 8 samples are compared. The cell wall is broken according to the above 7 methods, and then all the clear liquid is transferred to the sample well of the nucleic acid extraction reagent for nucleic acid purification according to the standard procedure, and all the genomic DNA obtained is collected, and then the concentration of gDNA is determined using a dsDNA detection kit. The results are shown in the table below:

[0045] Table 1 Comparison of the efficiency of extracting genomic DNA from common clinical algae using different methods

[0046]

[0047] It can be seen from the results in Table 1 that the concentrations of Prototheca genomic DNA extracted in Examples 1 to 3 are comparable, with Example 2 having the best effect. The concentration of genomic DNA extracted in Example 1 is 49 times that of Comparative Example 1, 23.85 times that of Comparative Example 2, and 6.21 times that of Comparative Example 3. The concentration of genomic DNA extracted in Example 2 is 51.1 times that of Comparative Example 1, 24.87 times that of Comparative Example 2, and 6.47 times that of Comparative Example 3. The concentration of genomic DNA extracted in Example 3 is 51.1 times that of Comparative Example 1, 24.86 times that of Comparative Example 2, and 6.47 times that of Comparative Example 3. Compared with the methods of Comparative Examples 1 to 3, the genomic DNA yield obtained by extracting three common clinical Prototheca using the methods of Examples 1-3 is higher, indicating that its cell wall breaking effect is far superior to the three methods used in Comparative Examples 1-3. At the same time, there is no significant difference in the yield of Prototheca genomic DNA between the methods of Examples 2 and 3 and the conventional homogenizer wall breaking method of Comparative Example 4, indicating that the methods of Examples 2 and 3 can achieve the same Prototheca wall breaking effect as that of a homogenizer by using a conventional constant temperature mixer.

[0048] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A simple method for breaking the cell wall of Prototheca, characterized in that: Different species of Prototheca are added into the chemical lysis solution and shaken and mixed at 60-80°C.

2. The method for breaking the cell wall of Prototheca according to claim 1, characterized in that: The temperature of the oscillation mixing treatment is 70-80°C.

3. The method for breaking the cell wall of Prototheca according to claim 1, characterized in that: The oscillation mixing time is 20 to 40 minutes.

4. The method for breaking the cell wall of Prototheca according to any one of claims 1 to 3, characterized in that: The rotation speed of the oscillating mixing is ≥1500 rpm.

5. The method for breaking the cell wall of Prototheca according to claim 1, characterized in that: Before the oscillation mixing, glass beads are added to the cell wall breaking reaction system.

6. The method for breaking the cell wall of Prototheca according to claim 5, characterized in that: The particle size of the glass beads is 0.3-0.7 mm.

7. The method for breaking the cell wall of Prototheca according to claim 1, characterized in that: The chemical lysis solution is prepared from guanidine thiocyanate and EDTA.

8. The method for breaking the cell wall of Prototheca according to claim 7, characterized in that: In the chemical lysis solution, the concentration of guanidine thiocyanate is 3-8 mol / L, and the concentration of EDTA is 20-30 mM.

9. The method for breaking the cell wall of Prototheca according to claim 1, characterized in that: The Prototheca is firstly prepared into a suspension, and the turbidity of the suspension is 1-5 MCF.

10. The method for breaking the cell wall of Prototheca according to claim 9, characterized in that: The volume ratio of the suspension of Prototheca to the chemical lysis solution is 1:1-3.

Images