RU2101354C1 - Method of bacillus anthracis nucleic acid preparing - Google Patents

Abstract

FIELD: biochemistry, biotechnology, bacteriology. SUBSTANCE: biomass is obtained at preliminary additional culturing microbe cells Bacillus anthracis under condition of the sealed flasks containing culture. Biomass is treated with lysozyme in presence of EDTA at concentration 20 mg/1 g biomass. The obtained suspension is frozen three times in liquid nitrogen followed by thawing at 60 C. Freezing time is 40-60 s. Then additional lysis with tris-SDS-buffer is carried out. Nucleic acids were purified with phenol and precipitated with cold ethanol. EFFECT: improved method of nucleic acid preparing. 3 tbl

Description

 The invention relates to biochemistry and bacteriology and can be used to obtain nucleic acids from the causative agent of anthrax and other spore-forming bacilli.

 The general principle of obtaining nucleic acids is to grow the maximum amount of biomass, complete destruction of bacterial cells, followed by the separation of nucleic acids from proteins, polysaccharides and other cellular elements.

 When working with the causative agent of anthrax, difficulties arise associated with the high resistance of this microorganism to the effects of physicochemical factors and the ability to spore formation (Instruction on the anti-epidemic regime of working with material infected or suspicious of infection with pathogens of infectious diseases of the II group. Saratov 1979) .

 For the destruction of cells, there are a number of methods: freezing and thawing, destruction by osmotic shock or using enzymes, exposure to lysing agents, as well as mechanical processing in a homogenizer, ultrasonic disintegrator. The choice of nucleic acid isolation method is mainly determined by the cellular structure of the microorganism (P. Zentbusch, Molecular and Cellular Biology. M. Mir, 1982, p. 50.).

 A known method of obtaining nucleic acids from Bac cells. cereusthuringiensis (Afrikyan E.K. Entomopathogenic bacteria and their significance, ed. by A.N.Armyanskoy SSR, 1973, p. 418).

A fresh bacterial culture from an agarized school was transferred to a meat-peptone broth and grown on a rocking chair in test tubes for 8 hours. The culture was then transferred to a flask with 1 liter of broth and incubated on a rocking chair at 37 ^o C. After 10 hours, glycine was added to the flask in final concentrations 2.5% sucrose 20% and cultivation is continued for 3 hours with moderate stirring, after which lysozyme is added at a rate of 200 μg / ml. Incubation at low speeds of the rocking chair continues for another 2 hours. Under microscopy, mass lysis of cells is noted. After centrifugation and suspension of the precipitate in a citrate mixture, lysozyme at the same concentration is again added in the presence of EDTA and TRIS buffer at pH 8.0. After 30 minutes of incubation, add a 2% solution of sodium lauryl sulfate, pH-8.0. Growth was continued for 15 minutes at 37 ^° C and citrate-salt buffer (2 M sodium chloride and 0.05 M sodium citrate) was added in a 4-fold volume and kept at 4 ^° C for 30 minutes. After centrifugation, a citrate-salt solution is added to the precipitate, incubated for 30 minutes, and after another centrifugation, 95% ethanol is added to the precipitate. The next step is that 0.9 M sodium chloride and 0.01 M sodium citrate are added to the resulting suspension and incubated at 37 ^° C. for 15 minutes. Then again add lysozyme at a concentration of 200 μg / ml in combination with EDHA and TRIS-buffer, pH 8.0 and placed in a thermostat for 30 min at 37 ^o C. After treatment with 2% sodium lauryl sulfate, pH 8.0 the mixture is maintained at 37 ^o C for 15 minutes and add 4 volumes of citrate-saline solution (2M sodium chloride and 0.05M sodium citrate). Incubation for 30 minutes at 37 ^° C. After centrifugation, a citrate-saline solution was added to the precipitate and incubated at 30 ^° C. Then, the suspension was centrifuged, ethanol was added and centrifuged again. The resulting precipitate is treated with a mixture of chloroform and octanol in a ratio of 5 to 1 and again centrifuged. RNA from the mixture was removed by adding RNAase at a concentration of 20 μg / ml for 20 min at room temperature. Then deproteinization is carried out with a mixture of chloroform and octanol. After this, ethanol precipitation and extraction in citrate-salt solution are carried out. To obtain purified DNA, a 4-fold deproteinization is carried out with a mixture of chloroform and octanol.

 The method allows to obtain purified DNA for further molecular genetic research. Its disadvantage is the duration and a large set of reagents used. It does not provide complete lysis of Bac cells. antharacis and, accordingly, guaranteed disinfection of the culture of anthrax microbe and increase the yield of the finished product.

The closest in technical essence to the invention is the selected as a prototype method for producing bacterial nucleic acids [1]
In this method, freshly precipitated cells in a stainless steel tube are mixed with lysozyme at a concentration of 2 mg / g and dissolved in saline EDGA (1 ml / g). The resulting mixture was kept in a thermostat for 20 min at 37 ^o C, the pH was adjusted to 9.0 by adding a solution of caustic soda. After the start of cell lysis, they are quickly frozen in a mixture of dry ice with acetone. This reaches a temperature of -20 ^o C. Exposure to 20 minutes Thawing is then carried out at 60 ^° C. Then, TRIS-SDS buffer is added to the suspension at a concentration of 10 ml per 1 g of biomass. The next step is added an equal volume of phenol, pH 9.0, and the resulting mixture is shaken in the cold (below -4 ^o C) for 20 min in a flask with a glass stopper. The resulting suspension is divided into 2 layers by centrifugation in a low speed centrifuge. The aqueous phase is sucked off and clarified by centrifugation (12000 rpm). Nucleic acids are precipitated by the addition of 3 volumes of cold ethanol. The filamentous precipitate is selected by winding on a glass rod. After drying, a dry powder is obtained containing nucleic acids.

 The prototype and the invention have the following similar essential features.

Preparation of biomass, processing it with lysozyme in the presence of EDTA for 20 min at 37 ^o C, pH 9.0, which is necessary for the destruction of the cell membrane.

 Freezing and thawing the suspension, providing a more complete lysis of cells.

 Additional cell lysis with TRIS-SDS buffer. Treatment of the cell suspension with phenol in a 1: 1 ratio in order to purify nucleic acids from proteins, polysaccharides and other cellular elements.

 Precipitation of nucleic acids by a triple volume of cold ethanol.

 The disadvantage of the prototype is the incomplete disinfection of Bac. antharacis associated with partial cell lysis and a lower yield of nucleic acids.

This drawback is due to the high resistance of the causative agent of anthrax to the effects of physico-chemical factors, the ability to spore formation. Therefore, the processing of Bac. antharacis according to the method of K. Miura does not allow to achieve their complete lysis, and therefore guaranteed disinfection. The amount of lysozyme used in this method (2 mg per 1 g of biomass) and the freezing temperature (-20 ^o C, even with a three-fold repetition of the operation do not allow to fulfill the above requirements, which reduces the yield of nucleic acids.

 The aim of the invention is to increase the yield of nucleic acids and ensuring guaranteed disinfection of pathogenic bacilli.

This goal is achieved in that the biomass was prepared with preliminary growth for 16-17 hours, subject to the conditions for sealing flasks. The biomass was treated with lysozyme at a concentration of 20-25 mg per 1 g of biomass in the presence of EDTA, kept in an incubator at 37 ^° C for 20 minutes. Then, the suspension was frozen three times in liquid nitrogen (-196 ^° C), followed by thawing at 60 ^° C. The freezing process lasted 40-60 s. Additional lysis was performed with TRIS-SDS buffer. Nucleic acids were purified with phenol and precipitated with a triple volume of cold ethanol.

 With respect to the prototype, the invention has the following distinctive features.

 The cultivation of biomass was carried out with preliminary growth for 16-17 hours under the condition of the tightness of the flasks, which ensures the receipt of the maximum possible amounts of microbial cells in the logarithmic growth phase with minimal sporulation.

 Table 1 shows comparative data on spore formation and biomass yield in various cultivation methods.

 An increase in the amount of lysozyme from 2 mg / g to 20-25 mg / g provides a more complete lysis of Bac cells. antharacis, and accordingly an increase in the yield of nucleic acids.

 In the table. 2 shows comparative data on the degree of cell lysis and the yield of nucleic acids from the concentration of lysozyme by analogue, prototype and invention.

Reducing the freezing temperature to -196 ^o C and reducing the time from 20-30 minutes to 40-60 s contributes to the complete lysis and increase the yield of nucleic acids, as shown in table 3.

 The possibility of carrying out the invention using the full totality of the claimed features is confirmed by examples of specific performance.

Example 1. In the experiment used a vaccine strain of Bac. antharacis STI-1. Sowing spore suspension was performed on 2 Petri dishes with Hottinger agar. They were grown in an incubator at 37 ^° C for 24 hours. The next day, typical colonies of anthrax microbe were selected and passaged in a loop of 250 ml. flasks with broth. The flasks were closed with cotton-gauze plugs and rubber caps on top of them. The latter were used to create an increased CO ₂ content in the flasks, since bicarbonate inhibits spore formation and stimulates the sensitivity of Bac cultures. antharacis to lysozyme (Chatterjee BR Williams RP j. Bact. 1985. vol. 89 p.1128-1133). The flasks were placed in a temperature-controlled shaker for 8 hours at 37 ^° C. After 6 hours, control smears were made. Spore forms of anthrax microbe were absent. Then, 20 ml of broth culture was reseeded in a 750 ml flask with 200 ml of broth and placed in a heat shaker for 9 hours at 37 ^° C. Again, rubber caps were used. Then the culture was centrifuged and the resulting biomass was washed three times from the medium with buffered nat. solution. The weight of wet biomass is 12 g. It was transferred to a plastic centrifuge cup and 240 mg of lysozyme (20 mg / g) and 12 ml of saline EDHA (1 ppm / g) were added. The contents of the cup were thoroughly mixed and left in a thermostat for 20 minutes at 37 ^° C. Then, the suspension was frozen in liquid nitrogen (-196 ^° C), and then thawed in an ultra-temperature bath at 60 ^° C for 7-10 minutes. This operation was repeated 3 times. After the last thawing, TRIS-SDS buffer was added in an amount of 96 ml (6 ml / g), mixed thoroughly and left in a thermostat for 20 min at 37 ^° C. Then 120 ml of freshly distilled phenol, pH 9.0, were added and shaken in the cold for 30 min. . The suspension was transferred to a glass centrifuge cup and divided into two layers in a centrifuge at 5000 rpm. The top layer was aspirated into a glass cylinder. The fluid volume was 90 ml. 270 ml of cold ethanol (triple volume) were carefully added and mixed. The obtained filamentous precipitate was removed by winding on a glass rod and transferred to a centrifuge cup. The precipitate was dried 3 times with alcohol, 3 times with a mixture of Nikiforov (alcohol + ether) and 3 times with ether. The weight of the obtained dry powder was 11.4 mg. A dry mixture of nucleic acids was sealed in ampoules and left for further research.

 The yield of nucleic acids was 0.95 mg per 1 g of biomass. Control seeding 30 minutes after lysozyme treatment showed a lack of growth, i.e. complete cell lysis.

 Example 2. It is carried out analogously to example 1, except that the cultivation with preliminary growth of microbial cells was performed for 16 hours, the concentration of lysozyme was 22 mg / g of biomass and the suspension of the cells was frozen for 40 s. The yield of nucleic acids also amounted to 0.95 mg per 1 g of biomass, and control seeding showed a lack of growth.

 Example 3. It was carried out analogously to examples 1 and 2. The difference was that the cultivation time was 16.5 hours, 22 mg of lysozyme per 1 g of biomass was used for lysis, and the suspension was frozen for 45 s. The nucleic acid yield was 0.95 mg / g biomass. There was a lack of growth during control seeding.

 As shown by experimental tests, reducing the cultivation time below 16 hours does not provide the maximum possible amounts of microbial cells, and therefore, reduces the yield of the finished product. An increase in this time leads to the formation of spores and, accordingly, does not provide guaranteed Bac disinfection. anthracis.

 A decrease in the lysozyme concentration of less than 20 mg / g of biomass does not ensure complete cell lysis, which reduces the yield of the finished product and does not guarantee cell disinfection. An increase in the amount of lysozyme over 25 mg / g biomass is impractical.

 The claimed time intervals for freezing a cell suspension in liquid nitrogen are necessary and sufficient, together with the remaining features of the invention, to achieve the goal.

 Thus, the invention is practicable, its use allows the production of nucleic acids from various spore-forming microorganisms with guaranteed disinfection and increase the yield of the finished product by more than 70% compared with the prototype.

Claims (1)

A method for producing Bacillus anthracis nucleic acids, including preparing biomass, treating it with lysozyme in the presence of EDTA for 20 minutes at 37 ^° C and pH 9.0, triple freezing and thawing the suspension, additional lysing with TRIS-CDC buffer, phenol treatment in a ratio of 1 1 and precipitation of nucleic acids with a triple volume of cold ethanol, characterized in that the biomass is prepared with the preliminary growth of microbial cells in compliance with the conditions for sealing flasks with the culture for 16 17 hours, treatment with lysozine concentration of 20 25 mg / g biomass, freezing is carried out in liquid nitrogen for 40 to 60 s.

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