RU2562841C2 - Method of preparing tick tissues for biological analysis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to method of preparing biological materials for carrying out analysis, in particular preparation of tick tissues for further determination of presence of tick-borne encephalitis virus in them, and can be used in healthcare, biotechnologies and immunology. Method of sample preparation of tick tissues includes washing tick tissues in 70-80% ethyl alcohol solution, its drying, placing into test tube with further grinding and centrifugation. Before grinding 100-200 mcl of lysing buffer are added into test tube, with grinding of tick tissues being carried out in solution in the same test tube. Obtained homogenate is centrifuged for 2-10 min at 2000-6000 rev/min.

EFFECT: method makes it possible to provide process manufacturability without application of expensive equipment.

5 cl, 3 ex, 1 tbl

Description

The invention relates to methods for preparing biological materials for research, in particular the preparation of tick tissues to further determine the presence of tick-borne encephalitis virus (TBEV) in them, and can be used in healthcare, biotechnology and immunology.

A known method of preparing biological material for PCR gene-amplification diagnostics, which consists in lysing samples with a NaI solution containing an RNA carrier, precipitating viral, bacterial and cellular DNA and RNA with isopropanol, centrifuging and purifying them (patent RU No. 2134871, IPC G01N 1/28, G01N 1/30, published on 08.20.1999).

The known method is very specific and cannot be used to prepare tick tissues, since in the process of its implementation the protein structure of the virus is destroyed, which is further necessary for the study.

As a prototype, a method for isolating TBEV from a tick was selected (Internet resource: http://www.bio-ua.com/index.php/prod?i=l66&PHPSESSIDID = 3fl72c88947d863e65bb76f2a0ff3bb2). This method consists in the fact that tick tissues are washed in a 70-80% ethanol solution, dried with filter paper, placed in an Eppendorf type tube and tightly closed, then placed in liquid nitrogen to freeze, then frozen tick tissues triturated with a pestle for homogenization and, adding a solution for breeding samples, centrifuged.

The main disadvantage of this method is its low technology, which consists in the use of liquid nitrogen in it, for the preparation and storage of which highly specialized equipment is required.

The objective of the proposed technical solution is to create a technologically advanced method of sample preparation of tick tissue.

The technical result obtained from the use of the invention is to provide a simple and reliable process for sample preparation of tick tissues without using expensive equipment and reducing labor costs at the stage of homogenization of the tick.

The technical result is achieved due to the fact that in the method of sample preparation of tick tissue, tick tissue is washed in a 70-80% solution of ethyl alcohol, dried with filter paper or any other paper towel with a dense texture and placed in a tube, then added to the tube with a tick 100-200 μl of lysis buffer and mite tissues are rubbed in solution in the same tube, and the resulting homogenate is centrifuged for 2-10 minutes at 2000-6000 rpm.

Drying can be done with filter paper or any paper towel.

Rubbing tick tissue can be done with a scalpel or pestle for homogenization.

Lysis buffer includes NaCl 145 mM, Na ₂ HPO ₄ 10 mM, KH ₂ PO ₄ 1.76 mM, pH 7.4, NP-40 0.5%, casein 2.5%, Proclin 300 0.1%.

A separate pestle should be used for each tick sample to avoid contamination. For repeated use, the pestle must be inactivated: moisten in 95% ethanol and set fire to the spirit lamp, then cool.

Mite tissue samples prepared for analysis can be stored for 3 hours at a temperature of 2-8 ° C or for 3 months at a temperature not exceeding minus 20 ° C.

The use of the claimed method of certain ranges of centrifugation modes is due to the achievement of the required parameters for the dissolution of tick tissues. The results of the experiments are shown in table 1.

Since one of the applications of the described sample preparation of the tick is a polymerase chain reaction, therefore, in addition to a visual assessment of the state of the supernatant and sediment, the obtained supernatant was checked by the polymerase chain reaction method. The analysis of the sample by polymerase chain reaction was carried out as follows: plasmid pQE30 containing the GFP gene was added to the homogenized mite sample. Next, 5 μl of 5 × PCR buffer, 0.5 μl of 10 μM dNTP, 0.75 μl of 100 mM MgCl ₂ , 1 μl of 5 μM forward primer, 1 μl of 5 μM reverse primer, 3 μl 5 were then added to 5 μl of the sample. μm fluorescently labeled sample, 1 unit DNA polymerase. The mixture was thoroughly mixed on a vortex and a polymerase chain reaction was performed with fluorescence detection in real time according to the program: 45 cycles: 95 ° C 15 sec., 60 ° C 30 sec. Fluorescence detection was carried out at 60 ° C. The threshold cycle (Ct) detected by the instrument was used to assess the state of the supernatant.

From the data obtained, it can be seen that at a centrifugation speed of 1000 rpm in the range of centrifugation duration from 2 to 10 min, incomplete deposition of tick fragments, the formation of a loose (mobile) sediment (which made it difficult to select a supernatant without sediment particles) were observed, which, in the end , led to a shift of Ct toward large cycles in the analysis of the sample by PCR.

In the range of centrifugation speeds from 2000 to 6000 rpm at times from 2 to 10 min, the formation of a transparent supernatant, a dense precipitate, acceptable Ct values were observed when analyzing samples by PCR.

At large (more than 6000 rpm) centrifugation values with a centrifugation duration of 2 to 10 min, no noticeable differences were observed between the results obtained and those obtained at a centrifugation speed of 6000 rpm at 2-10 min.

The simplicity of the described method is due to the fact that it requires 3 consecutive steps: 1. Rinse the tick in 70% alcohol, 2. Dry the tick with filter paper or a paper towel, 3. Homogenize the tick in a lysis buffer, while for implementation analogue methods require either a larger number of stages (for example, patent RU No. 2134871), or the involvement of specialized equipment for the operation and storage of liquid nitrogen, special disposable homogenizers designed to work with liquid az that is not always feasible in a laboratory (http://www.bio-ua.com/index.php/prod?i=166&PHPSESSID=3fl72c88947d863e65bb76f2a0ff3bb2).

The reliability of the method was evaluated by PCR. From the data shown in table 1, it is seen that in the specified range of centrifugation parameters, the samples were determined as positive.

Examples of specific performance of the method.

Example 1

The tick is washed with a 70% ethanol solution, spread on a clean paper towel and the tick is dried for 2 minutes. Place in a 1.5 ml Eppendorf tube. 150 μl of lysis buffer is added to the tick tube. Ticks in solution are thoroughly homogenized with a scalpel. The resulting suspension is centrifuged for 2 minutes at 3000 rpm.

Example 2

The tick is washed with a 75% ethanol solution, spread on a clean paper towel and the tick is dried for 2 minutes. Place in a 0.6 ml Eppendorf tube. 120 μl of lysis buffer is added to the tick tube. Ticks in the solution are thoroughly homogenized with a scalpel or pestle to homogenize the ticks (catalog number E-9259 of ZAO Vector-Best). The resulting suspension is centrifuged for 5 minutes at 2000 rpm.

Example 3

Ticks are captured with tweezers and placed in a container with 80% ethanol, then the ticks are transferred to clean, dry filter paper to dry the tick. The tick is transferred to a 1.5 ml disposable Eppendorf tube. 200 μl of lysis buffer is added to the tick tube. Ticks are thoroughly homogenized with a pestle to homogenize ticks (catalog No. E-9259 of ZAO Vector-Best).

The resulting suspension was stirred on Vortex for 30 seconds. The homogenizate is then centrifuged for 3 minutes at 4000 rpm.

Thus, the inventive method of sample preparation of tick tissue allows us to ensure the manufacturability of the process without the use of expensive equipment.

Claims (5)

1. A method of sample preparation of tick tissue, including washing tick tissue with 70-80% ethanol solution, drying it, placing it in a test tube, rubbing and centrifuging, characterized in that 100-200 μl of lysis buffer is added to the test tube before rubbing, the grinding of the tick tissue itself produced in solution in the same tube, and the resulting homogenate is centrifuged for 2-10 min at 2000-6000 rpm 2. The method of sample preparation of tick tissue according to claim 1, characterized in that filter paper is used for drying. 3. The method of sample preparation of tick tissue according to claim 1, characterized in that a paper towel is used for drying. 4. The method of sample preparation of tick tissue according to claim 1, characterized in that for grinding the tick use a pestle for homogenization. 5. The method of sample preparation of tick tissue according to claim 1, characterized in that a scalpel is used to rub the tick.