RU2624241C1 - Solid phase carrier for immobilization and/or storage of biological samples containing nucleic acids - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: solid phase carrier for immobilization and storage of samples containing nucleic acids is a porous cellulose base treated with a composition containing a detergent, a weak base, a chelating agent, and a disaccharide.

EFFECT: prolonged storage of samples at room temperature without destroying nucleic acids; also allows carrier sterilisation against impurity DNA or RNA without loss of carrier properties.

14 cl, 4 tbl, 4 ex

Description

Field of Technology:

The present invention relates to a solid-phase carrier for immobilization and subsequent long-term storage at room temperature of biological materials containing nucleic acids (DNA or RNA), which is a porous base treated with a composition containing a set of reagents.

The prior art:

The use of solid-phase carriers to preserve samples of biological material for the subsequent isolation and analysis of biomolecules has been widely known since the 1960s, when the first maps of this kind were created - the so-called Guthrie's cards [1, 2], designed for screening newborns to detect a genetic disease - phenylketonuria. Subsequently, numerous variants of such solid-phase carriers, in particular, cellulose-based carriers, ensuring the long-term preservation of samples of biological materials containing DNA or RNA at room temperature [3, 4], and requirements for such carriers, including criteria for their standardization [5, were developed ].

Known cards for storing samples Whatman ^® 903 Specimen Collection Paper [6-9], allowing for a long time retain DNA or RNA-containing biological material samples (such as blood samples, etc..) At -20 ° C in a hermetically sealed container together with a desiccant [9].

A solid-phase carrier for the long-term storage of samples of biological material containing DNA or RNA, including samples of genetic material isolated from blood, including a solid matrix made of solid cellulose (preferably filter paper), is known as the closest analogue of the present invention. ) or from a polymer, wherein said matrix contains a compound or composition deposited on the matrix or absorbed on the matrix and protecting DNA or RNA from degradation [10]. As a compound that protects DNA or RNA from degradation, the solid-phase carrier according to [10] uses uric acid together with a weak base to obtain a uric acid salt, urate, and to provide an alkaline pH in the range from 8.0 to 9.5.

Additionally, the solid phase carrier [10] may contain a chelating agent, such as EDTA, an anionic detergent, such as sodium lauryl sulfate (sodium dodecyl sulfate, SDS), and a slightly alkaline buffer, such as Tris-EDTA buffer. The disadvantages of the solid-phase carrier [10] include the fact that it requires impregnation in polystyrene for subsequent long-term storage at room temperature, or storage in a refrigerator at -15 ° C in the presence of a desiccant and several crystals of dry sodium carbonate, which increases the cost of storing samples by such a carrier.

In addition, the carrier in accordance with the prototype [10], like many other similar carriers, faces the problem of the presence of contaminating DNA or RNA of bacterial, viral, and other origin. A solid-phase carrier for storing biological samples for the purpose of subsequent extraction of DNA or RNA with optimal characteristics should not only provide the possibility of long-term storage of DNA-containing biological samples, but also provide the possibility of sterilization pretreatment in order to destroy contaminated DNA or RNA.

In connection with these shortcomings of the prototype [10], it remains an urgent task to create a solid-phase carrier based on porous cellulosic material that allows long-term storage of biological samples containing DNA or RNA at room temperature, without the use of additional measures, which, however, would not lose their properties as a result of sterilization pre-treatment.

In the framework of the present invention, it was unexpectedly shown that a porous cellulose-based solid phase carrier, impregnated or coated with a composition containing a weak base, an anionic detergent and a chelating agent, is characterized in that it additionally contains a disaccharide, effectively protects DNA or RNA contained in biological samples, from degradation due to ionizing radiation or ethylene oxide, at the same time allowing sterilization treatment of the carrier from “impurity” DNA or RNA, which concludes I unexpected technical result of the present invention.

In a preferred embodiment, the disaccharide is trehalose, sucrose, maltose, or a combination thereof; in a most preferred embodiment, the disaccharide is a combination of trehalose and sucrose in a mass ratio of from 1: 1 to 1: 4 or a mixture of trehalose and maltose in a mass ratio of from 1: 1 to 1: 4.

In a preferred embodiment, the pH of said composition is from 7.0 to 9.0.

In a more preferred embodiment of the invention, said composition contains from 0.2 to 0.4% detergent, from 0.1 to 0.2% weak base, from 0.02 to 0.04% chelating agent, and from 0.3 to 0 5% disaccharide.

In a preferred embodiment, the weak base is selected from the group consisting of Tris (hydroxymethyl) aminomethane, N- (2-hydroxy-1,1-bis (hydroxymethyl) ethyl) glycine, N, N-bis- (2-hydroxyethyl) glycine and sodium salt 4- (2-hydroxyethyl) 1-piperazineethanesulfonic acid.

In a preferred embodiment, the anionic detergent is selected from guanidine hydrochloride, guanidine isocyanate, urea, sodium lauryl sulfate (SDS), polyoxyethylene sorbitan monolaurate (Tween 20 or others), octylphenoxy-polyethoxyethanol (Nonidet P40 or others) and t-octylphenolphenol 100 or others).

In a preferred embodiment, the chelating agent is selected from the group consisting of trisodium nitrile triacetic acid, disodium salt of ethylenediaminetetraacetic acid, trisodium salt of N- (hydroxyethyl) ethylene diamine tetraacetic acid and pentasodium salt of diethylenetetramine pentaacetic acid.

The solid-phase carrier in accordance with the invention provides the possibility of long-term storage of biological samples containing nucleic acids (DNA or RNA) at room temperature without the use of additional means (such as impregnating cards into polystyrene, using a refrigerator, etc.), as well as the possibility of efficient sterilization pretreatment without loss of useful properties of the carrier.

Sterilization pretreatment can be carried out by ionizing radiation [11], for example, gamma radiation. Most preferably, the sterilizing dose of gamma radiation is from 10 to 25 kGy. Alternatively, sterilization can be carried out with a gas mixture containing ethylene oxide [12]. In the most preferred embodiment, the amount of ethylene oxide in the gas mixture is from 5 to 15%, while in the most preferred embodiment, the treatment with the gas mixture containing ethylene oxide is carried out for at least 4 to 8 hours.

Below are examples of the invention, clearly demonstrating the possibility of achieving the claimed technical result, without limiting the scope of the claims.

Examples of carrying out the invention

Example 1. Preparation of solid phase media.

40 g of sodium lauryl sulfate, 38.8 g of tris (hydroxymethyl) aminomethane and 5.8 g of EDTA were mixed with 50 g of a mixture of trehalose: sucrose (in a mass ratio of 1: 3), dissolved in distilled water, then concentrated hydrochloric acid was added with stirring to pH 8.5, and then the volume of the resulting solution was adjusted to 1 liter. Ashless filter paper type Whatman ^® 903 was impregnated with the resulting composition, and dried at 70 ° C for 4 hours. The obtained solid-phase carrier was cut into 5.0 × 5.0 cm cards. Solid-phase carriers were similarly prepared using 50 g of trehalose, 50 g of sucrose or 50 g of maltose and 50 g of a mixture of trehalose and maltose in a mass ratio of 1: 3 instead of 50 g mixtures of trehalose: sucrose in a mass ratio of 1: 3.

Similarly prepared control solid-phase media that do not contain disaccharide.

Example 2. The study of the storage of DNA on solid phase media.

To study the storage of DNA cards, the “accelerated aging” method was used.

85 μl of a capillary blood sample was placed on test and control media, as well as on reference media (FTA ™ Mini Card manufactured by GE Healthcare, Cat. No. WB120055) and dried at room temperature for 4 hours. Then part of the media was placed in an oven with a constant temperature of 60 ° C. The second part of the media was left at room temperature.

Samples of carriers stored at 60 ° C were examined every 7 days of storage, and samples of carriers stored at room temperature were examined every 21 days of storage.

For this, disks with a diameter of 2 mm were cut out from the carriers using a punch. Disks were placed in Eppendorf tubes and shaken with 0.2 μl Tris-EDTA buffer (pH 8.5) on a Vortex laboratory shaker for 5 minutes, then Tris-EDTA buffer was removed by decantation. The procedure was repeated twice. The discs were dried at 60 ° C for 30 minutes and placed in PCR tubes. 25 μl of the reaction mixture prepared according to the instructions for the AmpliSens ^® M. hominis-screen-titer-FL reagent kit was added to the tubes. The tubes were placed in an automatic amplifier and amplification was performed by real-time PCR using the following program (Table 1):

A fluorescent signal was detected starting from the sixth cycle. Detection was conducted via the JOE fluorophore channel (detection channel for an internal control specimen specific for human DNA).

The results are presented in table 2, where T60 is a solid-phase carrier according to the invention on a cellulose basis, prepared using trehalose as a disaccharide, stored at a temperature of 60 ° C;

S60 is a solid-phase carrier according to the invention on a cellulose basis, prepared using sucrose as a disaccharide and stored at 60 ° C;

M60 is a solid-phase carrier according to the invention on a cellulose basis, prepared using maltose as a disaccharide and stored at 60 ° C;

TS60 - a card prepared using a combination of trehalose and sucrose (in a mass ratio of 1: 3) as a disaccharide and stored at 60 ° C;

TM60 - a card prepared using a combination of trehalose and maltose (in a mass ratio of 1: 3) as a disaccharide and stored at 60 ° C;

O60 is a solid-phase carrier according to the invention on a cellulose basis, prepared without the use of a disaccharide and stored at 60 ° C;

F60 — FTA ™ Mini Card solid-state comparison carrier stored at 60 ° C;

T25 is a solid-phase carrier according to the invention on a cellulose basis, prepared using trehalose as a disaccharide and stored at room temperature (20-25 ° C);

O25 - a solid-phase carrier according to the invention on a cellulose basis, prepared without the use of a disaccharide and stored at room temperature (20-25 ° C);

F25 is an FTA ™ Mini Card solid-phase comparison medium stored at room temperature (20–25 ° C).

From Table 2 it follows that at room temperature the DNA samples are equally well stored on carriers with both disaccharides and without disaccharides (the results in the experimental group and in the comparison group were comparable), however, at elevated temperatures, the DNA is much better stored on solid-phase media according to the invention ; on the contrary, on cards without disaccharide, including on comparison media, DNA underwent complete destruction after 63 days of storage at 60 ° C.

Example 3. Sterilization pretreatment of carriers with a gas mixture containing ethylene oxide and gamma radiation.

In order to simulate contamination on solid-phase carriers of Example 1 containing a mixture of trehalose with sucrose (in a mass ratio of 1: 3), as well as similar carriers without disaccharides, washed human blood leukocytes or a DNA solution isolated from human blood leukocytes were applied. AmpliPrim RIBO-prep reagent kit was used to isolate DNA from leukocytes. After applying the white blood cells and the DNA solution, the cards were dried for 4 hours at room temperature, then packed in paper envelopes and sent for processing. Some cards were treated with a mixture of 10% ethylene oxide and carbon dioxide for 4 and 8 hours, and some with gamma irradiation (10 and 25 kGy). Control cards left sealed in an envelope without processing. After processing, the cards were removed from the envelopes and from the regions where the leukocyte and DNA samples were applied, 2 mm diameter disks were cut with a puncher. The residual DNA content was measured by real-time PCR according to example 2. The results are presented in table 3.

where EO4-LK - samples with disaccharides and coated with leukocytes treated with a gas mixture containing ethylene oxide for 4 hours;

OEO4-LK - samples without disaccharides and coated with leukocytes treated with a gas mixture containing ethylene oxide for 4 hours;

EO4-DNA — samples with disaccharides and a coated DNA solution, treated with a gas mixture containing ethylene oxide for 4 hours;

OEO4-DNA - samples without disaccharides and coated with a solution of DNA, treated with a gas mixture containing ethylene oxide for 4 hours;

EO8-LK - samples with disaccharides and coated with white blood cells, treated with a gas mixture containing ethylene oxide for 8 hours;

OEO8-LK - samples without disaccharides and coated with leukocytes treated with a gas mixture containing ethylene oxide for 8 hours;

EO8-DNA — samples with disaccharides and a coated DNA solution, treated with a gas mixture containing ethylene oxide for 8 hours;

OEO8-DNA - samples without disaccharides and coated with a DNA solution, treated with a gas mixture containing ethylene oxide for 8 hours;

GR10-LK - samples with disaccharides and coated with white blood cells, treated with gamma radiation with a dose of 10 kGy;

OGR10-LK - samples without disaccharides and with white blood cells, treated with gamma radiation with a dose of 10 kGy;

ГР10-DNA - samples with disaccharides and a coated DNA solution, treated with gamma radiation with a dose of 10 kGy;

OGR10-DNA - samples without disaccharides and coated with leukocytes, treated with gamma radiation with a dose of 10 kGy;

GR25-LK - samples with disaccharides and coated leukocytes, treated with gamma radiation with a dose of 25 kGy;

OGR25-LK - samples without disaccharides and with white blood cells, treated with gamma radiation with a dose of 25 kGy;

ГР25-DNA - samples with disaccharides and coated with a solution of DNA, treated with ionizing radiation (gamma radiation) with a dose of 25 kGy;

OGR25-DNA — samples without disaccharides and with a coated DNA solution, treated with ionizing radiation (gamma radiation) with a dose of 25 kGy;

K-LK - control samples with white blood cells;

K-DNA - control samples with applied DNA solution.

Example 4. Storage of DNA on cards treated with a gas mixture containing ethylene oxide and gamma radiation.

The solid-phase carriers of the invention prepared according to Example 1 using a mixture of trehalose with sucrose (1: 3) as a disaccharide, as well as similar carriers without disaccharides, were further treated with a mixture of 10% ethylene oxide for 4 hours or gamma radiation at a dose of 25 kGy. Then, blood samples of 85 μl were applied to the cards and DNA storage was studied using accelerated aging as in Example 2. As a comparison sample, we used DNA cards with trehalose and sucrose (in a mass ratio of 1: 3), not treated with a gas mixture containing ethylene oxide, or gamma radiation, as well as cards prepared without the use of disaccharides, but treated with a gaseous mixture containing ethylene oxide, or gamma radiation. The results are shown in table 4.

Where

TS60-EO is a solid-phase carrier according to the invention, prepared using a combination of trehalose and sucrose (in a mass ratio of 1: 3), as a disaccharide, further treated with a gas mixture containing ethylene oxide and stored at 60 ° C;

TS60-GR - solid-phase carrier according to the invention, prepared using a combination of trehalose and sucrose (in a mass ratio of 1: 3), as a disaccharide, additionally treated with gamma radiation and stored at 60 ° C;

O60-EO is a solid phase carrier according to the invention, prepared without the use of disaccharides, further treated with a gas mixture containing ethylene oxide, and stored at 60 ° C;

O60-GR - solid-phase carrier according to the invention, prepared without the use of disaccharides, additionally treated with gamma radiation and stored at 60 ° C;

TS60 is a solid phase carrier according to the invention, prepared using a combination of trehalose and sucrose (in a mass ratio of 1: 3) as a disaccharide and stored at 60 ° C.

Thus, the proposed solid-phase carrier for immobilization and storage of nucleic acid samples, which is a porous cellulose base treated with a composition containing a detergent, a weak base, a chelating agent and containing a disaccharide or a mixture of disaccharides, provides an unexpectedly high stability of immobilized biological samples containing nucleic acids including DNA and RNA, while at the same time providing the possibility of effective sterilizing pretreatment with ethylene oxide or ionizing radiation (without losing the beneficial properties of the carrier).

Bibliography:

1. R. Guthrie, A. Susi. A simple method for detecting phenylketonuria in large populations of newborn infants. // Pediatrics 32: 338-343, 1963. Available via the Internet (as of 03/16/2016); access mode: http://pediatrics.aappublications.org/content./pediatrics/32/3/338.full.pdf.

2. Julia A. McMillan, Ralph D. Feigin, Catherine DeAngelis, M. Douglas Jones (April 1, 2006). Oski's pediatrics: principles & practice. Lippincott Williams & Wilkins. pp. 162. Available via the Internet (as of 03/16/2016); access mode: https://books.google.com/books?id=VbjFQiz8aR0C&pg=PA162&hl=en#v=onepage&q&f=false.

3. M.C. Kline, D.L. Duewer. Polymerase chain reaction amplification of DNA from aged blood stains: quantitative evaluation of the "suitability for purpose" of four filter papers as archival media. // Analytical Chemistry, 2002, volume 74, No. 8, 1863-1869.

4. G.S. Makowski, F.L. Nadeau. Single tube multiplex PCR detection of 27 cystic fibrosis mutations and 4 polymorphisms using neonatal blood samples collected on Guthrie cards. // Annals of Clinical and Laboratory Science Journal, volume 33, No. 3, 243-250 (2003).

5. J.V. Mei, J.R. Alexander, et al. Use of Filter Paper for the Collection and Analysis of Human Whole Blood Specimens. // Journal of Nutrition, May 1, 2001, vol. 131, No. 5, 1631S-1636S. Available via the Internet (as of 03/16/2016); access mode: http://jn.nutrition.org/content/131/5/1631S.full.

6. Pak Yang Chum, Chas Andre. Direct PCR from Blood Preserved on Whatman FTA and 903 Cards using Thermo Scientific Phusion Blood Direct PCR Kit (2013). Available via the Internet (as of 03/16/2016); access mode: https://www.thermofisher.com/content/dam/LifeTech/global/brands/Documents/1114/app-note-direct-pcr-from-blood-preserved-on-whatman-fta-903-cards .pdf.

7. M.A. Harvey et al. Impregnated 903 ⁽ ™ ⁾ Blood Collection Paper: A Tool for DNA Prepartion from Dried Blood Spots for PCR Amplification. // Clinical Chemistry, 1995, volume 41, No. 6, S108.

8. M.A. Harvey, T.A. King, et al. Impregnated sample collection paper: A tool for preparation of DNA and RNA from dried blood spots for PCR amplification. // Clinical Chemistry, 1995, volume 41, No. 11, p. 1686, abstract # 041.

9. Whatman ™ Neonatal Screening cards. // GE Healthcare LifeSciences. Posted: 01/2013. Available via the Internet (as of 03/16/2016); access mode: https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/136076 4947442 / litdoc28984424_20130214002441.pdf.

10. US patent No. 5496562 A, published 05.03.1996; IPC C12N 15/10, C12Q 1/68.

Aparecida da Silva Aquino. Sterilization by Gamma Irradiation, Gamma Radiation (Edited by Prof. Feriz Adrovic), pp. 171-206. Дата публикации: 2012. Доступно (по состоянию на 16.03.2016); режим доступа: http://www.intechopen.com/books/gamma-radiation/sterilization-by-gamma-irradiation.eleven.

Aparecida da Silva Aquino. Sterilization by Gamma Irradiation, Gamma Radiation (Edited by Prof. Feriz Adrovic), pp. 171-206. Date of publication: 2012. Available (as of March 16, 2016); access mode: http://www.intechopen.com/books/gamma-radiation/sterilization-by-gamma-irradiation.

12. US patent No. 2075845 A, published 04/06/1937; IPC A61L 2/20.

Claims (14)

1. A solid-phase carrier for immobilizing and storing nucleic acid-containing samples, said carrier being a porous cellulose base treated with a composition containing a detergent, a weak base, a chelating agent and a disaccharide. 2. A solid phase carrier according to claim 1, characterized in that said detergent is selected from guanidine hydrochloride, guanidine isocyanate, urea, sodium lauryl sulfate (dodecyl sulfate), polyoxyethylene sorbitan monolaurate, t-octylphenoxypolyethoxyethanol and octyl. 3. A solid phase carrier according to claim 1, characterized in that said weak base is selected from tris (hydroxymethyl) aminomethane, N- (2-hydroxy-1,1-bis (hydroxymethyl) ethyl) glycine, N, N-bis- ( 2-hydroxyethyl) glycine and the 4- (2-hydroxyethyl) 1-piperazineethanesulfonic acid sodium salt. 4. The solid-phase carrier according to claim 1, characterized in that said chelating agent is selected from the group consisting of trisodium nitrile triacetic acid, disodium salt of ethylenediaminetetraacetic acid, trisodium salt of N- (hydroxyethyl) ethylenediaminetetraacetic acid and pententetrate acetic acid. 5. A solid phase carrier according to claim 1, characterized in that said disaccharide is trehalose, sucrose, maltose, or a combination thereof. 6. A solid phase carrier according to claim 5, characterized in that the said combination of disaccharides is a mixture of trehalose and sucrose in a mass ratio of from 1: 1 to 1: 4. 7. The solid phase carrier according to claim 5, characterized in that the said combination of disaccharides is a mixture of trehalose and maltose in a mass ratio of from 1: 1 to 1: 4. 8. A solid phase carrier according to claim 1, characterized in that said composition has a pH value of from 7.0 to 9.0. 9. A solid phase carrier according to claim 1, characterized in that said composition contains from 0.2 to 0.4% detergent, from 0.1 to 0.2% weak base, from 0.02 to 0.04% chelating agent and from 0.3 to 0.5% disaccharide. 10. A solid phase carrier according to any one of paragraphs. 1-9, characterized in that the said carrier is treated with a gas mixture containing ethylene oxide. 11. The solid-phase carrier according to claim 10, characterized in that the ethylene oxide content in said gas mixture is from 5 to 15%. 12. The solid-phase carrier according to claim 11, characterized in that the said gas mixture is processed from 4 to 8 hours. 13. A solid phase carrier according to any one of paragraphs. 1-9, characterized in that the said carrier is processed by gamma radiation. 14. The solid-phase carrier according to claim 13, characterized in that the dose of gamma radiation is from 10 to 25 kGy.