RU2535333C1 - Device for polymerase chain reaction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: device for polymerase chain reaction comprises at least four portions, each of which has two flat parallel surfaces perpendicular to a common axis, and holes. The portions are rotated in relation to each other and the common axis. The first portion has at least six through holes connecting its two flat surfaces, at least four holes of which are provided with hydrophilic filters mounted to contact the flat surface of the second portion. The second portion is configured with at least two through holes connecting its two flat surfaces, at least one hole of which comprises a sorbent fixed by the hydrophilic filters contacting the flat surfaces of the first and third portions. The third portion has at least two through holes connecting its two flat surfaces, at least one hole of which comprises the hydrophilic filter mounted to contact the flat surfaces of the second and fourth portions, and at least three blind holes, each of which comprises the hydrophilic filters mounted to contact the flat surface of the second portion. The fourth portion is provided with at least one blind optically transparent through hole, comprising the hydrophilic filter mounted to contact the flat surface of the third portion non contacting the bottom of the hole containing it.

EFFECT: enhancement involving carrying out the stages of nucleic acid recovery and PCR in the same apparatus with detecting reaction results and simplifying the device.

8 cl, 5 dwg

Description

The invention relates to medicine and biology and is intended for use in PCR studies.

Recent advances in bioanalytical microfluidic microarrays make their use in medicine and biology more and more realistic in the form of portable micro laboratories, the so-called “labs on a chip” (Manz A., Graber N., Widmer HM Miniaturized total chemical analysis systems: A novel concept for chemical sensing // Sensors and Actuators B: Chemical. - 1990. - Vol.1. - P.244-248).

The synonyms of “laboratories on a chip” are micro total analysis system (µ-TAS), microelectromechanical system (MEMS) (Chen J., Chen D., Yuan T., Chen X. Microfluidic PCR Chips // Nano biomedicine and engineering. - 2011 . - Vol.3, No. 4. - P.203-210).

Most biomedical tests involve the addition and removal of liquid reagents during a diagnostic test. Microchip control of microvolumes of liquids from various tanks, mixing in microreactors, separation, movement through channels, control of each of the stages of movement, detection of results, data conversion in the processor, display of results on a computer monitor in miniature repeats everything that the laboratory technician does in the process conducting a diagnostic test in the laboratory. That is, the entire laboratory process takes place on a 1-2 cm ² chip. Small volumes of regents, respectively low cost, reduced analysis time, high accuracy, all this attracts researchers to develop chips in the diagnosis of biochemical and clinical-biological analyzes (DeMello AJ Control and detection of chemical reactions in micro-fluidic systems // Nature. - 2006. - Vol.442, No. 7101. - P.394-402; Baek SH, Chang WJ, Baek JY, Yoon DS, Bashir R., Lee SW Dielectrophoretic technique for measurement of chemical and biological interactions // Analytical chemistry. - 2009. - Vol.81, No. 18. - P.7737-7742; Dittrich PS, Manz A. Lab-on-a-chip: microfluidics in drug discovery // Nature reviews. Drug discovery. - 2006. - Vol.5 , No. 3. - P.210-218).

Small volumes of liquids, their unique behavior in microvolumes, allows you to clearly control the concentration of reagents, reduces the cost of analysis, speeds up the analysis process, reduces the amount of environmental pollution, allows analysis outside of centralized laboratories.

PCR is one of the most important methods in diagnostics and one of the most convenient methods for microchipping. The first PCR chip was proposed in 1993 (North-up MA, Ching MT, White RM, Watson RT DNA amplification in a microfabricated reaction chamber // "Transducers '93", Seventh International Conference on Solid State Sensors and Actuators, Yokohama, Japan. - New York: IEEE, 1993. - P.924-927). Since then, various versions of microfluidic chips for PCR have been created (Chen J., Chen D., Yuan T., Chen X. Microfluidic PCR Chips // Nano biomedicine and engineering. - 2011. - Vol.3, No. 4. - P .203-210).

A typical microfluidic chip for PCR consists of three temperature zones (denaturation, elongation, annealing) and a microcapillary channel in which the PCR mixture passes through these zones.

In a microarray system for PCR, the uniformity of temperature distribution increases, the time for maintaining temperatures at the stages of DNA synthesis and denaturation is reduced, and the number of cycles from standard 35-45 to 20-25 is reduced, which speeds up PCR and increases the analytical sensitivity of the method (Fuchiwaki Y., Saito M ., Wakida S., Tamiya E., Nagai H. Ultra-fast and highly-efficient flow-through PCR microfluidics using vapor pressure and its application to rapid field detection // 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences. 3 -7 October 2010, Groningen, The Netherlands // Micro Total Analysis Systems. - 2010. - Vol.1. - P.148-150).

The following methods of promoting liquids are used in chips (Chen J., Chen D., Yuan T., Chen X. Microfluidic PCR Chips // Nano biomedicine and engineering. - 2011. - Vol.3, No. 4. - P.203 -210; RU 2432205, B01L 3/00, G01N 33/487).

1. The movement is provided by the pressure created by a mechanical pump or compressed gas.

2. Electrokinetic method.

3. Centrifugation.

4. Exposure to an external magnetic field.

5. Thermally convection-controlled chip based on the principle of Rayleigh-Benard convection.

The disadvantages of these analogues are the need to use an additional external unit, creating conditions for the promotion and mixing of reaction components, as well as the need to seal these components and the need to synchronize the operation of various units with an increase in the number of external factors that affect the quality of the reaction, which complicates the device.

The closest analogue of the claimed technical solution - the prototype - is a device for carrying out a molecular biological reaction (including real-time PCR or PCR), in which the individual necessary components of the reaction are located at the reaction site, but are spatially separated from each other before the reaction on the carrier . According to the formula of the prototype, the solid support is a porous support, preferably a porous filter. Reactions using this carrier according to the description of the prototype can be carried out using known methods in a separate container using another device after dissolving the reagents adsorbed on the carrier in a buffer that is not included in the prototype (WO 2012010708, C12Q 1/68).

The main disadvantage of the prototype is the inability to achieve a technical result - carrying out in one device the stages of isolation (purification) of nucleic acids and subsequent PCR with detection of the reaction results.

The main task solved by the claimed technical solution is to carry out in one device the steps of isolation (purification) of nucleic acids and subsequent PCR with detection of the reaction results while simplifying the device.

The problem is achieved due to the fact that the device for the polymerase chain reaction consists of at least four parts, each of which has two flat, parallel surfaces perpendicular to the common axis, relative to which the parts can rotate relative to each other, and holes, this first part has at least six through holes connecting its two flat surfaces, of which at least four holes are equipped with hydrophilic filters in contact with the flat surface of the second part, the second part is made with at least two through holes connecting its two flat surfaces, of which at least one hole contains a sorbent fixed by hydrophilic filters in contact with the flat surfaces of the first and third parts, the third part has at least two through holes connecting its two flat surfaces, of which at least one hole contains a hydrophilic filter in contact with the flat surfaces of the second and fourth parts, and, at least three through holes containing hydrophilic filters in contact with the flat surface of the second part, and the fourth part is made with at least one through, optically transparent hole containing a hydrophilic filter in contact with the flat surface of the third part, not in contact with the bottom of the hole containing it.

The number of holes, filters, sorbents and reagents for amplification and detection of nucleic acids in parts of the claimed device can be increased with an increase in the number of analyzed biological objects, and / or with an increase in the number of multiplex PNRs and / or uniplex (simplex) PCRs carried out in the device, and / or by increasing the number of nucleic acid purification steps. Additional purification of nucleic acids can also provide an increase in the number of parts of the device.

The basis of the claimed invention is based on a new set of original distinctive features providing a solution to the problem - this is a selected sequential arrangement of holes, hydrophilic filters and a sorbent in the device parts, which: firstly, makes it possible to carry out the stages of isolation (purification) of nucleic acids and subsequent multiplex in the device PCR and / or uniplex (simplex) PCR with detection of reaction results; secondly, it provides, without using an additional external unit, conditions for the promotion and mixing of reaction components as a result of soaking (including alternating) filters with liquid components; thirdly, it makes it possible to change the amount of liquid components used, which prevents the drying of the filters during the reaction, the leakage of liquids between flat surfaces and thus eliminates the need for sealing of the components.

The technical solution proposed as an invention was tested in various versions in the laboratory of immunology and virology, the group of molecular biology of the Institution of the Russian Academy of Medical Sciences of the Research Institute of Medical Primatology of the Russian Academy of Medical Sciences.

The essence of the proposed technical solution is illustrated by drawings of a variant of the device for analyzing one biological object using one multiplex or uniplex (simplex) PNR, where:

1, 2, 3 and 4 - parts of the device;

5, 6, 7, 8, 12, 14, 15, 16, 17, 18 and 20 - hydrophilic filters contained in the holes of the device;

9, 10, 11 and 19 - holes in the parts of the device that do not contain filters;

13 - sorbent contained in the opening of part 2 of the device;

21 is a through, optically transparent hole, in the open end of which there is a hydrophilic filter 20, not in contact with the bottom of the hole containing it;

22 - the common axis of the four parts of the device.

Figure 1 shows a variant of the sequence of the initial arrangement of holes 9, 10, 11, 19, not containing filters, holes 21, partially containing a filter 20, filters 5, 6, 7, 8, 12, 14, 15, 16, 17, 18 and 20 and sorbent 13 in parts 1, 2, 3 and 4 of the device.

Figure 2 shows a variant of the schematic appearance of the device with a common axis 22 of parts 1, 2, 3 and 4 and with a possible initial location of visible, filter-free openings 9 and 10 and filters 5, 6, 7 and 8 of part 1.

Figure 3 shows a variant of the schematic appearance of the device with a common axis 22 of parts 2, 3 and 4 and with a possible initial location of a visible, filter-free opening 11 and a visible filter 12 of part 2.

Figure 4 shows a variant of the schematic appearance of the device with a common axis 22 of parts 3 and 4 and with a possible initial location of a visible, filter-free opening 19 and visible filters 15, 16, 17 and 18 of part 3.

Figure 5 shows a variant of the schematic appearance of part 4 of the device with a possible initial location of the visible filter 20.

In this case, all filters and sorbent are contained in the holes. The filter 20 includes dried reagents for amplification and detection of nucleic acids, which can be included only in part of the filter.

Preferred are variants of the device in which, when turning their parts, the hole 10 is only aligned with the hole 11, the hole 11 is only aligned with the hole 10 and the hole 19, the hole 19 is only aligned with the hole 11 and the filter 20.

The configuration (form) shown in the diagrams and the proportional ratio of the sizes of parts, holes, filters and sorbent of the claimed device are due to the need for an informative description of the general principles of its operation and are not intended for practical use.

The above embodiment of the device shows a specific implementation of the claimed invention, but does not limit the scope of claims of the claims of the claimed invention.

From the patent technical literature and the practice of PCR studies, it is not known about a device for polymerase chain reaction, which would be identical to the claimed one.

Hence the conclusion that the claimed technical solution meets the criterion of "novelty" is legitimate.

The above set of essential features is necessary and sufficient to obtain a technical result — carrying out in one device the steps of isolation (purification) of nucleic acids and subsequent PCR with detection of the reaction results while simplifying the device. There is a causal relationship between the existing features and the problem to be solved, where each feature is necessary and influences the receipt of a technical result, and the combined features are sufficient to obtain it. The conclusion that the claimed technical solution meets the criterion of "inventive step" is legitimate.

The proposed device can be implemented many times, which means that the claimed technical solution meets the criterion of "industrial applicability".

The invention is illustrated by the following example, showing the possibility of carrying out in the above described embodiment of the claimed device the steps of isolation (purification) of nucleic acids and subsequent PCR with detection of the reaction results. In this case, the example device shows a specific implementation of the claimed invention, but does not limit the scope of claims of the claims of the claimed invention.

Before starting work, the following is performed:

- washing filters are introduced into filters 7 and 8;

- an eluting solution is introduced into the filter 5.

Then the following operations are performed.

A lysate of a biological object (a mixture of a biological object and a lysing solution) is introduced into hole 9 of part 1 of the device. In this case, the lysate of the biological object alternately (sequentially) impregnates the filter 12, sorbent 13, filter 14 and filter 18, and nucleic acids bind to the sorbent 13.

Part 2 of the device is rotated to combine filters 8 and 12, as well as filters 14 and 17. In this case, the washing solution from the filter 8 alternately (sequentially) impregnates the filter 12, sorbent 13, filter 14 and filter 17, washing the nucleic acids associated with the sorbent 13 .

Part 2 of the device is rotated to combine filters 7 and 12, as well as filters 14 and 16. In this case, the washing solution from the filter 7 alternately (sequentially) impregnates the filter 12, sorbent 13, filter 14 and filter 16, washing the nucleic acids associated with the sorbent 13 .

Part 2 of the device is rotated to combine filters 6 and 12. In this case, the filter 12, sorbent 13 and filter 14 are dried.

Part 2 of the device is rotated to combine filters 5 and 12, as well as filters 14 and 15. In this case, the eluting solution from filter 5 impregnates filter 12, sorbent 13, filter 14 and filter 15 and nucleic acids elute from sorbent 13 and transferred to the filter 15.

Part 4 of the device is rotated to combine filters 15 and 20. In this case, the eluted nucleic acids are impregnated with filter 20 and transferred to filter 20.

Part 4 of the device is rotated until the holes 10, 11, 19 and the filter 20 are combined. A solvent is introduced into the hole 10, which impregnates the filter 20 and flows with dissolved nucleic acids and reagents for amplification and detection of nucleic acids to the bottom of the hole 21.

Oil is introduced into hole 10 for PCR.

PCR is carried out using any base unit containing a light source, a fluorescence detector and a thermocouple, providing a cyclic adjustment of the temperature of the reaction mixture in hole 21 to 62-95 ° C and detection of nucleic acids in hole 21.

The device carries out multiplex PCR or uniplex (simplex) PCR.

With an increase in the number of holes, filters, and sorbents, two or more multiplex PCR and / or uniplex (simplex) PCR are carried out in the device.

An increase in the number of parts of the device allows for additional purification of nucleic acids.

Technical advantages of the claimed device consist in carrying out in one device the steps of isolation (purification) of nucleic acids and subsequent PCR with detection of the reaction results while simplifying the device in the form of a chip.

Claims (8)

1. A device for polymerase chain reaction, containing a filter, characterized in that it consists of at least four parts, each of which has two flat, parallel surfaces perpendicular to a common axis, relative to which the parts can rotate relative to each other, and holes, while the first part has at least six through holes connecting its two flat surfaces, of which at least four holes are equipped with hydrophilic filters in contact with the flat surface the second part, the second part is made with at least two through holes connecting its two flat surfaces, of which at least one hole contains a sorbent fixed by hydrophilic filters in contact with the flat surfaces of the first and third parts, the third part has at least two through holes connecting its two flat surfaces, of which at least one hole contains a hydrophilic filter in contact with the flat surfaces of the second and fourth parts, and, to at least three through holes containing hydrophilic filters in contact with the flat surface of the second part, and the fourth part is made with at least one through, optically transparent hole containing a hydrophilic filter in contact with the flat surface of the third part, not in contact with the bottom holes containing it. 2. The device according to claim 1, characterized in that its parts are made in the form of disks. 3. The device according to claim 1, characterized in that its filters are made of a material selected from the series: paper, cellulose, cellulose acetate, polyamide, polycarbonate, or mixtures thereof. 4. The device according to claim 1, characterized in that the sorbent of its second part is made in the form selected from the series: powder, granules, fiber, or mixtures thereof. 5. The device according to claim 1, characterized in that the sorbent of its second part is made of a substance selected from the series: silicon dioxide, aluminum oxide or a mixture thereof. 6. The device according to claim 1, characterized in that it further comprises at least one reagent for amplification and detection of nucleic acids. 7. The device according to claim 6, characterized in that it contains a reagent for amplification and detection of nucleic acids in dry form in at least one hole in the fourth part. 8. The device according to claim 6, characterized in that it contains a reagent for amplification and detection of nucleic acids in dry form on at least one filter in the hole of the fourth part.

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