RU2800875C9 - Cartridge for sterile storage of reagents - Google Patents

Abstract

FIELD: medicine; analytical medical equipment; in vitro diagnostics by molecular genetic methods.

SUBSTANCE: cartridge is made of a transparent material in the form of a cluster with conical cells. The wells vary in size and are located on concentric circles that correspond to different stages of the study — isolation of nucleic acids from cells and viruses, amplification of specific fragments and visualization of amplification products. The wells contain magnetic beads for nucleic acid extraction and other necessary reagents. The upper surface of the cluster is covered with a sealing film, in the center of the cluster there is a hole for fixing in the working position.

EFFECT: invention makes it possible to store various reagents under sterile conditions for several weeks in quantities strictly necessary for conducting a study of one clinical sample, namely, for isolating nucleic acids from a sample, carrying out 15 different single reactions of amplification of isolated nucleic acids, as well as carrying out chemical colorimetric or fluorescent reactions to visualize the results of amplification.

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Description

Field of technology to which the invention relates

The present invention relates to the field of medicine, and more specifically to analytical medical technology, in particular to in vitro diagnostics using molecular genetic methods for detecting nucleic acids of one or more pathogens.

State of the art

A search for similar storage devices revealed several similar developments, which are cartridges designed for specific analyzers. Thus, the published international application WO 2005/060432 protects a two-component cartridge for use with an electrochemical sensor. The cartridge is designed for a specific assay and cannot be used with any reagents other than sensor reagents built into a specifically configured transport component. Applications CA 2635268 C and JP 5254040 B2, for example, describe reagent containers for processing samples in a closed system and imply their use in a specific automatic analyzer.

The proposed device can be used both in an analyzer with an automatic dispenser operating according to a customizable algorithm, and in manual mode (if necessary) for the purpose of clinical diagnostic research using an isothermal amplification reaction. Transparent cells allow you to evaluate research results using colorimetric and luminescent analysis methods.

The closest analogues from the technical field have not been identified.

Disclosure of the invention

The technical result achieved by using the proposed invention is the ability to store various reagents under sterile conditions for several weeks in quantities strictly necessary to conduct the study of one clinical sample, namely to isolate nucleic acids from a sample, carry out 15 different singleplex amplification reactions of isolated nucleic acids, as well as performing chemical colorimetric or fluorescent reactions to visualize amplification results.

The technical result is achieved by the fact that the device is made in the form of a cluster with different-sized conical cells located in concentric circles, while one of the cells contains magnetic balls, and the others contain reagents for isolating nucleic acids from cells and viruses and detecting specific fragments of genomes . The cluster is equipped with a sealing protective film; in the center of the cluster there is a hole for fixing it in its working position inside the analyzer.

The arrangement of cells on concentric circles makes it possible to store a large number of reagents in isolation from each other and conduct multi-stage multiplex studies, as well as their distribution for different stages of the study on different rows from the center: on the first and second rows there are cells with solutions that provide amplification of specific fragments, the third row contains magnetic beads and reagents necessary for isolating nucleic acids from cells, viruses or clinical samples; the fourth - outer row - contains cells with reagents for a colorimetric reaction to visualize amplification products, as well as cells with positive and negative quality controls the reaction carried out.

The conical shape of the cells allows you to leave a minimum number of samples on the walls, and the variety of cell sizes ensures the storage of the exact amount of reagents required for one reaction.

The cluster is equipped with a protective sealing film, which prevents reagents from leaking and mixing. There is also a hole in the cluster for fixing it in the working position.

Brief description of drawings

The present invention is illustrated by drawings, where in Fig. 1 schematically shows the cartridge, top view, FIG. 2 schematically shows the cartridge, side view.

Cartridge 1 contains four concentric rows 2, 3, 4, 5 with cells for reagents located on them, as well as a cell for sample 6 on the third row and a cell for magnetic beads 7 on the third row. The cluster has a hole for fixation 8, the device is equipped with a protective sealing film 9.

Carrying out the invention

The proposed device operates as follows.

The cluster is filled with the reagents necessary for research and covered with a sealing film for storage and transportation.

Before starting the analysis, it is necessary to remove the sealing film 9 from the cartridge, which covers the entire upper surface of the cluster. The sample cell 6, located on the third concentric row from the center 4, is filled with the clinical sample for analysis. In a cell with magnetic balls 7, located on the same row, the necessary reagents are stored and nucleic acids are isolated and purified. The isolated nucleic acids are then dosed into 12 wells for singleplex or duplex amplification reactions, which are located on the first 2 and second 3 concentric rows, and after completion of the reaction, the amplification mixture is transferred to the cells with reagents for the colorimetric imaging reaction, located on the fourth row 5. Concentric arrangement cells with reagents, where each row contains reagents corresponding to a given stage of the study, allows you to avoid contamination of the sample, as well as increase the number of cells for singleplex reactions as part of a multiplex study.

Given that the device stores the exact quantities of reagents required for a single reaction, the researcher does not waste time aliquoting reagents and adding them to perform 15 different amplification reactions using different primers. And the outermost cells along the periphery of the device, where imaging reactions are carried out, are easily viewed after completion of the study to record the result of the colorimetric reaction.

The device can be used both in an analyzer with an automatic dispenser operating according to a customizable algorithm, and in manual mode (if necessary) for the purpose of clinical diagnostic research using an isothermal amplification reaction. Transparent cells allow you to evaluate the results of the study using colorimetric methods.

Examples of implementation of the invention

Example 1. Using a cartridge for molecular genetic research

To detect the main genetic markers of breast cancer (BC) in a clinical blood serum sample, sets of reagents were developed and a research method was proposed, which involves sample preparation in the form of isolation of nucleic acids (stage 1), staging 6 isothermal polymerase chain reactions (stage 2) and 6 subsequent reactions of visualization of amplification products (step 3).

To carry out stage 1 of the diagnosis, a blood serum sample was taken and the test tube with the sample was placed in the cell of the central circle of the cartridge. Next, using reagents from 4 tubes with reagents (magnetic beads, water, ethanol, proteinase and sodium dodecyl sulfate solution) placed on the central circumference of the cartridge, nucleic acids were sequentially isolated from the sample.

To carry out stage 2 of the diagnosis, 6 tubes with 6 reagent markers for breast cancer (carcinoembryonic antigen (CEA), CA15.3, MCA, CA549, CA27-29, BRMA) placed on the second concentric circle of the cartridge were used.

To carry out stage 3 diagnostics, 6 tubes containing imaging sensors placed on the peripheral circumference of the cartridge were used.

Example 2: Using a cartridge to identify infectious agents

To detect the main pathogens of neuroinfections in a clinical sample of cerebrospinal fluid, sets of reagents were developed and a research method was proposed, which involves sample preparation in the form of isolation of nucleic acids (stage 1), performance of 12 singleplex amplification reactions (stage 2) and 12 subsequent reactions of visualization of amplification products (stage 3).

To carry out stage 1 diagnostics, a sample of cerebrospinal fluid was taken and a test tube with the sample was placed in the cell of the central circle of the cartridge. Next, using reagents from 4 tubes with reagents (magnetic beads, water, ethanol, alkali and sodium dodecyl sulfate solution) placed on the central circle of the cartridge, nucleic acids were sequentially isolated from the sample.

To carry out stage 2 of the diagnosis, 12 tubes with 12 reagents were used to identify pathogens of neuroinfections, placed on the second and third concentric circles of the cartridge.

To carry out stage 3 diagnostics, 12 tubes containing imaging sensors placed on the peripheral circumference of the cartridge were used.

Claims (1)

A cartridge for the sterile storage of reagents, made in the form of a cluster with four rows of different-sized conical cells located in concentric circles, while the first and second rows contain cells with solutions that provide amplification of specific fragments; the third row contains magnetic beads and reagents necessary for the isolation of nucleic acids from cells, viruses or clinical samples, the fourth contains cells with reagents for a colorimetric reaction for the purpose of visualizing amplification products, as well as cells with positive and negative quality controls for the reaction performed, while one of the cells in the third row contains magnetic beads , and in others - reagents, a hole is made in the cluster to fix it in the working position, the cluster is covered with a sealing protective film.

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