JP7432815B2 - How to dissolve dry reagents - Google Patents

Description

The present invention relates to a method for dissolving dry reagents.

BACKGROUND ART Dried reagents are often used in automatic analyzers that analyze components in biological samples by mixing and reacting the biological samples with reagents. Dry reagents need to be dissolved in a solvent, which is usually done at a constant stirring time and temperature (for example, see Patent Document 1). If the dry reagent is not sufficiently dissolved, the reactivity of the reagent may decrease, leading to a false determination, but whether or not it is sufficiently dissolved can only be confirmed visually.

WO2017/149940 pamphlet

To provide a method for preventing defective dissolution of a dry reagent by checking the progress of dissolution in real time during dissolution of the dry reagent.

In order to solve the above problems, the present inventors have made extensive studies and have arrived at the present invention.
That is, the present invention includes the steps of adding a solvent to a dry reagent placed in a container, and detecting the fluorescence intensity by aiming the excitation light and fluorescence of a fluorescence detector so that the effective diameter covers all of the dry reagent. and stirring the container until the detected fluorescence intensity reaches an arbitrary threshold value.

The present invention will be explained in detail below.

A dry reagent is a reagent obtained by removing moisture from a liquid reagent by freeze-drying, natural drying, vacuum drying, or the like. Examples of nucleic acid amplification reagents include, but are not limited to, dry reagents containing an enzyme, a substrate, a primer, and a fluorescent label that specifically reacts with the amplification product. Regarding the shape, there is no problem whether it is granular, powder, or thin film, but thin film is particularly preferred.

There are no particular restrictions on the material, size, or shape of the container in which the dry reagent is placed, as long as it does not affect the dissolution reaction of the reagent or the amplification of the nucleic acid of the sample, but it should be made of plastic that is easily disposable. containers are preferred.

Purified water to which a measurement sample has been added is used as a solvent to dissolve the dry reagent. Depending on the reaction between the dry reagent and the measurement sample, an acid, salt, or surfactant may be further added to the solvent as appropriate.

After adding a solvent to the dry reagent placed in the container, the excitation light and fluorescence effective diameter of the fluorescence detector are aimed so as to cover all of the dry reagent, and detection of fluorescence intensity is started. Usually, the dry reagent has a higher specific gravity than the solvent, so if the container is large enough, the dry reagent will sink to the bottom of the container.

There is no problem with the fluorescence detector used if it is a general device equipped with an excitation light irradiation means for irradiating the sample with excitation light and a fluorescence detection means for detecting the fluorescence emitted from the sample. Fluorescence detectors are particularly suitable. Note that the effective apertures of excitation light and fluorescence refer to the effective apertures (see JIS B 7095:1997) of lenses provided in the excitation light irradiation means and the fluorescence detection means, respectively. In other words, a circle with an area equal to the cross-sectional area perpendicular to the optical axis of a bundle of parallel rays that exits from an object point at infinity on the optical axis of the lens and passes through a lens with an aperture corresponding to a given aperture scale. refers to the diameter of

The position of the fluorescence detector is not particularly limited as long as the effective diameter of the excitation light and fluorescence can cover all of the dried reagents, and an embodiment in which the incubator for nucleic acid amplification and the fluorescence detector are integrated is also possible. It is.

When detecting the fluorescence intensity by aiming the fluorescence detector so that the effective diameter of the excitation light and fluorescence covers all of the dry reagents, the fluorescence intensity is highest when dissolution has not yet begun (only the dry reagents), and the fluorescence intensity is highest when the dry reagents When is evenly dissolved in the solvent, the fluorescence intensity will be the lowest. That is, as the dry reagent dissolves, the fluorescence intensity gradually decreases.

Using the principles described above, an appropriate fluorescence intensity is set as a threshold. If the fluorescence intensity reaches an arbitrary threshold value, it can be determined that the dry reagent is evenly dissolved in the solvent, and therefore, stirring for promoting dissolution can be stopped at that point. Examples of the stirring method include a method using a stirring blade, a method of vibrating a container horizontally or vertically, a method of sucking and discharging using a dispensing mechanism, but there is no particular limitation.

This prevents poor dissolution of dry reagents in the container, eliminates unnecessary stirring, and contributes to overall time reduction.

FIG. 2 is a schematic diagram showing a method for dissolving a dry reagent of the present invention. 3 is a diagram showing the results of Example 1. FIG.

Specific embodiments will be described below, but it will be appreciated by those skilled in the art that the present invention is not limited to these examples, and that various modifications can be made within the scope of the invention described in the claims. Easy to understand.

Example 1
Using the fluorescence detector and stirring mechanism of the present invention shown in FIG. 1, it was verified whether the progress of dissolution of a dry reagent could be measured in real time using fluorescence. A colorless and transparent polypropylene container was used as the sample container 10, a nucleic acid probe modified with a fluorescent dye was used as the dry reagent 11, and a solvent mainly composed of purified water was used as the solvent 12. Three sets of excitation light irradiation section 14 and fluorescence detection section 15 were used: excitation light of 450 nm and fluorescence of 495 nm, excitation light of 500 nm and fluorescence of 545 nm, and excitation light of 590 nm and fluorescence of 645 nm. The three excitation light irradiation sections 14 were turned on in order (2.5 sec x 3, 2.5 sec off), and the corresponding fluorescence intensity of the fluorescence detection section 15 was measured. The solvent 12 was discharged into the sample container 10 by the dispensing mechanism 13, and then stirred by suction and discharge.

The results are shown in Figure 2. Before discharging the solvent 12, all the dry reagents were covered within the effective diameter of the fluorescence detector, so all three types of fluorescence detectors received high fluorescence intensity. The fluorescence intensity gradually decreased for 40 seconds after discharging the solvent 12. It can be seen that the dry reagent is dissolved in the solvent outside the effective diameter over time. After 40 seconds, the fluorescence intensity became constant. It can be seen that all of the dry reagents were dissolved in the solvent.

10: Sample container 11: Dry reagent 12: Solvent 13: Dispensing mechanism (stirring mechanism)
14: Excitation light irradiation section 14a: Excitation light source 14b: Optical filter (excitation light side)
14c: Excitation light effective diameter 15: Fluorescence detection section 15a: Light receiving element 15b: Optical filter (fluorescence side)
15c: Fluorescence effective diameter

Claims (3)

adding a solvent to the dry reagent placed in the container;
Aiming the excitation light of the fluorescence detector so as to cover the lower part of the container and starting to detect the fluorescence intensity;
stirring the container until the detected fluorescence intensity is below a predetermined threshold;
A method for dissolving a dry reagent comprising : 2. The method according to claim 1, wherein the dry reagent is in the form of a thin film. 3. The method according to claim 1, wherein the dry reagent is a nucleic acid amplification reagent.