JP2003506708A - Device capable of direct suction reaction and injection, and method of use - Google Patents

Abstract

SUMMARY OF THE INVENTION The present invention provides a chamber (2) having an open first end (3) and a closed second end (4); an open first end (9) and an open second end (10). And a sealing means for providing a seal between the elongate member and the chamber. The second end (10) of the elongate member is slidably movable from a first position in the chamber to a second position in the chamber such that the movement causes a pressure change in the chamber. Has become. The device according to the invention allows for sample collection and analysis in a single chamber. Devices and methods are illustrated where a wax and a reactive compound are contained within a chamber.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a device suitable for aspiration and analysis of biological samples and suitable for sample injection into biological material. The invention also relates to a method for detecting a target molecule in a sample.

[0002]

[Prior Art and Problems to be Solved by the Invention]

In medical diagnostic analysis, a biological sample is typically collected and then analyzed according to analytical procedures. Often, sample collection and subsequent analysis is performed at various locations in the laboratory or outside the laboratory. Therefore, it is necessary to carry a biological sample or an analytical reagent. Handling of such samples increases the risk of contamination with other biological materials.

Contamination risk refers to diffusion amplification reactions, such as the polymerase chain reaction (PCR), where the process can amplify a single-stranded nucleic acid (target nucleic acid) into millions of copies (amplification products). If it does, it becomes particularly important. Although the PCR technique has considerable potential utility in medical diagnostic laboratories, nucleic acid amplification reactions can easily contaminate unrelated nucleic acid molecules. For example, contamination can occur during sampling. In addition, the sample can be contaminated by amplification products (amplification products) of previous amplification reactions when the reaction chamber is opened, when standard pipetting techniques are performed, or when aerosols are formed. Such contamination can be amplified by the PCR reaction, which can detect false positive reactions of the substance and ultimately give a false diagnosis.

Because of the above problems, many reactions, especially sensitive PCR analyses, are performed over time and require a fairly skilled operator to ensure reliable results. To do.

Therefore, there has been a great need for products and procedures that can simplify sample collection and analysis and reduce the risk of contamination.

[0006]

[Means for Solving the Problems]

The present inventors have developed a device that can reduce the problems associated with performing multiple steps in a medical diagnostic analysis, thereby providing more reliable and accurate results. In particular, the device according to the invention allows sample collection and analysis in a single chamber, reducing the risk of contamination.

In a first aspect, the invention comprises a chamber having an open first end, a closed second end, an inner surface and an outer surface; an open first end, an open second end, an inner surface and an outer surface. An elongate member having; and a sealing means providing a seal between the elongate member and the chamber; a second end of the elongate member from a first position within the chamber to within the chamber. It is slidably movable between the second position and the first position to the second position.
Provided is a device adapted to cause a pressure change in the chamber by movement of the second end of the elongate member.

In a preferred embodiment of this first aspect, the chamber is elongated.

In a further preferred embodiment of the invention, the chamber is transparent to electromagnetic radiation. A lens is provided at the second end of the chamber for transmitting electromagnetic radiation. Preferably, the electromagnetic radiation is light such that the wavelength is 400-800 nm. More preferably, the electromagnetic radiation is fluorescent.

In a further preferred embodiment, the chamber is cylindrical. Preferably, the outer diameter of the chamber is at most 2.8 mm and / or the inner diameter is at most 2.5 mm. Preferably, the chamber length is in the range of 20-40 mm.

Preferably, the elongate member has an outer diameter of 2.5 mm at the maximum and an inner diameter of 0.2 mm.
The range is up to 1.2 mm.

In a further preferred embodiment, the first end of the elongate member has a shaping tip. This tip is capable of piercing biological tissue. In a preferred embodiment, the tip is on the order of 1 μm. However, the tip is 200 μm
The diameter can be up to, or even more.
Preferably, the tip diameter is 20 μm to 40 μm.

In yet another embodiment, the chamber and elongate member are formed from the same material. Preferably, this material is glass or plastic. However, in the preferred embodiment, the chamber is made of plastic and the elongate member is made of glass. Suitable plastics include, but are not limited to, polyethylene terephthalate, polypropylene, and polycarbonate.

In a preferred embodiment, the sealing means comprises a sleeve located on the outer surface of the elongate member near the second end of the elongate member. Preferably, the sleeve and the inner surface of the chamber form a seal. The sleeve can be of any suitable length. For example, the length of the sleeve can be 10-80 mm. However, it will be appreciated that longer sleeves are preferred as they may provide additional rigidity to the elongate member. The sleeve can abut the chamber inner surface over the entire length of the sleeve. However, it is preferred that the sleeve has a flared profile with a length of 0.2-2.0 mm at which it abuts the chamber inner surface to form a seal.

In a preferred embodiment, the chamber further has a collar near the open first end of the chamber. The collar has an inner surface and an outer surface.

The device may further comprise a first guide within the collar. The first guide preferably has an annular outer surface that engages the inner surface of the collar. As a result, a seal is formed between the first guide and the inner surface of the collar. Preferably, the first guide further has an opening for receiving the elongate member. The elongate member is received by the first guide through the opening such that a seal is formed between the elongate member and the first guide.

Preferably, the device further comprises a first member on the outer surface of the first end of the elongate member.
A second guide arranged near the edge is provided. By using this second guide, the long member can be held at a predetermined position during suction.

It is preferred to control the volume of sample used in the reaction as many biological reactions produce quantitative results. Therefore, in a further preferred embodiment, the device is used with a means for controlling the amount of sample aspirated. The means for controlling the sample volume can be, for example, a manual or motorized pipettor. It will be appreciated that currently available electric pipettors can be used with the device according to the invention. An example of such an electric pipettor is manufactured by World Precision Instruments,
There are UltraMicroPump-II (registered trademark) and Nanoliter 2000 (registered trademark).

In yet another preferred embodiment, one or both of the inner surface of the chamber and the inner surface of the elongated member are treated with an anti-reaction agent so that the sample does not adhere to the chamber or the elongated member. Has been done. The reaction inhibitor can be selected from the group consisting of bovine serum albumin, fetal calf serum, human serum albumin, polyvinyl alcohol, polyvinylpyrrolidone, and other suitable protein sources. Most preferably, the solution is a silicone compound sold under the trade name "Sigmacote" (Sigma).

In another preferred embodiment, the elongate member is filled with oil to facilitate sample uptake.

The present invention also provides a device capable of sequentially performing a series of discrete reactions in a chamber.

Accordingly, in a second aspect, the invention provides a device according to the first aspect, wherein the chamber comprises a first reaction compound; a first wax layer providing a seal on the first reaction compound; A second reaction compound disposed adjacent to the wax layer and separated from the first reaction compound via the first wax layer; and a second wax layer substantially covering the second reaction compound; And the second wax layer has a melting point lower than that of the first wax layer, and
Provided is a device in which the first wax layer is solid at the melting point of the second wax layer.

In a preferred embodiment of the second aspect, at least one of the first reaction compound and the second reaction compound, preferably both, is in liquid form at room temperature. However, the reaction compounds can also be solid at room temperature.
For example, the reaction compound can be lyophilic (emulsive).

[0024] In a third aspect, the invention provides a device according to the first aspect, wherein the chamber comprises a first reaction compound mixed with a first wax carrier; and a second reaction compound mixed with a second wax carrier. The second reaction compound is at least partially coated or covered or layered on the first reaction compound, and the second wax carrier is the first wax carrier. Has a melting point lower than that of the second
A device is provided in which the first wax carrier is a solid at the melting point of the wax carrier.

In a fourth aspect, the invention is a method for detecting a substance in a sample, comprising: (i) introducing a sample and a reactive compound into the chamber of the device according to the first aspect, A reactive mixture is formed from the sample and the reactive compound, in which case the reactive compound has at least one reactive agent capable of interacting with the substance to be detected, whereby it is released by the reactive mixture. The level of electromagnetic radiation,
Be different as compared to the level of electromagnetic radiation emitted by the reactive compound only; (ii) additionally exciting the reactive mixture by electromagnetic radiation; (iii) the chamber in which the reaction may take place. And (iv) detecting electromagnetic radiation emitted from the reactive mixture.

In a fifth aspect, the invention provides a method for amplifying at least one target nucleic acid molecule in a sample, comprising: (i) placing the sample and the reactive compound in a chamber of the device according to the first aspect. Introducing to form a reactive mixture from the sample and the reaction compound, where the reaction compound has a reagent for amplification of the target sequence; (ii) the chamber is used for amplification of the target sequence. Put it in a possible state;
Provide such a method.

[0027] In a sixth aspect, the invention provides a method for amplifying at least one target nucleic acid molecule in a sample, comprising: (i) placing the sample and the reactive compound in a chamber of the device according to the first aspect. Upon introduction, a reactive mixture is formed from the sample and said reactive compound, in which case the reactive compound reacts with the reactant for amplification of the target sequence and the level of electromagnetic radiation emitted by the reactive mixture. And at least one reagent capable of interacting with the amplified sequence so as to be different as compared to the level of electromagnetic radiation emitted by the compound alone; (ii) additionally Exciting the reactive mixture with electromagnetic radiation; (iii) bringing the chamber into a state where amplification of the target sequence can occur; (iv) reactivity Providing such a method; detecting electromagnetic radiation emitted from the compound.

In a seventh aspect, the invention is a method for performing a series of reactions in a single container, comprising: (i) introducing a sample into the chamber of the device according to the second aspect, Is located adjacent to the second wax layer; (ii) heating the chamber to a temperature at which the second wax layer is melted and the first wax layer remains solid; (iii) the sample Forming a reactive mixture by interacting with a second reactive compound; (iv) heating the chamber to a temperature that melts the first wax layer; (v) reacting the reactive mixture with the first reactive compound. Interact with each other;

In an eighth aspect, the invention is a method for performing a series of reactions in a single container, comprising: (i) introducing a sample into the chamber of the device according to the third aspect, Is positioned adjacent to the second reactive compound mixed with the second wax carrier; (ii) the chamber is brought to a temperature at which the second wax carrier is melted and the first wax carrier remains solid; Heating; (iii) forming a reactive mixture by interacting the sample with a second reactive compound; (iv) heating the chamber to a temperature at which the first wax carrier is melted; (v) reacting Interacting the sex mixture with the first reaction compound;

In a preferred embodiment of the fourth to sixth aspects, the sample is introduced into the chamber through the elongated member.

In a preferred embodiment of the fourth and sixth aspects, the electromagnetic radiation is
It is transmitted from an external radiation source through the chamber to the reactive mixture. Furthermore, the degree of difference in electromagnetic radiation from the reactive mixture is detected through the chamber.

In a preferred embodiment in the sixth aspect, the reactive compound comprises a reactive agent that allows fluorescence-based detection of the nucleic acid molecule product amplified by oligonucleotide hybridization.

In a preferred embodiment of the second and third aspects, the melting point of the first wax layer is about 70 ° C. Any wax having a melting point of about 70 ° C. can be used as appropriate. Preferably, this wax is a T wax (
EnerGene® or F wax (available from Schumann Sasol)
20% for paraflint G80N6 and 80% for T Wax).

In another preferred embodiment of the second and third aspects, the melting point of the second wax layer is about 55 ° C. All waxes having a melting point of about 55 ° C. can be used appropriately. Preferably, this wax is AmpliWax
(Registered trademark) (PE Applied Biosystems).

In yet another preferred embodiment of the second and third aspects, the oil layer covers the second wax layer. The presence of the oil layer assists in the transfer of the sample from the elongate member to the chamber, which maintains the sample as a mass adjacent to the second wax layer.
It can also provide multiple functions such as reducing pollution.

In still another preferred embodiment of the second and third aspects, the first reaction compound has a reaction agent necessary for amplification of the target nucleic acid molecule. More preferably, the first reaction compound has a reactive agent that enables fluorescence-based detection of nucleic acid molecule products amplified by oligonucleotide hybridization.

In yet another preferred embodiment of the second and third aspects, the second reactive compound is a cell degrading, protein digesting and / or altering, and / or DNA digesting and / or altering. It has a reactive agent that causes it.
Preferably, the second reaction compound has an alkaline buffer for enhancing cell degradation. More preferably, the second reaction compound has proteolytic enzyme K, or proteolytic enzyme in a buffer compatible with proteolytic enzyme K, or proteolytic activity.

In another preferred embodiment of the seventh and eighth aspects, in step (iii) and / or step (v), centrifugation of the elongated chamber is performed. Preferably, the centrifugal force is 5 to 200 g, more preferably 5 to 70 g.
It is said that However, as those skilled in the art will appreciate, the required centrifugal force is
It varies depending on various parameters such as chamber temperature, wax melting point and reactant viscosity.

It will be appreciated by those skilled in the art that the chambers in the device according to the second and third aspects may contain additional reactants so that they can cause three or more different reactions in the same chamber. Will

In a preferred embodiment of the present invention, the sample comprises biological material. Preferably, the biological material is cells or virus particles.

In another preferred embodiment of the invention, the sample is infected with erythrocytes, leukocytes, germ cells, bacteria, sperm, pollen, cell cultures, Papanicolaou smears, single cells, blood cell parasites, viruses. It is selected from the group consisting of single cells and fluids containing DNA or RNA or proteins.

The device according to the first aspect of the invention can be used for injection of a sample into biological tissue, or for transferring a sample from one sample source to another. It will be appreciated that it can be used.

Thus, in a ninth aspect, the invention provides a method of using a device according to the first aspect, such as using the device for injecting a fluid sample into a cell. Preferably, the fluid sample comprises genetic material such as DNA or sperm.

In a preferred embodiment of the ninth aspect, the device is adapted for use with a micromanipulator so that the sample introduction into the cell can be controlled.

As used herein, the terms “comprising” or “having” are intended to mean including the referenced members, numbers or steps without excluding other members, numbers or steps. I want you to understand.

[0046]

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described with reference to the accompanying drawings and experimental examples that do not limit the present invention.

With reference to FIGS. 1 to 4, a suction device (1) constructed according to a preferred embodiment of the present invention will be described. The device (1) comprises a chamber (2) having an open first end (3), a closed second end (4), an inner surface (5) and an outer surface (6). This chamber is cylindrical and has an outer diameter of 2.8 mm at maximum, an inner diameter of 2.5 mm at maximum, and a length of 20 to 40 mm. Preferably, the chamber (2) can hold a sample volume of up to 20 μl.

The chamber (2) is preferably made of a chemically inert, biocompatible and stable material. The term chemically inert means that the material is stable with respect to deformation, discoloration, cracking, separation, etc. when exposed to heat, autoclave treatment, extraction chemicals, diluents and other chemical solutions. is doing.
The term biocompatible means that the material does not bind to the biological material from solution and that it affects the stability, functionality or conformation of the biological material when in contact with it. It means that there is no application and that the components leaching out of the material placed in the biological solution do not contaminate the biological solution at all. The term stable means that the material retains all the above properties at room temperature for several years.

Preferred polymers include polypropylene, polyethylene terephthalate,
There is polycarbonate. The entire device can be molded from these polymers. Various grades of polypropylene are commercially available. Himont PD701
Neutral® (Hilmont USA, Wilmington, Delaware, USA)
Resins such as those manufactured by K.K.) are preferable in that they exhibit sufficient inertness and can be autoclaved. Examples of suitable polycarbonates include
Makrolon® (Bayer), calibre® (Dow Chemicals), lexan® (Germany), acrifix® (Rohm Chemical)
Fabrik). The entire device can be injection molded with high injection pressure. Alternatively, the elongate member can be formed from other materials such as glass.

The material is preferably transparent to electromagnetic waves. Alternatively or additionally, the closed second end (4) of the chamber has a lens (7) for transmitting electromagnetic radiation.
) (FIG. 2).

The device further comprises an open first end (9), an open second end (10) and an inner surface (11).
) And an outer surface (12). The elongate member is slidably movable in the elongate chamber (2) from a first position (13) to a second position (14). The first end (9) is the needle tip (15)
have. Preferably, the needle tips have a diameter in the range 1 μm to 200 μm. More preferably, the needle tips have a diameter in the range 1 μm to 50 μm. Preferably, the needle tip is capable of piercing living tissue, such as embryonic tissue. Therefore, it is preferable that at least the needle-shaped tip portion of the elongated member is made of glass. In one preferred embodiment, the elongate member (8) is entirely made of glass.

The sealing means (16) provides a seal between the outer surface (12) of the elongate member and the inner surface (5) of the chamber (2). The sealing means comprises a sleeve (17) on the outer surface (12) of the elongate member (8) near the second end (10).
Teflon® is preferably used as the material for the sleeve (17). However, other materials such as rubber, plastic or glass can also be used. The sleeve (17) is attached to the inner surface (5) of the chamber (2).
), Which results in a seal between the sleeve (17) and the inner surface (5) of the chamber. The sleeve can abut the chamber inner surface over the entire length of the sleeve or over a substantial portion of the length of the sleeve. In FIG. 5, the sleeve (29) has a flared shape (3
0), the flared shape (30) of which is the inner surface () of the chamber (32).
31) shows an alternative embodiment in which the seal is provided by abutting against 31).

In use, the fluid sample is passed through the elongated member (8) into the chamber (2) by moving the elongated member (8) from the first position (13) to the second position (14). Can be introduced. The movement of the elongate member (8) can be obtained by sliding the chamber (2) in a direction away from the first end (9) of the elongate member (8). Such movement of the chamber (2) with respect to the elongated member (8)
It will be appreciated that it causes a pressure decrease in the chamber (2), which sucks the sample through the elongated member (8) and into the chamber (2).

The chamber further comprises a flared collar (18) located on the chamber (2) near the open first end (3). The enlarged collar (18) has an inner surface (19) and an outer surface (20). The chamber (2) and the long member (8)
When moving in a direction away from or towards the first end (9) of the, the user can grip the outer surface (20).

The device further comprises a first guide (21) located within the expanding collar (18).
Is equipped with. The first guide (21) has an annular outer surface (22) which engages the inner surface of the flared collar (18). This forms a seal between the annular outer surface (22) and the inner surface (19). The first guide further has an opening (23) for receiving the elongate member (8). The elongated member (8) is received by the first guide (21) through the opening (23). Thereby, the long member (
8) is coaxially aligned with the chamber (2). A seal is formed by abutting the outer surface (12) of the elongated member (8) and the surface defining the opening (23). The first guide is preferably formed of silicone.

The device further comprises a second guide (24) located on the outer surface (12) of the first end (9) of the elongate member (8) and towards the first end (9). . This second
By using the guide (24), when the chamber (2) is slid in the direction toward and away from the first end (9) of the elongated member (8), the elongated member (8) is predetermined. Can be held in position.

[0057]   Hereinafter, a method of using the preferred device (1) according to the present invention will be described.

By using the device of the present invention, a liquid sample can be injected into cells. Sliding the chamber (2) towards the first end (9) of the elongate member (8) increases the pressure within the chamber (2), thereby causing the elongate member (8).
It will be appreciated that the fluid in the chamber (2) is evacuated through. Similarly, by using the device of the present invention, external genetic material such as DNA or semen can be injected into cells.

However, by using the device of the present invention, the sample can be stored in the chamber (2
A) and analyze the sample. This analysis can be performed by performing the desired reaction on the sample in the chamber. Within the scope of this embodiment, it is possible to introduce into the chamber the unit amount of reaction compound necessary to carry out the reaction. The term "unit dose" means that the reactive compound contains all or nearly all of the reactants needed to cause a reaction on the sample to be analyzed. Preferably, the user need only add the sample to initiate the reaction. The reactive compound can be introduced into the chamber (2) in advance before assembling the device. Instead of this,
The reaction compound can also be sucked into the chamber (2) through the elongated member (8). Preferably, the sample is individually introduced into the chamber (2) through the elongated member while the reaction compound is already introduced into the chamber. Then, the reaction compound and the sample react in the chamber (2) to form a reactive mixture.

One or both of the inner surface (5) of the chamber (2) and the inner surface (11) of the elongate member (8) react to facilitate introduction of the sample into the chamber (2). It can be treated with an inhibitor. The term "reaction inhibitor" refers to long members (8)
Agents that reduce or prevent the binding of biological materials in solution to the inner surface (11) of the chamber or to the inner surface (5) of the elongated chamber (2) Means By contacting the inner surface (11) of the elongate member (8) or the inner surface (5) of the chamber (2) with a reaction inhibitor, all the binding sites on each inner surface are absent before the sample is aspirated. It can be active. Alternatively or additionally, the reaction inhibitor can be added directly to the sample prior to aspiration. Due to the presence of the reaction inhibitor in the sample, the sample can be easily transported along the long member during suction. The reaction inhibitor may be a surfactant. Non-limiting examples of suitable reaction inhibitors include bovine serum albumin, fetal calf serum, human serum albumin, polyvinyl alcohol, polyvinylpyrrolidone and other suitable protein sources. Commercial products that can be suitably used as anti-reaction agents within the scope of the present invention will be known to the person skilled in the art. Suitable examples include "Sigmacote" (registered trademark) (Sigma) and "Vigro Retrieval" (
Registered trademark).

The elongated member (8) may be filled with oil in order to facilitate passage of the sample through the elongated member into the chamber.

Although various reactions can be carried out by using the device (1) according to the invention, in a preferred embodiment the device (1) collects a biological sample and targets within this sample. Used to analyze this sample for the presence of molecules. Preferably, the nature of the reactive compound is such that the presence of the target molecule within the biological material can be detected by a change in the emission level of electromagnetic radiation from the reactive mixture.

In a further preferred embodiment, the target molecule is a nucleic acid sequence. Preferably, the reaction compound has an agent capable of amplifying the target nucleic acid molecule. Preferably, the reactive compound has at least one agent capable of causing a change in the level of electromagnetic radiation from the reactive mixture by reacting with the amplified sequence.

An “amplification” reaction is one in which the enzymatic replication process is typically repeated for a number of cycles to produce multiple copies of the original nucleic acid sequence. The amplification process can be said to be exponential in terms of cycle number if additional copies can be formed from each of the duplicate copies formed in the previous cycle. Although exponential amplification is desirable in terms of improving analytical sensitivity, such enhanced sensitivity is
It is a disadvantage if the amplification product is not carefully packaged and causes contamination.

Techniques for amplifying nucleic acid molecules are well known to those of skill in the art. Examples of such techniques include US Pat. No. 4,683,202 and US Pat. No. 4,683,19.
Polymerase chain reaction (PCR) as disclosed in No. 5 (Mullis)
Ligase chain reaction (LCR) as disclosed in European Patent Publication No. 320 308 (Backman et al.); And International Patent Application No. 90
/ 01069 (Segev), European Patent Application Publication No. 439 182 (
Gap-filled LCRs, as disclosed in British Patent Application Publication No. 2,225,112 (Newton et al.) And International Patent Application No. 90/00447 (Birkenmeyer et al.). There are (GLCR) method and its modification method. Other amplification techniques include Q-beta replicase as disclosed in the literature; European Patent Application Publication No. 497242 (Walker), European Patent Application Publication No. 500224 (Walker et al.). , And Walker et al. Proc.
Strand Displacement Amplification (SDA) as disclosed in Nat. Acad. Sci. USA, 89: 392 (1992); Fahy et al. PCR Methods and Application 1:25 (1991).
Self-sustaining sequence replication (3SR) as disclosed in U.S.A .; and nucleic acid sequence based amplification (NASBA) as disclosed in the literature;
There is.

Some amplification reactions, eg PCR and LCR, are “thermocycled”.
Known as, has alternating cycles of hot and cold set temperatures. P
A CR or "polymerase chain reaction" is an amplification reaction in which a normally thermostable polymerase enzyme extends multiple primers using the original nucleic acid as a template to produce multiple copies of the original sequence. . PCR is disclosed in more detail in US Pat. No. 4,683,202 and US Pat. No. 4,683,195. LCR or "ligase chain reaction" means that usually a thermostable ligase enzyme is used to hybridize two or more oligonucleotide probes to a target.
A nucleic acid amplification reaction in which one or more oligonucleotide probes are ligated to produce multiple copies of the original sequence. LCRs, and their variants, gap LCRs, are described in EP-A 320 30
No. 8 (Backman et al.), European Patent Application Publication No. 439 182 (Backman).
Et al.), International Patent Application Specification No. 90/00447 (Birkenmeyer et al.), And the like.

In addition, amplification techniques for performing fluorescence-based detection and analysis are known to those of skill in the art. Examples of such techniques include molecular beacon analysis (Tyagi and
Kramer, 1996) and TaqMan® fluorescence energy transition analysis (Livak
Et al., 1995) and Foresta resonance energy transitions (Deniz AA et al.
Proc. Natl. Acad. Sci. USA 96: 3670 (1999)).

After introducing the reaction compound and the sample into the chamber (2), the elongated member (8) can be sealed. Preferably, the elongate member (8) can be sealed by cutting the elongate member (8) near the chamber (2) and then heat sealing. For example, the elongated member (8) can be cut and sealed at the same time by applying heat. Alternatively, the elongate member (8) can be removed from the chamber (2) and the open end (3) of the chamber (2) can be heat sealed or by applying a cap. it can.

The sealed chamber can then be placed in the thermal cycling means. As a result, the target nucleic acid molecule in the sample is amplified. At the same time, the emitted electromagnetic waves can be detected through the chamber wall or through the lens at the base of the chamber.

The device and method according to the present invention can be used, for example, for Taqman (Perkin-Elmer), molecular beacon analysis, LightCycler® fluorescent hybridized probe analysis (
It is suitable for use in various direct reaction detection techniques / chemistries such as Roche Molecular Systems).

A preferred system for real-time DNA amplification detection is the LightCycler® fluorescent hybridized probe assay. In this system, 3
Uses one of the basic building blocks. That is, two different oligonucleotides (
Labeled) and the amplification product. The first oligonucleotide has a fluorescein label at the third end and the second oligonucleotide has another label, LC Red 640 or LC Red 705, at the fifth end. There is. The sequence consisting of the two oligonucleotides is the amplified DN
These oligonucleotides are selected to hybridize to the A fragment in a head-to-tail configuration. When both oligonucleotides hybridize with this orientation, the two fluorescent dyes will be located close to each other. The first dye (fluorescein) is the LED of LightCycler
It is excited by a light source having a (light emitting diode) provided with a filter, and emits green fluorescence where it is slightly shifted to the long wavelength side. When the two dyes are located in close proximity to each other, the emission energy excites a second dye, LC Red 640 or LC Red 705, attached to the second hybridization probe, which causes the second dye to , Emits red fluorescence on the longer wavelength side. FRET
This energy transition, referred to as (Foresta resonance energy transition or fluorescence resonance energy transition), largely depends on the distance between two dye molecules. Energy is transferred with high efficiency only when both dye molecules are close to each other (distance of 1 to 5 nucleotides). LC Red 640 by selecting the appropriate detection channel
Alternatively, the intensity of light emitted from the second dye, LC Red 705, is filtered and measured by the optics in the thermal cycling means. The amount of fluorescence measured increases in proportion to the increase in the amount of DNA produced during the PCR process. A second dye, LC Red 640 and LC Red 705, emits light only when both oligonucleotides are hybridized, so that fluorescence measurements are taken after the annealing step. It is advantageous to use hybridized probes when examining samples with very low amounts of template molecule.
Not only is DNA quantification using a hybridized probe highly sensitive,
It is highly selective. This method can be compared to agarose gel electrophoresis combined with Southern blot analysis. However, there is no need to perform the constant consuming step, which was required in conventional analytical methods.

In the'TaqMan 'fluorescence energy transition analysis, a nucleic acid probe complementary to the internal segment of the target DNA is used. This probe is labeled with two fluorescent halves. In this case, the properties of the two fluorescent halves have been chosen such that the emission spectrum of one overlaps with the excitation spectrum of the other. As a result, the emission of the first fluorescent half is largely quenched by the second emitting half. This probe is present during PCR and when a PCR product is formed, the probe becomes subject to degradation by the fifth nuclease activity of Taq polymerase, which is unique for DNA hybridized to the template. The diffusive degradation of the probe causes the two fluorescent halves to separate in solution, which reduces the amount of quenching and increases the emission intensity.

The probes used as molecular beacons are based on the principle of single-stranded nucleic acid molecules with stem and loop structures. The loop portion of the molecule is a probe sequence that is complementary to a given sequence in the target nucleic acid. The stem is formed by annealing two complementary arm sequences located on either side of the probe sequence. The arm sequence is independent of the target sequence. The fluorescent half is attached to the end of one arm and the non-fluorescent quencher half is attached to the end of the other arm. The stem keeps these two halves in close proximity to each other. Thereby, the fluorescence from the fluorescent half is quenched by the fluorescence resonance energy transition.
The properties of the paired fluorescent and quenching halves that can be suitably used are such that the energy received by the fluorescent half is transferred to the quenching half and is not emitted as light in the quenching half. , Is supposed to be consumed as heat. As a result, the fluorescent half cannot emit light. When the probe encounters the target molecule, it forms a new hybrid of probe and target that is more stable than the hybrid formed by the arm sequence. Due to the relative rigidity of the nucleic acid double helix, the formation of the probe-target hybrid excludes the simultaneous presence of the hybrid formed by the arm sequence. Thus, the probe undergoes a spontaneous structural change that causes the arm sequences to be spaced apart from each other, thereby spacing the fluorescent half and quenching half from each other. In this case, the fluorescent half is no longer in close proximity to the quenching half so that the probe emits light when excited by a suitable light source. Such probes are called "molecular beacons" by emitting a fluorescent signal only when hybridized to a target molecule.

A variety of real-time fluorescence detection thermocycling means are currently available, with chemistries compatible with the above chemistries such that the final product emits fluorescence. Such thermal cycling means include Biosystems 77 by Perkin Elmer.
00 (registered trademark), Rotogene (registered trademark) by Cobett Research,
There is a Light Cycler® by Hoffman La Roche. In order to carry out the method according to the invention, it is envisaged to incorporate the device according to the invention in any of the above thermal cycling means.

In a preferred embodiment, the device according to the invention can be used to carry out a series of sequential reactions in a single chamber. In this embodiment, the elongated chamber (2) is pre-introduced with two or more reactive compounds, separated from each other by a wax layer.

FIG. 1 shows a device suitable for carrying out multiple reactions in sequence. The first reaction compound (25) is covered by the first wax layer (26). Second
The reactive compound (27) is introduced on the first wax layer (26) and is covered by the second wax layer (28). The first wax layer (26) has a higher melting point than the second wax layer (28). Oil (29) can be placed on the second wax layer (28).

As shown in FIG. 1, the chamber (2) into which a plurality of reaction compounds have been previously introduced is
It will be appreciated that the device (1) according to the invention can be stored before use. On storage, the open end of the chamber (3) can be sealed by a cap. Both wax layers, which have solidified in the chamber, are preferably left attached to the inner surface of the chamber during transport and handling.

As used herein, the term “wax” is a waxy organic material that is a much harder solid than grease at temperatures below about 40 ° C. and melts at slightly elevated temperatures. This means a substance that forms a liquid and has a lower density than water. Waxes can be composed of hydrocarbons, alcohols, fatty acids and esters. Waxes can be formed from plants, animals and minerals. Alternatively, it can be a synthetic material. Examples of inorganic waxes include petroleum wax and montan wax. Examples of synthetic waxes are Fischer-Tropsch waxes and polyolefin waxes. Preferably, the wax basically contains an ester of a fatty acid and a higher alcohol, an ester of a free fatty acid and an alcohol, and a saturated hydrocarbon. Typical pure compounds which are useful as waxes are eicosane, octacosane, cetyl palmitate and pentaerythritol tetrabehenate. Wax-like substances include, for example, polyester gels. Non-silicone thixotropic gel-like substances can be used as wax-like substances. For example, Amoco in Chicago, Illinois, USA
Polybutene H-100, a hydrocarbon gel-like material commercially available from Chemicals Corporation, can be suitably used. This polymer, which is listed as 12-H in the company's catalog, is a (85-98%) butylene polymer compound composed primarily of high molecular weight monoolefins. The balance can be isoparaffin when mixed with AEROSIL OX50®. It can also be a fused silica powder commercially available from the dye department of Degussa, Inc., New York, NY, USA. Other examples of effective hydrocarbon gel-like materials are ARCO Chemical, New York, NY, USA.
Poly bd® R-45HT available from Company. This polymer is listed in the company's catalog dated April 1976 as a hydroxyl group terminated butadiene homopolymer with a degree of polymerization of 50. Another example of a suitable gel-like material is a mixture of silicon fluid and ultrafine hydrophobic silicon dioxide powder.

Typical effective wax mixtures include paraffin and Paraplast (Sherwood
Medical), Ultraflex (registered trademark by Petrolite Corporation), and BeSquare 175 (registered trademark by Petrolite Corporation). Particularly useful waxes for the present invention include T wax (registered trademark) (Energens, Regensburg, Germany) and AmpliWax (registered trademark) (PE Applie).
d Biosystems) and polyester wax (Electron Microscopy Sciences
Company) Waxes are prepared by mixing pure waxes or mixed waxes with each other, or by mixing pure waxes or mixed waxes with grease or oil in any proportion while preserving the relative hardness and adhesive properties of the waxes. be able to. Preferably, the wax used in the present invention is
Before use, by using any known method effective for sterilization purposes,
Be sterilized.

The amount of wax required in the present invention is an amount that can sufficiently cover the surface of the reaction compound. This amount can be determined by routine experimentation. This amount will vary depending on various parameters including, for example, the size of the reaction chamber. For example, in a 0.2 ml microtube, 0.015-0.025
A wax amount of g is effective, and in a 0.5 ml microtube, a wax amount of 0.023 to 0.04 g is effective.

The term “oil” means a water-immiscible organic substance that is liquid at temperatures below about 40 ° C. and is less dense than water. "Inorganic oil", also known as liquid petroleum or paraffin oil, is colorless and optically transparent, has a density of 0.84 g / ml, is widely commercially available, and is a gas phase for PCR reaction. It is a high molecular weight hydrocarbon mixture commonly used as a barrier. Melting Point Bath Oil (registered trademark) by Sigma (
Silicon fluid) (Catalog No. M6884, density 0.96 g / ml, or Catalog No. M9389, density 1.05 g / ml) can also be used appropriately.

By moving the chamber (2) relative to the elongated member (8) as described above, the biological sample is sucked into the chamber (2) through the elongated member (8). can do. The sample thus introduced into the chamber (2) is
It will be appreciated that it will be introduced adjacent to the second wax layer (28).
The chamber (2) is then sealed and heated to a temperature such that the second wax layer (28) melts and the first wax layer (26) remains solid. This melting of the second wax layer (28) preferably allows the sample to be in fluid communication with the second reactive compound (27). At this point, chamber (2)
Can be centrifuged, which ensures that the sample and the second reactive compound (27) are mixed to form a reactive mixture. Centrifugal force is 5-200g
And preferably 5 to 70 g. This allows
The chamber (2) is in a state where it can trigger the desired reaction.

The chamber is then heated to a temperature at which the first wax layer (26) melts. This melting of the first wax layer (26) preferably allows the sample to be in fluid communication with the first reactive compound (25). At this point, the chamber (2) can be centrifuged in the same manner as described above, whereby the sample and the first reaction compound (25) can be reliably mixed. As a result, the chamber (2) is in a state where it can cause a desired reaction.

It is also envisioned that the chamber may be sealed and turned upside down before the wax layer is melted and the sample and reactant are mixed. For example, the sample can be drawn into the chamber, the open end of the chamber can be sealed, and then the chamber can be placed in the thermal cycling means with the chamber upside down. In this case, the collar is preferably removed from the chamber. In this embodiment, the same procedure as above is performed. In this case, the sample does not descend into the reaction compound, but the second reaction compound and the first reaction compound descend into the sample corresponding to the melting temperatures of the corresponding wax layers.

As mentioned above, in a preferred embodiment the device according to the invention can be used to detect target nucleic acid molecules in a biological sample. Therefore, it is preferable that the first reaction compound (25) has a reaction agent that enables amplification of the target nucleic acid molecule.

As will be appreciated by those skilled in the art, the presence of proteins, such as DNA-degrading enzymes, in biological samples will significantly interfere with the nucleic acid amplification reaction. Furthermore, it is important to release the nucleic acid from the cells, and it is also possible to release the nucleic acid from binding proteins such as histones. This allows
It ensures that the polymerase within the amplification reaction compound can access the nucleic acid.

Therefore, it is preferable that the second reaction compound (27) has a reaction agent capable of causing digestion or modification of protein and releasing nucleic acid molecule from cell elements. When the sample interacts with such a second reaction compound (27), cell decomposition occurs, proteins are digested, and nucleic acid molecules are released,
Thereby, the amplification reaction is performed. Furthermore, by heating the chamber to melt the first wax layer (26), all the proteolytic enzymes present in the second reaction compound can be made inactive to carry out the amplification reaction. Thus, in this embodiment, the user can perform efficient and reliable amplification reactions on biological samples. The risk of contamination is considerably reduced by the fact that the cell digestion reaction and the amplification reaction are triggered sequentially within the same chamber.

It will be appreciated that it is possible to place more than two reactive compounds in the chamber (2), separated from each other by a wax layer. For example, it is envisioned to segregate the reactive compounds by using multiple wax layers having different melting points. This allows three or more individual reactions to be sequentially triggered in the chamber.

It will be appreciated that the devices and methods according to the invention are useful in applications such as forensic analysis, genetic diagnostic tests, test gene extraction in single cells and sex determination tests. In particular, the reaction chambers and methods according to the present invention are suitable for reactions performed on small samples such as single cells, embryo cells, sperm, pollen, blood cell parasites and viruses infected single cells and DNA containing fluids. .

Experimental Example 1: Preparation of suction device The PCR reaction mixture (17 μl) was introduced into a chamber having a diameter of about 1.6 mm. 17 μl of the PCR reaction mixture was used as a reaction buffer containing DNA polymerase, nucleotides and MgCl ₂ (Taqman Universal PCR Master Mix-
PE Applied Biosystems) and two sets of oligonucleotide primers. Here, one set of oligonucleotide primers is human Y
The target region of the chromosome (SRY HMG Box) is specific, and the other set of oligonucleotide primers is specific to the autosomal target region G6PD on the human X chromosome. The PCR primers were 0.
Present at a final concentration of 3 μM. The reaction mixture is further 0.2 μM each
It contains two PCR product detection probes at a concentration of. One probe detects the product amplified from the human Y chromosome and the other probe detects the human X chromosome.
Detect the amplified product from the chromosome.

F wax with a melting point of about 76 ° C. (available from Schumann Sasol)
Paraflint G80N6 (20%, T Wax 80%) was heated to 90 ° C., 3.3 μl was aspirated using a Dolmond Capillary Positive Displacement Pipette, solidified, and then introduced into a capillary device. The device is then replaced with the Rotogene (Corbett
(Research) and centrifuged at 78 ° C. for 30 seconds. The device was then removed from the centrifuge and cooled to 4 ° C. This covered the PCR reaction mixture with a solid wax layer.

2.5 μl of proteolytic enzyme K mixture (2x Gold PCR buffer (Perkin Elmer
), 0.4 mg / ml of proteolytic enzyme K (Boehringer Mannheim), 18 μM SDS, and balance water) were layered onto the solidified F wax. Then, A wax (PE Applied Biosystems
AmpliWax Gem 50) manufactured by the same company was put into the device in the same manner as in the case of F wax. However, in this case the A-wax was melted at 75 ° C and then the device was centrifuged at 60 ° C for 30 seconds on a Rotogene.

Finally, 15 μl of inorganic oil (Sigma Chemicaks) was placed on the A wax.

Then, this chamber was assembled with a long glass member having needle-shaped tips to fabricate a device as shown in FIG.

Experimental Example 2: Detection of Target Nucleic Acid in Human Leukocytes Human leukocytes (WBC) from men and women and leukocytes from white blood cells (WBC).
Were collected into cell preparation tubes with sodium citrate (CRT) (Becton Dickinson, Catalog No. 362761) for separation. And a physiological saline solution containing phosphate as a buffer (0.01 M phosphate buffer, 0.0
Washed twice in 027M KCl and 0.137M NaCl). The final WBC pellet was resuspended in a "Vigro Retrival" (AB Technology). The "Vigro Retrival" functions to prevent the WBC from sticking to the tube wall. The WBC was then transferred into Petri dishes and oil coated for harvesting.

The desired number of WBCs was visually counted in 2.5 μl of “Vigro Retrival” preparation and aspirated using a device made as described in Experimental Example 1. As a control, male human DNA solution, female human solution, and a solution consisting only of "Vigro Retrival" (containing no DNA), which were made equal to each other, were sucked into respective separate devices. I decided.

WBC was sucked into the elongate member just below the oil and above the second wax layer. Then, the glass long member was removed. After mounting the cap, the device was placed in the Rotogene thermal cycling means. The device was heated at 60 ° C. for 10 minutes to initiate the initial protein digestion reaction.
At this point, the A wax melts and the F wax remains solid. After this reaction, the chamber was heated at 72 ° C. for 10 minutes to inactivate the protease K. After that, the chamber was subjected to the following PCR conditions. 1 × 94 ° C. for 10 minutes (melting the F wax layer); 50 × 93 ° C. for 15 seconds, 60 ° C. for 40 seconds,
15 seconds at 6093 ° C.

The final result of the PCR is shown in FIG. The x-axis clearly shows the type of sample. That is, it clearly indicates whether it is DNA or WBC, male or female, and the number of genes or WBCs applied to the reaction chamber.

For all control samples and 11 out of 12 samples analyzed, the expected results with respect to gender were obtained. In these samples, positive for the X internal reference, except for the control sample (Sample No. 3, 16), which gave an appropriate result that the gender signal, that is, the autosomal signal, was not obtained because it did not have DNA. Met. Sample No. Regarding 6, it failed to obtain a sex signal, that is, an autosomal signal.

In particular, a unique signal was detected for each of the male WBC and male DNA samples analyzed, and no false positive signal was detected in any of the female-based samples. Further, the sample No. As is clear in 10, 15, the method is sufficiently sensitive to detect a single copy of the target sequence present in a single WBC, in particular a single Y autosomal sequence.

The respective patent documents and the respective documents cited above are incorporated herein by reference in their entirety.

It will be appreciated by those skilled in the art that various modifications and variations can be made to the particular embodiments of the present invention without departing from the spirit and scope of the invention.
Therefore, the above embodiments can be considered to be illustrative in all aspects and not to limit the present invention.

[Brief description of drawings]

FIG. 1 is a perspective view showing a device according to a preferred embodiment of the present invention in an assembled state.

FIG. 2 is a cross-sectional view of a preferred device of the present invention showing the elongated member in the first position.

FIG. 3 is a cross-sectional view of a preferred device of the present invention showing the elongated member in the second position.

4 is an exploded cross-sectional view of the device of FIG.

FIG. 5 is a cross-sectional view of another preferred device of the present invention, showing (a) the assembled device and (b) the combination of the elongate member and the sealing means.

FIG. 6 is a graph showing the results of a sex determination analysis performed on human leukocytes by direct aspiration and one-step real-time amplification detection using a device according to a preferred embodiment of the present invention.

[Explanation of symbols]

  1 device   2 chamber   3 Open first end   4 Second end of blockage   5 Inner surface   6 outer surface   7 lenses   8 Long member   9 Open first end 10 Open second end 11 Inner surface 12 outer surface 13 First position 14 Second position 15 Needle-shaped tip (shape tip) 16 Sealing means 17 Sleeve 18 Expanding collar 21 First Guide (Guide) 23 opening 25 First reaction compound 26 First wax layer 27 Second reaction compound 28 Second wax layer 29 oil

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] May 28, 2001 (2001.5.28)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Contents of correction]

In a first aspect, the invention comprises a chamber having an open first end, a closed second end, an inner surface and an outer surface; an open first end, an open second end, an inner surface and an outer surface. An elongate member having; and a sealing means providing a seal between the elongate member and the chamber; a second end of the elongate member from a first position within the chamber to within the chamber. It is slidably movable between the second position and the first position to the second position.
By the movement of the second end of the elongated member, sufficiently to obtain uptake of fluid into the chamber through the elongated member to provide a device adapted to change in pressure in the chamber is caused.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/48 G01N 33/50 P // G01N 33/50 C12N 15/00 A (81) Designated country EP ( AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Brian William King Australia Queensland 4552 Mailney Stanley River Road (No house number) (72) Inventor Bruce Thomas Harrison Australia Queensland 4305 Eastern Heights Devon Strey・ 6F term (reference) 2G045 AA13 CA02 CA11 CB01 CB14 CB20 CB21 DA12 DA13 DA14 DA36 FA11 FB12 GC15 HA06 HA09 HA14 2G052 AA28 AB16 AB18 AC16 AD06 AD46 BA14 CA02 DA01 DA22 GA11 JA01 JA0 4B024 AA11 CA01 GA30 HA11 4 CC01 FA03 FA12

Claims (53)

[Claims] 1. A device having a chamber having an open first end, a closed second end, an inner surface and an outer surface; a device having an open first end, an open second end, an inner surface and an outer surface. An elongate member; and a sealing means for providing a seal between the elongate member and the chamber; the second end of the elongate member from the first position in the chamber. It is slidably movable to a second position in the chamber, and movement of the second end of the elongated member from the first position to the second position causes a change in pressure in the chamber. A device characterized by being triggered. 2. The device according to claim 1, wherein   A device wherein the chamber is elongated. 3. Device according to claim 1 or 2, characterized in that the sealing means provides a seal between the outer surface of the elongate member and the inner surface of the chamber. . 4. A device according to any one of claims 1 to 3, characterized in that the chamber allows the transmission of electromagnetic radiation. 5. The device according to any one of claims 1 to 3,   A device wherein the chamber is cylindrical. 6. The device according to claim 5, wherein the outer diameter of the cylindrical chamber is at most 2.8 mm. 7. Device according to claim 5 or 6, characterized in that the cylindrical chamber has an inner diameter of at most 2.5 mm. 8. A device according to any one of claims 5 to 7, characterized in that the cylindrical chamber has a length in the range of 20 to 40 mm. 9. The device according to claim 1, wherein the first end of the elongated member has a shaping tip. 10. The device according to claim 9, wherein the diameter of the shaping tip is in the range of 1 μm to 200 μm. 11. The device according to claim 1, wherein the chamber and the elongated member are made of the same material. 12. The device of claim 11, wherein   A device characterized in that the material is glass or plastic. 13. The device according to claim 1, wherein the chamber is made of plastic and the elongate member is made of glass, or the chamber is made of glass and the elongate member is made of glass. A device characterized in that the member is made of plastic. 14. The device according to claim 12 or 13, wherein the plastic is selected from the group consisting of polyethylene terephthalate, polypropylene and polycarbonate. 15. The device according to claim 1, wherein the sealing means is arranged on the outer surface of the elongated member in the vicinity of the second end of the elongated member. A device comprising: 16. The device according to claim 1, wherein the chamber further comprises a flared collar near the open end of the chamber. 17. The device according to claim 16, further comprising a guide in the diameter-expanding collar, the guide being provided with an opening for receiving the elongated member. And the device to be. 18. The device of claim 17, wherein the elongate member is received by the opening such that a seal is formed between the elongate member and the guide. . 19. The device according to claim 1, wherein at least part of one or both of the inner surface of the chamber and the inner surface of the elongated member is a reaction inhibitor. A device characterized by being coated with. 20. The device according to claim 19, wherein the reaction inhibitor is selected from the group consisting of bovine serum albumin, fetal calf serum, human serum albumin, polyvinyl alcohol, polyvinylpyrrolidone, and a silicone compound. A device containing at least one of the following: 21. The device according to claim 1, wherein the second end of the chamber is provided with a lens for transmitting electromagnetic radiation. 22. The device of any of claims 1-21, wherein the chamber comprises a first reaction compound; a first wax layer that provides a seal on the first reaction compound; the first wax layer. A second reaction compound disposed adjacent to the second reaction compound and separated from the first reaction compound via the first wax layer; and a second wax layer substantially covering the second reaction compound; The second wax layer has a melting point lower than that of the first wax layer, and thus the first wax layer is solid at the melting point of the second wax layer. Device characterized by. 23. The device of claim 22, wherein   The device wherein the melting point of the first wax layer is 70 ° C. 24. The device according to claim 22 or 23,   A device characterized in that the melting point of the second wax layer is 55 ° C. 25. The device according to any one of claims 22 to 24, wherein the first reaction compound has a reaction agent necessary for amplification of a target nucleic acid molecule. 26. The device according to any one of claims 22 to 25, wherein the second reaction compound causes cell degradation, protein digestion and / or alteration, or DNA digestion and / or alteration. A device having different reactive agents. 27. The device according to claim 26, wherein the second reaction compound has an alkaline buffer for enhancing cell degradation. 28. The device of any of claims 22-27, wherein the chamber further contains a third reactive compound disposed adjacent to the second wax layer. Device to do. 29. The device of claim 28, wherein the chamber further contains a third wax layer disposed adjacent to the third reactive compound. 30. The device of any of claims 1-21, wherein the chamber comprises a first reaction compound mixed with a first wax carrier; a second reaction compound mixed with a second wax carrier. Wherein the second reaction compound is at least partially coated or covered or layered on the first reaction compound, and the second wax carrier is A device having a melting point lower than that of the first wax carrier, so that at the melting point of the second wax carrier, the first wax carrier is solid. 31. The device according to claim 30, wherein the melting point of the first wax carrier is 70 ° C. 32. The device of claim 30 or 31, wherein the second wax carrier has a melting point of 55 ° C. 33. The device according to any one of claims 30 to 32, wherein the first reaction compound has a reaction agent necessary for amplification of a target nucleic acid molecule. 34. The device according to any one of claims 30 to 33, wherein the second reaction compound causes cell degradation, protein digestion and / or alteration, or DNA digestion and / or alteration. A device having different reactive agents. 35. The device according to claim 34, wherein the second reaction compound has an alkaline buffer for enhancing cell degradation. 36. The device of any of claims 30-35, wherein the chamber further contains a third reaction compound mixed with a third wax carrier, the third reaction compound being the third reaction compound. A device characterized in that it is at least partially coated or covered or layered on a bireactive compound. 37. A method for detecting a substance in a sample, comprising: (i) introducing a sample and a reactive compound into the chamber of the device according to any of claims 1-21. Forming a reactive mixture from the sample and the reactive compound, wherein the reactive compound has at least one reactive agent capable of interacting with the substance to be detected, whereby the reactive The level of electromagnetic radiation emitted by the mixture is different compared to the level of electromagnetic radiation emitted by the reactive compound alone; (ii) additionally exciting the reactive mixture by electromagnetic radiation (Iii) bringing the chamber into a state in which a reaction can occur; (iv) detecting electromagnetic radiation emitted from the reactive mixture; 38. A method for amplifying at least one target nucleic acid molecule in a sample, comprising: (i) a sample and a reactive compound in the chamber of the device of any of claims 1-21. To form a reactive mixture from the sample and the reaction compound, wherein the reaction compound has a reaction agent for amplification of the target sequence; (ii) the chamber The method is characterized in that amplification of the target sequence is possible. 39. A method for amplifying at least one target nucleic acid molecule in a sample, comprising: (i) a sample and a reactive compound in the chamber of the device of any of claims 1-21. To form a reactive mixture from the sample and the reactive compound, wherein the reactive compound is reacted with a reagent for amplification of the target sequence and an electromagnetic wave emitted by the reactive mixture. At least one reactant capable of interacting with the amplified sequence, such that the level of radiation is different compared to the level of electromagnetic radiation emitted by the reactive compound alone; (Ii) additionally exciting the reactive mixture with electromagnetic radiation; (iii) amplifying the chamber with the target sequence. A possible state; wherein the; (iv) detecting the electromagnetic radiation emitted from the reaction mixture. 40. The method according to claim 37 or 39, characterized in that electromagnetic radiation is transmitted from an external radiation source through the chamber to the reactive mixture. 41. The method according to claim 37 or 39, wherein the degree of difference of the electromagnetic radiation from the reactive mixture is detected through the chamber. 42. The method of any of claims 37-41, wherein the reactive compound is introduced into the chamber through the elongated member. 43. The method of any of claims 37-42, wherein the sample is introduced into the chamber through the elongate member. 44. A method for performing a series of reactions in a single container, comprising: (i) introducing a sample into the chamber of the device according to any of claims 22-27. Placing the sample in a position adjacent to the second wax layer; (ii) heating the chamber to a temperature that melts the second wax layer and leaves the first wax layer solid. (Iii) forming a reactive mixture by interacting the sample with the second reactive compound; (iv) heating the chamber to a temperature at which the first wax layer is melted; (v) ) Interacting the reactive mixture with the first reactive compound; 45. A method for performing a series of reactions in a single container, comprising: (i) introducing a sample into the chamber of the device according to any of claims 30-35. Placing the sample in a position adjacent to the second reactive compound; (ii) heating the chamber to a temperature that melts the second wax carrier and leaves the first wax carrier solid. (Iii) forming a reactive mixture by interacting the sample with the second reactive compound; (iv) bringing the chamber to a temperature at which the first wax carrier is melted;
Heating; (v) allowing the reactive mixture to interact with the first reaction compound; 46. The method according to claim 44 or 45, characterized in that said step (iii) and / or said step (v) are performed by centrifugation of said chamber. 47. The method according to any one of claims 37 to 46,   A method comprising providing the sample with a biological material. 48. The method of claim 47, wherein   A method characterized in that the biological material is cells or virus particles. 49. The method according to any one of claims 37 to 48, wherein the second reaction compound has a reaction agent for protein digestion and / or cell destruction. . 50. The method according to any one of claims 37 to 49, wherein the first reaction compound has a reaction agent for amplification of a target nucleic acid molecule. 51. The method according to any one of claims 37 to 50, wherein the sample is an erythrocyte, a leukocyte, an embryonic cell, a bacterium, a sperm, a pollen, a cell culture medium, a Pap smear, a single cell, or a blood cell parasite. Body, single cell infected with virus,
And a method of selecting from the group consisting of fluids containing DNA or RNA or proteins. 52. A method of use, characterized in that the device according to any one of claims 1 to 21 is used for injecting a fluid sample into cells. 53. The method of use according to claim 52,   A method of use, wherein the fluid sample is a genetic material.