JPH11271193A - Biological sample pretreatment device - Google Patents

Abstract

(57) [Summary] To automatically separate genes and nucleic acids in a biological sample from microorganisms and cells. A biological sample is transferred to a lysis sample tube,
A lysis step is carried out, transferred to an extraction sample tube 8, and an extraction step is carried out. During this time, the sample is moved by the dispensing nozzle incorporated in the dispensing arm. The sample tubes are installed so as to reduce contamination by the partition walls 14, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for pretreating a biological sample, and is used for measurement and preparation of trace genes and nucleic acids in blood and body fluid. It relates to labor saving and automation of preprocessing.

[0002]

2. Description of the Related Art Extraction of nucleic acids and genes from biological samples is a very important process in the fields of genetic engineering and clinical testing. In this case, the biological sample refers to body fluids, secretions, tissue cells, floating cells, and the like in an organism (animals including humans), and particularly refers to body fluids such as serum, blood, cerebrospinal fluid, lymph, and urine. Usually, a nucleated gene is surrounded by other organelles and the like in a cell, and is present in association with a nuclear protein and the like.
Virus and bacterial genes are also incorporated into the shell and cell wall.

[0003] In order to separate genes and nucleic acids from other components, it is necessary to destroy or dissolve cell walls, cell membranes, capsid proteins and envelopes. Therefore,
For this purpose, means such as physical destruction by ultrasonic waves or heating, and dissolution by proteases or surfactants are generally used. Hou ”Koji Shimonishi and others, Doujin Kagaku 1
996, pp. 1-17).

[0004] However, after performing these operations,
The process of isolating the DNA or RNA chain constituting the gene by the extraction operation is liable to cause contamination of the sample, and is usually performed manually by an operator. The standard method of extraction is chloroform
The phenol method is often used. However, the toxicity of chloroform and phenol, the trouble of waste disposal, the trouble of centrifugation, the infection from the sample, and the contamination of the sample are obstacles to the spread of daily work.

Therefore, as an alternative, a reagent kit that does not use an organic solvent (for example, QIAprep QIAprep)
kit and the like are commercially available, and patents have been devised (JP-A-7-236499, JP-A-9-327290). There is a method to perform the destruction process and the extraction process at the same time, but with good reproduction,
Not all samples can provide high-purity nucleic acids or genes. In particular, in order to extract bacterial genes, it is necessary to break down thick cell walls and separate proteins and the like that form a complex with the gene from the gene.

[0006] Therefore, it is general and reliable to combine the step of destroying microorganisms and cells and releasing the gene with the step of extracting the obtained gene. However, in the conventional method, the destruction / release step and the extraction / separation step are performed separately, and the separation and extraction of the gene cannot be performed consistently as a pretreatment. Infection and contamination of the sample. In addition, since this operation requires labor, the efficiency is low and the cost is high. There is also the possibility of pathogen transmission to workers.

[0007]

However, in the above-mentioned prior art, cross-contamination between samples is apt to occur due to manual operation.
May be inferior in reliability and misdiagnosis. In addition, since it depends on human labor, the restraining time of the worker is often very long. Therefore, in order to prevent contamination between samples, contamination from workers and the working environment, and infection to workers, the destruction process and the extraction process are incorporated in a series of continuous processes, and the movement and dispensing of samples are further performed. Could be solved by performing it automatically in a closed device. As a result of further study of a specific device for realizing such a problem, the present invention has been devised.

[0008]

According to the present invention, the steps of lysing bacteria and viruses in a sample to release fragments of genes and nucleic acids, attaching these to a solid surface, and subsequently eluting the same are automated. We devised that. The lysis and destruction of the cell walls and membranes surrounding the bacterial and viral genes is referred to as "lysis", and the lysis step and the step of isolating the gene (hereinafter referred to as "extraction" step) are continuous. And in the apparatus. The process of “lysis” is based on a conventionally known technique (Nippon Chemistry Experiment Guide 3 “Separation / Analysis of Nucleic Acids and Gene Experiments” edited by Yasushi Shimonishi et al., Edited by Kagaku Doujin 1996, pp. 1-17) Heating, ultrasonic vibration, microwave heating, enzyme dissolution, surfactant dissolution, etc. can be applied. The lysing step and the extracting step are performed in another continuous adjacent area on the apparatus, and an enclosure is provided around each sample container and the lysed sample container, thereby preventing errors due to contamination between the steps.

Further, in order to prevent the contamination of genes or the like from the outside air, the apparatus is designed such that the door blocks the inflow of air from the outside during operation. Use an unused disposable tip with a built-in breathable filter to move between samples.
I tried to prevent contamination. Contamination depends on the worker's body, breath,
Since it is generated from air, samples, waste, etc. inside and outside the device,
By dividing the process into sections and providing partition walls in each sample container, contamination is reduced.

[0010] In addition, by placing the final purified sample in a container that can be sealed with a lid, contamination after extraction is prevented. It is also effective to provide a sterilizing function using an ultraviolet lamp or the like in order to reduce contamination from the surface inside the apparatus. In order to restrict the air flow in the apparatus, it is also effective to weakly suck and exhaust the internal air.

That is, the (lysis) step is a step of destroying microorganisms and cells in a sample, and the DNA or RN as a gene is
This is performed in order to release A from unnecessary substances such as surrounding proteins and organelles. This allows DNA and RNA
Is converted into a free chain to facilitate extraction. The sample container has a structure suitable for the lysis method, is made of disposable plastic, and is made of a material that can be broken or dissolved.

A hydrophobic material that does not easily cause gene adsorption is preferable, but is not particularly limited. Tray for installing sample containers is provided with partition walls to separate each sample container, so that contamination by aerosol between samples (contamination)
Can be reduced. In particular, in the case of dispensing by the suction nozzle method, aerosol is easily generated when the nozzle tip moves up and down.

The (extraction) step is a step of separating genes and nucleic acids from unnecessary coexisting substances. The separation is carried out using a carrier capable of selectively adsorbing genes. It is effective to provide a partition between sample containers because aerosol contamination between samples easily occurs during extraction.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described based on embodiments.

(Embodiment 1) FIG. 1 shows an example of the configuration of a preprocessing apparatus according to the present invention. FIG. 2 shows a pretreatment process in the apparatus of the present embodiment. The main steps of the pretreatment device in the present embodiment are as follows. After the sample is put in the sample tube 6, it is set on the sample tray 5. After all the samples have been set, the cover of the pretreatment device is closed, and the pretreatment step is started. A certain amount of the sample is sucked by a dispensing nozzle incorporated in the dispensing arm 4 and introduced into the lysis sample tube 7.

The dispensing arm 4 can move freely along the moving rail 10 in the horizontal and vertical directions in the figure, and can further raise and lower the dispensing nozzle. The tip of the dispensing nozzle is equipped with a disposable gas filter built-in tip, which is replaced for each sample. A lysis reagent is added to the sample transferred to the lysis sample tube 7 with a dispensing nozzle.

An example using magnetic particles will be described below, but the present invention is not limited to this method. The lysis sample tube 7 to which the reagent has been added is held for a certain period of time, and then the sample is again transferred to the extraction sample tube 8 by a dispensing nozzle equipped with a disposable gas filter built-in tip. A fixed amount of magnetic fine particles coated with quartz is added to the sample, and a chaotropic reagent (for example, a 4M guanidine isothiocyanate solution) is added. The permanent magnet placed below the magnetic fine particles is raised, brought into close contact with the bottom of the tube, and collected at the bottom of the tube.

Next, using a dispensing nozzle equipped with a disposable pipette tip, a washing solution (5M guanidine isothiocyanate solution, 50 mM Tris-HCl buffer (pH 6.4)) is dispensed and washed, and finally sterilized. A small amount of pure water is added to the sample tube 8 and DN adsorbed on the quartz surface
A is eluted. The eluted DNA is sucked up by a disposable chip with a built-in ventilation filter and transferred to the extracted nucleic acid tube 9.

The DNA thus obtained is subjected to each analysis method. Quartz and glass are often used for the adsorption of genes and nucleic acids, and not only magnetic particles but also quartz or glass particles alone can be used similarly. In this case, the particle diameter was made larger than the tip diameter of the tip so that the particles were not removed by the nozzle tip. When extracting genes and nucleic acids,
Particles or material surfaces on which a nucleic acid probe specific to a specific gene sequence is immobilized can also be used.

As shown in FIG. 3, the upper end of each sample and nucleic acid sample tube is surrounded by a partition wall 14 on the four sides so as to reduce contamination between samples. Although the sample tube may have a lid, it is more suitable for the automation method to cover the purified sample at the end because the sample tube tends to hinder the dispensing. In RNA extraction, in particular, RNases are ubiquitous, and therefore, it is necessary to add an enzyme activity inhibitor or the like.

In the lysis step, in this embodiment, after treatment with a lysozyme solution, SDS (sodium dodecyl sulfate)-
The method of adding a NaOH solution was employed to recover the DNA of Escherichia coli. Other methods can be similarly applied to the lysis operation. As shown in FIG. 4, a method in which heated water is provided below the lysis sample tray and boiled by the electric heater 19 can also be applied.

In the case of ultrasonic waves, the element is placed at the bottom of a water tank so that bacterium can be lysed. In the case of microwaves, a microwave generator is installed above the lysis zone to enable lysis. The method used for lysis is not limited to the above method as long as it does not cause cleavage or decomposition of genes or nucleic acids.

An ultraviolet lamp (wavelength: 254 nm) is installed inside the apparatus.
It is effective to further prevent contamination if a UV lamp that radiates the strongest in the vicinity is installed, and sterilization and damage of the contaminated gene by UV light are performed before and after the pretreatment operation.

[0024]

According to the present invention, separation of genes and nucleic acids from biological samples containing genes and nucleic acids can be easily and automatically performed. In particular, contamination of the sample from the surrounding environment and other samples can be prevented, and high-quality nucleic acids and genes can be obtained. Further, it is possible to prevent the transmission of the pathogen from the sample to the worker.

[Brief description of the drawings]

FIG. 1 is a schematic plan view of a pre-processing apparatus according to an embodiment of the present invention as viewed from above.

FIG. 2 is a diagram showing a flow of a pre-processing process of the pre-processing device of the present invention.

FIG. 3 is a schematic diagram showing an installation state of a sample tube according to the present invention.

FIG. 4 is a schematic view of an example of another example of the lysis step of a sample according to the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Lysis sample tray, 2 ... Extraction sample tray, 3 ... Purified sample tray, 4 ... Dispensing arm, 5 ... Sample tray,
6 ... sample tube, 7 ... sample tube for lysis, 8 ... sample tube for extraction, 9 ... sample tube for purified sample, 10 ...
Rail for dispensing arm movement, 11 ... reagent bottle for lysis, 1
2: Extraction reagent bottle, 13: Dispensing nozzle tip disposal port, 14: Tube partition, 15: Tray plate, 16
... warming water, 17 ... sample solution, 18 ... heater.

Claims (6)

[Claims] The present invention comprises a first step of breaking down a gene or a nucleic acid in a biological sample by releasing microbial cells or cells, and a second step of separating the released gene or the nucleic acid from coexisting substances. In the biological sample pretreatment device having a pretreatment step, after the first step, the sample is transferred to another adjacent section, and the second step is performed.
A biological sample pretreatment device, wherein the gene or the nucleic acid is separated from the sample by performing the step of: 2. The biological sample pretreatment device according to claim 1, wherein the biological sample is a body fluid. 3. The method according to claim 1, wherein the compartment for the step of breaking and releasing and the compartment for the step of separating the released gene and the nucleic acid from coexistent substances are adjacent to each other to partition between sample containers. The biological sample pretreatment device according to claim 1. 4. The biological sample pretreatment apparatus according to claim 1, wherein a container used for destruction of the sample and a container for separating from the coexisting substance are replaced. 5. The biological sample pretreatment apparatus according to claim 1, wherein the step of breaking and releasing is any of heater heating, ultrasonic vibration, microwave heating, enzyme dissolution, and surfactant dissolution. . 6. A living organism having a pretreatment step comprising a step of breaking down microbial cells and cells of a gene or nucleic acid in a biological sample and releasing the same, and a step of separating the released gene or nucleic acid from coexisting substances. 2. The biological sample pretreatment device according to claim 1, wherein the compartment for performing both steps in the sample pretreatment device is closed by a door that shuts off the flow of air during the process.