WO2000077253A1

WO2000077253A1 - Apparatus and method for gene examination

Info

Publication number: WO2000077253A1
Application number: PCT/JP1999/003209
Authority: WO
Inventors: Hiroko Matsunaga; Katsuji Murakawa; Kazunori Okano; Yuji Miyahara
Original assignee: Hitachi, Ltd.
Priority date: 1999-06-16
Filing date: 1999-06-16
Publication date: 2000-12-21

Abstract

An apparatus (50) for preparing samples consisting of a sample preparation unit (100) having an upper opening, into which a buffer having cells suspended therein is poured, a lower opening, from which a waste liquor is discharged, and a channel connecting these openings, provided with a first filter (110) for capturing cells, a second filter (130) for capturing polynucleotides eluted from the cells with a denaturing agent poured into the upper opening and holding a PCR amplification reaction mixture containing a fluorescence-labeled primer for PCR and a PCR amplification reaction reagent for amplifying the copy of a desired partial base sequence in the polynucleotide, and a third hydrophobic filter (140), wherein these filters are arranged in this order from the upper opening toward the lower opening; a member (200) for holding the sample preparation unit; and a member (210) for controlling the temperature of the PCR amplification reaction mixture. By using this apparatus, a series of steps including the pretreatment of specimen samples, PCR amplification and the detection of the PCR product can be continuously performed. Thus, the contamination at each step can be prevented and a number of specimens can be easily treated simultaneously.

Description

Technical Description Arrest child testing device and genetic testing method

The present invention relates to a sample preparation apparatus for preparing a sample for performing a gene diagnosis using whole blood or a cell culture solution, a gene test apparatus for easily performing a process from sample preparation to detection of a target arrested child, and a gene. Related to inspection method. Background art

The human genome project was launched worldwide in the 1980s, and it is said that the entire genome sequence information will be available in the early 21st century. Once the whole genome sequence information is obtained, it is possible to compare and determine whether it is normal or abnormal at the arrested child level. It is likely to proceed. At present, the number of gene-related diseases is estimated to rise to 8,450. Despite this fact, the use of insurance in Japan at present is limited to some infectious disease tests. This is because diseases caused by distant gene abnormalities are more likely to be multifactorial than arrest diseases based on single abnormalities. For this reason, a wide variety of genetic tests are required, and expensive tests are required, which poses a medical economic problem. In addition, an inspection method has not been established, and a stable inspection cannot be performed anytime and anywhere.

The Po1ym erase Chain Reaction method (PCR method), a technology for analyzing arrested genes that is effective in detecting target genes, is a technology that can efficiently amplify trace amounts of distant genes. Can be dramatically improved. Several kits are available from QIAGEN (Germany) and STRATAGENE (US) for extracting DNA and RNA from tissues and whole blood. The work time required for extraction is about 30 minutes to 3 hours, depending on the kit. It is different. Automated nucleic acid extraction devices have been marketed by Toyobo, Kurashiki Spinning, and others. In addition, devices that detect PCR products in real time are AB IPRISM 7700 (Perkin Elmer (USA)) and Right Cycter ^™ (Roche Diagnostics (Germany)). And so on.

Japanese Patent Application Laid-Open No. 4-207195 discloses that a virus-adsorbing resin is added to a virus-containing suspension to capture the virus, the resin is separated on a filter, and a virus solution is passed through to contain a virus nucleic acid component. It is reported that the solution is collected and the nucleic acid components for virus testing are collected. The method for collecting nucleic acid components of viruses and the virus testing method are reported.

Japanese Patent Application Laid-Open No. 4-36198 reports a method for testing food poisoning bacteria. According to the test method described in Japanese Patent Publication No. 4-36198, a suspension containing bacteria is passed through a coarse first filter to separate insoluble solids. Next, a lysing agent is added to the filtrate to release the cell components. Then, finer insoluble solids are removed by the second filter. The nucleic acid component coagulant is added to the filtrate obtained here to precipitate the nucleic acid component, and the nucleic acid component is captured in the third filter. This is washed, and the aqueous solution flows into the third filter to elute the nucleic acid components. The eluted nucleic acid components are subjected to the polymerase chain reaction to amplify the DNA of the desired bacterium, thereby testing for the desired food poisoning bacterium.

JP-A-7-98314 discloses a fecal occult blood measuring device. In the fecal occult blood measurement device described in Japanese Patent Application Laid-Open No. 7-98314, the stool sample lysate is first removed of solid components by a separation filter. Thereafter, the stool sample solution from which the solid components have been removed is dropped onto the measurement filter. When fecal occult blood is present on the measurement filter, the Hb in the blood reacts with the first and second antibodies, and an immune complex is formed by the San German method. As a result, the measurement filter is colored. On the other hand, when there is no fecal occult blood, the measurement filter is uncolored. Therefore, the presence or absence of fecal occult blood can be easily determined by visually judging the presence or absence of coloring of the measurement filter. Disclosure of the invention

At present, there is no system that can fully automate the process from extraction of DNA or RNA from a sample to PCR and detection. The problem with manual operation is that there is a high possibility of contamination of contaminants between processes, and the inspection results may be completely different. The conventional devices and kits are far from being fully automated and only perform specific steps such as extraction only, PCR amplification, and PCR product detection, and are easily and stably anywhere, anytime. It did not satisfy the requirements of the inspection site to conduct the inspection. That is, there is a problem that the existing kit or device cannot perform from preprocessing to detection with a single application.

As described above, when using existing kits or equipment, the steps from pretreatment for extracting DNA or RNA to PCR amplification and detection of PCR products are performed using the same application unit. It was not possible to execute it, and several types of application units had to be used, and there was a problem that the possibility of contamination of contaminants between each process was extremely high.

An object of the present invention is to execute each step from a sample sample pretreatment step to a PCR amplification step and a PCR product detection step using the same application unit (sample preparation unit), and to perform the steps between each step. It is an object of the present invention to provide a genetic testing device, a genetic testing method, and a sample preparation device for diagnosing arrested children, which not only can prevent contamination, but also facilitate simultaneous processing of multiple samples.

In the present invention, a sample preparation device having a plurality of sample preparation units (application units) in which a cell capturing filter and a polynucleotide capturing filter are set in advance is used. Pour whole blood or cell culture solution plus cell buffer into each sample preparation unit, add denaturant, add PCR amplification reaction solution, and amplify and detect the target gene Can be performed continuously. The steps from sample sample pretreatment to PCR amplification and PCR product detection can be performed in the same sample preparation unit. Prevents mineralization and facilitates simultaneous processing of multiple samples.

A typical configuration of the present invention has the following features.

(C1) a flow path connecting an upper opening into which a buffer in which cells are suspended is injected, and a lower opening through which waste liquid flows out, wherein the flow path extends from the upper opening to the lower opening; A first filter that captures the cells, and a PCR primer that captures a polynucleotide eluted from the cells by a denaturant injected from the upper opening and is fluorescently labeled. A sample preparation unit comprising: a second filter for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of a desired nucleotide sequence portion of the polynucleotide; and a hydrophobic third filter. A sample preparation device comprising: a holding means for holding the sample preparation unit; a means for controlling the temperature of the PCR amplification reaction solution; and a label for the copy. Irradiating means for irradiating the PCR amplification reaction solution with a laser beam for exciting the fluorescent label from a direction substantially perpendicular or substantially parallel to the second filter; and a fluorescent light from the fluorescent label for labeling the copy. And a detecting means for detecting the signal from a direction substantially perpendicular to the second filter.

(C2) a flow path connecting an upper opening into which a buffer solution in which cells are suspended is injected and a lower opening through which waste liquid flows out, wherein the flow path is from the upper opening to the lower opening. A first filter for capturing the cells, and a primer for PCR, which is a fluorescence-labeled primer that captures a polynucleotide eluted from the cells by a denaturing agent injected from the upper opening. A second filter holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of a desired nucleotide sequence portion of the polynucleotide, and a third hydrophobic filter. Sample preparation unit to have.

(C3) a first filter and a second filter, each having a flow path connecting an upper opening and a lower opening, and being sequentially arranged in the flow path in a direction from the upper opening to the lower opening. A method for examining a gene using a sample preparation device having one or more sample preparation units having a hydrophobic third filter, and (1) Injecting a buffer in which cells are suspended into the upper opening of the sample preparation unit to capture the cells in the first filter; and (2) injecting a denaturant into the upper opening. (C) capturing the polynucleotide eluted from the cells by the second filter, and (3) including a fluorescently-labeled PCR primer in the upper opening; Injecting a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy, holding the PCR amplification reaction solution in the second filter, and amplifying the copy; 4) irradiating the PCR amplification reaction solution with a laser beam for exciting the fluorescent label for labeling the copy, and detecting the fluorescent light from the fluorescent label.

(C 4) a first member having a first opening formed in an upper portion into which a buffer solution in which cells are suspended is injected, and a first filter formed in a lower portion to capture the cells; A second opening for capturing a polynucleotide eluted from the cell with a denaturing agent injected from the first opening, and comprising a fluorescently labeled primer for PCR; A second member having a second filter at the bottom and a hydrophobic third filter are provided for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction. And a means for controlling the temperature of the PCR amplification reaction solution, and a plurality of samples arranged in this order from the top, the first member, the second member, and the third member A sample preparation device having a preparation unit.

(C5) a first member having a first opening formed at an upper portion, into which a buffer solution in which cells are suspended is injected, and a first filter formed at a lower portion to capture the cells; A polynucleotide having a second opening, wherein the polynucleotide eluted from the cell is captured by a denaturing agent injected from the first opening, and a primer for fluorescence-labeled PCR is contained; A second member having a second filter at a lower portion for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction to amplify a copy of a sequence portion, and a second member having a hydrophobic third filter The third member, a transparent fourth member having a through hole, and the PCR amplification reaction Means for controlling the temperature of the solution, wherein the first member, the fourth member, the second member, and the third member are arranged in this order from above, and the first filter A sample preparation device having a plurality of sample preparation units, wherein the second filter is opposed to the second filter via the through hole, and the second filter and the third filter are opposed to each other.

(C 6) a first through-hole, a first opening formed at an upper portion, into which a buffer solution for suspending cells is injected, and a second opening formed at a lower portion of the first opening to capture the cells.

A first member having a first filter; a second member having a second opening at an upper portion; and a polynucleotide denatured from the cells captured by a denaturing agent injected from the first opening, and a fluorescently labeled PCR A second filter having a lower portion for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of a portion of the desired nucleotide sequence of the polynucleotide containing a primer for PCR A member, a third member having a hydrophobic third filter, and means for controlling a temperature of the PCR amplification reaction solution, wherein, from above, the first member, the second member, The third member is arranged in this order, the first through hole and the second opening are opposed to each other, the second filter and the third filter are arranged to be opposed to each other, A flow path connecting the filter and the second filter A sample preparation device having a plurality of sample preparation units on which a sample is formed.

(C7) a first through-hole, a first opening formed in an upper part, into which a buffer solution for suspending cells is injected, and a first opening formed in a lower part of the first opening to capture the cells. A first member having a filter, and a polynucleotide eluted from the cells by a denaturing agent injected from the first opening, and comprising a fluorescent-labeled PCR primer. A second member having a second filter for holding a solution containing a PCR amplification reagent for a PCR amplification reaction for amplifying a copy of a desired base sequence portion, and a hydrophobic third filter. A third member, a transparent fourth member having a second through-hole and a second opening at an upper part, and means for controlling the temperature of the PCR amplification reaction reagent. Member, the fourth member, the second member, the third member The first filter and the second filter face each other via the second filter; and the second filter and the third filter are positioned opposite to each other. A sample preparation unit having a plurality of sample preparation units in which a flow path connecting the first filter and the second filter is formed.

(C8) A plurality of first regions are formed, each having a first opening formed at an upper portion into which a buffer solution in which cells are suspended is injected, and a first filter formed at a lower portion to capture the cells. A first member having a second opening at an upper portion, and a polynucleotide labeled with a denaturing agent injected from the first opening, the polynucleotide eluted from the cells being captured, and a fluorescently labeled primer for PCR. A plurality of second regions having a second filter at a lower portion for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of a desired base sequence portion of the polynucleotide containing the same A second member formed, a third member formed with a plurality of third regions having a hydrophobic third filter, and means for controlling the temperature of the PCR amplification reaction solution; From above, the first member, the second member, the A sample preparation unit having a plurality of sample preparation units arranged in the order of three members and having the first region, the second region, and the third region; An irradiation means for irradiating the PCR amplification reaction solution of each of the sample preparation units with laser light for exciting the fluorescent label for labeling the copy substantially simultaneously from a direction substantially perpendicular to the filter; A detector for detecting the fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of the unit from a direction substantially perpendicular to the second filter.

(C9) A plurality of first regions are formed, each having a first opening formed at an upper portion, into which a buffer solution in which cells are suspended is injected, and a first filter formed at a lower portion to capture the cells. A first member having a second opening at an upper portion thereof, and capturing a polynucleotide eluted from the cells by a denaturing agent injected from the first opening, and a fluorescently labeled PCR primer. Including a reagent for a PCR amplification reaction for amplifying a copy of the desired nucleotide sequence portion of the polynucleotide P A second member in which a plurality of second regions having a second filter holding a CR amplification reaction solution is formed at a lower portion, and a second member in which a plurality of third regions having a hydrophobic third filter are formed. A third member, a transparent fourth member having a plurality of fourth regions in which through holes are formed, and means for controlling the temperature of the PCR amplification reaction solution. A member, the fourth member, the second member, and the third member are arranged in this order; the first filter and the second filter face each other via the through hole; A plurality of sample preparation units having a first region, a second region, a third region, and the fourth region, wherein the filter and the third filter are configured to face each other; From each sample preparation unit, in a direction substantially parallel to the second filter, Irradiation means for irradiating the PCR amplification reaction solution of each sample preparation unit with laser light for exciting the fluorescent label for labeling the copy in the PCR amplification reaction solution of the sample at substantially the same time; Detecting means for detecting the fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of the sample preparation unit substantially simultaneously from a direction substantially perpendicular to the second filter of each of the sample preparation units. Genetic testing device to have.

(C10) a first through-hole, a first opening formed at an upper portion, into which a buffer for suspending cells is injected, and a lower portion formed at a lower portion of the first opening to capture the cells. A first member having a plurality of first regions having a first filter, and a first member having a second opening at an upper portion, the first member being eluted from the cells by a denaturing agent injected from the first opening. The PCR amplification reaction solution containing reagents for the PCR amplification reaction, which contains the primers for fluorescently labeled PCR and amplify a copy of the desired base sequence portion of the polynucleotide, is captured. A second member having a plurality of second regions formed below the second filter; a third member having a plurality of third regions formed having a hydrophobic third filter; Means for controlling the temperature of the amplification reaction solution. The

The first member, the second member, and the third member are arranged in this order, the first through hole and the second opening face each other, and the second filter and the third filter And a flow path connecting the first filter and the second filter is formed, and has a first area, a second area, and a third area. A plurality of sample preparation units and the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit from a direction substantially perpendicular to the second filter of each sample preparation unit. An irradiating means for irradiating the PCR amplification reaction solution of each sample preparation unit with laser light for exciting the sample almost simultaneously, and the fluorescence labeling the copy in the PCR amplification reaction solution of each sample preparation unit An arrestor inspection apparatus comprising: detection means for detecting fluorescence from a label substantially simultaneously from a direction substantially perpendicular to the second filter of each of the sample preparation units.

(C11) a first through-hole, a first opening formed at an upper portion thereof, into which a buffer solution for suspending cells is injected, and a second opening formed at a lower portion of the first opening to capture the cells. A first member in which a plurality of first regions having one filter are formed, and a polynucleotide eluted from the cells by a denaturing agent injected from the first opening, which is fluorescently labeled. A second region having a second filter that holds a solution containing a PCR amplification reaction reagent for a PCR amplification reaction that contains a primer for PCR and amplifies a copy of the desired nucleotide sequence portion of the polynucleotide A plurality of second members, a third member having a plurality of third regions having a hydrophobic third filter, and a fourth member having a second through hole and a second opening at an upper portion thereof; A transparent fourth member in which a plurality of regions are formed, and the PCR amplification reaction Means for controlling the temperature of a reagent, wherein the first member, the fourth member, the second member, and the third member are arranged in this order from above, and the first through hole is provided. And the second filter are opposed to each other via the second through-hole. The second filter and the third filter are arranged to face each other, and the first filter and the second filter are arranged opposite to each other. A plurality of sample preparation units having a first region, the second region, the third region, and the fourth region, wherein a flow path connecting the filter is formed; Mark the copy in the PCR amplification reaction solution of each sample preparation unit from a direction substantially parallel to the second filter of the unit. The laser light that excites the fluorescent label to be detected is applied to each of the sample preparation units.

Irradiating means for irradiating the PCR amplification reaction solution almost simultaneously; and irradiating the fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit with the fluorescence of the sample preparation unit. A detector for detecting a telegraph substantially simultaneously from a direction substantially perpendicular to the second filter.

(C12) a first member in which a plurality of first regions having a first opening formed in an upper part and a first filter formed in a lower part are formed; and a second member formed in an upper part. A second member having a plurality of second regions having a second filter underneath, and a third member having a plurality of third regions having a hydrophobic third filter; And arranged from above in the order of the first member, the second member, and the third member, and have the first region, the second region, and the third region. A method for genetic testing using a sample preparation device having a plurality of sample preparation units, comprising: (1) injecting a buffer solution in which cells are suspended into the first opening; (2) injecting a denaturing agent into the first opening to elute the polynucleotide eluted from the cells in the second opening; (3) a PCR amplification reaction comprising a PCR-primed PCR primer and a reagent for a PCR amplification reaction which includes a primer for PCR and amplifies a copy of the desired nucleotide sequence portion of the polynucleotide. Injecting a solution into the first opening, holding the PCR amplification reaction solution in the second filter, and amplifying the copy, (4) the second step of each sample preparation unit. Irradiating the PCR amplification reaction solution of each of the sample preparation units with a laser beam for exciting the fluorescent label for labeling the copy from a direction substantially perpendicular to the filter; Detecting the fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit from a direction substantially perpendicular to the second filter. Inspection method.

(C13) a first member in which a plurality of first regions having a first opening formed in an upper portion and a first filter formed in a lower portion are formed; and a second opening formed in an upper portion. And a second part in which a plurality of second regions having a second filter are formed below A third member having a plurality of third regions having a hydrophobic third filter, and a transparent fourth member having a plurality of fourth regions having through holes formed therein. , From above, the first member, the fourth member, the second member, and the third member are arranged in this order, the first region, the second region, the third region, A plurality of sample preparation units having the fourth region, wherein the first filter and the second filter face each other through the through hole, and the second filter and the third filter A method for genetic testing using a sample preparation device facing a filter, comprising: (1) injecting a buffer solution in which cells are suspended in the first opening, and capturing the cells in a first filter; (2) a polynucleotide eluted from the cells by injecting a denaturant into the first opening (3) a PCR amplification reaction that includes a fluorescence-labeled PCR primer in the first opening and amplifies a copy of a desired nucleotide sequence portion of the polynucleotide; Injecting a PCR amplification reaction solution containing reagents for the PCR, holding the PCR amplification reaction solution, and amplifying the copy;

(4) A laser beam for exciting the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit from a direction substantially parallel to the second filter of each sample preparation unit. Irradiating the PCR amplification reaction solution of each sample preparation unit with the PCR amplification reaction almost simultaneously; and (5) irradiating the fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit. Detecting each of the sample preparation units from a direction substantially perpendicular to the second filter at substantially the same time.

(C14) A plurality of first regions having a first through-hole, a first opening formed in an upper portion, and a first filter formed in a lower portion of the first opening are formed. A second member in which a plurality of second regions having a second opening in the upper part and a second filter in the lower part are formed, and a third member having a hydrophobic third filter. A third member in which a plurality of regions are formed, and the first member, the second member, and the third member are arranged in this order from above, and the first region is A second region, a plurality of sample preparation units having the third region, A first through hole faces the second opening, the second filter and the third filter are arranged facing each other, and connects the first filter and the second filter. A genetic test method using a sample preparation device in which a flow path is formed. (1) A buffer solution in which cells are suspended is injected into the first opening, and the cells are captured by a first filter. (2) injecting a denaturing agent into the first opening to capture the polynucleotide eluted from the cells in the second filter; and (3) fluorescently labeling the first opening. Injecting a PCR amplification reaction solution containing a PCR primer and a reagent for a PCR amplification reaction for amplifying a copy of the desired base sequence portion of the polynucleotide containing the PCR primer, holding the PCR amplification reaction solution, Amplifying the copy; (4) A laser beam for exciting the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit is applied from a direction substantially perpendicular to the second filter of the sample preparation unit. (5) irradiating the PCR amplification reaction solution of the sample almost at the same time; and (5) preparing the fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit. And detecting substantially simultaneously from a direction substantially perpendicular to the second filter of the unit.

(C15) a first through-hole, a first opening formed at an upper portion, into which a buffer solution for suspending cells is injected, and a second opening formed at a lower portion of the first opening to capture the cells. A first member in which a plurality of first regions having one filter are formed, and a polynucleotide eluted from the cells by a denaturing agent injected from the first opening, which is fluorescently labeled. A second region having a second filter that holds a solution containing a PCR amplification reaction reagent for a PCR amplification reaction that includes a primer for PCR and amplifies a copy of a portion of the desired nucleotide sequence of the polynucleotide A second member having a plurality of third regions having a hydrophobic third filter; a second member having a second through hole and a second opening at an upper portion thereof; And a transparent fourth member in which a plurality of regions are formed. Said first member, said fourth member, said second member, in the order of the third member A plurality of sample preparation units having the first area, the second area, the third area, and the fourth area, wherein the first through-hole and the second filter are provided. Oppose each other via the second through-hole, the second filter and the third filter are disposed opposite each other, and a flow path connecting the first filter and the second filter. A genetic test method using a sample preparation device in which is formed: (1) a step of injecting a buffer solution in which cells are suspended in the first opening and capturing the cells in a first filter (2) a step of injecting a denaturant into the first opening and capturing the polynucleotide eluted from the cells by the second filter; and (3) a step of applying a fluorescent label to the first opening. Of the desired nucleotide sequence of the polynucleotide, including a primer for PCR. Injecting a PCR amplification reaction solution containing reagents for the PCR amplification reaction, amplifying the copy, and holding the PCR amplification reaction solution to amplify the copy. (4) The sample preparation unit A laser beam for exciting the fluorescent label for labeling the copy in the PCR amplification reaction solution of each of the sample preparation units from a direction substantially parallel to the second filter of the sample preparation unit is applied to each of the sample preparation units. Irradiating the PCR amplification reaction solution almost simultaneously; and (5) applying the fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit to each of the sample preparation units. Detecting substantially simultaneously from a direction substantially perpendicular to the second filter.

(C 16) A first region having a first opening formed at an upper portion into which a buffer solution in which cells are suspended is injected, and a first filter formed at a lower portion to capture the cells, A plurality of first members, a second opening at the top, a polynucleotide eluted from the cells by a denaturing agent injected through the first opening, and a fluorescently labeled PCR A plurality of second regions having a second filter at a lower portion for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction containing a primer for the PCR and amplifying a copy of a portion of a desired base sequence of the polynucleotide A second member to be formed; a third member to form a plurality of third regions having a hydrophobic third filter; and control of the PCR amplification reaction solution. The first member, the second member, and the third member are arranged in this order from above, and the first region, the second region, and the third region are arranged from above. A sample preparation device having a plurality of sample preparation units having the following.

(C17) A plurality of first regions each having a first opening formed at an upper portion into which a buffer solution in which cells are suspended are injected, and a first filter formed at a lower portion to capture the cells. A first member to be formed, and a fluorescently labeled PCR having a second opening at an upper portion and capturing a polynucleotide eluted from the cells by a denaturing agent injected from the first opening. A second region having a second filter at the bottom that holds a PCR amplification reaction solution containing a PCR amplification reaction solution containing a primer for PCR amplification and a reagent for a PCR amplification reaction for amplifying a copy of the desired base sequence portion of the polynucleotide A plurality of second members, a third member having a plurality of third regions having a hydrophobic third filter, and a plurality of fourth regions having through holes formed therein; A fourth member and a means for controlling the temperature of the PCR amplification reaction solution. And from above, the first member, the fourth member, the second member, and the third member are arranged in this order, and the first filter and the second filter are arranged in this order. The second filter and the third filter are opposed to each other through a through-hole, and have a plurality of the first area, the second area, the third area, and the fourth area. A sample preparation apparatus having the sample preparation unit according to the above.

(C18) a first through-hole, a first opening formed at an upper portion thereof, into which a buffer for suspending cells is injected, and a second opening formed at a lower portion of the first opening to capture the cells. A first member having a plurality of first regions having a first filter, and a second member having a second opening at an upper portion, and a polyeluted from the cells by a denaturing agent injected from the first opening. A PCR amplification reaction solution containing a primer for PCR that captures nucleotides and containing a PCR amplification reaction that amplifies a copy of the desired base sequence portion of the polynucleotide, including a primer for PCR, is retained. A second member having a plurality of second regions formed at a lower portion thereof, a second member having a plurality of third regions having a hydrophobic third filter, and a third member having a plurality of third regions having a hydrophobic third filter. Means for controlling the amount of the PCR amplification reaction solution, and The first member, the second member, and the third member are arranged in this order, and the first through hole and the second opening face each other, and the second filter and the third filter A flow path connecting the first filter and the second filter is formed, and has a first region, a second region, and a third region. A sample preparation device having two or more sample preparation units.

(C19) a first through-hole, a first opening formed at an upper portion thereof, into which a buffer for suspending cells is injected, and a second opening formed at a lower portion of the first opening to capture the cells. A first member in which a plurality of first regions having one filter are formed; and a fluorescence-labeled PCR that captures a polynucleotide eluted from the cells by a denaturant injected from the first opening. A plurality of second regions having a second filter holding a solution containing a PCR amplification reaction reagent for a PCR amplification reaction for amplifying a copy of a portion of the desired nucleotide sequence of the polynucleotide containing primers for the polynucleotide are formed. A second member, a third member having a plurality of third regions having a third filter having a g¾ property, a fourth member having a second through-hole, and a transparent fourth member having a second opening at the top. Area force, the plurality of fourth members formed and the PCR Means for controlling the temperature of the reaction reagent, wherein the first member, the fourth member, the second member, and the third member are arranged in this order from above, and the first member The through-hole and the second filter face each other via the second through-hole, and the second filter and the third filter are arranged to face each other with a force, and the first filter and the second filter are arranged opposite to each other. A flow path connecting the second filter to the second filter; and a plurality of sample preparation units having the first area, the second area, the third area, and the fourth area. Sample preparation device.

(C 20) a flow path connecting an upper opening into which a buffer solution in which cells are suspended is injected, and a lower opening through which waste liquid flows out, wherein the flow path extends from the upper opening to the lower opening. A first filter that captures the cells, and a PCR primer that captures a polynucleotide eluted from the cells by a denaturant injected from the upper opening and is fluorescently labeled. PCR amplification reaction for amplifying a copy of the desired nucleotide sequence portion of the polynucleotide A sample preparation unit having a second filter for holding a PCR amplification reaction solution containing reagents for use in a sample, a hydrophobic third filter, holding means for holding the sample preparation unit, and the PCR amplification reaction solution Means for controlling the temperature of the sample. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a perspective view showing a configuration example of a sample preparation device according to a first embodiment of the present invention.

FIG. 2 is a sectional view showing an AA ′ section in FIG. 1 of the present invention. FIG. 3 is a cross-sectional view illustrating steps of a method of purifying a whole blood sample using the sample preparation device according to the first embodiment of the present invention.

FIG. 4 is a diagram showing the principle of fluorescence resonance energy transfer used in the first embodiment of the present invention.

FIG. 5 is a cross-sectional view showing a configuration example of an arrested child detecting device for detecting the amplification process of a target arrested child using an optical fiber in real time in the first embodiment of the present invention.

FIG. 6 is a cross-sectional view showing a configuration example of a gene detection device using an optical fiber covered with a sheath having a blade-shaped tip in the configuration example of FIG. 5 of the present invention. FIG. 7 is a cross-sectional view showing an example of the configuration of a gene detection device that irradiates a laser beam from below the sample preparation unit and detects the amplification process of a target gene in real time in the first embodiment of the present invention. It is.

FIG. 8 is a cross-sectional view showing an example of a configuration of a gene detection device that irradiates a laser from a lateral direction of a sample preparation unit and detects an amplification process of a target gene in real time in the first embodiment of the present invention. is there.

FIG. 9 is a perspective view showing a configuration example of a sample preparation device having a plurality of sample preparation units in the second embodiment of the present invention.

FIG. 10 is a cross-sectional view showing an example of the configuration of an arrested child detecting device for detecting the amplification process of a target gene in real time in the second embodiment of the present invention. Fig. 11 shows the configuration of an abductor detection device that irradiates a laser to a plurality of sample preparation units from the lateral direction and detects the amplification process of the target gene in real time in the second embodiment of the present invention. It is a perspective view showing an example.

FIG. 12 is a cross-sectional view showing a configuration example of a laser irradiation and fluorescence detection system for performing two-dimensional scanning used in the gene diagnosis apparatus according to the second embodiment of the present invention.

FIG. 13 is a cross-sectional view showing a configuration example of a laser irradiation and fluorescence detection system for performing one-dimensional scanning used in the gene diagnosis apparatus according to the second embodiment of the present invention.

FIG. 14 is a perspective view showing a configuration example of a sample preparation knit in the third embodiment of the present invention.

FIG. 15 shows a method of purifying a whole blood sample relating to a plurality of samples and a step of detecting an amplification product of a target gene in a fourth embodiment of the present invention using a cassette type sample preparation device. FIG.

FIG. 16 is a perspective view showing a configuration example of a sample preparation device having a sample preparation unit for simultaneously processing a plurality of samples in the fifth embodiment of the present invention.

FIG. 17 is a sectional view showing an AA ′ section in FIG. 16 of the present invention. FIG. 18 is a view showing the structure of each layer constituting a sample preparation apparatus according to a fifth embodiment of the present invention.

FIG. 19 is a perspective view showing a configuration example of a sample preparation device for simultaneously processing a plurality of samples in the sixth embodiment of the present invention.

FIG. 20 is a sectional view showing an AA ′ section in FIG. 19 of the present invention. FIG. 21 is a view showing the structure of each layer constituting a sample preparation apparatus according to a sixth embodiment of the present invention.

FIG. 22 is a perspective view showing a modified configuration example of the sample preparation device according to the sixth embodiment of the present invention.

FIG. 23 is a perspective view showing a configuration example of a sample preparation device for simultaneously processing a plurality of samples in the seventh embodiment of the present invention.

FIG. 24 is a sectional view showing an AA ′ section in FIG. 23 of the present invention. FIG. 25 shows the structure of each layer constituting the sample preparation apparatus according to the seventh embodiment of the present invention. FIG.

FIG. 26 is a plan view showing a configuration example of a sample preparation apparatus for simultaneously processing a plurality of samples in the eighth embodiment of the present invention.

FIG. 27 is a sectional view showing a configuration example of a sample preparation device according to an eighth embodiment of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments will be described in detail with reference to the drawings.

(First embodiment)

Pretreatment of sample blood>

Whole blood is collected from lung cancer patients or cancer screening examinees. Blood collection volume is 1 mL

(Milliliters) is sufficient, and the collected whole blood is subjected to anticoagulation with sodium citrate.

FIG. 1 is a perspective view showing a configuration example of a sample preparation apparatus according to an embodiment of the present invention, and FIG. 2 is a cross-sectional view showing a cross section taken along the line AA ′ in FIG. 1 of the present invention. Fig. 1,

As shown in Fig. 2, the sample preparation device 50 is composed of a sample preparation unit 100, a holding member 200 for holding the sample preparation unit 100, and a heat block 210 force. The sample preparation unit 100 has a rotationally symmetric axis and an upper hollow cylinder with a large first radius, a lower hollow cylinder with a small second radius, and a pot neck. The upper hollow cylindrical part and the bottle neck part of the sample preparation unit are stored in the hollow part formed in the holding member 200. The lower hollow cylindrical part of the sample preparation unit is connected to the hollow part formed in the heat block 210.

To be done. In the sample preparation unit shown in FIG. 1, a filter support 120 supporting the first filter 110, a filter support supporting the second filter 130, and a filter support supporting the third filter 140 are provided. The body 150 is omitted.

The sample preparation unit 100 has a flow path connecting an upper opening into which a buffer solution in which cells are suspended is injected, and a lower opening through which waste liquid flows out. A first filter (cell capture filter) 110 that captures target cells such as leukocytes, which are sequentially arranged in the channel in the direction of the mouth, and a denaturing agent injected from the upper opening. PCR amplification that captures polynucleotide eluted from cells and contains reagents for PCR amplification reaction, which includes a primer for PCR labeled with fluorescence and amplifies a copy of the desired base sequence portion of the polynucleotide It comprises a second filter (polynucleotide capture filter) 130 for holding the reaction solution, and a hydrophobic third filter (liquid holding filter for holding the PCR amplification reaction solution) 140.

To capture target cells such as leukocytes, the thickness of the first filter should be 0.05: 11111 to 0.5 mm, and the pore size should be 1 μπι to 3 μπι. In order to capture the polynucleotide eluted from the cells and retain the PCR amplification reaction solution, the thickness of the second filter is changed from 0.1 mm to 0.5 mm, the pore size is 10 μm, and the pore size is 40 μπι. I do. In order to hold the PCR amplification reaction solution, the thickness of the third filter should be 0.1 mm to 0.5 mm, and the pore size should be 10 μπι to 40 μπι. The first filter 110 is supported by a filter support 120, and the second filter 130 and the third filter 140 are supported by a filter support 150 made of plastic or the like. The filter supports 120, 140 maintain a constant flow path for the liquid poured from the top of the sample preparation unit, and when pressure is applied to the first, second, and third filters, Prevent damage to the second and third filters.

The first filter 110 and the filter support 120 are located in the upper hollow cylinder of the sample preparation unit, and the second filter 130, the third filter 140, and the filter support 150 are located in the lower part of the sample preparation unit. It is arranged in the hollow cylindrical part. The heat block 210 includes a peltier that raises and lowers the temperature of the solution held in the second filter 130 disposed in the lower hollow cylindrical portion of the sample preparation unit and in the upper space of the second filter 130. The device is embedded.

The dimensions of the sample preparation device 50 shown in Figs. 1 and 2 in the χ, y, and z directions are For example, they are 25 mm, 25 mm, and 60 mm, respectively. The dimensions of the holding member 200 and the heat block 210 in the z direction are 50 mm and 10 mm, respectively. The outer diameter and inner diameter of the upper hollow cylinder of sample preparation unit 100 are, for example, 20 mm and 18 mm, respectively, and the outer diameter and inner diameter of the lower hollow cylinder are 10 mm and 8 mm, respectively. is there.

FIG. 3 is a cross-sectional view showing the steps of a method for purifying a whole blood sample using the sample preparation device according to the embodiment of the present invention. In FIG. 3, the holding member 200 for holding the sample preparation knit 100, the third filter 140, and the heat block 210 (except for step-6) are omitted for simplicity. To destroy erythrocytes in the collected whole blood by osmotic pressure, add 9 mL of 50 mM NaCl solution to 1 mL of blood sample and stir. Immediately after stirring, pour 1 OmL of the solution 300 containing the disrupted red blood cells into the space above the first filter 110 of the sample preparation unit 100 (step-1).

Due to gravity, the solution 300 passes through the first filter 110 and flows down into the space below the first filter 110, but most of the solution 300 does not pass through the first filter 110 by gravity alone. Then, a pressure is applied from above the sample preparation unit 100 to pass the solution 300 through the first filter I10. At this time, only the white blood cells 302 in the solution 300 are captured by the first filter 110. The solution that has passed through the first filter 110 passes through the space below the first filter 110, the second filter 130, and the third filter 140, and flows out to the waste liquid tank. Instead of applying pressure for passing the solution through the first, second, and third filters, centrifugal force generation, suction from the lower opening, etc. can also be used. Subsequently, 3 mL of the denaturing agent 3I0 is poured into the upper space of the first filter 110 (step-2).

As in step-1, a pressure is applied from above the sample preparation unit 100 to pass the denaturant 310 through the first filter 110. At this time, the membrane force of the leukocyte 302 captured by the first filter 110 was 'disrupted, and the force of the component other than RNA320 was 304', remained in the first filter 110, and exited from the leukocyte 302. RNA3 20 passes through the lower space of the first filter 110 and is captured by the second filter 130. The denaturing agent 310 passes through the third filter 140 and flows out to the waste tank. For the second filter I30, use a thicker filter so that RNA can be sufficiently retained. For example, let the thickness of the second filter 130 be lmm. As described above, only the total RNA 320 can be captured by the second filter 130 from the collected whole blood (step-3).

Amplify target gene from t o t al RNA>

Using t0ta1 RNA purified from the collected whole blood, PCR amplification of the target arrested child for diagnosis is performed. According to the presence or absence of the amplified product of the gene, or the amount of amplification of the product, the diagnosis of the presence or absence or progress of cancer metastasis of the examinee can be performed. Prepare a PCR amplification solution containing a PCR buffer solution, two primers for amplifying the target gene, and four deoxynucleotides in advance. Pour 50 μL of the PCR amplification reaction solution 340 into the space above the first filter 110 (step-4).

Subsequently, pressure is applied from above the sample preparation knit 100. At this time, reduce the applied pressure and adjust the applied pressure so that the PCR amplification reaction solution has 340 power, 'the first filter 110 can pass,' and the second filter 130 does not pass. . Due to the adjustment of the applied pressure, the PCR amplification reaction solution is maintained at a pressure of 340 、, soaked in the second filter 130, and excessive PCR amplification reaction that cannot be retained in the second filter 130. The solution 330 in which the solution and the total RNA 320 are mixed is held in the lower space of the first filter and in the space above the second filter 130. The solution 330 containing the ttal RNA 320 does not leak out of the sample preparation unit due to the hydrophobic third filter 140 (step-5).

In the PCR, the heating block 210 controls the rise and fall of the temperature of the solution held in the second filter 130 and in the space above the second filter 130. In the solution 330 held in the space above the second filter 1 30, and the third filter 140, the PCR amplification of the target arrestor was performed. Is performed to obtain a solution containing PCR amplification product 335 (step-6). <Real-time detection of PCR products>

In the first method of real-time detection of PCR amplification products, a fluorescent substance that preferentially binds to newly synthesized (amplified) double-stranded DNA and greatly enhances the fluorescence intensity due to the binding is used as a PC. It is mixed in advance with the R amplification reaction solution 340. As the phosphor, for example, SYBR Green I (Molecubal Co., Ltd.) is used. Since SYBR Green I binds to newly synthesized (amplified) double-stranded DNA, the fluorescence intensity and DNA concentration are directly proportional, and the PCR amplification product can be detected quantitatively. The amplification process of the PCR product can be monitored in real time by detecting the fluorescence intensity during the PCR amplification reaction.

In the second method of real-time detection of PCR amplification products, the fluorescence resonance energy-transfer (Fl uor e s c e n c e Re c o r n an c e E n e r gy

T ran s f er (FRET) is used.

FIG. 4 is a diagram illustrating the principle of fluorescence resonance energy transfer used in the example of the present invention. FRET only occurs when the two probes hybridize to the amplification product in close proximity. The denatured DNA type 21 becomes denatured by denaturation, and the 3 'end is dyed with a donor dye (F 1) 22, and the labeled hybridization probe 23 anneals, and the hybridization probe 23 Adjacent to, a hybridization probe 25, labeled with an acceptor dye (F2) 24 at the 5 'end, hybridizes. The donor dye (F 1) 22 is excited by light 26 from an LED (Light Emitting Diode), and the donor dye (F 1) 22 is converted to an acceptor dye (F 1). 2) Emit light of a wavelength that excites 24. The secondary dye (F 2) 24 emits a longer wavelength light 27 by secondary excitation.

If the hybridization probes 23 and 25 do not have specific DNAs that hybridize adjacent to each other, the FRET phenomenon does not occur. As the PCR product increases, the amount of probe that hybridizes to type I DNA that has become single-stranded due to denaturation increases. The intensity of light 27 by FRET is Hybridization probe 23, 25 Force proportional to the amount of specific DNA PCR product that hybridizes adjacent to each other. By detecting light 27 by FRET, the amplification process of the PCR product can be monitored in real time.

FIG. 5 is a cross-sectional view showing a configuration example of an arrested child detecting device that detects an amplification process of a target gene in real time using an optical fiber in the first embodiment of the present invention. . The optical fiber 131 is inserted from the upper part of the sample preparation unit 100, pressure is applied to the center of the first filter 110, the filter is pierced, a hole is made, and the fiber 31 is passed through the upper part of the second filter 130. PCR amplification products retained in

Advance the optical fiber 31 to the point where the surface of the solution containing 335 is slightly above the level. Next, a laser 380 emitted from a light source 370 is applied to a dichroic mirror.

Reflect at 5 and irradiate the solution containing PCR amplification product 335 through fiber 31. Fluorescence is generated from the fluorescent substance that labels the PCR amplification product by the laser 380, and the fluorescence transmitted through the dichroic mirror 385 is transmitted to the CCD camera.

Detected by 400.

FIG. 6 is a cross-sectional view showing a configuration example of a far element detector using an optical fiber covered with a sheath having a blade-shaped tip in the configuration example of FIG. 5 of the present invention. The optical fiber 131 having an outer diameter of 100 zm is disposed inside a hollow cylinder of a sheath 42 having an outer diameter of 140 μπι and an inner diameter of 120 m, and the tip of the sheath 42 crosses the axis of the hollow cylinder. The surface is cut to form a blade-shaped tip 41. The configuration example shown in FIG. 6 is the same as the configuration example shown in FIG. 5, except that the optical fiber 31 shown in FIG. 5 is covered with a sheath 42 having a blade-shaped tip 41. Break the filter with a sheath 42 in the center of the first filter 110 and make a hole, and pass the sheath 42 to the top of the solution containing the PCR amplification product 335. In this state, the optical fiber 31 is protruded from the inside of the sheath 42 to the outside of the sheath 42, and the fiber 31 is brought close to the position where the surface of the solution containing the PCR amplification product 335 is slightly above. Detection of fluorescence generated by laser irradiation is performed in the same manner as in Fig. 5.

The amplification process of the target arrestor can be detected in real time without using optical fiber. Can also be issued.

FIG. 7 shows a configuration of a far-gene detector for irradiating a laser from below the sample preparation unit and detecting an amplification process of a target gene in real time in the first embodiment of the present invention. It is sectional drawing which shows an example. The laser 380 was applied from below the sample preparation unit 100 and the laser 380 was transmitted through the third filter 140. The PCR amplification product 3 held in the second filter 130 was used. Fluorescence from the fluorophore, which irradiates the solution containing 35 and labels the PCR amplification product, is released by the photomultiplier tube 39.0 placed below the lower opening of the sample preparation unit. Can be detected in real time.

Fig. 8 shows an example of the configuration of a gene detector that irradiates a laser beam from the side of the sample preparation unit and detects the amplification process of the target gene in real time in the first embodiment of the present invention. FIG. In the configuration example shown in Fig. 8, the holding member 200 "that holds the sample preparation unit 100 is made of a transparent material, and the laser 180 is used for the second filter 130. The solution containing the PCR amplification product 335 held in the upper part is irradiated. When the holding member 200 "is made of an opaque material, a hole through which the laser 380 passes is opened. It is good. As in the case of Fig. 7, the fluorescence 395 from the fluorescent substance labeling the PCR amplification product is supplied in real time by the photomultiplier tube 390 arranged below the lower opening of the sample preparation unit. Can be detected. The portion of the sample preparation unit 100 to be irradiated with the laser is made of a transparent material.

In FIGS. 7 and 8, the second filter 130 and the third filter 140 are desirably transparent. Even if the second filter 130 and the third filter 140 are not transparent, the laser 380 power passing through the hole of the third filter 140, ', the hole of the second filter 130 , The solution containing the PCR amplification product 335 is irradiated, and the fluorescence 395 from the fluorescent substance that labels the PCR amplification product, the pore of the second filter 130, and the third filter After passing through the hole of 140, the fluorescence power reaches the photomultiplier tube 39, so that the fluorescence can be easily detected in real time. Instead of detecting the PCR product in real time, the product may be detected after PCR amplification. Apply pressure from the top of the sample preparation unit and collect the solution after PCR amplification in the collection container located at the lower opening of the sample preparation unit. By irradiating the collection container with a laser and detecting the fluorescence from the fluorescent substance that labels the PCR amplification product, the PCR amplification product can be quantified. As explained above, by quantitatively analyzing the PCR amplification process of the target gene for diagnosis in real time, or by quantifying the amplification product after the PCR amplification reaction, the metastasis of lung cancer, etc., can be improved. It can be applied to the diagnosis of the presence or absence of metastasis.

(Second embodiment)

FIG. 9 is a perspective view showing a configuration example of a sample preparation device having a plurality of sample preparation units in the second embodiment of the present invention. The sample preparation device 50 'shown in Fig. 9 has a plurality of sample preparation units 50 shown in Figs. 1 and 2 in two crossing directions (Fig. 9 shows an example of 9 units for simplicity). ) It is an integrated configuration. The sample preparation device 50 'includes a holding member 200' for holding a plurality of sample preparation units 100 and a heat block 210 '. The upper hollow cylindrical part and the bottle neck part of each sample preparation unit 100 are stored in the hollow part formed in the holding member 200 '. The lower hollow cylindrical part of each sample preparation unit is housed in the hollow part formed in the heat block 210 '. The size of the sample preparation device 50 ', which has 10 sample preparation units 50 shown in Figs. 1 and 2 in the X and y directions, respectively, is 250 mm x 250 mm.

In the sample preparation device 50 'shown in Fig. 9, the purification of t0ta1 RNA from the blood of the sample, amplification of the target gene from total RNA, PCR Each process of real-time detection of products can be processed simultaneously for multiple samples.

Purification of total RNA from sample blood was carried out in the same manner as in the first embodiment, and the operation of purifying t0ta1 RNA from each sample blood was performed in parallel with each sample preparation unit 100 independently. Perform at the same time. Arrest for purpose from total RNA Amplification of the gene is performed in the same manner as in the first embodiment, in the liquid held above the second filter 130 and the third filter 140 of each sample preparation unit 100. Perform PCR amplification of the gene.

FIG. 10 is a cross-sectional view showing a configuration example of a gene detection device for detecting the amplification process of a target gene in real time. With the same configuration as in Fig. 5 or 6, each optical fiber 191, from the top of the sample preparation unit, is added to the solution containing the PCR amplification product 335 held in the second filter 130. Make contact. The solution containing the PCR amplification product 335 is irradiated with a laser 380 from a light source 370. The laser 380 is incident on each optical fiber 191 by a beam splitter composed of an array of dichroic mirrors 385. The fluorescence generated from the solution containing the PCR amplification product 335 is collected by the condenser lens 96 via the dichroic mirror 385 and detected by the CCD camera 400. The laser 380 shown in Fig. 10 is simultaneously irradiated on each of the three sample preparation units arranged in each of the three rows in the y direction shown in Fig. 9, and each sample in each row in the y direction is irradiated. Fluorescence from the preparation unit is detected simultaneously in real time.

FIG. 11 shows a second embodiment of the present invention in which a plurality of sample preparation units of a sample preparation apparatus 50 "are irradiated with a laser from a lateral direction to detect a gene amplification process of a target gene in real time. Fig. 9 is a perspective view showing an example of the configuration of a detection device, similarly to the example of the configuration shown in Fig. 8, the holding member 200 "holding sample preparation knit 100 is made of a transparent material. When the holding member 200 "is made of an opaque material, a hole through which the laser beam 180 passes may be formed. The portion of the sample preparation unit 100 to be irradiated with the laser beam may be used. It is composed of a transparent material.

The laser 380 is simultaneously irradiated by the dichroic mirror 385 onto three sample preparation knits 100 arranged in each of three rows parallel to the y-axis. The solution containing the PCR amplification product 335 held at the top of the second filter 130 is irradiated. In each sample preparation unit 100, the fluorescence 395 from the fluorescent substance labeling the PCR amplification product was measured by the sample preparation device 50 " It is detected in real time by the CCD camera 400 arranged below the lower opening of the camera. The CCD camera 400 may be provided to detect the fluorescence 395 from the upper opening of the sample preparation device 50 ".

In FIG. 11, the second filter 130 and the third filter 140 of each sample preparation unit 100 are desirably transparent. Even if the second filter 130 and the third filter 140 are not transparent, the laser 380 that has passed through the hole of the third filter 140, passes through the hole of the second filter 130, and the PCR amplification product 335 Irradiating the solution containing the fluorescein 395 from the fluorescent substance that labels the PCR amplification product, passes through the pores of the second filter 130 and the pores of the third filter 140, and the fluorescence is passed through the photomultiplier tube 390. , Fluorescence detection can be easily performed in real time.

Since the sample preparation unit 50 'shown in Fig. 11 can simultaneously irradiate a plurality of sample preparation units with laser at the same time, the PCR amplification process of the target gene for diagnosis in multiple sample preparation units can be performed in real time. Can be analyzed at the same time. Instead of the laser irradiation system including the light source 370 shown in Fig. 11 and the CCD camera 400 for fluorescence detection, the laser irradiation and fluorescence detection system shown in Fig. 12 is two-dimensionally scanned in the X and y directions, or The laser irradiation and fluorescence detection system shown in Fig. 13 and the fluorescence detection system are one-dimensionally scanned in the X or y direction, and a laser is irradiated from the upper opening of each sample preparation unit, and the fluorescence is emitted from each sample preparation unit. It can be detected from the upper opening. Needless to say, the plurality of photomultiplier tubes 390 shown in FIG. 13 may be replaced with the CCD camera 400.

As described above, the PCR amplification process of the target gene for diagnosis is quantitatively analyzed in real time for a plurality of samples, and the presence or absence of metastasis of lung cancer or the like, Applicable to diagnosis.

(Third embodiment)

Instead of the sample preparation knit 100 described in the first and second embodiments, a sample preparation knit 100 ′ shown in FIG. 14 can be used. Sample preparation unit 100 'has a rotationally symmetric axis and a square cross section with large sides It consists of an upper hollow cylinder, a lower hollow cylinder with a square cross section with small sides, and a neck connecting the upper hollow cylinder and the lower hollow cylinder. The upper hollow cylinder and neck of the sample preparation unit are housed in a hollow formed in the holding member. The lower hollow cylinder of the sample preparation unit is housed in the hollow formed in the heat block. The height of the sample preparation unit 100 'is the same as the height of the sample preparation unit 100, and the cross-sectional areas of the upper and lower hollow cylinders are 16 mm x 1 respectively. 6 mm, 5 mm X 5 mm.

(Fourth embodiment)

The pretreatment of the sample blood is performed in the same manner as in the first embodiment.

FIG. 15 shows a method of purifying a whole blood sample relating to a plurality of samples using a cassette type sample preparation apparatus and a step of detecting an amplification product of a target arrestor in the fourth embodiment of the present invention. FIG. To 1 niL of the collected blood sample for each sample, add 9 mL of a 50 mM NaCl solution and stir. Immediately after stirring, the solution for each sample was applied to the first filter 110, and the filter support 120 supporting the first filter, forming a plurality of gelkers attached to the bottom. Pour into separate wells of the first cassette 800. A separate sample is prepared for each well. Apply gas pressure from the top of the first cassette 800, or aspirate from the bottom of the first cassette 800, and capture only the leukocytes in the solution with the first filter.

Subsequently, the second filter, the third filter, and the filter support for supporting the second and third filters are provided at the bottom of the well formed corresponding to the well of the first cassette 800. Set the second cassette 8100 with the body 150 (not shown) mounted under the first cassette 800. Next, a denaturant is poured into the first cassette 800. Gas pressure is applied from the upper side of the first cassette 800 or suction is performed from the lower side of the second cassette 800 to pass the denaturant through the first and second filters. From the whole blood collected from each sample, the total RNA alone was collected independently for each sample from the second cassette 8100 in a separate well. The second filter 130 captures.

Amplify target arrested child from tota RNA>

Amplify the target gene for diagnosis using the purified t0ta1 RNA. After pouring the PCR amplification reaction solution into each well of the second cassette 810 in which the third cassette 820 for PCR and detection of amplified products is mounted at the bottom, gas was injected from the top of the second cassette 800 When pressure is applied or suction is performed from below the third force set 820, the RNA force captured by the second filter 130 of the second cassette 810, ', the second filter 1 30 When the PCR amplification reaction solution passes through, elute into the PCR amplification reaction solution and move to each well of the third cassette 820. Subsequently, only the third cassette 820 is set in the heat block 210 '(not shown in FIG. 15), and PCR of the target gene is performed.

Detection of PCR products>

After completion of PCR, each well of the third cassette 820 is irradiated with laser using the laser irradiation and fluorescence detection system shown in FIG. 12 or FIG. Detection and quantification of amplification products of arrested children.

(Fifth embodiment)

FIG. 16 is a perspective view showing a configuration example of a sample preparation apparatus having a sample preparation unit for simultaneously processing a plurality of specimens in the fifth embodiment of the present invention, and FIG. FIG. 18 is a cross-sectional view showing a cross section taken along the line AA ′ in FIG. 18, and FIG. It goes without saying that the configuration shown in FIGS. 16 to 18 may be configured as a sample preparation device having one sample preparation unit.

The sample preparation device 550-1 according to the fifth embodiment includes a first substrate 500, a second substrate 520, a third substrate 530, and a fourth # 540. In the sample preparation device 550-1, a plurality of sample preparation units are formed in a two-dimensional unit, and each sample preparation unit prepares a different sample.

The first substrate 500 contains a solution containing the destroyed red blood cells (the first Upper openings 5 11 1–1, 5 12–1, 5, 13–1, and 5 14–1, which are obtained by the same operation as in Example 1) are separately injected for each sample. A first filter and a force are formed at the bottom of each opening. In the second substrate 52 0, upper openings 5 11 1-1, 5 12-1, 5 13-1, 5 14-1, upper openings 5 11 1-2,

5 1 2—2, 5 1 3—2, 5 1 4—2, and a second filter and force are formed at the bottom of each opening. In the third substrate 5300, upper openings 5 1 1—2, 5 1 2−2,

A third filter is formed at the upper part of the bottom of each of the openings of 5 1 3—2 and 5 14−2 so as to face the second filter, and a lower opening of the bottom of each third filter 5 1 1—3 ,

5 1 2—3, 5 1 3—3, 5 1 4—3 are formed. The fourth 540 has through-holes 5 1 1–4, 5 1 2-facing the lower openings 5 1 1–3, 5 1 2–3, 5 1 3–3, 5 1 4–3 4, 5 1 3 -4, 5 1 4—4 ヽ are formed. The fourth substrate 540 includes a Peltier device for controlling the temperature of the solution held in each of the second filters and in the upper space of each of the second filters by increasing or decreasing the temperature. Force, 'embedded.

The first to fourth substrates can be obtained by processing with silicon liquid or gas phase etching. For example, the thickness of the first substrate is 500 μπι, and the thickness of the first filter is 1! 110 μπι, the first filter has a plurality of square holes with a cross section of (1! ~ 3 fim) X (1! ~ 3 m), and the thickness of the second substrate is 500 μπι ， The thickness of the second filter is 10! ~ 100 μπι, the second filter has a square cross section of about 10 mX and about 10 im, and the thickness of the third substrate is 50 O ^ m. The thickness is 1 Ο ^ π! 1100 βπι, The third filter has a hydrophobic surface and a plurality of square holes with a cross-sectional area of (2 μπ! ~ 3 m) X (2 μπ! ~ 3 ^ m). The areas of the bottoms of the openings of the first, second, and third substrates are 500 μπιχ 500 μπι, 500 Mm x 500 / m, 500 μm 500 μπι, respectively. The cross-sectional area of the through-hole of the substrate is 500 μπιΧ 500 μπι. The first, second, third, and fourth substrates are sequentially laminated and integrated. The size of the sample preparation device 550-1 composed of 20 sample preparation units in each of the X and y directions is 20 mm x 20mm.

Purification of total RNA from sample blood and amplification of the target gene from total RNA are performed in the same manner as in the first example. The solutions containing PCR amplification products 571, 572, 573, and 574 held in each second filter and in the space above each second filter were irradiated with the laser irradiation and fluorescence detection shown in Fig. 12. Scan the system two-dimensionally in the X and y directions, or scan the laser-irradiation and fluorescence detection system shown in Fig. 13 one-dimensionally in the _x or y direction to obtain

As shown in Fig. 7, laser 380 is irradiated, and the fluorescence generated from the solution containing the PCR amplification product is detected from the direction of laser 380 irradiation. Further, the solution containing the PCR amplification product held in the second filter and in the space above the second filter from each opening of the first から ^ is irradiated with the laser 380, and the fourth substrate is irradiated. Fluorescence may be detected from the opening of, or conversely, the laser 380 may be irradiated from each opening of the fourth ΔK to detect fluorescence from the opening of the first. Further, in the same manner as in the configuration shown in Fig. 5, 6, or 10, a hole is made in the first filter, and the optical fiber is held in the space above the second filter. The fluorescence may be detected in real time by irradiating the laser with the solution containing the product as close as possible.

(Sixth embodiment)

FIG. 19 is a perspective view showing an example of the configuration of a sample preparation apparatus for simultaneously processing a plurality of specimens in the sixth embodiment of the present invention, and FIG. FIG. 21 is a cross-sectional view showing a section taken along the line AA ′. FIG. 21 is a diagram showing the structure of each layer constituting the sample preparation apparatus of the sixth embodiment. It goes without saying that the configuration shown in FIGS. 19 to 21 may be configured as a sample preparation device having one sample preparation unit.

As in the fifth embodiment, in the sample preparation device 550-2, a plurality of sample preparation units are integrally formed in a two-dimensional manner, and each sample preparation unit performs sample preparation for a different sample. In the configuration of the sample preparation device 550-2 of the sixth embodiment, the first substrate 500 in the configuration of the sample preparation device 550-1 of the fifth embodiment is not used. A transparent fifth ¾ig510 is arranged between the second substrate 520 and the second substrate 520. The fifth ¾R5 10 has a through hole facing the first filter of the first substrate 500 and the upper openings 5 1 1—2, 5 12-2, 5 13-2, and 5 14-2 of the second substrate. It has holes 5 1 1-5, 5 12-5, 5 13-5, 5 14-5. The thickness of the fifth substrate 510 is 500 m, and the cross-sectional area of the through hole is δ Ο Ο μιηΧ δ Ο θ ίπι.

Purification of the total RNA from the sample blood and amplification of the target gene from the total RNA are performed in the same manner as in the first embodiment. Using the laser irradiation system shown in Fig. 11, a laser 380 is applied from the side of the fifth substrate 510 (from the y direction) to the space above each second filter as shown in Fig. 20. Irradiate the solution containing the retained PCR amplification product 57 1, 572, 573, 574

In the same manner as the configuration shown in Fig. 1, fluorescence 395 is detected from below the sample preparation device 550-2 by the CCD force camera 400 through the third and fourth openings. The fluorescence 395 may be detected from above the sample preparation device 550-12 by the CCD camera 400 through the first opening.

FIG. 22 is a perspective view showing a modified configuration example of the sample preparation device according to the sixth embodiment of the present invention. In the configuration of the sample preparation device 550-3 shown in Fig. 22, a plurality of sample preparation units are formed integrally in one dimension, and sample preparation of different samples is performed in each sample preparation unit. As shown in FIG. 22, a laser 380 is applied from the side of the fifth substrate 510 (from the X direction) to a solution 571,572 containing a PCR amplification product held in the space above each second filter. , 573, and 574, and the CCD camera 400 detects the fluorescence 395 from the side (from the y direction) of the fifth ¾K510, which crosses the irradiation direction of the laser. The fluorescence 395 may be detected from above or below the sample preparation device 550-3. In the configuration shown in Fig. 22, it goes without saying that the configuration of a sample preparation device having one sample preparation unit may be used.

(Seventh embodiment)

FIG. 23 shows a case where a plurality of specimens are simultaneously processed in the seventh embodiment of the present invention. Fig. 24 is a perspective view showing the structure of the sample preparation apparatus, Fig. 24 is a cross-sectional view showing the AA 'section in Fig. 23, and Fig. 25 is the configuration of the sample preparation apparatus of the seventh embodiment. FIG. 3 is a diagram showing the structure of each layer. It goes without saying that the configuration shown in FIGS. 23 to 25 may be configured as a sample preparation device having one sample preparation unit.

The sample preparation device 650-1 of the seventh embodiment includes a first substrate 600, a second substrate 620, a third substrate 630, and a fourth substrate 640. In the sample preparation device 650-1, a plurality of sample preparation units are formed in a two-dimensional integrated manner, and sample preparation for different samples is performed in each sample preparation unit.

On the first substrate 600, a plurality of upper openings 6 11 1—to which a solution containing destroyed red blood cells (obtained by the same operation as in the first embodiment for each sample) is separately injected for each sample. A first filter is formed at the bottom of each opening, with 1, 6 12-1, 6 13-1, 6 14-1. In the first substrate 600, through holes 601, 602, 603, and 604 are formed adjacent to the openings 611-1-1, 612-1, 613-1-1, 614-1, respectively. ing. The second substrate 620 has an upper opening 6 11 1 2, 6 11 -1, 6 13 2-1, 6 13-1, 6 14-1 facing the upper opening 6 1 1-2, 6 14-1 of the first substrate. 1 2, 2, 1 3, 2, 2, 14-2 and the through holes 60 1, 602, 603, 604 of the first substrate are respectively opposed to the first upper openings 6 1 1-2, 6 1 2-2, 6 1 3—2, 6 14—2 Second top opening 6 1 — 2, 6 1 2 '' — 2, 6 1 3 '1 2 with second filter at bottom , 6 14 '— 2 and force,' formed. The depth of the second upper opening is greater than the depth of the first upper opening. The third substrate 630 has an upper barrier 61 1 62,6 1 2 '—2,6 1 3 一 1, 2 1 61 して 2 facing the second filter at the bottom of each opening. A third filter force is formed in the upper part, and lower openings 611-1-3, 61-2-3, 613-3, 614-3 are formed in the lower part of each third filter. The fourth substrate 640 has through-holes 6 1 1—4, 6 1 2, and 6 1 1—3, 6 1 2—3, 6 13—3, 6 6 1 3-4, 6 14 —4 forces, 'formed. In addition, the fourth substrate includes the inside and the inside of each second filter. A Peltier element for controlling the temperature in the PCR amplification by raising and lowering the temperature of the solution held in the upper space of each second filter and the second filter is embedded.

The first to fourth substrates are obtained by processing by silicon liquid or gas phase etching. For example, the thickness of the first substrate is 500, and the thickness of the first filter is 1! 110 μπι, the first filter has a plurality of square holes with a cross-sectional area of (1 μπ! ~ 3 Mm) X (1 ^ 0 1113111), and the thickness of the second ¾g is 500 μπι And the thickness of the second filter is Ιθ ίπ! ~ Ι Ο Ο μπι, the second filter has a plurality of square holes with a cross-sectional area of about 10 μπ X X about 10 m, and the thickness of the third substrate is 500 μπι, The filter thickness is 10 ^ π! ~ 100 ^ m, the third filter has a hydrophobic surface and has a plurality of square holes with a cross-sectional area of (2βΐη ~ 3μ () X (^ !!!!! ~ ^ !!!) . The area of the bottom of the opening of the first substrate is 500 μπιχ50θίπι, and the cross-sectional area of the through hole of the first substrate is 500 μπαΧ500 0ππι. The size of the sample preparation device 650-1 which is composed of 20 sample preparation units in each of the χ and y directions is 2 Omm x 2 Omm.

Purification of the total RNA from the sample blood and amplification of the target gene from the total RNA are performed in the same manner as in the first embodiment. Using the laser irradiation and fluorescence detection system shown in Fig. 12 or Fig. 13, the laser is passed through the through holes 61, 602, 603, 604 of the first substrate, and The fluorescence is detected by irradiating the solution containing the PCR amplification product held in the upper space of the filter with the solution 571, 572, 573, 574. Using the laser irradiation system shown in Fig. 11, the laser is irradiated to the solution containing the PCR amplification product through the through holes 61, 62, 603, and 604, and the fluorescence is emitted to the fourth It may be detected by the CCD camera 400 through the through hole of the substrate.

In the sample preparation device 650-1 of the seventh embodiment, the first filter and the second filter are not opposed to each other, the area of the bottom of the upper opening of the second filter is small, and the depth is small. The solution containing the PCR amplification product is subjected to TO in a very small volume and the laser is directly irradiated on the solution containing the PCR amplification product to detect the fluorescence. It has the feature of being able to detect light directly and to simultaneously analyze the PCR amplification process of the target gene for high-sensitivity and quantitative diagnosis in real time.

(Eighth embodiment)

FIG. 26 is a plan view showing a configuration example of a sample preparation apparatus for simultaneously processing a plurality of specimens in the eighth embodiment of the present invention, and FIG. 27 is a sample preparation apparatus of the eighth embodiment. It is sectional drawing which shows the structural example of a device. It goes without saying that in the configuration shown in Figs. 26 and 27, the configuration of a sample preparation device having one sample preparation unit may be used.

In the sample preparation device 650-2 of the eighth embodiment, in the configuration of the sample preparation device 650-1 of the seventh embodiment, the second substrate 620 is replaced by a transparent ¾K620a and a substrate 620b. It consists of. Substrate 620a has a first upper opening 6 1 1 -2, 6 1 opposing the upper opening 6 1 1 — 1, 6 1 2 — 1, 6 13 -1, 6 14-1 of the first substrate. 1 2-2, 613—2, 614—2, and the through holes 60 1, 602, 603, and 604 of the first substrate, respectively, and the first upper opening 6 1 1 1 2, 6 1 There are formed through holes 6 1 1 '1, 2 6 1 2'-2, 6 1 3 '1 2, 6 14'-2 connected to 2-2, 6 13-2, 6 14-2. ¾¾ 602b has a second filter force, which is formed opposite the through-holes 6 1 -2, 6 1 2 '-2, 6 1 3' -2, 614 '-2 of the substrate 602a. . By integrating ¾K620a and S¾620b, a second upper opening 6 1 1 '— 2, 6 1 2' — 2, 6 1 3 '— above the second filter on substrate 602b — 2, 6 1 'One two forces,' formed.

As shown in Fig. 26 and Fig. 27, the laser 380 is irradiated from the side of the substrate 620a, and the PCR held in the space above the second filter of each sample preparation unit of the sample preparation device. The solution 571, 572, 573, 574 containing the amplification product is irradiated, and the fluorescence is detected by the CCD camera 400 through the through hole of the first substrate. The fluorescent light may be detected by the CCD camera 400 through the through hole of the fourth substrate.

In the sample preparation device of the eighth embodiment, a plurality of sample preparation units are simultaneously operated. Laser irradiation and direct detection of fluorescence from solutions containing PCR amplification products, and the PCR amplification process of the target gene for highly sensitive and quantitative diagnosis in multiple sample preparation units. There is a feature that can be analyzed simultaneously in real time.

In the fifth to eighth embodiments described above, since the silicon substrate can be used for photolithography and etching, the first, second, and third filters can be mounted on the silicon substrate. The hole diameter, the cross-sectional area of the through-hole, the depth of the opening, the low area of the opening, etc. can be formed with very high accuracy, and a sample preparation device having multiple fine preparation units with uniform performance can be provided at low cost. In addition, a diffusion layer can be formed in the silicon substrate to form a Pn junction for detecting heater resistance and temperature, so that the temperature cycle of the PCR amplification reaction can be performed without using an external temperature control heater. It can be performed only with a silicon chip, and a small sample preparation device can be manufactured. Furthermore, since silicon has a small heat capacity, it is possible to speed up the temperature cycle in the PCR amplification reaction.

The area occupied by each sample preparation unit is, for example, 1 mm XI mm, which is sufficient. Using a 1-inch silicon substrate, a sample preparation device with about 300 sample preparation units can be used. It can be created, and it will be possible to perform a telescope inspection using a small amount of sample, about 0.5.

In the above description, an example of extracting, amplifying, and detecting RNA derived from leukocytes in whole blood has been described. However, the sample to be tested to which the present invention is applicable is not limited to leukocytes in whole blood. Needless to say, RNA extracted from cells derived from various organs mixed in whole blood or RNA extracted from cells cultured in a cell culture medium may be used.

In the sample preparation device of the present invention, a buffer solution in which whole blood is suspended, a buffer solution in which cells cultured in a cell culture solution are suspended, a buffer solution in which tissue cells are suspended, etc. Pour into the sample injection opening of the sample preparation device having the second and third filters, and then add the reagent for cell disruption, the denaturant that elutes RNA from the cells, and the PCR amplification reaction solution in this order. To purify the total RNA, PCR amplify the target gene from tota 1 RNA, and detect the target gene. Since each series of operations is performed continuously, contamination between operations can be prevented, and simultaneous processing of multiple samples can be facilitated.

Claims

The scope of the claims

1. A flow path connecting an upper opening into which a buffer solution in which cells are suspended is injected, and a lower opening through which waste liquid flows out, and sequentially from the upper opening to the lower opening in the direction of the lower opening. A first filter (110) that captures the cells, and a polynucleotide for fluorescence-labeled PCR that captures a polynucleotide eluted from the cells by a denaturant injected from the upper opening; A second filter (130) holding a PCR amplification reaction solution containing a primer and a reagent for a PCR amplification reaction for amplifying a copy of the desired base sequence portion of the polynucleotide; A sample preparation unit (100, 100 ') having a third filter (140) having different properties, and holding means (200, 2) for holding the sample preparation unit. 00 ') and a means for controlling the temperature of the PCR amplification reaction solution. A sample preparation device (50, 50 ', 50 ") comprising (210, 210') and a laser beam (380) for exciting the fluorescent label for labeling the copy. The irradiation means (370, 380, 385, 31) for irradiating the PCR amplification reaction solution from a direction substantially perpendicular or substantially parallel to the filter 2 and the fluorescent label for labeling the copy Detecting means (385, 390, 400) for detecting the fluorescence (395) of the above from a direction substantially perpendicular to the second filter.

2. The arresting child inspection device according to claim 1, wherein the sample preparation device has a plurality of the sample preparation units arranged along a line, and the irradiating means includes the laser beam. From the direction substantially parallel to the second filter of each of the sample preparation units along the straight line to the PCR amplification reaction solution of each of the sample preparation units. The fluorescence from the fluorescent label, which labels the copy in the PCR amplification reaction solution of each sample preparation unit, is simultaneously emitted from a direction substantially perpendicular to the second filter of each sample preparation unit. A genetic test characterized in that a part of the holding means to be irradiated with the laser light is constituted by a member (200 ") transparent to the wavelength of the laser light and the wavelength of the fluorescence. apparatus.

3. The arresting child inspection device according to claim 1, wherein the sample preparation device has a plurality of rows of the plurality of sample preparation units arranged along a straight line, and Means for transmitting the laser beam along the straight line of each row from a direction substantially parallel to the second filter of each sample preparation unit of each row, And irradiating the PCR amplification reaction solution at substantially the same time with the PCR amplification reaction solution. Each of the sample preparation units in each row is detected almost simultaneously from a direction substantially perpendicular to the second filter, and the portion of the holding means to which the laser light is irradiated is determined by the wavelength of the laser light and the wavelength of the laser light. Material transparent to the wavelength of fluorescence 2 0 0 ") Todenko inspection apparatus characterized by being composed.

4. The genetic test device according to claim 1, wherein the sample preparation device has a plurality of the sample preparation units arranged along a line, and the irradiation unit emits the laser beam, The PCR amplification reaction solution of each sample preparation unit is irradiated almost simultaneously from a direction substantially perpendicular to the second filter of each sample preparation unit. A genetic test apparatus, wherein the fluorescence from the fluorescent label for labeling the copy in a solution is detected almost simultaneously from a direction substantially perpendicular to the second filter of each of the sample preparation units.

5. The arresting child inspection device according to claim 1, wherein the sample preparation device has a plurality of rows of the plurality of sample preparation units arranged along a straight line, and the irradiation means. Irradiates the laser light substantially simultaneously to the PCR amplification reaction solution of each of the sample preparation units of J from a direction substantially perpendicular to the second filter of each of the sample preparation units in each of the rows, The detecting means detects the fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of the sample preparation unit in each row, the fluorescence of the sample preparation unit in each row. An arrest child inspection device characterized in that detection is performed almost simultaneously from a direction substantially perpendicular to the second filter.

6. It has a flow path connecting an upper opening into which a buffer solution in which cells are suspended is injected, and a lower opening through which waste liquid flows out, and sequentially flows from the upper opening to the lower opening in the direction of the lower opening. A first filter (110) for capturing the cell, and a polynucleotide for capturing a polynucleotide eluted from the cell by a denaturing agent injected from the upper opening; A second filter (130) for holding a PCR amplification reaction solution containing a PCR primer and a PCR amplification reaction reagent for amplifying a copy of the desired base sequence portion of the polynucleotide; And a third filter (140).

7. The sample preparation unit according to claim 6, wherein a space for holding the PCR amplification reaction solution is provided between the first filter and the second filter. Preparation unit.

8. The sample preparation unit according to claim 6, wherein the area of the second filter is smaller than the area of the first filter.

9. The sample preparation unit according to claim 6, wherein the sample preparation unit is in contact with the second filter and the third filter.

10. The sample preparation unit according to claim 6, wherein the sample preparation unit is formed of a transparent member.

1 1. The sample preparation unit according to claim 6, wherein the sample preparation unit has a rotationally symmetric axis.

12. The sample preparation unit according to claim 11, wherein the outer shape of the sample preparation unit is a circle or a square in a cross section perpendicular to the rotational symmetry axis of the sample preparation unit. A sample preparation unit characterized in that:

13. A first filter (110) and a second filter, each having a flow path connecting an upper opening and a lower opening, and sequentially arranged in the flow path in a direction from the upper opening to the lower opening. (1 30) and the third hydrophobic filter (140) A genetic test method using a sample preparation device (50, 50 ', 50 ") including one or more sample preparation units (100, 100'), comprising: (1) the sample preparation unit Injecting a buffer solution in which cells are suspended into the upper opening to capture the cells in the first filter; and (2) injecting a denaturant into the upper opening to elute from the cells. A step of capturing a polynucleotide on the second filter; and (3) a PCR for amplifying a copy of a desired nucleotide sequence portion of the polynucleotide, wherein the upper opening includes a fluorescently labeled primer for PCR. Injecting a PCR amplification reaction solution containing a reagent for an amplification reaction, holding the PCR amplification reaction solution in the second filter, and amplifying the copy; and (4) labeling the copy. To excite the fluorescent label Irradiating the PCR amplification solution with the light to detect fluorescence from the fluorescent label.

14. The genetic test method according to claim 13, wherein the laser light is applied to the PCR amplification reaction solution from a direction substantially perpendicular or substantially parallel to the second filter, and the fluorescence is A gene inspection method characterized by being detected from a direction substantially perpendicular to the second filter.

15. The genetic test method according to claim 13, wherein the sample preparation device has a plurality of the sample preparation units arranged along a straight line, and the holding means includes a plurality of the sample preparation units. The portion to be irradiated is made of a member (200 ") transparent to the wavelength of the laser light and the wavelength of the fluorescent light, and the laser light is applied to the sample preparation unit along the straight line. The PCR amplification reaction solution of each of the sample preparation units is irradiated almost simultaneously from a direction substantially parallel to the filter 2, and the copy of the PCR amplification reaction solution of each sample preparation unit is labeled. Wherein said fluorescence from said fluorescent label is detected substantially simultaneously from a direction substantially perpendicular to said second filter of each of said sample preparation units.

16. The genetic testing method according to claim 13, wherein the sample preparation device has a plurality of rows of the plurality of sample preparation units arranged along a straight line. The portion of the holding means to be irradiated with the laser beam is made of a member (200 ") transparent to the wavelength of the laser beam and the wavelength of the fluorescent light. Irradiating the PCR amplification reaction solution of each sample preparation unit of each row substantially simultaneously from a direction substantially parallel to the second filter of each sample preparation unit of each row along the straight line. And the fluorescence from the fluorescent label that labels the copy in the PCR amplification reaction solution of the sample preparation unit in each row is the second fluorescence of the sample preparation unit in each row. An arrest child inspection method characterized in that detection is performed almost simultaneously from a direction substantially perpendicular to the filter.

17. The genetic test method according to claim 13, wherein the sample preparation device has a plurality of the sample preparation units arranged along a straight line, and the laser beam The PCR amplification reaction solutions of the sample preparation units are irradiated almost simultaneously from a direction substantially perpendicular to the second filter of the sample preparation unit, and the PCR amplification reaction solutions of the sample preparation units are irradiated with the PCR amplification reaction solutions of the sample preparation units. The method of claim 6, wherein the fluorescence from the fluorescent label for labeling the copy is detected substantially simultaneously from a direction substantially perpendicular to the second filter of each of the sample preparation units.

18. The genetic testing method according to claim 13, wherein the sample preparation device has a plurality of rows of the plurality of sample preparation units arranged along a straight line, and Irradiating the PCR amplification reaction solution of each sample preparation unit of each row substantially simultaneously from a direction substantially perpendicular to the second filter of each sample preparation unit of each row; The fluorescence from the fluorescent label, which labels the copy in the PCR amplification reaction solution of the sample preparation unit, is substantially perpendicular to the second filter of each sample preparation unit in each row. An arrest child inspection method characterized by being detected almost simultaneously from a direction. 1 9. The first opening (5 11-1-1, 512-1-1, 513-1, 5 14-4; 6 11 1-) formed in the upper part where the buffer solution in which cells are suspended is injected 1, 612—1, 6 13 −1, 614—4) and a first fan formed at the bottom to capture the cells. First member (500, 600) having a filter and a second opening (5 1 1 -2, 5 1 2-2, 5 1 3—2, 5 14-2; 6 1 1 -2 , 6 1 2—2, 6 13−2, 6 1−2; 6 1 1 ′ 1 2, 6 1 2 ′ — 2, 6 1 3 ′ 1 2, 614 ′ —2) The polynucleotide eluted from the cells is captured by the denaturing agent injected from the opening (1), and a copy of the desired nucleotide sequence portion of the polynucleotide is amplified, including a fluorescently labeled primer for PCR. A second member (520, 620) having a second filter at a lower portion for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction; and a third member having a hydrophobic third filter. A member (530, 630); and means (540, 640) for controlling the temperature of the PCR amplification reaction solution. The first member, the second member, and the third member are provided from above. Samples arranged in the order of A sample preparation device comprising a unit for producing a sample.

20. The sample preparation apparatus according to claim 19, wherein the first member, the second member, and the third member are formed from a silicon substrate, and the first filter, A second filter, wherein the holes of the second filter are holes formed in the silicon substrate.

21. The sample preparation device according to claim 19, wherein the area of said second filter is smaller than the area of said first filter.

22. First opening (5 11-1, 5 12-1, 5 13-1, 5 14-4) formed at the upper part and buffered to suspend cells, and at the lower part A first member (500) formed and having a first filter for capturing the cells, and a second opening (5 1 1—2, 5 1 2—2, 5 1 3—2, 5 1 at the top) 4-2) capturing the polynucleotide eluted from the cells by the denaturing agent injected from the first opening and containing a fluorescently labeled PCR primer, the desired nucleotide sequence of the polynucleotide A second member (520) having a second filter at a lower portion for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of the portion (5), and a hydrophobic third filter Third member having (530), a transparent fourth member (510) having through holes (5 1 1-5, 5 12-5, 5 13-3, 5 1-5); Means (540) for controlling the temperature of the amplification reaction solution, wherein the first member, the fourth member, the second member, and the third member are arranged in this order from above, A plurality of sample preparation units, wherein a first filter and the second filter face each other through the through hole, and the second filter and the third filter face each other. A sample preparation device characterized by the above-mentioned.

23. The sample preparation device according to claim 22, wherein the first member, the second member, and the third member are formed from a silicon substrate, and the first filter, the first filter, 2. A sample preparation apparatus comprising: a second filter; a pore force of the second filter; and a hole formed in the silicon substrate.

24. The sample preparation device according to claim 22, wherein the area of the second filter is smaller than the area of the first filter.

25. The first through hole (601, 602, 603, 604) and the first opening (61 1—1, 6 1 2 -

A first member (600) having a first filter formed at a lower portion of the first opening to capture the cells, and a first member (600) at an upper portion thereof. Opening of 2 (6 1 1–2, 6 1 2–2, 6 1 3—2, 6 14—2; 6 1 1 '— 2, 6, 1 '-2) capturing the polynucleotide eluted from the cells with a denaturing agent injected from the first opening, comprising a fluorescently labeled primer for PCR, A second member having, at the bottom, a second filter holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of a desired base sequence portion

(620), a third member (630) having a hydrophobic third filter, and means (640) for controlling the temperature of the PCR amplification reaction solution. The first member, the second member, and the third member are arranged in this order, and the first through-hole and the second opening (6 1 1′—2, 6 1 2′-2, 6 1 3 ' The second filter and the third filter are arranged to face each other, and the flow connecting the first filter and the second filter is arranged. A sample preparation device comprising a plurality of sample preparation units in which a passage is formed.

26. The sample preparation apparatus according to claim 25, wherein the first member, the second member, and the third member are formed from a silicon substrate, and wherein the first filter, the first filter, 2. The sample preparation apparatus according to claim 2, wherein the holes of the second filter and the second filter are holes formed in the silicon substrate.

27. The sample preparation apparatus according to claim 25, wherein the area of the second filter is smaller than the area of the first filter.

28. The first through hole (601, 602, 603, 604) and the first opening (61 1—1, 6 1 2 -

A first member (600) comprising: a first filter (600) formed below the first opening to capture the cells; PCR that captures the polynucleotide eluted from the cells with a denaturing agent injected from the opening of the above, and amplifies a copy of the desired nucleotide sequence portion of the polynucleotide including a primer for fluorescently labeled PCR. A second member (620b) having a second filter for holding a solution containing a PCR amplification reaction reagent for the amplification reaction, a third member (630) having a hydrophobic third filter, The second through hole (6 1 1, 1 2, 6 1 2 '— 2, 6 1 3' — 2, 614 '1 2) and the second opening (6 1 1-2, 6 1 2) -2, 613-2, 614-12), and a means (640) for controlling the temperature of the PCR amplification reaction reagent. From, before The first member, said fourth member, said second member, are arranged in order of the third member, the third

The first filter and the second filter face each other through the second through hole, and the second filter and the third filter are arranged to face each other, and the first filter and the second filter are arranged opposite to each other. A plurality of sample preparation units in which a flow path connecting to the second filter is formed; A sample preparation device characterized by having a knit.

29. The sample preparation apparatus according to claim 28, wherein the first member, the second member, and the third member are formed of a silicon substrate, and wherein the first filter, the first filter, 2. The sample preparation device according to claim 2, wherein the holes of the second filter and the second filter are holes formed in the silicon substrate.

30. The sample preparation device according to claim 28, wherein the area of the second filter is smaller than the area of the first filter.

3 1. The first opening (5 11-1, 5 12-1, 5 13-1, 5 13-1, 5 14-4; 6 11 -1, 6 1 2-1, 6 13-1, 6 14-4) and a first region having a plurality of first filters formed at the bottom and capturing the cells. Member (500, 600) and a second opening (5 1 1–2, 5 1 2–2, 5 1 3–2, 5 1–2; 6 1 1–2, 6 1 2-2) , 6 1 3-2, 6 1 -2; 6 1 1 '1 2,6 1 2' 1 2,6 1 3'—2,6 14'—2) and is injected from the first opening. For a PCR amplification reaction, the polynucleotide eluted from the cells is captured by a denaturing agent and a copy of the desired nucleotide sequence portion of the polynucleotide is amplified, including a fluorescently labeled primer for PCR. The second member (5) in which a plurality of second regions having a second filter at the lower part for holding a PCR amplification reaction solution containing 20, 620), a third member (530, 630) in which a plurality of third regions having a hydrophobic third filter are formed, and means (540) for controlling the temperature of the PCR width reaction solution. , 640), arranged from above in the order of the first member, the second member, and the third member, wherein the first region, the second region, and the third region A sample preparation apparatus having a plurality of sample preparation units having: a laser beam 380 for exciting the fluorescent label for labeling the copy from a direction substantially perpendicular to the second filter of each of the sample preparation units; Irradiation means for irradiating the PCR amplification reaction solution of each sample preparation unit almost simultaneously, and the PCR amplification reaction solution of each sample preparation unit A detection means for detecting fluorescence from the fluorescent label for labeling the copy in the substantially simultaneously from a direction substantially perpendicular to the second filter.

32. The first opening (5 11-1, 5 12-1, 5 13-1, 5 14-4) into which the buffer solution formed in the upper part is injected with the cell suspension, and the lower part A first member (500) having a plurality of first regions formed and having a first filter for trapping the cells, and a second opening (5 1 1 -2, 5 1 2-2 at the top); , 513-12, 51-2) and captures the polynucleotide eluted from the cells with the denaturing agent injected from the first opening, and prepares a fluorescently labeled primer for PCR. A plurality of second regions having a second filter at a lower portion for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of a desired base sequence portion of the polynucleotide are formed. A second member (520), a third member (530) in which a plurality of third regions having a hydrophobic third filter are formed, and a through hole (511). 5, 5 1 2—5, 5 13—5, 5 1 -5) A transparent fourth member (5 10) having a plurality of fourth regions in which the PCR amplification reaction solution is formed Means for controlling the first member, the fourth member, the second member, and the third member in this order from above, wherein the first filter and the third filter are arranged in this order. A second filter facing through the through hole, the second filter and the third filter facing each other, the first region, the second region, and the second region; A plurality of sample preparation units having an area 3 and the fourth area, and the PCR amplification of each of the sample preparation units from a direction substantially parallel to the second filter of each of the sample preparation units. The laser light that excites the fluorescent label that labels the copy in the reaction solution is applied to the PCR amplification reaction of each sample preparation unit. Irradiating means for irradiating the sample almost simultaneously with the liquid; and irradiating the fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit with the second filter of each sample preparation unit. Detecting means for detecting substantially simultaneously from a direction substantially perpendicular to the arrested child.

33. The first through hole (601, 602, 603, 604) and the first opening (61 1—1, 6 1 2 -1, 6 1 3-3, 6 14-4) and a first region having a plurality of first regions formed below the first opening and having a first filter for capturing the cells. Member (600) and a second opening (6 1 1-2, 6 1 2-2, 6 13-2, 6 14-2; 6 1 Γ-2, 6 L 2 '-2, 6 1 3 ′ —2, 6 1 ′ 1), which captures the polynucleotide eluted from the cells by the denaturant injected from the first opening, and is a fluorescently labeled primer for PCR. A plurality of second regions having a second filter at a lower portion for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of a desired base sequence portion of the polynucleotide containing Formed second member ( 620), a third member (630) in which a plurality of third regions having a hydrophobic third filter are formed, and means (640) for controlling the temperature of the PCR amplification reaction solution. The first member, the second member, and the third member are arranged in this order from the top, and the first through hole and the second opening (6 1 1′—2, 6 1 ), The second filter and the third filter are disposed opposite each other, and the first filter and the third filter are disposed opposite each other. A channel connecting the second filter is formed, a plurality of sample preparation units having the first region, the second region, and the third region; A laser that excites the fluorescent label that labels the copy in the PCR amplification reaction solution of each sample preparation unit from a direction substantially perpendicular to the filter An irradiating means for irradiating the PCR amplification reaction solution of each of the sample preparation units with light (380) substantially simultaneously; and a step of labeling the copy in the PCR amplification reaction solution of each of the sample preparation units. Detecting means for detecting the fluorescence from the fluorescent label substantially simultaneously from a direction substantially perpendicular to the second filter of each of the sample preparation units.

34. The first through hole (601, 602, 603, 604) and the first opening (6 1 1—1, 6 1 2- 1, 613-3, 614-4) and a first region having a plurality of first regions formed below the first opening and configured to capture the cells. A member (600), comprising a polynucleotide eluted from the cells by a denaturing agent injected from the first opening, comprising a fluorescently labeled primer for PCR, and a desired base of the polynucleotide. A second member having a plurality of second regions having a second filter holding a solution containing a PCR amplification reaction reagent for a PCR amplification reaction for amplifying a copy of the sequence portion (620 b) a third member having a plurality of third regions having a hydrophobic third filter;

(6 30) and the second through hole (6 1 1, 1 2, 6 1 2 '— 2, 6 1 3' — 2, 6 1 '-2) and the second opening (6 1 1 , A transparent fourth member (620a) in which a plurality of fourth regions having —2, 6 1 2—2, 6 13—2, 6 14-2) are formed, and the PCR amplification reaction reagent Means (640) for controlling the temperature of the first member, the first member, the fourth member, the second member, and the third member, which are arranged in this order from above, and The through-hole and the second filter face each other via the second through-hole, the second filter and the third filter are arranged to face each other, and the first filter and the third filter are arranged opposite to each other. A plurality of sample preparation units having the first region, the second region, the third region, and the fourth region; and a plurality of sample preparation units having the first region, the second region, the third region, and the fourth region. The second filter mentioned above From the direction of execution, a laser beam (380) for exciting the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit is irradiated with the PCR amplification reaction solution of each sample preparation unit. Irradiating means for irradiating the sample filters at substantially the same time, and applying the fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit to the second filter of each sample preparation unit. A detector for detecting a telegraph, comprising: a detection unit that detects a signal from a substantially vertical direction at substantially the same time.

35. The first opening formed in the upper part (5 1 1—1, 5 1 2-1, 1, 5 1 3—1, 5 1 4—4; 6 1 1—1, 6 1 2—1, 6 1 3- 1, 6 14-4) and a first filter having a first filter formed in the lower part. The member (500, 600) and the second opening (5 1 1—2, 5 1 2—2, 5 1 3—2, 5 1 4-2; 6 1 1—2, 6 1 2— 2, 6 1 3 -2, 6 14—

2; 6 1 1 '1 2, 6 1 2' — 2, 6 1 3 '—2, 614' —2) and a second region with a second filter at the bottom. Second part (52

0, 620) and a third member (530, 630) in which a plurality of third regions having a hydrophobic third filter are formed. From above, the first member, the second member The first member, the third member, and the third member;

An arrestor test method using a sample preparation device having a plurality of sample preparation units having the second region and the third region, wherein (1) a buffer solution in which cells are suspended in the first opening. Injecting the cells to capture the cells in a first filter;

(2) a step of injecting a denaturant into the first opening and capturing the polynucleotide eluted from the cells in the second filter; and (3) a primer for fluorescence-labeled PCR. A PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of a desired nucleotide sequence portion of a polynucleotide is injected into the first opening, and the PCR amplification reaction solution is poured into the second opening. Amplifying the copy by holding it in a filter; and (4) laser light 380 for exciting the fluorescent label for labeling the copy from a direction substantially perpendicular to the second filter of each of the sample preparation units. Irradiating the PCR amplification reaction solution of each sample preparation unit with the PCR amplification reaction almost simultaneously; and (5) irradiating the PCR label of each sample preparation unit with the fluorescent label for labeling the copy in the PCR amplification reaction solution.Genetic testing method characterized by a step of substantially simultaneously detecting light from a direction substantially perpendicular to the second filter.

36. A second filter having a first opening formed in the upper part (5 11-1, 5 12-1, 5 13-1, 5 1 -4) and a first filter formed in the lower part It has a first member (500) in which a plurality of regions (1) are formed, and a second opening (5 1 1—2, 5 1 2—2, 5 1 3—2, 5 14—2) at the top. A second member (520) in which a plurality of second regions having a second filter are formed at a lower portion, and a third member in which a plurality of third regions having a hydrophobic third filter are formed. Member (530) and through A transparent fourth member (5110) in which a plurality of fourth regions having holes (511-5, 512-5, 513-5, 514-5) are formed. The first member, the fourth member, the second member, and the third member are arranged in this order from above, and the first region, the second region, and the third member are arranged in this order. A plurality of sample preparation units having an area and the fourth area, wherein the first filter and the second filter face each other through the through hole, and the second filter and the second 3. A genetic test method using a sample preparation device in which the filter of 3 is opposed to: (1) Injecting a buffer solution in which cells are suspended in the first opening, and transferring the cells to the first filter. (2) a step of injecting a denaturing agent into the first opening and capturing the polynucleotide eluted from the cells in the second filter. (3) a PCR amplification reaction solution containing a reagent for a PCR amplification reaction in which the first opening contains a primer for fluorescently labeled PCR and amplifies a copy of a desired base sequence portion of the polynucleotide; And amplifying the copy by holding the PCR amplification reaction solution; and (4) preparing each of the sample preparations from a direction substantially parallel to the second filter of the unit preparation unit. A laser beam 380, which excites the fluorescent label for labeling the copy in the PCR amplification reaction solution of the unit, is irradiated to the PCR amplification reaction solution of each sample preparation unit almost simultaneously. (5) applying the fluorescence from the fluorescent label, which labels the copy in the PCR amplification reaction solution of each sample preparation unit, to the second filter of each sample preparation unit. Almost simultaneously from different directions A method of inspecting a distant gene.

37. The first through-hole (601, 602, 603, 604) and the first opening formed in the upper part (611-1-1,612-2-1,61-3-3,614) — 4) and a first member (600) in which a plurality of first regions having a first filter formed below the first opening are formed, and a second opening (61) is formed in the upper portion. 1−2, 6 1 2−2, 6 1 3−2, 6 14−2; 6 1 12, 6 1 2′—2, 6 1 3′—2, 614′—2) A second region with a second filter at the bottom, having a plurality of second §W (620) and a hydrophobic third filter A third member having a plurality of third members (630), the first member, the second member, and the third member being arranged in this order from above, A plurality of sample preparation units having a first region, the second region, and the third region, wherein the first through-hole and the second opening (6 1 —2, 6 12 ′ -2 , 6 13 3′—2, 6 14′—2) are opposed to each other, and the second filter and the third filter are arranged to face each other, and the first filter and the second filter are (1) Injecting a buffer solution in which cells are suspended into the first opening, and filtering the cells with a first filter (2) injecting a denaturant into the first opening and capturing the polynucleotide eluted from the cells in the second filter; (3) A PCR amplification reaction solution containing, in the first opening, a reagent for PCR amplification reaction that contains a fluorescently labeled PCR primer and amplifies a copy of the desired base sequence portion of the polynucleotide. A) amplifying the copy by holding the PCR amplification reaction solution; and (4) preparing each of the sample preparation units from a direction substantially perpendicular to the second filter of each of the sample preparation units. Irradiating the PCR amplification reaction solution of each of the sample preparation units with laser light (380) for exciting the fluorescent label for labeling the copy in the PCR amplification reaction solution of the unit at substantially the same time; 5) The fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of each of the sample preparation units is substantially simultaneously emitted from a direction substantially perpendicular to the second filter of each of the sample preparation units. Genetic testing method characterized by a step of leaving. 38. The first through-hole (601, 602, 603, 604) and the first opening (6 1 1—1, 6 1 2- 1, 61 3-3, 614-4) and a first member (1) in which a plurality of first regions having a first filter formed below the first opening and capturing the cells are formed. 600) and capturing a polynucleotide eluted from the cells with a denaturing agent injected from the first opening, comprising a fluorescently labeled primer for PCR, having a desired nucleotide sequence of the polynucleotide. Amplified copy of part A second region having a second filter holding a solution containing a PCR amplification reaction reagent for a PCR amplification reaction, a plurality of second members (620b) being formed, and a hydrophobic second A third member (630) in which a plurality of third regions having three filters are formed; 14'-2) and a fourth region having a plurality of fourth regions having second openings (6 1 1-2, 6 12-2, 6 13-2, 6 1 -2) at the top. A first member, a second member, and a third member, which are arranged in this order from the top, the first member, the fourth member, the second member, and the third member. A plurality of sample preparation units having a second area, a third area, and a fourth area, wherein the first through-hole and the second filter are connected through the second through-hole. Facing, the second filter and the third filter A method for inspecting an arrested child using a sample preparation device which is arranged to face a filter and is formed with a flow path connecting the first filter and the second filter, wherein: Injecting a buffer solution in which cells are suspended in the opening of the cell and capturing the cells in the first filter; and (2) injecting a denaturant into the first opening and eluted from the cell. (3) capturing, in the first opening, a copy of a desired base sequence portion of the polynucleotide, including a primer for fluorescence-labeled PCR, in the first opening; Injecting a PCR amplification reaction solution containing a reagent for a PCR amplification reaction to be amplified, holding the PCR amplification reaction solution, and amplifying the copy, and (4) amplifying the copy. From the direction almost parallel to the second filter, Irradiating the PCR amplification reaction solution of each sample preparation unit with laser light (380) for exciting the fluorescent label for labeling the copy in the PCR amplification reaction solution of the preparation unit at substantially the same time; (5) The fluorescence from the fluorescent label for labeling the copy in the PCR amplification reaction solution of each sample preparation unit is changed from a direction substantially perpendicular to the second filter of each sample preparation unit. Detecting the arrested child substantially simultaneously.

39. The first opening (5) formed at the top where the buffer in which the cells are suspended is injected 1 1—1, 5 1 2—1, 5 13—1, 5 14-4; 6 1 1—1, 6 1 2—1, 6 13—1, 6 14—4) A first member (500, 600) in which a plurality of first regions having a first filter formed and capturing the cells are formed, and a second opening (5 1 1—2, 5 1 2 -2, 5 1 3—2, 5 14-2; 6 1 1 -2, 6 1 2-2, 6 1 3—2, 6 1 -2; 6 1 Γ 1 2, 6 1 2 '1 2 , 613'1-2, 614'-2) for capturing a polynucleotide eluted from the cells by a denaturant injected from the first opening, and for fluorescently labeled PCR. A plurality of second regions having a second filter at the lower part that holds a PCR amplification reaction solution containing a reagent for a PCR amplification reaction and amplifying a copy of a portion of the desired base sequence of the polynucleotide containing the primers The formed second member (520, 620) and the third region having the hydrophobic third filter are A third member (530, 630) formed in number and means (540, 640) for controlling the temperature of the PCR amplification reaction solution; A sample preparation apparatus, comprising: a plurality of sample preparation units arranged in the order of two members, the third member, and having the first region, the second region, and the third region.

0. The first opening (5 11-1, 5 12-1, 5 13-1, 5 14-4) into which the buffer which is formed at the top and in which the cells are suspended is injected, and at the bottom A first member (500) having a plurality of first regions formed and having a first filter for trapping the cells, and a second opening (5 1 1 -2, 5 1 2-2 at the top); , 513-12, 51-2), comprising a polynucleotide eluted from the cells by a denaturing agent injected from the first opening, and comprising a fluorescently labeled PCR primer. A second region having a plurality of second regions formed below a second filter for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of a desired base sequence portion of a polynucleotide is formed. Member (520), a third member (530) in which a plurality of third regions having a hydrophobic third filter are formed, and a through-hole (511— Transparent fourth member (5110) having a plurality of fourth regions in which 5, 512-5, 513-5, 51-5) are formed 5 δ

And means for controlling the temperature of the PCR amplification reaction solution (540). From above, the first member, the fourth member, the second member, and the third member in this order. The first filter and the second filter are arranged through the through-hole, the second filter and the third filter are arranged, and the first region and the second region are arranged. A sample preparation apparatus, comprising: a plurality of sample preparation units having two regions, the third region, and the fourth region.

41. The first through hole (601, 602, 603, 604) and the first opening (61 1—1, 6 1 2 -1, 6 1 3-3, 6 1 -4) and a first region having a plurality of first regions formed below the first opening and having a first filter for capturing the cells. Member (600) and a second opening (6 1 1–2, 6 1 2-2, 6 1 3 -2, 6 14—2; 6 1 1 '— 2, 6 1 2' — 2 , 613′1-2, 614′1-2), and captures the polynucleotide eluted from the cells by a denaturant injected from the first opening, and obtains a fluorescently labeled PCR. A second filter having a PCR amplification reaction solution containing a reagent for a PCR amplification reaction at a lower portion thereof and a reagent for a PCR amplification reaction containing a primer for the desired base sequence of the polynucleotide, the primer comprising: The second member in which a plurality of regions are formed (620) A third member (630) in which a plurality of third regions having a hydrophobic third filter are formed, and means for controlling (640) the temperature of the PCR amplification reaction solution; From above, the first member, the second member, and the third member are arranged in this order, and the first through hole and the second opening (6 1 1 ′ —2, 6 1 2 ′- 2, 61 3 '-2, 614' -2) are opposed to each other, the second filter and the third filter are arranged to be opposed to each other, and the first filter and the second filter are arranged. A sample preparation unit having a plurality of sample preparation units formed with a first region, a second region, and a third region, wherein the flow passage is formed to connect the filter to a filter.

42. The first through hole (601, 602, 603, 604) and the first opening (61 1—1, 6 1 2 - 1, 613-3, 614-4) and a first member formed with a plurality of first regions having a first filter formed below the first opening and capturing the cells. (600), a polynucleotide eluted from the cells by a denaturing agent injected from the first opening, and a fluorescent-labeled primer for PCR, comprising a desired base sequence of the polynucleotide. A second region having a second filter holding a solution containing a PCR amplification reaction reagent for a PCR amplification reaction for amplifying a copy of the portion of the second member (620b) formed with a plurality of A third member in which a plurality of third regions having a hydrophobic third filter are formed;

(630), the second through-hole (6 1 1 '-2, 6 12' -2, 6 13 '-2, 61'-2) and the second opening (6 1 1 -2, 6 12-2, 613-2, 61-2), a transparent fourth member (620a) having a plurality of fourth regions formed therein, and means for controlling the temperature of the PCR amplification reaction reagent (640), and the first member, the fourth member, the second member, and the third member are arranged in this order from above, and the first through hole and the second member are arranged in this order. The second filter and the third filter are opposed to each other via the second through-hole, and are connected to each other, and connect the first filter and the second filter. A flow path is formed, comprising a plurality of sample preparation units having the first region, the second region, the third region, and the fourth region.

43. It has a flow path connecting an upper opening into which a buffer solution in which cells are suspended is injected, and a lower opening through which waste liquid flows out, and is sequentially arranged in the flow path from the upper opening to the lower opening. A first filter (110) for capturing the cells, and a primer for fluorescence-labeled PCR that captures a polynucleotide eluted from the cells by a denaturant injected from the upper opening. A second filter (130) for holding a PCR amplification reaction solution containing a reagent for a PCR amplification reaction for amplifying a copy of the desired base sequence portion of the polynucleotide comprising A sample preparation unit (100) having three filters (140); holding means (200) for holding the sample preparation unit; Means for controlling the temperature of the PCR amplification reaction solution (210)