Kit for preparing cancer cell detection sample and kit for cancer cell detection using the same

A kit for preparing a cancer cell detection sample which is highly safe, simple, and high in precision, and can detect a cancer cell quantitatively, and a kit for cancer cell detection using the same, as well as a method for diagnosing cancer using those kits, and a method for preparing a sample for cancer cell detection are provided. A kit for preparing a detection sample for detecting a cancer cell of the present invention, comprising a test container having an opening for receiving a biological sample collected from a subject, and a reagent inclusion part for accommodating a reagent containing a virus, a seal part for sealing the reagent inclusion part of the test container, a cap for closing the opening, and an opener for breaking the seal part.

FIELD OF THE INVENTION

The present invention relates to a kit for preparing a cancer cell detection sample and a kit for cancer cell detection using the same.

BACKGROUND

Cancer is a primary cause for mortality of Japanese, and it is said that if early treatment by early diagnosis is possible, the mortality can be remarkably decreased.

Currently, diagnosis of cancer is performed by discriminating a normal cell and a cancer cell by a morphological test of a cell in a test sample collected from a subject with microscopic examination generally by a cell testing technician. However, since this diagnosis method is a visual test by a cell testing technician, the method is not suitable for handling of a large amount of test samples as in group medical examinations, and there is a problem that unevenness occurs in test results depending on the status of a test sample, and the sample cannot be analyzed quantitatively.

In addition, a method for diagnosing cancer by detecting a tumor-derived DNA in plasma and serum of a subject is also being studied. Specifically, cancer is diagnosed by concentrating cells contained in blood collected from a subject, and biochemically analyzing a DNA and the like related to a blood free cancer cell in the concentrated cells. This method can relatively safely treat a large amount of test samples, but there is a problem that a step of concentrating blood free cancer cells is difficult, and simplicity is lacked.

On the other hand, in recent years, an anti-cancer agent (US2006239967) and a cancer cell detection reagent (US2006067890) using an oncolytic virus which specifically grows in a cancer cell are reported. The oncolytic virus is a virus which specifically grows in a cancer cell and, by infecting a cancer cell with the virus, the cancer cell can be directly destroyed and killed and, by incorporating a gene of a target protein into a genome thereof, it becomes possible to simply detect a cancer cell.

However, since the oncolytic virus has infectivity also on a normal cell apart from simple detection of a cancer cell, a facility at a P2 level becomes necessary for the detection. Therefore, there is a problem that it is difficult to actually use the virus in group medical examinations or the like.

SUMMARY

In view of such circumstances, the present inventors intensively studied and, as a result, found out a kit for preparing a cancer cell detection sample used in detection of a cancer cell with a reagent containing a virus, particularly, an oncolytic virus, which is not accompanied by contamination due to diffusion of the virus to the outside, which resulted in completion of the present invention.

That is, the present invention provides:

(1) A kit for preparing a detection sample for detecting a cancer cell, comprising a test container having an opening for receiving a biological sample collected from a subject, and a reagent inclusion part for accommodating a reagent containing a virus, a seal part for sealing the reagent inclusion part of the test container, a cap for closing the opening, and an opener for breaking the seal part;
(2) The kit for preparing a detection sample according to (1), wherein the cap has a virus-impermeable breathable filter;
(3) The kit for preparing a detection sample according to (1), wherein the opener breaks the seal part accompanying with an action of closing the opening by the cap;
(4) The kit for preparing a detection sample according to (1), wherein the cap has the opener;
(5) The kit for preparing a detection sample according to (1), wherein the virus in an interior portion of the test container is isolated from the outside when the opener breaks the seal part;
(6) The kit for preparing a detection sample according to (1), wherein the cap and the opener are integrally configured, the opener has a leading edge for breaking the seal part, and a middle part for connecting the cap and the leading edge, and the middle part is configured so as to be inserted into the opening in the state where it is adhered to the opening;
(7) The kit for preparing a detection sample according to (1), wherein a genome of the virus comprises a promoter of a carcinogenic gene and a gene encoding a target protein;
(8) The kit for preparing a detection sample according to (1), wherein the virus is an oncolytic virus;
(9) The kit for preparing a detection sample according to (1), wherein the virus is d12.CALP, d12.CALP delta RR, Telomelysin, Telomelysin-RGD, AxE1AdB-UPRT, AdSLP1.E1AdB, AxE1CAUP, AdE3-IAI.3B, Ad-MKAdMK, AdCEAp/Rep, AdAFPep/Rep, MMP-sub II SeV/delta M, or CD-MMP-sub II-SeV/delta M;
(10) The kit for preparing a detection sample according to (1), wherein the sample is collected from blood, sputum or uterus;
(11) A method for diagnosing cancer, comprising using the kit for preparing a detection sample as defined in (1);
(12) The diagnosis method according to (11), wherein the cancer is blood cancer, lung cancer or uterine cervical cancer;
(13) A kit for detecting a cancer cell, comprising a kit for preparing a detection sample for detecting a cancer cell comprising a test container having an opening for receiving a biological sample collected from a subject, and a reagent inclusion part for accommodating a reagent containing a virus, a seal part for sealing the reagent inclusion part of the test container, a cap for closing the opening, and an opener for breaking the seal part, and a slide glass comprising an insertion part for inserting the test container, a second opener for breaking a bottom of the test container by an action of inserting the test container into the insertion part, and a holding part for guiding a reaction solution of the biological sample and the reagent from the test container in which a bottom is broken with the second opener, and observably holding the reaction solution from the outside;
(14) The kit for cancer cell detection according to (13), wherein the cap has a virus-impermeable breathable filter;
(15) The kit for cancer cell detection according to (13), wherein the opener breaks the seal part accompanying with an action of closing the opening with the cap;
(16) The kit for cancer cell detection according to (13), wherein a virus in the interior portion is isolated from the outside when the second opener breaks the bottom of the test container;
(17) The kit for cancer cell detection according to (13), wherein a bottom of the test container has a second seal part which is broken with the second opener;
(18) A method for diagnosing cancer, comprising using the kit according to claim13for detection;
(19) The diagnosis method according to (18), wherein the cancer is blood cancer, lung cancer or uterine cervical cancer;
(20) A method for preparing a sample for detecting a cancer cell comprising steps of, adding a sample collected from a subject to a test container having an opening, and a reagent inclusion part for accommodating a reagent containing a virus in the state where it is sealed with a seal part, closing the opening with a cap, and breaking the seal part with an opener.

According to the present invention, since a reagent containing a virus is mixed and reacted with a sample without being diffused to the outside, and it becomes possible to observe the sample after the reaction, a kit for preparing a cancer cell detection sample which is highly safe, simple, and high in precision, and can detect a cancer cell quantitatively, and a kit for cancer cell detection using the same, as well as a method for diagnosing cancer using those kits, and a method for preparing a sample for cancer cell detection are provided.

When the kit and the method of the present invention are used, since it is not necessary to provide a large scale test facility at a P2 level, a group medical examination of cancer which is simple and high in precision becomes possible.

DETAILED DESCRIPTION OF THE EMBODIMENT

First, a construction of the kit for preparing a cancer cell detection sample relating to the present embodiment will be explained.FIG. 1is a side view showing a kit for preparing a cancer cell detection sample relating to one embodiment. The kit for preparing a cancer cell detection sample relating to the present embodiment includes a cap1and a test container3. The test container3is configured to be substantially cylindrical and, on an upper side thereof, as shown inFIG. 2, an opening4for receiving a biological sample collected from a subject is provided. The cap1which is inserted through the opening4includes an opener2which is substantially cylindrical, and a disk-like jaw part1aprovided on an upper end of the opener2. Further, the jaw part1ahas a fitting part lb which fits with an entire circumference of an upper end edge of the test container3when the cap1is abutted against the test container3(seeFIG. 5). The opener2has substantially the same diameter as an upper side inner side of the test container3, that is, a diameter of the opening4. Therefore, such opener2is inserted until the jaw part1ais abutted against an upper end of the test container3, in the state where the opener2is adhered to an entire circumference of the opening4of the test container3. In addition, two protrusions2aare provided on a lower side of the cylindrical opener2, that is, on a side opposite to the jaw of the cap1, and a first aluminum sheet5described later can be opened with this protrusion2a. In addition, at a central part of the cap1, a virus-adsorbable charcoal filter10is provided so as to close a cavity of the opener2. Thereby, also when the test container3is closed with the cap1, breathing into an interior space of the test container3from the outside air can be maintained (seeFIG. 5).

FIG. 3is an III-III cross-sectional arrow view shown inFIG. 2, of the test container3. In the present embodiment, in the test container3, the first aluminum sheet5partitioning the interior portion of the container is provided approximately at a center of the interior portion of the container. Further, on a bottom of the test container3, a second aluminum sheet8is provided, and a space held by the first aluminum sheet5and the second aluminum sheet8is a reagent inclusion part6isolated from the outside. In this reagent inclusion part6, a reagent7containing a virus is sealed therein. Like this, since the reagent7is sealed in the reagent inclusion part6which is a space isolated with the first aluminum sheet5and the second aluminum sheet8, the test container3can be carried safely without diffusion of a virus to the outside.

Then, a motion when a cancer cell detection sample is prepared using the kit for preparing a cancer cell detection sample relating to the present embodiment will be explained. First, a user injects a sample into the test container3. A cross-sectional view of the test container3into which a sample is injected is shown inFIG. 4. With the aforementioned construction, a part from the opening4of the test container3(upper end) to an approximately central position of the test container3, partitioned with the first aluminum sheet5(i.e. an upper side part of the test container3) is concave, and a sample9can be accommodated therein. The user injects the sample into a concave part of the test container3, for example, by using a pipette. The sample9injected through the opening4of the test container3is dammed with the first aluminum sheet5provided at a center of the test container, at the center of the interior of the container. For this reason, at injection of the reagent, it is not mixed with the reagent7containing a virus sealed into the aforementioned reagent inclusion part6. That is, at injection of the sample, the reagent7containing a virus is in the sealed state where it is isolated from the outside, and diffusion of the virus to the outside does not occur.

Then, the user closes the opening of the test container3with the cap1.FIG. 5shows a cross-sectional view of the test container3when the opening4is closed with the cap1. As described above, in the opener2of the cap1, a protrusion is provided on a tip thereof and, when the cap1is inserted into the test container3, the opener2can be inserted in the state where it is adhered to the opening4of the test container3. That is, at a position where a tip part of the opener2is inserted into the opening4, the opening of the test container3has been already closed, and an internal space of the test container3is isolated from the outside. The isolated state used herein refers to the state where a virus accommodated in the test container3is not diffused to the outside, and an internal space of the test container3is breathable with a fine pore for introducing the outside air for making a cell in a sample alive. Further, a full length of the opener2is configured to be longer than a length from an upper end of the test container3to the first aluminum sheet5at a central part of the test container3. Thereby, by inserting the cap1into the test container3until the jaw part1ais abutted against an upper end surface of the test container3, a protrusion of the opener3smashes the first aluminum sheet5, and can break the first aluminum sheet5. Further, in a middle part of the opener2, that is, between initiation of insertion of a part other than the protrusion to arrival of the protrusion of the opener2at the first aluminum sheet5, since the middle part of the opener2maintains the state where the part has been already adhered to the opening4over an entire circumference in a circumferential direction, the interior portion of the test container3is isolated from the outside. Therefore, upon mixing of a sample9and the reagent7, the interior portion of the test container3is in the state where it is isolated from the outside, and a virus is not diffused to the outside. In addition, by the charcoal filter10provided on an upper side of the cap1, breathability of the interior portion of the test container3is maintained without diffusion of a virus to the outside, and a cell contained in the sample9and a virus contained in the reagent7can be sufficiently reacted. Like this, by mixing the sample9and the reagent7in the interior portion of the test container3, a measurement sample (reaction solution) is prepared.

The reaction solution prepared by the test container3as described above is supplied to a slide glass11explained later, and is tested with microscopic examination. A construction of the slide glass11will be explained below.

FIG. 6is a perspective view of the slide glass11for microscopic examination relating to one embodiment. The slide glass11of the present embodiment is a flat, and is of a rectangular parallelepiped plate, and has an insertion part12for inserting the test container3at a position near a short side from a central part of a plane of a rectangle. The insertion part12is of a cylinder having substantially the same size of an internal diameter as an outer diameter of the test container3, and can be inserted through an opening at an upper part in the state where it is adhered to the test container3over an entire circumference. The test container3is inserted into this insertion part12from a bottom side having the second aluminum sheet8.

FIG. 7(a) shows a plane view of the slide glass11, andFIG. 7(b) shows a cross-sectional view of a side thereof. The slide glass is constructed by fusing two slide glasses, a first slide glass17and a second slide glass18. These first slide glass17and second slide glass18are fused so that water tightness is retained. In addition, as far as the first slide glass and the second slide glass are connected so as to retain water tightness, a connecting form is not limited, but for example, those slide glasses may be connected with an adhesive. In the first slide glass17, an opening is provided, and the insertion part12is provided so as to stand up from a periphery of the opening (seeFIG. 7(b)). In addition, in a place corresponding to the insertion part12of the second slide glass18, a second opener16is provided. This second opener16is provided at a substantially central position of the insertion part12, so as to be projected upwardly, and is approximately cone-like, an upper end being pointed. Thereby, the second aluminum sheet8provided on a bottom of the test container3which has been inserted into the insertion part12is smashed with the second opener16, and a reaction solution which is a content is introduced into the slide glass11.

At a part held by the first slide glass17and the second slide glass18of the slide glass11, a holding part13for holding a reaction solution to be subjected to microscopic examination is provided. The holding part13of the slide glass11is constructed of a microscopic examination part14for microscopically examining a reaction solution, and a reaction sample passageway part15for guiding a reaction solution flown into the insertion part12to the microscopic examination part14. The microscopic examination part14is formed as a flat space so that a supplied reaction solution can be observed by microscopic examination.

Then, a motion of use of the kit for cancer cell test relating to an embodiment of the present invention will be explained. First, a user injects a sample, and inserts the test container3in the state where an opening is closed with the cap1, into the insertion part12of the slide glass11. Thereby, the second aluminum sheet is broken with the second opener16, and a reaction solution accommodated in the test container3is supplied to the insertion part. Thereby, as shown in an arrow ofFIG. 7(a), the reaction solution is flown in the reaction sample passageway part15to reach the microscopic examination part14. In addition, as described above, since the insertion part12and the test container3can be inserted in the state where they are adhered without excess and deficiency, the holding part13is brought into the state where it is isolated from the outside, by insertion into the insertion part12of the test container3. Further, since a tip of the second opener16is positioned below an entrance upper side of the insertion part12, upon breaking of the second aluminum sheet8with the second opener16, the holding part13has been already brought into the state where it is isolated from the outside, that is, the state where a reaction solution containing a virus is isolated from the outside. With this construction, the virus in the reaction solution is not diffused to the outside also at microscopic examination, and diagnosis can be implemented safely.

An example of specific embodiments has been explained, but the present invention is not limited to these embodiments, and a variety of variations are possible.

The sample in the present invention is not particularly limited as far as it contains a cell derived from a living body collected from a subject, but examples include samples from blood, sputum and uterus. Particularly, samples of blood, sputum and uterus are preferable.

The virus used in the present invention is not particularly limited as far as it has the ability to infect a cell in a sample, but an oncolytic virus is exemplified, and specifically d12.CALP, d12.CALP delta RR, Telomelysin, Telomelysin-RGD, AxE1AdB-UPRT, AdSLP1.E1AdB, AxE1CAUP, AdE3-IAI.3B, Ad-MKAdMK, AdCEAp/Rep, AdAFPep/Rep, MMP-sub II SeV/delta M, or CD-MMP-sub II-SeV/delta M are preferable.

The seal part and the second seal part in the present invention are not particularly limited as far as a reagent containing a virus is not flown to the outside, and the virus is not diffused to the outside, but examples include an aluminum sheet.

The virus-impermeable breathable filter used in the present invention is not particularly limited as far as it dose not diffuse a virus in a reaction solution to the outside, and has breathability, but examples include a breathable anti-virus filter and a virus-adsorbable filter. Specifically, a charcoal filter is preferable.

Isolation in the present invention is not particularly limited as far as it is the state where a virus in not diffused to the outside, and a position with breathability rather than the complete closed state is preferable because a cell in a sample and a virus in a reagent can be reacted sufficiently.

The kit for preparing a cancer cell detection sample in the present invention is not particularly limited in its use as far as it is a test by which a cancer cell can be confirmed, and examples include a microscopic examination test and a test with a flow cytometer.

A variety of characteristics shown in the aforementioned embodiments can be combined mutually. When a plurality of characteristics are included in one embodiment, one or a plurality of characteristics among them can be appropriately extracted, and they can be adopted alone or in combination, in the kit for preparing a cancer cell detection sample, and the kit for cancer cell detection using the same, of the present invention.

EXAMPLES

The present invention will be specifically explained below using Examples, by referring to results of a method for detecting cancer which actually uses the kit for cancer cell detection of the present invention.

Detection of Lung Cancer Cell

A sputum collected from a healthy person was recovered into 1.5 ml of a cell culturing solution (Dulbecco's Modified Eagle Medium; DMEM), and pre-culturing was performed under the condition of 37° C. and 5% CO2for 60 minutes. The pre-cultured cell culturing solution was diluted with DMEM to 1.0×106cells/ml, and to this was added a solution of an A431 cell (purchased from ATCC) which is a human lung cancer cell strain to 1000 cells/300 μl, and this was used as a lung cancer sample.

Separately, as a control, a cell culturing solution (1.0×106cells/ml) before addition of an A431 cell solution, and an A431 cell solution which had been diluted with DMEM to 1000 cells 300 μl were prepared.

A reagent containing OBP-301 which is an oncolytic virus encoding a green fluorescent protein (GFP) was prepared by a known method described in CANCER RESEARCH 64, 6259-6265, Sep. 1, 2004. The prepared reagent was sealed into the reagent inclusion part6of the aforementioned test container3.

Each 250 μl of the control or the cancer sample was injected into the test container3in which the reagent containing OBP-301 was sealed into the reagent inclusion part6of the aforementioned kit for preparing a cancer cell detection sample. Thereafter, the cap1was inserted until the jaw part1awas abutted against an upper end of the test container3, and culturing was performed under the condition of 37° C. and 5% CO2for 24 hours, thereby, the sample was infected with a virus.

The test container3after virus infection was inserted into the insertion part12of the aforementioned slide glass11, and a reaction solution held by the microscopic examination part14was observed with a fluorescent microscope, and the results are shown in Table 1 andFIG. 8.

Sputum indicates a cell culturing solution (1.0×106cells/ml) before addition of an A431 cell, Sputum/A431 indicates a lung cancer sample, and A431 indicates an A431 cell solution which has been diluted with DMEM to 1000 cells/300 μl.

Detection of Uterine Cervical Cell

Using a sterilized swab, an oral cavity mucosa cell was recovered into 10 ml of a cell culturing solution (Dulbecco's Modified Eagle Medium; DMEM), and this was pre-cultured under the condition of 37° C. and 5% CO2for 60 minutes. The pre-cultured cell culturing solution was diluted with DMEM to 1.0×106cells/ml, and to this was added a Hela cell solution (purchased from ATCC) which is a human lung cancer cell strain to 1000 cells/300 μl, and this was used as a uterine cervical sample.

Separately, as a control, a cell culturing solution (1.0×106cells/ml) before addition of a Hela cell solution, and a Hela cell solution which had been diluted with DMEM to 1000 cells/300 μl were prepared.

According to the same procedure as that of Example 1 except for the aforementioned procedure, the sample was observed. The results are shown in Table 2 andFIG. 9.

Oral indicates a cell culturing solution (1.0×106cells/ml) before addition of a Hela cell, Oral/Hela indicates a uterine cervical sample, and Hela indicates a Hela cell solution which has been diluted with DMEM to 1000 cells/300 μl.

As apparent from Examples, a cancer cell can be actually detected using the kit for preparing a cancer cell detection sample of the present invention, and the kit for cancer cell detection using the same.

The foregoing detailed description and examples have been provided by way of explanation and illustration, and are not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments will be obvious to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.