JP2001221721A - Genetic diagnosis based on dry filter paper feces - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a dry filter paper feces sampling kit allowing fecal samples to be handled easily for genetic diagnosis, and assuring stabilization of DNA, and also provide a simple and practical DNA extraction method enabling extraction of DNA from a number of specimens. SOLUTION: The dry filter paper feces sampling kit includes filter paper to which a fecal sample is applied in a small amount in stamping fashion, with the filter paper being subsequently sealed in a plastic bag containing a desiccant to stabilize DNA and allow specimens to be easily and sanitarily handled, transported and stored. A method of extracting DNA from dry filter paper feces comprises the steps of directly boiling the dry filter paper feces in a solution of DNA extracts, and then adding chloroform thereto, thereby obtaining a DNA-containing water layer from the residues of feces and the filter paper.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a filter paper for genetic diagnosis comprising a filter paper carrying dry stool as a DNA material, a kit for collecting dried filter paper stool, and a method for collecting the filter paper for genetic diagnosis from a stool sample.
The present invention relates to a method for purifying and extracting DNA.

[0002]

2. Description of the Related Art Fecal samples are used as non-invasive DNA materials, and are used for general biological samples represented by blood, such as intestinal mucosal cells, blood cells, intestinal flora, or pathogenic microorganisms (bacteria and viruses). It has been noted that it has no unique information, and many reports have been made mainly on detection of pathogenic microorganisms and cancer-related genes of colorectal cancer. However, all of these reports use stool as it is, requiring freezing and storage with liquid nitrogen immediately after collection to prevent DNA degradation, and DNA extraction from stool samples was performed in addition to the usual deproteinization procedure. , The most widely used high-sensitivity detection method today: additional steps to remove co-existing anion polyhedrons that interfere with the polymerase chain reaction (PCR),
It is a very complicated and poorly practical method.

[0003]

DISCLOSURE OF THE INVENTION The present invention relates to a dry filter paper stool that is easy to handle, sanitary, can be stored for a long time, can be sent by mail, and ensures DNA stabilization for genetic testing of stool samples. The purpose is to develop a collection kit and a simple and practical DNA extraction method that enables DNA extraction of many samples. The present invention focuses on feces as a DNA material having unique biological information, and enables quick and accurate diagnosis and prevention of various genetic diseases using dry filter paper stool that is easy to handle. The method is characterized in that the DNA can be stabilized by the method, and the risk of contamination between samples can be reduced in the process of manipulating the samples. In feces, a large amount of intestinal mucosal cells, blood cells and intestinal flora, sometimes pathogenic microorganisms (bacteria and viruses), and intestinal neoplasia (including colorectal cancer and polyps) are present. By analyzing these DNAs, it is possible to collect comprehensive genetic information including not only the body components of the individual but also the internal environment mainly in the intestinal tract. As a specific example, a method for detecting K-ras gene mutation related to early diagnosis of colorectal cancer (international patent application filed separately)
However, the present invention is not limited to this example, and is widely used for DNs such as somatic cells, tumor cells, and pathogenic microorganisms.
A Includes general applications where diagnosis is considered useful.

[0004]

SUMMARY OF THE INVENTION The present invention provides a filter paper for genetic diagnosis comprising a filter paper carrying dry stool as a DNA material;
A kit for collecting dried filter paper stool for DNA testing, comprising a molded filter paper for collecting a fixed amount of stool sample from stool in the manner of stamping and preparing four spots of dried filter paper stool, and a plastic bag with a chuck containing a desiccant. And a method for purifying and extracting DNA from a stool sample, comprising subjecting the stool sample of the filter paper for gene diagnosis to boiling treatment in the presence of a cationic surfactant. Conventionally, cryopreservation with liquid nitrogen was required immediately after collection, because DNA degrading enzymes existed in fecal samples and DNA degradation gradually progressed. In the present invention, focusing on the fact that the activity of DNAse enzymes is extremely reduced in the dry state, a small amount of stool sample is applied to the filter paper in the manner of stamping to promote the removal of moisture, and is directly enclosed in a plastic bag containing a desiccant. By doing
We have developed a kit for collecting dried filter paper stool that stabilizes DNA and makes handling, transport and storage of samples simple and hygienic.

[0005] Regarding a simple DNA extraction method from a stool sample, dry stool is collected in a microtube together with filter paper, and DNA
Add a high salt extraction solution containing EDTA as a degrading enzyme inhibitor and a cationic surfactant for removing ion pairs of anionic polytetramers; cetyltrimethylammonium bromide (CTAB) and boil ( Boil) treatment. Next, chloroform is added and mixed, followed by centrifugation. The anion polysaccharide-CTAB pair, excess CTAB, fecal residue components and filter paper are transferred to the lower chloroform layer, and the target DNA solution is transferred to the upper aqueous layer. Could be separated. The aqueous layer was separated into a new tube, and the procedure of extraction with chloroform was repeated again, and 2-propanol was added to the obtained aqueous layer, followed by centrifugation to obtain a DNA precipitate. After washing the precipitate with 70% ethanol, Tris buffer-EDTA solution was added to obtain the final DNA solution. Hereinafter, embodiments of the present invention will be described in detail.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION From the collection of stool to the storage of stool on filter paper, a simple and efficient integrally formed paper assembly stamp is used. Details are shown in FIGS. 1, 2 and 3.
Advantech filter paper PKU-S type was used for the stamp material including the stool sample application part in consideration of physical strength and water retention, and the surface water-repellent coated paper was used for the surface part directly in contact with feces ( 1 and 2). 3m diameter on sample application part
m, 4 round punches with a depth of 0.5 mm, and a diameter of 5 m placed beneath it by pressing against the faeces surface and rotating
It is designed so that a substantially uniform stool sample corresponding to a hollow portion of a circular punching type can be collected on a circular filter paper of m (see FIG. 3). After stool collection, the stamp is turned upside down to promote water absorption and protect the stool collection area, and placed in a plastic bag with a desiccant-containing zipper to transport and store specimens while ensuring DNA stabilization. (See FIG. 4). At the time of sampling at the time of inspection, it is possible to simply and hygienically remove the sample application portion and pinch the edge of the circular filter paper (dry filter paper stool disc) with tweezers.

Next, the DNA extraction operation will be described. 0.5M Tris, 16mM EDTA, 1.4M NaCl, 0.5% CT
A solution having a composition of AB (pH 9.0) is prepared. Transfer a dry filter paper stool disc to a 0.5 ml centrifuge tube, add 0.2 ml of the extraction solution, cap, and boil at 95 ° C for 15 minutes. 1
After centrifugation at 5,000 rpm for 1 minute, add 0.2 ml of chloroform for 5 minutes.
Vortex vigorously -2 times per second. 15,000rpm-
Centrifuge for 5 minutes and transfer about 0.1 ml of the upper aqueous layer to another 0.5 m
l Transfer to a centrifuge tube. Add 0.2 ml of chloroform again, vortex twice for 5 seconds, centrifuge at 15,000 rpm for 5 minutes, and separate the resulting aqueous layer into another 0.5 ml centrifuge tube. Next, 0.1 ml of 2-propanol is added, and the mixture is allowed to stand at -20 ° C for 30 minutes. Centrifuge at 15,000 rpm for 10 minutes at -4 ℃, discard the supernatant, add 0.2 ml of 70% ethanol, and add 15,000 rpm for 5 minutes.
Centrifuge at 4 ° C. Discard the supernatant and add 0.05 ml of 10 mM Tris-1 mM EDTA buffer, pH 8.0 (hereinafter referred to as TE solution) to dissolve the DNA by vortexing to obtain the final DNA sample solution. It has been confirmed that it can be used for stool filter paper (Sanko Junyaku San Auto Hemo Kit) as well.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Dried filter paper stool collection kit and simple DN according to the present invention
Colorectal cancer K-ras using DNA samples obtained by A extraction method
An example in which a gene mutation was detected will be described. Allele-specific nested PCR for K-ras gene mutation in DNA from colon tumor exfoliated cells in dried stool samples
Method (hereinafter referred to as nested ASP), which has been developed by the applicants of the present invention for the purpose of applying to stool samples that require highly sensitive and highly specific results. This is described in detail in the specification of the patent application. To 2 μL of the stool sample DNA extract obtained by the above method, add the K-ras gene codon
Primer pair (K-rasF-1st:
5'-agg cct gct gaa aat gac tga ata, K-ras-R: 5'-ct
first stage PCR, including atg aaa atg gtc aga gaa acc)
Add 18 µL of the reaction mixture and perform PCR at an annealing temperature of 60 ° C-38 cycles. The DNA content of 10 μL of this first-step PCR product is confirmed by agarose minigel electrophoresis, and the remaining 10 μL is diluted by adding 90 μL of a TE solution. This diluent
2 μL contains three common mutations in the K-ras gene (codon 12: ggt →
gat, gtt and codon 13: ggc → gac, 12A, 12T respectively
And 13A) (K-ras12A-
3g: 5'-ctt gtg gta gtt gga ggt ga, K-ras12T-3g: 5 '
-ctt gtg gta gtt gga ggt gt, K-ras13A-1a: 5'-gtg gt
Add 18 μL of the second-stage PCR reaction mixture containing a gtt gga gct ggt aa and the above-mentioned K-ras-R), and set the annealing temperature.
Perform PCR at 60 ° C for 15 cycles. This second stage PCR product 1
0 μL is checked for the presence of an amplified band by agarose minigel electrophoresis. The presence or absence of the K-ras gene mutation was determined from the band intensity ratio of the second-stage and first-stage PCR products thus obtained. E across the mutated portion by 1st PCR
The xon1 region was amplified, and in 2nd PCR, a primer set was used that gave an amplified band if any of the three common mutations were present (international patent application filed separately). (FIG. 5).

[0009]

EFFECT OF THE INVENTION The use of the kit for collecting dried filter paper stool of the present invention facilitates the operation from collection of stool samples to DNA extraction and achieves more hygienic handling, and is an essential condition as a material for gene diagnosis. DNA stabilization is also possible. The genetic diagnosis described in the Examples requires a fairly advanced technique of detecting DNA having a small amount of K-ras gene mutation derived from colon cancer tissue under the condition that a large amount of DNA derived from bacteria or normal mucosal tissue is present. Things. Therefore,
It is clear that the results of direct detection of various bacterial DNAs or information on genetic polymorphisms in individuals can be obtained more easily and reliably. The fact that stool samples having unique information not found in other biological samples can now be practically used as non-invasive DNA materials is not only useful for diagnosis of various diseases, but is also a large number of operations. The ability to process specimens opens the way for application to various screenings for colorectal cancer and the like in normal populations. Therefore, contribution to preventive medicine, which is increasingly important in recent years, can be expected.

[Brief description of the drawings]

[Figure 1] A kit for collecting dried filter paper stool consisting of molded filter paper, a desiccant, and a plastic bag with a zipper for collecting a fixed amount of stool sample from stool by stamping and preparing four spots of dried filter paper stool FIG.

FIG. 2 is a side view of the kit for collecting dried filter paper stool.

FIG. 3 is a diagram showing the use of the kit for collecting dried filter paper stool.

FIG. 4 is a storage diagram of the kit for collecting dried filter paper stool.

Fig. 5 K-ras by DNA sample extracted from dried filter paper stool
An electrophoretogram showing an example of detecting a gene mutation.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Akihiro Yamaguchi 2-12-20 Shinkawa 2-chome, Kita-ku, Sapporo, Hokkaido Sapporo Immuno-Diagnostics Laboratory Co., Ltd. (72) Inventor Kenji Nakamura Kita, Sapporo, Hokkaido 2-12-12 Shinkawa-ku, Tokyo F-20 terms in Sapporo Immuno Diagnostics Laboratories Co., Ltd. QX02

Claims (3)

[Claims] 1. A filter paper for gene diagnosis comprising a filter paper carrying dry stool as a DNA material. 2. A kit for collecting dried filter paper stool for DNA testing, comprising a molded filter paper for collecting a fixed amount of stool sample from stool in the manner of stamping and preparing four spots of dried filter paper stool, and a desiccant containing Dry filter paper stool collection kit consisting of a plastic bag with a zipper. 3. A method for purifying and extracting DNA from a stool sample, the method comprising boiling the stool sample of the filter paper for gene diagnosis according to claim 1 in the presence of a cationic surfactant.