EP0653920A1 - Process for obtaining a non-liquid cell sample - Google Patents
Process for obtaining a non-liquid cell sampleInfo
- Publication number
- EP0653920A1 EP0653920A1 EP93919898A EP93919898A EP0653920A1 EP 0653920 A1 EP0653920 A1 EP 0653920A1 EP 93919898 A EP93919898 A EP 93919898A EP 93919898 A EP93919898 A EP 93919898A EP 0653920 A1 EP0653920 A1 EP 0653920A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- brush
- cells
- bristles
- membrane
- animal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/02—Instruments for taking cell samples or for biopsy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/0045—Devices for taking samples of body liquids
- A61B10/0051—Devices for taking samples of body liquids for taking saliva or sputum samples
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/02—Instruments for taking cell samples or for biopsy
- A61B2010/0216—Sampling brushes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
- A61B17/32—Surgical cutting instruments
- A61B2017/320004—Surgical cutting instruments abrasive
- A61B2017/320012—Brushes
Definitions
- cervicovaginal lavage was found to be superior to scrape, swab or brush methods for obtaining cervicovaginal cells (Goldberg, GL et. al., Am. J. Obstet. Gynecol. 161 : 1169-72 (1989); Vermund, SH et. al., Ann. J. Obstet. Gynecol. 160: 304-8 (1989)).
- cervicovaginal cells obtained by lavage are in a liquid format and therefore present the same problems with handling, processing and contamination as samples obtained by mouthwashing techniques.
- DNA has been obtained from epithelial cells, spun from urine (Gasparinin, P. et. al., N. Engl. J. Med. 320: 809 (1989)); and from root and surrounding sheath cells obtained from plucked hairs (Higuchi, R. et. al.. Nature 332:543-6 (1988)).
- samples so obtained often do not contain enough DNA for genetic analysis. Therefore hair and urine samples are not optimal for clinical analysis.
- a simple, noninvasive technique for obtaining a non-liquid cell sample having an appropriate amount of genetic material for clinical analysis would be useful.
- the invention relates to a simple, noninvasive process for obtaining a non- liquid cell sample from an animal for genetic analysis.
- the process involves inserting an appropriately sized brush into an animal's oral cavity, contacting the brush with an oral membrane, agitating the brush against the membrane so that cells are captured within the bristles of the brush, removing the brush, which contains the captured cells and isolating the cells from the bristles of the brush for use in genetic analysis.
- the brush is spiral, so that cells can be obtained by rotating the brush against an oral membrane.
- Cells so obtained can then be analyzed using any of a number of known genetic techniques. For example, cells obtained according to the disclosed method can be lysed and the cell's DNA can be contacted with a suitable labelled probe. In addition, DNA so obtained can first be amplified to increase the amount of genetic material available for analysis.
- a major advantage of using the process described herein for obtaining cells for genetic testing is that the non-liquid sample so obtained is easier to handle and to process than a liquid sample. Further a non-liquid sample is less prone to contamination (e.g by bacteria) and is more stable.
- the invention relates to a process for obtaining a non-liquid cell sample from an animal (e.g. a human or a domesticated mammal) for genetic analysis.
- the process employs a brush to obtain cells from within a animal's oral cavity.
- Examples of cells which can be obtained from an animal's oral cavity include epithelial cells of the inner cheek or tongue.
- a brush is inserted into an animal's oral cavity, contacted with an oral membrane and agitated (e.g. rotated or brushed) against the membrane for a sufficient period of time, so that cells are captured within the bristles of the brush.
- the brush, which contains captured cells is then removed from the animal's mouth and cells are isolated from the bristles of the brush for use in genetic analysis.
- a brush must be of an appropriate size to enable insertion into the animal's oral cavity.
- the brush bristles should be free of potentially contaminating material.
- a brush comprising mammalian hair should not be used in the subject method, because genetic material present within the hair could contaminate the sample and thereby interfere with the genetic analysis.
- Preferred brushes for use in the subject invention have synthetic (e.g. nylon or dacron) bristles.
- An especially preferred brush for use in the invention is the nylon bristle cytology brush, (Cytosoft Medical Packaging Corporation, Camarillo, CA), which is currently used for Pap Smear and Chiamydia testing.
- a brush which contains captured oral cells can be analyzed directly or transported and/or stored for subsequent genetic analysis.
- measures aimed at avoiding the dislodging of captured cells from a brush should be taken.
- the brush can be placed in a container (e.g. an envelope) during transportation and/or storage.
- Cells may be obtained from a brush by any method which avoids loss of captured cells.
- a preferred method of obtaining cells from the brush is by contacting the cells with a solution.
- the ceils can be obtained from the brush using a solution which lyses cells (e.g. sodium hydroxide, potassium hydroxide).
- Genetic material from oral cells so obtained can then be analyzed genetically using any of a number of known genetic techniques, such as digestion with restriction endonucleases, ultraviolet light visualization of ethidium bromide stained agarose gels, DNA sequencing, or hybridization with detectable nucleic acid sequences (e.g. labelled probes).
- the genetic material from cells obtained according to the disclosed method can be amplified using known amplification techniques, such as Q-beta-replicase and the poiymerase chain reaction (PCR) (Saiki, R.K. et. al.. Science 239:487-491 (1988); Wong, C, et. al.. Nature 330, 384-386 (1987) and U.S. Patent 4,683,202 to Muilis et. al.) to further increase the amount of genetic material available for genetic analysis.
- known amplification techniques such as Q-beta-replicase and the poiymerase chain reaction (PCR) (Saiki, R.K. et
- Example 1 Obtaining Cells From Oral Cavities Using either a Brush or a Wooden Spatula
- the oral cavities of five human subjects were scraped with wooden spatulas for about 30 seconds, and in five human subjects, the CytosoftTM nylon bristle cytology brushes (Medical Packaging Corporation; Camarilo, CA) were rotated against the inner side of their cheeks for about 30 seconds.
- the spatulas or brushes were soaked in test tubes containing 50mM NaOH for about 1 hour, after which one tenth volume of IM TRIS (pH8) was added to the test tubes to neutralize.
- the cheek cell lysates obtained using the spatulas or the brushes were used directly in a poiymerase chain reaction (PCR) to amplify one or more of the following exons of the Cystic Fibrosis transmembrane regulator gene: exons 4, 10, 1 1 , 20 and 21.
- Apropriate primers for the PCR were developed based on the nucleic acid sequence disclosed in Riordan, J.R. et al.. Science 245: 1066-1073 (1989).
- PCR amplification reactions were performed using AmpliTaq DNA poiymerase and a DNA Thermal Cycler (Perkin Elmer). Cheek lysates were amplified for 28 cycles of 10 seconds at 94°, 10 seconds 55°C, and 30 seconds at
Abstract
A simple, non-invasive process for obtaining a non-liquid cell sample for genetic analysis using a brush is described.
Description
PROCESS FOR OBTAINING A NON-LIQUID CELL SAMPLE
Background of the Invention
Advances in recombinant DNA technology have made possible the molecular analysis and prediction of several genetic disorders. At the present time, genetic analyses are carried out mainly using blood leukocytes, which are obtained from whole blood (e.g. via centrifugation). Whole blood, in turn must be obtained clinically by a physician or phlebotomist employing an invasive technique (e.g. venipuncture).
A non-invasive method for obtaining genetic material using a mouthwash has been described (Lench, N., et. al.. The Lancet 1 : 1356-8 (June 18, 1988)). In this method, an individual rinses his mouth with saline which is then emitted from the mouth into a container. The container is centrifuged and cells, which are pelleted upon centrifugation, are collected. The ease with which samples can be obtained is a major attraction of this method. However, samples so obtained are in a liquid format and therefore present problems with regard to handling, processing and contamination.
DNA for genetic analysis has also been obtained from cervicovaginal cells (Burk, .D. and C. Spitzer, Am. J. Obstet. Gynecol.162:652-4 (1990)). In a study which compared the quantative yield of cellular material obtained by various methods, cervicovaginal lavage was found to be superior to scrape, swab or brush methods for obtaining cervicovaginal cells (Goldberg, GL et. al., Am. J. Obstet. Gynecol. 161 : 1169-72 (1989); Vermund, SH et. al., Ann. J. Obstet. Gynecol. 160: 304-8 (1989)). However, cervicovaginal cells obtained by lavage are in a liquid format and therefore present the same problems with handling, processing and contamination as samples obtained by mouthwashing techniques.
For forensic purposes, DNA has been obtained from epithelial cells, spun from urine (Gasparinin, P. et. al., N. Engl. J. Med. 320: 809 (1989)); and from root and surrounding sheath cells obtained from plucked hairs (Higuchi, R. et. al.. Nature 332:543-6 (1988)). However, samples so obtained often do not contain enough DNA for genetic analysis. Therefore hair and urine samples are not optimal for clinical analysis.
A simple, noninvasive technique for obtaining a non-liquid cell sample having an appropriate amount of genetic material for clinical analysis would be useful.
Summary of the Invention
The invention relates to a simple, noninvasive process for obtaining a non- liquid cell sample from an animal for genetic analysis. The process involves inserting an appropriately sized brush into an animal's oral cavity, contacting the brush with an oral membrane, agitating the brush against the membrane so that cells are captured within the bristles of the brush, removing the brush, which contains the captured cells and isolating the cells from the bristles of the brush for use in genetic analysis. In a preferred embodiment, the brush is spiral, so that cells can be obtained by rotating the brush against an oral membrane.
Cells so obtained can then be analyzed using any of a number of known genetic techniques. For example, cells obtained according to the disclosed method can be lysed and the cell's DNA can be contacted with a suitable labelled probe. In addition, DNA so obtained can first be amplified to increase the amount of genetic material available for analysis.
A major advantage of using the process described herein for obtaining cells for genetic testing is that the non-liquid sample so obtained is easier to handle and to process than a liquid sample. Further a non-liquid sample is less prone to contamination (e.g by bacteria) and is more stable.
Detailed Description of the Invention
In general, the invention relates to a process for obtaining a non-liquid cell sample from an animal (e.g. a human or a domesticated mammal) for genetic analysis. The process employs a brush to obtain cells from within a animal's oral cavity. Examples of cells which can be obtained from an animal's oral cavity include epithelial cells of the inner cheek or tongue.
According to the method of the invention, a brush is inserted into an animal's oral cavity, contacted with an oral membrane and agitated (e.g. rotated or brushed) against the membrane for a sufficient period of time, so that cells are captured within the bristles of the brush. The brush, which contains captured cells is then removed from the animal's mouth and cells are isolated from the bristles of the brush for use in genetic analysis.
To be suitable for use in the subject invention, a brush must be of an appropriate size to enable insertion into the animal's oral cavity. In addition, the brush bristles should be free of potentially contaminating material. For example, a brush comprising mammalian hair should not be used in the subject method, because genetic material present within the hair could contaminate the sample and thereby interfere with the genetic analysis. Preferred brushes for use in the subject invention have synthetic (e.g. nylon or dacron) bristles. An especially preferred brush for use in the invention is the nylon bristle cytology brush, (Cytosoft Medical Packaging Corporation, Camarillo, CA), which is currently used for Pap Smear and Chiamydia testing.
Once removed from the mouth, a brush which contains captured oral cells can be analyzed directly or transported and/or stored for subsequent genetic analysis. At this point, measures aimed at avoiding the dislodging of captured cells from a brush should be taken. For example, the brush can be placed in a container (e.g. an envelope) during transportation and/or storage. Cells may be obtained from a brush by any method which avoids loss of captured cells. A preferred method of obtaining cells from the brush is by
contacting the cells with a solution. For example, if DNA is to be isolated from the cells for amplification, the ceils can be obtained from the brush using a solution which lyses cells (e.g. sodium hydroxide, potassium hydroxide).
Genetic material from oral cells so obtained can then be analyzed genetically using any of a number of known genetic techniques, such as digestion with restriction endonucleases, ultraviolet light visualization of ethidium bromide stained agarose gels, DNA sequencing, or hybridization with detectable nucleic acid sequences (e.g. labelled probes). In addition, the genetic material from cells obtained according to the disclosed method can be amplified using known amplification techniques, such as Q-beta-replicase and the poiymerase chain reaction (PCR) (Saiki, R.K. et. al.. Science 239:487-491 (1988); Wong, C, et. al.. Nature 330, 384-386 (1987) and U.S. Patent 4,683,202 to Muilis et. al.) to further increase the amount of genetic material available for genetic analysis.
The invention will now be illustrated in the following Example, which is not intended to be limiting in any way.
Example 1 : Obtaining Cells From Oral Cavities Using either a Brush or a Wooden Spatula
The oral cavities of five human subjects were scraped with wooden spatulas for about 30 seconds, and in five human subjects, the Cytosoft™ nylon bristle cytology brushes (Medical Packaging Corporation; Camarilo, CA) were rotated against the inner side of their cheeks for about 30 seconds. The spatulas or brushes were soaked in test tubes containing 50mM NaOH for about 1 hour, after which one tenth volume of IM TRIS (pH8) was added to the test tubes to neutralize.
The cheek cell lysates obtained using the spatulas or the brushes were used directly in a poiymerase chain reaction (PCR) to amplify one or more of the following exons of the Cystic Fibrosis transmembrane regulator gene: exons 4, 10, 1 1 , 20 and 21. Apropriate primers for the PCR were developed based on the nucleic acid sequence disclosed in Riordan, J.R. et al.. Science 245: 1066-1073 (1989).
PCR amplification reactions were performed using AmpliTaq DNA poiymerase and a DNA Thermal Cycler (Perkin Elmer). Cheek lysates were amplified for 28 cycles of 10 seconds at 94°, 10 seconds 55°C, and 30 seconds at
74°C, with a final soak for 10 minutes at 74°C. The reaction buffer used was described previously (Saiki, R.K. et al.. Science 239:487-491 (1988)).
Amplified material was then blotted onto nylon membranes (Biotrans Plus, ICN Biomedical) and PCR products were analyzed on 1% agarose gels. No PCR products were detected for any of the five samples in which cells were obtained using a wooden spatula. In contrast. PCR products were detected for all five samples obtained using a brush. These PCR products were hybridized to sequence specific probes to identify known mutations (Kerem, B. et al.. Science 245: 1073-1080 (1989); Lemna et al.. N. Eng. J. Med. 322:291-296 (1990); Kerem et al.. Proa Natl. Acad. Sci. USA 87:8447-8451 (1990)).
Equivalents:
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
Claims
1. A process for obtaining a non-liquid animal ceil sample comprising the steps of:
a) inserting a brush having bristles and capable of being inserted into an animal's oral cavity; b) contacting the bristles of the brush with an oral membrane comprising cells; c) agitating the brush against the membrane so that cells from the membrane are captured in the brush bristles; d) removing the brush containing captured cells from the oral cavity, thereby obtaining a non-liquid cell sample.
2. A process of Claim 1 wherein the brush is spiral.
3. A process of Claim 1 wherein the animal is a human.
4. A non-liquid cell sample obtained according to the method of Claim 1.
5. A process for conducting a genetic analysis of an animal comprising the steps of:
a) inserting a brush having bristles and capable of being inserted into an animal's oral cavity; b) contacting the bristles of the brush with an oral membrane comprising cells; c) agitating the brush against the membrane so that cells from the membrane are captured in the brush bristles; d) removing the brush containing captured cells from the oral cavity; and e) lysing the cells obtained in step d), thereby yielding genetic material; f) conducting a genetic analysis on the genetic material.
6. A process of Claim 5 wherein the animal is a human.
7. Genetic material obtained according to the method of Claim 5.
8. A process for detecting the presence of a particular nucleic acid sequence of interest in genetic material contained in cells obtained from an animal, comprising the steps of :
a) inserting a brush having bristles and capable of being inserted into a mammal's oral cavity; b) contacting the bristles of the brush with an oral membrane comprising cells; c) agitating the brush against the membrane so that cells from the membrane are captured in the brush bristles; d) removing the brush containing captured cells from the oral cavity; e) lysing the cells obtained in step d), thereby yielding genetic material; g) contacting the genetic material with a detectable nucleic acid sequence complementary to the nucleic acid sequence of interest; h) allowing the genetic sequences and the detectable nucleic acid sequence to hybridize; and i) detecting hybridization as an indication of the presence of the nucleic acid sequence of interest.
9. A process of Claim 8 wherein the animal is a human.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US92987192A | 1992-08-07 | 1992-08-07 | |
US929871 | 1992-08-07 | ||
PCT/US1993/007382 WO1994003111A1 (en) | 1992-08-07 | 1993-08-05 | Process for obtaining a non-liquid cell sample |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0653920A1 true EP0653920A1 (en) | 1995-05-24 |
Family
ID=25458604
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP93919898A Withdrawn EP0653920A1 (en) | 1992-08-07 | 1993-08-05 | Process for obtaining a non-liquid cell sample |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0653920A1 (en) |
JP (1) | JPH08501228A (en) |
CA (1) | CA2137282A1 (en) |
WO (1) | WO1994003111A1 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995019447A1 (en) * | 1994-01-14 | 1995-07-20 | The Jockey Club | Non-invasive sampling method for nucleic acid analysis |
EP0956083A4 (en) * | 1995-11-15 | 2000-07-05 | Genelink Inc | Non-invasive identification system |
PT1155381E (en) * | 1999-02-23 | 2010-10-11 | Cdx Lab Inc | Minimally invasive apparatus and method for testing lesions of the oral cavity and similar epithelium |
US9968247B2 (en) | 2014-05-02 | 2018-05-15 | United States Endoscopy, Inc. | Cleaning device for an endoscopic device |
JP2016045102A (en) * | 2014-08-25 | 2016-04-04 | 日東電工株式会社 | Sample sampling material for high-performance liquid chromatography |
JP2016045103A (en) * | 2014-08-25 | 2016-04-04 | 日東電工株式会社 | Sample sampling material for gas chromatography-mass spectrometry |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2839049A (en) * | 1954-03-25 | 1958-06-17 | Kenneth S Maclean | Abrasive cytologic brush |
GB2159420B (en) * | 1984-05-29 | 1988-05-11 | Medscand Ab | Endocervical sampling brush and smear method |
EP0507904B1 (en) * | 1990-10-26 | 1999-01-20 | Sangtec Medical Ab | Method for evaluating the adequacy of clinical specimens for hybridization assays and kit for performing the evaluation |
-
1993
- 1993-08-05 JP JP6505572A patent/JPH08501228A/en active Pending
- 1993-08-05 WO PCT/US1993/007382 patent/WO1994003111A1/en not_active Application Discontinuation
- 1993-08-05 CA CA002137282A patent/CA2137282A1/en not_active Abandoned
- 1993-08-05 EP EP93919898A patent/EP0653920A1/en not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of WO9403111A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1994003111A1 (en) | 1994-02-17 |
CA2137282A1 (en) | 1994-02-17 |
JPH08501228A (en) | 1996-02-13 |
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