U.S. patent application number 12/521402 was filed with the patent office on 2011-01-13 for method and materials for triggered release of a biological sample.
This patent application is currently assigned to Qiagen GmbH. Invention is credited to Daniel Groelz, Thomas Hanselle, Christain Lenz, Uwe Oelmueller, Markus Sprenger-Haussels.
Application Number | 20110008771 12/521402 |
Document ID | / |
Family ID | 37735807 |
Filed Date | 2011-01-13 |
United States Patent
Application |
20110008771 |
Kind Code |
A1 |
Hanselle; Thomas ; et
al. |
January 13, 2011 |
METHOD AND MATERIALS FOR TRIGGERED RELEASE OF A BIOLOGICAL
SAMPLE
Abstract
The invention relates to a method for releasing a biological
sample from a solid matrix substantially without disintegration of
biomolecules comtained in said biological sample by at least
partially transferring the solid state of said matrix into a
dissolved or liquid state by changing at least one physico-chemical
property of the environment of said matrix.
Inventors: |
Hanselle; Thomas; (Hilden,
DE) ; Sprenger-Haussels; Markus; (Hilden, DE)
; Lenz; Christain; (Hilden, DE) ; Groelz;
Daniel; (Hilden, DE) ; Oelmueller; Uwe;
(Hilden, DE) |
Correspondence
Address: |
Baker Donelson Bearman, Caldwell & Berkowitz, PC
920 Massachusetts Ave, NW, Suite 900
Washington
DC
20001
US
|
Assignee: |
Qiagen GmbH
Hilden
DE
|
Family ID: |
37735807 |
Appl. No.: |
12/521402 |
Filed: |
December 27, 2007 |
PCT Filed: |
December 27, 2007 |
PCT NO: |
PCT/EP2007/064561 |
371 Date: |
August 28, 2009 |
Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
A61B 10/0051 20130101;
A61B 2010/0074 20130101; C12N 15/1003 20130101; Y10S 436/808
20130101; B01L 3/50825 20130101; G01N 2035/00267 20130101; B01L
2300/046 20130101; B01L 3/5029 20130101; B01L 2300/042 20130101;
G01N 1/4022 20130101; G01N 2001/028 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 29, 2006 |
EP |
06027074.1 |
Claims
1. A method for releasing a biological sample directly collected on
a solid state matrix from said matrix substantially without
disintegration of biomolecules contained in said biological sample
by at least partially transferring the solid state of said matrix
into a dissolved and/or liquid state by changing at least one
physico-chemical property of an environment of said matrix.
2. A method for releasing a biological sample from a solid state
matrix substantially without disintegration of biomolecules
contained in said biological sample by at least partially
transferring the solid state of said matrix into a dissolved and/or
liquid state by changing at least one physico-chemical property of
an environment of said matrix in the presence of at least one
chaotropic substance, optionally in the presence of at least one
substance of high ionic strength.
3. The method according to claim 1, wherein said change of said at
least one physico-chemical property of said environment consists of
a temperature increase.
4. The method according to claim 1, wherein said change of said at
least one physico-chemical property of said environment consists of
addition of a solvent and optionally addition of at least one
compound selected from the group consisting of reagent(s),
enzyme(s), acid(s), and base(s).
5. The method according to claim 1, wherein said change of at least
physico-chemical property of said environment consists of an
addition of at least one compound selected from the group
consisting of reagent(s), enzyme(s), acid(s), and base(s), and
optionally an addition of at least one liquid.
6. A method for releasing a biological sample from a solid matrix
comprising at least one selected from the group consisting of one
of waxes, high molecular weight alkane hydrocarbons, monoterpenes,
sucrose, agarose, starch, chitin, peptides, proteins, pectins,
agar, acidic polymethylacrylates, cellulose acetate, cellulose
acetate phthalate, vinylacetate, polyvinyl acetate phthalate,
hydroxypropyl methylcellulose-acetate succinate, alginates,
copolymers of methacrylic acid and methylmethacrylate, copolymers
of vinylacetate and methacrylic acid, and
polyvinylacetatephthalate, said method being conducted
substantially without disintegration of biomolecules contained in
said biological sample and said method comprising: adding at least
one enzyme and at least one liquid to said matrix in the presence
of at least one chaotropic substance, and optionally in the
presence of at least one substance of high ionic strength.
7. The method of claim 6, further comprising adding at least one
further compound selected from the group consisting of reagent(s),
solvent(s), enzyme(s), acid(s), and base(s).
8. A kit comprising a dissolvable solid matrix and at least one
compound selected from the group consisting of reagent(s),
enzyme(s), acid(s), and base(s), liquid(s), and solvent(s).
9. The kit of claim 8, further comprising at least one one
chaotropic substance and optionally at least one substance of high
ionic strength.
10. The kit of claim 8 comprising a dissolvable solid matrix
comprising at least one selected from the group consisting of wax,
high molecular weight alkane hydrocarbons, paraffin, monoterpenes,
sucrose, agarose, starch, chitin, peptides, proteins, pectins,
agar, acidic polymethylacrylates, cellulose acetate, cellulose
acetate phthalate, vinylacetate, polyvinyl acetate phthalate,
hydroxypropyl methylcellulose-acetate succinate, alginates,
copolymers of methacrylic acid and methylmethacrylate, copolymers
of vinylacetate and methacrylic acid, and base(s), and/or
liquid(s), thereof.
11. A solid matrix for direct collection of a biological sample
said matrix comprising at least one selected from the group
consisting of waxes, high molecular weight alkane hydrocarbons,
monoterpenes, sucrose, agarose, starch, chitin, peptides, proteins,
pectins, agar, acidic polymethylacrylates, cellulose acetate,
cellulose acetate phthalate, vinylacetate, polyvinyl acetate
phthalate, hydroxypropyl methylcellulose acetate succinate,
alginates, copolymers of methacrylic acid and methylmethacrylate,
copolymers of vinylacetate and methacrylic acid, and
polyvinylacetatphthalate.
12. The method according to claim 2, wherein said change of said at
least one physico-chemical property of said environment consists of
a temperature increase.
13. The method according to claim 2, wherein said change of said at
least one physico-chemical property of said environment consists of
addition of a solvent, and optionally addition of at least one
compound selected from the group consisting of reagent(s),
enzyme(s), acid(s), and base(s).
14. The method according to claim 2, wherein said change of at
least physico-chemical property of said environment consists of an
addition of at least one compound selected from the group
consisting of reagent(s), enzyme(s), acid(s), and base(s), and
optionally an addition of at least one liquid.
Description
TECHNICAL FIELD
[0001] The invention relates to a method and materials for
releasing a biological sample from a matrix and to a kit for
releasing a biological sample from a matrix.
STATE OF THE ART
[0002] The analysis of biological material has long been of
paramount importance in the diagnosis and treatment of disease, in
food and environmental analysis and in forensic investigations, in
particular using histological and pathological techniques. Recent
technological advances have broadened the scope of such
investigations by facilitating analysis of nucleic acids and
proteins, which has opened up a large number of further
possibilities. Gene activity can, for example, be determined
directly by analysis of RNA, in particular the messenger RNA (mRNA)
in cells. Quantitative analysis of transcription templates (mRNA
templates) in cells by means of modern molecular biological
methods, such as real time reverse transcriptase polymerase chain
reaction (real time RT-PCR) or gene expression chip analysis
enables, for example the identification of incorrectly expressed
genes, so that, for example, metabolic disorders, infections or the
presence of cancer can be detected. The analysis of DNA from cells
by molecular biological methods, such as PCR (polymerase chain
reaction), RFLP (restriction fragment length polymorphism), ALFP
(amplified fragment length polymorphism) or sequencing permits, for
example, the detection of genetic defects or the determination of
the HLA (human leukocyte antigen) type as well as other genetic
markers. The analysis of genomic DNA and RNA is also used as direct
evidence for infectious agents, such as viruses, bacteria, and the
like.
[0003] As the benefits of being able to analyse certain components
of biological samples, for example nucleic acids or proteins, have
become known, and as the analyses themselves have become more
accurate and more accessible, such analyses have become important
and frequently used tools available not only to the medical and
veterinary professions, but also in a wide range of other areas,
such as in the analysis of forensic materials, pharmaceutical
products and intermediates, foods and environmental materials. In
many of these areas it is important to maintain the integrity of
the molecular structure of a sample.
[0004] Current procedures for the collection and/or handling of
biological samples such as blood, saliva or cells employ solid
matrices such as cellulose- or cotton-based papers or swabs which
differ significantly in their constituents and size.
[0005] For buccal or vaginal smears, swabs with a head made of
cellulose, cotton or polymer fibres are frequently used. Swabs with
heads made from cotton or synthetic fibre and swabs with ejectable
paper heads, for example, are available from various commercial
suppliers. After collection of the biological sample with such
swabs, the swab is usually dried, stored and transported in a dried
form, or stored and transported in a medium containing nutrients,
antibiotics or other preservatives.
[0006] In order to recover the biological sample from the swab, it
is generally necessary to wash the sample away from the swab
support material. The washing away of the sample, which is often
done by contacting the swab with a lysis reagent, is generally
inefficient, incomplete and cumbersome. To maximise the amount of
sample recovered, the support material of the swab head needs first
to be separated from its shaft. Depending on the type of swab, this
is accomplished by hand, by cutting with scissors, with a scalpel
or a razor blade, or by bending or ejection. Separation processes
involving cutting require the cutting device to be thoroughly
cleaned and sterilised or disposed of after each use to avoid
cross-contamination. Cross-contamination is a problem for all
analytical methods, but particularly when working with samples
destined for analysis of the nucleic acids, especially when
employing an amplification process such as the polymerase chain
reaction (PCR). Cross-contamination can, in the worst case, lead to
distortion or even total falsification of the analysis results. The
cutting process also represents a significant risk of injury to the
operator, which can result in incorporation of foreign, potentially
infectious material.
[0007] Ejectable swabs, where the head of the swab is separated
from the shaft by bending or pressing the swab shaft, represent an
expensive alternative.
[0008] Both types of swab have in common that the separated swab
head carrying the biological material necessarily remains in the
sample tube after separation from the swab shaft. The separated
swab head then absorbs the transportation and/or storage solution,
decreasing the accessible volume in the tube and hindering an
efficient recovery of the sample with a minimal amount of liquid.
Since often only very small amounts of sample are concerned, often,
for example, on a nanogram scale, it is desirable to avoid as far
as possible any loss or over-dilution of the sample.
[0009] Other biological samples such as blood are generally
collected and stored using papers or cards, which can be treated or
untreated. Treated papers are usually treated with several
different agents who inactivate pathogens and prevent microbial
growth and DNA degradation. In order to separate target
biomolecules it is necessary to punch out smaller pieces of paper
or card from the paper carrying the dried sample material. This
process requires the cleaning and sterilisation of the punching
device after each punching action in order to avoid
cross-contamination. Disposable punching equipment is commercially
available, but is cumbersome and expensive. In addition, the small
weight of the paper punch together with static electricity and
normal air movement make it difficult to handle the punch and to
transfer it to the bottom of the sample preparation vessel.
[0010] Furthermore, for optimal yield of the target biomolecule it
is necessary to cut the support material into small pieces before
introducing it into the process to recover the biological sample.
The cutting process is cumbersome and difficult to automate and
represents another potential source of cross-contamination and risk
of injury of the operator. Not cutting the support material results
in reduced yields of recovered biological sample and compromised
sensitivity in the downstream application, such as analysis of the
sample.
[0011] After transfer of the solid matrix into the recovery
reagent, which is usually a lysis reagent, the target biomolecules
are usually eluted from the solid support. The elution of target
biomolecules from the solid matrix is inefficient, cumbersome and
tends to suffer from retention of a proportion of the biomolecules
on the solid matrix. Elution can be carried out chemically,
physically or enzymatically, or by a combination thereof. For
further isolation of the target biomolecule it is necessary to
separate the solid matrix from the liquid containing the
biomolecule. This is done by removal of the liquid by pipetting, or
by removal of the solid matrix. Both processes are inefficient and
can lead to loss of sample material unless large quantities of
solvent are used. They are also difficult to automate, because the
solid matrix can interfere with a liquid handling system. It is
also difficult to design an automated system that is able to remove
blood spots or swabs reliably from a vessel. Another problem is the
amount of liquid that remains trapped in the solid matrix. Since
this liquid comprises the target biomolecules, its removal with the
solid matrix causes decreased yield and sensitivity.
[0012] A number of different solid matrices in the form of a blood
card or swabs, for example swabs with heads made from cellulose,
cotton, Dacron.RTM. or other polymeric fibres is known and
commercially available. There is also a choice of several recovery
and purification methods. There is, however, a lack of a standard
procedure for collection, storage, stabilisation and purification
of biological samples. This makes it necessary to carry out a lot
of optimisation work in order to optimise the yield and performance
of a given combination of support material and sample preparation
method.
[0013] WO 01/60517 describes a method for taking a blood sample
using a receptacle for receiving a sample, preferably blood,
containing a solution for stabilising nucleic acids and a solid
phase capable of binding nucleic acids. The sample, once
transferred to the vessel, subsequently needs to be removed from
the solid phase by repeated washing. Furthermore, the collection of
the sample requires the use of a cannula to transfer the liquid
sample from its source into the receptacle, which has been placed
under a low pressure. This prevents the use of the receptacle for
small amounts of sample, for example blood from a finger prick or a
heel prick from an infant, and for samples which cannot easily be
transferred via cannula, or only with some discomfort for the
sample donor, such as saliva, urine or cerebrospinal fluid. The
receptacle is also not suitable for solid or semi-solid samples
since a solid or semi-solid sample does not mix sufficiently with
the stabilising solution in the presence of the solid phase.
[0014] EP 819 696 A2 describes a method of isolating nucleic acid
from a biological sample and proposes the use of a nucleic
acid-binding solid phase, preferably silica, which is mixed with
the sample together with a chaotropic substance. The method
requires a large number of washing, drying and elution steps in
order to first isolate the solid phase with the thereto-bound
nucleic acid from the solution and then isolate the nucleic acids
from the solid phase. Furthermore, too large an amount of silica
can be saturating, such that beyond a given amount of silica no
further nucleic acid is obtained from the sample.
[0015] WO 02/072870 A2 describes a method and a device for storing
genetic material. The head portion of the device is composed of a
solid matrix for adsorbing genetic material and a preserving
mechanism to protect the genetic material from degradation. The
device can, however, only be used for liquid samples. Furthermore,
the solid matrix must be cut into smaller portions in order to
carry out analysis of the genetic material, resulting in loss of
material and risk for the operator.
[0016] US 2006/0099567 and US 2005/0276728 propose a storage device
for biological materials comprising dissolvable or dissociable
matrix material which coats a sample well in a sample plate. The
matrix material coating together with the sample is dried and
subsequently rehydrated for sample recovery. The proposed system is
particularly intended for use in high throughput systems. The
biological sample is, however, first collected using conventional
techniques and as such the proposed system does not address the
above-mentioned problems associated with recovery of the sample
from the collecting device. In addition, these documents are silent
about the benefits of dissolving the matrix material in the
presence of a chaotropic salt.
[0017] It is an object of the present invention to overcome the
disadvantages known from the prior art.
[0018] Another object of the present invention is to enable a
simpler and faster recovery of a biological sample from a device
used to collect the sample, compared to sample recoveries known
from the prior art.
[0019] It is also an object of the present invention to minimise
the loss of biological material when recovering a biological sample
from a device used to collect the sample, without needing to use
large quantities of solvent.
[0020] It is a further object of the present invention to avoid the
use of cutting and/or punching devices in the recovery of a
biological sample from a collecting device.
[0021] Another object of the invention is to provide a means of
collecting and handling a biological sample which can be applied to
a range of different types of biological sample.
[0022] It is a further object of the present invention to provide
an integrated sampling or collecting and storage device and a
method for using or further processing such integrated device.
[0023] It is a further object of the present invention to provide a
method for dissolving or liquefying a sampling device without
substantial degeneration of the biomolecules contained in said
biological sample.
[0024] It is a further object of the present invention to provide a
method for dissolving or liquefying a sampling device wherein the
interaction of water molecules with biomolecules contained in said
biological sample is modified without substantial degeneration of
the biomolecules contained in said biological sample.
BRIEF DESCRIPTION OF THE INVENTION
[0025] A contribution to solving the problems arising from present
techniques and to at least partly overcoming their disadvantages is
made according to the present invention by a method for releasing a
biological sample from a solid matrix substantially without
disintegration of said biological sample by at least partially
transferring the solid state of said matrix into a dissolved or
liquid state by changing at least one physico-chemical property of
the environment of said matrix, preferably in the presence of at
least one chaotropic substance. Preferably, the solid matrix has
been used for direct collection of said biological sample.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The biological sample can be a sample collected from any
biological source and is preferably a biological sample of any
human, animal and plant material. As biological samples according
to the invention are generally considered, without limitation,
cell-free and cell-comprising sample material, plasma, bodily
fluids, such as blood, sputum, saliva, urine, cerebrospinal fluid,
sperm, serum as well as cells, leukocyte fractions, crusta
phlogistica (buffy coat), faeces, swabs, aspirates, tissue samples
of any sort, tissue fragments and organs, vaccines, food or
environmental samples which comprise free or bound nucleic acids or
nucleic acid-carrying cells, plants, plant extracts and plant
parts, bacteria, viruses, yeasts and other fungi, or extracts
therefrom, other eukaryotes and prokaryotes, among others, in
particular all possible tissue samples, tissue fragments, organs,
whole organisms and individual cells. Preferred biological samples
comprise a biological material such as any bodily fluid, such as
blood, saliva, urine, semen, sputum or cerebrospinal fluid, an
epithelial cell, a smear, a bacterium, a virus, a vaccine, a fine
needle aspirate, a biopsy, a forensic sample, an isolated cell, a
fungus or a part or an extract of a fungus, a plant or a part or an
extract of a plant. Direct collection is understood to describe the
fact that said matrix has been used directly during the collection
of the biological sample. A non-limiting example of such use would
be a so called swab that would be brought in close contact with
said biological sample, e.g. by rubbing at the mucous membrane in
the interior of the mouth or the like. Non-direct collection in
contrast would be if another collection device was used for
collecting the biological sample and the biological sample would
have been then deposed by that collection device on the matrix
according to the invention, e.g. by washing it from said collection
device on said matrix.
[0027] Biomolecules are organic molecules present in a biological
fluid such as nucleic acids, proteins, enzymes, and the like. In a
preferred embodiment the biological sample comprises a protein
and/or nucleic acid or nucleic acids. The term "nucleic acid" is
here used in its broadest sense and comprises ribonucleic acids
(RNA) and deoxyribonucleic acids (DNA) from all possible sources,
in all lengths and configurations, such as double stranded, single
stranded, circular, linear or branched. All sub-units and sub-types
are also comprised, such as monomeric nucleotides, oligomers,
plasmids, viral and bacterial nucleic acids, as well as genomic and
non-genomic DNA and RNA from animal and plant cells or other
eukaryotes or prokaryotes, messenger RNA (mRNA) in processed and
unprocessed form, transfer RNA (tRNA), heterogeneous nuclear RNA
(hn-RNA), ribosomal RNA (rRNA), complementary DNA (cDNA), genomic
DNA (gDNA), siRNA, miRNA, as well as all other conceivable nucleic
acids.
[0028] Substantially without disintegration according to the
invention is preferably understood to mean that the biomolecule
remains substantially intact and does not disintegrate into its
component parts when the solid matrix is at least partially
transferred from the solid state into a dissolved or liquid state.
It can, however, also mean that at least one component of the
biological sample remains substantially intact and does not
disintegrate into its respective component parts when the solid
matrix is at least partially transferred from the solid state into
a dissolved or liquid state.
[0029] Modification of the interaction of water molecules with
biomolecules contained in said biological sample is being
understood as decreasing or totally destroying any interaction of
water with biomolecules, preferably nucleic acid, such as formation
of a hydrate shell or any other hydrogen bond structures between
the biomolecules contained in said biological sample and water
molecules. Surprisingly, it has been found that said modification
of the interaction of water molecules with biomolecules does result
in higher yield and or better performance of the method according
to the invention.
[0030] It is preferred that the at least one solid matrix is at
least partially transferable from the solid state into a dissolved
or liquid state for at least partially releasing the collected
biological sample when at least one physico-chemical property is
changed, preferably in the presence of at least one chaotrope. The
term "solid state" is understood to mean a physical state of the
solid matrix. The dissolved or liquid state of the solid matrix
according to the present invention, on the other hand, can be in
the form of a solution, dispersion, suspension, emulsion or melt.
The terms "transferable" and "at least partially transferable"
according to the invention should be understood as meaning that at
least part of, preferably most of and most preferably all of the
solid matrix comprised in the method according to the invention can
be transferred from the solid state into a dissolved or liquid
state. It is preferred according to the invention that changing at
least one physico-chemical property triggers at least a partial,
preferably to at least 10 wt. %, more preferably to an amount of at
least 50 wt. %, even more preferably to an amount of at least 75
wt. % and most preferably a full transfer into said liquid or
dissolved state. The transformation from the solid state into a
dissolved or liquid state is preferably in the form of a
dispersing, an emulsifying, a suspending, a dissolving or a
melting, preferably a dispersing, a dissolving or a melting, and
preferably occurs by changing at least one physico-chemical
property according to the invention.
[0031] Physico-chemical properties which can be changed to result
in an at least partial transfer of the solid state of the matrix to
a dissolved liquid state comprise, for example, contacting the
solid matrix with a liquid in which it at least partially
dissolves, or contacting the solid matrix with a reagent or with a
composition which comprises at least one component which enhances
or causes its dissolution, or subjecting the solid matrix to a
temperature or pressure at which it melts. These physico-chemical
properties can have an effect alone or in combination with each
other or with other physico-chemical properties which, when
changed, effect, catalyse or in any way enhance a transfer of the
solid matrix into a liquid state.
[0032] It is preferred according to the present invention that the
solid matrix should be at least partially solid, preferably
substantially solid, more preferably entirely solid, under the
collection conditions employed for collecting the biological
sample. This is particularly preferable if the sample is to be
collected directly at its source. The collection conditions can be
physiological conditions when collecting a sample from a living
organism, for example a sample from a human, animal or plant.
Physiological conditions are generally characterized as the typical
conditions of physico-chemical properties such as temperature, pH,
concentration of electrolytes, presence of enzymes or other
components, among others, which are present in the organism, in
particular in the part of the organism which is to be collected as
a sample, in its natural state.
[0033] Thus for a healthy human, for example, physiological
temperatures fluctuate but generally range from 36.degree. C. to
38.degree. C. These temperatures can be altered by several degrees
in an ill human, for example reaching as high as 44.degree. C. or
as low as 30.degree. C. Physiological pH in a human is generally
considered to be 7.4. pH can, however, vary depending on the
location in the body, whereby pH in the stomach, for example,
generally lies in the range of from 1.5 to 3.
[0034] It is particularly preferred according to the invention that
the biological sample is applied to the solid matrix by means of
directly contacting the solid matrix with the biological sample and
preferably by collecting the biological sample directly from its
source using the solid matrix (see direct collection). The
biological sample is preferably not poured onto the solid matrix
according to the invention.
[0035] The transfer of the at least one solid matrix from the solid
state into a dissolved or liquid state, preferably in the presence
of at least one chaotrope, preferably occurs in the course of at
least one of stabilization, transport, or storage of the solid
matrix carrying the biological sample, or in the course of
processing the biological sample, for example by purification of
target biomolecules from the solid matrix. It is advantageous that
the biological sample does not dry on the solid matrix. Thus, for
example, a stabilizing material, preferably in the form of a
stabilizing liquid which is applied to stabilize the biological
sample can itself effect or enhance an at least partial dissolving
of the solid matrix to at least partially release the sample or a
component thereof, or can comprise an agent which effects or
enhances a dissolving of the solid matrix to at least partially
release the sample or a component thereof. This at least partial
dissolving of the solid matrix can then occur while the solid
matrix comprising the biological sample is in contact with the
stabilizing material, preferably the stabilizing liquid, during
stabilization, transport, handling or storage of the sample.
[0036] Another possibility is that an agent which causes or
enhances the dissolving of the solid matrix is applied, preferably
but not necessarily in the form of a liquid, in the course of
processing the biological sample, for example in the course of
isolation and/or preparation of the biological sample or a
component thereof for analysis, or isolation and/or purification of
target biomolecules. It is also possible that the temperature is
increased or decreased, preferably increased, during at least one
of stabilization, transport, processing and storage of the solid
matrix which is in contact with the biological sample. This would
cause a meltable solid matrix to be at least partially transferred
from the solid state to a liquid state and preferably to at least
partially release the biological sample or a component thereof
substantially without disintegration of the biological sample
according to the invention.
[0037] In one preferred embodiment of the method according to the
invention, the change of physico-chemical property of the
environment of the matrix consists of a temperature increase,
preferably in the presence of at least one chaotrope.
[0038] It is advantageous if the solid matrix is solid at the
temperature of collection of the biological sample. If a sample is
to be collected from a living person or animal, it is preferred
that the solid matrix is solid at physiological temperatures, and
that the solid matrix gets transferred to the liquid state above
physiological temperatures, preferably at temperatures above
45.degree. C., preferably at temperatures within the range from
45.degree. C. to 95.degree. C., preferably at temperatures within
the range from 45.degree. C. to 85.degree. C., more preferably at
temperatures within the range from 45.degree. C. to 75.degree. C.,
even more preferably at temperatures within the range from
45.degree. C. to 70.degree. C., most preferably at temperatures
within the range from 45.degree. C. to 65.degree. C. It is
preferred that the biological sample can be released without being
damaged or degraded. The solid matrix is thus preferably meltable
at a temperature at which the biological sample and its components
are not destroyed or degraded. If a sample is to be collected from
a dead person or animal, or from a plant, fungus, food, or from the
environment, the solid matrix can melt at a lower temperature,
provided that it is solid at the temperature of collection of the
sample. The solid matrix preferably has a sharp melting point, i.e.
it melts over a small temperature range, preferably over a
temperature range of less than 5.degree. C., more preferably over a
temperature range of less than 4.degree. C., even more preferably
over a temperature range of less than 3.degree. C., most preferably
over a temperature range of less than 2.degree. C.
[0039] Materials which are suitable as meltable solid matrices
according to the invention are, for example, fats which are solid
at physiological temperatures, such as cocoa butter or palm fat. A
further preferred meltable solid matrix according to the invention
is a wax with a melting point above 45.degree. C. and a plastic
structure at normal ambient temperatures. Suitable waxes can be
animal and/or insect waxes, for example beeswax. Chinese wax
produced by scale insects coccus ceriferus, shellac wax from the
lac insect coccus lacca, spermaceti from the head cavities and
blubber of the sperm whale and lanolin (wool wax) from the
sebaceous glands of sheep are mentioned as examples. Examples of
suitable vegetable waxes are bayberry wax from the surface of the
berries of the bayberry shrub, candelissa wax from the Mexican
shrubs Euphorbia cerifera and E. antisyphilitica, carnauba wax from
the leaves of the carnauba palm, castor wax, catalytically
hydrogenated castor oil, esparto wax, a byproduct of making paper
from esparto grass, Japan wax, a vegetable tallow (not a true wax)
from the berries of Rhus and Toxicodendron species, jojoba oil
pressed from the seeds of the jojoba bush, a replacement for
spermaceti, ouricury wax from the Brazilian feather palm and rice
bran wax obtained from rice bran. Mineral waxes are also suitable
as meltable solid matrix according to the invention, such as
ceresin waxes, montan wax extracted from lignite and brown coal,
ozocerite found in lignite beds and peat waxes. Another group of
materials suitable as solid matrix according to the invention is
high molecular weight alkane hydrocarbons such as paraffin, an
odourless, tasteless petroleum wax with a typical melting point
between 47.degree. C. and 65.degree. C. Also suitable are synthetic
waxes such as polyethylene waxes based on polyethylene,
Fischer-Tropsch waxes, or chemically modified waxes--usually
esterified or saponified, substituted amide waxes and polymerised
.alpha.-olefins. Further possible solid matrices are monoterpenes
such as thymol, found in oil of thyme and extracted as a white
crystalline substance of a pleasant aromatic odour and strong
antiseptic properties, with a melting point of 48-52.degree. C., or
camphene, a bicyclic monoterpene with a melting point of
51-52.degree. C. Another group of meltable solid matrix comprises
sugar, i.e. sucrose in combination with an aqueous lysis buffer. A
further possible solid matrix is low melting agarose with a melting
point below 65.degree. C.
[0040] In another embodiment of the device according to the present
invention, the solid matrix is at least partially soluble,
preferably fully soluble, for at least partially releasing the
collected biological sample, preferably in the presence of at least
one chaotrope. In this embodiment, the change of physico-chemical
property of the environment of the matrix can consist of addition
of a solvent and optionally at least one compound selected from
reagent(s), enzyme(s), acid(s), and base(s). It is also possible
that the change of physico-chemical property of the environment
consists of addition of at least one compound selected from
reagent(s), enzyme(s), acid(s) and base(s) and optionally one or
more liquids.
[0041] The solid matrix can thus preferably be dissolved, for
example, by at least one of contacting it with a liquid in which it
dissolves and contacting it with a reagent or with a composition
which effects, catalyses or otherwise enhances its dissolution,
preferably in the presence of at least one chaotrope. It is also
conceivable that, for example, the solid matrix can be contacted
with a liquid in which it does not dissolve and then is at least
partially dissolved by further contacting with a reagent or
composition. It is also possible that the solid matrix is first
contacted with a reagent or composition and then contacted with a
liquid. The reagent or composition can be in solid or liquid form.
The term "liquid form" is understood to mean at least one solvent,
or at least one solution in any conceivable solvent or mixture of
solvents which is considered suitable by the person skilled in the
art, or reagents or compositions which are liquid at the desired
operating temperature, as well as melts.
[0042] In one aspect of the invention the solid matrix can be at
least partially dissolved by at least one organic solvent as
reagent, preferably in the presence of at least one chaotrope. In
this case the solid matrix is preferably a polymer, preferably a
polymer selected from polymers comprising at least one carboxylic
acid-comprising monomer, polymers comprising at least one
carboxylic acid group-comprising monomer and at least one
ethylenically unsaturated monomer, polymers comprising at least one
monomer comprising at least one acid group and at least one
ethylenically unsaturated group, polymers comprising at least one
monomer comprising at least one ring system, preferably at least
one ring comprising at least one hetero-atom, preferably at least
one oxygen-comprising ring, polymers comprising at least one ester
group. Examples of preferred polymers are acidic polymethacrylates,
which are soluble in iso-propanol, cellulose acetate phthalate,
which is soluble in acetone, crotonic acid copolymers, such as
vinyl acetate, which are well soluble in dichloromethane and very
well soluble in acetone, polyvinylacetate phthalate, which is well
soluble in 90% ethanol, or hydroxypropylmethlcellulose-acetate
succinate, which is well soluble in acetone.
[0043] The reagent according to the invention can comprise at least
one chelator or salt thereof. The solid matrix can thus be at least
partially transferred into a liquid or dissolved state with
assistance from at least one chelator or salt thereof as reagent,
in the presence of a solvent or optionally one or more liquids as
defined above. A suitable chelator according to the invention
comprises at least 2, 3, 4, 5, 6, 7 or 8 coordinating sites,
preferably 2 to 6, more preferably 4 to 6 binding sites.
[0044] It is especially preferred that the solid matrix is at least
partially transferred into a liquid or dissolved state in the
presence of at least one chaotropic substance (chaotrope),
optionally in the presence of at least one solution of a high ionic
strength, as reagent. The ionic strength, I, of a solution is a
function of the concentration of all ions present in a solution. On
a molality basis,
I m = 1 2 .SIGMA. m B z B 2 ##EQU00001##
where the sum goes over all the ions B. .about.B is the charge
number of ion B. (see e.g. IUPAC Compendium of Chemical
Terminology, Electronic version,
http://goldbook.iupac.org/I03180.html).
[0045] Solutions of high ionic strength are known to the skilled in
the art and do have an ionic strength in the range of 0.1 to 50,
preferably 5-30, especially preferred 7-24. Chaotropic substances
are known from U.S. Pat. No. 5,234,809 and other related patents
which for the purposes of the US-patent practice are herewith
incorporated by reference. A chaotrope is an agent which causes
molecular structure to be disrupted, in particular molecular
structures formed by non-covalent forces such as hydrogen bonding,
salt bridges, and the hydrophobic effect. As chaotrope, guanidinium
salts are preferred, whereby guanidinium isothiocyanate or
guanidinium hydrochloride are particularly preferred. The at least
one chaotrope is preferably present in solution, preferably in
aqueous solution, at a concentration within the range from 0.01-15
(mol/l), preferably 1 to 6 M (mol/l), more preferably within the
range from 2 to 5.9 M, even more preferably within the range from 3
to 5.8 M and most preferably within the range from 4 to 5.7 M. In
this aspect of the method according to the invention, therefore,
the solid matrix can be at least partially transferred into a
liquid or dissolved state by being contacted with a substance or a
composition that effects, catalyses or otherwise enhances the
disruption of the water structure.
[0046] It is also conceivable according to a further aspect of the
present invention that the solid matrix can be at least partially
transferable into a liquid or dissolved state by contacting with at
least one enzyme, preferably in the presence of at least one
chaotrope. The solid matrix can thus comprise at least one material
which can be at least partially transferred from the solid state
into a dissolved or liquid state in the presence of at least one
enzyme.
[0047] The enzyme is preferably at least one enzyme selected from
the group consisting of proteases, cellulases, amylases,
glycanases, chitinases, lysozymes, lipases, esterases, pectinases,
pectolyases, agarases which can be present alone or in combination
with at least one other enzyme, reagent, acid or base.
[0048] It is preferred according to the invention that the solid
matrix comprises at least one of a peptide, an ester, a
polysaccharide and a cellulose and/or a derivative of at least one
thereof. These preferred materials are particularly advantageous if
the solid matrix is to be transferred into a dissolved or liquid
state in the presence of at least one enzyme. They are also
preferably capable of being at least partially transferred into a
liquid or dissolved state when at least one other physico-chemical
property is changed. Examples of preferred solid matrices are
materials linked by peptide bonds that are degradable by proteases,
materials linked with ester bonds that are degradable by esterases
and/or by changing pH; polysaccharides linked by glycosidic bonds
that are degradable by polysaccharide-degrading enzymes such as
amylases, chitinases, cellulases, or by other enzymes or
reagents.
[0049] A preferred solid matrix according to the invention
comprises or has a surface comprising a cotton-based material. By
"cotton" according to the invention is preferably understood a
natural polymer of cellulose, preferably a polysaccharide composed
of 100-10,000 .beta.-D-glucose molecules connected via one to four
glycosidic bonds. Cellulose according to the invention is insoluble
in aqueous solutions but can be solubilised by degrading the
polymer to .beta.-D-glucose. This degradation is preferably
mediated by the enzyme cellulase, for example as commercially
available from Novozyme, or Sigma.
[0050] A further preferred solid matrix according to the invention
comprises or has a surface comprising a starch-based material. By
"starch" according to the invention is preferably understood a
combination of two polymeric carbohydrates (polysaccharides),
preferably a combination of amylose and amylopectin. Starches
according to the invention are insoluble in water. They can be
digested by hydrolysis, preferably catalysed by enzymes called
amylases, which can break the glycosidic bonds between the
"alpha-glucose" components of the starch polysaccharide. The
resulting sugars can then be processed, for example, by further
enzymes such as maltase.
[0051] Another solid matrix preferred according to the invention
comprises or has a surface comprising a polyester-based material.
The polyester-based material can be solubilised by incubation with
esterases or lipases.
[0052] A further solid matrix preferred according to the invention
comprises or has a surface comprising a material based on a chitin
or a chitin derivative. By "chitin" according to the invention is
preferably understood a material constructed from units of
acetylglucosamine, in particular N-acetyl-D-glucos-2-amine, linked
together in .beta.-1,4 fashion. Chitin can be digested, for example
by enzymatic incubation with lysozyme or chitinase, for example
from Bacillus sp. PI-7S.
[0053] In another preferred aspect of the solid matrix according to
the invention, it can comprise or have a surface comprising at
least one di-, oligo- or polypeptide material. The peptide-based
material can be degraded and solubilised by incubation with a
protease, for example Proteinase K. Proteinase K is an endolytic
protease that cleaves peptide bonds at the carboxylic sides of
aliphatic, aromatic or hydrophobic amino acids. Other proteases,
for example collagenases, plasmin, subtilisins may be employed,
depending on the basis or surface of the solid matrix.
[0054] The solid matrix can also comprise or have a surface
comprising at least one pectin or derivative thereof. Pectin is
preferably understood as primarily comprising an
.alpha.-(1,4)-polygalacturonic acid backbone which can be randomly
acetylated and methylated. The pectin-based material can be
degraded and solubilised by incubation with pectinase and/or
pectolyase. Pectinase catalyses the random hydrolysis of
1-4-.alpha.-D-galactosiduronic linkages in pectin and other
galacturonans. Pectolyase catalyses the eliminative cleavage of
(1,4)-.alpha.-D-galacturonan methyl ester to give oligosaccharides
with 4-deoxy-6-O-methyl-.alpha.-D-galact-4-enuronosyl groups at
their non-reducing ends.
[0055] A further preferred solid matrix according to the invention
comprises or has a surface comprising an agar or agarose or a
derivative thereof. Agar-based materials can be solubilised by
incubation with agarase (agarose 3-glycanohydrolase).
[0056] It is preferred according to the invention that the solid
matrix is composed of at least one material that can be transferred
into a dissolved or liquid state by changing the pH of its
environment, preferably in the presence of at least one chaotrope.
It is preferred that the solid matrix is at least partially soluble
at a pH other than 7, preferably at a pH within the range from 1 to
6, preferably within the range from 2 to 6, more preferably within
the range from 3 to 6, yet more preferably within the range from 4
to 6, even more preferably within the range from 5 to 6, or at a pH
within the range from 8 to 14, preferably within the range from 8
to 13, more preferably within the range from 8 to 12, yet more
preferably within the range from 8 to 11, even more preferably
within the range from 8 to 10, again more preferably within the
range from 8 to 9. The solid matrix which is at least partially
soluble at a pH other than 7 can thus be at least partially
dissolvable through contact with a reagent that induces and/or
catalyses the disintegration or dissolving of the solid matrix
through the pH of the reagent. In a preferred embodiment the solid
matrix is insoluble in acidic media and soluble in neutral and/or
basic media. In another embodiment, the solid matrix is insoluble
in basic media and soluble in neutral and/or acidic media. In a
further embodiment the solid matrix is insoluble in neutral or
physiological-pH media and soluble at acidic and/or basic pH.
Numerous pH-dependent reactions triggered by the presence of acid
or base, such as by the concentration of H.sub.3O.sup.+ or OH.sup.-
ions, are known to those skilled in the art.
[0057] Suitable solid matrices which can be transferred into a
dissolved or liquid state in a pH-dependent manner are, for
example, metastable materials which are stable or almost stable in
a first pH range for at least 30 seconds and which dissolve in a
different pH range. Preferred materials of this type according to
the invention are polymers such as those already known for the
encapsulation and release, preferably delayed release, of
pharmaceuticals. Polymers which are suitable as solid matrix
according to the present invention are preferably copolymers
comprising ethylenically unsaturated monomers such as methacrylic
acid, acrylic acid, methyl methacrylate, ethyl acrylate, vinyl
acetate, as well as derivatives or salts thereof, as well as
copolymers comprising naturally occurring polymers, such as
celluloses, or derivatives or salts thereof. Preferred are
copolymers comprising two or more of the above monomers, for
example copolymers comprising two or more ethylenically unsaturated
monomers and/or derivatives or salts thereof, copolymers comprising
two or more naturally occurring polymers and/or derivatives or
salts thereof, copolymers comprising at least one ethylenically
unsaturated monomer and/or at least one derivative or salt thereof
with at least one naturally occurring polymer and/or at least one
derivative or salt thereof. Particularly preferred are copolymers
of methacrylic acid and methyl methacrylate in molar ratio 1:1 or
1:2, as, for example, available under the respective trade names
Eudragit.RTM. L100 with release pH of 6.0 or Eudragit.RTM. S100
with release pH of 7.0 (Rohm GmbH), copolymers of methacrylic acid
and ethyl acrylate in molar ratio 1:1, such as, for example, the
polymer available under the trade name Eudragit.RTM. L100-55 with
release pH 5.5 (Rohm GmbH), hydroxypropylmethyl cellulose acetate
succinate, such as distributed under the trade name Aqoat.RTM. with
release pH dependent on the chain size, for example with release pH
of 5.0 (HPMCAS-LF), of 5.5 (HPMCAS-MF) or of 7.0 (HPMCAS-HF)
(ShinEtsu Synthapharm), cellulose derivatives such as cellulose
acetate phthalate, for example cellulose derivatives available
under the trade name Aquateric.RTM. with release pH within the
range from 6.2 to 6.5 (FMC Corp), copolymers of vinyl acetate and
methacrylic acid in a ratio 9:1, for example as available under the
trade name Kollicoat.RTM. VAC with release pH within the range
5.8-6.0 (BASF), or polyvinyl derivatives such as polyvinylacetate
phthalate, for example as available under the trade name
Sureteric.RTM. with release pH within the range 4.5-5.5 (Colorcon
Ltd.). The release pH is the pH where the polymer mentioned starts
to dissolve. Dissolving at the release pH is not a fast process, so
the materials could be used for a swab even if the release pH is
below the pH of saliva (pH 6 to pH 8). At pH values below the
release pH the polymers are substantially insoluble.
[0058] According to a further aspect of the present invention it is
also possible for a reaction which effects an at least partial
transfer of the solid state of the matrix into a dissolved of
liquid state to be catalyzed by both acid and base, preferably in
the presence of at least one chaotrope. For example, the acid- or
base-catalyzed hydrolysis of amides, esters and the like can be
employed if the solid matrix comprises amide and/or ester linkages.
Furthermore, many polysaccharides such as starch and cellulose are
insoluble in water. Both cellulose and starch can, however, be
degraded into water-soluble mono- and oligosaccharides by acidic
and by basic hydrolysis. Accordingly, a pH shift from neutral pH to
acidic or basic pH leads to dissolving of starch or cellulose.
[0059] The pH is preferably regulated by use of a suitable buffer
system for the pH selected. Buffer systems for given pH ranges and
which are preferably compatible with a biological sample are known
to the skilled person.
[0060] It is particularly preferred that the solid matrix can be
transferred into a dissolved or liquid state using at least one of
the above-described solvents, reagents enzymes, acids, bases, or
any combination thereof which appears suitable to the person
skilled in the art and which preferably allows the release of the
biological sample without its being disintegrated, damaged or
degraded or otherwise disadvantageously altered with respect to its
further treatment in the presence of at least one chaotropic
substance and optionally at least one substance of high ionic
strength.
[0061] It is also possible that the method according to the
invention further comprises the step of transporting the biological
sample material which has been contacted with the device according
to the invention. In a preferred aspect of the invention the
biological sample or at least one component thereof is at least
partially released from the solid matrix during transport,
preferably by at least one of changing one or more physico-chemical
properties and contacting the solid matrix with at least one
releasing aid.
[0062] The invention also relates to a method for releasing a
biological sample from a solid matrix comprising one or more of
cotton or a cotton derivative, polyester or a polyester derivative,
paper, cellulose derivative substantially without disintegration of
the biological sample, by adding at least one enzyme and at least
one liquid to the matrix. The at least one liquid can be a solvent,
preferably a solvent as defined above, a substance or a composition
dissolved in at least one solvent, or a substance or composition
which is itself present in liquid form. It is also possible in this
aspect of the method according to the invention that at least one
further compound selected from reagent(s), solvent(s), enzyme(s),
acid(s) and base(s) is added. With respect to the materials
comprised in the matrix, as well as the at least one liquid
reagent(s), solvent(s), enzyme(s), acid(s) and base(s), reference
is made to the details given above for the matrix, liquid(s),
reagent(s), solvent(s), enzyme(s), acid(s) and base(s). It is
preferred to release said biological sample in the presence of at
least one chaotropic substance and optionally at least one
substance of high ionic strength.
[0063] According to the invention, for stabilization, transport,
storage and/or purification the solid matrix can be in solid,
liquid or dissolved state and is preferably in a liquid or
dissolved state. It can be advantageous if the solid matrix is
transferable from the solid state into a liquid or dissolved state
during at least one of stabilization, transport, storage and
purification.
[0064] It is preferred that the solid matrix is transferable from
the solid state into a liquid state during transport of a
biological sample collected onto the device according to the
invention, preferably in the presence of at least one chaotrope.
Transport in this context can be transport from the source of the
sample to a place where the sample can be analysed and/or stored or
otherwise prepared or processed. The transport can thus be, for
example, simply transport from a patient to a work station in the
same or a nearby room. It could also be transport from the source
of the sample to a central analysis and/or storage facility, which
might involve a delay and/or a journey of several hours or days,
although preferably less than 24 hours.
[0065] An at least partial releasing of the biological sample
during at least one of stabilization, transport, storage and
purification, preferably during stabilization or transport, more
preferably during transport, represents a particular advantage of
the method according to the invention in terms of efficiency and
time-saving.
[0066] The at least partial releasing of the biological sample
and/or a component of the biological sample, such as a biomolecule
or a cell, preferably occurs by at least partially transferring the
solid matrix of the device of the invention from the solid state
into a liquid or dissolved state. It is preferred to release said
biological sample in the presence of at least one chaotropic
substance and optionally at least one substance of high ionic
strength.
[0067] Isolation of one or more biological components from the
biological sample can occur during or after the releasing of the
sample, preferably after the releasing of the sample. All isolation
and purification techniques known to the skilled person are also
considered according to the invention for the isolation and/or
purification of the biological sample.
[0068] The solid matrix in both the solid state and in a liquid or
dissolved state is preferably stable to storage at ambient
conditions of temperature and humidity, in order to avoid as far as
possible the necessity for special packaging to protect the device,
and for an extended shelf life of the device. Any necessary
packaging preferably maintains the sterility of the device, in
particular of the solid matrix.
[0069] A solution to the above-mentioned problems is also provided
by a kit comprising at least one compound selected from reagent(s),
enzyme(s), acid(s), and base(s), liquid(s), and solvent(s) for
releasing a biological sample from a solid matrix substantially
without disintegration of said biological sample by at least
partially transferring the solid state of said matrix into a
dissolved or liquid state by changing at least one physico-chemical
property of the environment of said matrix. It is preferred to
release said biological sample in the presence of at least one
chaotropic substance and optionally at least one substance of high
ionic strength and as such said kit does preferably further
comprise at least one chaotropic substance and optionally at least
one substance of high ionic strength.
[0070] Concerning the at least one compound selected from
reagent(s), enzyme(s), acid(s), and base(s), liquid(s), and
solvent(s), reference is made to the details regarding the
reagent(s), enzyme(s), acid(s), and base(s), liquid(s), and
solvent(s) already mentioned in connection with the method
according to the invention.
[0071] In a preferred embodiment of the kit according to the
invention, the kit further comprises at least one solid matrix for
collecting a biological sample. The solid matrix can have any form
or shape which is suitable for the collection of a biological
sample. In a preferred embodiment of the device according to the
invention, the solid matrix is in the form of a round or elongated
ball, a bulb, a mesh, a stick, a woven bulb, a paper, a card, a
particle, a bead, a rod, a plug or a filter. For details concerning
the material of the solid matrix, reference is made to the
discussion above regarding the method according to the
invention.
[0072] It is preferred according to the invention that the kit
comprises at least one enzyme for releasing a biological sample
from a solid matrix comprising one or more of waxes, high molecular
weight alkane hydrocarbons like paraffin, monoterpenes, sucrose,
agarose, starch, chitin, peptides, proteins, pectins, agar, acidic
polymethylacrylates, cellulose acetate, cellulose acetate
phthalate, vinylacetate, polyvinyl acetate phthalate, hydroxypropyl
methylcellulose-acetate succinate, alginates, copolymers of
methacrylic acid and methylmethacrylate, copolymers of vinylacetate
and methacrylic acid, and polyvinylacetatephthalate, substantially
without disintegration of the biological sample by adding at least
one enzyme and optionally at least one further compound selected
from reagent(s), enzyme(s), acid(s), and base(s), liquid(s), and
solvent(s) to the matrix. The at least one further compound is
preferably at least one compound selected from reagent(s),
enzyme(s), acid(s), and base(s), liquid(s), and solvent(s) referred
to above in the details regarding the reagent(s), enzyme(s),
acid(s), and base(s), liquid(s), and solvent(s) mentioned in
connection with the method according to the invention. It is
preferred to release said biological sample in the presence of at
least one chaotropic substance and optionally at least one
substance of high ionic strength and as such said kit does
preferably further comprise at least one chaotropic substance and
optionally at least one substance of high ionic strength.
[0073] In a particularly preferred aspect, the kit according to the
invention further comprises at least one further compound selected
from reagent(s), enzyme(s), acid(s), and base(s), liquid(s), and
solvent(s). Concerning these reagent(s), enzyme(s), acid(s), and
base(s), liquid(s), and solvent(s), reference is made to the
details regarding the reagent(s), enzyme(s), acid(s), and base(s),
liquid(s), and solvent(s) already mentioned in connection with the
method according to the invention. It is preferred to release said
biological sample in the presence of at least one chaotropic
substance and optionally at least one substance of high ionic
strength and as such said kit does preferably further comprise at
least one chaotropic substance and/or at least one substance of
high ionic strength.
[0074] It is also possible that the at least one further compound
comprises at least one additive, preferably at least one additive
which helps to maintain the integrity of the biological sample or
of at least one component thereof, such as the cell or cells or
components thereof, the nucleic acids or the proteins. An additive
can also be any additive which enhances the stabilization or
processing of the biological sample or which affects a property of
the solid matrix in solid, liquid or dissolved state or of the
biological sample. Examples of such additives include, but are not
limited to, inhibitors which inhibit the decomposition of nucleic
acids and/or proteins, inhibitors that can effect inhibition of
agents that damage macromolecules and modification agents which act
against, or reduce the activity of proteases, RNases, DNases or
other enzymes for degradation reactions of the biological sample
and/or a component thereof. An additive could also be a protein
modification agent, such as an acetylating agent, a halogenating
agent, a nucleotide, a nucleic acid analogue, an amino acid or an
amino acid analogue, a carbodiimide or an imide, a haloacetate, a
haloacetamide, acetylsalicylic acid or an acid anhydride.
[0075] Other additives are also conceivable, such as ionic or
non-ionic detergents, preferably cationic detergents, reducing
agents, such as 2-mercaptoethanol, dithiothreitol, ascorbic acid,
antimicrobials, chelating agents, such as ethylene diamine
tetraacetic acid (EDTA) and buffer substances other than those used
as processing agents, such as HEPES or MOPS.
[0076] The kit according to the invention can be, but need not
necessarily be, so devised that it can be portable, for example for
use both in and outside medical or veterinary facilities or out of
doors, for example in hospitals, in doctor's surgeries, in
pharmacies, in workplaces, in the home, in law enforcement, in
forensics, in environmental sample collecting, in food sample
collecting, in plant sample collecting. The at least one compound
can comprise one or more compositions suitable for stabilising
and/or treating a broad range of biological samples and/or for
transferring the solid matrix of the device according to the
invention from the solid state into a liquid or dissolved state. It
is also possible that the at least one compound comprises a
specialized composition for a particular sample or type of sample.
A portable kit preferably also comprises a suitable carrier for
carrying the kit, as well as optionally one or more portable
devices capable of heating or cooling the kit, in particular the
kit comprising the sample. The kit according to the invention
preferably also comprises instructions for using the kit. It is
also possible that the kit comprises further components, such as
those which might be necessary to fulfil legal purposes in law
enforcement, establishment of genetic relationships or workplace
testing. Such components can include tape or other tamper-proof
sealing element and/or legal documents.
[0077] It is conceivable that the system and/or the kit according
to the invention is compatible with an at least partially automated
system, which can be an automated system or a semi-automated system
or, for example, a high throughput system. The at least partially
automated system can relate to at least one of storage, retrieval,
processing, purification and analysis of a biological sample.
[0078] The embodiments and aspects of the invention can of course
be combined with each other in any way which appears suitable to
the skilled person to achieve the objects of the invention.
[0079] The invention is further illustrated by the following
non-limiting figures and examples.
BRIEF DESCRIPTION OF THE FIGURES
[0080] FIG. 1 shows different forms of a rod-like unit comprising a
closed holding element and a solid matrix: [0081] a) round head;
[0082] b) elongated head; [0083] c) bulb-shaped head; [0084] d)
paper; [0085] e) teat-shaped head [0086] f) elongated head with a
hollow rod containing a fluid or a solid [0087] g) with a closed
circuit passing through the rod and the solid matrix.
[0088] FIG. 2 shows different forms of the rod-like unit comprising
a holding element in the form of a hollow rod open to a solid
matrix: [0089] a) the basic structure; [0090] b) the basic
structure with a fluid flowing through the hollow rod;
[0091] The embodiments of FIG. 1 represent the preferred forms of
the system 1 according to the invention. The solid matrix 3 is
attached to the holding element 4 in the form of a rod, at the tip
5 of the rod. The rod may be solid or hollow and is, if hollow,
closed to the solid matrix 3 of the head.
[0092] FIG. 1 f) exemplifies a system with, as holding element, a
hollow rod which has a larger opening 6 in the rod at the opposite
end to the head, to allow ingress of a fluid 7, for example for
cooling or heating, and optionally to act as a reservoir for the
fluid 7.
[0093] FIG. 1 g) shows an alternative embodiment, whereby a circuit
9 which is closed to the solid matrix 3 is provided by or through
the rod, for example for flow of a cooling or heating agent or for
passage of a current to warm the solid matrix 3 of the head.
[0094] The further aspects depicted in FIG. 2 a) and b) show a
hollow rod as holding element which is open to the solid matrix 3
of the head. The cavity 8 provided within the hollow rod can be
used to effect or assist a transfer of the solid matrix 3 from the
solid state into a liquid or dissolved state, for example by
providing a releasing aid.
[0095] FIG. 2 b) shows the flow of a fluid 7, for example a cryogen
or a releasing aid, through the rod and at least into, preferably
through the head.
Examples
Example 1
Extraction of DNA from a Soluble Buccal Swab Using a Chaotropic
Solution
[0096] Cellulose acetate fibers in the form of a swab were used for
taking buccal swabs from human donors. Swabs were cut into two
equally sized pieces (A and B) and DNA was extracted using
different procedures.
Procedure 1
[0097] Cellulose acetate swab part A was dissolved in 800 .mu.l of
Buffer AVL (high salt buffer from QIAGEN containing guanidinium
salt) over five minutes at room temperature with gentle agitation.
560 .mu.l of the suspension were transferred into a 2 ml
microcentrifuge tube and 560 .mu.l of ethanol added to the tube
containing the suspension. Mixing was carried out by vortexing and
the sample was transferred onto a column with a membrane (QIAamp
Mini Spin column from QIAGEN). The column was centrifuged at 8,000
rpm for one minute to bind nucleic acids to the membrane. The
column was washed a first time with 500 .mu.l of AW1 buffer
(guanidinium salt and ethanol containing buffer from QIAGEN),
followed by centrifugation at 8000 rpm for one minute. The column
was then washed a second time with 500 .mu.l of AW2 Buffer (ethanol
containing buffer from QIAGEN), followed by centrifugation at 8000
rpm for one minute. In a drying step the column was transferred
into a new collection tube and centrifuged for three minutes at
14,000 rpm. For elution, 150 .mu.l of AE buffer (low salt elution
buffer from QIAGEN) were dispensed onto the dried membrane followed
by centrifugation for one minute at 14,000 rpm.
[0098] Genomic DNA in the eluate was quantified using real time
PCR. The mean yield for procedure 1 was 8.7 ng of DNA.
Procedure 2
[0099] Cellulose acetate swab part B was dissolved in 800 .mu.l of
Buffer AVL (high salt buffer from QIAGEN containing guanidinium
salt) over five minutes at room temperature with gentle agitation.
400 .mu.l of the suspension were transferred into a 2 ml
microcentrifuge tube. 720 .mu.l of deionized water and 20 l of
proteinase K (from QIAGEN) were added to the tube containing the
suspension. Mixing was carried out by vortexing and the sample was
incubated at 56.degree. C. for 10 minutes. A further 456 .mu.l of
Buffer AVL from QIAGEN) and 800 .mu.l of ethanol were added and
mixing was carried out by vortexing. The sample was then
transferred onto a column (QIAamp.RTM. Mini Spin column from
QIAGEN). The column was centrifuged at 8,000 rpm for one minute to
bind nucleic acids to the membrane. The column was washed a first
time with 500 .mu.l of Buffer AW1 (from QIAGEN), followed by
centrifugation at 8000 rpm for one minute. The column was then
washed a second time with 500 .mu.l of Buffer AW2 (from QIAGEN),
followed by centrifugation at 8000 rpm for one minute. In a drying
step the column was transferred into a new collection tube and
centrifuged for three minutes at 14,000 rpm. For elution, 150 .mu.l
of Buffer AE (low salt elution buffer from QIAGEN) were dispensed
onto the dried membrane followed by centrifugation for one minute
at 14,000 rpm.
[0100] Genomic DNA in the eluate was quantified using real time
PCR. The mean yield for procedure 2 was 50.9 ng of DNA.
Example 2
Solubility of Cellulose Acetate Phthalate in Different Buffers
[0101] The solubility of cellulose acetate phthalate (CAP) in
different buffers was investigated.
[0102] 50 mg (+/-1 mg) of CAP were transferred into a 2 ml
microcentrifugation tube. 1 ml buffer was added. The tube was
closed and subjected to pulse vortexing for 10 seconds to mix the
contents. The tube was then observed at room temperature for 30
hours.
[0103] The buffers used were ATL (a lysis buffer for bacteria), AVL
(a lysis buffer), MTL (a lysis and binding buffer), ML (a lysis and
binding buffer), and RLT (a lysis buffer), all of which are
commercially available buffers from QIAGEN.
[0104] The results are given in the following table 1.
TABLE-US-00001 TABLE 1 Buffer 30 min 1 h 3 h 10 h 24 h 30 h ATL CAP
Not Not Only few CAP CAP beads swollen, swollen, small beads beads
unaffected little significantly beads totally totally dissolved
dissolved visible dissolved dissolved AVL CAP Significantly Nearly
CAP CAP CAP beads dissolved totally beads beads beads stick
dissolved totally totally totally together, dissolved dissolved
dissolved swollen MTL CAP Significantly CAP CAP CAP CAP beads
dissolved beads beads beads beads stick totally totally totally
totally together, dissolved dissolved dissolved dissolved swollen
ML CAP Significantly CAP CAP CAP CAP beads dissolved beads beads
beads beads stick totally totally totally totally together,
dissolved dissolved dissolved dissolved swollen RLT CAP CAP Thick,
Thick, Thick, Thick, beads beads viscous viscous viscous viscous
stick stick layer on layer on layer on layer on together, together,
buffer buffer buffer buffer swollen swollen RLT RLT RLT RLT
Example 3
Extraction of DNA from a Meltable Swab Using a Chaotropic
Solution
[0105] Swabs with a head made of Paraffin (Paraplast-XTRA, melting
point 52.degree. C., McCormick Scientific) were used for taking
samples from HeLa cells. Swabs were melted at 70.degree. C. in
different solutions with or without chaotropic salts. DNA was
extracted using the QIAamp DNA (QIAGEN) procedure.
Procedure
[0106] HeLa cells grown as monolayer were washed twice with PBS.
Cells samples were taken with swabs with a head made of
Paraplast-XTRA shaped as a scraper. The head of the swabs were
dissolved in a 2 ml microcentrifuge tube within 500 .mu.l of
different reagents with or without a chaotropic salt (Tab. 2,
reagent composition). Melting of the swab head was achieved by
incubation for 10 min at 72.degree. C. with gentle agitation. After
centrifugation at full speed for 1 min (14,000 rpm) the paraplast
constituted a solid layer on top of the liquid solution. 400 .mu.l
of the liquid solution were retrieved and transferred into a new 2
ml microcentrifuge tube.
[0107] After adding 20 .mu.l of proteinase K and incubation for
10min at 56.degree. C., 200 .mu.l of ethanol (99.5%) was added.
Mixing was carried out by vortexing and the sample was transferred
onto a column with a membrane (QIAamp Mini Spin column from
QIAGEN). The column was centrifuged at 8,000 rpm for one minute to
bind nucleic acids to the membrane. The column was washed a first
time with 500 .mu.l washing buffer AW1 (QIAamp DNA, QIAGEN),
followed by centrifugation at 8000 rpm for one minute. The column
was washed a second time with 500 .mu.l washing buffer AW2 (QIAamp
DNA, QIAGEN), followed by centrifugation at 8000 rpm for one
minute. For a drying step the column was transferred into a new
collection tube and centrifuged for one minute at 14,000 rpm. For
elution, 100 .mu.l aqueous buffer AE (QIAamp DNA, QIAGEN) were
dispensed onto the dried membrane followed by centrifugation for
one minute at 10,000 rpm.
[0108] The integrity and size of total DNA was analysed by agarose
gel electrophoresis. 10 .mu.l of eluate were mixed with 4 .mu.l
loading buffer (containing 50% glycerol and bromphenol blue). The
samples were applied to a 1% agarose gel in 1.times. TBE buffer.
Electrophoresis was run for 150min and approximately 2 Volt per cm
length of the electrophoresis chamber. DNA was visualised by
ethidium bromide staining (Figure Experiment 3).
[0109] Performance in PCR was analysed in a quantitative, real-time
PCR on an ABI Sequence Detection System ABI PRISM 7700 (Tab. 2, CT
values and standard deviations from two independend extractions
running in triplicates). 25 .mu.l assay with 2 .mu.l sample as
template contained primer and probes for amplification of a 294 by
fragment within exon 3 (forward primer 5''-TCA CCC ACA CTG TGC CCA
TCT ACG A-3'', reverse primer 5''-CAG CGG AAC CGC TCA TTG CCA ATG
G-3'', probe 5''-(FAM)ATG CCC TCC CCC ATG CCA TCC TGC GT(BHQ)-3'')
of the human .beta.-actin gene using the QuantiTect Probe PCR kit
(QIAGEN).
[0110] DNA extracted from swab heads melted within a solution
containing chaotropic salt was of high molecular weight with a
fragment length of about 23 kb, running as a distinct band on an
agarose gel (Figure Experiment 3, rows 1-5). These DNA samples
could be amplified by real time PCR leading to CT values in the
range between 27 to 29 (Table 2, A to C).
[0111] In contrast no DNA band was visible when the swab head was
melted in a solution without chaotropic salt and isolated with the
method described above (Figure Experiment 3, rows 6 and 7).
Consistent with this result CT values in real time PCR were 6 to 7
cycles later in case of reagent solution D or there was no
amplification at all in case of reagent solution E (Table 2).
TABLE-US-00002 TABLE 2 Beta-Actin Reagent qPCR solution Reagent
composition CT-value A Buffer AL (lysis buffer cont. 27.5 +/- 0.6
guanidinium salt, QIAamp DNA, QIAGEN), mixed 1:1 with PBS B Buffer
AL mixed 3:2 with PBS 28.6 +/- 0.3 C Buffer AL mixed 3.5:1.5 with
PBS 28.5 +/- 0.4 D Phosphate buffered saline (PBS) 34.4 +/- 1.0 E
Xylol 40.0 +/- 0.0
[0112] Agarose gel electrophoresis with eluates from paraplast
swabs melted in reagent A (1), B (2 and 4), C (3 and 5), D (6) and
E (7). M: Lambda Hind III molecular weight marker.
Example 4
Extraction of Nucleic Acids from a Soluble Swab Using a Chaotropic
Solution
[0113] Swabs with a head made of cellulose acetate were spiked with
saliva and a mixture containing different viral and bacterial
pathogens. Swabs were dissolved either in acetone or in a buffer
containing chaotropic agents (buffer AVL, QIAGEN). Nucleic acids
were extracted using the QIAamp Viral RNA (QIAGEN) procedure.
Procedure
[0114] A pathogen mixture containing Chlamydia trachomatis and
Hepatitis A Virus (HAV) was prepared. 5 ml of saliva was collected
in a 50 ml tube. 100 .mu.l aliquots of the saliva and 40 .mu.l
aliquots of the pathogen mixture were spiked onto the cellulose
acetate swabs. Swabs were transferred into 15 ml tubes containing
either 3 ml acetone or 3 ml of AVL Buffer. After inverting 3 times
samples were stored for 24 hours at room temperature.
[0115] After storage 560 .mu.l of ethanol (99.5%) was added to 700
.mu.l sample. Mixing was carried out by vortexing and 630 .mu.l of
the sample was transferred onto a column with a membrane (QIAamp
Mini Spin column from QIAGEN). The column was centrifuged at 8,000
rpm for one minute to bind nucleic acids to the membrane. The
remaining sample was transferred onto the QIAamp Mini Spin column
and the column was centrifuged at 8,000 rpm for one minute. The
column was washed a first time with 500 .mu.l washing buffer AW1
(QIAGEN), followed by centrifugation at 8000 rpm for one minute.
The column was washed a second time with 500 .mu.l washing buffer
AW2 (QIAGEN), followed by centrifugation at 8000 rpm for one
minute. For a drying step the column was transferred into a new
collection tube and centrifuged for three minutes at 14,000 rpm.
For elution, 100 .mu.l aqueous buffer AVE (QIAGEN) was dispensed
onto the dried membrane. A 1 Minute incubation was followed by
centrifugation for one minute at 10,000 rpm.
[0116] The PCR-performance of the extracted nucleic acid was
analysed in different real-time PCRs.
[0117] Chlamydia trachomatis-DNA extracted from dissolved swabs was
analysed using the artus C. trachomatis TM PCR Kit (QIAGEN) on an
ABI Sequence Detection System ABI PRISM 7900 as described by the
manufacturer.
[0118] HAV-RNA extracted from dissolved swabs was analysed with
artus HAV LC RT-PCR Kit (QIAGEN) in a Light Cycler 1.0 from Roche
as described by the manufacturer.
[0119] All samples dissolved within one hour in acetone as well as
AVL. Mean CT values and standard deviations from four samples each
extracted in triplicates are shown in table 3.
[0120] CT values for C. trachomatis target DNA was lower for
samples dissolved in Buffer AVL compared to those dissolved in
acetone (delta CT=-2.70). CTs for internal controls spiked into the
PCR reaction were equal, indicating that inhibition of the PCR is
not the reason for the higher target CT values of the acetone
samples.
[0121] Comparable with the C. trachomatis data, in the artus HAV LC
RT-PCR the CT values were lower for the samples dissolved in AVL
compared to the acetone samples (delta CT=-1.87).
TABLE-US-00003 TABLE 3 Real-time PCR results artus artus artus C.
trachomatis C. trachomatis HAV LC Reagent TM PCR Kit TM PCR Kit
RT-PCR Kit composition target DNA internal control target RNA
Acetone 34.4 +/- 1.0 25.3 +/- 0.2 35.25 +/- 1.0 AVL 31.7 +/- 0.5
25.4 +/- 0.1 33.38 +/- 0.3
* * * * *
References