CA 02437473 2003-08-18
PATENT
P-5063P1
TITLE OF THE INVENTIDN
METHOD FOR PRESERVATION OF CELLS AND NUCLEIC ACID TARGETS
BACI~GR~IJND ~F THE INVENTI~N
The field of the present invention broadly relates to retaining nucleic acid
integrity
in order that such nucleic acid is amenable to certain diagnostic processes,
such as
hybridization and amplification. More specifically, the present inventic'n
relates to the use
of compositions that cause an inhibitory affect on proteolytic agents .and/or
nucleic acid
degradative agents.
Diagnostic processes that utilize nucleic acid molecules include nucleic~acid
probe
hybridization to determine the presence andlor amount of a target nucleic
acid, nucleic acid
primer hybridization for nucleic acid amplification processes and enzymatic
activity
including nucleic acid extension, nicking an~lor cleavage. Nucleic acid
amplification
processes such as strand displacement amplification (SDA), polymerase chain
reaction
(PCR), ligase chain reaction (LCR), nucleic acid sequence based amplification
(NASBA),
transcription mediated amplification (TMA) and others are used to create
multiple copies
of a particular nucleic acid sequences) of interest (target sequence) 'which
is present in
lesser copy number in a sam~ie:
A known method for inactivating DNases .and proteolytic en:~yrnes is to heat a
.
sample at 100°C for fifteen minutes (gee, for example, Sambrook, J.,
Fritsh, E. F., and
Maniatis, T. Molecular Clo~ia~g: ~4 Laboratory Manual. 2ncl, el., Cold
S'prircg Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,
1989). Other
methods for inactivating proteoiytic enzymes~use chaotropic agents, such as
guanidinium
thiocyanate or sodium thiocyanate, to denature these enzymatic protein;9 (see,
for .example,..
LJ.S. Pateilt:No. 4,84.3,155, incorporated herein.by reference). . In audition
to inactivating
1
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PATE1VT
P-5063P 1
nucleases and proteolytic enzymes, chaotrapic agents are also known to promote
lysis of
cell walls in a wide range of biological materials (IJ.S. Patent No.
5,234,809, incorporated
herein by reference). The use of chaotropic agents to promote inactivation of
proteases or
nucleases should thus be avoided when preservation of cell walls is also
desired.
A more general description of a method for preserving nucleic acids is
provided by
PCT Publication WO 00/50640 to Exact Laboratories wherein the use o f EDTA and
EGTA
as nuclease inhibitors in methods for extracting DNA from exfoliated human
epithelial
cells in stool samples is disclosed. Also, Kyoto Ikagaku Kenkyusho: KK own two
Japanese
patent disclosures relating to methods for preserving cells in urine. In Jl'
04118557 A2, the
use of citric acid as a buffering agent in addition to EDTA as an
antibacterial drazg is
disclosed, and JP 05249104 A2 discloses the use of sodium fluoride as a
fluorine
compound in addition to citric acid and EDTA. Sierra, Diagnostics i.n PCT
Publication
WO 99/29904 disclose the use of ethylenediaminetetraacetic acid (E,DTA),
ethylenebis
(oxyethylenenitrilo) tetraacetic acid (EGTA), 1,2-bis (2-aminophenoxy;) ethane-
N,N,N',N'-
tetraacetic acid (BAPTA), or salts thereof in conjunction with at least one of
the "chelator
enhancing components" lithium chloride, guanidine, sodium salicylate, sodium
perchlorate
or sodium thiocyanate. Lithium chloride, guanidine, andl sodium thi~~cyanate
are known
chaotropes to those skilled in the art. Effective concentrations of c~elating
enhancing
components range from about 0.1 M to about 2.~ M:
Many of . these methods are .time consuming, labor intealsive andlor have
cumbersome safety requirements associated therewith. Another problenn with
methods that
utilize relatively severe processing steps or conditions is the loss of ;>ome
target nucleic .
acid sequence. Despite the ability of nucleic acid amplification processes to
make multiple
copies of .target sequence (amplicoris) from very few original targets,
amplification
efficiency and detection ability are improved if there are greater numbers of
original targets
in the sample. The greafer detection ability can be very important when
processing
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PL~TEl~T
P-S063P I
particularly difficult to detect samples such as acid fast ~acildus (AFB)
smear negative
Mycobacterium tuberculosis samples.
Another common problem with samples to be subjected to a molecular diagnostic
process is the stability of the sample over time. Stability of the sample
becomes more
important when samples are taken at one location, but are then transported to
another
location such as a centralized laboratory for molecular dia~ostic processing.
For example, many clinically relevant organisms do not maintain their
integrity in
urine samples and vaginal and cervical swabs for more than about twenty-four
(24) hours at
room temperature. Thus, refrigeration of such samples during transport to
centralized
IO laboratories and/or during storage has become a necessity. Dne analyte that
is commonly
tested from urine samples and swabs and is notoriously unstable in samples
stored at room
temperature is Neisseria gonorrhoecze.
SEMMA~Y DF TIIE IhtVEhITIDhT
IS In order to address the problems associated with maintaining th.e integrity
of target
nucleic acid and maintaining the stability of a sample and thus, achieve the
benefits of
improved detection of target nucleic acid sequences, the present invention
provides a
method for preserving cells and. nucleic acids in a. sample by exposing the
sample t~ a
composition that causes an inhibitory ' affect on proteolytic. . agents and/or
nucleic acid
20 degradative agents in the sa~riple. Such compositions that: are useful ixi
the method of the
present invention include chelating agents such as sodiur~a citrate, sodium
borate, sodium
fluoride, and EDTA, that will bind trace metals necessary for proteolytic
activity of
proteolytic agents and/or nuclease activity of nucleic acid. degradative
agents in a sample.
These compositions lack effective concentrations of ''chelator enhancing
components" or
25 chaotropic agents:
3
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PATENT
P-5063P 1
DETAILED DESCRIPT"I~N CAF THE INVENTI~N
As stated above, the present invention relates to a method for preserviing
cells and
nucleic acids in a sample by exposing the sample to a composition that lacks
effective
concentrations of "chelating enhancers" or chaotropic agents but nevertheless
causes an
inhibitory affect on proteolytic agents and/or nuclease agents in the sample.
In the method,
the sample is exposed to such a composition prior to lysis of cells in the
sample such that
cells containing nucleic acid will remain stable in the sample.
The types of samples which may be subjected to the method of the present
invention include virtually all human and veterinary clinical samples such as
sputum
samples, blood samples, urine samples, cerebrospinal fluid ("CSF") sa~.mples,
vaginal and
cervical swabs and others, environmental samples such as water, air anal soil
samples, and
food samples. The samples which may be subjected to the method of tl~e present
invention
are suspected of containing cells with a target nucleic aciid sequence to be
subjected to a
diagnostic process which may include hybridization, such as direct probe
hybridization or
primer hybridization for initiation of an amplification process.
The proteolytic agents typically found in such samples include proteolytic
enzymes,
such as proteases including endopeptidases such as renin, exopeptidases such
as trypsin
and chymotrypsin, and cathepsins. Wh.en.present in a sample, proteolytic
agents will cause
degradation of cell' walls, thus releasing nucleic acid, including any target
nucleic acid for
diagnosis. . Such released nucleic acid is then subj ect to the :actions of
other agents that
adversely affect diagnostic processes, including degradative enzymes, such as
DIVases and
RNases, and substances that inhibit nucleic acid hybridization and/or
amplification
processes, such as porphyrin compounds derived from hems and hexriatin, serum
enzymes
such as aprotinin, leupeptin Ph~ISF .and pepstatin, and urea. The exposure of
nucleic acid,
particularly a target nucleic acid, to such agents adversely affects any
diagnostic process to
be performed with the nucleic acid. Thus, 'prevention of contact of tl:ie
nucleic acid and
CA 02437473 2003-08-18
PATENT
P-5063P1
such agents by inhibiting the premature lysis of cell walls allows for the
maintenance of a
stable sample i:or a longer period before a diagnostic process may be;
performed on the
sample.
The method of the present invention involves the exposure of the sample to a
S composition that lacks effective concentrations of chelating enhancers o~r
chaotropic agents
and causes an inhibition of proteolytic agents and/or nucleic acid
degrasiative agents in the
sample. This f;xposure may occur at any time prior to the lysis of cells to
release target
nucleic acid. T'he exposure results in a reaction mixture containing the
sample of cells and
a composition that binds at least one co-factor necessary for activity of a
proteolytic agent
and/or a nucleic acid degradative agent
Many such compositions are useful in the method of the present invention.
Examples of s~zch useful compositions include chelating agents such as sodium
citrate,
sodium borate, sodium fluoride and EDTA. Such chelating agents bind trace
metals
necessary for proteolytic activity of proteolytic agents in a sample. Thus,
the proteolytic
agents can not ;act as efficiently, or at all, to degrade cell walls. This
allows the cell walls
to remain intacvt, and the integrity of nucleic acid molecules within the
cells- is maintained,
because such nucleic acid molecules are not exposed to agents that cause;
their degradation.
The compositions that cause the inhibition of proteolytic agents may be used
in any
form or state. :For example, such:cornpositions may be exposed to a sample as
a liquid or '
as a solid in a dry grar<ular.form,. a compressed tablet, a dissolvable
capsule containing the
composition or in a permeable vehicle such as a sank coaataining the
composition. Also,
the composition, in any form, may be added to a container holding the sample
or
incorporated in the container to which the sample is added.
The concentrations of such compositions that are useful for the methods of the
-present invention range from about 2.Sm~~I to about 350mIVI, with a preferred
range of
S
CA 02437473 2003-08-18
Pr~TENT
P-50631'1
about SmM to about 100mM. The temperatures at which the methods of the present
invention are effective range from about 5°C to about 60°C.
Other compositions useful in the methods of the present invention can be
identified
by one of ordinary skill in the art with a reasonable expectation of success
by performing
routine screening assays directed towards the optimal characteristics of such
compositions,
e.g., causing the inhibition of proteolytic agents.
The Examples set forth herein provide a simple, straight-forward, routine
screening
assay by which one of ordinary skill in the art can quickly determine whether
a particular
composition will be likely to be useful in the methods of the present
invention.
When the composition and the sample are exposed to one another, trace metals,
such as magnesium, calcium, zinc and manganese that are rieces:~ary co-factors
for
proteolytic agents, such as proteolytic enzymes, and nucleic acid de,gradative
enzymes,
such as DNases and RNases, are bound by the composition: In such a bound
state, the
trace metals are not available for their co-factor function. Thus, the
degradative action of
I S proteolytic agents on cell walls, and nucleic acid degradative enzymes on
nucleic acids is
inhibited.
The amount of time for exposure of the composition and the sample to one
another
. for the method of the present invention to be effective is generally
minimal, without any
maximum time. The binding of trace metals begins almost immediately upon
exposure of
. . 20 the composition and the sample to one another, and such binding does
not diminish
appreciably over time. As a general rule, the exposure of composition and
sample to one
another should not be less than about thirty X30) minutes for flee method of
the present
invention to be effective.
Optionally, following such exposure, the composition and sample may be
25 separated. Such separation may be conducted by a variety of means such as
centrifiigation,
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PATENT
P-5063P 1
filtering, or if a permeable vehicle is used, then the pern eable vehicle; can
be physically
removed from the sample or the treated sample can simply be removed by
pipetting.
A variety of processes are currently used to prepare target nucleic acids in
samples
for hybridization or amplification. For example, sputum samples that are
processed to
S amplify mycobacterial nucleic acid sequences are typically subj ected to a
NALClNaOH
process. Similarly, other types of clinical samples are subjected to other
well-known
standard processes, for example, centrifugation for large volume sample s
such. as blood and
urine. The method of the present invention may be used before, as part of, or
after those
standard processes.
In addition to utility of the method of the present invention for preserving
cell wall
integrity and nucleic acid integrity, the method also stabilizes the sample
for transport at
room temperature. The inhibition of proteolytic agents and/or nucleic acid
degradative
agents resulting from the method ~f the present invention permits room
temperature
storage and transport of the sample over time periods greai;er than those
presently available
using traditional sample preservation methods. For example, using mixed bed
ion
exchange resins, such as those disclosed in European Patent Application
Publication No.
0915171A2, permit room temperature storage of urine sample for as long as four
days,
whereas the method of the present invention as shown in the Examples below,
permits
room temperature storage of urine samples for at least fourteen (14.) days.
The following examples illustrate specific embodix~nents of the iinvention
described
herein. As would be apparent to skilled artisans, various changes an<i
modifications are
possible and are contemplated within the scope of the invention described.
EN.AtVfPLE 1
7
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PATEloIT
P-5063P 1
Protection of N. ~onorrhoeae DNA from De:~,radation in l:Jrine
The purpose of this Example was to determine if a method of the present
invention
eliminates the degradation of N. gonorrhoeae plasmid DNA in human urine.
MATERIALS
BDProbeTecTM ET Neisseria gonorrhoeae priming microwells (BD)
BDProbeTecTM ET IVeissericz gonorrhoecze amplification microwells (Bl~)
BDProbeTecTM ET Neisseria gonorrhoe~e plasrnid DNA (BD)
BDProbeTecTM ET instrument and instrument plates
BDProbeTecTM ET lysing heater
BDProbeTecTM ET priming and warming heater
BDProbeTecTM ET pipettor and pipette tips
Sodium citrate, trisodium salt (Sigma)
Ethylenediaminetetraacetic acid, disodium salt "EDTA" (Sigma)
Negative pooled urine
Glycine HCl (Sigma)
Ferrosoferric oxide (Pea Ridge Iron ~re)
Dimethyl sulfoxide "D1VIS~" (Sigma)
Bicine (Sigma)
Glycerol (Sigma)
Potassium hydroxide "KGH" (Sigma)
PR~CEDURE:
Twenty-milliliter.volumes of negative pooled urine were prepared with 10, 40,
and 100 .
mlVl sodium citrate {final c~ncentration); 10, 20, and 40 mM EDTA (final
concentration);
and without chemical additive. In addition, a 20-ml volume of negative :pooled
urine was
heated for 30 minutes (heated-treated urine). The samples were incubated for
60 minutes
at room ternpefature. After the incubation step, the samples were spiked with
N
gonorrhoeae plasmid DNA (2500 DNA copieslml). The samples were :incubated for
60
minutes at room temperature. After the incubation step, two-ml volumes from
each sample
(eight replicates) were heated for 30 minutes at 114°C to denature the
DNA. . .
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PATENT
P-5063P1
The heat-denatured DNA was isolated from th:e samples with paramagnetic
particles in an acidic environment as described in lJnited States Patent No.
5,973,138. A 1-
ml volume of the 8 replicates as described above was added to ferrosofernc
oxide. A 100-
u1 volume of 6 M giycine HCl was added to each sample. The DNA-ferrosoferric
oxide
complexes were separated on a magnet and washed two times with a 900-ul volume
of
1mM glycine HCl to reduce the ionic strength and remove nonspecifically bound
materials.
The DNA was released from the ferrosoferric oxide with 480-ul volume of
elution buffer
(88.3 mM KOH, 177.8 mM bicine, 12.1% (v/v) glycerol;, and 11.0% (v/v) DMSO
[final
concentration] ).
Strand Displacement Amplification (SDA) was performed according to the
manufacturer's instructions (BDProbeTecTMET Chlamydta trachomatis and N.
gonorrhoeae .Amplified DNA Assay Package Insert L-000203 [November 1999]). A
150-
ul volume of each sample was added into a priming microwell. The priming
microwell
plate was incubated for 20 minutes at room temperature, after wluch the plate
was
incubated at 72.5°C. The amplification microwell plate vvas incubated
at 54°C. After 10
minutes, a 100-u1 volume from each priming microwell was added into the
corresponding
amplification microwell. The amplification microwell plate was place in a BD
ProbeTec
ET instrument. Data was reported as MOTA value (metric other than
acceleration).
- 20 RESULTS ~ ' . . . . . . .
The results are provided in the table below as the average amplification MOTA
value (eight replicates) from each processed sample.
9
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PATENT
P-5063P1
Sam 1e _ Average MOTH 1/alue
No additive 26
mM sodium citrate 6673
40 mM sodium citrate 38684
100 mM sodium citrate 24069
10 mM EDTA 16103
mM EDTA 16178
40 mM EDTA 20007
CONCLUSIONS
5
The data of this Example indicate that sodium citrate at 10, 20, and 40 rnM
and
EDTA at 10, 40, 100 mM protected N gonorrhoecze plasmid DNA from degradation
in
human urine by endogenous nucleases. The data also show that heat-treated
urine
prevented the degradation of N gonorrhoeae plasmid DNA.
E~~AMPLE 2
Stability of Cells of N. ~onorrhoeae in Urine with. Sodium Citrate
The purpose of this Example was to determine the stability of intact cells of
N. gonorrhoeae in hurrian urine for a period of 14 days at ~.-oom
temperavture.
MATERIALS
Amplification reagents and equipment were the same as described in Example 1.
Sodium citrate (Sigma)
BSA/PBS (0.2 % bovine serum albumin, 10 mM potassium phosphate buffer [pH
7.6), and
150 mM sodium chloride)
Negative pooled urine
. 25 ~ CT/GC diluent (BD)
N. gonorrhoeae ATCC 1 X424
Dynac II centrifuge (BD)
PROCEDURE
CA 02437473 2003-08-18
PATENT
P-5063P1
Eighty-milliliter volumes of negative pooled urine were prepared with and
without
100mM sodium citrate (final concentration). The samples were spiked with cells
of
N. gonorrhoecze a final concentration 2500 cells/ml. Each sample was incubated
for 14.
S days at room temperature. At specific time points (day 0, 6, and 14), 2.0-ml
volumes of
each sample (eight replicates) was centrifuged at 2000 x g for 30 minutes at
room
temperature. The supernate was decanted and the cell pellet was resuspended
with a 2.0-
ml volume of BSAIPBS, and then centrifuged at 2000 x g f~r 30 minutes at room
temperature. Again, the supernatant was decanted and the cell pellet was
resuspended with
a 2.0-ml volume of CT/GC diluent. Each sample was heated for 30 minutes at
114°C to
lyse the cells. SDA was performed on the cell lysate as described in Example
1.
RESULTS.
The results are presented in the table below as the average amplificaf.ion
MOTA value
(eight replicates) from each processed sample for 14 days.
Average M~TA Vs~lue
No additive 26683 9540 3424 4990
CONCLUSIONS .
The data of this Example demonstrate that the addition of 100 mM sodium
citrate
to human urine inhibited the lysis of intact cells of N. gonorrhaeae sand
maintained their
stability for minimum of l4 days at room temperature. In contrast, the cells
were not stable
in urine without sodium citrate and had the tendency to undergo lysis . prior
to
centrifugation, as indicated by decreased MOTA values >~elative-to day 0.
1i
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PATENT
P-5063PI
E~~AMPLE 3
Stability of Cells of N. ,~onorrhoeae in Urine with Other Chemical Additives
The purpose of this example was to determine whether or not other chemical
additives can maintained the stability of intact cells oFN. gonorrhoeae in
human urine.
MATERIALS
Amplification reagents and equipment were the same as described in Example 1.
BSA/PBS {0.2% bovine serum albumin, 10 mM potassium phosphate buffer [pH 7.6],
and
150 mM sodium chloride)
Negative pooled urine
CTIGC diluent (BD)
N. gonorrhoeae ATCC 19424
Sodium citrate (Sigma)
Sucrose (Sigma)
Polyvinylpyrrolidone "PVP" [average molecular weight 40,000] {Sigma) .
Magnesium chloride "MgCl2'°
Sodium fluoride (Sigma)
Sodium formate (Sigma)
Sodium borate (Sigma)
Ethylenediaminetetraacetic acid, disodium salt "EDTA" (Sigma)
Ethylenebis(oxyethylenenitrilo) tetraacetiG acid " EG'~A" (Sigma) . . .
Dynac II centrifuge (BD)
PROCEDURE
. Forty-milliliter volumes of negative urine was prepared with 100 mM sodium
citrate (final concentration); S, 10, and I5% (vlv) sucrose (final
concentration); 5~, 10, and
15% (v/v) PVP (final concentration); 5, 10, and I5 mM magnesium chloride
(final
concentration); 10 and 100 mM sodium fluoride (final concentration); l.O.and
100 mM
I2
CA 02437473 2003-08-18
PATENT
P-S063P 1
sodium formate (final concentration); 10 and 100 mM sodium borate (final
concentration);
O.S and S mM EDTA (final concentration); O.S and S mM EGTA (final
concentration); and
without chemical additive. The samples were spiked with cells of N.
gonorrhoeae at a
final concentration of 2500 cells/ml. Eacb sample was incubated for 6 days at
room
S temperature. At specific time points (day 0 and 6), the samples were
processed by
centrifugation at 2000 x g as described in Example 2. Each sample was heated
for 30
minutes at 114°C to lyse the cells. SDA was performed on the cell
lysate as described in
Example 1.
RESULTS
The data are presented in the table below as the average amplification Mt7TA
score
(eight replicates) from each processed sample for 6 days.
I3
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PATENT
P-5063P 1
Average MOTH Value
No additive 31886 2710
100 mM sodium citrate 27310 26394
5% sucrose 28427 501
10% sucrose 29548 5555
15~ sucrose 27391 8497
5% PVP 22234 4361
10% PVP 26537 2404
15% PVP 20306 'i63
mM MqCi2 28072 1507
mM MqCl2 27157 4547
mM MqCl2 35926 4868
10 mM sodium fluoride 32162 10894
100 mM sodium fluoride 26385 24559
10 mM sodium formate 34446 2211
100mM sodium formate 29357 2,139
10 mM sodium borate 26170 15464
100 rnM sodium borate 24607 28908
0.5 mM EDTA 35950 t~50
~5 mM EDTA 32392 34445
x'0.5 mM EGTA 32611 1150
5 mM EGTA 34504 6931
CONCLUSIONS
5
The data of this Example indicate that the average amplification MOTA values
for
cells of N. gonorrhoeue with 1p0 ml\rI sodiurn.citrate, 101 mlVl sodium
fluoride, 100.rnM
sodium ,borate, .and 5 mM EFTA at day 0 and 6 were not significantly
different. These
data indicate that the stability of intact cells of N. gonorrhoeae was
maintained in human
10 urine with these chemical stabilizers. Furthermore, the results demonstrate
that these
chemical stabilizers performed in a similar manner as sodium citrate to
prevent the lysis of
intact cells of N. gonorrhoeae.
14
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PATENT
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EXAMPLE 4
Stability of Cells of N. ~onorrhoeae in Urine at Different Temperatures
The purpose of this Example was to determine whether or not stability of
intact
cells of N. gonorrhoeae can be maintained in urine with sodium citrate and the
other
chemical stabilizers as described in Example 3 for 6 days at 5, 25, 35, and
45°C.
MATERIALS
Amplification reagents and equipment were the same as described in Example 1
BSA/PBS (0.2% bovine serum albumin, 10 mM phosphate buffer [pIY 7.6], and 150
mM
sodium chloride)
Negative pooled urine
CTIGC diluent (BD)
N. gonorrhoeae ATCC 19424
Sodium citrate (Sigma)
Sodium fluoride (Sigma)
Sodium borate (Sigma)
Ethylenediaminetetraacetic acid "EDTA" (Sigma)
Dynac II centrifuge
PROCEDURE
Seventy milliliter volumes of negative pooled urine was prepared with 100 mM
sodium citrate (final concentration); 100 mM sodium fluoride (final
concentration); 100
mM sodium borate .(final concentration); 5mM EDTA (final concentration); and
without
chemical additive. The samples were spiked with cells of l~T. gonorrhoeae at a
final
. . concentration of 2500 cells/ml. Each sample was incubated ,for 6 days at
5, 25, 35, and
45°C. At specific time points (day 0 and 6), the samples were processed
by centrifugation
CA 02437473 2003-08-18
PEITEIVT
P-5063P1
at 2000 x g as described in Example 2. Each sample was heated for 30 minutes
at 114°C to
lyse the cells. SDA was performed on the cell lysates as described in Ex<~mple
1.
RESULTS
The data are presented in the table below as the average MOTA value (eight
replicates) from each sample for 6 days at 5, 25, 35, and 45°C.
No additive 58904 59755 62552 1383 55505 8129 81772 14725
100 mM sodium 56773 57701 59703 51791 54238 51468 59944 63623
atrate
100 mM sodium 43807 59543 44945 47391 41608 47347 45741 48043
fluoride
100 mM sodum 41940 40325 49622 50745 60308 60296 63343 62283
borate
CONCLUSION
The data of this Example indicate that there was no significant difference in
the
average MOTA values for intact cells of N. gonorrhoeae in human urine with 100
mM
15_ ,_ sodium citrate, 100 mM sodium fluoride, 100 mM sodium borate, and 5mM
EDTA for day
0 and 6 at 5, 2S, 35, and 45°C. These results indicate that these
chemical stabilizers gave
good stabilization to the intact cells of N. gonorrhoeae in urine and
prevented cell lysis for
a minimum of 6 days. In contrast, the cells were not stable in urine without a
chemical
additive and had the tendency to undergo lysis prior tc> centrifugati'an
within 6 days at
temperatures 5, 25, 35, and 45°C; as indicated by decreased M~TA.values
relative today
0.
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EXAMPLE 5
Stabilit ~~N_gonorrhoeae DNA in Urine
The purpose of this Example was to determine whether N. gonorrhoeae plasmid
DNA can be maintain~l in human urine with the chemical additives as described
in
Example 3 for 3 days at room temperature.
MATERIALS
Amplification reagents and equipment were the described in Example 1
BDProbeTec ET Neisseria gonorrhoeae plasmid DNA (BD)
Ethylenediaminetetraacetic acid, disodium salt "EDTA" (Sigma)
Negative pooled urine
Ferrosoferric oxide (Pea Ridge Iron Ore)
Dimethyl sulfoxide "DMSO" (Sigma)
Bicine (Sigma)
Glycerol (Sigma)
Potassium hydroxide "KOH" (Sigma)
PROCEDURE
Sixty-milliliter volumes of negative pooled. urine were prep~~red with 100 mM
sodium citrate (final concentration); I00 mM sodium fluoride (final
concentration); 100
xnM sodium borate (final concentration); 10 and 20 mM EDTA (final
concentration); and
no chemical additive. The samples were incubated for 60 minutes at roam
temperature.
After the incubation step, the samples were spiked with N, gonorrhoeae plasmid
DNA at a
final concentration of 5000 DNA copies/ml. The samples were incubated for 3
days at
' room temperature. At specific time points (day 0-and-3), 2-ml volumes from
each sample
17
CA 02437473 2003-08-18
PATENT
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(eight replicates) were heated for 30 minutes at 114°C to denature the
DNA. The heat-
denatured DNA was isolated from the samples with paramagnetic particles in an
acidic
environment as described in Example I. SDA was performed on the isolated DNA
as
described in Example 1.
S
RESULTS
The results are presented in the table below as the average IdIOTA value
(eight
replicates) from each sample after incubation for 3 days at room temperature.
I0
No additive 16339 20
100 mM sodium citrate 26852 30978
100 mM sodium fluoride 27811 42689
100 mM sodium borate 23263 34089
mM EDTA 32177 30445
CONCLUSIONS
The data of this Example demonstrate that 100 mM sodium citrate, 100 mM
sodium fluoride, I00 mM sodium borate, IO_ rnM EDTA, and 20 mM EDT.A protects
N.
gonor~rhoeae plasmid DNA from degradation in urine from endogenous nucleases
for a:
minimum of three days at room temperature. In the absence of a chemical
additive in the
urine, N. gonorrhoeae plasmid DNA was rapidly degraded. ,
EXAMPLE 6
1~
CA 02437473 2003-08-18
PATENT
P-5063P1
Stability of Cells of N. gonorrhoeae in Urine with EDTA at Different
Temperatures
Over Time and Comparison of EDTA to a Commercialh~ Available Urine
Preservative
The purpose of this Example was to determine whether or not stability of
intact
S cells of N. gonorrhoeae can be maintained in urine with EDTA for 6 and 14
days at 5, 27,
33, 45 and 60°C and to compare the effectiveness of EDTA versus a
commercially
available urine preservative, DNA/RNA Protects from Sierra Diagnostics, LLC.
MATERIALS
Amplification reagents and equipment were the same as described in Example 1.
Ethylenediaminetetraacetic acid, dipotassium salt "EDTA" (Sigma)
DNA/RNA Protects (Sierra Diagnostics)
BSAIPBS (0.2 % bovine serum albumin, 10 mM potassium phosphate puffer [pI-
17.6], and
150 mM sodium chloride)
Negative pooled urine
CT/GC diluent (BD)
N. gonorrhoeae ATCC 19424
Dynac II centrifuge (BD)
PROCEDURE
i) Potassium EDTA: Seventy milliliter volumes of pooled negative urine were
prepared with .10 mM potassium EDTA (final concentration). The samples were
spiked
with cells of N. gonorrhoeae at a final concentration of 2000 cells/ml.. Each
sample was
incubated for I4 days at S, 25, 33, 4S, and 60°C. At specific time
points (day 0, 6 and 14),
the samples were processed by centrifugation at 2000 x g as described in
Example 3. Each
sample was heated for 30 minutes at 114°C to lyse the cells. SDA was
performed on the
~ cell lysates as described in Example 1.
19
CA 02437473 2003-08-18
PATENT
P-5063P 1
ii) DNA/RNA Protect: Seventy-milliliter volumes of pooled negative urine
were prepared with 10% (v/v) DNA/RNA Protects (final concentration.). The
samples
were spiked with cells of N. gonorrhaeae at a final concentration of 2000
cells/mI. Each
sample was incubated for 14 days at S, 25, 33, 45, and 60°(~. At
specific; time points (day
0, 6 and 14), the samples were processed by centrifugation at 2000 x g as
described in
Example 3. Each sample was heated for 30 minutes at 114°C to lyre the
cells. SDA was
performed on the cell lysates as described in Example 1.
RESULTS
The data are presented in the table below as fhe average IdIOTA value (eight
replicates) from each sample for 6 and 14 days at 5, 27, 33, 45 and
60°C.
CA 02437473 2003-08-18
r ~ O
~t7
M
o O ~ O
n N
O ~ N
M r
O ~
M
'-1 ~ ' ~
N
M
W ~O
m-
O
~ d'
r
a4
o O M O
IV' ~
O
r
O ~
n ~
M
N
N
~'oo N
N
G~M
I-
U coN
D
m M
m
v
M
N
O
r ~
Q M
U corn_
. . . .
. N fC~ CD
O N
M
O ~ ~
~
N ~
N
~'O a0
O CD
N O)
N CO
GO
~ M ~
U ~ c'~
~
M
~ c~u
.. _ ' '
a~ H
..
.'-. o Q
z o Q .
CA 02437473 2003-08-18
PATENT
P-5063P 1
CONCLUSION
The data of this Example demonstrate that the addition of 10 m:~I EDTA to
human
urine inhibited the lysis of intact cells of N. gonorrhoeae and maintained
their stability for
minimum of 14 days at 5, 27, 33, 45, and 60°C. In contrast, the cells.
were not stable in
urine without EDTA at 27°C and had the tendency to undergo lysis prior
to centrifugation,
as indicated by decreased MOTA values relative to day 0. Also, in the DNA/RNA
Protect-treated human urine, the cells were not stable by day 6 at 27and
33°C and by day
14 at 5 and 60°C.
While the invention has been described with some specificity, modifications
apparent to those with ordinary skill in the art may be made without departing
from the
scope of the invention. Various features of the invention are set forth in the
following
claims.
22
