DESCRIPTION
The present invention relates to a method for
selective isolation of microorganisms of interest and/or
5 of nucleic acids of interest in a liquid biological sample
containing or likely to contain, notably, numerous
untargeted cells and/or numerous nucleic acids of
untargeted cells. It also relates to the use thereof,
diagnostic tests based on a method .for isolating
10 microorganisms of interest or on a method for isolating
the nucleic acids of microorganisms of interest and kits
for isolating microorganisms of interest and/or the
nucleic acids of microorganisms of interest in a liquid
biological sample comprising or likely to comprise,
15 notably, microorganisms of interest, untargeted cells and,
optionally, debris of microorganisms of interest and/or of
untargeted cells.
The prior art consists of a certain number of
20 scientific publications and patents. Thus, in 1992, Baker
et al. described the use of a reagent called saponin for
lysing human cells differentially and isolating the
Plasmodium pathogen from whole blood. They demonstrated
that saponin with a low final concentration, of the order
25 of 0.015%, can lyse human cells in 20 pL of citratebuffered
whole blood and supply enough clean lysate for
performing PCR detection.
Patent application EP-A-0,745,849 revisited this
differential approach of using saponin for larger volumes
30 of whole blood (5 mL) with a final concentrated saponin
solution of the order of 0.020-0.125%.
Another patent application EP-A-2,185,681 proposes a
method of preparing the saponin solutions, using a more
concentrated saponin solution, i.e. with a final
2
concentration between 2 and 10% for a volume of whole
blood of average size, i.e. 5 mL. This invention proposes
using hypotonic or physiological buffers for saponin
solutions of this kind.
5
However, at present, no rapid solution is available
for isolating and identifying a very small amount of
pathogenic cells within voluminous biological samples. For
example, for septicaemia, it is necessary .to be able to
10 identify from 1 to 10 colony-forming units (CFU) of
pathogens in 10 mL of whole blood within less than 6
hours, or in the most difficult cases 1 fg of nucleic acid
of targeted pathogen in more than 800 ug of untargeted
(human) nucleic acids.
15 The problems encountered in diagnostics of infectious
diseases are therefore:
1) complexity of the matrix sample ordinarily including
very few cells of pathogens, on the one hand, and a
very large number of human cells, and other cells or
20 particles, on the other hand, which must be removed
so that they do not interfere with isolation and
identification of the pathogens, and
2) the physical limitations to isolation of a very small
number of pathogens from a very large sample volume.
25 To summarize, the challenge is to remove the maximum
amount of untargeted elements (cells and/or particles)
while retaining the maximum amount of pathogenic targets,
few of which were present initially, in order to increase
their concentration artificially and facilitate their
30 detection.
The invention described here is a method that can
isolate and identify a small number of pathogens from
large volumes of biological samples (for example of the
3
order of 10 ml) in a relatively short time. These
pathogens are, non-exhaustively, bacteria, viruses and
fungi. The invention makes it possible to identify
quantities as small as 3 CFU in 10 mL of whole blood (i.e.
5 0.3 CFU/mL), with a limit of detection never previously
described in the literature. Its novelty is based on:
1. An attractive limit of detection never attained
previously, of the order of 0.3 CFU/mL, relative to
10 the clinical requirement, which is from 0.1 to 1
CFU/mL.
2. The ability to use various whole .bloods buffered with
EDTA or with citrate, but also with heparin. Heparin
15 is a reagent that is much used in hospitals, but is
often rejected in molecular biology approaches owing
to its capacity for preventing amplification of
nucleic acids and reactions based on enzyme(s). This
invention can manage blood buffered with heparin,
20 which is not generally the case with the molecular
methods described in the literature.
3. Capacity for using large volumes of whole blood of
10 mL or more.
25
4. The use of polyethylene glycol (PEG) for improving
precipitation of the pathogens. The precipitation of
DNA or of viruses by PEG is described very well in
the literature. However, the use of PEG for
30 precipitating bacterial cells or fungi such as
.yeasts is absent from the state of the art. Since
saponin is a reagent similar to detergents, the
pathogenic cells or particles may float inside the
tube or experience difficulties in precipitating to
4
the bottom of a tube under the effect of
centrifugation. Even if it is not required for
certain treated samples, addition of PEG guarantees
precipitation of the targets that is constant
without the risk of floating. Moreover, this
addition of PEG improves the adhesion of pellets
containing pathogens on the plastic tube and the
resistance to surface washings.
5. Combining differential lysis with saponin with
treatments based on highly concentrated nucleases;
in other words, lysis of non-target cells, which
will release their nucleic acids, which on a second
occasion will be degraded by treatment 'with
nucleases. This differential lysis releases a large
amount of human nucleic acids. Typically 880 ug of
human DNA can be extracted from 10 ml of whole
blood. These human nucleic acids are the source of
considerable problems when the aim is to detect a
very low copy number of pathogenic nucleic acids:
i) they compete during purification of the
pathogenic target nucleic acids, saturating the
nucleic acid capture reagents owing to their
excess (for example silica membranes, magnetic
silica beads, chromatography or affinity
matrices, etc.),
ii) they interfere during amplifications of the
pathogenic molecules. With maximum removal of
human nucleic acids, the limits of detection are
improved. According to these critical points,
the invention uses a large amount of nucleases
to remove a maximum of human nucleic acids after
lysis with saponin. Combining saponin with a
• 5
treatment with highly concentrated nucleases has
never been described in the literature.
6. Finally, we have demonstrated that buffered saponin
5 solution, with a pH between 6 and 9, can prevent
precipitation of undesirable elements of the blood,
particularly red blood cells. Previously, scientists
have used buffers close to pH 7 for the saponin
solution, but without ever correlating the effect of
10 this pH with the stability of the sample.
To summarize, the present invention combines
different uses of chemicals for attaining a limit of
detection of pathogen compatible with the values of
detection of a clinical range (0.1 to 1 CFU/mL) . The
15 combined use of saponin with PEG and at least one nuclease
has never been described and allows a technical
improvement that was unknown hitherto. Combining of these
reagents may be complete (saponin + PEG + nuclease) or
partial (saponin + nuclease or saponin + PEG only or else
20 PEG only) for preparing the sample for analysis for
identification or for detection.
For this purpose, the present invention relates to a
method for selective isolation of microorganisms of
25 interest in a liquid biological sample comprising or
likely to comprise, notably:
•microorganisms of interest whose cell membrane or
capsid does not contain cholesterol, and
•untargeted elements, i.e.:
30 - untargeted cells whose cell membrane contains
cholesterol, and
- optionally, viruses with envelopes containing
cholesterol, and
• 6
optionally mycoplasmas containing cholesterol,
and
- optionally, debris of microorganisms of interest
and/or of untargeted cells,
5 said method comprising the following steps:
a) bringing the liquid biological sample into
contact with a saponin formulation, in order to
destabilize the cell membranes containing
cholesterol or the viral envelopes containing
10 cholesterol or the mycoplasma membranes containing
cholesterol,
b) carrying out osmotic shock of the untargeted
cells in order to lyse them specifically,
c) adding a solution of at least one enzyme able to
35 lyse the free nucleic acids (DNA and/or RNA) derived
from the untargeted elements lysed in solution in
the sample, allowing the microorganisms of interest
to be obtained selectively.
20
The present invention also covers a method for selective
isolation of microorganisms of interest in a liquid
biological sample comprising or likely to comprise,
notably:
25 •microorganisms of interest whose cell membrane or
capsid does not contain cholesterol, and
•untargeted elements, i.e.:
untargeted cells whose cell membrane contains
cholesterol, and
30 - optionally, viruses with envelopes containing
cholesterol, and
- optionally mycoplasmas containing cholesterol,
and
7
- optionally, debris of microorganisms of interest
and/or of untargeted cells,
said method comprising the following steps:
a) bringing the liquid biological sample into contact
5 with a saponin formulation, in order to destabilize
the cell membranes containing cholesterol and/or the
viral envelopes containing cholesterol and/or the
membranes of the mycoplasmas containing cholesterol,
b) performing osmotic shock of the untargeted cells in
10 . order to lyse them specifically,
c) adding an agent for precipitating the unlysed
microorganisms of interest in solution in the
sample, allowing the microorganisms of interest to
be obtained selectively.
15
According, to a third embodiment, the invention covers a
method for selective isolation of microorganisms of
interest in a liquid biological sample comprising or
likely to comprise, notably:
20 •microorganisms of interest whose cell membrane or
capsid does not contain cholesterol, and
•untargeted elements, i.e.:
- untargeted cells whose cell membrane contains
cholesterol, and
25 - optionally, viruses with envelopes containing
cholesterol, and
optionally mycoplasmas containing cholesterol,
and
- optionally, debris of microorganisms of interest
30 and/or of untargeted cells,
said method comprising the following steps:
a) bringing the liquid biological sample into contact
with a saponin formulation, in order to destabilize
the cell membranes containing cholesterol and/or the
8
viral envelopes containing cholesterol and/or the
membranes of mycoplasmas containing cholesterol,
b) performing osmotic shock of the untargeted cells in
order to- lyse them specifically,
5 c) adding an agent for precipitating the unlysed
microorganisms of interest in solution in the
sample, and
d) adding a solution of at least one enzyme able to
lyse the free nucleic acids {DNA and/or RNA) derived
10 from the untargeted elements lysed in solution in
the sample, allowing the microorganisms of interest
to be obtained selectively.
Step c) of this last-mentioned method may be carried out
15 independently of step a) , after steps a) and b) or after
step d ).
The present invention also relates to a method for
selective isolation of nucleic acids of interest in a
20 liquid biological sample comprising or likely to comprise,
notably:
•microorganisms of interest whose cell membrane or
capsid does not contain cholesterol, and
•untargeted elements, i.e.:
25 - untargeted cells whose cell membrane contains
cholesterol, and
- optionally, viruses with envelopes containing
cholesterol, and
- optionally mycoplasmas containing cholesterol,
30 and
- optionally, debris of microorganisms of interest
and/or of untargeted cells,
said method comprising the following steps:
9
a) bringing the liquid biological sample into contact
with a saponin formulation, in order to destabilize
the cell membranes containing cholesterol and/or
viral envelopes containing cholesterol and/or the
5 membranes of mycoplasmas,
b) performing osmotic shock of the untargeted cells in
order to lyse them specifically,
c) adding a solution of at least one enzyme able to
lyse the free nucleic acids {DNA and/or RNA) derived
10 from the untargeted elements lysed in solution in
the sample,
d) inactivating the enzyme added in step (c), and
e) making accessible the nucleic acids of
microorganisms of interest not degraded by the
35 enzyme of step (c) .
According to a fifth embodiment, the invention covers
a method for selective isolation of nucleic acids of
interest in a liquid biological sample comprising or
20 likely to comprise, notably:
•microorganisms of interest whose cell membrane or
capsid does not contain cholesterol, and
•untargeted elements, i.e.:
- untargeted cells whose cell membrane contains
25 cholesterol, and
- optionally, viruses with envelopes containing
cholesterol, and
optionally mycoplasmas containing cholesterol,
and
30 • - optionally, debris of microorganisms of interest
and/or of untargeted cells,
said method comprising the following steps:
a) bringing the liquid biological sample into contact
with a saponin formulation, in order to destabilize
10
the cell membranes containing cholesterol and/or the
viral envelopes containing cholesterol and/or the
membranes of mycoplasmas containing cholesterol,
b) performing osmotic shock of the untargeted cells in
5 order to lyse them specifically,
c) adding an agent for precipitating the unlysed
microorganisms of interest in solution in the
sample, and
d) making accessible the nucleic. acids of
10 microorganisms of interest not accessible by the
action of steps a) and b ) .
According to a sixth embodiment, .the invention covers
a method for selective isolation of nucleic acids of
15 interest in a liquid biological sample comprising or
likely to comprise, notably:
•microorganisms of interest whose cell membrane or
capsid does not contain cholesterol, and
•untargeted elements, i.e.:
20 - untargeted cells whose cell membrane contains
cholesterol, and
- optionally, viruses with envelopes containing
cholesterol, and
optionally mycoplasmas containing cholesterol,
25 and
- optionally, debris of microorganisms of interest
and/or of untargeted cells,
said method comprising the following steps:
a) bringing the liquid biological sample into contact
30 with a saponin formulation, in order to destabilize
'the cell membranes containing cholesterol and/or the
viral envelopes containing cholesterol and/or the
membranes of mycoplasmas containing cholesterol,
II
b) performing osmotic shock of the untargeted cells in
order to lyse them specifically,
c) adding an agent for precipitating the unlysed
microorganisms of interest in solution in the
5 sample, and
d) adding a solution of at least one enzyme able to
lyse the free nucleic acids (DNA and/or RNA) derived
from the untargeted elements lysed in solution in
the sample,
10 e) inactivating the enzyme added in step (d),
f) obtaining the nucleic acids of microorganisms of
interest:
•not degraded by the enzyme of step (d), and
•not accessible by the action of steps a) and b).
35
Step c) of this last-mentioned method may be carried out
independently of step a) , after steps a) and b) or after
step d).
20 According to a seventh aspect, the present invention
also relates to an improved method of precipitation of the
microorganisms of interest selected from bacteria and
fungi (preferably yeasts), consisting of adding at least
one precipitant, in liquid biological samples, if
25 necessary treated beforehand with saponin.
Addition of precipitant(s) makes it possible to
improve the efficiency of precipitation of the
microorganisms contained in a liquid biological sample.
30
.Whatever the embodiment, the method of isolation is
characterized in that the saponin is in a volume at least
greater than or equal to the volume of the sample at the
same concentration.
12
The final saponin concentration is above 0.02% and
less than or equal to 20%, preferably between 0.05% and
20%, more preferably between 0.5 and 20% and even more
5 preferably between 0.08 and 4%.
Osmotic shock of the untargeted cells in order to
lyse the latter specifically is a property inherent in
bringing the liquid biological sample into contact with
10 the saponin formulation as described in step a) . This
saponin formulation is used in.large volume, which has the
effect of:
- weakening or destabilizing the cell membranes containing
cholesterol, namely the membranes of the untargeted cells,
15 and at the same time
inducing an osmotic pressure, which will lead to
turgescence of the untargeted cells and lysis thereof.
In other words, steps a) and b) may be rewritten as a
single step consisting of bringing the liquid biological
20 sample into contact with a saponin formulation in order to
lyse specifically the cell membranes of the untargeted
cells containing cholesterol and the viral envelopes
containing cholesterol and the membranes of the
mycoplasmas containing cholesterol.
25
The method of isolation is also characterized in
that the saponin consists of a triterpenoid.
Saponin is specific to membranes or envelopes containing
cholesterol.
30
In the context of the method for isolating
microorganisms of interest and of the improved method of
precipitation of microorganisms of interest according to
the invention, the precipitant used is selected from PEG,
13
glycogen and the nucleic acids (DNA/tRNA etc.). It is also
possible to use a mixture thereof.
The precipitant preferably used is PEG.
5 In the context of the method for isolating nucleic
acids of microorganisms of interest, the precipitant may
consist of polyethylene glycol (PEG).
The concentrations of precipitant ,. used in the
10 context of the invention are between 0.1% and 20%,
preferably between 1% and 20%.
At the end of the method . for isolating the
microorganisms of interest, whatever the embodiment
15 thereof, it is possible to treat the medium obtained for
further isolation of the microorganisms of interest, thus
allowing detection thereof. This is done using
conventional cellular techniques, for example cytology
techniques (flow cytometry or others), immunology
20 techniques (technology with antibodies or phages for
detection by immunoassay) or classical cell culture
techniques.
Thus, it is possible to apply the medium obtained for
conventional culture on a suitable medium, preferably on a
25 solid medium (for example a nutrient agar medium) in a
Petri dish for at least 24h, for example 48h for yeasts,
at a suitable temperature, for example 30CC for yeasts,
preferably 37 °C, or any medium suitable for growth of the
microorganisms of interest. Any known technique and
30 protocol allowing growth of the microorganisms and then
individualization/isolation thereof may also be used.
14
10
According to certain variants of carrying out the
method of isolation, the enzyme able to lyse the free
nucleic acids is an enzyme which is then inactivated:
•chemically by adding EDTA and/or EGTA and/or DTT
and/or ' p-mercaptoethanol and/or DEPC, and/or
guanidine, and/or
•physically by increasing the temperature between 4 0
and 100 °C in the presence or in the absence of
detergents, such as sodium dodecyl sulphate (SDS).
in cases when the method of isolation according to
the invention .only comprises a single enzyme able to lyse
the free nucleic acids (DNA and/or RNA) derived from the
untargeted elements lysed in solution in the sample, this
15 enzyme is a DNase.
According to certain variants of carrying out the
method of isolation where at least one enzyme is added
that is able to lyse the free nucleic acids, i.e. during
20 step (c) when the method used does not comprise a step of
adding precipitant or during step (d) when the method
comprises a step of adding precipitant, the enzyme able to
lyse the free nucleic acids is an enzyme that may be
inactivated:
25 •chemically by adding EDTA and/or EGTA and/or DTT
and/or [3-mercaptoethanol and/or DEPC, and/or
guanidine, and/or
•physically by increasing the temperature between 4 0
and 100 °C in the presence or in the absence of
30 detergents, such as sodium dodecyl sulphate (SDS).
In the methods for isolating microorganisms of
interest and for isolating nucleic acids of microorganisms
of interest according to the invention comprising a step
15
of adding precipitant and a step of adding at least one
enzyme able to lyse the free nucleic acids (DNA and/or
RNA) derived from the untargeted elements lysed in
solution in the sample, these two steps do not have a
5 defined order and may be reversed in the execution of the
method.
In the context of the method for isolating nucleic acids
of microorganisms of interest according to the invention
comprising a step of inactivating the enzyme able to lyse
10 the free nucleic acids, the precipitant may be added after
this inactivation step.
The invention also relates to a .method for isolating
microorganisms of interest or for isolating nucleic acids
15 of microorganisms of interest or for precipitating
microorganisms of interest, in a liquid biological sample,
preferably of blood,_ characterized in that during the
method, the pH is maintained in a range between 5 and 10,
preferably between 6 and 9, by adding a solution that is:
20 »basic if the pH is below 5, preferably below 6,
•acidic if the pH is above 10, preferably above 9,
so that the pH is within the range.
According to a variant of use, the invention uses a
25 saponin formulation leading to a final concentration above
0.02% and less than or equal to 20%, preferably above
0.05% and less than or equal to 20% and/or a precipitating
agent (or precipitant) at a concentration from 0.1 to 20%,
preferably 0.1 to 4%, even more preferably from 0.5 to 4%,
30 and/or an enzyme able to lyse the free nucleic acids (DNA
and/or RNA) containing between 500 and 20000 enzyme units.
According to another of its aspects, the invention
covers the use of a saponin formulation and of a solution
16
of at least one enzyme able to lyse the free nucleic acids
(DNA and/or RNA) and optionally at least one precipitant,
preferably selected from PEG, glycogen and the nucleic
acids (preferably DNA, tRNA), for isolation of
5 microorganisms of interest or of nucleic acids of
microorganisms of interest, in a liquid biological sample.
According to another of its aspects, the present
invention covers the use of PEG for precipitation of
10 microorganisms of interest, preferably of bacteria and
fungi (preferably yeasts), in' liquid biological samples,
if necessary treated beforehand with saponin.
The invention further relates to a diagnostic test
15 based on a method for isolating microorganisms of
interest, as described above, or on the uses for isolation
of the microorganisms of interest, as described above, or
on a method of precipitation of microorganisms as
described above.
20
The invention also relates to a diagnostic test based
on a method for isolating nucleic acids of microorganisms
of interest, as described above, or on the uses for
isolation of the nucleic acids of microorganisms of
25 interest, as described above.
The diagnostics may be carried out by conventional
techniques known by a .person skilled in the art,
employing, for example, classical techniques of detection
30 used in microbiology, immunoassay techniques or classical
molecular biology techniques such as PCR, NASBA etc.
The invention also relates to a diagnostic kit for
isolating microorganisms of "interest in a liquid
17
biological sample comprising or likely to comprise,
notably, microorganisms of interest, untargeted cells and,
optionally, viruses with envelopes, mycoplasmas and/or
debris of microorganisms of interest and/or of untargeted
5 cells, said kit comprising:
(a) a container, and
(b) at least one saponin formulation, and
(c) at least one solution of a precipitant, such as
polyethylene glycol (PEG).
10
The invention finally relates to a diagnostic kit for
isolating the nucleic acids of microorganisms of interest
in a liquid biological sample comprising or likely to
comprise, notably, microorganisms of interest, untargeted
15 cells and, optionally, enveloped viruses, mycoplasmas
and/or debris of microorganisms of interest and/or of
untargeted cells, said kit comprising:
(a) a container, and
(b) at least one saponin formulation, and
20 (c) at least one solution of a precipitant, such as
polyethylene glycol (PEG).
The two kits described above may further comprise
(d) at least one solution of at least one enzyme able to
25 lyse the nucleic acids.
The kit further comprises:
(c) or (d1) at least one acid solution and/or at
least one basic solution, and/or
30 (d) EDTA and/or EGTA and/or DTT and/or (3-
mercaptoethanol and/or DEPC, and/or guanidine and/or
(e) at least one detergent or an anionic agent.
18
In the present description, the enzyme or enzymes
allowing lysis of the nucleic acids are enzymes that are
known and are used conventionally by a person skilled in
the art, such as DNAse I, RNAself etc. , and the step
5 consisting of making accessible the nucleic acids of the
microorganisms of interest is carried out in a
conventional manner that is known by a person skilled in
the art, for example by mechanical lysis and then
purification of the nucleic acids and PCR amplification or
10 any other known technique.
In the rest of this patent application, the terms
used have the following definitions:
- "Viral envelope" means the envelope of enveloped
15 viruses, which contains cholesterol, in contrast to
the capsid of capsid viruses, which does not contain
cholesterol. The viral envelope is therefore sensitive
to saponin.
- "Destabilize the cell membranes and/or viral envelopes
20 and/or the membranes of mycoplasmas" means the
physicochemical mechanisms leading to a loss of
regulation of osmolarity at the membrane level and/or
the level of the viral envelope and/or of the membrane
of a mycoplasma, disturbance of membrane transport and
25 the potential appearance of pores at the level of the
cell membrane, mycoplasma membrane or viral envelope.
- The term "microorganisms of interest" comprises all
microorganisms that do not contain accessible
cholesterol, which are potentially pathogenic, notably
30 for humans. These microorganisms include viruses (with
the exception of the enveloped viruses), bacteria,
fungi (yeasts), but also microscopic animals.
- "Nucleic acids of interest" correspond to the nucleic
acids (DNA and RNA) contained in the cells or
19
particles of the "microorganisms of interest" defined
above.
- "Untargeted cells" are to be understood as all cells
of living organisms that do not contain "nucleic acids
5 of interest" as defined above.
- The abbreviation "EDTA" corresponds to
ethylenediaminetetraacetic acid.
- The abbreviation "EGTA" corresponds to ethylene glycol
tetraacetic acid.
10 - The abbreviation "DTT" corresponds to dithiothreitol.
- The abbreviation "DEPC" corresponds to
diethylpyrocarbonate.
- CFU stands for colony-forming unit..
- The term "detergents" means all classes of molecules
15 that may induce a physicochemical modification of
other molecules. These detergents may be of a chemical
nature such as SDS, Tween-20, Triton X-100, brij97, or
may be enzymatic.
- "Liquid biological sample" is to be understood as a
20 liquid sample that may contain the "microorganisms of
interest" selected from the following group: amniotic
fluid, aqueous humour, bile, blood, mammary secretion,
bronchoalveolar wash, cerebrospinal fluid, chyle,
chyme, faeces, interstitial fluid, lymph, menstrual
25 fluid, mucus, plasma, pleural fluid, pus-, saliva,
sebum, sperm, serum, sputum, sweat, synovial fluid,
tears, urine and vitreous humour.
Moreover, it has to be clear that the method for selective
30 isolation of nucleic acids of interest as described above
and the use thereof allow isolation of the microorganisms
of interest, to the extent that the nucleic acids of
interest correspond to the nucleic acids (DNA and/or RNA)
contained in the cells or particles of the microorganisms
' 20
of interest. In other words, selective isolation of the
nucleic acids of interest allows isolation of the
microorganisms of interest from which the nucleic acids of
interest are derived.
5
The accompanying examples are presented for
demonstrating the efficacy of the method according to the
invention and are given for purposes of illustration and
are not exhaustive.
10
Example 1: Detection of 5 and 10 CFU of Pseudomonas
aeruginosa, in 10 mL of whole blood treated with 4% saponin
solution
15 In a 50-mL plastic tube, 2 0 mL of whole blood treated with
EDTA was inoculated with 20, 10, 2 and 0 (negative
control) CFU of Pseudomonas aeruginosa. The number of CFU
inserted in the blood was verified by streaking on agar
media in a Petri dish. The 20 mL of inoculated blood was
20 divided into two and each 10-mL volume was deposited in
two 50-mL plastic tubes to provide duplicates. Forty
millilitres of filtered 4% saponin solution, 50 mM Tris-
HC1 at pH 8.0 and 4% of PEG-8000 were added to the
inoculated blood. The tubes were agitated by inverting
25 twice, incubated at room temperature for 5 minutes and
centrifuged at 12000 g for 10 minutes. The supernatant was
removed and the adhering pellet was washed three times
with 15 mL of 4% PEG-8000, which prevents detachment of
the pellet. A volume of 200 pL of 10 mM Tris-HCl at a pH
30 of 7.5 was added to the pellet.
Next, '10 uL of DNAse I (500 u/ul; Roche) and 2 uL RNAse If
(50 u/uL; New England Bio labs) were put in the tubes. The
pellets were digested with the ( enzymes for 10 minutes,
agitating twice by vortexing after 2 minutes and 4 minutes
2J
of incubation. After digestion with the nucleases, 10 uL
of EDTA at 0.5 M and pH 8.0 .was added to the tubes. The
samples were transferred to microtubes with a capacity of
1.5 mL, containing 200 mg of glass beads with a diameter
5 of 1 mm and 50 mg of zirconium beads with a diameter of
0.1 mm. The microtubes were heated for 10 minutes at 80°C.
Then 20 uL of proteinase K (Novagen) and 40 uL of 10% SDS
were added to the tubes. The microtubes were incubated for
5 minutes at room temperature and heated for 5 minutes at
10 80°C.
Mechanical lysis of the Pseudotaonas cells was performed by
agitating the tubes containing the beads for 20 minutes
using a vortex. The DNA present in the lysis supernatant
was purified using the Nucleospin Blood® kit from Macherey-
15 Nagel. Quantitative PCR amplification was performed using
the full eluate, or 40 ul. The samples with 5 CFU of
Pseudomonas aeruginosa were detected well on a replica by
means of the invention, and partial detection of one
replica out of two was observed for 1 CFU inserted, as is
20 quite clear from Table I below:
22
Table I: Evaluation of the limit of detection for
Pseudomonas aeruginosa using the method according to the
invention
5 Example 2: Detection of 28 and 140 CFU of Candida albicans
in 10 mL of whole blood treated with 4% saponin solution
In a 50-mL plastic tube, 20 mL of whole blood treated with
EDTA was inoculated with 140, 28 and 0 (negative control)
10 CFU of Candida albicans. The number of CFU inserted in the
blood was verified by streaking on agar media in a Petri
dish. The 20 mL of blood inoculated was divided into two
and each 10-mL volume was deposited in two 50-mL plastic
tubes to provide duplicates. Forty millilitres of filtered
15 4% saponin solution, 50 mM Tris-HCl at pH 8.0 and. 4% of
PEG-8000 were added to the inoculated blood. The tubes
were agitated by inverting twice, incubated at room
temperature for 5 minutes and centrifuged at 12000 g for
10 minutes. The supernatant was removed and the pellet was
20 washed three times with 15 mL of 4% PEG-8000, which
prevents detachment of the pellet. A volume of 200 uL of
10 mM Tris-HCl at a pH of 7.5 was added to the pellet.
Next, 10 uL of DNAse I (500 u/pL; Roche) and 2 pL RNAse If
(50 p/pL; New England Biolabs) were put in the tubes. The
25 pellets were digested with the enzymes for 10 minutes,
agitating twice by vortexing after 2 minutes and 4 minutes
of incubation. After digestion with the nucleases, 10 uL
of EDTA at 0.5 M and pH 8.0, a volume of 40 pi of 10% SDS
and 20 pi of proteinase K were added to the tubes. The
30 samples were transferred to microtubes with a capacity of
1.5 mL, containing 200 mg of glass beads with a diameter
of 1 mm and 50 mg of zirconium beads with a diameter of
0.1 mm. The microtubes were heated for 60 minutes at 80°C.
23
Mechanical lysis of the Candida albicans cells was
performed by agitating the beads for 20 minutes using a
vortex. The DNA present in the lysis supernatant was
purified using the Nucleospin Blood® kit from Macherey-
5 Nagel and purified a second time using the gDNA Clean-up
XS® kit from Macherey-Nagel. Quantitative PCR amplification
was performed using the full eluate, i.e. 4 0 uL. The
samples with 2 8 CFU of Candida albicans were detected on a
replica by means of the invention, as is clearly shown in
10 Table 2 below:
Table 2: Detection of Candida albicans using the method
15 according to the invention
Example 3: Detection of 20000 virions of human Adenovirus
5 in 10 mL of whole blood treated with 4% saponin solution
20 In a 50-mL plastic tube, 10 mL of whole blood treated with
EDTA was inoculated with 2 0000 virions of human Adenovirus
5. Forty millilitres of filtered 4% saponin solution, with
50 mM Tris-HCl at pH 8.0 and 4% of PEG-8000 were added to
the inoculated blood. The tubes were agitated by inverting
25 twice, incubated at room temperature for 5 minutes and
24
centrifuged at 12000 g for 10 minutes. The supernatant was
removed and the pellet was washed three times with 15 mL
of 4% PEG-8000, which prevents detachment of the pellet. A
volume of 200 uL of Tris-HCl at 10 mM and at a pH of 7.5
5 was added to the pellet. Next, 10 pL of DNAse I (500 p/pl;
Roche) and 2 pL RNAse If (50 u/uL; New England Biolabs)
were put in the tubes. The pellets were digested with the
enzymes for 10 minutes, agitating twice by vortexing after
2 minutes and 4 minutes of incubation. After digestion
10 with the nucleases, 10 uL of 0.5 M EDTA, pH 8.0 and 40 pi
of 10% SDS were added to the tubes. The samples were
transferred to microtubes with a capacity of 1.5 ml,
containing 200 mg of glass beads with a diameter of 1 mm
and 50 mg of zirconium beads with a diameter of 0.1 mm.
15 The microtubes were heated for 10 minutes at 80°C.
Mechanical lysis of the virions was performed by agitating
the tubes containing the beads for 20 minutes using a
vortex. The DNA present in the lysis supernatant was
purified using the Nucleospin Blood® kit from Macherey-
20 Nagel. The nucleic acids were eluted with 40ul.
Quantitative PCR amplification was performed using 10 pi
of eluate. The 20000 virions were detected well by means
of the invention, see Table 3 below;
Table 3: Evaluation of the limit of detection of
human Adenovirus 5 by means of the invention
Example 4: Efficacy of lysis of human blood cells with
30 various concentrations of saponin
25
In a 50-mL plastic tube, 10 mL of whole blood treated with
EDTA was inoculated with 24 and 0 (negative control) CFU
of Pseudomonas aeruginosa. The number of CFU inserted in
the blood was verified by streaking on agar media • in a
5 Petri dish. Forty mi11Hitres of filtered saponin
solution, 50 mM Tris-HCl at pH 8.0 and 4% of PEG-8000 were
added to the inoculated blood. The final saponin
concentrations were 0.005%, 0.02%, 0.08% and 0.4%. Each
concentration was tested in duplicate. The tubes were
10 agitated by inverting three times, incubated at room
temperature for 10 minutes and centrifuged at 12000 g for
10 minutes. The supernatant was removed and the adhering
pellet was washed three times with 15 mL of 4% PEG-8000,
which prevents detachment of the pellet. A volume of
15 400 pL of 10 mM Tris-HCl at a pH of 7.5, 2.5 mM MgCl2,
0.5 mM CaCl2, DNAse I (Roche) 5000u, RNAse If (New England
Biolabs) 10Ou was added to the pellet. The pellets were
incubated at 32°C with stirring for 90 minutes. The
samples were transferred to microtubes containing 10 pL of
20 0.5 M EDTA at pH 8.0 and 400 pi of buffer B3 (Macherey-
Nagel) . The microtubes were heated for 10 minutes at 80°C
and then cooled for 5 min in ice. 5 pg of lysine was added
to the tubes. The microtubes were incubated for an
additional 10 minutes in ice and then were treated using
25 the Nucleospin Blood® kit from Macherey-Nagel according to
the supplier's conditions but doubling the amounts of
proteinase K and of ethanol, in keeping with the ratios of
the reagents. Quantitative PCR amplification was performed
using 2pl of eluate for detecting human DNA and 38 pL for
30 the PCRs for detecting the DNA of Pseudomonas aeruginosa.
Most of the tubes were positive for Pseudomonas aeruginosa
(the negative controls, uninoculated bloods, treated with
0.4% of saponin being definitely negative). The example
26
shows that it is possible to detect Pseudomonas aeruginosa
for saponin concentrations well below 0.4%.
The amplifications for the final saponin concentrations
greater than or equal to 0.08% did not succeed in
5 detecting human DNA or extremely small quantities (Table
4). In contrast, for the final saponin concentrations less
than or equal to 0.02%, the amounts of human DNA detected
are very high (Cq around 28). This demonstrates that at a
final saponin concentration less than or equal to a value
10 of 0.02%, the white blood cells are no longer correctly
lysed whereas they are lysed very well with a final
saponin concentration of 0.08% or 0.4%.
15
27
Example 5: Detection of 21 CFU of Pseudomonas aeruginosa
in 10 mL of whole blood treated with 0.4% saponin solution
In a 50-mL plastic tube, 10 mL of whole blood treated with
5 EDTA was inoculated with 21 (5 tubes) and 0 {negative
control, 1 tube) CFU of Pseudomonas aeruginosa. The number
of CFU inserted in the blood was verified by streaking on
agar media in a Petri dish. Forty millilitres of filtered
0-. 4% saponin solution, 50 mM Tris-HCl at pH 8.0 and 4% of
10 PEG-8000 were added to the inoculated blood. The tubes
were agitated by inverting three times, incubated at room
temperature for 10 minutes and centrifuged at 12000 g for
10 minutes. The supernatant was removed and the adhering
pellet was washed three times with 15 mL of 4% PEG-8 000,
15 which prevents detachment of the pellet. A volume of
800 pL of 10 mM Tris-HCl at a pH of 7.5, 2.5 mM MgCl2,
0.5 mM CaCl2, DNAse I (Roche) 5000u, RNAse If (New England
Biolabs) lOOu was added to the pellet. The pellets were
incubated at 32 °C with stirring for 90 minutes. 10 pL of
20 0.5 M EDTA at pH 8.0 and 400 pi of buffer B3 (Macherey-
Nagel) were added and then the tubes were heated for 10
minutes at 80°C and were then cooled for 5 min in ice.
5 pg of lysine was added to the tubes. The tubes were
incubated for an additional 10 minutes in ice' and then
25 were treated using the Nucleospin Blood® kit from Macherey-
Nagel according to the supplier's conditions but
quadrupling the amounts of proteinase K and of ethanol, in
keeping with the ratios of the reagents. Quantitative PCR
amplification was performed using 2pl of eluate for
30 detecting human DNA and 38 pL for the PCRs for detecting
the DNA of Pseudomonas aeruginosa.
*: no fluorescence signal was detected after 50 amplification cycles.
¥able_5: Quantitative PCR for detecting the DNA of P.
5 aeruginosa.
Example 6: Detection by growing 117 CFU of Candida
albicans on solid medium starting from 10 mL of whole
10 blood treated with 4% saponin
In a 50-mL plastic tube, 10 mL of whole blood treated with
EDTA was inoculated with 117 CFU of Candida albicans. The
number of CFU inserted in the blood was verified by
15 streaking on agar media in a Petri dish. Forty millilitres
of filtered 4% saponin solution, 50 mM Tris-HCl at pH 8.0
and 4% of PEG-8000 were added to the inoculated blood. The
tubes were agitated by inverting twice, incubated at room
temperature for 5 minutes and centrifuged at 12000 g for
20 10 minutes. The pellets obtained were resuspended with
212ul of Tryptone salt (AES) or 212 ul of mix of nucleases
(Tris 10 mM pH7.5; DNAse I (Roche) 5000 u; RNAse If (New
England Biolabs) lOOu), incubated for 10 minutes at room
temperature and then streaked on"a solid medium in a Petri
29
dish SDC (bioMerieux) . The number of colonies was then
counted after incubating at 30 °C for 48 hours.
Table 6 shows that on average 66% of CFU were found after
treatment with saponin with or without treatment with
5 nucleases.
*: no fluorescence signal was detected after 50 amplification cycles.
10 Table 6: Petri dish count of colonies of Candida, albicans
after treatment with 4% saponin
15 Example 7: Facilitating effect of PEG on the precipitation
by centrifugation of Pseudomonas aeruginosa present in
blood
200ul of whole blood treated with EDTA was distributed in
20 a 1.5-mL plastic tube. 1 mL of 10 mM MgCl2 or 1 mL of MgCl2
supplemented to final 4% of PEG was added to the tubes.
Next, these tubes were inoculated with 12 9 CFU of
Pseudomonas aeruginosa. The tubes were vortexed for 5
seconds and then centrifuged for 10 minutes at 5000g. The
25 pellet's were then re suspended with lOOpl of Tryptone salt
and then streaked on solid medium in a Petri dish TSA
30
(bioMerieux) . The dishes were incubated for 24h at 37 °C
prior to counting.
Addition of PEG contributed to a 23.5% improvement in
precipitation yield (Table 7).
5
*: no f l u o r e s c e n c e s i g n a l was d e t e c t e d a f t e r 50 a m p l i f i c a t i o n c y c l es
Table 7: P e t r i d i s h count of c o l o n i e s of Pseudomonas
aeruginosa a f t e r c e n t r i f u g a t i o n with or without the
10 presence of PEG

CLAIMS
1. Method for selective isolation of microorganisms of
interest in a liquid biological sample comprising or
5 likely to comprise, notably:
•microorganisms of interest whose cell membrane or
capsid does not contain cholesterol, and
•untargeted elements, i.e.:
- untargeted cells whose cell membrane contains
10 cholesterol, and
- optionally, viruses with envelopes containing
cholesterol, and
optionally mycoplasmas containing cholesterol,
and
15 - optionally, debris of microorganisms of interest
and/or of untargeted cells,
said method comprising the following steps:
a) bringing the liquid biological sample into
contact with a saponin formulation, in order to
20 destabilize the cell membranes containing
cholesterol or the viral envelopes containing
cholesterol or the membranes of mycoplasmas
containing cholesterol,
b) performing osmotic shock of the untargeted cells
25 in order to lyse them specifically,
c) adding a solution of at least one enzyme able to
lyse the free nucleic acids (DNA and/or RNA) derived
from the untargeted elements lysed in solution in
the sample, allowing the microorganisms of interest
30 to be obtained selectively.
2. Method for selective isolation of microorganisms of
interest in a liquid biological sample comprising or
likely to comprise, notably:
32
•microorganisms of interest whose cell membrane or
capsid does not contain cholesterol, and
•untargeted elements, i.e.:
- untargeted cells whose cell membrane contains
5 cholesterol, and
- optionally, viruses with envelopes containing
cholesterol, and
- optionally mycoplasmas containing cholesterol,
and
10 - optionally, debris of microorganisms of interest
and/or of untargeted cells,
said method comprising the following steps:
a) bringing the liquid biological sample into
contact with a saponin formulation, in order to
15 destabilize the cell membranes containing
cholesterol and/or the viral envelopes containing
cholesterol and/or the membranes of the mycoplasmas
containing cholesterol,
b) performing osmotic shock of the untargeted cells
20 in order to lyse them specifically,
c) adding an agent for precipitating the unlysed
microorganisms of interest in solution in the
sample, allowing the microorganisms of interest to
be obtained selectively.
25
3. Method for selective isolation of microorganisms of
interest in a liquid biological sample, according to Claim
1, further comprising a step consisting of adding an agent
for precipitating the unlysed microorganisms of interest
30 in solution in the sample, said step being carried out in
step a), after steps a) and b) or after step c ).
33
4. Method for selective isolation of nucleic acids of
interest in a liquid biological sample comprising or
likely to comprise, notably:
•microorganisms of interest whose cell membrane or
5 capsid does not contain cholesterol, and
•untargeted elements, i.e.:
- untargeted cells whose cell membrane contains
cholesterol, and
- optionally, viruses with envelopes containing
10 cholesterol, and
optionally mycoplasmas containing cholesterol,
and
- optionally, debris of microorganisms of interest
and/or of untargeted cells,
15 said method comprising the following steps:
a) bringing the liquid biological sample into contact
with a saponin formulation, in order to destabilize
the cell membranes containing cholesterol and/or the
viral envelopes containing cholesterol and/or the
20 membranes of mycoplasmas containing cholesterol,
b) carrying out osmotic shock of the untargeted cells
in order to lyse them specifically,
c) adding a solution of at least one enzyme able to
lyse the free nucleic acids (DNA and/ or RNA) derived
25 from the untargeted elements lysed in solution in the
sample,
d) inactivating the enzyme added in step (c) , and
e) making accessible the nucleic acids of
microorganisms of interest not degraded by the enzyme
30 of step (c) .
5. Method for selective isolation of nucleic acids of
interest in a liquid biological sample comprising or
likely to comprise, notably:
34
•microorganisms of interest whose cell membrane or
capsid does not contain cholesterol, and
•untargeted elements, i.e.:
- untargeted cells whose cell membrane contains
5 cholesterol, and
- optionally, viruses with envelopes containing
cholesterol, and
- optionally mycoplasmas containing cholesterol,
and
10 - optionally, debris of microorganisms of interest
and/or of untargeted cells,
said method comprising the following steps:
a) bringing the liquid biological sample into
contact with a saponin formulation, in order to
15 destabilize the cell membranes containing
cholesterol and/or the viral envelopes containing
cholesterol and/or the membranes of the mycoplasmas
containing cholesterol,
b) carrying out osmotic shock of the untargeted
20 cells in order to lyse them specifically,
c) adding a precipitant of the unlysed
microorganisms of interest in solution in the
sample, and
d) making accessible the nucleic acids of
25 microorganisms of interest not accessible by the
action of steps a) and b).
6. Method for selective isolation of nucleic acids of
30 interest in a liquid biological sample, according to Claim
4, further comprising a step consisting of adding an agent
for precipitating the unlysed microorganisms of interest
in solution in the sample, said step being carried out in
35
step a), after steps a) and b) or after step c) or after
step d).
7. Method of isolation according to any one of Claims 1
5 to 6, characterized in that the saponin is in a volume at
least greater than or equal to the volume of the sample at
the same concentration.
8. Method of isolation according to any one of Claims 1
10 to 7, characterized in that the final saponin
concentration is above 0.02% .and less than or equal to
20%.
9. Method of isolation according to any one of Claims
15 2, 3, 5 to 8, characterized in that the precipitant
consists of polyethylene glycol (PEG).
10. Method of isolation according to any one of Claims 1
to 9, characterized in that the saponin consists of a
20 triterpenoid.
11. Method of isolation according to any one of Claims
1, 3, 4, 6 to 10, characterized in that the enzyme able to
lyse the free nucleic acids is then inactivated:
25 •chemically by adding EDTA and/or EGTA and/or DTT
and/or p-mercaptoethanol and/or DEPC and/or
guanidine
and/or
•physically by increasing the temperature between 40
30 and 100°C in the presence or in the absence of
detergents, such as sodium dodecyl sulphate (SDS).
12. Improved method of precipitation of the
microorganisms of interest selected from bacteria and
36
fungi, consisting of adding at least one precipitant, in
liquid biological samples, if necessary treated beforehand
with saponin.
5 13. Method according to Claim 12, characterized in that
the precipitant is selected from PEG, glycogen, the
nucleic acids.
14. Method according to any one of Claims 1 to 13,
10 characterized in that during the method, the pH is
maintained in a range between 5 and 10, preferably between
6 and 9, by adding a solution that is:
•basic if the pH is below 5, preferably below 6,
•acidic if the pH is above 10, preferably above 9,
15 so that the pH is within the range.
15. Use of a saponin formulation and of a solution of at
least one enzyme able to lyse the free nucleic acids (DNA
and/or RNA) and optionally of at least one precipitant for
20 isolation of microorganisms of interest or of nucleic
acids of microorganisms of interest, in a liquid
biological sample.
16. Use according to Claim 15, characterized in that it
25 uses a saponin formulation leading to a final
concentration above 0.02% and less than or equal to 20%,
and/or a precipitant at a concentration from 0.1 to 20%,
and/or an enzyme able to lyse the free nucleic acids (DNA
and/or RNA) containing between 500 and 20000 enzyme units.
30
17. Use of PEG and/or of glycogen and/or of nucleic acids
for precipitation of microorganisms of interest in
biological samples, the sample being brought into contact
with the saponin beforehand, if necessary.
37
18. Diagnostic test based on a method according to any one
.of Claims 1 to 14, or on a use according to any one of
Claims 15 to 17.
5
19. Diagnostic kit .for isolating the microorganisms of
interest and/or for isolating the nucleic acids of
microorganisms of interest in a liquid biological sample
comprising or likely to comprise, notably, microorganisms
10 of interest, untargeted cells and, optionally, enveloped
viruses, mycoplasmas and/or debris of microorganisms of
interest and/or of untargeted cells, said kit comprising:
(a) a container, and
(b) at least one saponin formulation, and
15 (c) at least one solution of a precipitant, such as
polyethylene glycol (PEG).
20. Kit, according to Claim 19, further comprising:
(d) at least one enzyme able to lyse the nucleic
20 acids.
21. Kit, according to any one of Claims 19 to 20,
characterized in that the kit further comprises:
d or d' ) at least one acid solution and/or at least
25 one basic solution, and/or
e) EDTA and/or EGTA and/or DTT and/or [3-
mercaptoethanol and/or DEPC and/or guanidine and/or
f) 'at least one detergent or an anionic agent.